Immune cytokines for cancer treatment

Abstract

This invention relates to immune cytokines comprising anti-CTLA-4 antibodies fused with a Treg-specific IL-2 receptor antagonist, and their use in selectively depleting tumor-associated Tregs, particularly for the treatment of cancer.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy. Specifically, it relates to immune cytokines comprising anti-CTLA-4 antibodies fused to a regulatory T-cell (Treg)-specific IL-2 receptor antagonist, and their use in selectively depleting tumor-associated Tregs, particularly for the treatment of cancer. Background Technology

[0002] Regulatory T cells (Tregs) are key components of the tumor microenvironment in several tumor types that prevent effector T cell and NK cell-mediated immune responses. Depletion of Tregs from the tumor microenvironment can restore immune responses and contribute to tumor clearance (e.g., Curiel et al., Nat. Med., 2004, 10, 942-949; Hiraoka et al., Clin. CancerRes., 2006, 12, 5423-5434; Stirm et al., Journal for ImmunoTherapy of Cancer2023;11:e006263; and a review in Shan et al., Trends in Cancer, November 2022, Vol.8, No. 11 https: / / doi.org / 10.1016 / j.trecan.2022.06.008). Given the role of Tregs in the prevention of autoimmune diseases, immune interventions aimed at depleting Treg cells must selectively deplete only tumor-infiltrating Tregs (Fontenot et al., Nat. Immunol., 2003, 4, 330-336).

[0003] Treg cell survival depends on interleukin-2 (IL-2), which is mainly produced by effector T cells and NK cells and transmits signals in Treg cells via a high-affinity IL-2 receptor (IL-2R) complex composed of an α chain (IL-2-RA or CD25), a β chain (IL-2-RB or CD122), and a γ chain (IL-2-RG or γc or CD132). The α chain has a low affinity for IL-2 and does not participate in signal transduction. The β and γ chains combine to form an intermediate-affinity receptor, expressed on effector T cells (conventional T cells (Tconv) (CD4+Foxp3-), CD8+) and NK cells responsible for cell-mediated immune responses; all three receptor chains together form a high-affinity receptor, constitutively expressed on immunosuppressive regulatory T cells (CD4+Foxp3+), and transiently or transiently expressed on activated effector T cells and NK cells.

[0004] To regulate the biological activity of IL-2 for cancer treatment, various IL-2 variants (IL-2 mutant proteins) have been constructed (see Leon et al., Seminars in Oncology, 2018, 45, 95-104 for a review). The α-mutant protein is an agonist of IL-2R signaling, but its ability to amplify Tregs in vivo is reduced (Carmenate et al., J. Immul., 2013, 190, 6230-6238; Rojas et al., J. Mol. Recognit., 2015, 25, 261-268). The β+ mutant protein (plus-beta mutein), or IL-2 superkine, is an agonist of IL-2R signaling, preferentially expanding CD8+ T cells and NK cells in vivo, and also has some effect on Treg cells (Levinet et al., Nature, 2012, 484, 529-533). The γ-free mutant protein is an antagonist of IL-2R signaling, exhibiting a certain preferential affinity for Treg cells (Carmenate et al., J. Immunol., 2018, 200, 3475-3484). In mice, the α-free and β+ mutant proteins exhibit higher antitumor activity and lower toxicity than wild-type IL-2. The γ-free mutant protein demonstrates antitumor activity in mice similar to that of anti-CD25 monoclonal antibodies. Other antagonists of IL-2 signaling are disclosed in WO 2020 / 201095, which can inhibit Treg division by blocking wild-type IL-2 signaling while preserving active IL-2 signaling (IL-2V1; IL-2V4; IL-2V5; IL-2V6) in CD8+ T cells.

[0005] Antitumor immune responses are also regulated by various immune checkpoint proteins (PD-1, PD-L1, CTLA-4, LAG-3), which have become targets for blocking monoclonal antibodies (immune checkpoint inhibitors) and can currently be used alone or in combination to treat certain types of cancer. CTLA-4, or CTLA4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152, is a protein receptor that functions as an immune checkpoint and downregulates the immune response. CTLA-4 is constitutively expressed in regulatory T cells, but is only upregulated in regular T cells after activation. When it binds to CD80 and CD86 on the surface of antigen-presenting cells, it acts as a "switch off". Several CTLA-4 inhibitors, such as ipilimumab and trimemumab (both FDA-approved), have been developed for the treatment of various cancers, including metastatic melanoma, malignant pleural mesothelioma, metastatic non-small cell lung cancer (NSCLC), prostate cancer, renal cell carcinoma, and hepatocellular carcinoma (review in Oncology Live, June 22, 2023, 24, 11, 39-). Although some patients respond to these therapies (whether as monotherapy or in combination), their high incidence of adverse reactions limits their efficacy.

[0006] In order to improve cancer treatment, there is a need for therapies that aim to selectively deplete tumor-invasive Tregs. Summary of the Invention

[0007] The inventors used single-cell RNA sequencing data from cancer patients and validated it at the protein level using FACS, discovering that tumor-associated Tregs selectively overexpress interleukin-2 receptor α chain (IL2-RA or CD25) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152) compared to peripheral Tregs or other T cell populations (Tconv and CD8 T cells) in tumors or blood. Figure 1 ).

[0008] Based on these findings, the inventors developed a novel immune cytokine (ICK) that targets the CTLA-4 and CD25 membrane proteins preferentially expressed on tumor-associated Tregs. This immune cytokine combines an anti-CTLA-4 checkpoint inhibitor (e.g., ipilimumab (humanized IgG1)) with a therapeutic approach that selectively targets the IL-2 mutant protein in CD25-expressing cells, as disclosed in WO 2020 / 201095.

[0009] The inventors have demonstrated that this immunocytokine can induce CD25 endocytosis, a property not found in previously disclosed IL-2 mutant proteins, providing an additional mechanism for interfering with CD25-mediated survival signaling in Treg cells. Therefore, compared to using anti-CTLA-4 antibodies or IL-2 mutant proteins alone or in combination, this immunocytokine selectively and efficiently kills Treg cells. In vitro experiments confirmed that this immunocytokine selectively depletes Treg cells in representative human tumor microenvironments. Thus, this specific combination of antibody and IL-2 mutant protein in the immunocytokine provides key properties for clearing tumor-associated Treg cells and promoting effective antitumor responses. Among the immunocytokines tested, those derived from one of the IL-2 mutant proteins (IL-2V5) also exhibited superior development performance (higher production titers, higher purity, and higher stability), while showing lower immunogenicity (in silico) and better pharmacokinetic parameters (mice and in vitro) compared to immunocytokines derived from other IL-2 mutant proteins (IL-2V1 and IL-2V6). Given these results, this novel immune cytokine represents a particularly promising candidate for developing new drugs to treat cancer.

[0010] Therefore, the present invention relates to a fusion protein (immunocytokine) comprising an anti-CTLA-4 antibody and an IL-2 variant, the IL-2 variant being a Treg-specific IL-2 receptor antagonist comprising substitutions of: K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A, and further comprising substitutions of: (i) S127K, (ii) Y31P and S127K, or (iii) K49Q, E52S, R81E, D84N, T131R and L132S, the positions of which are determined by comparison with human IL-2 (SEQ ID NO: 1), and wherein the N-terminus of the IL-2 variant is fused to the C-terminus of the antibody heavy chain.

[0011] Regulatory T cell (Treg) specific IL-2 receptor antagonists may further include a deletion of at least one amino acid at a position selected from S4, S5 or S6, especially a deletion of one amino acid.

[0012] The present invention also relates to a fusion protein comprising an anti-CTLA-4 antibody and an IL-2 variant, the IL-2 variant being a regulatory T cell (Treg)-specific IL-2 receptor antagonist, the regulatory T cell (Treg)-specific IL-2 receptor antagonist comprising a deletion of an amino acid at a position selected from S4, S5, or S6, and: (a) Replace K9E, L12E, H16R, M23L, N26K, Y31P, K49Q, E52S, R81E, D84N, S87N, V91K, E95K, T131R and L132S, or (b) Replace K9E, L12E, H16R, L19R, M23L, N26K, Y31P, K49Q, E52S, R81E, D84N, S87N, E95K, T131R and L132S, or (c) Replace H16R and V91K, or (d) Replacement of H16R, L19R and V91K The position shown was determined by comparison with human IL-2 (SEQ ID NO: 1), wherein the N-terminus of the IL-2 variant is fused to the C-terminus of the antibody heavy chain.

[0013] Regulatory T cell (Treg) specific IL-2 receptor antagonists can have at least 85% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, especially at least 90% sequence identity, and even more especially at least 95% sequence identity.

[0014] The immune cytokines of this invention induce CD25 endocytosis.

[0015] In some embodiments, the immune cytokines of the present invention induce Treg apoptosis.

[0016] In some embodiments, the immune cytokines of the present invention selectively deplete tumor-associated Tregs.

[0017] In some implementations, the anti-CTLA-4 antibody is ipilimumab, trimemumab, or a functional variant thereof.

[0018] In some specific embodiments, the anti-CTLA-4 antibody comprises H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 3, H-CDR3 of SEQ ID NO: 4, L-CDR1 of SEQ ID NO: 5, L-CDR2 of SEQ ID NO: 6, and L-CDR3 of SEQ ID NO: 7, or variants thereof, said variants further having one or more conserved substitutions on one or more of these CDRs. In some more specific embodiments, the anti-CTLA-4 antibody comprises a VH domain having at least 85% sequence identity with SEQ ID NO: 8 and a VL domain having at least 85% sequence identity with SEQ ID NO: 9.

[0019] In some implementations, the anti-CTLA-4 antibody comprises a human IgG1 Fc domain and / or a human Ig kappa light chain constant domain, or is a single-domain antibody.

[0020] In some implementations, the IL-2 variant contains an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 13 to 15.

[0021] In some embodiments, the immune cytokines of the present invention further comprise a linker between the C-terminus of the antibody heavy chain and the N-terminus of the IL-2 variant; preferably, the linker comprises the sequence SEQ ID NO: 10.

[0022] In some specific embodiments, the fusion protein of the present invention comprises: - An anti-CTLA-4 antibody heavy chain fused to an IL-2 variant, wherein the heavy chain fusion has at least 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 11; and - An anti-CTLA-4 antibody light chain having at least 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 12.

[0023] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of the present disclosure. The present invention also relates to pharmaceutical compositions of the present disclosure for the treatment of cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, malignant pleural mesothelioma, esophageal cancer, head and neck squamous cell carcinoma, urothelial carcinoma, primary Hodgkin's lymphoma, gastric cancer, large B-cell lymphoma, cervical cancer, Merkel cell carcinoma, endometrial cancer, squamous cell carcinoma of the skin, triple-negative breast cancer, invasive breast cancer, pancreatic adenocarcinoma, thymoma, prostate adenocarcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, and sarcoma. In some embodiments, the pharmaceutical composition is used in combination with at least one immune checkpoint inhibitor; preferably anti-PD-1 and / or anti-PDL-1. Invention Details

[0025] This invention relates to immune cytokines and their use for selectively depleting tumor-associated regulatory T cells, particularly for the treatment of cancer, wherein the immune cytokines comprise an anti-CTLA-4 antibody fused to a mutant IL-2 protein, the mutant IL-2 protein having Treg-specific IL-2 receptor antagonistic activity.

[0026] definition

[0027] As used in this article, "regulatory T cells", "Tregs" or "Tregs" refers to CD3+CD4+Foxp3+ T cells, including CD3+CD4+Foxp3+CD25+ and CD3+CD4+Foxp3+CD25- cells.

[0028] As used in this article, "effect cells" refers to effector T cells and NK cells. Effector T cells (Teff or Teffs) refer to one or more conventional T cells (Tconv) and CD8+ T cells. Tconv are CD3+CD4+Foxp3- cells. CD8+ T cells are CD3+CD8+ cells. NK cells are CD3-CD16+ cells.

[0029] As used herein, “tumor-associated Treg” refers to activated Tregs within tumor-infiltrating Tregs. In some specific implementations, “tumor-associated Treg” refers to a distinct and segregated population (or group, subpopulation, or cluster) of CD4+Foxp3+ cells that differs from heterogeneous pools of Treg cells in that: (i) it increases in tumors and eventually in tumor-draining lymph nodes; (ii) it is enriched in diseased tissues for clonal expansion of TCR specificity; and (iii) it is enriched for transcriptomic features of T-cell receptor (TCR) triggering, cell activation, and expansion, as disclosed in WO 2021 / 165546.

[0030] In this article, the term "antibody" is used with the broadest definition, encompassing both polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments such as antigen-binding fragments (Fab), Fab', Fab'-SH, F(ab')2 fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding antigens, single-chain antibody fragments (including single-chain variable fragments (scFv)), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobodies). The term "antibody" covers any portion containing CDR3. The term also encompasses recombinant and / or other modified forms of immunoglobulins, such as intracellular antibodies, peptibody antibodies, chimeric antibodies, fully human antibodies, heavy chain-only antibodies (composed of VHH, CH2, and CH3 domains), humanized antibodies, heteroconjugated antibodies, multispecific antibodies (e.g., bispecific antibodies), biantibodies, triantibodies, and tetraantibodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass its functional antibody fragment. The term also encompasses complete or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD. A full-length or complete antibody molecule is a complete immunoglobulin comprising two immunoglobulin (Ig) heavy chains and two Ig light chains linked by disulfide bonds. The antibodies of the present invention comprise a heavy chain containing a heavy chain variable region.

[0031] As used herein, the terms "specific binding" or "specific to" for antibodies refer to an antibody or antibody fragment recognizing and binding to a specific antigen, but not recognizing or binding in significant amounts (detectable binding) to other molecules present in the sample or other molecules that the antibody may encounter in vivo, while being able to detectably bind to its specific antigen. The specific binding of an antibody to its antigen can be determined by standard assays such as immunoassays. An antibody is considered to have specific binding to its antigen when its dissociation constant (KD) is equal to or less than 1 µM in a standard KD assay. KD values ​​are expressed as molar concentration (M); the KD of antibodies is typically determined using the Biacore surface plasmon resonance method.

[0032] The term "cytotoxic T-lymphocyte-associated protein 4" (CTLA4, CTLA-4, CD152) refers to proteins derived from the mammalian genome. CTLA4The protein encoded by the gene. CTLA4 is a member of the immunoglobulin superfamily, encoding a protein that transmits inhibitory signals to T cells. This protein contains a V domain, a transmembrane domain, and a cytoplasmic tail. Multiple transcriptional splicing variants encoding different isoforms have been characterized. The membrane-bound isoform functions as a disulfide-linked homodimer, while the soluble isoform functions as a monomer. Representative examples of CTLA4 include, but are not limited to, humans (NCBI gene ID: 1493), mice (NCBI gene ID: 12477), and other functional homologs. The amino acid sequence of human FAP is 223 amino acids, UniProtKB / Swiss-Prot P16410.

[0033] CTLA-4 is a membrane receptor known to be transported from the cell surface to the cytoplasm, where it can undergo endocytosis and degradation or be re-expressed via receptor cycling or de novo synthesis. This cycling defines an important mechanism of Treg cell inhibition: Treg cells capture the CTLA-4 ligand CD80 from the surface of antigen-presenting cells and clear CD80 through transendocytosis and endocytosis-mediated degradation (Qureshi et al, Science, 2011, 332, 600-603).

[0034] CTLA-4 is a receptor preferentially expressed on tumor-associated Tregs. Receptors preferentially expressed on tumor-associated Tregs are cell surface receptors that are overexpressed (or upregulated) on tumor-associated Tregs compared to peripheral Tregs from the blood, Tconvs from tumors or the blood, and CD8 T cells. Figure 1 As shown.

[0035] The term interleukin-2, or IL-2, also known as TCGF or lymphokine, refers to the protein encoded by the IL-2 gene in the mammalian genome. IL-2 is expressed as a precursor containing an N-terminal signal peptide (20 amino acids), which is cleaved to produce the mature protein (IL-2). Representative examples of IL-2 include, but are not limited to, human (Gene ID: 3558), rat (Gene ID: 116562), cat (Gene ID: 751114), and mouse (Gene ID: 16183) forms. As used herein, IL-2 refers to wild-type IL-2. The human IL-2 precursor has a 153-amino acid sequence, UniProtKB / Swiss-Prot: P60568.1. Mature IL-2 has a 133-amino acid sequence from position 21 to position 153 of the precursor and corresponds to SEQ ID NO: 1.

[0036] The term IL-2 mutant protein refers to an IL-2 mutant protein or IL-2 variant that contains one or more mutations (insertions, deletions, substitutions) compared to wild-type IL-2. IL-2 mutant proteins are IL-2 variants whose biological activity is altered compared to wild-type IL-2. Human IL-2 mutant proteins are preferred.

[0037] The term Treg-specific IL-2 receptor antagonist or IL-2 mutant protein with Treg-specific IL-2 receptor antagonistic activity refers to an IL-2 mutant protein of a dominant-negative IL-2 molecule that cannot induce IL-2-mediated STAT-5 phosphorylation in cells carrying IL-2R and competes with wild-type IL-2 for signal transduction via the trimer α, β, γ IL-2R. The Treg-specific IL-2 receptor antagonistic activity of the IL-2 mutant protein of the present invention has been described in the embodiments of this application.

[0038] As used herein, the term "functional" when describing the anti-CTLA-4 antibodies (including their variants) disclosed herein refers to anti-CTLA-4 antibodies that specifically bind to CTLA-4. The activity of this antibody can be determined using a standard immunoassay using the CTLA-4 protein or its antigen.

[0039] As used herein, the term "functional" when describing the IL-2 mutant proteins (including their variants) disclosed herein refers to IL-2 mutant proteins that possess Treg-specific IL-2 receptor antagonistic activity. The antagonistic activity of IL-2 mutant proteins against the IL-2 receptor can be determined by standard assays, such as STAT5 phosphorylation assays using peripheral blood samples that compete with Proleukin, as described in the examples.

[0040] As used herein, the term “functional” in describing the immune cytokines of the present invention refers to an immune cytokine that, as disclosed herein, combines the CTLA-4 binding activity of an anti-CTLA-4 antibody with the Treg-specific IL-2 receptor antagonistic activity of an IL-2 mutant protein, and further induces CD25 endocytosis, as illustrated in the examples.

[0041] In the following description, amino acid residues are represented by standard single-letter amino acid codes. The positions shown were determined by alignment with a reference sequence. The reference sequence for the IL-2 mutant protein is the human IL-2 sequence (SEQ ID NO: 1). For example, K9 is the lysine residue at position 9 in SEQ ID NO: 1. In this document, substitution is indicated by a single-letter amino acid code followed by the single-letter amino acid code of the substituted residue; K9E indicates that the lysine (K) residue at position 9 in SEQ ID NO: 1 is substituted by a glutamic acid (E) residue.

[0042] As used herein, the term "variant" refers to a polypeptide containing an amino acid sequence having at least 85% sequence identity with the native sequence. The term "variant" also refers to a functional variant that exhibits activity of the native sequence. The activity of a variant or fragment can be assessed using methods well known to those skilled in the art, such as those disclosed in the embodiments of this application.

[0043] The variant comprises one or more amino acid mutations, wherein the mutation can be an insertion, deletion, or substitution with a conserved or non-conserved amino acid. A conserved substitution refers to the replacement of an amino acid with an amino acid having similar chemical or physical properties (size, charge, or polarity), and such substitution generally does not adversely affect the biochemical, biophysical, and / or biological properties of the protein (antibody or IL-2 mutant protein). The conserved substitution is advantageously selected from one of the following five groups: Group 1 – small aliphatic nonpolar or weakly polar residues (A, S, T, P, G); Group 2 – polar residues with negative charges and their amides (D, N, E, Q); Group 3 – polar residues with positive charges (H, R, K); Group 4 – large aliphatic nonpolar residues (M, L, I, V, C); and Group 5 – large aromatic residues (F, Y, W).

[0044] The percentage of amino acid or nucleotide sequence identity is defined as the percentage of amino acid residues or nucleotides in the compared sequence that are identical to a reference sequence, after alignment and, if necessary, the introduction of vacancies to achieve maximum sequence identity, without taking any conserved substitutions of the amino acid sequence into account. Alignments used to determine the percentage of amino acid sequence identity can be performed using a variety of methods known to those skilled in the art, such as publicly available computer software like the GCG (Genetics Computer Group, Program Manual for the GCGPackage, Version 7, Madison, Wisconsin) pileup program, or any sequence comparison algorithm such as BLAST (Altschul). et al (e.g., J. Mol. Biol., 1990, 215, 403-), FASTA, or CLUSTALW. When using such software, it is preferable to use the default parameters, such as space penalty and extension penalty. The BLASTP program uses a word length (W) of 3 and an expected value (E) of 10 by default. Alignment is typically performed over the entire length of the reference sequence.

[0045] As used herein, the term "cancer" refers to any member of a class of diseases or conditions characterized by the uncontrolled division of cells and their ability to invade other tissues, either by direct invasion into adjacent tissues or by metastasis to distant sites. Metastasis refers to the stage in which cancer cells are transported via the bloodstream or lymphatic system. The term cancer in this invention also includes cancer metastasis and cancer recurrence. Cancers are classified based on the similar cell types of the tumor and the tissue from which it is inferred to be the origin of the tumor. For example, carcinomas are malignant tumors originating from epithelial cells. This group represents the most common cancers, including common breast cancer, prostate cancer, lung cancer, and colon cancer. Lymphomas and leukemias include malignant tumors originating from blood and bone marrow cells. Sarcomas are malignant tumors originating from connective tissue or mesenchymal cells. Mesotheliomas are tumors originating from the mesothelial cells of the superficial layers of the peritoneum and pleura. Gliomas are tumors originating from glial cells (the most common type of brain cells). Germ cell tumors are tumors originating from germ cells, typically found in the testes and ovaries. Choriocarcinoma is a malignant tumor that originates from the placenta. In this article, "cancer" refers to any type of cancer, including solid tumors and liquid tumors.

[0046] In this document, the terms "subject" and "patient" are used interchangeably and refer to both humans and non-human animals. As used herein, the term "patient" refers to mammals, such as, but not limited to, rodents, felines, canines, bovines, sheep, equines, and primates. Preferably, the patient in this invention is a human.

[0047] As used herein, the term "treatment" means reversing, alleviating, or inhibiting the progression of the disease or condition referred to by the term, or preventing the disease or condition, or reversing, alleviating, or inhibiting the progression of the disease or condition, or preventing one or more symptoms of the disease or condition referred to by the term. As used herein, the term "treatment" includes both preventative or preventive treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of being exposed to the disease, and patients who have or have been diagnosed with a disease or medical condition, and includes inhibiting clinical relapse. Treatment may be applied to patients who have or are likely to develop a medical condition to prevent, cure, delay the onset of the disease or recurrent disease, reduce its severity, improve one or more symptoms, or prolong the patient's survival beyond the expected survival without such treatment.

[0048] "Treating cancer" includes, but is not limited to, reducing the number of cancer cells in a patient's body or shrinking the size of a tumor; slowing the progression of cancer to a more aggressive form (i.e., maintaining cancer in a form sensitive to therapeutic drugs); reducing cancer cell proliferation or slowing tumor growth; killing cancer cells; reducing cancer cell metastasis in the subject or lowering the likelihood of cancer recurrence. As used herein, "treating a subject" refers to any type of treatment that can benefit a subject who has cancer, is at risk of developing cancer, or is facing cancer recurrence. Treatment includes improving the subject's condition (e.g., one or more symptoms), delaying disease progression, delaying the onset of symptoms, slowing the progression of symptoms, etc.

[0049] Unless the context clearly indicates otherwise, “a,” “an,” and “the” all include plural references. Therefore, the terms “a” (or “an”), “one or more,” or “at least one” are used interchangeably in this document; unless otherwise stated, “or” means “and / or.”

[0050] Immune cytokines

[0051] This invention relates to a fusion protein comprising an anti-CTLA-4 antibody and an IL-2 variant, the IL-2 variant being a regulatory T-cell-specific IL-2 receptor antagonist comprising the following substitutions: K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A; and further comprising the following substitutions: (i) S127K, (ii) Y31P and S127K, or (iii) K49Q, E52S, R81E, D84N, T131R, and L132S, the positions of which are determined by alignment with human IL-2 (SEQ ID NO: 1), and wherein the N-terminus of the IL-2 variant is fused to the C-terminus of the antibody heavy chain.

[0052] The protein described in this invention is an immune cytokine formed by fusing an anti-CTLA-4 antibody with a variant of the IL-2 cytokine. This paper names regulatory T cell-specific IL-2 receptor antagonists as IL-2 antagonists or IL-2 mutant proteins.

[0053] The immune cytokines of this invention induce CD25 endocytosis and Treg apoptosis, as illustrated in the embodiments of this application. These properties enable them to selectively deplete tumor-associated Tregs.

[0054] The immune cytokines described in this invention can be derived from known anti-CTLA-4 antibodies, particularly therapeutic anti-CTLA-4 antibodies. Alternatively, novel anti-CTLA-4 antibodies can be prepared using standard methods known in the art, as disclosed herein.

[0055] For example, the antibodies of this disclosure can be prepared by immunizing experimental animals with an antigen (receptor protein or a fragment thereof, ultimately conjugated to a vector), thereby inducing B cells of the mammal to produce antibodies; the antibodies are then recovered from the serum of the immunized animals. To obtain monoclonal antibodies, B cells are isolated from the spleen of the immunized animals and immortalized according to standard hybridoma production techniques. The antibodies of this disclosure can also be obtained by screening phage display libraries. Specifically, VH and VL fragments of antibodies can be screened from a phage display library using peptide antigens, and recombinant antibodies can be produced according to standard techniques known in the art. The antibody VH and VL sequences can be obtained by sequencing single-cell B cell receptors using high-throughput sequencing technology, as described in Goldstein et al., Communications Biology, 2019, 2, 304. The antibodies of this disclosure can also be produced in host cell transfection tumors, for example, using a combination of recombinant DNA techniques and gene transfection methods known in the art (Morrison, Science, 1985, 229, 1202-1207). To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. When a recombinant expression vector encoding an antibody gene is introduced into host cells (particularly eukaryotic cells, such as mammalian cells), the antibody is produced by culturing the host cells for a period sufficient for antibody expression within the host cells and (optionally) secreting the antibody into the culture medium in which the host cells are growing. The antibody can be recovered and purified from the culture medium after secretion, for example, using standard protein purification methods (Shukla et al., Journal of Chromatography, 2007, 848, 28-39).

[0056] The antibody as defined in this invention specifically binds to the CTLA-4 receptor, which is preferentially expressed on tumor-associated Tregs. In some embodiments, the antibody may further inhibit signal transduction through this receptor, meaning that the antibody is a blocking, inhibitory, or antagonistic antibody.

[0057] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), preferably human or humanized VH and VL. In some specific embodiments, the antibody further comprises an Ig constant region, including a native sequence Ig constant region and a variant Ig constant region. The Ig constant region is preferably human. The Ig constant region comprises at least one Ig constant domain; particularly comprising at least CH1 and CL, more particularly comprising CL and CH1, and further comprising an Fc domain (CH2 and / or CH3 domain). The Fc region can be derived from any Ig class or isotype, preferably human IgG isotype. The Fc region can be a native sequence Fc region or a variant Fc region. An example of a variant Fc region is a silent Fc region; a silent IgG1 Fc region may contain N297A or L234A and L235A mutations. Another example of variant Fc regions includes mutations designed to enhance ADCC, particularly those enhancing FcγR affinity, such as S298A / E333A / K334A, S239D / I332E, and P247I / A339Q (see van der Horst et al., Cancers, 2020, 12, 3041 for a review). Variant human Fc constant regions may contain M428L and N434S substitutions (LS) to increase antibody half-life. Fc region residues are numbered using the Kabat EU index.

[0058] In some specific embodiments, the antibody comprises a human IgG1 Fc domain and / or a human Ig kappa light chain constant domain.

[0059] In some specific embodiments, the antibody is a full-length human antibody or a humanized antibody. The antibody is preferably IgG, especially IgG1. The corresponding immune cytokine has a classic Ig structure, comprising two Ig heavy chains and two Ig light chains, and a copy of an IL-2 variant fused to the C-terminus of each heavy chain.

[0060] In some implementations, the antibody is a single-domain antibody (or nanobody), such as VHH.

[0061] In some implementations, the antibody is a heavy chain-only antibody, particularly an antibody containing only fully human heavy chains.

[0062] Various anti-CTLA-4 antibodies have been described in the art, which can be used in the immune cytokines of the present invention. Non-limiting examples of known anti-CTLA-4 antibodies have been disclosed in the following references, including monoclonal antibodies and their chimeric, fully human, and fully humanized versions, as well as heavy chain-only antibodies, particularly fully human heavy chain-only antibodies: ipilimumab (patents US 7,605,238, US 6,984,720, and US 8,017,114), trimemumab (patents US 6,682,736, US 7,109,003, and US 8,143,379), and international patent applications WO 0037504, WO 01 / 14424, WO 2019 / 174603, WO2021 / 129775, WO 2021 / 13102, WO 2022 / 017428, WO 2022 / 169269, WO 2022 / 184155, WO2022 / 184155, WO 2022 / 222961, Patent US8,491,895, Single-chain anti-CTLA4 antibody (International patent applications WO1997 / 020574, WO 2007 / 123737, WO 2019 / 233413, Gan et al., PNAS, 2022, 119,e2200879119), Oncology Live, June 22, 2023, 24, 11, 39-; J Clin Oncol. 2023;41(suppl 16)). Other anti-CTLA4 antibodies that can be used in the immune cytokines of this invention include: ADG 116; botentimab (AGEN1181); BMS-986249 and BMS-986288; HBM4003; quarvolimumab (MK-1308; Peretset al., Ann Oncol. 2021;32(3):395-403. doi:10.1016 / j.annonc.2020.11.020); zefulimab (AGEN1884); Gotistobart (ONC-392); XmAb22841 and XTX101; and the anti-CTLA4 antibodies shown in Table 3. Alternatively, novel anti-CTLA4 antibodies may also be obtained by standard methods known in the art as disclosed herein.

[0063] In some specific implementations, the anti-CTLA-4 antibody is a therapeutic antibody, such as ipilimumab (human IgG1), trimemumab (human IgG2a), or a functional variant thereof.

[0064] In some specific embodiments, the anti-CTLA-4 antibody comprises H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 3, H-CDR3 of SEQ ID NO: 4, L-CDR1 of SEQ ID NO: 5, L-CDR2 of SEQ ID NO: 6, and L-CDR3 of SEQ ID NO: 7, or variants having one or more conserved substitutions on one or more of these CDRs. In particular, the monoclonal antibody or its antigen-binding fragment described herein may have 1, 2, 3, 4, 5, 6, or more changes in the amino acid sequence of 1, 2, 3, 4, 5, 6, or 6 CDRs of the polyclonal antibody provided herein, especially in the CDRs of SEQ ID NO: 2 to 7. In some specific embodiments, the anti-CTLA-4 antibody comprises a VH domain having at least 85% sequence identity with SEQ ID NO: 8 and a VL domain having at least 85% sequence identity with SEQ ID NO: 9. Preferably, the VH and / or VL domains have at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence, and even more preferably, at least 95%, 96%, 97%, 98%, or 99% identity with the sequence. Preferably, the VH domain has H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 3, and H-CDR3 of SEQ ID NO: 4, and / or the VL domain has L-CDR1 of SEQ ID NO: 5, L-CDR2 of SEQ ID NO: 6, and L-CDR3 of SEQ ID NO: 7.

[0065] In some specific embodiments, the anti-CTLA-4 antibody comprises an IgG Fc region, including the native sequence Fc region and variant Fc regions disclosed herein. In some specific embodiments, the anti-CTLA-4 antibody comprises a human IgG1 Fc domain and / or a human Igkappa light chain constant domain.

[0066] In some specific implementations, the anti-CTLA-4 antibody is a single-domain antibody (or nanobody), such as VHH.

[0067] In some embodiments, the anti-CTLA-4 antibody is a heavy chain-only antibody, particularly an antibody containing only fully human heavy chains. In some embodiments, the anti-CTLA-4 antibody is bispecific or multispecific. For example, the antibody may contain an antigen-binding site specific to another protein, particularly another surface molecule specific to Treg cells, as disclosed herein. More particularly, this surface molecule is preferentially expressed on tumor-associated Tregs. An antibody specific to another protein may target any receptor preferentially expressed on tumor-associated Tregs. Examples of such receptors are known in the art and disclosed, for example, in WO 2021 / 165546. The bispecific or multispecific antibodies of the present invention may target cell surface receptors of any tumor-specific Tregs disclosed in WO 2021 / 165546, particularly the cell surface receptors disclosed in Table 1 or Table 2 of WO 2021 / 165546.

[0068] In various implementations, the IL-2 mutant protein may include the following substitutions: K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A, and S127K; K9E, L12E, H16R, L19R, M23L, N26K, Y31P, S87N, V91K, E95K, N119K, T123A, and S127K; or K9E, L12E, H16R, L19R, M23L, N26K, K49Q, E52S, R81E, D84N, S87N, V91K, E95K, N119K, T123A, T131R, and L132S. In some preferred embodiments, the IL-2 mutant protein comprises the following substitutions: K9E, L12E, H16R, L19R, M23L, N26K, Y31P, S87N, V91K, E95K, N119K, T123A, and S127K.

[0069] In various embodiments, the IL-2 mutant protein may or may not contain at least one additional amino acid mutation (insertion, deletion, substitution). In some embodiments, the IL-2 mutant protein does not contain an additional amino acid mutation.

[0070] In some other embodiments, the IL-2 mutant protein comprises at least one additional amino acid mutation (insertion, deletion, or substitution). The IL-2 mutant protein preferably comprises at least one amino acid deletion, more preferably located at a position selected from S4, S5, or S6, particularly comprising a single amino acid deletion at a position selected from S4, S5, or S6, the position of which is determined by comparison with SEQ ID NO:1. This amino acid deletion may be combined with or replaced by at least one additional substitution selected from: amino acid substitutions from 3T to A for controlling potential O-glycosylation in the T residue; and substitutions from 125C to A for controlling free 125-cysteine ​​residues that may induce disulfide bonds. The IL-2 mutant protein may comprise one, two, or all of the above deletions and / or additional substitutions, as well as combinations thereof. All such possible combinations are specifically covered. The IL-2 mutant protein is at least 125 amino acids in size. Preferably, the IL-2 variant is 125 to 135 amino acids in size. In some preferred embodiments, the IL-2 mutant protein is a human IL-2 mutant protein.

[0071] In some specific embodiments, the IL-2 mutant protein has at least 85% amino acid identity with SEQ ID NO: 1. Preferably, the IL-2 mutant protein has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence, and even more preferably, at least 95%, 96%, 97%, 98%, or 99% identity with the sequence.

[0072] In some preferred embodiments, the IL-2 mutant protein does not contain any substitutions at the following positions: 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 126, 129, 130, and 133; 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, and 75. 76, 80, 85, 86, 88, 92, 110, 125, 126, 129, 130 and 133 bits, or positions 4, 8, 10, 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 38, 42, 45, 48, 62, 67, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 90, 92, 110, 125, 126, 128, 129, 130 and 133.

[0073] In some preferred embodiments, the IL-2 mutant protein has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 13, 14, or 15; preferably, it has at least 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 13, 14, or 15; more preferably, it has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14; even more preferably, it has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14; and even more preferably, it has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14. 14 has at least 95%, 96%, 97%, 98%, or 99% identity; more preferably, the IL-2 mutant protein does not contain any substitutions at the following positions: 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 126, 129, 130, and 133; 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 6 Bits 8, 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 125, 126, 129, 130, and 133, or bits 4, 8, 10, 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 38, 42, 45, 48, 62, 67, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 90, 92, 110, 125, 126, 128, 129, 130, and 133.

[0074] The IL-2 mutant protein of the fusion protein of the present invention may comprise: (i) a deletion of an amino acid at a position selected from S4, S5, or S6; and (ii) Replace: (a) K9E, L12E, H16R, M23L, N26K, Y31P, K49Q, E52S, R81E, D84N, S87N, V91K, E95K, T131R and L132S replace: or (b) K9E, L12E, H16R, L19R, M23L, N26K, Y31P, K49Q, E52S, R81E, D84N, S87N, E95K, T131R, and L132S shall replace; or (c) Replace with H16R and V91K; or (d) Replaced by H16R, L19R and V91K; The location shown was determined by comparison with human IL-2 (SEQ ID NO: 1).

[0075] The IL-2 mutant protein of the fusion protein of the present invention has at least 85% amino acid identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. Preferably, the IL-2 mutant protein has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and even more preferably, it has at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

[0076] The IL-2 mutant protein is fused to the C-terminus of the antibody heavy chain, meaning that the N-terminus of the IL-2 mutant protein is fused to the C-terminus of the antibody heavy chain. The IL-2 mutant protein can be fused directly to the antibody or fused via a linker or spacer used to physically separate the two protein domains. Suitable linkers are known in the art and include, for example, linkers containing glycine and serine residues. Examples of suitable linkers include the sequence (GGGGS)n, where n preferably does not exceed 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, the fusion protein comprises a linker with the sequence (GGGGS)3 (corresponding to SEQ ID NO: 10).

[0077] In some specific embodiments, the fusion protein of the present invention is derived from a complete antibody molecule, preferably a complete human or humanized antibody molecule, such as human or humanized IgG1 and IgG4, and the N-terminus of the IL-2 mutant protein is fused to the C-terminus of the antibody heavy chain via a linker (e.g. (GGGGS)n, more preferably (GGGGS)3).

[0078] In some preferred embodiments, the fusion protein of the present invention comprises: - An anti-CTLA-4 antibody heavy chain fused to an IL-2 variant, wherein the heavy chain fusion has at least 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 11; and - An anti-CTLA-4 antibody light chain having at least 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 12.

[0079] Preferably, the VH domain of the anti-CTLA-4 antibody heavy chain has H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 3, and H-CDR3 of SEQ ID NO: 4, and / or the VL domain of the anti-CTLA-4 antibody light chain has L-CDR1 of SEQ ID NO: 5, L-CDR2 of SEQ ID NO: 6, and L-CDR3 of SEQ ID NO: 7.

[0080] Fusion proteins may contain additional portions and / or be further modified. This invention covers immunocytokines having one or more amino acid residues, peptide bonds, N-terminals, and / or C-termini with one or more chemical modifications, provided the modified immunocytokines are functional. These modifications can be introduced into the fusion protein using conventional methods known to those skilled in the art and include, in a non-limiting manner: conjugation to a target molecule or reagent, such as PEG (polyethylene glycolation), to prolong the biological (e.g., serum) half-life of the immunocytokines; substitution of native amino acids with non-protein amino acids (D-type amino acids or amino acid analogs); modification of peptide bonds, particularly with trans or trans-bonds or bonds other than peptide bonds; cyclization; and addition of chemical groups to the amino acid side chains or the N-terminus and / or C-terminus of the immunocytokines, particularly for conjugation of the immunocytokines to molecules or agents of interest.

[0081] In some implementations, the IL-2 variant is provided in combination with at least a target agent, such as in the form of a complex, a particle, or a conjugate. The target agent includes, but is not limited to, any therapeutic agent, including cells, such as the patient's chimeric antigen receptor (CAR) T cells.

[0082] As used herein, the term "immunocytokine" encompasses the various forms of immune cytokines disclosed herein, such as immune cytokines that are or are not modified and that are or are not bound to at least one target agent in the form of a complex or conjugate, as described above.

[0083] The immune cytokines of the present invention can be prepared using conventional techniques in the art, particularly by expressing recombinant DNA in a suitable cell system (eukaryotic or prokaryotic) and screening for their activity (i.e., ability to induce CD25 endocytosis and / or induce Treg apoptosis) using the assays described herein or other similar assays.

[0084] Production of nucleic acids, vectors, host cells, and immune cytokines

[0085] This article also discloses nucleic acid molecules encoding the immune cytokines disclosed herein.

[0086] Typically, the nucleic acid is a recombinant, synthetic, or semi-synthetic nucleic acid that can be expressed in host cells suitable for the expression or production of immune cytokines, particularly human immune cytokines. The host cell can be a cell used for the production of recombinant immune cytokines or a patient cell used for the production of immune cytokines in vivo. Typically, the nucleic acid can be DNA, RNA, or a mixture of molecules and can be further modified and / or integrated into any suitable expression vector. As used herein, the terms "vector" and "expression vector" refer to a medium that can introduce a DNA or RNA sequence (e.g., a foreign gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Recombinant vectors can be eukaryotic or prokaryotic expression vectors, such as plasmids, bacteriophages for bacterial introduction, YACs capable of transforming yeast, viral vectors (especially retroviral vectors), or any expression vector. The expression vectors defined herein are selected to produce immune cytokines in vitro or in vivo.

[0087] Examples of nucleic acid molecules include those encoding amino acid sequences of the immunocytokines disclosed above. In some specific embodiments, the nucleic acid molecule encodes the heavy and light chain amino acid sequences of the immunocytokines disclosed above. In some more specific embodiments, the nucleic acid molecule is selected from nucleotide sequence pairs SEQ ID NO: 16 and 17, which encode immunocytokines comprising the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 12.

[0088] This disclosure also relates to nucleic acids encoding the immune cytokines of this disclosure, said immune cytokines having a nucleotide sequence having at least 80%, such as at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, identity with any of the nucleotide sequences defined above.

[0089] This disclosure also relates to nucleic acid molecules or constructs derived from the aforementioned sequences that have been optimized for protein expression in host cells, particularly eukaryotic cells, preferably mammalian cells such as CHO or HEK cell lines or human cells. Codon optimization is used to enhance protein expression levels in living organisms by improving the translation efficiency of target genes. Appropriate methods and software for codon optimization in target hosts are well known in the art and are publicly available (see, for example, the GeneOptimizer software suite in Raab et al., Systems and Synthetic Biology, 2010, 4, (3), 215-225).

[0090] In some embodiments, the nucleic acid molecule is a eukaryotic (preferably mammalian) expression cassette in which an immune cytokine coding sequence is operatively linked to a suitable regulatory sequence for expression in immune cytokine-producing cells or patient cells. Such sequences known in the art include, in particular, promoters, and other regulatory sequences capable of further controlling transgene expression, such as, but not limited to, enhancers, terminators, and introns. The promoters may be tissue-specific, ubiquitous, constitutive, or inducible promoters and are functional in immune cytokine-producing cells. Such promoters are well known in the art, and their sequences are available in public sequence databases.

[0091] Another object of this disclosure relates to a vector containing at least one nucleic acid encoding an immune cytokine of this disclosure.

[0092] This invention can utilize any vector suitable for delivering and expressing nucleic acids into individual cells, particularly for nucleic acid therapy. Such vectors known in the art include viral vectors and non-viral vectors. Non-viral vectors include a variety of (non-viral) agents commonly used to introduce or maintain nucleic acids in individual cells. Agents used to introduce nucleic acids into individual cells by various means include, in particular, polymer-based, particle-based, lipid-based, peptide-based delivery media or combinations thereof, such as, but not limited to, cationic polymers, dendritic polymers, micelles, liposomes, liposome complexes, exosomes, microparticles, and nanoparticles, including lipid nanoparticles (LNPs) and virus-like particles; and cell-penetrating peptides (CPPs). Agents used to maintain nucleic acids within individual cells include, in particular, naked nucleic acid vectors, such as plasmids, transposons, and microcircles. Viral vectors are inherently capable of penetrating cells and delivering target nucleic acids into the cell; this process is called viral transduction. As used herein, the term "viral vector" refers to a non-replicating, non-pathogenic virus engineered for the delivery of genetic material into cells. In a viral vector, viral genes essential for viral replication and virulence are replaced by expression cassettes of the target transgene. Therefore, the viral vector genome contains a transgenic expression cassette flanked by the viral sequence required for viral vector production. As used herein, the term "recombinant virus" refers to a virus, particularly a viral vector, produced using standard recombinant DNA techniques known in the art. As used herein, the term "viral particle" refers to the extracellular form of a nonpathogenic virus, particularly a viral vector, which consists of genetic material formed from DNA or RNA encased in a protein coat called a capsid, and in some cases also includes an envelope derived from the host cell membrane and containing viral glycoproteins. As used herein, a viral vector refers to a viral vector particle. These vectors have minimal eukaryotic sequences to minimize the likelihood of chromosomal integration. Furthermore, these methods can be advantageously combined to introduce and maintain the nucleic acids of the present invention within individual cells.

[0093] In specific embodiments, the carrier is a particle or vesicle, particularly a lipid-based microvesicle or nanovesicle or particle, such as a liposome or lipid nanoparticle (LNP). In more specific embodiments, the nucleic acid is RNA, particularly mRNA, and the carrier is a particle or vesicle as described above, particularly an LNP.

[0094] In other embodiments, the nucleic acid is DNA, preferably contained in an expression vector (e.g., a plasmid or a viral vector). The vector can be a recombinant integrative or non-integrative viral vector. Examples of recombinant viral vectors include, but are not limited to, vectors derived from retroviruses, adenoviruses, adeno-associated viruses (AAVs), herpesviruses, poxviruses, and other viruses. Retroviruses particularly include lentiviral vectors, such as human immunodeficiency virus (HIV), including HIV type 1 (HIV1) and HIV type 2 (HIV2) vectors.

[0095] The polynucleotides disclosed herein can be prepared using conventional methods known in the art. For example, they can be prepared by amplifying nucleic acid sequences via PCR or RT-PCR, screening genomic DNA libraries by hybridization with homologous probes, or by full or partial chemical synthesis. Recombinant vectors can be constructed and introduced into host cells using conventional recombinant DNA and genetic engineering techniques known in the art.

[0096] Another object of this disclosure relates to host cells that have been transfected, infected, or transformed by the nucleic acids and / or vectors of the present invention. As used herein, the term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell, causing the host cell to express the introduced gene or sequence to produce a desired substance (typically a protein or enzyme encoded by the introduced gene or sequence). Transformation can be transient or time-stable. Stable transformation can be achieved by integrating nucleic acids into the host cell genome. Host cells that receive and express the introduced DNA or RNA have been "transformed."

[0097] The host cell can be a prokaryotic cell (e.g., bacteria) or a eukaryotic cell (e.g., yeast, insect cells, or mammalian cells). Mammalian cells can be ape, human, dog, or rodent cells. Mammalian host cells used to express the antibodies of this disclosure particularly include Chinese hamster ovary cells (CHO cells), including dhfr-CHO cells (as described in Urlaub and Chasin, 1980) and CHOK1dhfr+ cell lines using DHFR selection markers (as described in Kaufman and Sharp, 1982). In a preferred embodiment, the host cell is a CHO cell. In another preferred embodiment, the host cell is an insect cell.

[0098] The polynucleotides, vectors, or cells disclosed herein can be used to produce the proteins of this invention using known recombinant DNA technologies. As disclosed below, the polynucleotides or vectors can also be used in nucleic acid therapy.

[0099] Another aspect of the invention relates to a method for producing the immune cytokines of the present disclosure, comprising: (i) culturing host cells of the present disclosure to express the immune cytokines; and optionally, (ii) recovering the immune cytokines; and (iii) purifying the immune cytokines.

[0100] The immunocytokines disclosed herein can be produced in host cells transfected with tumors using, for example, a combination of recombinant DNA techniques and gene transfection methods, as is well known in the art (Morrison et al., Science, 1985, 229, 1202-1207). To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of commonly used techniques for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. When a recombinant expression vector encoding an immunocytokine gene is introduced into a host cell (particularly a eukaryotic cell, such as a mammalian cell), the production of immunocytokines can be achieved by culturing the host cells for a period sufficient to express the immunocytokines within the host cells and optionally secrete them into the culture medium in which the host cells grow. After secretion, immune cytokines can be recovered and purified from the culture medium using standard protein purification methods (Shukla et al., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 2007, 848, 28-39). Methods for purifying peptides are well known in the art, such as chromatography (e.g., ion exchange chromatography, gel permeation chromatography, and reversed-phase chromatography).

[0101] Pharmaceutical Compositions and Therapeutic Uses

[0102] The immune cytokines, polynucleotides, and / or carriers of the present invention can be used to treat cancer.

[0103] The immunocytokines, polynucleotides, and / or carriers of the present invention, when administered to a patient, inhibit or eliminate tumor-infiltrating Tregs, particularly tumor-associated Tregs, thereby relieving the inhibition of immune cells (B cells, NK cells, CD4+ or CD8+ T cells; dendritic cells; macrophages, etc.) by Tregs. The immunocytokines, polynucleotides, carriers, and / or cells of the present invention are used to selectively deplete tumor-infiltrating Tregs, particularly tumor-associated Tregs, by depriving them of IL-2, without affecting effector immune cells (particularly NK cells, CD4+ and CD8+ T cells) and allowing their proliferation. In a specific embodiment, the immunocytokines, polynucleotides, carriers, and / or cells of the present invention are used to selectively deplete tumor-infiltrating Tregs, particularly tumor-associated Tregs. In a specific embodiment, the immunocytokines, polynucleotides, carriers, and / or cells of the present invention are used to stimulate anti-tumor CD8+ T cell responses.

[0104] This invention relates to a pharmaceutical composition comprising the immune cytokines, polynucleotides and / or carriers of the present invention as active substances, and at least one pharmaceutically acceptable medium and / or carrier.

[0105] A "pharmaceutically acceptable carrier" is a medium through which a therapeutic drug is administered, provided that such medium does not produce adverse reactions, allergic reactions, or other adverse effects when administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0106] Preferably, the pharmaceutical composition comprises a carrier that is pharmaceutically acceptable for injectable formulations. These carriers may in particular be isotonic, sterile saline solutions (sodium monophosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dry, particularly lyophilized compositions that can be formulated into injectable solutions by adding sterile water or physiological saline as appropriate.

[0107] Suitable drug forms for injection include sterile aqueous solutions or suspensions. These solutions or suspensions may contain additives compatible with the active agent. In all cases, the drug form must be sterile and flowable to a degree that facilitates syringe manipulation. It must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. Suitable examples of solutions include buffers, such as phosphate-buffered saline (PBS) or lactated Ringer's solution.

[0108] The pharmaceutical composition is formulated for use in various routes of administration, including but not limited to oral, parenteral, and topical administration. The drug carrier is a carrier suitable for the intended route of administration known in the art. The composition may be in the form of one or more dosage units.

[0109] In some embodiments, the pharmaceutical composition is a liquid formulation, preferably a concentrated liquid formulation containing immune cytokines. In other embodiments, the pharmaceutical composition is a lyophilized formulation.

[0110] The pharmaceutical composition comprises a therapeutically effective amount of immune cytokines, polynucleotides, and / or carriers. In this invention, a therapeutically effective amount refers to a dose sufficient to reverse, alleviate, or inhibit the progression of the disease or condition referred to in the term, or to reverse, alleviate, or inhibit the progression of one or more symptoms of the disease or condition referred to in the term. The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the intended effect. A therapeutically effective amount is sufficient to demonstrate a positive medical response in the individual receiving the administration. A positive medical response refers to a reduction in subsequent (preventative) or established (therapeutic) disease symptoms. A positive medical response includes partial or complete suppression of disease symptoms. A positive medical response can be determined by measuring various objective parameters or criteria, such as objective clinical signs of the disease and / or prolongation of survival. The medical response of the compositions of this invention can be readily verified in suitable animal disease models well known in the art.

[0111] Determining and adjusting the effective dose depends on a variety of factors, such as the composition used, route of administration, the physiological characteristics of the individual considered (e.g., sex, age, and weight), concomitant medications, and other factors known to a medical professional. The effective dose can be determined using standard clinical techniques. Additionally, in vivo and / or in vitro assays may be selectively used to help predict the optimal dose range.

[0112] In some specific implementation schemes, the therapeutically effective dose of immune cytokines is at least 0.05 mg / kg.

[0113] In some embodiments, the pharmaceutical composition comprises another active agent, wherein the active agent is a drug or therapeutic agent capable of preventing, treating, or improving cancer in humans or animals. The active agent may be a protein (including antibodies), oligonucleotide (including antisense oligonucleotides), peptide nucleic acid (PNA), small interfering RNA, locked nucleic acid (LNA), phosphoryldiamine morpholino oligonucleotide (PMO), and decoy DNA molecules, plasmids, aptamers (including DNA, RNA, or peptide aptamers), small or large chemical drugs, or mixtures thereof. Specifically, the active agent may be an immunomodulator, such as an immune checkpoint inhibitor. The active agent may also be an anticancer agent or a tumor antigen.

[0114] The present invention also provides immune cytokines, polynucleotides, carriers or pharmaceutical compositions of the present invention for use as medicines or for treatment.

[0115] The present invention also provides immune cytokines, polynucleotides, carriers or pharmaceutical compositions of the present invention for the prevention or treatment of cancer.

[0116] The present invention also provides the use of the immune cytokines, polynucleotides, carriers or pharmaceutical compositions of the present invention for the prevention or treatment of cancer.

[0117] The present invention also provides the use of the immune cytokines, polynucleotides, carriers or pharmaceutical compositions of the present invention in the preparation of medicaments for the prevention or treatment of cancer.

[0118] The present invention also provides a pharmaceutical composition for treating cancer, comprising the immune cytokines, polynucleotides and / or carriers of the present invention as active components.

[0119] This invention also provides a pharmaceutical composition comprising the immune cytokines, polynucleotides, and / or carriers of this invention for the treatment of cancer. As used herein, the term "cancer" refers to any cancer that may affect any of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lips; salivary glands; tongue; gums; oral cavity; palate; tonsils; larynx; trachea; bronchi, lungs; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs, such as stomach, intrahepatic bile ducts, bile ducts, pancreas, small intestine, colon; rectum; urinary organs, such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin. Bones; joints, articular cartilage of the limbs; eyes and their appendages; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and all parts of the central nervous system; connective tissue, subcutaneous tissue and other soft tissues; retroperitoneum, peritoneum; adrenal glands; thyroid gland; endocrine glands and related structures; female reproductive organs, such as ovaries, uterus, cervix; uterine body, vagina, vulva; male reproductive organs, such as penis, testes and prostate; hematopoietic system and reticuloendothelial system; blood; lymph nodes; thymus.

[0120] The term "cancer" in this invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, non-Hodgkin's lymphoma, neuroblastoma, glioma, adenocarcinoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and terminal mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, undifferentiated thyroid carcinoma, type 2A multiple endocrine neoplasia, type 2B multiple endocrine neoplasia, familial medullary thyroid carcinoma, pheochromocytoma, and accessory neuron. Cancers include: skin cancers (including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, keratoacanthoma, nevus, dysplastic nevus, lipoma, hemangioma, and dermatofibroma), nervous system cancers, brain cancers (including astrocytoma, medulloblastoma, glioma, low-grade glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, spinal neurofibroma, glioma, or sarcoma), and skull cancers (including osteoma, hemangioma, sarcoma). Osteitis externa, xanthomas, or osteitis deformans; meningeal cancer (including meningioma, meningeal sarcoma, or glioma); head and neck cancer (including squamous cell carcinoma of the head and neck and oral cancer (e.g., buccal carcinoma, lip cancer, tongue cancer, oral cavity cancer, or pharyngeal cancer)); lymph node cancer; gastrointestinal cancer; liver cancer (including hepatocellular carcinoma, hepatocellular carcinoma, bile duct cancer, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma); colon cancer; stomach cancer; esophageal cancer (including squamous cell carcinoma, laryngeal cancer, adenocarcinoma, leiomyosarcoma, or lymphoma); colorectal cancer; bowel cancer; small bowel cancer (e.g., adenocarcinoma). Lymphoma, neoplasms, Kaposi's sarcoma, leiomyomas, hemangiomas, lipomas, neurofibromas or fibromas), colorectal cancer (e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma or leiomyoma), pancreatic cancer (including ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, neoplasms or vipoma), ear, nose and throat (ENT) cancer, breast cancer (including HER2-enriched breast cancer, luminal type A breast cancer, luminal type B breast cancer and triple-negative breast cancer), uterine cancer (including endometrial cancer,Examples of cancers include endometrial cancer, endometrial stromal sarcoma and malignant mixed Müllerian tumor, uterine sarcoma, leiomyosarcoma and gestational trophoblastic disease, ovarian cancer (including ovarian dysgerminoma, granulosa cell-theca cell tumor and Sertoli-Leydig cell tumor), cervical cancer, vaginal cancer (including squamous cell vaginal carcinoma, vaginal adenocarcinoma, clear cell vaginal adenocarcinoma, vaginal germ cell tumor, vaginal botryoid sarcoma and vaginal melanoma), and vulvar cancer (including squamous cell vulvar carcinoma, verrucous vulvar carcinoma, vulvar melanoma, etc.). Basal vulvar cancer, Bartholin's gland cancer, vulvar gland cancer, and Keira's proliferative erythema; urogenital tract cancers; kidney cancer (including clear cell renal cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma, or leukemia); adrenal cancer; bladder cancer; urethral cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma); prostate cancer (e.g., adenocarcinoma or sarcoma); and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma). Tumors, adenomatous tumors or lipomas), lung cancer (including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) (including squamous cell lung cancer, lung adenocarcinoma (LUAD) and large cell lung cancer), bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, pulmonary sarcoma, chondrosarcoma, and pleural mesothelioma), sarcomas (including Askinoma, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant angioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcoma), soft tissue sarcomas (including alveolar soft tissue sarcoma, ... Angiosarcoma, Phyllostachys sarcoma, Dermatofibrosarcoma protuberans, Desmoid tumor, Connective tissue proliferative small round cell tumor, Epithelioid sarcoma, Extraosseous chondrosarcoma, Extraosseous osteosarcoma, Fibrosarcoma, Gastrointestinal stromal tumor (GIST), Hemangiopericytoma, Angiosarcoma, Kaposi's sarcoma, Leiomyosarcoma, Liposarcoma, Lymphangiosarcoma, Lymphosarcoma, Malignant peripheral nerve sheath tumor (MPNST), Neurofibrosarcoma, Pleuroid histiocytoma, Rhabdomyosarcoma, Synovial sarcoma and undifferentiated pleomorphic sarcoma, Cardiac cancer (including sarcoma),Examples include angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyosarcoma, fibroma, lipoma and teratoma), bone cancer (including osteosarcoma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma and reticulum cell sarcoma, multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma, osteocartilaginous exostoses, benign chondroma, chondroblastoma, chondromycinoid fibroma, osteoid osteoma and giant cell tumor), hematologic and lymphatic cancers, blood cancers (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma and myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma, and hairy cell and lymphatic diseases and their metastases). Lymphoma can be, for example, T-cell lymphoma, such as anaplastic T-cell lymphoma.

[0121] The cancer may be a solid cancer or a liquid cancer, particularly selected from any of the cancers disclosed herein.

[0122] In some specific implementations, the cancer is selected from the group consisting of: melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, malignant pleural mesothelioma, esophageal cancer, squamous cell carcinoma of the head and neck, urothelial carcinoma, primary Hodgkin's lymphoma, gastric cancer, large B-cell lymphoma, cervical cancer, Merkel cell carcinoma, endometrial cancer, squamous cell carcinoma of the skin, triple-negative breast cancer, invasive breast cancer, pancreatic adenocarcinoma, thymoma, prostate adenocarcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, and sarcoma.

[0123] The present invention also provides a method for treating cancer, comprising: administering a therapeutically effective amount of the immune cytokines, polynucleotides, carriers, and pharmaceutical compositions of the present invention to a patient.

[0124] The immunocytokines, polynucleotides, carriers, and pharmaceutical compositions of the present invention are typically administered according to known procedures, dosages, and administration times, effectively producing therapeutic effects in an individual. The immunocytokines, polynucleotides, carriers, and pharmaceutical compositions can be administered via any convenient route, such as infusion or bolus injection in a non-restrictive manner, absorption through the epithelial or mucocutaneous skin layer (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), inhalation, transdermal application, or intratumoral administration. Administration can be systemic, local, or a combination of systemic and local administration; systemic administration includes parenteral and oral administration, and local administration includes local, local-regional, and intratumoral administration. Systemic administration is preferably parenteral, such as subcutaneous (SC), intramuscular (IM), intravascular (IV), or intra-arterial; intraperitoneal (IP); intradermal (ID), epidural, or other routes. Parenteral administration is advantageously performed by injection or infusion. In some specific embodiments, administration is parenteral and / or intratumoral, preferably intravascular, such as intravenous (IV), intratumoral, or a combination of IV and intratumoral administration.

[0125] A “treatment regimen” refers to a mode of treatment for a disease, such as the mode of medication administration during treatment. Treatment regimens may include induction regimens and maintenance regimens. The phrase “induction regimen” or “induction period” refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. The overall goal of an induction regimen is to provide the patient with a high level of medication in the initial phase of the treatment regimen. An induction regimen may be a (partial or complete) “loading regimen,” which may include a higher dose of medication, a higher frequency of medication administration, or both than that used by the physician during a maintenance regimen. The phrase “maintenance regimen” or “maintenance period” refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient’s condition during the treatment of a disease, for example, to keep the patient in remission for a long period (months or years). Maintenance regimens may be continuous treatment (e.g., medication administration at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent treatment (e.g., treatment interruption, intermittent treatment, treatment upon relapse, or treatment upon reaching specific predetermined criteria [e.g., pain, disease symptoms, etc.]).

[0126] In some implementations, the immune cytokines, polynucleotides, carriers, and pharmaceutical compositions are used for human prevention or treatment.

[0127] The immune cytokines, polynucleotides, carriers, and pharmaceutical compositions of the present invention are advantageously used in combination with other cancer therapies. Specifically, the immune cytokines, polynucleotides, carriers, and / or pharmaceutical compositions of the present invention can be used in combination with targeted therapies, immunotherapies (e.g., immune checkpoint therapy and immune checkpoint inhibitors, co-stimulatory antibodies, and CAR-T cell therapy), anticancer agents (including therapeutic agents (chemotherapy) and anticancer vaccines), and / or radiotherapy. Combination therapies can be administered individually, simultaneously, and / or sequentially.

[0128] Immune checkpoint therapies, such as checkpoint inhibitors, include, but are not limited to, programmed death receptor-1 (PD-1) inhibitors, programmed death ligand-1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte activation gene 3 (LAG-3) inhibitors, T cell immunoglobulin-containing and mucin-domain-containing protein 3 (TIM-3) inhibitors, T cell immune receptor with Ig and ITIM domains (TIGIT) inhibitors, B and T lymphocyte attenuator (BTLA) inhibitors, V domain T cell activation Ig inhibitor (VISTA) inhibitors, indoleamine 2,3-dioxygenase (IDO) inhibitors, cytotoxic cell immunoglobulin-like receptor (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors, and carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM-1) inhibitors. Specifically, checkpoint inhibitors include anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-TIM-3, and anti-LAG3 antibodies. Co-stimulatory antibodies deliver positive signals via immunomodulatory receptors including, but not limited to, ICOS, CD137, CD27, OX-40, and GITR.

[0129] Examples of anti-PD1 antibodies include, but are not limited to: nivolumab, cimiprilmab (REGN2810 or REGN-2810), tislelizumab (BGB-A317), spartalizumab (PDR001 or PDR-001), ABBV-181, JNJ-63723283, BI 754091, MAG012, TSR-042, AAGEN2034, Pidilizumab, Nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335 or Opdivo), Pembrolizumab (MK-3475, MK03475, Lambolizumab, SCH-900475 or Keytruda) and antibodies described in the following international patent applications: WO2004004771, WO2004056875, WO2006121168, WO2008156712, WO2009014708, WO2009114335, WO2013043569 and WO2014047350.

[0130] Examples of anti-PD-L1 antibodies include, but are not limited to, LY3300054, atezolizumab, durvalumab, and averumab.

[0131] Examples of anti-VISTA antibodies are described in U.S. patent application US20130177557.

[0132] Examples of inhibitors of the LAG3 receptor are described in U.S. Patent 5,773,578.

[0133] An example of a KIR inhibitor is IPH4102, which targets KIR3DL2.

[0134] Targeted therapy consists of drugs designed to interfere with specific molecules essential for tumor growth and progression. For example, therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors. In some cases, monoclonal antibodies are conjugated with radioactive isotopes or toxins to specifically deliver these cytotoxic agents to target cancer cells. Small molecules can penetrate cell membranes and interact with intracellular targets. Small molecules are often designed to interfere with the enzymatic activity of target proteins, such as proteasome inhibitors, tyrosine kinase inhibitors, cyclin-dependent kinase inhibitors, and histone deacetylase inhibitors. Targeted therapy may also utilize cytokines. Examples of such targeted therapies include, but are not limited to, adorantrolactam (HER2) and afatinib (EGFR). (HER1 / ERBB1), HER2), interleukin (Proleukin), alectinib (ALK), alemtuzumab (CD52), axitinib (kit, PDGFRβ, VEGFR1 / 2 / 3), belimumab (BAFF), belistat (HDAC), bevacizumab (VEGF ligand), belintoomeb (CD19 / CD3), bortezomib (proteasome), brentuximab velituximab (CD30), besutinib (ABL). Brigatinib (ALK), Cabozantinib (FLT3, KIT, MET, RET, VEGFR2), Cananumab (IL-1β), Carfilzomib (Proteasome), Ceritinib (ALK), Cetuximab (EGFR), Coffitinib (MEK), Crizotinib (ALK, MET, ROS1), Dabrafenib (BRAF), Daramumab (CD38), Dasatinib (ABL), Denoxinumab (RANKL), Dinutuximab (B4GALNT1) (GD2)), Elotuzumab (SLAMF7), Enxidipine (IDH2), Erlotinib (EGFR), Everolimus (mTOR), Gefitinib (EGFR), Tiimomab (CD20), Smoothened, Sipuleucel-T, Stutuzumab (IL-6), Sorafenib (VEGFR, PDGFR, KIT, RAF), Tocilizumab (IL-6R), Tansirolimus (mTOR), Tofacitinib (JAK3), Trametinib (MEK), Tosimomab (CD20), Trastuzumab (HER2), Vandetanib (EGFR), Vemolfenib (BRAF), Venexlaxel (BCL2), Vemodegumab (PTCH, Smoothened), Vorinostat (HDAC), Ziv-Aflibercept (PIGF, VEGFA / B).

[0135] In some embodiments, the immune cytokines, polynucleotides, carriers, and / or pharmaceutical compositions of the present invention are administered to a patient in combination with chemotherapy. As used herein, the term "chemotherapy" has its common meaning in the art and refers to a treatment method in which a chemotherapeutic agent is administered to a patient. Chemotherapy agents include, but are not limited to, alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, propylthiophene, and piperidonium; aziridines, such as phenyldopa, carboquinone, metedopa, and utetadopa; ethyleneimine and methylmelamines, including atrazamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylmelamine; acetyl compounds (especially boletine and bolete ketone); camptothecins (including synthetic analogs such as topotecan); lichenin; callystatin; CC-1065 (including its synthetic analogs adozelecin, cazelecin, and bizelecin); cryptophytes (especially cryptophytes). Cryptocoryne 1 and Cryptocoryne 8); Saccharin; Ducamycin (including synthetic analogs KW-2189 and CB1-TM1); Erut Robin; Panclastatin; Botulinum toxin; Sponge statin; Nitrogen mustards, such as chlorambucil, naphazoline, chophosphatamide, estradiol, ifosfamide, nitrogen mustard, nitrogen mustard hydrochloride, melphalan, novibixine, phenethylamine, prednimustine, trophosphatamide, uracil mustard; Nitrosoureas, such as carmustine, chlorzotocin, formustine, lomustine, nimustine, and ranimustine; Antibiotics, such as enediyne antibiotics (e.g., calichimycin, especially calichimycin γ and calichimycin ω); Motrin, including motrin Medicinal fungicide A; bisphosphonates, such as clophosphonates; esparmycin; and neocachinosine chromophores and related chromophores, enediyne antibiotic chromophores, aclarubicin, actinomycin, otralamycin, azaserine, bleomycin, carclomycin, carbimycin, carmomycin, cachinofelline, chromomycin, actinomycin, daunorubicin, ditocin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, macelomycin, mitomycin C, mycophenolic acid, nogaramycin, olivomycin Drugs containing: pepromycin, porphyromycin, puromycin, quercetin, rhodoxorubicin, streptomycin, streptozotocin, tuberculin, ubenmex, zinosstatin, zorubicin; antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (such as dinotrexate, methotrexate, pterin, trimesartan); purine analogs (such as fludarabine, 6-mercaptopurine, thiamine, thioguanine); pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmoflurane, cytarabine, dideoxyuridine, doxorubicin, enoxatabine, fluorouridine; androgens, such as calusone, drotaldone propionate, epiandrone, mepistonone, testosterone lactone.Antiadrenergic drugs, such as aminoglutethimide, mitotane, and trilostertan; folic acid supplements, such as floridine; acetylglucosinolate; aldehyde phosphoramide glycoside; aminolevulinic acid; enuracil; acridine; betabucil; bisaxatine; edastrasartan; desofol; colchicine; diazinon; irizophenone; efemin; eletate; epomycin; etoglobulin; gallium nitrate; hydroxyurea; lentinan; lonidanine; maytansine compounds, such as maytansine and salamisine; mitoxurone; mitoxuron; mopiperol; nitrobenzamide; pentazocine; fenitromethorphan; pirarubicin; loxoanthrone; podophyllic acid; 2-ethylhydrazine; methylhydrazine derivatives, including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex; razorbenzan; rhizomycin; cizofuran; spirogermanium; ternozolidine; triadimefon; 2,2',2"-Trichlorotriethylamine; trichothecene compounds (especially T-2 toxin, veraculin A, roridin A, and anguidine); ethyl carbamate; vindesine; dacarbazine; mannomustine; mitorabrinol; mitorabrol; piperbbromide; cytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and docetaxel; Chlorobutazone; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum-based coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Norenone; Teniposide; Idatraxa; Daunorubicin; Aztreon; Xeloda; Ibandronate sodium; Irinotecan (e.g., CPT-1) 1) Topoisomerase inhibitors RFS 2000; difluoromethylmimetamine (DMFO); retinoids, such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, podophyllotoxins, enzymes such as L-asparaginase; anthraquinones; hormones and antagonists, including corticosteroid antagonists, such as prednisone and its equivalents, dexamethasone and aminoglutethimide; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens, such as diethylstilbestrol and ethinylestradiol equivalents; antiestrogens, such as tamoxifen; androgens, including testosterone propionate and flumethasone / equivalents; antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs and leuprorelin; and nonsteroidal antiandrogens, such as flutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above drugs.

[0136] In some embodiments, the immune cytokines, polynucleotides, carriers, and / or pharmaceutical compositions of the present invention are administered to a patient in combination with radiotherapy. Suitable examples of radiotherapy include, but are not limited to, external beam radiotherapy (e.g., superficial X-ray therapy, positive voltage X-ray therapy, megavolt-level X-ray therapy, radiosurgery, stereotactic radiotherapy, fractionated stereotactic radiotherapy, cobalt therapy, electron therapy, fast neutron therapy, neutron capture therapy, proton therapy, intensity-modulated radiotherapy (IMRT), three-dimensional conformal radiotherapy (3D-CRT), etc.); brachytherapy; unsealed source radiotherapy; tomographic radiotherapy, etc. Gamma rays are another form of photon used in radiotherapy. Certain elements (e.g., radium, uranium, and cobalt-60) spontaneously produce gamma rays by releasing radiation during decomposition or decay. In some embodiments, the radiotherapy may be proton radiotherapy or proton microbeam radiotherapy. Proton radiotherapy is a form of ultra-precise radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Controlin RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun 1;104(2):266-271. doi: 10.1016 / j.ijrobp.2019.01.080;Prezado Y, Jouvion G, Patriarca A,Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapywidens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7;8(1):16479. doi: 10.1038 / s41598-018-34796-8).Radiation therapy can also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation. FLASH radiotherapy uses ultra-rapid delivery of radiation, with a dose rate several orders of magnitude higher than that of conventional clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. MedSci (Paris) 2015; 31: 121-123. DOI: 10.1051 / medsci / 20153102002); Patriarca A., Fouillade CM, Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016 / j.ijrobp.2018.06.403. Epub 2018 Jul 11).

[0137] In some specific embodiments, the immune cytokines, polynucleotides, carriers and / or pharmaceutical compositions of the present invention are administered in combination with immune checkpoint therapy, particularly PD-1 and / or PDL-1 inhibitors.

[0138] Unless otherwise stated, the present invention will be practiced using conventional techniques known to those skilled in the art. These techniques have been described in detail in the literature.

[0139] The present invention will be described through the following embodiments, which are not limiting, and reference will be made to the following figures: Attached Figure Description

[0140] Figure 1 Compared with peripheral Treg cells, tumor-associated Treg cells overexpress CD25 and CTLA-4.

[0141] A / B: Single-cell RNA sequencing data from NSCLC (internal data and Guo et al, Nat Med, 2018, 24, 978-985) and C / : Single-cell RNA sequencing data from hepatocellular carcinoma (Zheng et al, Cell, 2017, 169, 1342-1356) were used to generate violin plots to represent the expression levels of CD25 (IL-2RA) and CTLA-4 transcripts in hematopoietic Treg cells and matched tumor-associated Treg cells. D / E: Quantitative analysis of CD25 and CTLA-4 protein expression levels in intratumoral and peripheral Treg cells, CD4+ T conventional (Tconv) and CD8+ T cells by flow cytometry (n=5 NSCLC patients).

[0142] Figure 2 Stability of IPI-V5 under stress conditions.

[0143] PBMCs from healthy volunteers were stimulated for 10 minutes at room temperature with the following substances: IL-2 at the indicated concentration (Proleukin®), or IL-2 + 500 nM IPI-V5 (Pro+IPI-V5), or IL-2 + 500 nM IPI-V5 treated as follows: A / After 3 consecutive freeze-thaw cycles (Pro+IPI-V5_3xFT); B / Incubated at 25°C for 2 weeks (Pro+IPI-V5_25C) or at 50°C for 1 week (Pro+IPI-V5_50C); C / Incubated at room temperature with gentle stirring at 300 rpm for 48 hours (Pro+IPI-V5_stirred); D / Incubated at 37°C with 0.01% H2O2 for 3 days (Pro + IPI-V5_OxH2O2), or at 37°C with 1 mM Cells were incubated with AAPH for 1 week (Pro+IPI-V5_OxAAPH); or E / at room temperature at acidic pH 3.5 for 24 hours (Pro+IPI-V5_acid), or at 37°C at alkaline pH for 24 hours (Pro+IPI-V5_alkaline). After fixation and permeabilization, cells were stained with antibodies against CD3, CD4, CD25, CD56, FoxP3, and pSTAT5, and analyzed by flow cytometry. pSTAT5 (%) represents the proportion of all viable cells stained positive for the antibodies. Data were obtained from a representative PBMC donor.

[0144] Figure 3 : Identify CD4+ T cell epitopes derived from immune cytokine candidates, commercial antibodies, and Proleukin through computer simulation.

[0145] The netMHCIIpan software was used to identify CD4+ T cell epitopes (x-axis) targeting Ipi-V1, Ipi, V5, and Ipi-V6 (orange dots) and targeting marketed and developing therapeutic molecules (green diamonds). Similarly, the figure also shows the number of peptide-HLA-II pairings (y-axis), which represents the number of HLA-II alleles predicted to bind to all epitopes.

[0146] Figure 4 Interactions between different constructs and human FcRn.

[0147] The binding affinity of FcRn to FcRn in vitro was assessed by ELISA using the LUMIT™ FcRn Binding Immunoassay Kit (Promega) and directly compared with ipilimumab. An Ab control, consisting of a strong FcRn-binding antibody, was used as a positive control.

[0148] Figure 5 PK curves of mice after administration of different compounds.

[0149] C57BL6 / NRj mice were intravenously injected (retroorbital) with 6 mg / kg IPI-V1, IPI-V5, IPI-V6, or 5 mg / kg ipilimumab; blood was repeatedly drawn from the tail vein over 72 hours. Plasma concentrations were assessed using a non-GLP human IgG ELISA kit (Abcam).

[0150] Figure 6 CTLA-4 receptor occupancy.

[0151] hCTLA-4 transfected Raji cells were analyzed by flow cytometry to assess dose-receptor occupancy. Raji cells were incubated at 4°C for 30 min. All compounds were detected using FITC-labeled anti-human Fc secondary antibody. After cell fixation, cells were analyzed using an LSR Fortessa (Becton Dickinson) flow cytometer. The same analysis of untransduced Raji cells showed no binding (data not shown).

[0152] Figure 7 Inhibition of STAT-5 signaling in Treg cells.

[0153] Fresh, healthy donor PBMCs were stimulated at room temperature for 10 minutes with either the indicated concentration of IL-2 (Proleukin®) or a combination of IL-2 (Proleukin®) and a fixed dose of 500 nM (90 µg / ml) of the indicated compound. Cells were fixed, permeabilized, and stained with antibodies against CD3, CD4, CD25, CD56, FoxP3, and pSTAT-5, and analyzed by flow cytometry. pSTAT5 (%) represents the proportion of all viable cells positive for each antibody subtype.

[0154] Figure 8 CD25 endocytosis mediated by immune cytokines.

[0155] Human Treg cells expanded in vitro were cultured at 37°C for 60 minutes with culture medium, Fc-V5 (a non-targeting IL-2V5), ipilimumab, or IPI-V5. The cells were then fixed, permeabilized, stained with anti-CD25 fluorescent antibody (red) or anti-human Fc fluorescent antibody (green, showing the localization of Fc-V5, ipilimumab, or IPI-V5), and analyzed by confocal fluorescence microscopy. Scale bar: 4µM. Dashed boxes show intracellular vesicles. Yellow indicates co-localization of red and green.

[0156] Figure 9 Immunocytokines preferentially mediate apoptosis in expanded human Tregs. A to D. Expanded human Tregs and Tconvs were co-cultured at 37°C with cell culture medium containing different concentrations of Ipi-V5 or ipilimumab. After 3 days, apoptosis and Treg markers were assessed. (A, B) Apoptosis was quantitatively analyzed by flow cytometry based on annexin V staining. The percentage of annexin V-positive cells (A) or MFI (B) in Tregs and Tconvs was assessed. CD25 (C) and human Fc (D) staining was performed on treated expanded Tregs and Tconvs to assess the behavior of CD25 and Ipi-V5 on the cell surface. Data are presented as mean ± SEM. E. Expanded human Treg cells were cultured in vitro for 3 days with 0.9 ng / ml IL-2 (Proleukin®) and indicated concentrations of Fc-V5, ipilimumab, Ipi-V5, or Fc-V5 combined with ipilimumab. The resulting Treg cells were then analyzed by flow cytometry. The percentage of annexin V+ cells corresponds to the proportion of all phylogenetic Treg cells that were positively stained with fluorescent annexin V reagent.

[0157] Figure 10 Immune cytokines induce CD25 endocytosis in Treg cells.

[0158] In vitro expanded Treg and Tconv cells were seeded and treated with three different concentrations (0.312, 3.12, and 31.2 nM) of IPI, IPI-V5, Fc-V5, or a combination of IPI and Fc-V5. Endocytosis kinetics were measured at different time points. The mean fluorescence intensity (MFI) of CTLA-4, CD25-specific antibodies, and anti-human Fc expression on Treg and Tconv cells was quantitatively analyzed by flow cytometry (n=2).

[0159] Figure 11 Treg depletion in the microenvironment of breast and lung cancer in vitro.

[0160] Breast and lung cancer samples were isolated from six patients and maintained in vitro in media containing ipilimumab or Ipi-V5. Cells were collected after 72 hours, and CD45-positive cells were analyzed by flow cytometry. A: Treg cells (CD3+CD4+FoxP3+ gated); B: CD4 normal T cells (CD3+CD4+FoxP3- gated); C: CD8+ T cells (CD3+CD8+ gated); D: NK cells (CD3-CD56+ gated); E: Frequency of CD25+ cells in each cell population. Data shown are individual records from six patients / samples in three independent experiments. The nonparametric Mann-Whitney test showed p=0.0066.

[0161] Example

[0162] Example 1: Principles and Design of Novel Immune Cytokines

[0163] Materials and Methods

[0164] Raw counting data from three single-cell RNAseq datasets (Tosello & Richer, unpublished; Guo et al, PMID: 29942094; and Zheng et al, PMID: 28622514) were downloaded from the Gene Expression Omnibus (GEO). The counting matrices for each dataset were imported into Seurat software and analyzed independently using standard procedures. Briefly, the gene expression levels for each single cell in blood and tumor tissue were normalized using the "NormalizeData" function with default parameters. Then, 3000 highly variable genes were identified using the "FindVariableFeatures" function and the "vst" method. After removing heterogeneity associated with mitochondrial contamination, the gene expression matrices were scaled and centered using the "ScaleData" function. For cluster analysis, the ElbowPlot function was used to compute relevant principal components to determine the data dimensions. Relevant principal components were selected using the "FindNeighbors" function to construct a shared nearest neighbor (SNN) graph, and the Louvain algorithm was used to determine the clusters. Finally, based on the aforementioned SNN graph, the Uniform Manifold Approximation and Projection (UMAP) method was applied to visualize the single-cell transcription profile in 2D space. Clusters were annotated using marker genes and published gene features. Missing values ​​in the counting matrix were imputed using the Adaptive Threshold Low-Rank Approximation (ALRA, PMID: 35017482). A violin plot showing the expression values ​​after ALRA imputation was used to illustrate the expression of CTLA4 and CD25.

[0165] Tumor samples were surgically cut into small pieces and digested for 30 minutes in CO2-free medium (Gibco) containing 0.1 mg / ml Liberase TL (Roche) and 0.1 mg / ml DNase (Roche). Cells were then mechanically separated using a 2.5 mL syringe plunger on a 40 μm cell sieve (BD) to obtain a single-cell suspension. The tumor cell suspension was washed with PBS and incubated with LIVE / DEAD Fixable Cell Dead Stain (Invitrogen) at room temperature for 15 minutes. Next, the cells were washed and incubated with fluorescently labeled antibodies at 4 °C for 30 minutes. The following antibodies were used for surface staining: CD3 R718 (clone UCHT1), CD4 BV786 (clone RPA-T4), CD25 BUV737 (clone 2A3), and CD8 BUV496 (clone RPA-T8) from BD. For nuclear staining, cells were fixed, permeabilized, and stained using Foxp3 / transcription factor staining buffer (eBioscience). CTLA4 BB700 (clone BNI3) and FOXP3 PE-CF594 (clone 236A / E7) were used for intracellular staining. Cells were collected on a Fortessa flow cytometer (BD) and analyzed using FlowJo software (V.10). Treg cells were identified as CD3+CD4+FOXP3+, Tconv cells as CD3+CD4+FOXP3-, and CD8 cells as CD3+CD8+. The median CD25 and CTLA4 intensities were calculated for all cell populations of interest.

[0166] result

[0167] Treg cell survival depends on interleukin-2 (IL-2), which is primarily produced by effector T cells and NK cells and transmits signals in Treg cells via a high-affinity IL-2 receptor (IL-2R) complex composed of α, β, and γ chains (CD25, CD122, and CD132, respectively). A key data point for developing novel immune cytokines is the observation of higher levels of CD25 and CTLA-4 transcripts in tumor-associated Treg cells compared to hematologic Treg cells and other lymphocyte types. Figure 1 AC) and protein ( Figure 1D and E) show selectively higher differential expression levels. In contrast, in the tumor microenvironment, effector T cells and NK cells are in a state of chronic activation, primarily expressing intermediate-affinity IL-2R composed of β and γ chains. Therefore, tumor-associated Treg cells differ from tumor-associated effector T cells and peripheral Treg cells in their higher CD25 (IL2RA) expression levels, which has been observed in both non-small cell lung cancer (NSCLC) and liver cancer samples. Figure 1 The diagrams above, from A to 1C, and Figure 1 D). CD25 overexpression allows tumor-associated Treg cells to be preferentially targeted. Antitumor immune responses are also regulated by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Although CTLA-4 is constitutively expressed in both cancerous tissues and Treg cells outside of cancerous tissues, its detection rate on the cell surface of circulating and lymphoid organ Treg cells is minimal. Figure 1 Figures A to 1C below, and Figure 1 E). In contrast, tumor-associated Treg cells express high levels of CTLA-4 (E) on their surface. Figure 1 E).

[0168] Based on these findings, the inventors developed a novel immunocytokine (ICK) designed to efficiently and selectively deplete tumor-infiltrating Tregs, particularly tumor-associated Tregs, by targeting preferentially expressed CTLA-4 and CD25 membrane proteins, while minimizing impact on effector cells (CD8+ T cells, CD4+ Tconv cells, and NK cells). This novel immunocytokine further deprives Tregs of IL-2 by downregulating CD25, thereby selectively targeting tumor-associated Tregs to restore anti-tumor responses.

[0169] To this end, we designed, generated, and evaluated six different ICKs fused from anti-CTLA-4 antibodies with different IL-2 mutant proteins, and assessed their bioactivity and exploitability. The anti-CTLA-4 antibody was a recombinant humanized IgG1 monoclonal antibody, ipilimumab (US Patent No. 7,605,238 B2). The IL-2 mutant proteins were Treg-specific IL-2R antagonists and their derivatives previously disclosed in WO 2020 / 201095, comprising 3T to A amino acid substitutions (to control potential O-glycosylation in the T residue) and 125C to A substitutions (to control free 125-cysteine ​​residues that may induce disulfide bonds), namely IL-2V1, IL-2V1_T3A-C125A, IL-2V5, IL-2V5_T3A-C125A, IL-2V6, and IL-2V6_T3A-C125A. IL-2V1 includes K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A, and S127K as replacements; IL-2V5 includes K9E, L12E, H16R, L19R, M23L, N26K, Y31P, S87N, V91K, E95K, N119K, T123A, and S127K as replacements; IL-2V6 includes K9E, L12E, H16R, L19R, M23L, N26K, K49Q, E52S, R81E, D84N, S87N, V91K, E95K, N119K, T123A, T131R, and L132S as replacements. The (GGGGS)3 linker connects the C-terminal amino acid of the antibody heavy chain to the N-terminal amino acid of the IL-2 mutant protein. The resulting immunocytokine is a 180 kDa homodimer formed by fusing the anti-CTLA-4 antibody with two IL-2 mutant proteins via the (GGGGS)3 linker. These immunocytokines are indiscriminately named IPI-Vno., where no. is the IL-2 variant number, such as 1, 5, or 6.

[0170] Example 2: Identification and Characterization of Immune Cytokines

[0171] 1. Manufacturability

[0172] Materials and Methods

[0173] Expression vectors SLXplasmid_220_Puro_BT+ and SLXplasmid_221_Hygro+ (Selexis, Plan-Les-Ouates, Swizerland) are used to directly obtain heavy and light chains (one light chain and six heavy chain variants) of immune cytokines. These expression vectors contain the bacterial β-lactamase gene (AmpR) from transposon Tn3 (conferring ampicillin resistance) and the bacterial ColE1 origin of replication. As a derivative of pGL3 Control (Promega), the terminator region of the Selexis expression vector carries an SV40 enhancer downstream of the BGH polyadenylation signal. Furthermore, these vectors also contain human X_29 SGE downstream of the expression cassette, as well as an integrated puromycin or hygromycin resistance gene under the control of the SV40 promoter. The expression vectors encode the target gene under the control of the hEF-1α promoter and the CMV enhancer. The plasmid was used to transform 50 µL of competent DH5α cells (Invitrogen, catalog number 18265-017). A bacterial clone was amplified in a 300 mL Erlenmeyer conical flask containing 150 mL LB + 100 µg / mL ampicillin. Plasmid DNA was extracted in bulk using the NucleoBond Xtra MidiPlus EF Medium-Quantity Extraction Kit (Macherey-Nagel) according to the manufacturer's instructions. Prior to transfecting CHO cells, 70 µL of the medium-quantity plasmid preparation was digested with Pvul-HF (NEB) to linearize the plasmid DNA. The validated digestion products were subjected to agarose gel electrophoresis. DNA bands containing the mammalian expression cassette of the target gene were excised from the gel, purified using standard methods, and extracted with NanoDrop. TM One C Quantification was performed using Thermo Fisher. The transgenic sequence was validated using Sanger sequencing technology.

[0174] These monocistronic vectors (containing puromycin and hygromycin resistance cassettes) were used to generate cell lines: the target gene was transfected into the CHO-M cell line using microwell technology, followed by two rounds of cloning to obtain high-yield clonal cell lines. The wild-type CHO-K1 cell line (lot number 4765275) was derived from a subclone of the parental CHO cell line provided by Dr. T. Puck of the University of Chicago and was adapted in BalanCD medium supplemented with 6 mM L-glutamine. To generate cell lines, BalanCD medium (lot number 91128210913) supplemented with 6 mM L-glutamine (lot number 7511020) and 1 / 400 penicillin / streptomycin (lot number 0000115461) was preheated by inoculating 1 mL into one well of a 24-well plate (per microwell group) and incubated at 37°C and 5% CO2. Linearized DNA (3 μg total) was pre-prepared in sterile reaction tubes for each microcell group (plus one GFP control) into monocistronic vectors carrying puromycin and hygromycin resistance cassettes, named Puro_BT+_SLX-595X_SP51H_V5_Hc_Sc and Hygro_BT+_SLX-595X_SP51L_Lc_Sc, respectively. The MicroPorator (Invitrogen Neon™ Transfection System) was set up by injecting 3 mL of electrolyte buffer E (lot number 2B17161) into a dedicated tube (included in the Invitrogen Neon™ Transfection System Kit MP-100) and setting specific pulse conditions for CHO-M cells (1130V, 20 ms, 3 pulses). To avoid reducing cell viability and transfection efficiency, cells were prepared shortly before transfection. CHO-M cells were counted, and cell viability was determined using trypan blue staining (cell viability of seedtrain-cultured cells encoding S-22-CHOM-2#180-8 was 97.6%). The required number of cells (3.4 × 10⁵ cells) were centrifuged (400 g, 5 min, room temperature). The cell pellet was gently resuspended in resuspension buffer R (batch number 2B17161) to a cell concentration of 3.4 × 10⁷ cells / mL. 10 μL of the cell suspension was immediately transferred to a DNA tube and carefully mixed. The cell-DNA mixture was aspirated using a dedicated MicroPorator pipette and placed in a pipette workstation. After microporization, cells were transferred to pre-prepared 24-well plates and incubated overnight in a static humidified incubator at 37°C and 5% CO₂. Parallel experiments were performed using a GFP-expressing vector as a transfection efficiency control (normal transfection efficiency of 50-70% was observed under a microscope the following day).

[0175] Two days after transfection, a portion of the transfected cells from each group were transferred at a concentration of 5000 cells / mL to a semi-solid medium in an Omnitray culture plate (WFI (L201015 and L210117), CloneMedia CHO Growth A (batch number 99901210801), supplemented with 6 mM L-glutamine (batch numbers AG29701757 and 7511020), CloneDetect (batch number CDU0520A), 3 μg / mL puromycin (batch number 039M4114V) and 250 μg / mL hygromycin (batch number HGG-41-06)). Ten days later, the seeded cells were screened using ClonePix2, and candidate cells were selected and transferred to 96-well plates (BalanCD medium (batch number 91128210913), supplemented with 6 mM L-glutamine (batch number 7511020), 1 / 100 penicillin / streptomycin (batch number 0000115461), 5 μg / mL puromycin (batch number 039M4114V), and 500 μg / mL hygromycin (batch number HGG-41-06)). After all candidate cells were transferred to 96-well deep-well plates, cell selection was performed using the fed-batch culture method in 96-well deep-well plates.

[0176] result

[0177] Twelve candidate products (six variants, two transfection ratios) were first generated, purified, and analyzed in Ambr15 medium and then in 1-liter shake flasks. Results of transient transfection (LC / HC ratio 1:3) are not listed due to unsatisfactory productivity. Results of transient transfection (LC / HC ratio 1:1.3) are described below. Fed-batch Ambr15 cultures were prepared to evaluate production yield, monomericity of purified protein A, and purity on LabChip capillary SDS-PAGE under both reducing and non-reducing conditions.

[0178] SDS-Page Materials System: LabChip GXII Touch 24 protein characterization system (Perkin Elmer).

[0179] Chip: LabChip® GXII Touch™ measured (Perkin Elmer, CLS 150338).

[0180] Reagent: ProteinEXact assay kit (Perkin Elmer, CLS 150466).

[0181] Sample: Monoclonal antibody.

[0182] Reducing agent: DTT (from the kit).

[0183] Non-reducing buffer: from the kit.

[0184] SDS-PAGE program

[0185] Pre-treat the LabChip GXII Touch chip according to the instructions in the assay kit manual. Prepare the gel dye solution and load it into the chip. Dilute the purified protein with the provided DTT-free sample buffer, mix gently, and incubate at 70°C for 10 minutes. For reduction conditions, dilute the protein with the provided DTT-containing sample buffer, mix gently, and incubate at 70°C for 10 minutes. Then, load the samples into the designated wells of a 96-well plate according to the instrument requirements and place it in the LabChip GXII Touch system.

[0186] Table 1: Small-scale production of immune cytokines

[0187] All productivity data support variant V5, which exhibits higher production yields, lower fragmentation, and fewer aggregates. No significant differences were observed between V5 and the V5 (mutated) variant. However, the T3A-C125A double mutation appears to adversely affect the productivity of both V1 and V6 variants.

[0188] Twelve 1-liter pools were generated and purified using protein A capture chromatography to assess their commercial viability. The results are shown below.

[0189] Protein A capture

[0190] Column: HiTrap MabSelect PrismA 5 mL

[0191] Applied to columns: Approximately 1 L SN

[0192] Buffer A: PBS, pH 7.4

[0193] Buffer solution Bi: 0.1 M sodium citrate, pH 3.5

[0194] Buffer B2: 0.1 M glycine-HCl, pH 2.7

[0195] Flow rate: 1.0 m / min

[0196] Washing: 5 CV PBS, 850 mM NaCl

[0197] Wash-off: 100% B1 -> 100% B2

[0198] Neutralization: Adjust the pH to 5-6 using 1.5 M Tris pH 8.8.

[0199] Materials for size exclusion chromatography (SEC) HPLC Column: Waters BioSuite 250 UHR SEC, 4 µm, 4.6 × 300 mm.

[0200] Mobile phase (buffer A): 0.2 M potassium phosphate, 2.5 M KCl, pH 6.2.

[0201] Sample: 1.0 µg monoclonal antibody (diluted with buffer A).

[0202] HPLC system: equipped with a UV detector (e.g., 280 nm).

[0203] Flow rate: 0.35 mL / min.

[0204] Injection volume: 7 µL.

[0205] SEC HPLC analysis procedure

[0206] Equilibrate the SEC column with 5 column volumes (approximately 8 mL) of buffer A at a flow rate of 0.35 mL / min until the baseline stabilizes. Dilute 1.0 µg of elution buffer with buffer A and filter through a 0.22 µm filter membrane. Load 7 µL of sample (containing 1.0 µg of protein) onto the column. Set the flow rate to 0.35 mL / min and run for 30 minutes.

[0207] Integrate the chromatogram to quantitatively analyze the main peak and any impurities, such as aggregates or fragments. Calculate the percentage of each substance based on the peak area.

[0208] Table 2: Large-scale production of immune cytokines

[0209] The results were consistent with observations from small-scale experiments, with all productivity data supporting variant V5, which exhibited higher production yields, less fragmentation, and fewer aggregates. No significant differences were observed between V5 and the V5 (mutated) variant. The T3A-C125A double mutation appeared to be detrimental to the productivity of both V1 and V6 variants. Protein A titers in the CHO pool indicated that the V5 candidate had the best productivity and product properties. Ipi-V5 (with or without the aforementioned two mutations) appeared to exhibit better properties. Furthermore, for Ipi-V1 and Ipi-V6, variants carrying the aforementioned mutations appeared to be detrimental to productivity (more aggregates, more fragments).

[0210] In summary, these production data indicate that IPI-V5 produces better results and exhibits more uniform biochemical properties compared to other ICK candidates containing different IL-2V.

[0211] 2. Stability study under stress conditions

[0212] All six ICK candidates underwent stability studies after being subjected to different stress conditions (freeze / thaw, temperature, stirring, pH, oxidation). Biochemical analyses, including monomericity analysis, were then performed. The data showed that the selected candidate drug IPI-V5 remained stable under different conditions. Subsequently, the bioactivity of IPI-V5 after stress testing was evaluated using a STAT-5 phosphorylation competition assay. Figure 2 In short, fresh, healthy donor PBMCs were stimulated for 10 minutes at room temperature with either IL-2 (Proleukin®) at the indicated concentration or a combination of IL-2 (Proleukin®) and a fixed dose (90 µg / ml) of IPI-V5 at 500 nM. Cells were fixed, permeabilized, and stained with antibodies against CD3, CD4, CD25, CD56, FoxP3, and pSTAT-5, and analyzed by flow cytometry. pSTAT5 (%) corresponds to the proportion of all viable cells positively stained for each antibody subtype.

[0213] The following conditions were tested: IPI-V5, 3 freeze / thaw cycles => 75±10°C (at least 30 minutes) / room temperature IPI-V5, 25°C, 2 weeks IPI-V5, 50°C, 1 week IPI-V5, stirred at 300 rpm for 48 hours at room temperature. IPI-V5 was subjected to oxidative stress at 37°C with 0.01% H2O2 for 3 days. IPI-V5 was subjected to oxidative stress at 37°C with 1 mM AAPH for one week. IPI-V5 under acidic pH stress for 24 hours at pH 3.5 (room temperature). IPI-V5, alkaline pH stress (37°C) for 24 hours Summary of results on the inhibition of WT IL-2-induced STAT-5 phosphorylation of IPI-V5: IPI-V5 was stable after three freeze / thaw cycles, two weeks at 25°C, and 48 hours of stirring. The effect of H2O2 oxidative stress remains unclear.

[0214] 3. Computer-simulated immunogenicity studies

[0215] Materials and Methods

[0216] HLA-II allele data were obtained from the Allele Frequency Net database (Gonzalez-Galarza, FF et al., Nucleic Acids Res., 2020, 48, D783–D788), derived from HLA-II genes relevant to cord blood and / or anthropological studies. HLA alleles with increased frequency in Caucasian populations were selected for binding prediction. The binding of 15 amino acid peptides (15mers) derived from candidate immune cytokine molecules, antibodies, and cytokines (all sequences publicly available) to HLA-II molecules was predicted using netMHCIIpan version 4 (Reynisson, B. et al., Nucleic Acids Res., 2020, 48, W449–W454). Self-peptides from the query protein, present in the human proteome GRCh38 or any of the 29 immunoglobulin germline genes, were excluded. Standard quantitative estimates of peptide binding were used as thresholds to screen for strongly binding peptides (ranked in the top 2% by binding affinity compared to a random pool of natural peptides), weakly binding peptides (ranked between the top 2% and 10% by binding affinity), and non-binding peptides (ranked below 10% by binding affinity). All compounds were characterized by the following: the total number of peptides binding to any HLA-II isotype, the number of unique peptide-HLA pairs, and an immunogenicity score derived by multiplying the binding strength (Score_EL from netMHCIIpan) by the median frequency of HLA-II alleles.

[0217] result

[0218] Immunogenicity was assessed by computational interference from the presence of potential CD4+ T cell epitope sequences presented by all MHC class II molecules. Results showed that the overall immunogenic potential of IPI-V5 (number of potential epitopes and number of potential peptide-HLA pairs) was at the same level as currently used clinical antibodies (such as atezolizumab, trastuzumab, omalizumab, or rituximab); and its immunogenicity was lower than that of IPI-V1 and IPI-V6 versions. Figure 3 ).

[0219] 4. Pharmacokinetic Studies

[0220] The alternative pharmacokinetic (PK) properties were evaluated by comparing the binding of the anti-CTLA-4 antibody ipilimumab and different IPI-IL-2 variants to the neonatal Fc receptor (FcRn). Data showed that the Ipi-Vx variant exhibited comparable or superior FcRn binding ability to the comparative drug ipilimumab, predicting an extended Fc-mediated half-life. Figure 4 ).

[0221] Then, mice were intravenously injected (once on day 0, 6 mg / kg) with IPI-V1, IPI-V5, IPI-V6, or ipilimumab, and blood was repeatedly drawn from the tail vein over 72 hours. The levels of IPI variants and ipilimumab in the blood were detected by ELISA. Figure 5 ).

[0222] For IPI-V5, data shows its C max The concentration was 32 µg / ml, approximately 40% of the theoretical value calculated considering only plasma distribution (based on a mean blood volume of 78 ml / kg). This suggests that the drug distribution may target sites present in the blood and blood-related tissues (lymphoid organs). In fact, even without competition for mouse IL-2, Ipi-V5 was still able to bind to mouse IL-2R. After this initial distribution / retention period, the terminal elimination half-life (T1 / 2) of Ipi-V5 was approximately 72 hours, within the expected half-life range of human monoclonal antibodies in mice. In contrast, ipilimumab distributed within the expected blood volume due to the absence of interaction with mouse CTLA-4, C max It was approximately 85 µg / ml. The T value was measured after IV administration of ipilimumab. 1 / 2 Comparable to Ipi-V5. While these data need to be confirmed in non-human primates, they suggest that Ipi-V5 has a T... 1 / 2 It may be similar to the T1 / 2 of ipilimumab (15.4 days in humans).

[0223] 5. Bioactivity

[0224] - Evaluate the binding activity of immune cytokine candidates to CTLA-4.

[0225] Receptor occupancy of different immune cytokine candidates (IPI-V1, IPI-V5, IPI-V6) was assessed in vitro on Raji C4 cells expressing human CTLA-4 using a combination assay. No significant differences were observed in dose-receptor occupancy results. Figure 6 ).

[0226] - The IL-2 antagonistic activity of the IL-2V moiety in immune cytokine candidates was evaluated using a competitive assay.

[0227] A STAT5 phosphorylation competition assay was performed to determine whether the IL-2V moiety of immune cytokine candidates (IPI-V1, IPI-V5, IPI-V6) could competitively induce STAT-5 phosphorylation in human Treg cells against exogenous IL-2. No significant differences were observed among the tested variants. Figure 7 ).

[0228] Overall, all candidate drugs performed similarly in both assays (i.e., CTLA-4 binding and IL-2V antagonistic activity). No effect of the "facilitating production" mutation was observed.

[0229] - Immune cytokine candidates mediate CTLA-4-dependent CD25 endocytosis

[0230] A specific mechanism of action of immune cytokine candidates has been discovered, namely, they mediate CTLA-4-dependent CD25 endocytosis in primary human Treg cells.

[0231] CTLA-4 is a membrane receptor known to be transported from the cell surface to the cytoplasm, where it undergoes endosomal degradation or can be re-expressed via receptor cycling or de novo expression. This cycling defines a key mechanism of Treg cell inhibition: Treg cells capture the CTLA-4 ligand CD80 from the surface of antigen-presenting cells and clear CD80 through trans-endocytosis and endocytosis-mediated degradation (Qureshi et al, Science, 2011, 332, 600-603). Ipi-V5, which binds to CTLA-4, and anti-CTLA-4 antibodies (ipilimumab) are also readily endocytosed and degraded along with CTLA-4. However, Ipi-V5 binds to both CTLA-4 and CD25, and may therefore drive CD25 endocytosis. To verify whether Ipi-V5 can drive CD25 endocytosis, a confocal microscopy experiment was performed. In this experiment, Treg cells were incubated with either ipilimumab or Ipi-V5 at 37°C for 60 minutes, after which the localization of CD25, Ipi-V5, and ipilimumab was analyzed. Figure 8 ).

[0232] Observations revealed that CD25, originally present only on the plasma membrane of Treg cells, appeared in the cytoplasm after the addition of Ipi-V5; however, CD25 did not appear in the cytoplasm after the addition of the anti-CTLA-4 antibody ipilimumab. The addition of a non-targeting IL-2 mutant protein control molecule (Fc-V5, which contains two IL-2 mutant proteins but lacks anti-CTLA-4 activity) did not alter the localization of CD25. In the cytoplasm, CD25 co-localized with Ipi-V5. These data indicate that Ipi-V5 binds to CTLA-4 and CD25 on the surface of Treg cells, and the Ipi-V5 / CTLA-4 / CD25 complex is subsequently internalized. Unbound by theory, the inventors believe that CD25 endocytosis can be observed using any other immune cytokine derived from any anti-CTLA-4 antibody other than ipilimumab, provided that: anti-CTLA-4 antibodies induce receptor crosslinking and CTLA-4 molecule aggregation through endocytosis, or promote the formation and subsequent endocytosis of clathrin-coated vesicles, or trigger conformational changes in the CTLA-4 cytoplasmic tail (which serves as an internalization signal to recruit endocytosis mechanisms), or the binding of anti-CTLA-4 antibodies disrupts lipid rafts, thereby enhancing the internalization of the ICK-receptor complex.

[0233] This CTLA-4-dependent CD25 endocytosis represents another mechanism of action of Ipi-V5, reducing CD25 expression on the surface of Treg cells and potentially impairing the ability of Tregs to receive IL-2-mediated survival signals. This specific mechanism of action, based on endocytosis and reduced CD25 expression on the Treg surface, is crucial for clearing Tregs and promoting effective antitumor responses.

[0234] - Immunocytokine candidates primarily mediate apoptosis in human Treg cells.

[0235] An assay was performed to evaluate the ability of IPI-V5 to induce apoptosis in expanded human T regulatory cells (eTreg) and expanded conventional T cells (eTconv) after IL-2 amplification. Additionally, to assess marker expression on Treg cells, cells were stained using a panel of antibodies. PBMCs were obtained from fresh blood from healthy donors on day -7 and magnetically separated on a MACS column using CD25-coated beads. The CD25+ fraction was further stained against CD127 and CD25 for sorting on a Beckman Coulter Cytoflex sorter to obtain Treg (CD4+CD8-CD25+CD127lo). Following CD4 selection, Tconv was purified from the CD25- fraction. Cells were cultured for 6 days under activation conditions using anti-CD3 / anti-CD28 beads. One day prior to the start of the assay, beads were removed from Treg and Tconv and incubated for 24 hours in X-vivo medium supplemented with 300 iU / ml Proleukin. On day 0, beads were added to cells and treated with different concentrations of the anti-CTLA-4 antibody ipilimumab or Ipi-V5. Three days later, cells were stained to assess apoptosis (annexin V) and phenotype (Treg markers).

[0236] After 3 days of Ipi-V5 treatment, compared with eTconv, the percentage of connectin V positive cells observed in the membrane of eTreg increased ( Figure 9 A) and increased MFI of annexin V ( Figure 9 B) indicates that Ipi-V5-induced apoptosis mainly occurs in the eTreg cell population. Furthermore, the induction of apoptosis is dose-dependent. The anti-CTLA-4 antibody ipilimumab induced lower levels of apoptosis, which were similar between eTreg and eTconv cells. Apoptosis induced by ipilimumab alone, by the IL-2 mutant protein (Fc-V5) alone, or by a combination of ipilimumab and IL-2 mutant protein (Fc-V5) was lower in eTreg cells than by ipi-V5 alone. Figure 9 E).

[0237] CD25 expression was downregulated in a concentration-dependent manner, and this downregulation was more pronounced in eTreg than in eTconv. Figure 9 C). In contrast, human Fc (which stains the Fc portion of IPI and Ipi-V5) can detect a more stable presence of this compound and indirect CTLA-4 on the cell membrane surface in Ipi-V5-treated eTreg cells. Figure 9 D).

[0238] endocytosis kinetics of immune cytokine candidates

[0239] We performed a second assay to evaluate the kinetics of CD25 and CTLA4 endocytosis following treatment of eTreg and eTconv with Ipi-V5. Briefly, eTreg and eTconv were seeded and treated with three different concentrations of IPI, IPI-V5, Fc-V5, or a combination of IPI and Fc-V5 (at -48 h, -24 h, -4 h, -2 h, and -0.5 h) in the presence of aCD3 / aCD28 beads. Cells were collected at different time points, stained, and analyzed by flow cytometry.

[0240] Compared with Tconv cells and cells treated with the anti-CTLA-4 antibody ipilimumab, Treg cells treated with Ipi-V5 showed decreased expression of surface CD25, highlighting the role of Ipi-V5 in CD25 endocytosis. Figure 10 (Left figure). CTLA-4 undergoes a rapid cycling process on the cell surface. Compared to other treatment groups, the increased CTLA-4 surface expression after Ipi-V5 treatment indicates that CTLA-4 expression on the cell surface was stabilized. Figure 10 (See middle image). Anti-human Fc staining showed that ipilimumab was rapidly lost from the cell membrane, following a CTLA-4 circulation pattern. Ipi-V5 was detected on the cell surface in a dose-dependent manner, with its intensity decreasing slowly over 48 hours, indicating a lower rate of endocytosis than ipilimumab. Figure 10 (See right image).

[0241] Although Ipi-V5 binds to both CD25-expressing activated eTconv and eTreg cells, it only induces apoptosis in Treg cells, accompanied by a decrease in CD25 surface expression. Simultaneously, Ipi-V5 stabilizes CTLA-4 on the plasma membranes of both Treg and Tconv cells, and its endocytic kinetics are slower compared to the anti-CTLA-4 antibody ipilimumab.

[0242] The ability of immune cytokine candidates to reduce the proportion of Tregs in TILs from human NSCLC and BC tumor samples under in vitro conditions

[0243] To investigate the activity of immune cytokine candidates in representative human tumor microenvironments, isolated fresh breast cancer (n=2) and lung cancer (n=4) tumor samples were cultured in vitro for up to 72 hours. Tumor samples were obtained from the Institut Curie in Paris, and all samples were approved by the ethics committee with informed consent from the patients. Isolated samples were cultured individually in culture medium or co-cultured with different concentrations of the anti-CTLA-4 antibody ipilimumab or Ipi-Vx, and then analyzed by flow cytometry. Data showed that the frequencies of Treg cells, Tconv cells, CD8+ T cells, or NK cells did not change at 24 and 48 hours (data not shown). However, at 72 hours, the percentage of Treg cells among total CD3+ T cells significantly decreased with doses ranging from 1 nM (0.09 µg / ml) to 100 nM (9 µg / ml) of Ipi-Vx. Figure 11 In contrast, the number of Tconv (CD4+CD25-), CD8+ T cells, or NK cells did not change significantly. Figure 11 (BD). In samples treated with Ipi-Vx, CD25 expression in Treg cells was significantly reduced, while CD25 levels were undetectable in other cell subsets. Figure 11 This in vitro experiment demonstrated that Ipi-Vx can selectively deplete Treg cells in a representative human tumor microenvironment and showed that in the presence of Ipi-Vx, Treg cells may take up to 3 days to die due to IL-2 deficiency.

[0244] 6. Conclusion

[0245] In vitro characterization of six immunocytokines composed of an anti-CTLA-4 monoclonal antibody (ipilimumab or IPI) and two identical IL-2 variants (IL-2V) molecules bound to it (IL-2V1, IL-2V1_T3A-C125A, IL-2V5, IL-2V5_T3A-C125A, IL-2V6, and IL-2V6_T3A-C125A) showed that all IPI-IL-2Vs retained the biological activity of both components (i.e., the anti-CTLA-4 monoclonal antibody and the IL-2V molecule), including CTLA-4 binding and Treg-specific IL-2R antagonistic activity. However, IPI-V5 exhibited superior pre-CMC performance (higher production titers, higher purity yields, and higher stability) while possessing lower immunogenicity (in silico) and optimal pharmacokinetic parameters (in mice and in vitro).

[0246] Notably, the immunocytokines can induce CD25 endocytosis, a property not present in previously disclosed IL-2 variants, providing an additional mechanism for interfering with CD25-mediated survival signaling in Treg cells. Therefore, compared to using anti-CTLA-4 antibodies or IL-2 mutant proteins alone or in combination, these immunocytokines can selectively and efficiently kill Treg cells. In vitro experiments confirmed that these immunocytokines can selectively deplete Treg cells in a representative human tumor microenvironment. This specific combination of antibody and IL-2 mutant protein in the immunocytokines produces key properties for clearing tumor-associated Treg cells and promoting effective anti-tumor responses. Among the immunocytokines tested, IPI-V5 is a particularly promising candidate for clinical development of novel cancer therapies.

[0247] Sequence Description

[0248] SEQ ID NO: 1 Human IL-2

[0249] SEQ ID NO: 2 Ipilimumab HCDR1

[0250] SEQ ID NO: 3 Ipilimumab HCDR2

[0251] SEQ ID NO: 4 Ipilimumab HCDR3

[0252] SEQ ID NO: 5 Ipilimumab LCDR1

[0253] SEQ ID NO: 6 Ipilimumab LCDR2

[0254] SEQ ID NO: 7 Ipilimumab LCDR3

[0255] SEQ ID NO: 8 Ipilimumab VH

[0256] SEQ ID NO: 9 Ipilimumab VL

[0257] SEQ ID NO: 10 Connector

[0258] SEQ ID NO: 11 IPI-V5 heavy chain

[0259] SEQ ID NO: 12 IPI-V5 Light Chain

[0260] SEQ ID NO: 13 IL-2V1

[0261] SEQ ID NO: 14 IL-2V5

[0262] SEQ ID NO: 15 IL-2V6

[0263] SEQ ID NO: 16 IPI-V5 heavy chain coding sequence

[0264] SEQ ID NO: 17 IPI-V5 light chain coding sequence

[0265] SEQ ID NO: 18 IL-2 V12

[0266] SEQ ID NO: 19 IL-2 V13

[0267] SEQ ID NO: 20 IL-2 V9

[0268] SEQ ID NO: 21 IL-2 V10

[0269] Table 3: Developed anti-CTLA4 antibodies

[0270] (Source: TABS Antibody Database, November 14, 2023)

Claims

1. A fusion protein comprising an anti-CTLA-4 antibody and an IL-2 variant, said IL-2 variant being a regulatory T cell (Treg) specific IL-2 receptor antagonist, comprising substitutions of: K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A, and further comprising substitutions of: (i) S127K, (ii) Y31P and S127K, or (iii) K49Q, E52S, R81E, D84N, T131R and L132S, the positions of which were determined by comparison with human IL-2 (SEQ ID NO: 1), and wherein the N-terminus of said IL-2 variant is fused to the C-terminus of said antibody heavy chain.

2. The fusion protein of claim 1, wherein the regulatory T cell (Treg) specific IL-2 receptor antagonist further comprises a deletion of at least one amino acid selected from S4, S5 or S6, particularly a deletion of one amino acid.

3. The fusion protein according to claim 1 or 2, which induces CD25 endocytosis.

4. The fusion protein according to any one of claims 1 to 3, which induces Treg apoptosis.

5. The fusion protein according to any one of claims 1 to 4, which selectively depletes tumor-associated Tregs.

6. The fusion protein according to any one of claims 1 to 5, wherein the anti-CTLA-4 antibody comprises H-CDR1 of SEQ ID NO: 2, H-CDR2 of SEQ ID NO: 3, H-CDR3 of SEQ ID NO: 4, L-CDR1 of SEQ ID NO: 5, L-CDR2 of SEQ ID NO: 6, and L-CDR3 of SEQ ID NO: 7, or variants thereof, wherein the variants have one or more conserved substitutions on one or more of these CDRs.

7. The fusion protein according to claim 6, wherein the anti-CTLA-4 antibody comprises a VH domain having at least 85% identity with SEQ ID NO: 8 and a VL domain having at least 85% identity with SEQ ID NO:

9.

8. The fusion protein according to any one of claims 1 to 7, wherein the anti-CTLA-4 antibody is ipilimumab, trimemumab, or a functional variant thereof.

9. The fusion protein according to any one of claims 1 to 8, wherein the antibody comprises a human IgG1 Fc domain and / or a human Ig kappa light chain constant domain, or the antibody is a single-domain antibody.

10. The fusion protein according to any one of claims 1 to 9, wherein the IL-2 variant has at least 85% sequence identity with any one of SEQ ID NO: 13 to SEQ ID NO:

15.

11. The fusion protein according to any one of claims 1 to 10, further comprising a linker between the C-terminus of the antibody heavy chain and the N-terminus of the IL-2 variant; preferably, the linker comprises the sequence SEQ ID NO:

10.

12. The fusion protein according to any one of claims 1 to 11, comprising: - An anti-CTLA-4 antibody heavy chain fused to an IL-2 variant, wherein the heavy chain fusion has at least 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 11, and - An anti-CTLA-4 antibody light chain having at least 85%, 90%, 95% or 98% sequence identity with SEQ ID NO:

12.

13. A fusion protein comprising an anti-CTLA-4 antibody and an IL-2 variant, said IL-2 variant being a regulatory T cell (Treg)-specific IL-2 receptor antagonist, said regulatory T cell (Treg)-specific IL-2 receptor antagonist comprising a deletion of an amino acid at a position selected from S4, S5, or S6, and: (a) K9E, L12E, H16R, M23L, N26K, Y31P, K49Q, E52S, R81E, D84N, S87N, V91K, E95K, T131R and L132S replace: or (b) K9E, L12E, H16R, L19R, M23L, N26K, Y31P, K49Q, E52S, R81E, D84N, S87N, E95K, T131R, and L132S shall replace; or (c) Replace with H16R and V91K; or (d) Replaced by H16R, L19R and V91K; The position shown was determined by comparison with human IL-2 (SEQ ID NO: 1), where the N-terminus of the IL-2 variant is fused to the C-terminus of the antibody heavy chain. In particular, the regulatory T cell (Treg) specific IL-2 receptor antagonist has at least 85% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, especially at least 90% sequence identity, and more particularly at least 95% sequence identity.

14. A pharmaceutical composition comprising a therapeutically effective amount of the fusion protein according to any one of claims 1 to 13.

15. The pharmaceutical composition according to claim 14, for treating cancer.

16. The pharmaceutical composition according to claim 15, wherein the cancer is selected from the group consisting of: melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, malignant pleural mesothelioma, esophageal cancer, squamous cell carcinoma of the head and neck, urothelial carcinoma, primary Hodgkin's lymphoma, gastric cancer, large B-cell lymphoma, cervical cancer, Merkel cell carcinoma, endometrial cancer, squamous cell carcinoma of the skin, triple-negative breast cancer, invasive breast cancer, pancreatic adenocarcinoma, thymoma, prostate adenocarcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, and sarcoma.

17. The pharmaceutical composition used according to claim 15 or 16, in combination with at least one immune checkpoint inhibitor, preferably anti-PD-1 and / or anti-PDL-1.

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