Drug conjugates targeting CTLA4, products comprising same and therapeutic uses thereof

Abstract

The present invention relates to a novel drug conjugate comprising a CTLA4 targeting molecule, at least one cytotoxic agent and a linker linking the CTLA4 targeting molecule and the cytotoxic agent, and to compositions and kits comprising such a drug conjugate, and uses thereof, in particular for treating cancer and / or preventing cancer relapse in a subject in need thereof.

Description

Technical Field

[0001] This invention relates to a novel pharmaceutical conjugate and combinations, compositions, and kits comprising such pharmaceutical conjugates, the pharmaceutical conjugates comprising a cytotoxic T-lymphocyte antigen 4 (CTLA4) protein-targeting molecule (preferably an anti-CTLA4 monoclonal antibody), at least one cytotoxic agent, and a linker connecting the CTLA4-targeting molecule to the cytotoxic agent, and relates to its use, particularly for the treatment of cancer and / or the prevention of cancer recurrence in subjects in need of such treatment. Background Technology

[0002] Following observed treatment successes in melanoma and lung cancer (Antonia et al., 2017; Eggermont et al., 2015, 2016, 2018, 2020; Gao et al., 2020; Luke et al., 2022), anti-PD(L)1 and anti-CTLA4 immunotherapies are currently being developed for primary (localized) tumors in neoadjuvant and adjuvant settings, as well as for metastatic settings in most cancers. However, improvements in recurrence-free survival and overall survival in localized and metastatic cancers come at the cost of serious adverse autoimmune or inflammatory toxicities of grade 3 to 5 according to the Common Terminology Criteria for Adverse Events (CTCAE), also known as immune-related adverse events (or irAEs). This toxicity has been observed in a higher proportion of patients with localized cancerous tumors than in patients with metastatic tumors (>30%).

[0003] The mechanism of action of anti-CTLA4 therapy depends on CTLA4 immune checkpoint blockade (antagonism of CD80 and CD86) and CTLA4+ lymphocyte depletion induced by antibody-derived cytotoxicity (ADCC) or antibody-derived cellular phagocytosis (ADCP).

[0004] Currently, the FDA and EMA have approved two anti-CTLA4 monoclonal antibodies: ipilimumab (IgG1; Yervoy®, Bristol Myers Squibb) and tremelimumab (IgG2; Imjudo®, AstraZeneca). Intravenous anti-CTLA4 immunotherapy is currently approved for the treatment of localized and metastatic melanoma (“MM”), renal cell carcinoma (RCC), MSI-H / MMRd (microsatellite high instability / mismatch repair deficient) colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (“NSCLC”), and malignant pleural mesothelioma, but at the cost of significant intravascular coagulation (irAEs).

[0005] Regarding cancer treatment involving intravenously administered products, the insufficient antitumor efficacy of these products is often related to the limited systemic doses available due to on-target detachment adverse events. In fact, the approved doses and regimens for these products are typically determined in Phase I clinical trials with dose-escalation components, which are then stopped at the maximum tolerated dose when treated patients experience dose-limiting toxicities. Furthermore, combinations of immunotherapies are limited by their additional and sometimes synergistic effects on systemic toxicity. Last but not least, the use of systemic immunotherapy for localized cancers (Stages I-II, + / - III) is limited by the irreversible and sometimes fatal effects of intravenously administered immunotherapy, where the benefit / risk ratio is not comparable to that observed in patients with advanced, recurrent, or refractory cancers.

[0006] Therefore, achieving superior therapeutic effects while further improving the safety of cancer treatment remains a goal of oncology.

[0007] The inventors present novel therapeutic tools that can be advantageously used in the context of cancer treatment. Summary of the Invention

[0008] The inventors have developed and, for the first time herein, advantageously describe CTLA4-targeting drug conjugates. These drug conjugates comprise i) a cytotoxic T-lymphocyte antigen 4 (CTLA4) protein-targeting molecule, preferably an anti-CTLA4 monoclonal antibody, ii) at least one cytotoxic agent, and iii) a linker connecting said CTLA4-targeting molecule and said cytotoxic agent. The specific drug conjugate described herein is an anti-CTLA4 antibody-drug conjugate (“ADC”).

[0009] In one particular aspect, the anti-CTLA4 ADC comprises i) ipilimumab or trimemumab as the anti-CTLA4 monoclonal antibody, ii) any cytotoxic agent as described herein, and iii) any adapter as described herein connecting the anti-CTLA4 monoclonal antibody and the cytotoxic agent.

[0010] Patent application WO2020 / 092155 describes specific polypeptides having heavy chain variable regions and / or light chain variable regions that specifically bind to the CTLA4 protein, as well as antibodies and fragments containing them. Byrne et al. describe a “CTLA-4-targeted engineered toxin body (ETB)” (“MT-8421”) that can be used alone or after treatment with an αPD-1 monoclonal antibody. However, none of these documents describe or propose pharmaceutical conjugates comprising anti-CTLA4 monoclonal antibodies (mAbs) such as ipilimumab or trimemumab according to the present invention. Instead, when describing combination therapies, they direct those skilled in the art away from anti-CTLA-4 mAbs described as causing adverse events and recommend more selective approaches involving the use of highly specific anti-CTLA4 polypeptides.

[0011] Compared with the effects observed in treatment involving (intravenous or intratumoral) administration of a single anti-CTLA4 agent, the CTLA4-targeting drug conjugates described in this article demonstrate superior therapeutic efficacy with significantly reduced toxicity.

[0012] In one particular aspect, the cancer subject to be treated is a subject whose tumor or tumor microenvironment (TME) contains CTLA4+ cells, preferably containing CTLA4+ tumor cells and / or CTLA4+ immune cells.

[0013] In another specific aspect, the cancer subject is a subject with (primary or secondary) resistance to (naked, i.e., unmodified, unconjugated, or unlinked to any other agent) anti-CTLA4 and / or anti-PD(L)1 agents, particularly a subject whose tumor and / or TME do not show myeloid cells, show low levels or dysfunctional myeloid cells, such as macrophages, which do not express or express at low levels Fcγ receptor I (“CD64”), Fcγ receptor IIIa (“CD16a”) and / or Fcγ receptor IIIb (“CD16b”).

[0014] For example, subjects to be treated are those whose tumors or TMEs do not contain Fcγ receptor I (“CD64”), Fcγ receptor IIIa (“CD16a”) and / or Fcγ receptor IIIb (“CD16b”) positive myeloid cells (especially macrophages), or whose proportion of Fcγ receptor I (“CD64”), Fcγ receptor IIIa (“CD16a”) and / or Fcγ receptor IIIb (“CD16b”) positive myeloid cells, especially macrophages, is lower than that detected in cancer patients or cancer patient populations who have the same cancer and have been identified as having a durable clinical benefit (DCB) from anticancer therapy, preferably particularly involving anti-CTLA-4 monoclonal antibodies such as ipilimumab or trimemumab [used alone or in combination with additional conventional anticancer agents such as anti-PD1 (e.g., nivolumab)].

[0015] The inventors also describe herein a pharmaceutical combination or composition comprising at least one pharmaceutical conjugate of the present invention, such as at least two different pharmaceutical conjugates, the first conjugate comprising a first cytotoxic agent, the second conjugate comprising a different cytotoxic agent, the composition further comprising a pharmaceutically acceptable load, excipient, carrier, or diluent. A pharmaceutical combination or composition is also described comprising at least one pharmaceutical conjugate of the present invention and different therapeutic agents, the therapeutic agents being selected, for example, from immune checkpoint targets, anti-angiogenic agents, cytotoxic agents, hormones, and any combination thereof, the composition further comprising a pharmaceutically acceptable load, excipient, carrier, or diluent.

[0016] A kit is also described. This kit comprises at least two pharmaceutical conjugates of the present invention, or at least one pharmaceutical conjugate of the present invention, and various therapeutic agents, said therapeutic agents being, for example, selected from immune checkpoint targets, anti-angiogenic agents, cytotoxic agents, hormones, and any combination thereof.

[0017] This document also describes the first use of the pharmaceutical conjugates, combinations, compositions, and kits disclosed herein for the treatment of cancer or the prevention of cancer recurrence in subjects in need, as well as any cancer prevention or treatment method that includes the step of administering the pharmaceutical conjugate, a combination or composition comprising it to a subject in need. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter herein pertains.

[0019] The following definitions are used to understand the implementation methods provided herein.

[0020] Unless otherwise stated, the terms “cancer,” “cancerous tumor,” “malignant tumor,” “tumor,” “tumor formation,” “cancer disease,” “malignant disease,” or “proliferative disease” are used interchangeably herein. These terms refer to or describe physiological disorders in mammals typically characterized by uncontrolled cell proliferation that may be associated with tumor formation and growth and / or systemic dissemination (metastasis) of proliferating cells. As used herein, “cancer” means any malignant and / or invasive growth or tumor caused by abnormal cell proliferation. As used herein, “cancer” means solid tumors named after the type of cells that form them, as well as cancers of the blood, bone marrow, or lymphatic system. Examples of solid tumors include, but are not limited to, melanoma, sarcoma, and carcinoma. Examples of blood cancers include, but are not limited to, leukemia, lymphoma, and myeloma. The term “cancer” includes, but is not limited to, primary cancers originating in a specific part of the body. The term cancer also includes: metastatic cancer, which has spread from its primary site to other parts of the body, such as the brain, bones, lungs, or liver; recurrence of the original primary cancer after remission; and a second primary cancer in people with a history of cancer, which is a new primary cancer of a different type than the former.

[0021] In one particular aspect, the cancer is characterized by the presence of malignant tumors and / or metastases in the brain, bone, lungs, or liver.

[0022] In the context of this invention, "tumor cell" or "cancer cell" refers to a tumor or tissue obtained from a subject who has cancer or is at risk of developing cancer, particularly from at least one cancer identified herein, such as melanoma, sarcoma, or carcinoma, and exhibiting well-known characteristics of cancer cells, such as persistent proliferation signaling, evasion of growth inhibitors, resistance to cell death, ability to replicate immortality, induction of angiogenesis, and activation of invasive and metastatic cells. It should be understood that the expression "tumor cell" used to identify cells obtained from a subject's tumor is also used in this specification to identify circulating tumor cells, cells obtained from liquid tumor biopsies, cells obtained from a tumor bed, or cells obtained from metastases. In addition to cancer cells, the term "tumor cell" also refers to any cell present in a tumor, such as stromal cells (e.g., fibroblasts or vascular cells) or immune cells.

[0023] In one specific aspect, the tumor is a malignant (cancerous) tumor. In another specific aspect, the tumor is a "premalignant" (precancerous) tumor.

[0024] In the context of this invention, "conventional cancer treatment" (also referred to herein as "standard care" or "primary cancer treatment modality") means treatment that is routinely applied, or a therapy that, while not routinely applied, is appropriate and at least recommended by health authorities. Oncologists select "conventional" treatments based on the specific cancer to be prevented or treated. Conventional cancer treatment may involve, for example, cytotoxic agents, anti-angiogenic agents, anti-hormonal agents (hormone therapy), immunotherapeutic agents (immunotherapy), and / or tumor exposure to radiation (radiotherapy).

[0025] The term "adjuvant therapy" refers to additional treatment given after the primary treatment modality, particularly after surgical removal of the primary tumor.

[0026] The terms “neoadjuvant” and “neoadjuvant treatment” refer to treatments performed before surgery.

[0027] In recent years, scientific and clinical advances have improved our understanding of the role and phenotype of regulatory T lymphocytes (Tregs) in cancer immune tolerance.

[0028] The inventors identified tumor antigen-specific Treg phenotypes in other tumor-infiltrating lymphocytes, demonstrating that their presence in the tumor microenvironment (“TME”) is crucial for the efficacy of immunotherapies targeting CTLA4 and PD-1 immune checkpoints. Specifically, the inventors identified high expression of membrane CTLA-4 by these tumor antigen-specific Tregs and demonstrated that, upon immobilization with an anti-CTLA-4 agent such as ipilimumab, CTLA-4 is internalized along with the anti-CTLA-4 agent into the cytoplasm of CTLA-4+ cells.

[0029] Based on their work, the inventors herein provide novel therapeutic compounds, combinations of compounds, and pharmaceutical compositions comprising these compounds, and describe their use in oncology. More specifically, they have developed and, for the first time described herein, CTLA4-targeting drug conjugates (also referred to herein as "drug conjugates") that exhibit significantly higher cytotoxicity to CTLA-4-expressing cells than anti-CTLA-4 alone, and are much less toxic to patients, due to effective concentrations that are, for example, more than 6 times lower than the standard concentrations used for ipilimumab. Therefore, the conjugates of the present invention offer a favorable benefit outweighing the risks of treatment compared to the effects observed with treatment involving (systemic or intratumoral) administration of a single anti-CTLA4 agent. The inventors specifically demonstrate herein that the CTLA4-targeting drug conjugates advantageously induce selective destruction of specific CTLA4+ cell populations, thereby allowing for more effective cancer treatment while reducing the toxicity of said treatment to subjects.

[0030] Drug conjugates

[0031] The inventors hereby describe for the first time a pharmaceutical conjugate comprising i) a cytotoxic T-lymphocyte antigen 4 (CTLA4) protein-targeting molecule, preferably an anti-CTLA-4 monoclonal antibody, ii) at least one anticancer agent, preferably at least one cytotoxic agent, and iii) a linker connecting the CTLA4 targeting molecule and the cytotoxic agent.

[0032] CTLA4 targeting molecules

[0033] CTLA-4, or CTLA4 (Cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (Cluster of Differentiation 152), is a protein receptor that acts as an immune checkpoint and downregulates the immune response. CTLA-4 is constitutively expressed in regulatory T cells (Tregs), but upon activation, it is upregulated only in regular T cells, a phenomenon particularly pronounced in cancer. CTLA4 acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. The CTLA4 protein is synthesized in mice by… Ctla-4 Gene encoding (Brunet JF, Denizot F, Luciani MF, Roux-Dosseto M, Suzan M, Mattei MG, Golstein P (1987). A new member of the immunoglobulin superfamily: CTLA4 immunoglobulin superfamily-CTLA4) "Nature. 328 (6127): 267-70), in humans it is encoded by the CTLA-4 gene (Dariavach P, Mattéi MG, Golstein P, Lefranc MP (December 1988)." Human Ig superfamily CTLA-4 gene: Chromosomal localization and protein sequence specificity between mouse and human CTLA-4 cytoplasmic domains (Human Ig superfamily CTLA-4 gene: chromosomal localization and ientity of protein sequence between murine and human CTLA-4 cytoplasmic domains) ”. European Journal of Immunology. 18 (12): 1901-5).

[0034] CTLA-4 is a member of the immunoglobulin superfamily, expressed by activated T cells, and transmits inhibitory signals to T cells. The CTLA-4 receptor downregulates the immune system. CTLA-4 is homologous to the T cell costimulatory protein CD28; both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 has a greater affinity for both CD80 and CD86 than CD28, thus outcompeting CD28 in ligand competition. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Activation of T cells via the T cell receptor and CD28 leads to increased CTLA-4 expression. In particular, CTLA-4 has been found to be a receptor expressed at the membrane level after regulatory T cells (Tregs) bind to their TCR receptors (Rowshanravan et al., 2018) and contributes to their inhibitory function. The mechanism of action of CTLA-4 in T cells remains controversial.

[0035] The CTLA4 protein contains an extracellular domain, a transmembrane domain, and a cytoplasmic tail. Alternating splice variants encoding different isoforms have been characterized. The membrane-bound isoform functions as a homodimer linked by disulfides, while the soluble isoform functions as a monomer. The intracellular domain, similar to that of CD28, lacks intrinsic catalytic activity and contains a YVKM motif capable of binding PI3K, PP2A, and SHP-2, and a proline-rich motif capable of binding SH3-containing proteins. CTLA-4's primary role in suppressing T cell responses appears to be directly mediated through the dephosphorylation of SHP-2 and PP2A by proximal TCR signaling proteins such as CD3 and LAT. CTLA-4 can also indirectly influence signal transduction by competing with CD28 for CD80 / 86 binding. CTLA-4 can also bind PI3K, but the importance and outcome of this interaction remain uncertain.

[0036] In the context of this invention, a CTLA4-targeting molecule is a molecule that recognizes and binds to CTLA4 on the cell surface, such as recognizing the extracellular domain of CTLA4, before being internalized into the cytoplasm by CTLA4+ cells. CTLA4+ cells are, for example, tumor cells or lymphocytes, particularly activated T lymphocytes, and even more particularly regulatory T cells (Tregs).

[0037] The CTLA4 targeting molecule is preferably an anti-CTLA-4 monoclonal antibody, such as ipilimumab or trimemumab.

[0038] Another CTLA4-targeting molecule described in this article is a molecule that can target and bind to CTLA4, or in other words, a molecule that targets CTLA4.

[0039] CTLA4 targeting molecules can be selected from, for example, molecules that can directly or indirectly regulate, preferably inhibit or reduce T cell function (especially the aforementioned inhibitory signaling to T cells).

[0040] Specific CTLA4-targeting molecules can selectively destroy CTLA4+ cells, especially CTLA4+ cancer cells or CTLA4+ lymphocytes, preferably CTLA4+ Tregs, and even more preferably CTLA4+ tumor antigen-specific Tregs (“tumor Tregs”).

[0041] Another specific CTLA4-targeting molecule can mimic or enhance the biological function of CTLA4 on Tregs. A specific and preferred CTLA4-targeting molecule is an anti-CTLA4 molecule that induces the depletion of Tregs in the subject, preferably the depletion of tumor antigen-specific Tregs (“tumor Tregs”), and even more preferably the specific depletion of tumor Tregs and Tregs present in the tumor microenvironment (TME), thereby excluding other tumor-infiltrating lymphocytes or any other lymphocytes present in the subject.

[0042] In one particular aspect, the CTLA4-targeting molecule is an anti-CTLA4 antibody (immunoglobulin), any (functional) fragment thereof (including single-chain antibodies) that would be considered equivalent by a person skilled in the art, or any (functional) variant thereof.

[0043] In the context of this invention, the term "antibody" (or "immunoglobulin") refers to any kind of antibody, such as a monoclonal antibody, a multispecific antibody (i.e., an antibody containing a first antigen-binding site and at least one distinct second antigen-binding site; for example, a bispecific antibody), or a single-chain antibody. The term also covers any (functional) variants or (functional) fragments thereof.

[0044] A typical antibody consists of a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region (abbreviated hereinafter as CH). Heavy chains are classified as γ, μ, α, δ, or ε, and antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, respectively. The heavy chain constant regions of immunoglobulins IgG, IgD, and IgA (γ, δ, and α chains, respectively) contain three domains (CH1, CH2, and CH3) and a hinge region for increased flexibility, while the heavy chain constant regions of immunoglobulins IgM and IgE contain four domains (CH1, CH2, CH3, and CH4). Depending on the structure of its heavy chain, the antibody of the present invention can be an IgG, IgM, IgA, IgD, and IgE isotype. However, in a preferred embodiment, the antibody of the present invention is an IgG isotype, i.e., its heavy chain is γ-type.

[0045] IgG antibodies are classified into four distinct subtypes according to their abundance in serum: IgG1, IgG2, IgG3, and IgG4 (IgG1 being the most abundant). The structure of the hinge region in the γ-chain endows each of these subtypes with unique biological characteristics (even though their Fc regions share approximately 95% similarity, the structures of their hinge regions are relatively different). The antibody of the present invention can be an IgG1, IgG2, IgG3, or IgG4 subtype. However, in a preferred embodiment, the antibody of the present invention is an IgG1 or IgG2 subtype.

[0046] Each light chain comprises a light chain variable region (abbreviated as VL herein) and a light chain constant region CL containing only one domain. There are two types of light chains in mammals: the κ chain encoded by the immunoglobulin κ locus on chromosome 2; and the λ chain encoded by the immunoglobulin λ locus on chromosome 22. In a preferred embodiment, the antibody of the present invention has a κ light chain. The VH and VL regions can be further subdivided into hypervariable regions, called “complementarity-determining regions” (CDRs), which are primarily responsible for binding epitopes of the antigen, and interspersed with more conserved regions called “framework regions” (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The functional ability of the antibody to bind a specific antigen depends on the variable region of each light / heavy chain pair and is primarily determined by the CDRs.

[0047] The heavy chain variable regions of antibodies produced by different B cells differ, but the heavy chain variable regions of all antibodies produced by a single B cell or a B cell clone (or hybrid group) are identical. In contrast, the constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0048] As used herein, the term "antibody fragment" refers to Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, single-chain antibodies, dimers, microantibodies, nanobodies, biantibodies, and their multimers and bispecific antibody fragments. Antibody fragmentation can be performed using conventional techniques. Various techniques have been developed to generate antibody fragments. Traditionally, these fragments are derived from the proteolytic digestion of intact antibodies. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges, thereby generating Fab' fragments. Papain digestion leads to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, microantibodies, nanobodies, biantibodies, bispecific antibody fragments, and other fragments can also be synthesized using recombinant technologies.

[0049] Typically, the antibody fragments of the present invention are functional fragments, that is, antibody fragments that can bind to and preferably inhibit or neutralize the activity of target molecules, just like the antibodies from which they are derived.

[0050] In a particular and preferred embodiment, the antibody of the present invention is a monoclonal antibody. As used herein, "monoclonal antibody" refers to an antibody derived from a nearly homogeneous group of antibodies. More specifically, the antibodies in a given subject are identical, but contain a small number of potentially naturally occurring mutations, which may be present in a very small proportion. In other words, a monoclonal antibody consists of homogeneous antibodies grown from a single cell clone (e.g., hybridoma, eukaryotic host cells transfected with DNA molecules encoding homogeneous antibodies, prokaryotic host cells transfected with DNA molecules encoding homogeneous antibodies, etc.), and is typically characterized by one and only one isotype and subtype of heavy chain and only one type of light chain. Furthermore, unlike polyclonal antibody formulations, each monoclonal antibody targets a single epitope of the antigen.

[0051] To generate monoclonal antibodies, antibody-producing cells (lymphocytes) can be collected from immunized animals as described above and fused with myeloma cells using a standard somatic cell fusion procedure, thereby immortalizing these cells and generating hybridoma cells. These techniques are well-known in the art (e.g., the hybridoma technique originally developed by Kohler and Milstein (1975)), as well as other techniques such as human B-cell hybridoma technology, EBV hybridoma technology for generating human monoclonal antibodies, and screening of combinatorial antibody libraries. Hybridoma cells can be immunochemically screened to generate antibodies that specifically react with target peptides, thereby isolating only monoclonal antibodies that bind to said peptides.

[0052] The antibodies or fragments thereof of the present invention may be human, chimeric, humanized, mouse, CDR transplanted, phage-displayed, bacterial-displayed, yeast-displayed, transgenic mouse-produced, mutagenic, or randomized antibodies or fragments.

[0053] Chimeric antibodies are molecules whose different parts come from different animal species, such as molecules that have a variable region from mouse monoclonal antibodies (mAbs) and a constant region from human immunoglobulins.

[0054] The humanized form of the antibody of the present invention is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. To a large extent, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the hypervariable region of the receptor are replaced with residues from the hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate, thereby achieving the desired specificity, affinity, and capacity. In some cases, the frame region (FR) residues of the human immunoglobulin (receptor antibody) are replaced with corresponding non-human residues of the donor antibody. Furthermore, the humanized antibody may contain residues not found in the receptor antibody or donor antibody. Generally, the humanized antibody may contain substantially all at least one, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of the non-human immunoglobulin (the donor antibody with the desired specificity, affinity, and capacity), and all or substantially all FRs are FRs of the human immunoglobulin sequence. Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "input" residues and are typically derived from the "input" variable region. Humanization is essentially achieved by replacing the corresponding sequence in a human antibody with a hypervariable region sequence. Thus, such a humanized antibody is chimeric, where a variable region essentially smaller than the complete human variable region is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies, with some hypervariable region residues and possibly some FR residues replaced by residues at similar sites in rodent antibodies. Other approaches typically involve conferring the binding affinity of the donor CDR to the antibody receptor variable region framework. One approach involves simultaneously transplanting and optimizing the binding affinity of the variable region binding fragment. Another approach involves optimizing the binding affinity of the antibody variable region.

[0055] The antibodies or fragments thereof of the present invention may have other agents conjugated thereto, such as drugs, toxins or radioactive atoms.

[0056] In one particular aspect, the CTLA4 targeting molecule is DARPIN (a designed ankyrin repeat protein), such as MP0250 DARPin®, MP0317 DARPin®, or MP0533 DARPin®.

[0057] In another specific aspect, the CTLA4 targeting molecule is an aptamer, specifically an oligonucleotide DNA or RNA sequence that specifically binds to CTLA4, such as pegaptanib.

[0058] The CTLA4 targeting molecule is preferably an anti-CTLA4 antibody, or even more preferably an anti-CTLA4 monoclonal antibody, such as quavonlimab, ipilimumab or trimemumab, with ipilimumab or trimemumab being preferred, and ipilimumab being even more preferred.

[0059] Ipilimumab is a monoclonal antibody that works by targeting CTLA-4, thereby activating the immune system. Cytotoxic lymphocytes (CTLs) can recognize and destroy cancer cells. However, inhibitory mechanisms can interrupt this destruction. Ipilimumab shuts down these inhibitory mechanisms and enhances the body's immune response to cancer cells (i.e., allowing lymphocytes to continue destroying cancer cells). Ipilimumab was approved by the U.S. Food and Drug Administration (FDA) in March 2011 for the treatment of melanoma. It is also approved in combination with nivolumab for the treatment of advanced renal cell carcinoma, microsatellite instability (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), and malignant pleural mesothelioma.

[0060] A major drawback of ipilimumab therapy is its association with severe and potentially fatal immune adverse effects due to T-cell activation and proliferation, occurring in 10% to 20% of patients. Serious adverse effects include stomach pain, abdominal distension, constipation, diarrhea, fever, dyspnea, and urinary problems. Checkpoint inhibitor-induced colitis occurred in 5.7% to 9.1% of individuals receiving ipilimumab. Individual cases of severe neurological disorders, including acute inflammatory demyelinating polyneuropathy, ascending motor paralysis, and myasthenia gravis, have been observed following ipilimumab use.

[0061] Trimelimumab is a fully human monoclonal antibody used to treat hepatocellular carcinoma (a type of liver cancer) and is designed to attach to and block CTLA-4. When used in combination with durvalumab, the most common side effects include rash, pruritus (itching), diarrhea, abdominal pain, elevated liver enzyme levels, fever, hypothyroidism, cough, peripheral edema (especially swelling of the ankles and feet), and elevated levels of lipase (an enzyme that helps digest fats, primarily produced by the pancreas).

[0062] Trimelimumab blocks the binding of antigen-presenting cell ligands B7.1 and B7.2 to CTLA-4, thereby inhibiting the downregulation of B7-CTLA-4-mediated T cell activation. Subsequently, B7.1 or B7.2 can interact with another T cell surface receptor protein, CD28, resulting in B7-CTLA-4-mediated inhibition not antagonizing B7-CD28-mediated T cell activation. Unlike ipilimumab, which is an IgG1 isotype, trimemumab is an IgG2 isotype.

[0063] Therefore, the specific drug conjugates described herein are antibody-drug conjugates (“ADCs”) or their pharmaceutically acceptable polymorphs, enantiomers, stereoisomers, salts, solvates, or tautomers. ADCs are a class of biopharmaceuticals designed as targeted therapies for cancer treatment. Unlike chemotherapy, ADCs aim to target and kill tumor cells while sparing healthy cells. ADCs are complex molecules composed of antibodies linked to a biologically active payload, typically a cytotoxic (anticancer) agent or drug.

[0064] In the context of this invention, the ADC preferably comprises a monoclonal anti-CTLA4 antibody, such as ipilimumab or trimemumab, with ipilimumab being preferred.

[0065] Effective payload of drug conjugate

[0066] Many of the payloads of tumor drug conjugates (typically ADCs) are based on natural products.

[0067] Payloads include, for example, microtubule inhibitors such as monomethylolpropamine (MMAE), monomethylolpropamine F (MMAF), or maytansine-like substances (such as DM1 or DM4); DNA damaging agents such as chaziomycin; and topoisomerase 1 inhibitors such as SN38 (the active metabolite of irinotecan) or exatecan. Alternatives to small molecule payloads, such as siRNA, have also been investigated.

[0068] In a preferred aspect, the payload is an anticancer agent, preferably a cytotoxic agent, typically a small molecule.

[0069] The cytotoxic agent may be selected from, for example, microtubule inhibitors (MTIs), DNA damaging agents such as alkylating agents or platinum complexes, cytotoxic antibiotics, antimetabolites, topoisomerase I inhibitors, RNA polymerase inhibitors, antimitotic agents, and any combination thereof.

[0070] The microtubule inhibitor (MTI) may be, for example, taxanes (e.g., paclitaxel or docetaxel); vinca alkaloids (e.g., vinca alkaloids (VBL), vinorelbine (VRL), vincristine (VCR), or vindesine (VDS)); or epothilone (e.g., epothilone A, epothilone B, ixabepilone, or semi-synthetic analogs of epothilone B, epothilone C, epothilone D, epothilone E, or epothilone F).

[0071] In one particular aspect, the microtubule inhibitor is selected from maytansine-like substances (such as DM1 or DM4), olistatin and its derivatives, such as monomethylolistatin E (MMAE) or monomethylolistatin F (MMAF).

[0072] MMAE and MMAF are synthetic antitumor agents that inhibit cell division by blocking microtubule polymerization. Due to their high toxicity, MMAE and MMAF cannot be used as single-dose chemotherapy drugs.

[0073] The olistatin derivative is preferably MMAF.

[0074] A particular conjugate comprises i) ipilimumab, ii) a microtubule inhibitor as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0075] A particular conjugate comprises i) trimelimumab, ii) a microtubule inhibitor as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0076] A particular conjugate comprises i) ipilimumab, ii) maytansine as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0077] A particular conjugate comprises i) trimemumab, ii) maytansine as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0078] A particular and preferred conjugate comprises i) ipilimumab, ii) DM1 as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0079] A particular and preferred conjugate comprises i) trimelimumab, ii) DM1 as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0080] A particular and preferred conjugate comprises i) ipilimumab, ii) DM4 as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0081] A particular and preferred conjugate comprises i) trimelimumab, ii) DM4 as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0082] A particular conjugate comprises i) ipilimumab, ii) olarestatin or an olarestatin derivative as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0083] A particular conjugate comprises i) trimelimumab, ii) olarestatin or an olarestatin derivative as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0084] A particular conjugate comprises i) ipilimumab, ii) a DNA damaging agent as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0085] A particular conjugate comprises i) trimelimumab, ii) a DNA damaging agent as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0086] A particular conjugate comprises i) ipilimumab, ii) kazimidam as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0087] A particular conjugate comprises i) trimelimumab, ii) kazimidam as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0088] A particular and preferred conjugate comprises i) ipilimumab, ii) a topoisomerase 1 inhibitor as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0089] A particular and preferred conjugate comprises i) trimelimumab, ii) a topoisomerase 1 inhibitor as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0090] A particular and preferred conjugate comprises i) ipilimumab, ii) SN38 as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0091] A particular and preferred conjugate comprises i) trimelimumab, ii) SN38 as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0092] A particular and preferred conjugate comprises i) ipilimumab, ii) esanotecan as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0093] A particular and preferred conjugate comprises i) trimelimumab, ii) ethatecan as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0094] A particular and preferred conjugate comprises i) ipilimumab, ii) a cytotoxic agent, preferably MMAF, and iii) a cleavable linker, preferably comprising a valine-citrulline dipeptide (“Vc” dipeptide as described below) or a phenylalanine-lysine dipeptide.

[0095] A particular and preferred conjugate comprises i) trimelimumab, ii) MMAF as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0096] A particular and preferred conjugate comprises i) ipilimumab, ii) MMAE as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0097] A particular and preferred conjugate comprises i) trimelimumab, ii) MMAE as a cytotoxic agent, and iii) a cleavable linker comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

[0098] In another specific aspect, the cytotoxic agent is a DNA damaging agent, such as an alkylating agent or a platinum complex.

[0099] Examples of alkylating agents include nitrogen mustards (e.g., bendamustine, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan); nitrosoureas (e.g., carmustine, lomustine, streptozocin); alkyl sulfonates (e.g., busulfan); triazines (e.g., dacarbazine, temozolomide); and ethyleneimines (e.g., altretamine, thiotepa).

[0100] Examples of platinum complexes include cisplatin, carboplatin, and oxaliplatin.

[0101] In another specific aspect, the cytotoxic agents are cytotoxic antibiotics, such as bleomycin, kazidromycin, daunorubicin, doxorubicin, dactinomycin D, epirubicin, idarubicin, mitoxantrone, and mitomycin.

[0102] In another specific aspect, the cytotoxic agents are antimetabolites, such as 6-mercaptopurine, fludarabine, 5-fluorouracil, gemcitabine, cytarabine, pemetrexed, and methotrexate.

[0103] In another specific aspect, the cytotoxic agents are topoisomerase inhibitors, such as irinotecan (CPT-11) and topotecan.

[0104] In another specific aspect, the cytotoxic agents are RNA polymerase inhibitors, such as rubitectedin and CX-5461.

[0105] In another specific aspect, the cytotoxic agents are antimitotic agents, particularly non-taxane site microtubule stabilizers, such as peloruside A (PLA) or laulimalide.

[0106] The payload can be combined with several different cytotoxic agents, such as any combination of the aforementioned anticancer agents, and in particular any combination of the aforementioned cytotoxic agents.

[0107] In the context of this invention, the drug-to-antibody ratio (DAR), i.e., the number of effective payload molecules per antibody, can vary. For example, the DAR can be two (DAR=2) or four (DAR=4).

[0108] connector

[0109] The CTLA4-targeting molecule, such as a CTLA4 antibody, preferably a CTLA-4 monoclonal antibody, is linked to the cytotoxic (anticancer) agent via a linker, thereby ensuring reduced cytotoxic payload shedding before reaching tumor cells, thus improving safety and limiting dosage. Linker stability is particularly important when the drug conjugate is administered to the desired subject via a systemic route (typically intravenously).

[0110] Linkers are based on chemical motifs, including disulfides, hydrazones or peptides (cleavable) or thioethers (uncleavable). Cleavable and uncleavable linkers have been shown to be safe in preclinical and clinical trials [see, for example, brentuximab vedotin, which includes an enzyme (cathepsin)-sensitive cleavable linker for delivering the antimicrotubule agent MMAE to human-specific CD30+ malignant cells, and trastuzumab emtansine, which is a combination of a microtubule formation inhibitor and a trastuzumab antibody and uses a stable uncleavable linker].

[0111] The emergence of better, more stable linkers alters the function of chemical bonds. The type of linker (cleavable or uncleavable) endows the (cytotoxic) payload with specific properties. For example, an uncleavable linker holds the drug within the cell. As a result, the entire CTLA4-targeting molecule (e.g., a CTLA4 antibody, including a CTLA4 monoclonal antibody), the linker, and the payload enter the targeted CTLA4+ cells, particularly tumor-bearing Treg cells, where the CTLA4-targeting molecule is degraded. The resulting complex (CTLA4-targeting molecule, linker, and cytotoxic agent) is considered the active drug. Conversely, cleavable linkers are cleaved by enzymes within the cell. The (cytotoxic) payload can then escape from the target cell and attack neighboring cells in a process known as "bystander killing."

[0112] A specific and preferred type of connector of the present invention is a cuttable connector, such as an acid-unstable connector, an enzyme-cuttable connector, a lysosomal protease-sensitive connector, a disulfide connector, or a β-glucuronide connector.

[0113] A preferred acid-unstable linker is cleavable at acidic pH conditions present in vivo within cells.

[0114] A specific and preferred enzyme-cleavable linker comprises a valine-citrulline dipeptide (also referred to as the “Vc” dipeptide in the experimental section) or a phenylalanine-lysine dipeptide.

[0115] A particular and preferred conjugate comprises i) ipilimumab, ii) a cytotoxic agent as described herein, and iii) a cleavable linker comprising a valine-citrulline dipeptide.

[0116] A particular and preferred conjugate comprises i) ipilimumab, ii) a cytotoxic agent as described herein, and iii) a cleavable linker comprising a phenylalanine-lysine dipeptide.

[0117] Another particular conjugate contains i) trimemumab, ii) a cytotoxic agent as described herein, and iii) a cleavable linker comprising a valine-citrulline dipeptide.

[0118] Another particular conjugate contains i) trimemumab, ii) a cytotoxic agent as described herein, and iii) a cleavable linker comprising a phenylalanine-lysine dipeptide.

[0119] Pharmaceutical composition or combination

[0120] A particular pharmaceutical composition of the present invention comprises at least one pharmaceutical conjugate according to the invention, such as at least two, three or four different pharmaceutical conjugates, wherein the first conjugate comprises a first payload (or cytotoxic agent), the second or other (additional) conjugates comprise different payloads (or cytotoxic agents), and a pharmaceutically acceptable load, excipient, carrier or diluent.

[0121] "Pharmaceutical composition" means a mixture of one or more therapeutic agents described herein or their pharmaceutically acceptable salts, solvates, hydrates, or prodrugs as active ingredients, and at least one pharmaceutically acceptable load, excipient, carrier, and / or diluent. Therefore, "pharmaceutical composition" generally refers to one or more active ingredients and one or more inert components constituting a carrier, as well as any product directly or indirectly produced by the combination, compounding, or aggregation of any two or more components, or by the dissociation of one or more components, or by other types of reactions or interactions of one or more components. Therefore, the pharmaceutical compositions of the present invention comprise any composition prepared by mixing the compounds of the present invention (typically a therapeutically effective amount), preferably at least one pharmaceutical conjugate, and at least one pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may additionally contain one or more other compounds as active ingredients, such as one or more additional therapeutic (preferably anticancer) compounds of the present invention, or prodrug compounds or other known active substances, preferably active anticancer substances.

[0122] As used herein, an “effective dose” or “effective amount” of a compound (e.g., a pharmaceutical conjugate, combination, or composition) is an amount sufficient to achieve any one or more beneficial or desired outcomes (including biochemical, histological, and / or behavioral symptoms) of a disease (typically cancer) and its complications.

[0123] For therapeutic purposes, a "therapeutic effective amount" refers to the amount of a compound, such as a pharmaceutical conjugate, combination, or composition, applied that will alleviate one or more symptoms of the treated condition to a certain extent. In the treatment of cancer, a therapeutic effective amount refers to an amount that has the following effects: reduces tumor size; inhibits (i.e., slows down, preferably terminates) tumor metastasis; inhibits (i.e., slows down, preferably terminates) tumor growth or invasion to a certain extent; alleviates (or preferably eliminates) one or more signs or symptoms related to cancer or the treatment of cancer itself to a certain extent; reduces the dosage of other drugs required to treat the disease; or mitigates secondary adverse (toxic) effects of cancer treatment.

[0124] The effective dose can be administered once or multiple times. For the purposes of this invention, the effective dose of a drug, compound, combination, or composition is an amount sufficient to directly or indirectly achieve preventive (in the case of preventing cancer recurrence) or therapeutic treatment. As understood in clinical context, the effective dose of a drug, compound, combination, or composition may be achieved with or without another drug, compound, combination, or composition.

[0125] As used herein, "pharmaceuticalally acceptable loading agents, excipients, carriers, or diluents" refer to loading agents, excipients, carriers, or diluents that do not cause significant irritation to organisms and do not eliminate the biological activity and properties of the active compound or therapeutic agent. Pharmaceutically acceptable loading agents, excipients, carriers, or diluents can comprise any conventional drug loading agents, excipients, carriers, or diluents. The selection of loading agents, excipients, carriers, or diluents will largely depend on a variety of factors, such as the specific route of administration, the effect of the loading agent, excipient, carrier, or diluent on solubility and stability, and the nature of the dosage form. Suitable drug carriers include inert diluents or fillers, water, and various organic solvents (such as hydrates and solvates). If desired, the pharmaceutical composition may contain additional ingredients such as flavoring agents, binders, etc.

[0126] Another specific pharmaceutical composition of the present invention comprises a pharmaceutical conjugate, various therapeutic agents and pharmaceutically acceptable loads, excipients, carriers or diluents.

[0127] Different treatment agents

[0128] As taught above, in a preferred aspect, in addition to the CTLA4-targeting drug conjugate, the combination, composition, or kit described below further comprises at least one (i.e., one or more additional) different therapeutic agents, preferably anticancer agents.

[0129] The various therapeutic agents are typically anticancer agents. These anticancer agents can be selected from, for example, immune checkpoint inhibitors, anti-angiogenic agents, cytotoxic agents, hormones, aromatase inhibitors, glucocorticoids, and any combination thereof, wherein the amounts are preferably used together to effectively treat cancer.

[0130] The immune checkpoint targeting agent may be an anti-PD1 agent [i.e., an agent that targets programmed cell death-1 receptor (PD1)], such as nivolumab, pembrolizumab, cemiplimab, retifanlimab, toripalimab, or dostarlimab; an anti-PDL1 (programmed cell death-1 receptor ligand) agent, such as atezolizumab, duvarulimab, or avelumab; an anti-LAG3 (lymphocyte activation gene 3) agent, such as relatlimab or eftilagimod; or an anti-TIGIT (T cell immune receptor with Ig and ITIM domains) agent, such as tiragolumab or domvanalimab.

[0131] The anti-angiogenic agent may be a tyrosine kinase inhibitor, such as sorafenib, sunitinib, vendetanib, lenvatinib, axitinib, or cabozantinib.

[0132] The anti-angiogenic agent may be a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway (ligand or receptor), such as bevacizumab or aflibercept.

[0133] The cytotoxic agent may be an anticancer chemotherapeutic agent, such as alkylating agents, like nitrogen mustards (e.g., bendamustine, chlorambucil, cyclophosphamide, ifosfamide, metronidazole, and melphalan); nitrosoureas (e.g., carmustine, lomustine, streptozotocin); alkyl sulfonates (e.g., busulfan); triazines (e.g., dacarbazine, temozolomide); ethyleneimines (e.g., hexamethylmelamine, thiotepa); antimetabolites. Such as 6-mercaptopurine, fludarabine, 5-fluorouracil, gemcitabine, cytarabine, pemetrexed, methotrexate; topoisomerase inhibitors, such as irinotecan (CPT-11) or topotecan; antibiotics, such as bleomycin, daunorubicin, doxorubicin, actinomycin D, epirubicin, idarubicin, mitoxantrone, and mitomycin; or antimitotic agents, especially non-taxane site microtubule stabilizers, such as piroxicam A (PLA) or lamivudine.

[0134] Hormonal agents are those that inhibit the estrogen or progesterone pathway, such as tamoxifen.

[0135] The anticancer agent may be an aromatase inhibitor, such as anastrozole (Arimidex) or exemestane (aromasin).

[0136] The anticancer agent may be a glucocorticoid pathway target, such as prednisone, prednisolone, triamcinolone, methylprednisolone, or dexamethasone.

[0137] In some embodiments, the additional anticancer agent is selected from oncolytic viruses (e.g., T-VEC), oncolytic peptides (e.g., LTX315), and protein kinase C agonists (e.g., tigilanol tiglate).

[0138] In a preferred embodiment, the at least one different therapeutic agent, preferably an anticancer agent, is selected from immune checkpoint targets, cytotoxic agents (especially anticancer chemotherapeutic agents), antiangiogenic agents, hormones, and any mixtures or combinations thereof.

[0139] Route of administration and formulation

[0140] The (pharmaceutical) combinations or (pharmaceutical) compositions of the present invention include those suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular, pulmonary (nasal or oral inhalation), or nasal administration, but the most suitable route in any given case will depend on the nature and severity of the disease being treated and the nature of the active ingredient. An effective dose of the active compound of the present invention can be delivered to mammals, particularly humans, via any suitable route of administration.

[0141] In the context of this invention, the active compound is preferably administered intravenously and / or intratumorally. The preferred route of administration for the pharmaceutical conjugates described herein is intratumorally.

[0142] Active ingredients can be readily available in unit dosage forms and prepared using any method well-known in the pharmaceutical field. Dosage forms include, for example, tablets, dispersants, suspensions, solutions, capsules, creams, ointments, aerosols, etc.

[0143] The (drug) combination or pharmaceutical composition may be in the form of, for example, tablets, capsules, pills, powders, sustained-release formulations, solutions or suspensions suitable for oral administration; sterile solutions, suspensions or emulsions suitable for parenteral injection; ointments or creams suitable for topical application; or sterile solutions, suspensions, emulsions or slow-release chemical agents suitable for intratumoral application, thereby allowing the drug conjugate, particularly CTLA4 ADC, to have a prolonged residence time and slow release in the tumor microenvironment.

[0144] The combination or pharmaceutical composition may be a unit dosage form suitable for a precise amount to be administered in a single dose.

[0145] Combinations or pharmaceutical compositions of therapeutic agents suitable for delivering the combination therapies of the present invention, and methods of their preparation, will be apparent to those skilled in the art. Such compositions and methods of their preparation can be found, for example, in "Remington's Pharmaceutical Sciences," 19th edition (Mack Publishing Company, 1995), the disclosure of which is incorporated herein by reference in its entirety.

[0146] Suitable modified release formulations are described in U.S. Patent No. 6,106,864. Details of other suitable release technologies, such as high-energy dispersions and permeation-coated particles, can be found in Verma et al., Pharmaceutical Technology On-line, 25(2), 1-14 (2001). The disclosures of these references are incorporated herein by reference in their entirety.

[0147] In practical use, the compounds of the present invention can be used as active ingredients and tightly mixed with drug loading agents, excipients, carriers, and / or diluents using conventional drug mixing techniques. As described above, the loading agents, excipients, or carriers can take various forms depending on the desired formulation for administration, such as oral or parenteral (including intravenous or intratumoral).

[0148] Dosimetry

[0149] When the drug conjugate is used to treat or prevent the cancers mentioned herein, satisfactory results are generally obtained when the drug conjugate is administered at a dose of about 1.5 mg / kg to about 5.5 mg / kg, possibly as a single intravenous dose once every 3 weeks (Q3W) IV.

[0150] The daily dose of the anti-CTLA4 or CTLA4-targeting drug conjugate administered to the subject is, for example, about 0.01 mg / kg to about 10 mg / kg, preferably about 0.1 mg / kg to about 1 or 3 mg / kg, and even more preferably about 0.1 mg / kg to about 1 mg / kg.

[0151] The effective dose of the CTLA4-targeting drug conjugate of the present invention is generally in the range of about 0.01 mg to about 5 or about 10 mg / kg body weight, preferably about 0.01 to about 1 mg / kg body weight, in the form of a single dose or divided doses. This dose can be administered daily, weekly, every two weeks, or every three weeks to the subject in need. This dosage regimen can be adjusted by an oncologist to provide the patient with the best therapeutic response.

[0152] In some cases, a dosage level of the drug conjugate at the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may be used without causing any harmful side effects, provided that this larger dose is first divided into several smaller doses administered throughout the day. The dose may be administered as a single dose, or optionally further divided into smaller doses suitable for twice-daily, three-daily, or four-daily administration.

[0153] The therapeutically effective dose of a compound depends on many factors, including, for example, the age and weight of the (mammal) subject, the exact condition requiring treatment and its severity, the nature of the formulation and the method of administration, and is ultimately determined by the attending physician or veterinarian. However, the effective dose of the CTLA4-targeting drug conjugate according to the invention is generally in the range of 0.01 to 5 or 10 mg / kg body weight per day for the recipient (mammal subject), particularly typically in the range of 0.01 to 1 mg / kg body weight per day. Therefore, the practical daily dose for a 70 kg adult is typically between 0.7 and 70 mg, which can be administered as a single daily dose or typically in a series of fractionated doses per day (e.g., twice, three times, four times, five times, or six times) to ensure a consistent total daily dose.

[0154] The effective dosage of any active ingredient described herein can vary depending on the specific compound used, the administration method, the disease being treated, and the severity of the disease. Such dosage can be readily determined by those skilled in the art, typically by oncologists.

[0155] In one particular aspect, the pharmaceutical conjugate comprises i) ipilimumab or trimemumab, ii) a cytotoxic agent, such as a tubulin inhibitor, maytansin-like substance, DNA binding agent, or topoisomerase inhibitor, particularly such as MMAE, MMAF, DM1, DM4, chalcogenide, SN38, or esanotecan, preferably any one of DM1, DM4, MMAE, or MMAF, even more preferably MMAF, and iii) a cleavable linker, preferably comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide, and the pharmaceutical conjugate is present in the combination or composition at a dose of about 0.01 mg to about 10 mg, preferably about 0.1 mg to about 1 mg, for example 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, or 0.9 mg, more preferably about 0.3 mg.

[0156] The effective dose of ipilimumab is usually in the range of about 1 to about 10 mg / kg body weight, preferably about 3 to about 10 mg / kg, such as 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg or 9 mg / kg, and is usually administered daily in the form of a single dose or divided doses.

[0157] In one particular aspect, the pharmaceutical conjugate comprises i) ipilimumab or trimemumab, ii) a cytotoxic agent, such as a tubulin inhibitor, maytansin-like substance, DNA binding agent, or topoisomerase inhibitor, particularly such as MMAE, MMAF, DM1, DM4, chalcogenide, SN38, or esanotecan, preferably any one of DM1, DM4, MMAE, or MMAF, and iii) a cleavable linker, preferably comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide, and the pharmaceutical conjugate is present in the combination or composition at a dose of about 0.01 mg to about 10 mg, preferably about 0.1 mg to about 1 mg, for example 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, or 0.9 mg, more preferably about 0.3 mg.

[0158] The effective dose of ipilimumab is usually in the range of about 1 to about 10 mg / kg body weight, preferably about 3 to about 10 mg / kg, such as 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg or 9 mg / kg, and is usually administered daily in the form of a single dose or divided doses.

[0159] Reagent test kit

[0160] The kits described herein may be particularly suitable for administering different dosage forms, such as oral and parenteral (usually intravenous or intratumoral, preferably intratumoral), for administering a single active (particularly therapeutic) agent of a combination or composition at different dose intervals, or for titrating the active (particularly therapeutic) agents of a combination or composition with each other. To aid compliance, the kits typically include instructions for use. The kits may also contain other materials for administering the medication, such as diluents, filters, intravenous bags and tubing, needles, and syringes.

[0161] The kit of the present invention comprises at least two drug conjugates as described herein, or at least one drug conjugate as described herein and different therapeutic agents as described herein, said therapeutic agents being selected, for example, from immune checkpoint targets, anti-angiogenic agents, cytotoxic agents, hormones, and any combination thereof.

[0162] A particular kit contains a) at least one pharmaceutical conjugate containing a CTLA4-targeting molecule as described herein, particularly a pharmaceutical conjugate, especially an anti-CTLA4 ADC, said pharmaceutical conjugate comprising i) a CTLA4-targeting molecule, preferably an anti-CTLA-4 monoclonal antibody, ii) at least one cytotoxic agent, and iii) a linker connecting said CTLA4-targeting molecule and said cytotoxic agent, and b) various therapeutic agents as described herein, said therapeutic agents being selected, for example, from immune checkpoint targets, anti-angiogenic agents, cytotoxic agents, hormones, and combinations thereof.

[0163] Another specific kit contains a Pickering emulsion of a CTLA4-targeting molecule, particularly an anti-CTLA4 ADC, formulated in polylactic-co-glycolic acid (PLGA) nanoparticles with or without radiopaque ethyl iodine oil, to allow the drug conjugate to have prolonged residence and slow release in the injected tumor.

[0164] Therapeutic uses of drug conjugates, combinations, compositions and kits

[0165] The inventors also describe combinations or compositions as described herein for use as medicaments. Combinations or compositions of the present invention comprise at least one CTLA4-targeting molecule as described herein, preferably a pharmaceutical conjugate comprising i) a CTLA4-targeting molecule, preferably an anti-CTLA-4 monoclonal antibody, ii) at least one cytotoxic agent, such as a tubulin inhibitor, maytansine-like substance, DNA binding agent, or topoisomerase inhibitor, particularly such as MMAE, MMAF, DM1, DM4, kazidromycin, SN38, or ethatecan, and iii) a linker connecting the CTLA4-targeting molecule and the cytotoxic agent, preferably a cleavable linker as described herein, such as a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide. Combinations or compositions of the present invention typically comprise additional (different) therapeutic agents. Several different (active) agents may be used sequentially, simultaneously, or in parallel in subjects in need.

[0166] As used herein, the term "combination" or "combination therapy" refers to each therapeutic agent of the combination therapy of the present invention being administered, alone or in the form of a pharmaceutical composition or medicine, sequentially, in parallel or simultaneously.

[0167] As used herein, the terms "sequential" or "arranged" refer to the administration of each therapeutic agent of the combination therapy of the present invention, either alone or in the form of a drug, one after another, wherein each therapeutic agent may be administered in any order. Sequential administration may be particularly useful when the therapeutic agents in the combination therapy are in different dosage forms, for example, one therapeutic agent is a tablet and the other is a sterile liquid, and / or when the therapeutic agents are administered according to different dosing regimens, for example, one therapeutic agent is administered daily and the second therapeutic agent is administered at a lower frequency, such as weekly.

[0168] As used herein, the term "parallel" means that each therapeutic agent in the combination therapy of the present invention is administered alone or as a separate drug, a second therapeutic agent is administered immediately after the first therapeutic agent, and the therapeutic agents are administered in any order. In a preferred embodiment, the therapeutic agents are administered in parallel.

[0169] As used herein, the term "simultaneously" means that each therapeutic agent of the combination therapy of the present invention is administered in the same drug. As will be understood by those skilled in the art, the combination therapy can be effectively administered to a subject at different stages of treatment.

[0170] The pharmaceutical conjugates, compositions, or kits of the present invention are used in subjects in need to treat (or in the case of treatment) cancer, or to prevent (or in the case of prevention) cancer recurrence.

[0171] In the context of this invention, the terms "patient" and "subject" are synonyms and refer to any single subject who requires treatment or participates in a clinical trial, epidemiological study, or serves as a control, including human and mammalian veterinary patients.

[0172] "Subject" or "patient" is usually a mammal. The subject can be a human or a non-human mammal, such as a rodent, for example a mouse or rat; a rabbit; a primate, such as a monkey; a dog, a cat, a bovine, an equine, such as a horse; or a genetically modified species thereof.

[0173] In one particular and preferred aspect, the mammal is a human, regardless of its age or sex. In one particular aspect, the subject is an adult subject. In another particular aspect, the subject is a human child aged from birth to 18 years of age.

[0174] In a particular aspect of each of the methods, combinations and uses described herein, the pharmaceutical conjugates of the invention are administered to a subject, particularly a subject diagnosed with cancer who has not previously received treatment (cancer), i.e., has not undergone cancer treatment.

[0175] In another specific aspect of each of the methods, combinations, and uses described herein, the pharmaceutical conjugates of the invention are administered to a subject suffering from cancer who has been exposed to at least one prior therapy using an anticancer agent, particularly prior therapy containing an anti-CTLA-4 monoclonal antibody as described herein, especially ipilimumab or trimemumab [used alone or in combination with additional conventional anticancer agents such as anti-PD1 (e.g., nivolumab), or whose tumor has been surgically removed, i.e., a subject who has undergone treatment.

[0176] In a particular and preferred aspect of each of the methods, combinations, and uses described herein, the pharmaceutical conjugates of the invention are administered to a subject, particularly a subject diagnosed with cancer, preferably for neoadjuvant treatment (i.e., prior to any surgery). Thus, in a preferred aspect, the malignant tumor, particularly a primary malignant tumor, and / or draining lymph nodes, has not yet been surgically removed.

[0177] In another specific aspect, the subject has been exposed to, for example, as part of a complete routine treatment regimen, such as at least one cycle of the entire planned treatment regimen.

[0178] When disease progression and / or unacceptable therapy-induced toxicity are observed or anticipated, patients are preferably treated with the combinations or compositions of the present invention.

[0179] In a particular aspect of each of the pharmaceutical conjugates, compositions, kits, combinations, methods, and uses described herein, the subject's cancer is a cancer as defined above, particularly a solid carcinoma selected from melanoma, renal cell carcinoma (RCC), colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), mesothelioma (MM) (particularly pleural mesothelioma), or a hematologic malignancy selected from lymphoma and leukemia.

[0180] In the context of the pharmaceutical conjugates, compositions, kits, combinations, methods, and uses described herein, the subject's cancer is early-stage cancer, such as a localized primary tumor, or late-stage cancer or cancer with a poor prognosis, such as locally advanced cancer, (surgically) unresectable cancer, metastatic cancer, recurrent cancer, or cancer resistant to conventional treatment.

[0181] In one particular aspect of the invention, the pharmaceutical conjugate, composition, combination, or kit is intended for use in combination with radiotherapy, i.e., in combination with a subject’s tumor exposure to radiation.

[0182] The radiotherapy may be, for example, external beam radiotherapy, brachytherapy, whole-body radiotherapy, and especially radioligand-based radiotherapy or proton therapy.

[0183] This invention is particularly beneficial to subjects suffering from cancer whose tumor or cancer tumor microenvironment (TME) comprises CTLA4+ cells (i.e., CTLA4-expressing cells), especially CTLA4+ tumor cells and / or CTLA4+ immune cells, preferably CTLA4+ Tregs. Such tumors are considered herein to be in the “immunoediting stage,” i.e., tumors that are particularly sensitive to (or able to benefit from) the anti-CTLA4 drug conjugates of this invention.

[0184] This invention is also particularly beneficial to subjects who, in addition to having cancers in which the tumor microenvironment (TME) contains CTLA4+ cells (especially CTLA4+ tumor cells and / or CTLA4+ immune cells, preferably CTLA4+ Tregs), have not or will not demonstrate a “durable clinical benefit” (“DCB” means a complete or partial response or stable disease state at 6 months after treatment initiation according to RECIST 1.1 criteria) following (naked) anti-CTLA4 and / or anti-PD(L)1 therapy. These patients are also referred to herein as “no durable clinical benefit” (or “no DCB”) patients.

[0185] In other words, these subjects or patients are either patients who have not received anticancer treatment, or patients who have been exposed to anticancer treatment but have shown resistance to the treatment, particularly to (naked) anti-CTLA4 and / or anti-PD(L)1 (primary or secondary resistance, preferably primary resistance).

[0186] In one particular aspect, the untreated (anti-cancer) subject is a subject who is expected to be resistant to (naked) anti-CTLA4 and / or anti-PD(L)1 agents.

[0187] Specific subjects or patients expected to be "DCB-free" are those whose tumors do not contain myeloid cells, contain low levels or dysfunctional myeloid cells (especially macrophages), and whose myeloid cells do not express or express low levels of Fc. Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc Receptor IIIb (“CD16b”).

[0188] In the context of this invention, Fc expressed by myeloid cells Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc Low levels of receptor IIIb (“CD16b”) are insufficient to allow ADCC or ADCP activity.

[0189] Dysfunctional myeloid cells are defined herein as cells incapable of performing antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP). In vitro ADCC / ADCP assays can be performed by those skilled in the art using known methods (see Yamashita, M., Kitano, S., Aikawa, H. et al., Anovel method for evaluating antibody-dependent cell-mediated cytotoxicity by flow cytometry using cryopreserved human peripheral blood mononuclear cells). Sci Rep 6, 19772 (2016)https: / / doi.org / 10.1038 / srep19772).

[0190] Another specific type of expected "DCB-free" subject or patient is one whose tumor contains Fc Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc The proportion of receptor IIIb (“CD16b”) positive myeloid cells (especially macrophages) was lower than that detected in subjects with the same cancer who were identified or expected to have a durable clinical benefit from anticancer therapy, preferably involving anti-CTLA-4 monoclonal antibodies such as ipilimumab or trimemumab [used alone or in combination with additional conventional anticancer agents such as anti-PD1 (e.g., nivolumab).

[0191] Therefore, in the context of this invention, "DCB-free" subjects are preferred subjects. In fact, these DCB-free patients are particularly likely to benefit from the ADC of this invention because the ADC is capable of producing clinical benefit in these patients, or in other words, capable of converting "DCB-free" subjects or patients into "DCB" subjects or patients. In these DCB patients, due to this invention, disease stabilization or partial or complete remission will be advantageously observed.

[0192] The specific CTLA4-expressing cells sensitive to the anti-CTLA4 drug conjugates described herein are regulatory T cells, preferably CD4+FOXP3+CTLA4+ T cells and / or CD4+CD25+CD39+ T cells. These Treg cells are Treg cells that have undergone tumor antigen-specific clonal proliferation.

[0193] Preferred subjects are those with cancer whose tumors and / or TMEs contain CTLA4-expressing cells. These subjects are more likely to benefit from administration of the pharmaceutical conjugates of the present invention or compositions containing such pharmaceutical conjugates, particularly those comprising: i) a cytotoxic T-lymphocyte antigen 4 protein (CTLA4) targeting molecule, preferably an anti-CTLA-4 monoclonal antibody, ii) at least one cytotoxic agent, and iii) a linker connecting the CTLA4 targeting molecule and the cytotoxic agent.

[0194] Therapeutic targeting of such tumor or immune cell subsets, particularly of tumor antigen-specific Tregs, allows for a favorable specific anti-tumor immune response against cancer while maintaining (i.e., lower toxicity) in healthy tissues, typically surrounding the tumor, especially when the anti-CTLA4 drug conjugate or a composition containing the drug conjugate is administered intratumorally.

[0195] Therefore, in a particular aspect of the invention, the cancer subject to be treated is a subject whose tumor or tumor microenvironment (TME) contains CTLA4+ cells, preferably CTLA4+ tumor cells and / or CTLA4+ immune cells, particularly CTLA4+ Tregs.

[0196] In another respect, the subject is a subject resistant to (naked) anti-CTLA4 and / or anti-PD(L)1 agents.

[0197] In another specific aspect of the invention, the cancer subject to be treated is a subject whose tumor and / or TME does not show myeloid cells, shows low levels or dysfunctional myeloid cells, said myeloid cells not expressing or expressing low levels of Fc. Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc Receptor IIIb (“CD16b”), especially in tumors or TMEs that do not express or express low levels of Fc. Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc Subjects with myeloid cells, particularly macrophages, of receptor IIIb (“CD16b”).

[0198] On the other hand, the subject to be treated is the Fc contained within it. Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc The proportion of receptor IIIb (“CD16b”) positive myeloid cells (particularly macrophages) was lower than that detected in subjects with the same cancer who were identified or expected to have a durable clinical benefit (DCB) from anticancer therapy, preferably particularly involving anti-CTLA-4 monoclonal antibodies such as ipilimumab or trimemumab [used alone or in combination with additional conventional anticancer agents such as anti-PD1 (e.g., nivolumab).

[0199] This article also describes a method for selecting / identifying subjects (typically patients) who are most likely to be sensitive to or more responsive to the cancer treatments described herein, particularly those who become sensitive to or more responsive to cancer treatment again, for example, after showing resistance to anticancer treatment or having been identified or predicted as “DCB-free” subjects. The method preferably includes step i) determining the presence of CTLA4+ cells (also referred to as “intratumoral CTLA4+ cells”) in the tumor or TME, and the presence of myeloid cells (also referred to as “intratumoral myeloid cells”), particularly macrophages, and / or said myeloid cells expressing Fc. Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc The level of receptor IIIb (“CD16b”), and step ii) identifying the subject as a subject who will particularly benefit from the treatment described herein, which includes administration of the pharmaceutical conjugate of the present invention.

[0200] CTLA4+ cell detection can be readily performed by those skilled in the art using any commercially available anti-CTLA4 staining antibody, such as Biolegend catalog number 369612 (BD Biosciences catalog number 557301), in vitro, ex vivo, or in vivo.

[0201] Similarly, those skilled in the art can readily express Fc in vitro from fixed or frozen baseline tumor biopsies from cancer patients. Receptor I ("CD64"), Fc Receptor IIIa (“CD16a”) and / or Fc The detection and quantification of receptor IIIb (“CD16b”) in myeloid cells (particularly macrophages) using transcriptomic evaluation methods, such as RNAseq or qRT-PCR for detecting or quantifying the expression levels of any of the following genes: CD68, CD163, CSF1R, CD16a, CD16b, and CD64, or any combination thereof, particularly CD64, CD16a, and / or CD16b.

[0202] The pharmaceutical conjugates or compositions of the present invention may preferably be administered to subjects as neoadjuvant therapy as a monotherapy or in combination with different anticancer agents prior to any partial or complete surgical resection of the tumor or destruction of the lesion by any cytoreductive strategy, and preferably by local delivery, including intratumoral (IT), intravascular (IV) or local routes, by a single administration or by repeated administration.

[0203] The pharmaceutical conjugates of the present invention can be advantageously administered intratumorally to cancer patients, particularly those with localized solid carcinoma, preferably prior to any cancer treatment, especially before surgery.

[0204] In a preferred aspect, the intratumorally administered anti-CTLA4 drug conjugates, particularly anti-CTLA4 ADCs, described herein maximize bioavailability and thus maximize the local efficacy of cancer treatment, particularly the anti-cancer payload, by inducing CTLA4+ immune cell exhaustion in the tumor and / or tumor microenvironment while avoiding any systemic toxicity of anticancer therapy (through significantly reduced systemic exposure). As described above, the drug conjugates of the present invention can also be advantageously administered to cancer patients with metastatic malignancies.

[0205] The inventors have advantageously described above pharmaceutical conjugates for use as medicines, preferably for the treatment of cancer. Corresponding therapeutic uses are also described herein, particularly methods for treating cancer or preventing cancer recurrence in subjects in need. These methods include the step of administering the pharmaceutical conjugate of the invention, or a composition comprising said pharmaceutical conjugate, alone or in combination with one or more different therapeutic compounds (as described herein), to a subject in need. Preferred pharmaceutical conjugates comprise i) a CTLA4-targeting molecule, particularly an anti-CTLA4 monoclonal antibody, such as ipilimumab or trimemumab; ii) at least one cytotoxic agent, such as a tubulin inhibitor, maytansine-like substance, DNA binding agent, or topoisomerase inhibitor, particularly, for example, MMAE, MMAF, DM1, DM4, kazimycin, SN38, or essanotecan; and iii) a linker connecting said CTLA4-targeting molecule and said cytotoxic agent, preferably a cleavable linker as described herein, such as a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide. Attached Figure Description

[0206] Figure 1 The effect of anti-CTLA4 ipilimumab (IPI) plus anti-PD1 nivolumab (NIVO) on complete response rate in metastatic melanoma is cancer stage dependent. This is a summary of the 1082MO5 year characterization of complete responses in patients with advanced melanoma who received nivolumab plus ipilimumab (Nivo+Ipi) or nivolumab alone. Ann. Oncol. 31, S734-S735 (2020) Summary results of the CheckMate 066, 067, 069 trials.

[0207] Figure 2 CTLA4 membrane expression on T cells within the tumor.

[0208] (a) The proportion and mean fluorescence intensity (“MFI”) of membrane CTLA4 expression on T cell populations; (b) The proportion of CTLA4+ Tregs in multiple cancer types (MM: metastatic melanoma; NSCLC: non-small cell lung cancer; RCC: renal cell carcinoma; HNSCC: head and neck squamous cell carcinoma; EOC: epithelial ovarian cancer); (c) The proportion of CTLA4+ cells among FoxP3+ CD4+ T cells in tumors; and (d) The proportion of CTLA4+ cells among CD25+ FoxP3+ CD4+ T cells in freshly resected and dissected human tumors after co-incubation with ipilimumab or its isotype. (e) Description of the proportion of CTLA4+ FOXP3+ CD4+ T cells and CTLA4 MFI from a single tumor. All measurements were obtained by flow cytometry via non-competitive staining of CTLA4.

[0209] Figure 3 Ipilimumab cell distribution at 37°C compared to 4°C, as examined by confocal microscopy.

[0210] At 37°C, ipilimumab was internalized and localized to subcellular intracellular vesicles on CTLA4-expressing cells within 1 hour. Diffused ipilimumab intracellular staining was observed after 24 hours.

[0211] Figure 4Intracytic FoxP3 and membrane CTLA4 labeling were performed by flow cytometry in newly dissociated melanoma tumors in the CD4+CD25+CD39+ population (double positive, single positive, or double negative).

[0212] Figure 5 Following intratumor ipilimumab treatment, the proportion of CD4+CD25+CD39+ intratumoral Tregs was significantly reduced.

[0213] Left image: Immunocytography of fresh biopsy samples shows that in melanoma patients who will receive durable clinical benefit from treatment (DCB: disease control lasting more than 6 months), baseline (before any treatment) CD4+CD39 levels were higher. 高 CD25 高 Patients with a high proportion of T cells showed significantly more abundant T cells. Middle and right figures: When ipilimumab was administered intratumorally to melanoma patients (IT group), the intratumoral CD4+CD25+CD39+ T cell population was significantly reduced after ipilimumab administration, and the reduction was even lower when ipilimumab was administered intravenously to melanoma patients (IV group).

[0214] Figure 6 Single-cell sequencing of CD45+ enriched cells from five newly resected tumors. (a) Clonality among four major subsets of T cells; (b) Clusters of specific T cells; (c) Subsets of specific T cells.

[0215] Figure 7 Compared with intravenous ipilimumab, intratumoral ipilimumab produces less toxicity in patients while maintaining the same antitumor efficacy.

[0216] Based on the proportion of treatment-related adverse events in the treatment group (a) and the best response according to recist1.1 (b).

[0217] Figure 8 The generation of three ipilimumab antibody-drug conjugates.

[0218] (a) Chemical formulas of the three payloads; (b) The principle of UV light binding of the payloads to ipilimumab via the valine-citrulline linker; (c) SDS-PAGE reduction gel confirming the molecular weight change after ipilimumab binds to the payloads. Lanes from left to right: 1: DM1, 2: DM1 + ​​ipilimumab, 3: DM1 + ​​rituximab, 4: VcMMAE, 5: VcMMAE + ipilimumab, 6: VcMMAE + rituximab, 7: VcMMAF, 8: VcMMAF + ipilimumab, 8: VcMMAF + rituximab, 10: gradient band, 11: ipilimumab, 12: rituximab.

[0219] Figure 9 Phenotypes of Raji hCTLA4 and Raji cell lines.

[0220] CTLA-4 expression at the membrane of Raji cells and RaijhCTLA-4 cells.

[0221] Figure 10 Cytotoxicity induced in vitro by ipilimumab ADC treatment on cell lines overexpressing hCTLA-4 (a, b, c) and control cell line not expressing CTLA4 (d).

[0222] (a) RajihCTLA4 cell lines after 48 hours of treatment (n=3), (b) RajihCTLA4 cell lines after 72 hours of treatment (n=4), (c) RajihCTLA4 cell lines after 96 hours of treatment (n=3), and (d) RajihCTLA4 cell lines (Rajih without CTLA4 expression) after 96 hours of treatment (n=3) at different time points and concentrations of ADC treatment (DM1_ipilimumab, VcMMAE_ipilimumab, VcMMAF_ipilimumab).

[0223] Figure 11: Lateral scattering diagram of RajihCTLA4 cells treated with increased concentrations of ipilimumab-VcMMAF, rituximab-VcMMAF, ipilimumab, rituximab, and VcMMAF.

[0224] (a) FSC / SSC of Raji hCTLA4; (b) Percentage of dead cells in the two populations; (c) CTLA4 expression in the two populations; (d) FSC / SSC dot plot of Raji hCTLA4 cells treated with ipilimumab-VcMMAF, rituximab-VcMMAF, ipilimumab, rituximab and VcMMAF during 72 hours.

[0225] Figure 12 Patients resistant to ipilimumab have a tumor microenvironment lacking sufficient macrophages. Specifically, patients resistant to ipilimumab have a tumor microenvironment lacking sufficient macrophages (CD68 and / or CD163 and / or CSF1R) and FcgRI (CD64) and / or FcgRIIIa (CD16a) and / or FcgRIIIb (CD16b). The bar graph represents the comparison of CD68, CD163, CSF1R, CD16a, CD16b, and CD64 per million transcripts (TPM) at baseline between patients with clinical benefit (DCB) and those without clinical benefit (no DCB). The results presented here are from metastatic melanoma patients who participated in a phase 1 study and received an initial combination of ipilimumab and nivolumab. DCB = 6-month clinical benefit.

[0226] Throughout this application, various references describe the current state of the art to which this invention pertains. The disclosures of these references are incorporated herein by reference.

[0227] Other features and advantages of the invention are set forth in the following experimental section (see reference). Figures 1 to 12 The experimental portion described herein should be considered illustrative and not limiting of the scope of this application.

[0228] Experimental Section

[0229] Development of anti-CTLA4 drug conjugates for intratumoral administration

[0230] Materials and methods

[0231] Antibody drug conjugates

[0232] Following the manufacturer's (oYoLink®, Alpha Thera) protocol, ipilimumab and rituximab were labeled as three different cytotoxic drug payloads (DM1, VcMMAE, VcMMAF). Each different payload is linked via a lysosomal cleavable dipeptide valine-citrulline (“vc” or “Vc”) linker.

[0233] cell culture

[0234] Raji hCTLA-4 cell lines were grown in Iskov modified Durbeco medium (Sigma Life Sciences, I3390-500 mL) supplemented with 10% complement-depleted SVF Hyclone (research grade, catalog number: SV30160.03), 1% L-glutamine (Gibco, catalog number: 25030-024), 25 nM Hepes (Gibco, catalog number: 15630-056), 1% penicillin / streptomycin (Gibco, catalog number: 15140-122), and 10 µg / ml blast fungicide (Invivogen, catalog number: ant-bl-1). Raji cell lines were grown in IMDM (Sigma Life Sciences, I3390-500mL) supplemented with 10% complement-depleted SVF Hyclone (research grade, catalog number: SV30160.03), 1% L-glutamine (Gibco, catalog number: 25030-024), and 1% penicillin / streptomycin (Gibco, catalog number: 15140-122).

[0235] Cytotoxicity assay

[0236] The proportion of dead cells was assessed by flow cytometry using zombie water (Biolegend) staining, and expressed as a percentage of dead cells across all events (excluding cell debris). Cells were incubated at room temperature for 20 minutes and then washed with 2 ml of PBS.

[0237] To evaluate the most effective concentration of anti-CTLA4 drug conjugates, the inventors compared the percentage of dead cells induced at different time points: 48 hours, 72 hours, and 96 hours, with concentrations of 0 to 1 µM ipilimumab-drug conjugates, rituximab-drug conjugates, ipilimumab, rituximab, and the individual payloads.

[0238] They provided an additional control by treating Raji cell lines that do not express CTLA4 for 96 hours with the same experimental conditions (the same concentrations of ipilimumab drug conjugate, rituximab drug conjugate, rituximab, ipilimumab, and individual payloads).

[0239] result

[0240] CD4+FOXP3+ T cells, known as regulatory T cells or “Tregs,” are a subset of lymphocytes that play a crucial role in the development of immune system tolerance (Lucca and Dominguez-Villar, 2020; Plitas and Rudensky, 2020). Their presence, overactivity, or deficiency is associated with autoimmune diseases or cancer, respectively (Tay et al., 2023). CTLA4 is a co-inhibitory (“checkpoint”) receptor expressed at the membrane level by Tregs upon binding to their TCR receptors (Rowshanravan et al., 2018). The consensus regarding the mechanism of action of anti-CTLA4 molecules in mice suggests specific depletion of tumor Tregs (Marabelle et al., 2013; Selby et al., 2013; Simpson et al., 2013). However, in humans, the question of whether CTLA4 antagonists or Treg destruction plays a role remains controversial (Ferrara et al., 2019; Sharma et al., 2019). Recently, the complete response rate of anti-PD1+ / - anti-CTLA4 in melanoma has also demonstrated the impact of disease progression level (“cancer stage”) on the efficacy of anti-CTLA4 therapy. Figure 1 ).

[0241] Recently, the inventors analyzed freshly removed tumor tissue using flow cytometry. Figure 2 )prove: i) CTLA-4 in humans is primarily expressed by Treg cells in terms of both its relative proportion to other immune populations and its absolute number on the surface of immune cells (mean fluorescence intensity, "MFI"). Figure 2 a) and ii) Proportion of Treg CTLA4+ cells ( Figure 2 c and Figure 2 d) CTLA4 decreased after ipilimumab treatment, and for some samples, the decrease was more pronounced than in most tested samples. By examining the samples individually, the inventors observed a shift in this population towards the CTLA4-negative side in some samples. Figure 2 e).

[0242] The observation that CTLA4 levels decreased after ipilimumab exposure led the inventors to hypothesize that ipilimumab could be internalized along with CTLA4 after immobilization.

[0243] They confirmed this hypothesis through confocal microscopy examination of transgenic cell lines expressing human CTLA4: after co-incubating ipilimumab conjugated with a fluorescent dye with CTLA4+ transgenic cell lines at 37°C for 1 hour, ipilimumab was internalized into intracellular membranous vesicles, and diffuse intracellular staining was observed after 24 hours at 37°C. Figure 3 No internalization was observed at 4°C.

[0244] Furthermore, the inventors demonstrated in freshly excised and dissected human tumors that this CD4+FOXP3+CTLA4+ population can be readily detected in CD4+ cells by dual labeling with CD25+ and CD39+, thereby facilitating the detection of Tregs in single, fresh tumor biopsy tissues where intracytoplasmic FOXP3 labeling is not feasible due to limited cell availability. Figure 4 ).

[0245] Furthermore, the inventors demonstrated in a clinical trial (NIVIPIT trial; NCT02857569) that metastatic melanoma patients (DCB patients: durable clinical benefit) who benefited from the combination of anti-PD1 (nivolumab) + anti-CTLA4 (ipilimumab) as first-line therapy were those with CD4+ CD25+ CD39+ T cell populations in their tumors. Figure 5 (Left). In patients who received treatment and achieved durable clinical benefit (DCB), this population of Treg-rich cells decreased with treatment. The inventors also demonstrated that the reduction observed with intravenous administration of anti-CTLA4 ( Figure 5 Compared to the right side, when high concentrations of anti-CTLA4 are injected locally, this reduction favorably increases (the reduction in the right side). Figure 5 ;middle).

[0246] The inventors also demonstrated through single-cell sequencing (single-cell RNA-seq) of dissociated human tumors that a second cluster (blue-green) composed of Tregs (CD4+FOXP3+CD25+CD39+) accounted for 79.5% of a cell population expressing high CTLA4 and undergoing clonal proliferation. This clonal nature of tumor-invasive Tregs indicates that Tregs proliferate after recognizing antigens from the tumor microenvironment. Figure 6 ).

[0247] In the NIVIPIT clinical trial (NCT02857569), the inventors also demonstrated that intratumoral injection of a low dose of the anti-CTLA4 drug ipilimumab (0.3 mg / kg) significantly reduced the drug's toxicity in the treatment of metastatic melanoma, while advantageously maintaining efficacy levels comparable to those achieved at FDA and EMA approved doses. Figure 7 ).

[0248] The inventors now describe for the first time in this paper a pharmaceutical conjugate containing a CTLA4-targeting molecule, particularly an intratumoral anti-CTLA4 antibody-drug conjugate (ADC) that provides highly advantageous efficacy over IT and IV administration of anti-CTLA4 with significantly reduced toxicity. This conjugate is particularly beneficial for cancer patients whose tumors are still in the immune editing phase involving CTLA4+ Tregs.

[0249] Construction of anti-CTLA4 antibody drug conjugates

[0250] As proof of principle, the inventors generated three ipilimumab antibody-drug conjugates using Alpha Thera's oYoLink® technology, in which the anti-CTLA4 antibody was conjugated to three different payloads: DM1 (emtansine), VcMMAE (monomethylolpropamine E), and VcMMAF (monomethylolpropamine F). Given that ipilimumab follows intracellular transport of CTLA4 (Khailaie et al., 2018), they selected a lysosomal cleavable dipeptide valine-citrulline linker (Alpha Thera's oYoLink technology). Figure 3 Since the CTLA4+ transgenic cell line is derived from the CD20+ Raji lymphoma cell line, anti-CD20 rituximab antibody was conjugated with these same drugs as a positive control group.

[0251] The inventors verified using SDS gel that three ipilimumab ADCs (ipilimumab-DM1, ipilimumab-MMAE, and ipilimumab-MMAF) correctly bind to their payloads via the valine-citrulline linker. Figure 8 ).

[0252] Ipilimumab-MMAF anti-CTLA4 drug conjugate

[0253] To evaluate the cytotoxicity induced by ipilimumab-DM1, ipilimumab-MMAE, and ipilimumab-MMAF, the inventors performed in vitro assays on the genetically engineered Raji hCTLA4 cell line (Invivogen) selectively expressing human CTLA4 under blast fungicide selection and the Raji control cell line not expressing CTLA4. Figure 9 ).

[0254] Different ipilimumab-ADC concentrations were tested between 0 nM and 1 M. To assess the most potent anti-CTLA4 drug conjugate concentration, they compared the percentage of cell death induced by ipilimumab-drug conjugate versus rituximab-drug conjugate, ipilimumab, rituximab, and each of the three payloads alone at different time points: 48 hours ( Figure 10 a) 72 hours ( Figure 10 b) and 96 hours ( Figure 10 c). They provided an additional control by treating Raji cell lines that do not express CTLA4 for 96 hours with the same experimental conditions (the same concentrations of ipilimumab drug conjugate, rituximab drug conjugate, rituximab, ipilimumab, and individual payloads).

[0255] Cytotoxicity was evaluated by flow cytometry using a marker of dead cells (zombie water staining). Data showed that within 72 hours ( Figure 10 b) and 96 hours ( Figure 10 c) Following this, ipilimumab-MMAF and ipilimumab-DM1 induced greater cytotoxicity. At 10 nM, ipilimumab-MMAF induced 50% cell death, the same as ipilimumab-DM1. At 10 nM, MMAF alone did not induce cytotoxicity, while DM1 alone induced 30% cell death after 72 hours of treatment. Treatment with Raji cell lines confirmed that ipilimumab-MMAF does not induce cytotoxicity in cell lines without CTLA4 expression. Figure 10 d).

[0256] Ipilimumab-MMAF selectively targets CTLA4-expressing cells.

[0257] Forward-scattered (FSC) and side-scattered (SSC) flow cytometry analysis revealed two populations in the Raji hCTLA4 cell line (Fig. 11a). Some cells lost the CTLA4 transgene, resulting in two cell populations. Population 2, which did not express CTLA4, gradually died due to blast fungicide (Fig. 11b). The Raji hCTLA4 phenotype showed specific CTLA4 staining in population 1 (Fig. 11c).

[0258] By comparing the FSC / SSC ratio of cells treated with different concentrations of ipilimumab-VcMMAF, rituximab-VcMMAF, rituximab, ipilimumab, and VcMMAF, the inventors observed that population 2 gradually died as the concentration of ipilimumab-VcMMAF treatment increased.

[0259] in conclusion

[0260] The inventors demonstrated through confocal microscopy that ipilimumab, after being immobilized to CTLA4, is internalized together with CTLA4. This observation led them to investigate the possibility of generating drug conjugates containing CTLA4-targeting molecules and to evaluate their efficacy.

[0261] The experimental results described for the first time in this paper surprisingly demonstrate that ipilimumab drug conjugates exhibit stronger cytotoxicity against CTLA4+ cells compared to the payload or ipilimumab alone. Treatment with 10 nM ipilimumab-MMAF for 72 hours induced a 50% mortality rate. In vitro experiments by the inventors showed that the effective concentration of ipilimumab conjugate (10 nM) was 6.6 times lower than the standard concentration for ipilimumab treatment. This illustrates the benefit of using such drug conjugates (e.g., anti-CTLA4 drug conjugates, such as the ipilimumab-MMAF conjugate) to treat cancer. By targeting tumor-specific Tregs, the drug conjugates of this invention provide a new and highly advantageous treatment option, thereby allowing for increased therapeutic efficacy and reduced toxicity to patients.

[0262] References

[0263] - Antonia, SJ, Villegas, A., Daniel, D., Vicente, D., Murakami, S., Hui, R., Yokoi, T., Chiappori, A., Lee, KH, de Wit, M., et al. (2017). Durvalumab after Chemoradiotherapy in Stage III Non-Small-Cell Lung Cancer. N. Engl. J. Med. 377, 1919-1929.

[0264] Intratumoral Immunotherapy: From Trial Design to Clinical Practice. Clin. Cancer Res. 27, 665–679 (2021).

[0265] - Eggermont , AMM , Chiarion-Sileni , V. , Grob , J.-J. , Dummer , R. , Wolchok , JD , Schmidt , H. , Hamid , O. , Robert , C. , Ascierto , PA , Richards , JM Person (2015). Adjuvant ipilimumab versus placebo after complete resectionof high-risk stage III melanoma (EORTC 18071): a randomized, double-blind,phase 3 trial. The Lancet. Oncol. 16 , 522-5

[0266] - Eggermont , AMM , Chiarion-Sileni , V. , Grob , J.-J. , Dummer , R. , Wolchok , JD , Schmidt , H. , Hamid , O. , Robert , C. , Ascierto , PA , Richards , JM Person (2016). Prolonged Survival in Stage III Melanoma with IpilimumabAdjuvant Therapy. N. Engl. J. Med. 375 , 1845-1855.

[0267] - Eggermont, A.M.M., Blank, C.U., Mandala, M., Long, G. V., Atkinson, V., Dalle, S., Haydon, A., Lichinitser, M., Khattak, A., Carlino, M. S. et al. (2018). Adjuvant Pembrolizumab versus Placebo in Resected Stage III Melanoma. N. Engl. J. Med. 378 , 1789 - 1801.

[0268] - Eggermont, A.M.M., Blank, C.U., Mandala, M. and Long, G. V (2020). Longer Follow - Up Con fi rms Recurrence - Free Survival Bene fi t of Adjuvant Pembrolizumab in High - Risk Stage III Melanoma: Updated Results From the EORTC 1325 - MG / KEYNOTE - 054 Trial abstract. 38 .

[0269] - Ferrara, R., Susini, S. and Marabelle, A. (2019). Anti - CTLA - 4 Immunotherapy Does Not Deplete FOXP₃ + Regulatory T Cells (Tregs) in Human Cancers—Letter. Clin. Cancer Res. 25 , 3468 - 3468.

[0270] - Gao, J., Navai, N., Alhalabi, O., Siefker-Radtke, A., Campbell, M.T., Slack Tidwell, R., Guo, C.C., Kamat, A.M., Matin, S.F., Araujo, J.C. et al. (2020) Neoadjuvant PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable high-risk urothelial carcinoma. Nat. Med。

[0271] - Khailaie S., Rowshanravan, B., Robert, P. A., Waters E., Halliday, N., Badillo Herrera, J. D., Walker L. S. K., Sansom D. M., Meyer-Hermann M. (2018) Characterization of CTLA4 Trafficking ans Implications for its Function. B. J. 115, 1330-1343 (2018)。

[0272] - Lucca L. E. and Dominguez-Villar, M. (2020). Modulation of regulatory T cell function and stability by co-inhibitory receptors. Nat. Rev. Immunol。

[0273] - Luke, J.J., Rutkowski, P., Queirolo, P., Del Vecchio, M., Mackiewicz, J., Chiarion-Sileni, V., de la Cruz Merino, L., Khattak, M.A., Schadendorf, D., Long, G. V et al. (2022). Pembrolizumab versus placebo as adjuvant therapy in completely resected stage IIB or IIC melanoma (KEYNOTE-716): a randomised, double-blind, phase 3 trial. Lancet (London, England) 1-12。

[0274] - Marabelle, A., Kohrt, H., Sagiv-Barfi, I., Ajami, B., Axtell, R.C., Zhou, G., Rajapaksa, R., Green, M.R., Torchia, J., Brody, J. et al. (2013). Depleting tumor-specific Tregs at a single site eradicates disseminated tumors. J. Clin. Invest. 123 , 2447-2463。

[0275] - Marabelle, A., Tselikas, L., de Baere, T. and Houot, R. Intratumoral immunotherapy: using the tumor as the remedy. Ann. Oncol. 28, xii33-xii43(2017)。

[0276] - Marabelle, A. et al., Starting the fight in the tumor: expert recommendations for the development of human intratumoral immunotherapy (HIT-IT). Ann. Oncol. Off. J. Eur. Soc. Med. Oncol.29, 2163 - 2174 - (2018).

[0277] - Plitas, G. and Rudensky, A.Y. (2020). Regulatory T Cells in Cancer. Annu. Rev. Cancer Biol. 4 , 459 - 477.

[0278] - Rowshanravan, B., Halliday, N. and Sansom, D.M. (2018). CTLA - 4: A moving target in immunotherapy. Blood 131 , 58 - 67.

[0279] - Selby, M.J., Engelhardt, J.J., Quigley, M., Henning, K.A., Chen, T., Srinivasan, M. and Korman, A.J. (2013). Anti - CTLA - 4 Antibodies of IgG2a Isotype Enhance Antitumor Activity through Reduction of Intratumoral Regulatory T Cells. Cancer Immunol. Res. 1 , 32 - 42.

[0280] - Sharma, A., Subudhi, S.K., Blando, J., Scutti, J., Vence, L., Wargo, J., Allison, J.P., Ribas, A. and Sharma, P. (2019). Anti - CTLA - 4 immunotherapy does not deplete Foxp3 þ regulatory T cells (Tregs) in human cancers. Clin. Cancer Res. 25 , 1233 - 1238.

[0281] - Simpson, T.R., Li, F., Montalvo-Ortiz, W., Sepulveda, M.A., Bergerhoff, K., Arce, F., Roddie, C., Henry, J.Y., Yagita, H., Wolchok, J.D. et al. (2013). Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. 210 , 1695 - 1710.

[0282] - Tay, C., Tanaka, A. and Sakaguchi, S. (2023). Tumor-infiltrating regulatory T cells as targets of cancer immunotherapy. Cancer Cell 41 , 450 - 465.

[0283] - Tselikas, L. et al., Interventional radiology for local immunotherapy in oncology. Clin. Cancer Res. clincanres.4073.2020 (2021) doi:10.1158 / 1078 - 0432.ccr - 19 - 4073.

[0284] - Tselikas, L. et al., Interventional radiology for local immunotherapy in oncology. Clin. Cancer Res. clincanres.4073.2020 (2021) doi:10.1158 / 1078 - 0432.ccr - 19 - 4073.

[0285] - Tselikas, L. et al. (2020). Pickering emulsions with ethiodized oil and nanoparticles for slow release of intratumoral anti-CTLA4 immune checkpoint antibodies. J. Immunother. Cancer 8, e000579。

Claims

1. A pharmaceutical conjugate comprising i) an anti-cytotoxic T-lymphocyte antigen 4 protein (anti-CTLA4) monoclonal antibody, ii) at least one cytotoxic agent, and iii) a linker connecting a CTLA4 targeting molecule and said cytotoxic agent.

2. The drug conjugate according to claim 1, wherein the anti-CTLA4 monoclonal antibody is ipilimumab or trimemumab.

3. The pharmaceutical conjugate according to claim 1 or 2, wherein the linker is a cleavable linker, such as an acid-unstable linker, an enzyme-cleavable linker, a lysosomal protease-sensitive linker, a disulfide linker, or a β-glucuronide linker.

4. The pharmaceutical conjugate according to any one of claims 1 to 3, wherein the cytotoxic agent is selected from microtubule inhibitors, DNA damaging agents such as alkylating agents or platinum complexes, cytotoxic antibiotics, antimetabolites, topoisomerase inhibitors, RNA polymerase inhibitors, antimitotic agents, and combinations thereof.

5. The drug conjugate according to claim 4, wherein the microtubule inhibitor is selected from methylolamine (DM1), olamine and its derivatives such as monomethylolamine E (MMAE) or monomethylolamine F (MMAF), preferably MMAF.

6. The pharmaceutical conjugate according to claim 2, wherein the conjugate comprises i) ipilimumab; ii) a cytotoxic agent, preferably MMAF; and iii) a cleavable linker, preferably comprising a valine-citrulline dipeptide or a phenylalanine-lysine dipeptide.

7. A pharmaceutical composition comprising at least one pharmaceutical conjugate as claimed in any one of claims 1 to 6, such as at least two different pharmaceutical conjugates, wherein the first conjugate comprises a first cytotoxic agent and the second conjugate comprises a different cytotoxic agent, or, for example, at least one pharmaceutical conjugate and a different therapeutic agent selected from immune checkpoint targets, anti-angiogenic agents, cytotoxic agents and hormones, and a pharmaceutically acceptable load.

8. A kit comprising at least two pharmaceutical conjugates as described in any one of claims 1 to 6, or at least one pharmaceutical conjugate as described in any one of claims 1 to 6 and a different therapeutic agent selected from immune checkpoint targets, anti-angiogenic agents, cytotoxic agents, hormones, and any combination thereof.

9. The pharmaceutical conjugate according to any one of claims 1 to 6, the pharmaceutical composition according to claim 7, or the kit according to claim 8, for treating cancer or preventing cancer recurrence in subjects in need.

10. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to claim 9, wherein the cancer is a solid cancer selected from melanoma, renal cell carcinoma (RCC), colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), mesothelioma (MM), or a hematologic malignancy selected from lymphoma and leukemia.

11. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to claim 9 or 10, wherein the cancer cells or cancer tumor microenvironment (TME) comprises CTLA4-expressing cells.

12. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to claim 11, wherein the CTLA4-expressing cells are regulatory T cells, preferably CD4+FOXP3+CTLA4+ T cells and / or CD4+CD25+CD39+ T cells, comprising Treg cells that have undergone tumor antigen-specific clonal proliferation.

13. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to any one of claims 9 to 12, wherein the pharmaceutical conjugate or pharmaceutical composition is administered to the subject as a neoadjuvant therapy, preferably as a monotherapy or in combination with different anticancer agents prior to any partial or complete surgical resection of the tumor or destruction of the lesion by any cytoreductive strategy, and preferably by local delivery, including intratumoral (IT), intravascular, or local routes.

14. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to any one of claims 9 to 13, wherein the pharmaceutical conjugate is administered to the subject at a concentration of about 0.01 mg to about 1 mg per kilogram of the subject's body weight.

15. The pharmaceutical conjugate, pharmaceutical composition or kit for use according to any one of claims 9 to 14, wherein the subject is a mammal, preferably a human.

16. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to any one of claims 9 to 15, wherein the subject is a subject whose tumor or tumor microenvironment (TME) contains CTLA4+ tumor cells and / or CTLA4+ immune cells.

17. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to claim 16, wherein the subject is a subject resistant to naked anti-CTLA4 and / or anti-PD(L)1 agents.

18. The pharmaceutical conjugate, pharmaceutical composition, or kit for use according to claim 16 or 17, wherein the subject is a subject whose tumor and / or TME does not show myeloid cells, shows low levels or dysfunctional myeloid cells, said myeloid cells not expressing or expressing low levels of Fcγ receptor I ("CD64"), Fcγ receptor IIIa ("CD16a"), and / or Fcγ receptor IIIb ("CD16b").

Images