Anti-CTLA-4 antibody for treatment of patients with prostate cancer and combination therapy with a radioligand therapeutic agent

Abstract

The invention provides an anti-CTLA-4 antibody or a nucleic acid encoding the anti-CTLA-4 antibody for use in treating prostate cancer in a subject, optionally in combination with a radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan. The invention further provides treatment methods for prostate cancer in a subject in need thereof, comprising administering the anti-CTLA-4 antibody or the nucleic acid encoding the anti- CTLA-4 antibody, optionally in combination with a radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan.

Description

[0001] Anti-CTLA-4 antibody for treatment of patients with prostate cancer and combination therapy with a radioligand therapeutic agent

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an antibody having the ability to bind to CTLA-4 for the treatment of prostate cancer as well as a method of treating prostate cancer comprising the anti-CTLA antibody. The invention further provides a medical preparation comprising the anti-CTLA antibody and optionally a radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan. The invention further relates to a method of administering the anti- CTLA antibody, a combination therapy, and a method of treating cancer in a subject in need thereof as well as the anti-CTLA antibody for use in these methods.

[0004] BACKGROUND OF THE INVENTION

[0005] Prostate cancer (PCa) is the second most common cancer among males and the second- leading cause of death in man. In 2012 an estimated 1.1 million men worldwide were diagnosed with the disease, 70% (759,000) in (more) developed countries. Surgical or medical castration represent a major advance in prostate cancer care. Nevertheless, most tumors will eventually develop into castration-resistant prostate cancer (CRPC) or metastatic castration-resistant prostate cancer (mCRPC). mCRPC is the final stage of PCa progression and the major cause of death. Effective treatment to prostate cancer represents a significant unmet medical need.

[0006] Once it is developed, patients commonly only have the choice between cytotoxic chemotherapy, abiraterone acetate, enzalutamide, SiPuleucel-T, radium-223 or PARP inhibitors to prolong survival. However, these therapies are still associated with certain drawbacks, such as poor tissue selectivity, high systemic toxicity and drug resistance. Survival benefit is generally limited to less than 6 months, highlighting the need for novel therapies to sustain response. In recent years, targeted tumor therapy has exhibited significant superiority to traditional therapies. Progress has been made in PCa treatment based on prostate-specific membrane antigen (PSMA), which is expected to achieve a more accurate treatment of PCa.

[0007] PSMA, a transmembrane glycoprotein located on the cell membrane, is expressed at an extremely low level in non-prostatic tissues such as lacrimal gland, nervous system, duodenum, and in normal prostatic tissues. However, its expression in PCa tissues increases by 100-1000 times compared with that in normal tissues. Particularly, the expression level is much higher in the poorly differentiated, metastatic, and castration-resistant PCa tissues.

[0008] Recently, the radioligand therapeutic lutetium177LU vipivotide tetraxetan (marketed as PLUVICTO® by Novartis AG) has been approval for treatment of PSMA-positive prostate cancer, specifically metastatic castration-resistant prostate cancer. It is also known as177Lu vipivotide tetraxetan, which, when used in combination with physician’s choice of care, showed improvement in both progression-free survival (8.7 vs 3.4 months) and overall survival (15.3 vs 11.3 months). By binding to PSMA-expressing tumor cells,177Lu vipivotide tetraxetan represents a PSMA-positive prostate cancer-targeting radiotherapy. Radioligand therapy (RTL) is a treatment by injecting a certain therapeutic dose of radionuclide-labeled ligand into the body; after specifically binding to the targeted cells, the radionuclide releases alpha (a) particles, beta (0) particles or Auger electrons, which act on biological macromolecules and produce free radicals, thus inducing DNA single- or double-strand break to achieve aging, apoptosis or necrosis of targeted cells. Different from conventional external radiotherapy, RLT targets diseases at a cellular level rather than at a gross anatomical level.

[0009] The development highlights the potential of targeted radiation therapy. Radiotherapy increases regulatory T cells which represent a major mechanism of tumor evasion of host immunity. Preclinical models have demonstrated that local radiotherapy selectively expands and functionally activate regulatory T cells in the tumor microenvironment.

[0010] Despite extensive effort, no immunotherapy has been approved for prostate cancer. An initial phase 3 study of the anti-CTLA-4 antibody ipilimumab 10 mg / kg versus placebo after a single dose of palliative radiation therapy in post-chemotherapy mCRPC revealed a trend towards an overall survival advantage with an increase in long-term responders in the ipilimumab arm. A subsequent long-term analysis of this trial confirmed the increased numbers of long-term responders. A second phase 3 study of ipilimumab 10 mg / kg versus placebo for chemotherapy -naive mCRPC without visceral metastases revealed a 23% objective response rate with ipilimumab but no overall survival advantage, possibly related to a 40% grade 3 AE (adverse event) rate leading to a 29% rate of toxicity-related treatment discontinuation. A phase 2 study of ipilimumab 3 mg / kg monotherapy had a best overall response of stable disease, possibly due to the lower dose. A phase 2 study of ipilimumab 3 mg / kg plus the anti- PD-1 antibody nivolumab 1 mg / kg continued to show an objective response rate (up to 25%) and a 42-53% grade 3-4 treatment-related adverse event rate with an 8% drug toxicity related death rate.

[0011] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD 152 (cluster of differentiation 152), is a cell surface protein receptor that interacts with B7-1 (CD80) and B7- 2 (CD86) to ensure proper function of regulatory T cells (Tregs) and protect host against autoinflammatory diseases. CTLA-4 is shown to be an immune checkpoint and a target for cancer therapy in animal models.

[0012] Unlike other checkpoint inhibitors such as anti-PD-l / PD-Ll antibodies, the anti-CTLA-4 antibody, ipilimumab (YERVOY®), has gained market approval for one indication (melanoma) as a monotherapy. The toxicity profile significantly limits its dose and exposure that are required for achieving higher efficacy benefit. The less optimal dose may explain the consistently lower response rate than an anti-PD-1 antibody in head-to-head comparison studies in melanoma and its failure as a monotherapy in multiple Phase III clinical trials in other cancer indications. Despite its approval for multiple cancer indication as a combination therapy with nivolumab (Opdivo®, an anti-PD-1 antibody), the incidence of grade 3 / 4 immunotherapy-related adverse effects (irAEs) (e.g., up to 73-90% of patients with melanoma receiving ipilimumab / nivolumab as a neo-adjuvant therapy) remains high. CTLA-4 remains a valid and attractive immunotherapy target, however, the less favorable safety profile significantly limits its clinical usage.

[0013] The molecular basis underlying irAEs and cancer immunotherapeutic effects (CITE) of anti- CTLA-4 antibodies is traditionally viewed as antagonizing the endogenous function of CTLA-4. In both mice and humans, genetic inactivation of CTLA-4 caused severe autoimmune diseases; therefore, an effective antagonist of CTLA-4 is expected to likely induce autoimmune diseases. If inactivation of CTLA-4 is necessary, then irAEs is expected to be a necessary price for cancer immunity.

[0014] There is thus an unmet medical need to improve treatment options for subjects with prostate cancer.

[0015] SUMMARY OF THE INVENTION

[0016] Against the aforementioned background, it is an object of the present invention to provide effective treatment options for subjects with prostate cancer. It is a further object of the present invention to provide safe treatment options for subjects with prostate cancer.

[0017] These objects are achieved by the invention as disclosed herein.

[0018] The present disclosure demonstrates surprising effectiveness of a particular treatment of diseases, disorders, and conditions, in particular prostate cancer such as PSMA-positive prostate cancer or metastatic castration-resistant prostate cancer. The present disclosure demonstrates benefits when a subject receives a treatment regimen comprising an anti-CTLA- 4 antibody.

[0019] The treatment comprises an antibody therapy. The treatment can be a combination treatment comprising an antibody therapy and a radioligand therapy. In some embodiments, the anti- CTLA-4 antibody is administered to a subject who is receiving or has received a radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan. In some embodiments, the radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan is administered to a subject who is receiving or has received the anti-CTLA-4 antibody.

[0020] Combination treatment as used herein includes but is not limited to concomitant administration. In some embodiments, the combination of the anti-CTLA-4 antibody and the radioligand therapeutic agent is individually administered to the subject within one, two or three or more weeks of each other. In some embodiments, the combination is individually administered to the subject within one, two or three or more months of each other. In some embodiments, the radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan is administered for a longer period of time compared to the anti-CTLA-4 antibody. In some preferred embodiments, the anti-CTLA-4 antibody is administered for a longer period of time compared to the radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan.

[0021] The treatment as disclosed herein surprisingly results in an effective treatment. The effect can be seen in longer overall survival, by longer progression-free survival, by increased radiographic progression free survival, by longer freedom from disease progression (stable disease state), reduction of tumor-related symptoms, and / or reduction of need for pain medications during and / or following the therapy (antibody therapy or combination therapy). The effect can be reflected in an improved quality of life, such as mobility, strength of appetite, and / or psychological status.

[0022] In some embodiments, the effect can be observed by the Kaplan-Meier method to provide estimates of the radiographic progression free survival (rPFS) curves among with minimum and maximum survival times. The Kaplan-Meier method is well known and for example described in Goel MK, Khanna P, Kishore J. Understanding survival analysis: Kaplan-Meier estimate. Int J Ayurveda Res. 2010 Oct;l(4):274-8. doi: 10.4103 / 0974-7788.76794. PMID: 21455458; PMCID: PMC3059453, which disclosure is incorporated in its entirety for the purpose described herein.

[0023] The invention further provides a method of treating prostate cancer in a subject in need thereof, comprising administering the anti-CTLA-4 antibody or the combination of the anti- CTLA-4 antibody and the radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan as disclosed herein. In some embodiments, the prostate cancer is a PSMA-positive prostate cancer.

[0024] The invention further provides a medical preparation for treating prostate cancer comprising the anti-CTLA-4 antibody or the combination of the anti-CTLA-4 antibody and the radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan as disclosed herein, preferably in form of a kit comprising a first container including the anti-CTLA-4 antibody or a nucleic acid encoding the anti-CTLA-4 antibody and optionally a second container including the radioligand therapeutic agent such as lutetium (177LU) vipivotide tetraxetan. DETAILED DESCRIPTION

[0025] Although certain embodiments of the present invention are described in detail below, it is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0026] In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

[0027] Definitions

[0028] The terms indicated for explanation of the invention have the following meaning, unless otherwise indicated in the description or the claims. Additional definitions are set forth throughout the detailed description.

[0029] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. K51bl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0030] The practice of the present invention will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 4thEdition, M.R. Green, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012).

[0031] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e., the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. The terms “a”, “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0032] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0033] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0034] The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0035] An "adjuvant” is a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund’s adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF a, INF-y, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose- 6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0036] An adverse effect (AE) is the development of an undesirable medical condition or the deterioration of a pre-existing medical condition following or during exposure to a pharmaceutical product, whether or not considered causally related to the product. The term AE is used to include both serious and nonserious Aes. A serious adverse event (SAE) can be an AE that meets any of the following outcome criteria: results in death, is immediately life-threatening, requires inpatient hospitalization >24 hours or prolongation of existing hospitalization, results in persistent or significant disability / incapacity or substantial disruption of the ability to conduct normal life functions, is a congenital abnormality or birth defect, is an important medical event that may j eopardize the patient or may require medical intervention to prevent one of the outcomes listed above. Assessment of severity for all Aes (serious and non-serious) will be made according to the NCI CTCAE v5.0. Any adverse event that changes NCI CTCAE grade over the course of a given episode can have each change of grade recorded. Grade 1 : Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not indicated. Grade 2: Moderate; minimal, local, or noninvasive intervention indicated; limiting age-appropriate instrumental activities of daily living (ADLs). Grade 3 : Severe or medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care ADLs. Grade 4: Life threatening consequences; urgent intervention indicated. Grade 5: Death related to AE.

[0037] In its broadest sense, as used herein, the term “amino acid” is a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-C00H. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0038] As used herein, an “analog” is a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0039] As used herein, an "antibody" is a molecule that possesses an antigen-binding site. The term encompasses functional antibody fragments, such as a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody and a single-domain antibody (sdAB). The antibody can comprise a variable region. The term "variable region" is intended to distinguish such domain of the antibody from domains that are broadly shared by antibodies (such as e.g., an antibody Fc domain). The variable region comprises a "hypervariable region" whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; ref. 44) and / or those residues from a "hypervariable loop" (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain. "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes antigen-binding fragments, monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies of the invention). In particular, such antibodies include immunoglobulin molecules of any type (e g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. Antibodies described herein may be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig, and human.

[0040] As used herein, the term "antigen binding fragment" or “antibody binding portion” of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity determining regions ("CDRs") and optionally the framework residues that comprise the antibody's "variable region" antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Examples of binding fragments encompassed within the term “antigen binding fragment” or “antigen binding portion” of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., 1989, Nature 341 :544-546), which consist of a VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988, Science 242:423-426; and Huston et al., 1988, Proc. Natl. Acad Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" or “antibody binding portion” of an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding-domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939.

[0041] These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. “Antigen binding fragment" or “antibody binding portion” may further include fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.). As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0042] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fdlers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline. Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0043] The term “CTLA-4” relates to cytotoxic T lymphocyte antigen-4. CTLA-4 is expressed on the surface of cells and, if it is located at the surface of said cells, is accessible to binding by CTLA-4 specific antibodies. “Cell surface” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. For example, a transmembrane protein having one or more extracellular portions is considered as being expressed on the cell surface.

[0044] A “disease” is any pathological state, including cancer, in particular those forms of cancer described herein. Any reference herein to cancer or particular forms of cancer also includes cancer metastasis thereof. In some preferred embodiments, a disease to be treated according to the present application involves cells expressing PSMA. “Diseases associated with cells expressing PSMA” or similar expressions means as disclosed herein that is expressed in cells of a diseased tissue or organ. In some embodiments, expression of PSMA in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. An increase refers to an increase by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In some embodiments, expression is only found in a diseased tissue, while expression in a corresponding healthy tissue is repressed. For example, PSMA can be expressed in prostate cancer tissue while expression is not detectable in non-cancerous prostate tissue. According to the disclosure, diseases associated with cells expressing PSMA are cancer diseases. Furthermore, according to the disclosure, cancer diseases preferably are those wherein the cancer cells express PSMA.

[0045] As used herein, a “cancer disease” or “cancer” includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and / or migration. By “cancer cell” is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Preferably, a “cancer disease” is characterized by cells expressing PSMA and a cancer cell expresses PSMA. A cell expressing PSMA preferably is a cancer cell, preferably of the cancers described herein.

[0046] An antibody is “derived from” a particular germline sequence if the antibody is obtained from a system by immunizing an animal or by screening an immunoglobulin gene library, and wherein the selected antibody is at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, an antibody derived from a particular germline sequence may display no more than 10 amino acid differences, more preferably, no more than 5, or even more preferably, no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0047] A "diluent" is a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and / or mixing media. Examples of suitable diluents include ethanol, glycerol, and water. Herein, "DNA" is a nucleic acid molecule which is entirely or at least substantially composed of deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" is a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0048] As used herein, the term “effective amount” refers to an amount of a given substance that is sufficient in quantity to produce a desired effect, including an improvement or remediation of the disease, disorder, or symptoms of the disease or condition. For example, an effective amount of a composition for use in treating prostate cancer in a subject is an amount capable to achieve a detectable effect or benefit upon administration to the subject.

[0049] As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA- dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or a single- stranded RNA (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA. As is understood in the art, the phrase “nucleic acid encoding a peptide or protein” means that the nucleic acid, if present in the appropriate environment, for example within a cell and / or in a cell-free translation system, can direct the assembly of amino acids to produce the peptide or protein via a process of translation.

[0050] An “epitope” is a part of an antigen that as used herein, refers to an agent that elicits an immune response; and / or an agent that binds to a T cell receptor (e g., when presented by an MHC molecule) or to an antibody. For example, epitopes are the discrete, three-dimensional sites of an antigen, which are recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Preferably, the term refers to an immunogenic portion of an antigen comprising the epitope. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein.

[0051] An "excipient" is a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0052] A “gene” is a DNA sequence in a chromosome that codes for a protein. In some embodiments, a gene includes a coding sequence (i.e., sequence that encodes a particular protein); in some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type- specific expression, inducible expression, etc.).

[0053] A "humanized antibody" is an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin. The nonhuman immunoglobulin providing the CDR's is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor." Constant regions need not be present, but if they are, they preferably can be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR's, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human. One says that the donor antibody has been "humanized," by the process of "humanization," because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDR's. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an Fc.gamma.RIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations).

[0054] As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules.

[0055] As used herein, these terms “increased”, “induced”, or “reduced” or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0056] An “antigenic fragment” is a peptide capable of eliciting an immune response in a subject.

[0057] The term "antigen" or "immunogen" refers to any peptide or protein that is a target of an immune response and / or that will elicit an immune response. In particular, an "antigen" is any substance that reacts specifically with, i.e., binds to antibodies or T-lymphocytes (T-cells), in particular T-cell receptors. The term "antigen" encompasses antigenic fragments, i.e., the antigen can be an antigenic fragment.

[0058] A “mutation” is a change or alteration in the sequence of a polynucleotide or polypeptide. In some embodiments, the mutation can be a single nucleotide substitution, deletion, or addition. In some embodiments, the mutation can be a substitution, deletion, or addition or of a plurality of nucleotides. In some embodiments, the mutation can be a single amino acid substitution, deletion, or addition. In some embodiments, the mutation can be a substitution, deletion, or addition or of a plurality of amino acids.

[0059] "Nucleoside” are compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'- deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA).

[0060] Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG. A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is, in some embodiments, modified by one or more alkyl groups, e.g., one or more Cl-4 alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5- alkyl-cytosine, 5 -alkyl -uracil, and N(l)-alkyl-uracil, such as N7-Ci-4 alkyl-guanine, N6-CI-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(l)-Ci-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(l)- m ethyl -uracil. The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject.

[0061] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. In certain embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring. The term “pharmaceutically acceptable” refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition. The term “carrier” refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application. The term “carrier” also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a subject. The term “excipient” when used herein is intended to indicate all substances which may be present in a pharmaceutical composition and which are not active ingredients such as, e g., carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.

[0062] The terms "polynucleotide” and “nucleic acid” can be used interchangeably herein to refer to polymers of nucleotides. The term " polynucleotide" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a polynucleotide is DNA. In some embodiments, a polynucleotide is RNA. In some embodiments, a polynucleotide is a mixture of DNA and RNA. A polynucleotide may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A polynucleotide can be isolated. The term "isolated polynucleotide " means, according to the present disclosure, that the polynucleotide (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0063] The term “PSMA-positive prostate cancer” means a cancer involving cancer cells expressing PSMA, preferably on the surface of said cancer cells. “Cell surface” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. For example, a transmembrane protein having one or more extracellular portions is considered as being expressed on the cell surface.

[0064] The term "RNA" relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). In some embodiments, "RNA" refers to mRNA.

[0065] As used herein, a “subject” is a human, preferably a male human. The subject may be of any age, but preferably may be at least 18 years old. In some preferred embodiments, the subject is a patient suffering from prostate cancer, preferably PSMA-positive prostate cancer. In some embodiments, the prostate cancer is metastatic castration resistant prostate cancer. The metastatic castration resistant prostate cancer preferably can be PSMA-positive metastatic castration resistant prostate cancer.

[0066] The term "treating" when used in the context of a disease or disease condition means ameliorating, improving or remedying a disease, disorder, or symptom of a disease or condition associated with the disease, or can mean completely or partially stopping, on a molecular level, the biochemical basis of the disease, such as halting replication of a virus, etc. It describes an act that leads to the elimination, reduction, alleviation, reversal, or prevention or delay of onset or recurrence of any symptom of a disease.

[0067] With regard to nucleotide and amino acid sequences, the term “variant” refers, in particular, to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular those which are naturally present. An allelic variant relates to an alteration in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies numerous allelic variants for a given gene. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence. The term “variant” shall encompass any post-translationally modified variants and conformation variants.

[0068] The anti-CTLA-4 antibody for use in treating prostate cancer in a subject

[0069] The present invention provides an anti-CTLA-4 antibody or a nucleic acid encoding the anti- CTLA-4 antibody for use in treating prostate cancer (e.g., PSMA-positive prostate cancer) in a subject.

[0070] The anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody can be used in a method of treating prostate cancer in a subject as disclosed herein. Features described herein in more detail in connection with the “antibody or nucleic acid for use in treating” embodiments equally apply to the corresponding method of treatment embodiments.

[0071] In some embodiments, the prostate cancer is a castration-resistant prostate cancer. In some embodiments, the prostate cancer is a metastatic castration-resistant prostate cancer. Preferably the prostate cancer is a PSMA-positive prostate cancer, even more preferably a PSMA-positive metastatic castration-resistant prostate cancer.

[0072] In some particularly preferred embodiments, the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody is used in treating prostate cancer in the subject in combination with the radioligand therapeutic agent. In some embodiments, the prostate cancer is a PSMA-positive prostate cancer and the radioligand therapeutic agent is a PSMA-targeting radioligand. The PSMA-targeting radioligand therapeutic agent preferably is lutetium (177LU) vipivotide tetraxetan.

[0073] Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a regulator of adaptive immune responses, having a role in the maintenance of peripheral tolerance and in shaping the repertoire of emergent T cell responses. Without wishing to be bound by theory, CTLA-4 can be recycled between the cell surface and endosomes, where it is usually prevented from lysosomal degradation and can recycle back to the cell surface by binding to the lipopolysaccharideresponsive and beige-like anchor (LRBA) protein. The anti-CTLA-4 antibodies disclosed herein have reduced autoimmune side effects when used to enhance immune responses and are particularly suitable for treating prostate cancer in subjects.

[0074] In some embodiments, the anti-CTLA-4 antibody is an antigen-binding fragment thereof, preferably selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody and a single-domain antibody (sdAB), and variants thereof.

[0075] In some embodiments, the radioligand therapeutic agent is comprised in a composition. In some embodiments, the anti-CTLA-4 antibody or a nucleic acid encoding the anti-CTLA-4 antibody, preferably the anti-CTLA-4 antibody, is comprised in a composition. The anti- CTLA-4 antibody and the radioligand therapeutic agent described herein may be administered in the form of any suitable pharmaceutical composition. Pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se. The pharmaceutical composition may e.g., be in the form of a solution or suspension. The pharmaceutical composition may comprise salts, buffer substances, preservatives, carriers, diluents and / or excipients all of which are preferably pharmaceutically acceptable. Salts which are not pharmaceutically acceptable may be used for preparing pharmaceutically acceptable salts and are included in the invention. Pharmaceutically acceptable salts of this kind comprise in a non-limiting way those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0076] Suitable buffer substances for use in a pharmaceutical composition include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt. Suitable preservatives for use in a pharmaceutical composition include benzalkonium chloride, chlorobutanol, paraben and thimerosal. An injectable formulation may comprise a pharmaceutically acceptable excipient such as Ringer lactate.

[0077] Possible carrier substances for parenteral administration are e.g., sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers.

[0078] The agents (such as the anti-CTLA-4 antibody and / or the radioligand therapeutic agent) and compositions described herein may be administered via any conventional route, such as by parenteral administration including by injection or infusion. Administration is preferably parenterally, e.g., intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. Compositions suitable for parenteral administration usually comprise a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient. Examples of compatible carriers and solvents are Ringer solution and isotonic sodium chloride solution. In addition, usually sterile, fixed oils are used as solution or suspension medium.

[0079] In some embodiments, one or more antibody or functional fragment disclosed herein is in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an antibody or functional fragment as provided herein and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

[0080] The compositions disclosed herein may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0081] In some preferred embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is comprised in a composition further comprising acetic acid, sodium acetate, gentisic acid, sodium ascorbate, pentetic acid, and water for injection. The composition preferably comprises 900-1,100 MBq / mL of the radioligand therapeutic agent, 0.1-1.0 mg / mL acetic acid, 0.1 -1.0 mg / mL sodium acetate, 0.1 -1.0 mg / mL gentisic acid, 40.0- 60.0 mg / mL sodium ascorbate, 0.05-0.20 mg / mL pentetic acid, and water for injection (q.s. to 1 mL) at pH 3.5-8.0. Most preferably, the composition comprises about 1,000 MBq / mL (27 mCi / mL) of the radioligand therapeutic agent, 0.30 mg / mL acetic acid, 0.41 mg / mL sodium acetate, 0.39 mg / mL gentisic acid, 50.0 mg / mL sodium ascorbate, 0.10 mg / mL pentetic acid, water for injection (q.s. to 1 mL), at pH 4.5-7.0.

[0082] In some embodiments, the anti-CTLA-4 antibody is comprised in a composition which has been diluted with 2-15% dextrose solution to a final concentration of about 0.1 to 5.0 mg / mL, from a formulation containing 1.0 to 40.0 mg / mL anti-CTLA-4 antibody, a histidine buffer, trehalose dihydrate, and PS80. Preferably the composition has been diluted with 4-6 % dextrose solution to a final concentration of about 0.3 to 4.0 mg / mL, from a formulation containing 4.0 to 30.0 mg / mL anti-CTLA-4 antibody, 10-50 mM histidine buffer, 5-15% (w / v) trehalose dihydrate, and 0.01-0.10 % (w / v) PS80, at pH 5.0-7.0. In some particularly preferred embodiments, the composition has been diluted with about 5% dextrose solution to a final concentration of about 0.5 to 3.0 mg / mL, from a formulation containing about 5.0 or about 30.0 mg / mL anti-CTLA-4 antibody, about 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and about 0.06% (w / v) PS80, at pH about 6.0.

[0083] In some embodiments, the anti-CTLA-4 antibody or the nucleic acid encoding the anti- CTLA-4 antibody, and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) are administered separately to the subject.

[0084] Methods of administering the anti-CTLA-4 antibody disclosed herein and / or the radioligand therapeutic agent disclosed herein include, but are not limited to, parenteral administration (e g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e g., intranasal and oral routes). In some embodiments, the antibodies of the invention are administered intramuscularly, intravenously, or subcutaneously. In some preferred embodiments, the antibodies and / or the radioligand therapeutic agent are administered intravenously. The antibodies and / or the radioligand therapeutic agent may be administered by any convenient route, for example, by infusion or bolus injection, and may be administered together with other biologically active agents. Administration can be systemic or local. In some particularly preferred embodiments, the anti-CTLA-4 antibody is administered via an IV infusion. In some particularly preferred embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered via an IV injection or IV infusion. In some particularly preferred embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered via an IV infusion.

[0085] The agents (such as the anti-CTLA-4 antibody and / or the radioligand therapeutic agent) and compositions described herein are administered in effective amounts. An “effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. An effective dose may be an amount that achieves the desired reaction or desired effect, e g., intended therapeutic result, when administered in accordance with a treatment regimen. In the case of treatment of a particular disease or of a particular condition, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the prostate cancer and, in particular, interrupting or reversing the progress of the prostate cancer. An effective amount of an agent or composition described herein may depend on the severeness of the prostate cancer, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the agents described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0086] In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of up to 100 mg / kg, up to 50 mg / kg, up to 40 mg / kg, up to 30 mg / kg, up to 20 mg / kg, up to 15 mg / kg, up to 10 mg / kg, up to 6 mg / kg, or up to 3 mg / kg. Preferably the anti-CTLA-4 antibody is administered repeatedly at a dose of 0.1 to 20 mg / kg, preferably 0.5 to 15 mg / kg, more preferably 0.6 to 10 mg / kg, 1 to 10.0 mg / kg, 1 to 6 mg / kg, or 1.0 to 3 mg / kg.

[0087] In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of up to 15 mg / kg, up to 10 mg / kg, up to 6 mg / kg, up to 3 mg / kg, or up to 1 mg / kg, such as 0.1 to 15 mg / kg, 0.3 to 12 mg / kg, 0.5 to 11 mg / kg, 0.6 to 10.5 mg / kg or about 1 to 10 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg or any combination thereof. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg. In some embodiments, the anti- CTLA-4 antibody is administered repeatedly at a dose of about 10 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 6 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 3 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 1 mg / kg.

[0088] During therapy, the doses can be independently selected from the group comprising a dose of up to 10 mg / kg, up to 6 mg / kg, up to 3 mg / kg, or up to 1 mg / kg, such as 0.1 to 10 mg / kg, 0.3 to 6 mg / kg, 0.5 to 3 mg / kg, or 0.6 to 1 mg / kg. During therapy, the doses can be independently selected from the group comprising a dose of up to 15 mg / kg, up to 10 mg / kg, up to 6 mg / kg, up to 3 mg / kg, or up to 1 mg / kg, such as 0.1 to 15 mg / kg, 0.1 to 12 mg / kg, 0.3 to 12 mg / kg, 0.5 to 11 mg / kg, 0.6 to 10 mg / kg or about 1 to 10 mg / kg. In some embodiments, the dose is administered according to any of the above embodiments. In some embodiments, during therapy, the doses can be independently selected from a dose of about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg. In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 10 mg / kg. In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 6 mg / kg. In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 1 mg / kg. In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 3 mg / kg.

[0089] The antibody or functional fragment of the antibody can be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0090] In some embodiments, the anti-CTLA-4 antibody (or the nucleic acid encoding the anti- CTLA-4 antibody) is administered once a week, once every 2 weeks, once every 4 weeks, once every 6 weeks, or once every 2 months. In some embodiments, the anti-CTLA-4 antibody (or the nucleic acid encoding the anti-CTLA-4 antibody) is administered once every 4 weeks. The time between administration of anti-CTLA-4 antibody (or the nucleic acid encoding the anti-CTLA-4 antibody) can depend on the dose. For example, if the anti-CTLA- 4 antibody (or the nucleic acid encoding the anti-CTLA-4 antibody) is administered at a dose of up to 6 mg / kg, the anti-CTLA-4 antibody (or the nucleic acid encoding the anti-CTLA-4 antibody) may be administered once every 3 weeks starting on day 1 of the therapy. If the anti-CTLA-4 antibody (or the nucleic acid encoding the anti-CTLA-4 antibody) can be administered at a dose of up to 3 mg / kg, the anti-CTLA-4 antibody (or the nucleic acid encoding the anti-CTLA-4 antibody) may be administered once every 2 weeks starting on day 1 of the antibody therapy.

[0091] The dosing regimen (e.g., dosing, administration interval, and treatment duration) can depend on the severeness of the prostate cancer (e.g., PSMA-positive prostate cancer), the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the regime described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial regime, short administration periods and / or higher doses may be used.

[0092] In some embodiments, the anti-CTLA-4 antibody is administered repeatedly once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 15 weeks, once every 16, weeks, or once every 17 weeks. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly once every 4 weeks or once every 6 weeks. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly once every 4 weeks. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly once every 6 weeks.

[0093] It is understood that the frequency can change during the combination therapy. In some embodiments, the administration frequency can be independently selected from once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 15 weeks, once every 16, weeks, and once every 17 weeks. For example, the anti- CTLA4 antibody may be administered once every 4 weeks following by once every 6 weeks, or the anti-CTLA4 antibody may be administered once every 6 weeks following by once every 4 weeks.

[0094] During therapy, the anti-CTLA-4 antibody doses can be independently selected from the group comprising a dose of up to 15 mg / kg, up to 10 mg / kg, up to 6 mg / kg, up to 3 mg / kg, or up to 1 mg / kg, such as 0.1 to 15 mg / kg, 0.1 to 12 mg / kg, 0.3 to 12 mg / kg, 0.5 to 11 mg / kg, 0.6 to 10 mg / kg, or about 1 to 10 mg / kg, wherein the anti-CTLA-4 antibody dose is administered repeatedly once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 15 weeks, once every 16, weeks, or once every 17 weeks. During therapy, the anti-CTLA-4 antibody doses can be independently selected from the group comprising a dose of up 10 mg / kg or about 1 to 10 mg / kg, wherein the anti-CTLA- 4 antibody dose is administered repeatedly once every 4 weeks or once every 6 weeks.

[0095] In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 10 mg / kg once every 6 weeks (e.g., to a total of 9 doses). In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 6 mg / kg once every 6 weeks (e g., to a total of 9 doses). In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 1 mg / kg once every 6 weeks (e g., to a total of 9 doses). In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 3 mg / kg once every 4 weeks (e g., to a total of 13 doses).

[0096] In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of 10 mg / kg once every 4 weeks. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly at a dose of 6 mg / kg once every 3 weeks. In some embodiments, the anti-CTLA-4 antibody is administered repeatedly (e.g., up to 5 times, up to 4 times, up to 3 times, or up to 2 times) at a dose of 10 mg / kg once every 4 weeks, followed by a dose of 6 mg / kg once every 3 weeks (e.g., to a total of 13 doses). Preferably, the anti-CTLA-4 antibody can be administered repeatedly at a dose independently selected from 10 mg / kg, 6 mg / kg, or 1 mg / kg once every 6 weeks (e.g., to a total of 9 doses). In some embodiments, the total number of doses depend on the overall anti-CTLA-4 antibody dosing period (i.e., the anti-CTLA-4 antibody treatment period). Preferably, the overall anti-CTLA-4 antibody dosing period is approximately 1 year, preferably 48 to 58 weeks or 50 to 54 weeks. For example, if the anti-CTLA-4 antibody is administered once every 4 weeks, a total of up to 13 doses can be preferably administered over an overall anti-CTLA-4 antibody dosing period of 52 weeks. If the anti-CTLA-4 antibody is administered once every 6 weeks, a total of up to 9 doses can be preferably administered over an overall anti-CTLA-4 antibody dosing period of 54 weeks. In some embodiments, the anti-CTLA-4 antibody or the nucleic acid encoding the anti- CTLA-4 antibody is administered for up to 13 doses or approximately up to 1 year. In some embodiments, the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody is administered for up to 9 doses or approximately up to 1 year.

[0097] In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 10 mg / kg once every 6 weeks for up to 9 doses. In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 6 mg / kg once every 6 weeks for up to 9 doses. In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 1 mg / kg once every 6 weeks for up to 9 doses. In some embodiment, the anti-CTLA-4 antibody is administered repeatedly at a dose of about 3 mg / kg once every 4 weeks for up to 13 doses.

[0098] In some embodiments, the anti-CTLA-4 antibody or the nucleic acid encoding the anti- CTLA-4 antibody is administered for a minimum of 15 minutes, a minimum of 30 minutes, a minimum of 60 minutes, or a minimum of 90 minutes For example, if the anti-CTLA-4 antibody is administered at a dose of up to 3 mg / kg, the administration may take a minimum of 15 minutes or a minimum of 30 minutes. If the anti-CTLA-4 antibody can be administered at a dose of up to 3 mg / kg, the administration may take a minimum of 60 minutes.

[0099] In some embodiments, the anti-CTLA-4 antibody is used in treating prostate cancer (e.g., PSMA-positive prostate cancer) in a subject. In some embodiments, the nucleic acid encoding the anti-CTLA-4 antibody is used in treating prostate cancer (e.g., PSMA-positive prostate cancer) in a subject. The anti-CTLA-4 antibody may be encoded by one or more nucleic acids, preferably by two nucleic acids. In some such embodiments, the one or more nucleic acids are optimized for expression in mammalian cells, preferably human cells.

[0100] In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.0 to 8.0 GBq. The radioligand therapeutic agent preferably is a targeted radioligand therapeutic agent, such as a PSMA-targeting radioligand therapeutic agent. Lutetium (177LU) vipivotide tetraxetan is a radioligand therapeutic agent composed of PSMA- 617, a human prostate-specific membrane antigen-targeting ligand, conjugated to the betaemitting radioisotope lutetium177Lu. Upon intravenous administration of lutetium (177LU) vipivotide tetraxetan, vipivotide tetraxetan can target and bind to PSMA-expressing tumor cells. Upon binding, PSMA-expressing tumor cells are destroyed by lutetium177Lu through the specific delivery of beta particle radiation. PSMA, a tumor-associated antigen and type II transmembrane protein, is expressed on the membrane of prostatic epithelial cells and overexpressed on prostate tumor cells.

[0101] Lutetium (177LU) vipivotide tetraxetan refers to a lutetium compound that is complexed to a

[0102] PSMA-targeting ligand of the following formula:

[0103] Lutetium (177LU) vipivotide tetraxetan is also marketed under the name of PLUVICTO®, formerly known as177Lu-PSMA-617.

[0104] In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.4 GBq (200 mCi).

[0105] In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered once every 2 weeks, once every 4 weeks, once every 6 weeks or once every 2 months.

[0106] In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered once every 6 weeks. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered for up to 6 doses.

[0107] In some embodiments, the anti-CTLA-4 antibody is administered in an amount of 1-10 mg / kg once every 4 weeks starting on day 1 of the treatment and the radioligand therapeutic agent (e g., lutetium (177LU) vipivotide tetraxetan) is administered once every 6 weeks at a dose of

[0108] 7.4 GBq (200 mCi) on day 1 or later. Preferably, the anti-CTLA-4 antibody can be administered for a maximum of 13 cycles or up to 1 year and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) can be administered for up to 6 doses. In some embodiments, the anti-CTLA-4 antibody is administered in an amount of 1-10 mg / kg once every 6 weeks starting on day 1 of the treatment and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered once every 6 weeks at a dose of

[0109] 7.4 GBq (200 mCi) on day 1 or later. Preferably, the anti-CTLA-4 antibody can be administered for a maximum of 9 cycles or up to 1 year and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) can be administered for up to 6 doses. In some embodiments, the radioligand therapeutic agent (e g., lutetium (177LU) vipivotide tetraxetan) is administered after the anti-CTLA-4 antibody, such as after the infusion of the anti-CTLA-4 antibody. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered several days after the anti-CTLA-4 antibody.

[0110] In preferred embodiments, the anti-CTLA-4 antibody is administered at 10 mg / kg as IV infusion, once every 28 days (4 weeks), for up to 13 doses. In preferred embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered at

[0111] 7.4 GBq (200 mCi) as IV infusion, once every 6 weeks, for up to 6 doses.

[0112] In some embodiments, the anti-CTLA-4 antibody is capable of binding human CTLA-4. The term “binding” preferably relates to a specific binding. An antibody usually is capable of binding to a predetermined target if it has a significant affinity for said predetermined target and binds to said predetermined target in standard assays. “Affinity” or “binding affinity” is often measured by equilibrium dissociation constant (KD). Preferably, the term “significant affinity” refers to the binding to a predetermined target with a dissociation constant (KD) of 10'5M or lower, IO'6M or lower, 10'7M or lower, 10'8M or lower, or 10'9M or lower. An antibody is not (substantially) capable of binding to a target if it has no significant affinity for said target and does not bind significantly, in particular does not bind detectably, to said target in standard assays. For example, if the KD for binding of an antibody to the target to which the antibody is capable of binding is 10'9M, the KD for binding to a target for which the antibody has no significant affinity would be is at least around 10'8M, 10'7M, 10'6M, 10'5M, 10'4M, 10’3M, 10’2M, or 10-1M.

[0113] Binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., Antibody- Antigen Interactions In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and methods described herein. Affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data may be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51 :660 (1949). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, e.g., KD, IC50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer For example, the affinity of an antibody can be evaluated by Octet. Multi-concentration kinetic experiments can be performed on the Octet Red96 system (ForteBio). Anti-hlgG Fc biosensors (ForteBio, #18-5064) can be hydrated in sample diluent (0.1% BSA in PBS and 0.02% Tween 20) and preconditioned in pH 1.7 glycine. The antigen can be diluted using a 7-point, 2-fold serial dilution starting at 600 nM with sample diluent. The antibody to be tested can be diluted to 10 pg / mL with sample diluent and then immobilized onto anti-hlgG Fc biosensors for 120 seconds. After baselines are established for 60 seconds in sample diluent, the biosensors can be moved to wells containing the antigen at a series of concentrations to measure the association. Association can be observed (e g., for 120 seconds) and dissociation can be observed (e.g., for 180 seconds) for each protein of interest in the sample diluent. The binding affinities can be characterized by fitting the kinetic sensorgrams to a monovalent binding model (1 : 1 binding).

[0114] The anti-CTLA-4 antibody disclosed herein is specific for CTLA-4 if it is capable of binding to CTLA-4 but is not (substantially) capable of binding to other targets. The anti-CTLA-4 antibody disclosed herein preferably does not inhibit binding of human CTLA-4 to the B7.1 (CD80) and B7.2 (CD86) ligands of the Antigen Presenting Cell. In some embodiments, the level of B7.1 and B7.2 on immune cells following anti-CTLA-4 treatment is used as a biomarker for measuring the biological activity of anti-CTLA-4 antibodies in vivo and monitoring responses to anti-CTLA-4 treatment by measuring the level B7.1 and / or B7.2 expression on immune cells, and comparing the level of expression before and after treatment. In some embodiments, the level ofB7.1 and / or B7.2 expression is monitored over time during a course of therapy. The therapeutic effect of CTLA-4 antibodies disclosed herein is preferably achieved through antibody -mediated depletion of Tregs specifically within tumor microenvironment. The anti-CTLA-4 antibodies disclosed herein are preferably not capable of blocking B7-CTLA-4 interactions under physiological conditions.

[0115] A fundamental question for the generation of safe and effective anti-CTLA-4 antibodies is whether CITE and irAE are intrinsically linked. The classical checkpoint blockade hypothesis stipulated that anti-CTLA-4 antibodies promote cancer immunity by blocking a negative signal of B7-CTLA-4 interactions to promote naive T cell activation in the lymphoid organ. According to this model, therapeutic antibodies are antagonists that functionally inactivate CTLA-4-B7 interactions. Since genetic inactivation of CTLA-4 expression leads to autoimmune diseases in mouse and human, it was assumed that the irAE would be a necessary price for CITE. However, rather than blocking B7-CTLA-4 interactions, the therapeutic effect of anti-mouse CTLA-4 antibodies requires antibody-mediated depletion of Tregs specifically within tumor microenvironment. It is not relevant whether an antibody is capable of blocking B7-CTLA-4 interactions under physiological conditions for the induction of CITE. In some embodiments, the anti-CTLA-4 antibody disclosed herein can induce CITE without blocking B7-CTLA-4 interactions.

[0116] In some embodiments, the anti-CTLA-4 antibody is not ipilimumab (marketed as YERVOY®). In particularly preferred embodiments, the antibody having the ability of binding to CTLA-4 is described in International Patent Application Publication No., WO 2017 / 106372, which is incorporated herein in its entirety.

[0117] In some embodiments, the anti-CTLA-4 antibody comprises a light chain variable region comprising (i) a complementarity determining region 1 (CDR1) region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 1, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 2, 3 or 4, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 5.

[0118] In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable region comprising (i) a CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 6, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 7, 8 or 9, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 10.

[0119] In some embodiments, the anti-CTLA-4 antibody comprises: a. a light chain variable region comprising (i) a complementarity determining region CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 1, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 2, 3 or 4, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 5; and, b. a heavy chain variable region comprising (i) a CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 6, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 7, 8 or 9, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 10.

[0120] A CDR refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL - sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Rabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., 1977, J. Biol. Chem. 252:6609-6616; Kabat, 1978, Adv. Prot. Chem. 32: 1-75). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved P-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901- 917). Both terminologies are well recognized in the art. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948; Morea et al., 2000, Methods 20:267-279). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al- Lazikani et al., supra). Such nomenclature is similarly well known to those skilled in the art.

[0121] The anti-CTLA-4 antibody may preferably comprise the CDRs listed in table 1.

[0122] Table 1: exemplary CDR sequences of the anti-CTLA-4 antibody

[0123] In some preferred embodiments, the anti-CTLA-4 antibody comprises: a. a light chain variable region comprising (i) a complementarity determining region CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 1, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 3, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 5; and, b. a heavy chain variable region comprising (i) a CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 6, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 8, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 10.

[0124] In some embodiments, the anti-CTLA-4 antibody can comprise heavy chain framework regions 1-4 (FR1, FR2, FR3, and FR4) preferably comprising (i) a heavy chain FR1 amino acid sequence as set forth in SEQ ID NO: 21, (ii) a heavy chain FR2 amino acid sequence as set forth in SEQ ID NO: 22, (iii) a heavy chain FR3 amino acid sequence as set forth in SEQ ID NO: 23, 24, or 25, and (iv) a heavy chain FR4 amino acid sequence of as set forth in SEQ ID NO: 26 or 27.

[0125] In some embodiments, the anti-CTLA-4 antibody can comprise light chain framework regions 1-4 (FR1, FR2, FR3, and FR4) preferably comprising (i) a light chain FR1 amino acid sequence as set forth in SEQ ID NO: 28, (ii) a light chain FR2 amino acid sequence as set forth in SEQ ID NO: 29, 30, or 31 (iii) a light chain FR3 amino acid sequence as set forth in SEQ ID NO: 32 or 33, and (iv) a light chain FR4 amino acid sequence of as set forth in SEQ ID NO: 34, 35, or 36.

[0126] The anti-CTLA-4 antibody may preferably comprise the CDR and framework region sequences listed in table 2.

[0127] Table 2: exemplary CDR and framework region sequences of the anti-CTLA-4 antibody

[0128] In some embodiments, the antibody having the ability to bind to CTLA-4 can be a polyclonal, monoclonal antibody or a chimeric antibody, optionally having an IgG or IgM isotype of any subclass, such as subclass I. Preferably, the anti-CTLA-4 antibody is an IgG or IgM antibody, preferably an IgG antibody, more preferably an IgGl antibody. The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity. In some embodiments, the monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a non-human animal, e g., mouse, fused to an immortalized cell. In some preferred embodiments, the anti-CTLA-4 antibody is a humanized anti-CTLA-4 antibody, preferably a humanized anti-CTLA-4 IgGl monoclonal antibody. In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain comprising a Fc region of a human Ig protein, preferably IgGl protein. In some preferred embodiments, the anti-CTLA-4 antibody is a glycoprotein and the constant region of each heavy chain contains 1 N-linked glycan site at residues N305 based on the reference sequence SEQ ID NO: 11.

[0129] In some embodiments, the anti-CTLA-4 antibody can comprise a Fc region, for example as set forth in SEQ ID NO: 12 or 13.

[0130] The anti-CTLA-4 IgGl antibody can be a mutated anti-CTLA-4 IgGl antibody, comprising an Fc region. Relative to the sequence of the IgGl backbone in SEQ ID NO: 12, the mutation may be M135Y, S137T, T139E, S181A, E216A, or K217A, or a combination thereof. These mutations are contemplated to lead to increased ADCC activity and increased half-life of the antibody in vivo. Preferably, the Fc region of the antibody may comprise all six mutations Preferably the Fc region can have the sequence set forth in SEQ ID NO.: 13.

[0131] In some embodiments, the heavy chain variable domain is selected from the sequence set forth in SEQ ID NOs: 14, 17, and 19. In some embodiments, the light chain variable domain is selected from the sequence set forth in SEQ ID NOs: 15, 16, and 20. In some embodiments, the heavy chain variable domain is selected from the sequence set forth in SEQ ID NOs: 14, 17, and 19 and the light chain variable domain is selected from the sequence set forth in SEQ ID NOs: 15, 16, and 20.

[0132] In some particularly preferred embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0133] In some particularly preferred embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15, and an Fc region comprising the sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0134] In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 16, and an Fc region comprising the sequence set forth in SEQ ID NO: 13.

[0135] In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0136] In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 17, a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15, and an Fc region comprising the sequence set forth in SEQ ID NO: 13.

[0137] In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18.

[0138] In some embodiments, the anti-CTLA-4 antibody is a pH-sensitive anti-CTLA-4 antibody. For example, the pH-sensitive anti-CTLA-4 antibody can dissociate from CTLA-4 at pH 6.5 or below, more preferably pH 5.5 or below. In some preferred embodiments, binding to CTLA-4 is reduced at an endosomal pH of 5.5 by more than 50% relative to binding at neutral pH (pH 7.0). Such a reduction may reach more than 75% at lysosomal pH 4.5 as compared to pH 7.0. The antibody- antigen complex preformed at pH 7.0 may dissociate under an acidic environment of pH 4.5-6.0. The reduction in binding may also be in comparison to a reference antibody which may be considerably less pH sensitive using the same standard. The reference antibody may be an antibody known in the art such as Ipilimumab or Tremelimumab. In the context of an engineered antibody, the changes may also be in comparison to a wild-type antibody which may be considerably less pH sensitive using the same standard. Without wishing to be bound by theory, it is contemplated that a pH-sensitive antibody is not only safer but also more effective in Treg depletion and tumor rejection than a pH-insensitive CTLA-4 antibody (e.g., Ipilimumab). Meaning, Ipilimumab can bind to CTLA-4 at a pH of 4- 7 and no dissociation can be observed at pH 4-7. pH-insensitive antibodies can cause downregulation of CTLA-4 through lysosomal degradation. CTLA-4 down-regulation can cause autoimmune diseases; while in the tumor CTLA-4 down-regulation can reduce ADCC activity and thus anti-cancer efficacy. The sensitivity to pH can be measured by any method known to the skilled person. For example, human or monkey-CTLA-4-Fc (0.5 ug / ml) can be coated on ELISA plates at 4°C overnight. Biotinylated anti-CTLA-4 antibodies can be added at 1 pg / ml in 1% BSA PBS with pH 4.5-7.0. Two hours later, antibodies binding with CTLA- 4 can be measured by using HRP-labeled streptavidin. A pH-sensitive anti-CTLA-4 antibody can comprise, for example, following CDR sequences: a. a light chain variable region comprising (i) a complementarity determining region CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 1, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 2, 3 or 4, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 5, and, b. a heavy chain variable region comprising (i) a CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 6, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 7, 8 or 9, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 10.

[0139] Although the following provides considerations regarding the mechanism underlying the therapeutic efficacy of antibodies of the disclosure it is not to be considered as limiting to the invention in any way. In some embodiments, the antibodies described herein are contemplated to be a highly selective, humanized monoclonal immunoglobulin G1 (IgGl)-kappa isotype antibody against CTLA-4 with a robust anti-tumor activity and lower autoimmune toxicity in comparison to ipilimumab. The disclosed antibodies preferably can dissociate from CTLA-4 under low pH in endosomes to allow both CTLA-4 and the antibody to escape from lysosomal degradation and recycle to the cell surface. Unlike ipilimumab that down-regulates CTLA-4 expression on Treg cells, the antibodies can keep a high-level expression of CTLA-4 on Treg cells through this recycling mechanism and makes Treg cells a better target for antibody-dependent cellular cytotoxicity, particularly in the tumor microenvironment (TME). The selective elimination of Treg cells in the tumor microenvironment and maintenance of CTLA-4 expression in Treg cells in the peripheral tissues by the anti-CTLA-4 antibody is contemplated to form the cellular and molecular basis for more potent tumor rejection and low toxicity. For example, the anti-CTLA-4 antibody described herein dissociates from CTLA-4 in endosomes, allows normal recycling of both antibodies and CTLA-4, which lead to a much-reduced autoimmune toxicity. The preservation of the recycling of both CTLA-4 and the anti-CTLA-4 antibody such facilitates more potent antibody dependent cellular cytotoxicity (ADCC) to eliminate Treg cells in the tumor microenvironment and induces strong CITE. ADCC preferably occurs when antibodies bind to antigens such as CTLA-4 on Treg cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells. This FcR-mediated depletion of Treg cells in the tumor microenvironment can lead to tumor rejection in subjects having prostate cancer.

[0140] In some embodiments, the anti-CTLA-4 antibody is an antibody selected from the group consisting of (i) an antibody which is a chimerized or humanized form of the antibody defined by the sequence identifiers above, (ii) an antibody having the specificity of the antibody defined by the sequence identifiers above, and (iii) an antibody comprising the antigen binding portion or antigen binding site, in particular the variable region, of the antibody defined by the sequence identifiers above or variant thereof and preferably having the specificity of the antibody defined by the sequence identifiers above.

[0141] In some embodiments, the anti-CTLA-4 antibody comprises one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs together with their intervening framework regions. Preferably, the framework regions portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of antibodies made by recombinant DNA techniques may result in the introduction of residues N- or C-terminal to the variable regions encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join variable regions of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels. In one embodiment an antibody comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs in a human antibody framework.

[0142] It will be appreciated by those skilled in the art that in particular the sequences of the CDR, hypervariable and variable regions can be modified without losing the ability to bind CTLA- 4. For example, CDR regions will be either identical or highly homologous to the regions of antibodies specified herein. By “highly homologous” it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable and variable regions may be modified so that they show substantial homology with the regions of antibodies specifically disclosed herein.

[0143] It will be appreciated by those skilled in the art that the anti-CTLA-4 antibody can comprise variants of the sequence(s) disclosed herein without losing the ability to bind CTLA-4.

[0144] Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

[0145] Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place.

[0146] Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and / or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0147] Preferably, the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. In preferred embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS “needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0148] “Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. The term “percentage identity” is intended to denote a percentage of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by “window of comparison” in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. U SA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis ).

[0149] The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared and multiplying the result obtained by 100 to obtain the percentage identity between these two sequences.

[0150] Antibodies described herein and useful in the methods described herein can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, 1975, Nature 256:495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, e g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes.

[0151] In some embodiments, an animal system for preparing hybridomas that secrete monoclonal antibodies may be a murine system. Hybridoma production in the mouse is a very well- established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0152] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and the rabbit system (e.g., described in Spieker-Polet et ah, 1995, Proc. Natl. Acad. Sci. U.S.A. 92:9348; see also Rossi et ah, 2005, Am. J. Clin. Pathol. 124: 295).

[0153] Yet another strategy for generating monoclonal antibodies is to directly isolate genes encoding antibodies from lymphocytes producing antibodies of defined specificity. For details of recombinant antibody engineering see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Bemiy K.C. Lo Antibody Engineering ISBN 1 - 58829- 092-1.

[0154] To generate antibodies, mice can be immunized with carrier-conjugated peptides derived from the antigen sequence, i.e., the sequence against which the antibodies are to be directed, an enriched preparation of recombinantly expressed antigen or fragments thereof and / or cells expressing the antigen, as described. Alternatively, mice can be immunized with nucleic acid encoding the antigen or fragments thereof. In the event that immunizations using a purified or enriched preparation of the antigen do not result in antibodies, mice can also be immunized with cells expressing the antigen, e g., a cell line, to promote immune responses.

[0155] The immune response can be monitored over the course of the immunization protocol with plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusions. Mice can be boosted intraperitonealy or intravenously with antigen expressing cells 3 days before sacrifice and removal of the spleen to increase the rate of specific antibody secreting hybridomas.

[0156] To generate hybridomas producing monoclonal antibodies, splenocytes and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody secreting hybridomas. By Immunofluorescence and FACS analysis using antigen expressing cells, antibodies with specificity for the antigen can be identified. The antibody secreting hybridomas can be re-plated, screened again, and if still positive for monoclonal antibodies can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization. Antibodies also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as are well known in the art (Morrison, 1985, Science 229: 1202).

[0157] For example, in some embodiments, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used by the GS gene expression system disclosed in International Patent Application Publication Nos. WO 87 / 04462 and WO 89 / 01036 and EP 338 841 A or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO cells, NS / 0 cells, HEK293T cells or HEK293 cells or alternatively other eukaryotic cells like plant derived cells, fungal or yeast cells. The method used to introduce these genes can be methods described in the art such as electroporation, lipofectine, lipofectamine or others. After introduction of these antibody genes in the host cells, cells expressing the antibody can be identified and selected. These cells represent the transfectomas which can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0158] Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g., E. coli. Furthermore, the antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or in eggs from hens, or in transgenic plants; see e.g., Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R , et al (1999) Biol. Chem. 380: 825-839.

[0159] Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al, 1998, Nature 332:323-327; Jones etal, 1986, Nature 321 :522-525; and Queen et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V (D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region.

[0160] The ability of antibodies to bind an antigen can be determined using standard binding assays (e g., ELISA, Western Blot, Immunofluorescence and flow cytometric analysis).

[0161] To purify antibodies, selected hybridomas can be grown in two-liter spinner- flasks for monoclonal antibody purification. Alternatively, antibodies can be produced in dialysis-based bioreactors. Supernatants can be filtered and, if necessary, concentrated before affinity chromatography with protein G-sepharose or protein A-sepharose. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0162] To determine if selected monoclonal antibodies bind to unique epitopes and / or to characterize one or more binding properties, site-directed or multi-site directed mutagenesis can be used.

[0163] To determine the isotype of antibodies, isotype ELISAs with various commercial kits (e g., Zymed, Roche Diagnostics) can be performed. Wells of microtiter plates can be coated with anti-mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either mouse IgGl, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg / ml) and analyzed at OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) may be used as described by the manufacturer.

[0164] In order to demonstrate presence of antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing antigen, flow cytometry can be used. Cell lines expressing naturally or after transfection antigen and negative controls lacking antigen expression (grown under standard growth conditions) can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1 % FBS, and can be incubated at 4 °C for 30 min. After washing, the APC- or Alexa647-labeled anti IgG antibody can bind to antigen-bound monoclonal antibody under the same conditions as the primary antibody staining. The samples can be analyzed by flow cytometry with a FACS instrument using light and side scatter properties to gate on single, living cells. In order to distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, the method of co-transfection can be employed. Cells transiently transfected with plasmids encoding antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. As the majority of transfected cells express both transgenes, antigen- specific monoclonal antibodies bind preferentially to fluorescence marker expressing cells, whereas non-specific antibodies bind in a comparable ratio to non-transfected cells. An alternative assay using fluorescence microscopy may be used in addition to or instead of the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy.

[0165] In order to demonstrate presence of antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing antigen, immunofluorescence microscopy analysis can be used. For example, cell lines expressing either spontaneously or after transfection antigen and negative controls lacking antigen expression are grown in chamber slides under standard growth conditions in DMEM / F12 medium, supplemented with 10 % fetal calf serum (FCS), 2 raM L-glutamine, 100 lU / ml penicillin and 100 pg / ml streptomycin. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with monoclonal antibodies against the antigen for 30 min. at 25°C. After washing, cells can be reacted with an Alexa555-labelled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy.

[0166] Cell extracts from cells expressing antigen and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate. Antibodies can be further tested for reactivity with antigen by Immunohistochemistry in a manner well known to the skilled person, e.g., using paraformaldehyde or acetone fixed cryosections or paraffin embedded tissue sections fixed with paraformaldehyde from noncancer tissue or cancer tissue samples obtained from patients during routine surgical procedures or from mice carrying xenografted tumors inoculated with cell lines expressing spontaneously or after transfection antigen. For immunostaining, antibodies reactive to antigen can be incubated followed by horseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the vendors instructions. The testing of monoclonal antibody activity in vitro will provide an initial screening prior to testing in vivo models.

[0167] The subject can be administered, in addition to the anti-CTLA-4 antibody and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan), other chemotherapeutic agents or combinations of chemotherapeutic agents such as cytostatic agents. Chemotherapeutic agents may affect cells in one of the following ways: (1) damage the DNA of the cells so they can no longer reproduce, (2) inhibit the synthesis of new DNA strands so that no cell replication is possible, (3) stop the mitotic processes of the cells so that the cells cannot divide into two cells.

[0168] In some embodiments, the subject is a male subject, preferably a male adult subject. The adult subject is a subject of 18 years or older. In some embodiments, the subject is a human.

[0169] In some embodiments, the subject has a prostate-specific membrane antigen (PSMA) positive PET / CT scan prior the antibody or combination therapy. In some embodiments, the subject has >1 metastatic lesion prior the antibody or combination therapy that is present on baseline CT, MRI, or bone scan imaging. In some embodiments, the subject has prior orchiectomy and / or ongoing androgen-deprivation therapy and / or a castrate level of serum testosterone (<50 ng / dL or <1.7 nmol / L).

[0170] In some embodiments, the prostate cancer (e.g., PSMA-positive prostate cancer) progressed after androgen receptor pathway inhibition. The invention further provides a method of treating a prostate cancer (e.g., PSMA-positive prostate cancer) in a subject in need thereof, comprising administering the anti-CTLA-4 antibody disclosed herein or the nucleic acid encoding the anti-CTLA-4 antibody as disclosed herein. In some embodiments, the method comprises administering the anti-CTLA-4 antibody in combination with a radioligand therapeutic agent, preferably a PSMA-targeting radioligand, more preferably lutetium (177LU) vipivotide tetraxetan. In some embodiments, the anti-CTLA-4 antibody, and the radioligand therapeutic agent, preferably a PSMA- targeting radioligand, more preferably lutetium (177LU) vipivotide tetraxetan, are administered separately to the subject.

[0171] The invention further provides a medical preparation for treating prostate cancer (e g., PSMA- positive prostate cancer) in a subject, comprising the anti-CTLA-4 antibody as disclosed herein or the nucleic acid encoding the anti-CTLA-4 antibody as disclosed herein. In some embodiments, the medical preparation comprises the anti-CTLA-4 antibody in combination with radioligand therapeutic agent, preferably a PSMA-targeting radioligand, more preferably lutetium (177LU) vipivotide tetraxetan. In some embodiments, the medical preparation is a kit. In some embodiments, the medical preparation is present in form of a kit comprising a first container including the anti-CTLA-4 antibody or a nucleic acid encoding the anti-CTLA-4 antibody, and optionally a second container including the radioligand therapeutic agent, preferably a PSMA-targeting radioligand, more preferably lutetium (177LU) vipivotide tetraxetan.

[0172] In some embodiments, the medical preparation comprises printed instructions for use of the medical preparation or treatment of the prostate cancer (e.g., PSMA-positive prostate cancer).

[0173] In some embodiments, the invention provides a combination for use in treating prostate cancer e.g., PSMA-positive prostate cancer, the combination comprises the anti-CTLA-4 antibody disclosed and described herein and the radioligand therapeutic agent, preferably a PSMA-targeting radioligand, more preferably lutetium (177LU) vipivotide tetraxetan, as disclosed and described herein.

[0174] The invention further provides a method of administering an anti-CTLA-4 antibody to a subject, the method comprising administering one or more doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg. The method of administering an anti-CTLA-4 antibody to a subject con comprise administering one or more doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg.

[0175] The radioligand therapeutic agent (such as lutetium (177LU) vipivotide tetraxetan) disclosed herein can be used in a “method of administering an anti-CTLA-4 antibody” as disclosed herein. The anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody disclosed herein can be used in a “method of administering an anti-CTLA-4 antibody” as disclosed herein. Features described herein in more detail in connection with the “antibody or nucleic acid for use in treating” embodiments equally apply to the corresponding method of administering embodiments.

[0176] In some embodiments, each dose of the anti-CTLA-4 antibody is administered once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks. In some embodiments, each dose of the anti- CTLA-4 antibody is administered once every 6 weeks. In some embodiments, each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg. In other embodiments, each dose is about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg In some embodiments, each dose is independently 10 mg / kg, 6 mg / kg, or 1 mg / kg. In other embodiments, each dose is 10 mg / kg, 6 mg / kg, or 1 mg / kg. In some embodiments, the anti- CTLA-4 antibody is administered for up to 9, up to 10, up to 11, up to 12, up to 13, or up to 14 doses. In some embodiments, up to 9 doses of the anti-CTLA-4 antibody can be administered to the subject.

[0177] In some embodiments, the anti-CTLA-4 antibody is administered to a subject who is receiving or has received a radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan. In some embodiments, the radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan is administered to a subject who is receiving or has received the anti- CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) are administered separately to the subject. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.0 to 8.0 GBq. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.4 GBq (200 mCi). In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered once every 6 weeks. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered for up to 6 doses.

[0178] In some embodiments, a method of administering an anti-CTLA-4 antibody is provided, comprising administering up to 10, in particular up to 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg or about 1 mg / kg and is administered once every 6 weeks. In some embodiments, a method of administering an anti-CTLA-4 antibody is provided, comprising administering up to 10, in particular up to 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg or about 1 mg / kg and is administered once every 6 weeks.

[0179] Without wishing to be bound by theory, it has been surprisingly found that the anti-CTLA-4 antibody disclosed herein has a relatively long half-life, allowing for an administration only once every 6 weeks, or even less frequently, for up to 9 doses. This is advantageous compared to prior art anti-CTLA-4 antibodies, which have shorter half-lives, requiring a shorter dosage frequency, e.g., every 3 to 4 weeks. The present invention therefore surprisingly allows particularly beneficial synchronisation of the dosage regimes with co-therapy, in particular with radioligand therapeutic agents such as lutetium (177LU) vipivotide tetraxetan, which is administered every 6 weeks. Accordingly, the invention has the benefits of increasing patient compliance, acceptance, feasibility and ease of combination therapy of an anti-CTLA-4 antibody and a radioligand therapeutic agent. Patient may only need to be treated once every cycle, i.e., the anti-CTLA4 antibody and co-therapy, such as radioligand therapeutic agents, can be administered on the same day, without the need of administering the anti-CTLA4 antibody and co-therapy on separate days. This dosage frequency further beneficially allows co-administration of the anti-CTLA4 antibody and co-therapy, meaning administration sequentially one after another or together, in a single composition.

[0180] In some embodiments, the subject has cancer. The cancer may be a solid tumor. The cancer may be advanced or metastatic. The subject may have previously exhibited failure or intolerance to standard of care for the cancer. The cancer may be refractory or resistant to anti-PD-I / PD-LI treatment. The cancer may be melanoma, metastatic melanoma, PD(L flrefractory melanoma, non-small cell lung adenocarcinoma, metastatic NSCLC, NSCLC with driver mutations (for example, EGFR / ALK mutations or other targetable mutations), PD-I- refractory NSCLC, head and neck cancer, adenoid cystic carcinoma (which may be RIM), squamous carcinoma, triple negative (basal-type) breast cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, colon cancer, hepatocellular carcinoma, other solid tumors, or metastatic colorectal cancer (which may have microsatellite instability). Preferably the cancer is prostate cancer.

[0181] In some embodiments, the anti-CTLA-4 antibody is administered intravenously, e.g., via an IV infusion. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered via an IV infusion.

[0182] The invention further provides a combination therapy, wherein an anti-CTLA4 antibody and a radioligand therapeutic agent (such as lutetium (177LU) vipivotide tetraxetan) are administered to a subject having cancer, wherein both the anti-CTLA4 antibody and the radioligand therapeutic agent are administered on the same day and at the same dosage frequency. In some embodiments, this combination therapy includes synchronization for only part of the therapy.

[0183] The radioligand therapeutic agent (such as lutetium (177LU) vipivotide tetraxetan) disclosed herein can be used in a combination therapy as disclosed herein. The anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody disclosed herein can be used in a combination therapy as disclosed herein. Features described herein in more detail in connection with the “antibody or nucleic acid for use in treating” embodiments equally apply to the corresponding combination therapy embodiments.

[0184] In some embodiments, each dose of the anti-CTLA-4 antibody is administered once every 6 weeks. In some embodiments, each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg. In other embodiments, each dose is about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or 1 about mg / kg. In some embodiments, each dose is independently about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg. In other embodiments, each dose is about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered for up to 9, up to 10, up to 11, up to 12, up to 13, or up to 14 doses. In some embodiments, up to 9 doses of the anti-CTLA-4 antibody can be administered to the subject.

[0185] In some embodiments, the anti-CTLA-4 antibody is administered to a subject who is receiving or has received a radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan. In some embodiments, the radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan is administered to a subject who is receiving or has received the anti- CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) are administered separately to the subject. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.0 to 8.0 GBq. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.4 GBq (200 mCi). In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered once every 6 weeks. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered for up to 6 doses.

[0186] In some embodiments, a method of administering an anti-CTLA-4 antibody is provided, comprising administering up to 10, in particular up to 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg or about 1 mg / kg and is administered once every 6 weeks. In some embodiments, a method of administering an anti-CTLA-4 antibody is provided, comprising administering up to 10, in particular up to 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg or about 1 mg / kg and is administered once every 6 weeks.

[0187] In some embodiments, a method of treating cancer in a subject in need thereof is provided, comprising administering up to 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg or about 1 mg / kg and administered once every 6 weeks, further comprising administering a radioligand therapeutic agents such as lutetium (177LU) vipivotide tetraxetan every 6 weeks on the same day as the anti-CTLA4 antibody.

[0188] The cancer may be a solid tumor. The cancer may be advanced or metastatic. The subject may have previously exhibited failure or intolerance to standard of care for the cancer. The cancer may be refractory or resistant to anti-PD-I / PD-LI treatment. The cancer may be melanoma, metastatic melanoma, PD(L )-I-refractory melanoma, non-small cell lung adenocarcinoma, metastatic NSCLC, NSCLC with driver mutations (for example, EGFR / ALK mutations or other targetable mutations), PD-I-refractory NSCLC, head and neck cancer, adenoid cystic carcinoma (which may be RIM), squamous carcinoma, triple negative (basal-type) breast cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, colon cancer, hepatocellular carcinoma, other solid tumors, or metastatic colorectal cancer (which may have microsatellite instability). In some embodiments, the subject has prostate cancer. In some embodiments, the anti-CTLA-4 antibody is administered intravenously.

[0189] The invention further provides a method of treating cancer in a subject in need thereof, comprising administering one or more doses of the anti-CTLA-4 antibody to the subject, wherein each dose is independently selected from about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg. The method of treating cancer in a subject in need thereof can comprise administering one or more doses of the anti-CTLA-4 antibody to the subject, wherein each dose is independently selected from about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg.

[0190] The radioligand therapeutic agent (such as lutetium (177LU) vipivotide tetraxetan) disclosed herein can be used in a “method of treating cancer” as disclosed herein. The anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody disclosed herein can be used in a “method of treating cancer” as disclosed herein. Features described herein in more detail in connection with the “antibody or nucleic acid for use in treating” embodiments equally apply to the corresponding “method of treating cancer” embodiments.

[0191] In some embodiments, each dose of the anti-CTLA-4 antibody is administered once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks. In some embodiments, each dose of the anti- CTLA-4 antibody is administered once every 6 weeks. In some embodiments, each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg. In other embodiments, each dose is about 10 mg / kg, about 6 mg / kg, about 3mg / kg, or about 1 mg / kg. In some embodiments, each dose is independently about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg. In other embodiments, each dose is about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg. In some embodiments, the anti-CTLA-4 antibody is administered for up to 9, up to 10, up to 11, up to 12, up to 13, or up to 14 doses. In some embodiments, up to 9 doses of the anti- CTLA-4 antibody can be administered to the subject.

[0192] In some embodiments, the anti-CTLA-4 antibody is administered to a subject who is receiving or has received a radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan. In some embodiments, the radioligand therapeutic agent such as lutetium177LU vipivotide tetraxetan is administered to a subject who is receiving or has received the anti- CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody and the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) are administered separately to the subject. In some embodiments, the radioligand therapeutic agent (e g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.0 to 8.0 GBq. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered repeatedly at a dose of 7.4 GBq (200 mCi). In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered once every 6 weeks. In some embodiments, the radioligand therapeutic agent (e.g., lutetium (177LU) vipivotide tetraxetan) is administered for up to 6 doses.

[0193] In some embodiments, a method of administering an anti-CTLA-4 antibody is provided, comprising administering up to 10, in particular up to 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg or about 1 mg / kg and is administered once every 6 weeks. In some embodiments, a method of administering an anti-CTLA-4 antibody is provided, comprising administering up to 10, in particular up to 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg or about 1 mg / kg and is administered once every 6 weeks. In some embodiments, a method of treating cancer in a subject in need thereof is provided, comprising administering 9 doses of the anti-CTLA-4 antibody to a subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg and administered once every 6 weeks, further comprising administering a radioligand therapeutic agents such as lutetium (177LU) vipivotide tetraxetan every 6 weeks on the same day as the anti-CTLA4 antibody.

[0194] The cancer may be a solid tumor. The cancer may be advanced or metastatic. The subject may have previously exhibited failure or intolerance to standard of care for the cancer. The cancer may be refractory or resistant to anti-PD-I / PD-LI treatment. The cancer may be melanoma, metastatic melanoma, PD(L )-I-refractory melanoma, non-small cell lung adenocarcinoma, metastatic NSCLC, NSCLC with driver mutations (for example, EGFR / ALK mutations or other targetable mutations), PD-I-refractory NSCLC, head and neck cancer, adenoid cystic carcinoma (which may be RIM), squamous carcinoma, triple negative (basal-type) breast cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, colon cancer, hepatocellular carcinoma, other solid tumors, or metastatic colorectal cancer (which may have microsatellite instability). In some embodiments, the subject has prostate cancer. In some embodiments, the anti-CTLA-4 antibody is administered intravenously.

[0195] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein.

[0196] FIGURE DESCRIPTION

[0197] Figure 1 depicts a simulated concentration-time profile at different dosage frequencies. #1 refers to PP4637 at a dosage frequency of 6 mg / kg Q3W (once every 3 weeks); #2 refers to PP4637 at a dosage frequency of 3 mg / kg Q3W (once every 3 weeks); #3 refers to PP4637 at a dosage frequency of 6 mg / kg Q6W (once every 6 weeks); #4 refers to ipilimumab refers to 3 mg / kg Q3W (once every 3 weeks).

[0198] This application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing file is named 231286WO_Sequence Listing.xml and 34 KB in size. SEQ ID NOs: 1-10 are exemplary heavy and light chain CDR1-3 sequences of the anti- CTLA-4 antibody disclosed herein.

[0199] SEQ ID NO: 11 is an exemplary heavy chain of the anti-CTLA-4 antibody disclosed herein.

[0200] SEQ ID NOs: 12 and 13 are exemplary unmutated and mutated Fc regions, respectively, suitable for the anti-CTLA-4 antibody disclosed herein.

[0201] SEQ ID NOs: 14, 17, and 19 are exemplary heavy chain variable regions of the anti-CTLA-4 antibody disclosed herein.

[0202] SEQ ID NOs: 15, 16, and 20 are exemplary light chain variable regions of the anti-CTLA-4 antibody disclosed herein.

[0203] SEQ ID NO: 18 is an exemplary light chain of the anti-CTLA-4 antibody disclosed herein.

[0204] SEQ ID NOs: 21-36 are exemplary framework regions of the anti-CTLA-4 antibody disclosed herein.

[0205] EXAMPLE

[0206] Example 1

[0207] Treating metastatic castration resistant prostate cancer with lutetium (177LU) vipivotide tetraxetan and an anti-CTLA-4 antibody having the ability to bind to CTLA-4.

[0208] A clinical study is performed comparing the therapeutic effect of the use of the lutetium (177LU) vipivotide tetraxetan as a monotherapy or in combination with the anti-CTLA-4 antibody in the treatment of metastatic castration resistant prostate cancer, in patients with metastatic castration resistant prostate cancer who progressed on androgen receptor targeting agents (ARTA). The patients are subdivided into the following groups: • Group A: A Q6 weeks (“Q6W”, every 42 days) monotherapy schedule employing a single dose of lutetium (177LU) vipivotide tetraxetan administered.

[0209] • Group B: A Q4 weeks ( “Q4W”, every 28 days) or Q6 weeks (“Q6W”, every 42 days) schedule of the anti-CTLA-4 antibody sequentially at either 3 mg / kg Q4W for up to 13 cycles (cohort Al), 10 mg / kg Q6W for up to 9 doses (cohort A2), 6 mg / kg Q6W for up to 9 doses (cohort A-l), or 1 mg / kg Q6W for up to 9 doses (cohort A-2) in combination with a Q6W schedule of lutetium (177LU) vipivotide tetraxetan. The lutetium (177LU) vipivotide tetraxetan is administered at a dose of 7.4 GBq (200 mCi).

[0210] Group B, after completing of the combination therapy some subjects are administered the antibody having the ability to bind CTLA-4 as a monotherapy for up to 9 cycles or up to 13 cycles or up to 1 year.

[0211] A number of patients in Group B are administered the anti-CTLA-4 antibody at 1 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg in combination with the lutetium (177LU) vipivotide tetraxetan.

[0212] The results from this combination treatment trial with an antibody that binds to CTLA-4 and lutetium (177LU) vipivotide tetraxetan surprisingly show an effective and safe treatment with additive to synergistic effects compared to lutetium (177LU) vipivotide tetraxetan treatment as a monotherapy.

[0213] Example 2

[0214] Pharmacokinetic (PK) modeling was performed on the available and measured in-patient PK observation data to gain a better understanding of optimal dosage frequency of the anti- CTLA-4 antibody (i.e., PP4637). Such PK simulations were performed for PP4637 and ipilimumab.

[0215] For PP4637, the PK parameters were calculated by an updated population PK modeling with 1488 measured PK observations from 277 patients with advanced cancer including 216 patients having received PP4637 at a dose range of 0.1 to 10 mg / kg Q3W (once every 3 weeks) or Q4W (once every 4 weeks) as a single agent, and 61 patients having received PP4637 at dose range of 3 to 6 mg / kg Q3W (once every 3 weeks) in combination with pembrolizumab (Pembro). For ipilimumab, the PK parameters were obtained from the journal article entitled “Model-based clinical pharmacology profiling of ipilimumab in patients with advanced melanoma” (Feng et al. Br J Clin Pharmacol. 2014). The data used for the PK simulation are summarized in Table 3.

[0216] Table 3: PP4637 dosage information obtained from 277 subjects with 1488 PK observations for PP4637 population PK model development from study PRESERVE- 001 bTwo doses of 10 mg / kg followed by 6 mg / kg maintenance dose

[0217] The following PK parameters were used for the PK simulation (see Table 4). Table 4: PK parameter values used for PK simulation.

[0218] 1: Population PK Modeling results based on clinical data in 277 subjects with 1488 PK observation

[0219] 2: Feng et al. Br J Clin Pharmacol. 2014

[0220] The projected Cmax and Cmin at cycle 3 are shown in Table 5.

[0221] Table 5: Simulated cycle 3 Cmax and Cmin values for PP4637 and ipilimumab (Ipi).

[0222] Fig. 1, the simulation provides a comparison of the concentration-time profile for PP4637 administered at a dosage frequency of 3 mg / kg Q3W, 6 mg / kg Q3W, and 6 mg / kg Q6W, versus ipilimumab administered at dosage frequency of 3 mg / kg Q3W, repeatedly to cancer patients. It is noted that the drug peak concentration (Cmax) and trough concentration (Cmin. i.e., the concentration reached by the drug immediately before the next dose is administered) are projected to be higher following the PP4637 dosage frequency than that for ipilimumab dosage frequency. Particularly, the projected higher Cmax and Ctroug at PP4637 dosage frequency of 6 mg / kg Q6W, demonstrates the surprising potential of the anti-CTLA-4 antibody disclosed herein being used in beneficial Q6W schedules.

Claims

C l a i m s1. An anti-CTLA-4 antibody or a nucleic acid encoding the anti-CTLA-4 antibody for use in treating prostate cancer in a subject.

2. The anti-CTLA-4 antibody for use according to claim 1, for use in treating prostate cancer in the subject in combination with a radioligand therapeutic agent.

3. The anti-CTLA-4 antibody for use according to claim 2, wherein the prostate cancer is a PSMA-positive prostate cancer and the radioligand therapeutic agent is a PSMA- targeting radioligand therapeutic agent.

4. The anti-CTLA-4 antibody for use according to claim 3, wherein the radioligand therapeutic agent is lutetium (177LU) vipivotide tetraxetan.

5. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the prostate cancer is a metastatic castration resistant prostate cancer.

6. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is an antigen-binding fragment or a variant thereof.

7. The anti-CTLA-4 antibody for use according to claim 6, wherein the antigen-binding fragment is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB) and variants thereof.

8. The anti-CTLA-4 antibody for use according to any one of claims 2-7, wherein the radioligand therapeutic agent, preferably lutetium (177LU) vipivotide tetraxetan, is comprised in a composition further comprising acetic acid, sodium acetate, gentisic acid, sodium ascorbate, pentetic acid, and water for injection, preferably wherein thecomposition comprises 900-1,100 MBq / mL of the radioligand therapeutic agent, 0.1- 1.0 mg / mL acetic acid, 0.1-1.0 mg / mL sodium acetate, 0.1-1.0 mg / mL gentisic acid, 40.0-60.0 mg / mL sodium ascorbate, 0.05-0.20 mg / mL pentetic acid, and water for injection (q.s. to 1 mL) at pH 3. -8.0, even more preferably wherein the composition comprises 1,000 MBq / mL (27 mCi / mL) of the radioligand therapeutic agent, 0.30 mg / mL acetic acid, 0.41 mg / mL sodium acetate, 0.39 mg / mL gentisic acid, 50.0 mg / mL sodium ascorbate, 0.10 mg / mL pentetic acid, water for injection (q.s. to 1 mL), at pH 4.5-7.0.

9. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is comprised in a composition which has been diluted with 2-15% dextrose solution to a final concentration of about 0.1 to 5.0 mg / mL, from a formulation containing 1.0 to 40.0 mg / mL anti-CTLA-4 antibody, a histidine buffer, trehalose dihydrate, and PS80, preferably wherein the composition has been diluted with 4-6 % dextrose solution to a final concentration of about 0.3 to 4.0 mg / mL, from a formulation containing 4 0 to 30.0 mg / mL anti-CTLA-4 antibody, 10-50 mM histidine buffer, 5-15% (w / v) trehalose dihydrate, and 0.01-0.10 % (w / v) PS80, at pH 5.0-7.0.

10. The anti-CTLA-4 antibody for use according to claim 9, wherein the composition has been diluted with 5% dextrose solution to a final concentration of about 0.5 to 3.0 mg / mL, from a formulation containing 5.0 or 30.0 mg / mL anti-CTLA-4 antibody, 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80, at pH 6.0.

11. The anti-CTLA-4 antibody for use according to any one of claims 2-10, wherein the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody, and the radioligand therapeutic agent are administered separately to the subject.

12. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is administered repeatedly at a dose of 0.1 to 20 mg / kg, preferably 0.5 to 15 mg / kg, more preferably 1 to 10 mg / kg.

13. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is administered repeatedly at a dose of up to 10 mg / kg, up to 6 mg / kg, up to 3 mg / kg, or up to 1 mg / kg, preferably at a dose of about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg, more preferably at a dose of about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg.

14. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody is administered once a week, once every 2 weeks, once every 4 weeks, once every 6 weeks, or once every 2 months, preferably once every 6 weeks.

15. The anti-CTLA-4 antibody for use according claim 14, wherein the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody is administered once every 4 weeks.

16. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is administered repeatedly at a dose of 10 mg / kg once every 4 weeks.

17. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is administered repeatedly at a dose of 6 mg / kg once every 3 weeks.

18. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody is administered for up to 13 doses or approximately 1 year, preferably wherein the anti-CTLA-4 antibody is administered for up to 9 doses, preferably wherein up to 9 doses of the anti-CTLA-4 antibody are administered every 6 weeks.

19. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody is administered via an IV infusion.

20. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody is administered for a minimum of 30 minutes or a minimum of 60 minutes.

21. The anti-CTLA-4 antibody for use according to any one of claims 2-20, wherein the radioligand therapeutic agent is administered repeatedly at a dose of 7.0 to 8.0 GBq.

22. The anti-CTLA-4 antibody for use according to claim 21, wherein the radioligand therapeutic agent is administered repeatedly at a dose of 7.4 GBq (200 mCi).

23. The anti-CTLA-4 antibody for use according to any one of claims 2-22, wherein the radioligand therapeutic agent is administered once every 2 weeks, once every 4 weeks, once every 6 weeks or once every 2 months.

24. The anti-CTLA-4 antibody for use according to claim 23, wherein the radioligand therapeutic agent is administered once every six weeks.

25. The anti-CTLA-4 antibody for use according to any one of claims 2-24, wherein the radioligand therapeutic agent is administered for up to 6 doses.

26. The anti-CTLA-4 antibody for use according to any one of claims 2-25, wherein the radioligand therapeutic agent is administered via an IV injection or IV infusion.

27. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is capable of binding human CTLA-4.

28. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is a humanized anti-CTLA-4 antibody.

29. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody is an IgG or IgM antibody, preferably an IgG antibody, more preferably an IgGl antibody.

30. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody comprises: a. a light chain variable region comprising (i) a complementarity determining region CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 1, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 2, 3 or 4, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 5; and, b. a heavy chain variable region comprising (i) a CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 6, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 7, 8 or 9, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 10.

31. The anti-CTLA-4 antibody for use according to claim 30, wherein the anti-CTLA-4 antibody comprises: a. a light chain variable region comprising (i) a complementarity determining region CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 1, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 3, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 5; and, b. a heavy chain variable region comprising (i) a CDR1 region, wherein the amino acid sequence of the CDR1 region is set forth in SEQ ID NO: 6, (ii) a CDR2 region, wherein the amino acid sequence of the CDR2 region is set forth in SEQ ID NO: 8, and (iii) a CDR3 region, wherein the amino acid sequence of the CDR3 region is set forth in SEQ ID NO: 10.

32. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody comprises a heavy chain comprising a Fc region of a human Ig antibody.

33. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti-CTLA-4 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15, and an Fc region comprising the sequence set forth in SEQ ID NO: 13.

34. The anti-CTLA-4 antibody for use according to any one of the preceding claims, wherein the anti- CTLA-4 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 18.

35. A method of treating prostate cancer in a subject in need thereof, comprising administering the anti-CTLA-4 antibody as defined in any one of claims 1-34.

36. The method according to claim 35, comprising administering the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody in a combination therapy with a radioligand therapeutic agent, preferably wherein the radioligand therapeutic agent is a PSMA-targeting radioligand therapeutic agent, more preferably wherein the radioligand therapeutic agent is lutetium (177LU) vipivotide tetraxetan.

37. The method according to claim 36, wherein the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody, and the radioligand therapeutic agent are administered separately to the subject.

38. A medical preparation for treating prostate cancer in a subject, comprising the anti- CTLA-4 antibody for use according to any one of claims 1-34.

39. The medical preparation according to claim 38, comprising the anti-CTLA-4 antibody or the nucleic acid encoding the anti-CTLA-4 antibody in combination with radioligand therapeutic agent, preferably a PSMA-targeting radioligand therapeutic agent, more preferably lutetium (177LU) vipivotide tetraxetan.

40. The medical preparation according to claim 38 or 39, which is present in form of a kit comprising a first container including the anti-CTLA-4 antibody or a nucleic acidencoding the anti-CTLA-4 antibody and optionally a second container including the radioligand therapeutic agent, preferably lutetium (177LU) vipivotide tetraxetan.

41. The medical preparation according to any one of claims 38-40 further including printed instructions for use of the medical preparation or treatment of the prostate cancer.

42. A method of administering an anti-CTLA-4 antibody to a subject in need thereof, comprising administering one or more doses of the anti-CTLA-4 antibody to the subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, about 3 mg / kg, or about 1 mg / kg.

43. The method according to claim 42, wherein one dose of the anti-CTLA-4 antibody is administered once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks, preferably once every 6 weeks.

44. The method according to claim 42 or 43, wherein the anti-CTLA-4 antibody is administered for up to 9, up to 10, up to 11, up to 12, up to 13, or up to 14 doses, preferably wherein up to 9 doses of the anti-CTLA-4 antibody are administered.

45. The method according to any of claims 42-44, wherein the subject has cancer, preferably a solid tumor.

46. The method according to any of claims 42-45, wherein the anti-CTLA-4 antibody is administered intravenously.

47. A method of treating cancer in a subject in need thereof, comprising administering one or more doses of the anti-CTLA-4 antibody to the subject, wherein each dose is independently about 10 mg / kg, about 6 mg / kg, or about 1 mg / kg.

48. The method according to claim 47, wherein one dose of the anti-CTLA-4 antibody is administered once every 6 weeks, once every 7 weeks, once every 8 weeks, onceevery 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks, preferably once every 6 weeks.

49. The method according to claim 47 or 48, wherein the anti-CTLA-4 antibody is administered for up to 9, up to 10, up to 11, up to 12, up to 13, or up to 14 doses, preferably wherein 9 doses of the anti-CTLA-4 antibody are administered.

50. The method according to any of claims 47-49, wherein the subject has a solid tumor.

51. The method according to any of claims 47-50, wherein the anti-CTLA-4 antibody is administered intravenously.

52. A combination therapy, wherein an anti-CTLA4 antibody and a radioligand therapeutic agent are administered to a subject having cancer, wherein both the anti- CTLA4 antibody and the radioligand therapeutic agent are administered on the same day and at the same dosage frequency.

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