Highly effective adoptive T cell therapy

Abstract

The present invention relates generally to the field of anti-cancer and autoimmune therapies, and in particular to the use of adoptive T cell transfer therapy. More specifically, the present invention relates to compositions, pharmaceutical compositions or methods using IL-4, fragments or variants thereof, which are partially fused, for enhancing the effectiveness of anti-cancer immunotherapy or autoimmune therapy.

Description

Technical Field

[0001] The present invention relates generally to the field of cancer therapy and autoimmune therapy, and more particularly to the use of adoptive T cell transfer therapy. More specifically, the present invention relates to a composition, pharmaceutical composition or method using a partially fused IL-4, fragment or variant thereof to enhance the effectiveness of anti-cancer immunotherapy or autoimmune therapy.

Background Art

[0002] Adoptive T cell transfer therapy, which injects ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) or biotechnologically engineered T cells into cancer patients, has proven impressive treatment outcomes in patients with certain subtypes of B cell leukemia, lymphoma, and multiple myeloma in recent years. Nevertheless, the effectiveness of ACT in most solid tumors is severely limited by numerous biophysical and biochemical barriers. Once they enter the tumor tissue and are stimulated by persistent antigens, these injected CD8+ T cells are gradually driven to differentiate into progenitor exhausted CD8+ T cells and terminally exhausted CD8+ T cells. Terminally exhausted CD8+ T cells, characterized by a highly tumor cell-lytic ability, are extremely important for eliminating solid tumor cells, but these subsets tend to die due to survival defects. In contrast to progenitor exhausted T cells that respond to immune checkpoint inhibition therapy, terminally exhausted CD8+ T cells with unique transcriptional and epigenetic features do not respond to most current therapeutic agents.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Therefore, there is still an urgent need for interventions aimed at enhancing the survival and lifespan of terminally exhausted CD8+ T cells in order to increase the effectiveness of ACT against solid tumors.

Means for Solving the Problems

[0004] The present invention relates to a composition, pharmaceutical composition or method using IL-4, a fragment or variant thereof fused to a moiety, for enhancing the effectiveness of anti-cancer immunotherapy or autoimmune therapy.

Brief Description of the Drawings

[0005]

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Mode for Carrying Out the Invention

[0006] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All patent application publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The patent application publications and applications discussed herein are provided only for their disclosures prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0007] In case of conflict, the specification, including definitions, will control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this specification belongs. The following definitions are provided to facilitate understanding of the present invention when used herein.

[0008] The terms "comprise / comprising" are generally used in the sense of include / including, i.e., to allow for the presence of one or more features or components. The terms "comprise(s)" and "comprising" also encompass the more restrictive "consist(s)", "consisting", and "consist / consisting essentially of".

[0009] As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0010] As used herein, "at least one" means "one or more", "two or more", "three or more", etc. For example, one or more structures refer to one structure, two structures, three structures, etc.

[0011] As used herein, the terms "subject" / "subject in need thereof" or "patient" / "patient in need thereof" are well recognized in the art and are used interchangeably herein to refer to mammals including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some cases, the above subject is a subject in need of treatment (treatment) or a subject with a disease or disorder. However, in other embodiments, the above subject can be a healthy subject. This term does not represent a specific age or gender. Therefore, it is intended that both male and female, adult and neonatal subjects are included. Preferably, the subject is a human, and most preferably, a human who may be at risk of developing cancer.

[0012] According to the present invention, the cancer is a solid cancer or a liquid cancer. In one embodiment, the cancer is a solid cancer. Preferably, the solid cancer is selected from the non-limiting group including lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain tumor and skin cancer (melanoma), especially melanoma, or a combination of one or more of these.

[0013] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to polymers of any kind of deoxyribonucleotides (e.g., DNA, cDNA, etc.) or ribonucleotides (e.g., RNA, mRNA, etc.), or polymers of combinations of deoxyribonucleotides and ribonucleotides (such as DNA / RNA, etc.), which are in linear or cyclic configurations and in single-stranded or double-stranded forms. These terms are not construed restrictively with respect to the length of the polymer and can include known analogs of natural nucleotides, as well as nucleotides in which the base, sugar and / or phosphate moieties are modified (e.g., phosphorothioate backbone). Generally, analogs of a particular nucleotide have the same base pairing specificity. For example, an analog of A base pairs with T.

[0014] As used herein, the term "vector" refers to a viral vector, or a nucleic acid (DNA or RNA) molecule such as a plasmid or other vehicle, which contains one or more heterologous nucleic acid sequences of the present invention and is preferably designed for the purpose of transfer (introduction) and / or amplification between different host cells.

[0015] In some embodiments, the vector is an expression vector, a gene delivery vector or a gene therapy vector.

[0016] The terms "expression vector", "gene delivery vector" and "gene therapy vector" refer to any vector effective to incorporate and express one or more nucleic acids of the present invention, preferably under the control of a promoter, into a cell. A cloning vector or an expression vector may include additional elements such as, for example, regulatory elements and post-transcriptional regulatory elements in addition to the promoter.

[0017] As used herein, "at least one (kind)" means "one (kind) or more", "two (kinds) or more", "three (kinds) or more", etc.

[0018] As used herein, interleukin-4 (IL-4) refers to a member of the cytokine family. IL-4 is a compact globular cytokine of 14 kDa stabilized by three internal disulfide bonds. It was first identified as a B cell activating factor in the early 1980s and exhibits many biological and immunomodulatory functions. As an important regulatory factor in humoral and adaptive immunity, IL-4 acts as a lymphocyte growth factor and survival factor, stimulating the proliferation of activated B cells and T cells. In preclinical studies, recombinant non-targeted murine IL-4 as a therapeutic agent showed promising antitumor activity in various cancer mouse models, but only minimal antitumor activity was observed in some clinical trials. Preferably, IL-4 is the human sequence shown in SEQ ID NO: 1, a fragment or variant thereof.

[0019] In one aspect, IL-4 is the mouse sequence shown in SEQ ID NO: 20, a fragment or variant thereof.

[0020] In one aspect, the terms "variant" and "fragment" apply to all sequences of the invention, regardless of the polynucleotide or polypeptide described herein. The term "variant", when referring to IL-4, means, for example, one or more biologically active derivatives of IL-4, preferably the human IL-4 sequence of the invention. Generally, the term "variant" refers to a molecule that has a native sequence and has one or more additions, substitutions (generally, conservative in nature) and / or deletions compared to the native molecule, but whose modification does not destroy its biological activity and is "substantially homologous" to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). Generally, the sequences of such variants have a high degree of sequence homology or identity to the reference sequence, e.g., when the two sequences are aligned, more than 25%, generally more than 50% to 70%, and even more specifically 80% or more, e.g., at least 85%, at least 90% or 95% or more sequence homology or sequence identity.

[0021] As used herein, for example, a "fragment" of IL-4 of the present invention, preferably human IL-4, refers to a sequence containing fewer amino acids or nucleotides in length than the respective polypeptide sequence or nucleic acid sequence. Preferably, this sequence contains less than 90%, preferably less than 60%, particularly less than 30% amino acids or nucleotides in length than the respective polypeptide sequence or nucleic acid sequence (SEQ ID NO: 1).

[0022] While focusing on the development of a novel and efficient approach for treating cancer using CAR-engineered T cells, the inventors have surprisingly shown that IL-4 fused to a moiety that extends the half-life of IL-4 and / or stabilizes IL-4 significantly increases the antitumor efficacy of anti-cancer immunotherapies such as ACT in a mouse model bearing multiple syngeneic solid tumors. These results indicate the great potential of IL-4 fused to a moiety that promotes the efficacy of anti-cancer immunotherapy.

[0023] Accordingly, the present invention provides a composition and a pharmaceutical composition, i) an interleukin-4 polypeptide (IL-4) fused to a moiety, a fragment or a variant thereof, ii) an anti-cancer immunotherapy comprising a pharmaceutically acceptable carrier, diluent and / or excipient, wherein the moiety is a molecule that extends the half-life of the IL-4, its fragment or variant, and / or stabilizes the IL-4, its fragment or variant and provides a composition and a pharmaceutical composition.

[0024] A pharmaceutical composition for use in the treatment and / or prevention of a disease in a subject in need thereof, i) an interleukin-4 polypeptide (IL-4) fused to a moiety, a fragment or a variant thereof, ii) an anti-cancer immunotherapy comprising, wherein the moiety is a molecule that extends the half-life of the IL-4, its fragment or variant, and / or stabilizes the IL-4, its fragment or variant A pharmaceutical composition is also provided.

[0025] In one aspect of the invention, the disease is selected from the group comprising solid cancer, liquid cancer, and autoimmune diseases.

[0026] Solid cancer is selected from the non-limiting group comprising lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, and brain tumors, and skin cancer, particularly melanoma, or combinations of one or more of these.

[0027] Autoimmune diseases are selected from the non-limiting group comprising rheumatoid arthritis (RA), multiple sclerosis (MS), endometriosis, inflammatory bowel disease (IBD), psoriasis, and psoriatic arthritis.

[0028] As used herein, the terms "subject" / "subject in need thereof" or "patient" / "patient in need thereof" are well recognized in the art and are used interchangeably herein to refer to mammals including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, most preferably humans. In some cases, the subject is a subject in need of treatment or a subject having a disease or disorder. However, in other aspects, the subject can be a healthy subject. This term does not denote a particular age or gender. Thus, it is intended to encompass male and female, adult and neonatal subjects. Preferably, the subject is human, most preferably a human who may be at risk of developing cancer or an autoimmune disease.

[0029] In one embodiment, the moiety is selected from the group consisting of polyethylene glycol (PEG), natural polysaccharides and semi-synthetic polysaccharides such as O-linked oligosaccharides and N-linked oligosaccharides, dextran, hydroxyethyl starch (HES), polysialic acid and hyaluronic acid, albumin (e.g., human serum albumin (HSA)), an antibody or fragment thereof, protein polymers such as homoamino acid polymers, elastin-like polypeptides, XTEN and PAS. In a preferred embodiment, the moiety is selected from the group consisting of albumin (e.g., HAS shown in SEQ ID NOs: 17-19) and an antibody or fragment thereof.

[0030] As shown in the exemplary sequences, the moiety may be covalently fused to the IL-4 polypeptide, fragment or variant thereof directly, or via a linker or indirectly by a linker, at the C-terminus or N-terminus (e.g., moiety-IL-4 or IL-4-moiety). In one embodiment, the moiety can be covalently fused to the IL-4 polypeptide, fragment or variant thereof at the N-terminus or C-terminus by a polypeptide linker.

[0031] As used herein, "antibody" is a protein molecule that reacts with a specific antigenic determinant or epitope and belongs to one or five different classes based on structural characteristics: IgA, IgD, IgE, IgG and IgM. The antibody may be a polyclonal antibody (e.g., polyclonal serum) or a monoclonal antibody, examples of which include, but are not limited to, fully assembled antibodies, single-chain antibodies, antibody fragments, and chimeric antibodies, humanized antibodies, provided that these molecules are still biologically active and still need to bind to at least one peptide of the present invention.

[0032] Typical antibodies are composed of two immunoglobulin (Ig) heavy chains and two Ig light chains. There are several different types of heavy chains, which define the class or isotype of the antibody. These heavy chain types vary between different animals. All heavy chains contain a series of immunoglobulin domains and usually have one variable (VH) domain, which is important for antigen binding, and several constant (CH) domains. Each light chain is composed of two consecutive (tandem) immunoglobulin domains, namely one constant (CL) domain and one variable domain (VL) important for antigen binding.

[0033] An "antigen fragment" includes a part of a full-length antibody. Examples of fragments include Fab, Fc, Fab', F(ab')2, and Fv fragments, diabodies, minibodies, nanobodies, linear antibodies (Zapata et al. (1995) Protein Eng. 8(10):1057-1062), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Preferably, the antibody fragment is the Fc domain of IgG, preferably the silent Fc domain of immunoglobulin (Ig) G, most preferably the silent Fc domain of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain of human IgG is selected from the group consisting of IgG1 Fc (SEQ ID NO: 2), IgG2 Fc (SEQ ID NO: 8), IgG3 Fc (SEQ ID NO: 11), and IgG4 Fc (SEQ ID NO: 14), sequences comprising fragments, variants, or combinations of one or more of these sequences. In one embodiment, the Fc domain is a mouse IgG Fc domain selected from the group consisting of sequences comprising the mouse IgG Fc (SEQ ID NO: 21), fragments or variants thereof.

[0034] Typically, IL-4, its fragments or variants are covalently fused directly or indirectly, via a linker, preferably a polypeptide linker, to the N-terminus or C-terminus of the Fc domain. In one embodiment, this polypeptide linker consists essentially of a stretch of Gly and Ser residues (a "GS" linker, such as (GGS)n or (GGGGS)n, where n is between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), for example, those shown in SEQ ID NO: 4 or SEQ ID NO: 5.

[0035] Any anti-cancer immunotherapy known in the art can be suitable for the present invention. In one embodiment of the present invention, the anti-cancer immunotherapy is selected from the group consisting of adoptive T cell transfer therapy (ACT), immune checkpoint inhibition therapy, cytokine therapy, cancer vaccine therapy, bispecific antibody therapy and other cancer immunotherapies, or combinations of one or more of these.

[0036] ACT is a type of immunotherapy in which T cells are given to a patient to help the body fight a disease, such as cancer. In cancer treatment, T cells are usually taken from the patient's own blood or tumor tissue, grown in large numbers in the laboratory, and then returned to the patient to help the immune system fight the cancer. Sometimes, the T cells are modified in the laboratory so that they can better target the patient's cancer cells and kill them. Non-limiting types of adoptive cell transfer include T cell receptor (TCR)-based adoptive therapy (TCR-T), chimeric antigen receptor T cell (CAR-T) therapy, tumor infiltrating lymphocytes (TIL) and natural killer (NK) cell therapy, CAR-NK cells, CAR-NKT cells, TCR-transgenic NK cells, TCR-transgenic NK-T cells, CAR-macrophages or any synthetic tumor-specific immune cells, or combinations of one or more of these. ACT is also called adoptive cell therapy, cellular adoptive immunotherapy, and T cell transfer therapy.

[0037] Preferably, in immune checkpoint inhibition therapy (ICBT), the inhibitor is selected from the non-limiting group consisting of or comprising a CTLA-4 inhibitor, a TIM3 inhibitor, a TIGIT inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor or a combination of one or more thereof, such as a PD-1 / PD-L1 inhibitor or a TIM3 / PD-1 / PD-L1 inhibitor.

[0038] Non-limiting examples of PD-1 inhibitors include nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, dostarlimab, and atezolizumab.

[0039] Non-limiting examples of PD-L1 inhibitors include atezolizumab, avelumab, AMP-224, MEDI-0680, RG-7446, GX-P2, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014, CX-072, and BMS-936559.

[0040] Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy®) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly ticilimumab, CP-675,206).

[0041] An example of a TIM3 inhibitor is cobolimab.

[0042] As shown in the examples, the pharmaceutical composition of the present invention enhances the efficacy of anti-cancer therapy, particularly immunotherapy, by enhancing the survival and lifespan (and thus their number) of T cell effectors to enhance the effectiveness of immunotherapy. In one aspect, the pharmaceutical composition of the present invention specifically enriches tumor-specific PD-1+TIM-3+CD8+ T cells in the tumor microenvironment by enhancing the survival and lifespan of terminally exhausted T cells and also rescuing them from necrosis and extending their survival.

[0043] In one aspect, a pharmaceutical composition comprising IL-4, a fragment or variant thereof fused to a moiety of the present invention increases the effectiveness of anti-cancer immunotherapy by an increase of about 2% or more, about 5% or more, about 20% or more, about 40% or more, about 60% or more, about 500% or more compared to the effectiveness of anti-cancer therapy in the absence of IL-4, a fragment or variant thereof fused to the moiety.

[0044] The present invention further contemplates methods for treating and / or preventing diseases.

[0045] The term "treatment" or "treating" means any administration to a subject for the purpose of (i) inhibiting a disease, i.e., arresting the progression of clinical symptoms, and / or (ii) alleviating the disease, i.e., causing regression of clinical symptoms, of compositions, pharmaceutical compositions, therapeutic agents, compounds, combinations of compounds, etc. of the present disclosure. (i) inhibiting a disease, i.e., arresting the progression of clinical symptoms, and / or (ii) alleviating the disease, i.e., causing regression of clinical symptoms of the present disclosure.

[0046] As used herein, the term "prevention" or "preventing" means any administration to a subject for the purpose of (i) preventing a disease, i.e., preventing the onset of clinical symptoms of the disease, of compositions, pharmaceutical compositions, therapeutic agents, compounds, combinations of compounds, etc. of the present disclosure. (i) preventing a disease, i.e., preventing the onset of clinical symptoms of the disease of the present disclosure.

[0047] In the context of the present invention, the disease is a cancer or an autoimmune disease as described herein.

[0048] In one aspect, the present invention is a method for treating and / or preventing cancer or an autoimmune disease, the method comprising administering to a subject in need thereof: i) a therapeutically effective amount of at least one IL-4, fragment or variant thereof fused to a moiety that is a molecule that extends the half-life of said IL-4, fragment or variant or stabilizes said IL-4, fragment or variant; and ii) simultaneously, concurrently or sequentially, a therapeutically effective amount of an anti-cancer therapy or an autoimmune disease therapy.

[0049] In one aspect, a method for treating and / or preventing cancer or an autoimmune disease in a subject in need thereof comprises: (i) removing and isolating immune cells, preferably native T cells, from said patient or subject; (ii) genetically engineering said T cells with one or more recombinant constructs encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor targeting motif or antigen; (iii) ex vivo expanding said T cells into a larger engineered T cell population; and (iv) reintroducing said engineered T cells into said subject in need thereof, simultaneously, concurrently or sequentially with a therapeutically effective amount of at least one IL-4, fragment or variant thereof fused to a moiety that is a molecule that extends the half-life of said IL-4, fragment or variant or stabilizes said IL-4, fragment or variant.

[0050] In one aspect, the method for treating and / or preventing the cancer or autoimmune disease comprises: (i) removing and isolating immune cells, preferably natural T cells, from the patient or subject, or providing immune cells, preferably natural T cells; (ii) genetically engineering the T cells with at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor targeting motif or antigen; (iii) expanding ex vivo into a larger population of engineered T cells; (iv) reintroducing the engineered T cells into the patient or subject; and (iv) reintroducing the engineered T cells into the subject in need thereof, simultaneously, concurrently, or sequentially, with at least one IL-4, fragment, or variant thereof fused to a moiety that extends the half-life of the IL-4, fragment, or variant, or stabilizes the IL-4, fragment, or variant, wherein the moiety is a molecule that extends the half-life of the IL-4, fragment, or variant, or stabilizes the IL-4, fragment, or variant.

[0051] In one aspect, the method for treating and / or preventing the cancer or autoimmune disease comprises: (i) removing and isolating immune cells, preferably natural TIL cells, from the patient or subject, or providing immune cells, preferably natural TIL cells; (ii) expanding ex vivo into a larger population of TIL cells; and (iii) reintroducing the TIL cells into the subject in need thereof, simultaneously, concurrently, or sequentially, with at least one IL-4, fragment, or variant thereof fused to a moiety that extends the half-life of the IL-4, fragment, or variant, or stabilizes the IL-4, fragment, or variant, wherein the moiety is a molecule that extends the half-life of the IL-4, fragment, or variant, or stabilizes the IL-4, fragment, or variant.

[0052] In one aspect, the method for treating and / or preventing cancer or autoimmune disease in the above subject comprises administering the pharmaceutical composition of the present invention to a subject in need thereof.

[0053] In one aspect, a therapeutically effective amount of the fusion IL-4 and a therapeutically effective amount of an anti-cancer therapy or an autoimmune disease therapy are administered, to a greater or lesser extent, simultaneously, concurrently, or sequentially, i.e., co-administered.

[0054] In another aspect, a therapeutically effective amount of the fusion IL-4 and a therapeutically effective amount of an anti-cancer therapy or an autoimmune disease therapy are continuous.

[0055] In another example, a first pharmaceutical composition is typically administered first, and then a second pharmaceutical composition is typically administered within several weeks (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 10 weeks, about 20 weeks, about 30 weeks). In this case, the administration of the first pharmaceutical composition is stopped, for example, usually after 24 to 36 weeks when one or more therapeutic effects are detected, but the second composition continues to be administered.

[0056] In one aspect, the above-described method of treatment and / or prevention can further comprise administering at least one additional therapeutic agent or therapy, preferably an anti-cancer agent or anti-cancer therapy (i.e., when the disease is cancer), more preferably a therapeutically effective amount or therapeutically effective dosage of an anti-cancer agent or anti-cancer therapy. The one or more anti-cancer agents or anti-cancer therapies are selected from the non-limiting group comprising radiation therapy, chemotherapy, immune checkpoint inhibitors, immunotherapy, and hormone therapy, or a combination of one or more of these.

[0057] As used herein, the term "therapeutically effective amount" means an amount of an agent, therapy, compound, or combination of compounds that is high enough to significantly modify the symptoms and / or condition being treated in a favorable direction within the scope of sound medical judgment, but low enough to avoid serious side effects (with a reasonable risk / benefit ratio).

[0058] The present invention further contemplates an IL-4 fused to a moiety that extends the half-life of IL-4 or stabilizes IL-4, selected from the group consisting of, or comprising, the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22, fragments of any of these sequences, or variants thereof.

[0059] The present invention also contemplates a nucleic acid sequence encoding one or more IL-4s fused to a moiety that extends the half-life of IL-4 or stabilizes IL-4, as described herein. In a preferred embodiment, this nucleic acid sequence encodes one or more IL-4s fused to a moiety shown by the amino acid sequences selected from SEQ ID NO: 6, 7, 9, 10, 12, 13, 15, 16, 18 and 19.

[0060] The present invention further provides a plasmid or vector comprising a nucleic acid sequence encoding one or more recombinant constructs as described herein.

[0061] Any vector known in the art can be suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector (such as pMSGV, etc.), a DNA vector, a murine leukemia virus vector, an SFG vector, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus vector (AAV), a lentiviral vector (such as pGAR, etc.), or any combination thereof.

[0062] The present invention further provides a method for promoting tumor regression mediated by ACT, which comprises administering, simultaneously, concurrently or sequentially with ACT administration, at least one IL-4, fragment or variant thereof fused to a moiety in a therapeutically effective amount, wherein the moiety is a molecule that extends the half-life of the IL-4, fragment or variant thereof or stabilizes the IL-4, fragment or variant thereof.

[0063] The invention described herein may be subject to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications as long as they do not depart from the spirit or essential characteristics thereof. The invention individually or collectively includes all of the processes, features, compositions and compounds referred to or shown herein, as well as any and all combinations or any two or more of the above processes or features. Therefore, the present disclosure should be considered in all aspects to be illustrative and not restrictive, and the scope of the invention is indicated by the appended claims, and all changes falling within the meaning and scope of the equivalents are intended to be included therein. Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety. The above description will be more fully understood with reference to the following examples.

Examples

[0064] Materials and Methods Mice. Female CD45.2, 6 - 8 weeks old + Thy1.2 +C57BL / 6 (C57BL / 6J) mice were purchased from Charles River Laboratories (Lyon, France). TCR transgenic Thy1.1+ pmel-1 (PMEL) mice (B6.Cg-Thy1a / Cy Tg(TcraTcrb)8Rest / J) and TCR transgenic OT-I mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / J) were originally purchased from the Jackson Laboratory. All mice were maintained in the animal facility of the Center of PhenoGenomics at the Ecole Polytechnique Federale de Lausanne (EPFL). Experimental procedures in mouse research were approved by the Swiss authorities (Canton of Vaud, animal protocol IDs 3206 and 3533) and were conducted according to the guidelines from the Center of PhenoGenomics at EPFL.

[0065] Human blood samples. All human blood samples (prepared as buffy coats) were purchased from Interregional Blood Transfusion SRC Ltd. with informed consent from anonymous healthy donors and were genetically engineered with ethical approval from the Federal Office of Public Health, Switzerland (Notification 4182209 / 2).

[0066] Cells and tumor models. B16F10 melanoma cells were originally obtained from the American Type Culture Collection. The YUMM1.7-OVA mouse melanoma cell line and the HER2-transduced MC38 mouse colon cancer cell line (MC38-HER2) were generated as previously reported. All mouse tumor cells were cultured in DMEM (Gibco / Thermo Fisher Scientific) supplemented with complete DMEM, fetal bovine serum (FBS) (10% v / v, Gibco / Thermo Fisher Scientific), HEPES (pH 7.2 - 7.5, 1% v / v, Gibco / Thermo Fisher Scientific), and penicillin / streptomycin (1% v / v, Gibco / Thermo Fisher Scientific). B16F10, YUMM1.7-OVA, or MC38-HER2 tumor cells (5×10 5 or 1×10 6 or as indicated) were subcutaneously implanted into the right flank of Thy1.2 + C57BL / 6 WT mice to establish syngeneic tumor models.

[0067] Production of mouse Fc-IL-4 and human Fc-IL-4 proteins. Both mouse Fc-IL-4 and human Fc-IL-4 fusions were expressed by FreeStyle 293-F cells (Gibco / Thermo Fisher Scientific) at the EPFL Protein Expression Core Facility (Protein Expression Core Facility). First, the supernatant of the culture medium containing the recombinant protein was filtered through a 0.22 μm membrane to obtain a clear solution. This recombinant protein was captured on a HiTrap protein A affinity chromatography column in an AKTA pure 25 (GE Healthcare) and eluted with an elution buffer (0.05 M sodium citrate, 0.3 M sodium chloride, pH 3.0). The eluted protein was immediately recovered in a neutralization buffer (1 M Tris-HCl, pH 10.0) and then concentrated by tangential flow ultrafiltration (molecular weight cut-off 10 kDa) in a Vivaspin (GE Healthcare). The concentrated protein solution was further purified on a Superdex 200 Increase size exclusion chromatography column (GE Healthcare). The purified protein was aliquoted and stored at -80 °C until use. The purity of the recombinant protein was confirmed by SDS-PAGE.

[0068] PMEL, WT OT-I, and human CD8 +Preparation of T cells. Spleens from PMEL or OT-I mice were mechanically meshed through a 70 μm strainer (Fisher Scientific). Red blood cells (RBC) were lysed with ACK lysis buffer (2 ml per spleen, Gibco / Thermo Fisher Scientific) for 3 minutes at room temperature. After washing twice with cold PBS (Gibco / Thermo Fisher Scientific), the splenocytes were then resuspended in complete RPMI medium ((RPMI-1640) (Gibco), FBS (10% v / v), HEPES (pH 7.2 - 7.5, 1% v / v), penicillin / streptomycin (1% v / v), sodium pyruvate (1% v / v, Gibco / Thermo Fisher Scientific) and 2-mercaptoethanol (0.1% v / v, Gibco / Thermo Fisher Scientific)) supplemented with mouse IL-2 (10 ng / ml) and IL-7 (1 ng / ml, PeproTech), and either human gp10025-33 or OVA257-264 peptide (1 μM, GenScript) for PMEL or OT-I T cells, respectively, at a cell density of 2×10 6 / ml. After 3 days of culture, viable cells were enriched by density gradient centrifugation over Ficoll-Paque PLUS (GE Healthcare), and then 0.5 - 1.0×10 6Cultured for an additional 2 days at a cell density of / ml to obtain activated CD8+ T cells with a purity of >95% (flow cytometry analysis). Human peripheral blood mononuclear cells (prepared as buffy coats) from anonymous healthy donors were activated in vitro for 2 days in the presence of human IL-2 (10 ng / ml) using coated anti-human CD3 (2 μg / ml, OKT3, BioLegend) and CD28 (2 μg / ml, CD28.2, BioLegend) antibodies. Activated human CD8+ T cells were isolated by Ficoll density gradient separation and magnetic-activated cell sorting (MACS) using a human CD8+ T cell isolation kit (Miltenyi Biotec) for in vitro assays.

[0069] Preparation of mouse CAR-T cells. For the preparation of mouse CAR-T cells, the calcium phosphate method was used to transfect Phoenix-Eco cells with the HER2 CAR-bearing plasmid and the pCL-Eco packaging plasmid. The virus-containing supernatant was collected every 24 hours after transfection. Spleen cells derived from WT mice were stimulated for 1 day with mouse T activator CD3 / CD28 Dynabeads and then transduced with the virus-containing supernatant using the spin transduction method. Briefly, the virus-containing supernatant was dispensed into non-tissue culture-treated 6-well plates and centrifuged at 2,000 g for 2 hours at 32 °C. The 6-well plates were first coated with protamine (10 μg / ml, Sigma-Aldrich) overnight at 4 °C and then blocked with PBS containing FBS (v / v 1%) for 20 minutes before use. Activated T cells suspended in T cell culture medium were added to the virus-loaded 6-well plates immediately after aspirating the supernatant and centrifuged at 2,000 g for 0.5 hour at 32 °C. The cells were further passaged every 24 hours for an additional 4 days before use. Forty-eight hours after transduction, the transduction efficiency was determined by staining the surface HER2 CAR using biotinylated human HER2 / ErbB2 Protein (Acro Biosystems) and avidin-Alexa Fluor 488 conjugate (Invitrogen / Thermo Fisher Scientific). Non-transduced T cells activated by Dynabeads were used as a control.

[0070] Analysis of tumor-infiltrating immune cells. B16F10 tumor-bearing thy1.2 + C57BL / 6 mice were PMEL CD8+ T cells (5×10 6) were subjected to i.v. adoptive transfer, followed by four doses of Fc-IL-4 (20 μg) or PBS control (or as indicated) given every other day by peritumoral (p.t.) injection. For the BrdU experiment, mice were administered BrdU (1 mg, Sigma-Aldrich) by i.p. injection 1 day prior to tumor harvest. After the above treatments, tumors were harvested, weighed, minced mechanically, and stirred at 37 °C for 60 min at 1,000 r.p.m. in RPMI-1640 medium containing collagenase type IV (1 mg / ml, Gibco / Thermo Fisher Scientific), dispase II (100 μg / ml, Sigma-Aldrich), hyaluronidase (100 μg / ml, Sigma-Aldrich), and DNaseI (100 μg / ml, Sigma-Aldrich) for digestion. RBC lysis was performed on the digested tumor samples using ACK lysis buffer. Tumor-infiltrating leukocytes were then enriched (concentrated) by density gradient centrifugation on Percoll (GE Healthcare), resuspended in PBS containing BSA albumin (0.2%, w / v, Sigma-Aldrich), stained with the indicated antibodies, and analyzed by flow cytometry.

[0071] Flow cytometry analysis. For surface marker staining, cells collected in a U-bottom 96-well plate were first blocked with anti-mouse CD16 / 32 antibody (BioLegend), incubated with the indicated antibodies for 20 minutes at 4°C, and subsequently subjected to live / dead staining with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) or Zombie Aqua Fixable Dye (BioLegend). The cells were then washed with PBS containing BSA (0.2%, w / v) and resuspended in the same buffer for flow cytometry analysis. For intracellular staining, cells were first stained for surface markers and Aqua Fixable Dye as described above. Next, the cells were fixed and permeabilized using the Cytofix / Cytoperm Fixation / Permeabilization Solution Kit (BD Biosciences) for cytokine staining or the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) for BrdU, active caspase-3, and transcription factor staining according to the manufacturer's instructions, and then incubated with the indicated antibodies for intracellular staining. For intracellular cytokine staining, cells were first stimulated with Cell Stimulation Cocktail (Invitrogen / Thermo Fisher Scientific, containing a protein transport inhibitor) at 37°C for 4 - 6 hours. The cells were then processed as described above for surface marker staining and intracellular staining. Data were collected using an Attune NxT Flow Cytometer (Invitrogen / Thermal Fischer Scientific) equipped with Attune NxT Software v.3. Analysis was performed using FlowJo 10.6.1 (Tree Star). Gate margins were determined by isotype controls and fluorescence minus one controls (controls).

[0072] Antibodies and reagents for flow cytometry. The following antibodies or staining reagents were purchased from BioLegend: CD16 / 32 (93, 101302), Thy1.1 (OX-7, 202529), Thy1.2 (30-H12, 105343), CD45.1 (A20, B218971), CD45.2 (104, 109814), CD8α (53-6.7, 100714), CD8β (YTS256.7.7, 126606), CD4 (RM4-5, 100526), NK1.1 (PK136, 108740), F4 / 80 (BM8, 123108), CD3ε (17A2, 100306), CD19 (6D5, 115520), CD44 (IM7, 103006), CD11c (N418, 117348), I-A / I-E (MHC-II, M5 / 114.15.2, 107643), Siglec-F (S17007L, 155508), CD80 (16-10A1, 104734), CD86 (GL-1, 105006), Foxp3 (MF-14, 126406), CD11b (M1 / 70, 101228), Ki67 (16A8, 652424), BrdU (3D4, 364104), Granzyme B (GB11, 515403), IFN-γ (XMG1.2, 505826), TNF-α (MP6-XT22, 506308), IL-2 (JES6-5H4, 503822), IL-10Rα (1B1.3a, 112705), CD69 (H1.2F3, 104512), Gr-1 (RB6-8C5, 202519), PD-1 (29F.1A12, 135216), TIM-3 (RMT3-23, 119706). TCF-1 (C63D9, 2203S) was obtained from Cell Signaling Technology. Active caspase-3 (C92-605) was obtained from BD Biosciences. Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (A-11008), MitoTracker Green FM, MitoTracker Deep Red FM, and MitoSOX Red mitochondrial superoxide indicator were obtained from Thermo Fisher Scientific.

[0073] In vitro restimulation of CD8+ T cells. Activated PMEL CD8 at the resting stage (day 7 of culture) + T cells were restimulated for 2 days with a dimerized anti-CD3 antibody (prepared by mixing anti-CD3 antibody (17A2, Bio X Cell) with goat anti-rat IgG (Invitrogen / Thermo Fisher Scientific) at a molar ratio of 2:1) in complete RPMI medium containing IL-2 (10 ng / ml). The recovered cells were phenotyped by flow cytometry based on the expression levels of surface inhibitory receptors (PD-1 and TIM-3). Live CD8 + T cells were analyzed by flow cytometry or isolated by Ficoll density gradient separation for the Seahorse assay. PD-1 + TIM-3 + CD8 + T cell subsets were sorted for in vitro co-culture assays or Seahorse assays.

[0074] In vitro co-culture of T cells and tumor cells. B16F10 tumor cells were cultured in the above complete DMEM. B16F10 tumor cells (0.16 M in 2 ml complete DMEM medium) were seeded in each well of a 6-well plate at 37 °C overnight. After aspiration of the tumor culture medium, activated PMEL CD8 at the resting stage + T cells were added to the tumor cell culture at a T cell / tumor cell ratio of 1:2. After co-culturing for another 2 days, all cells were recovered for flow cytometry analysis. To determine the lysis of target cells, the viability of tumor cells from the co-culture was measured by DAPI staining and flow cytometry.

[0075] Seahorse assay. To measure the OCR and ECAR of T cells, the Seahorse assay was performed. Sorted subsets of Pan CD8+ T cells or mouse CD8+ T cells under different treatment conditions (3×10 5The ( / well) was seeded at 37°C for 40 minutes in a Seahorse culture plate (Seahorse Bioscience) in a non-CO2 incubator. OCR and ECAR were measured by an XF96 Seahorse Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer's instructions. During the Seahorse assay, the cells were treated with oligomycin (1 μM, Sigma-Aldrich), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 2 μM, Sigma-Aldrich), rotenone (0.5 μM, Sigma-Aldrich), antimycin A (0.5 μM, Sigma-Aldrich), glucose (10 mM, Sigma-Aldrich), and 2-DG (50 mM, Sigma-Aldrich). Each condition was performed in 3 - 6 replicates in one experiment.

[0076] Metabolic inhibitor treatment. Activated PMEL CD8+ T cells at the above-mentioned resting stage (day 7 of culture) were restimulated for 2 days with a dimerized anti-CD3 antibody in complete RPMI medium containing IL-2 (10 ng / ml) and the indicated inhibitors (SF-31, 5 μM; 3-BP 10 μM; sodium fluoride, 1 mM; LDHAi, 16 μM; 6-AN, 5 mM; oligomycin, 1 μM; etomoxir (Sigma-Aldrich), 200 μM; BPTES (Sigma-Aldrich), 20 μM; and UK5099 (Sigma-Aldrich), 100 μM) in the presence or absence of Fc-IL-4 (20 ng / ml). The number of viable CD8+ T cells was determined by flow cytometry analysis.

[0077] Antitumor therapy and re-challenge experiment. Mice bearing established tumors with an area of approximately 25 - 60 mm 2 (on day 6 after inoculation or as indicated) were treated with activated PMEL T cells, OT-I T cells, or HER2 CAR T cells (5×10 6Treated by adoptive transfer (as shown), and then, starting on day 6, Fc-IL-4 (20 μg) or PBS control was administered p.t. every other day (or as shown) (a total of 4 or 8 doses as shown). Mice receiving only PBS control, only Fc-IL-4, or p.t. administration of non-transduced T cells were used as controls. Tumor area and body weight were measured every other day. Tumor area was calculated from caliper measurements of two orthogonal diameters using the formula area = length × width. Mice were euthanized when weight loss exceeded 15% of the pre-treatment weight, when the tumor area reached 150 mm 2 (as a predetermined endpoint), or when the animal became moribund. In the tumor cell re-challenge experiment, B16F10 (1 × 10 5 ), YUMM1.7-OVA (5 × 10 5 ), or MC38-HER2 (5 × 10 5 ) cells were re-transplanted subcutaneously into the left flank of surviving mice from the treatment group on day 90 after the primary inoculation. The same number of tumor cells as the control was subcutaneously inoculated into age-matched naive WT mice. The survival of the re-challenged mice was monitored for at least an additional 60 days.

[0078] In vivo immune cell depletion study. Mice (day 6 after inoculation) bearing established B16F10 tumors with an area of approximately 25 mm 2 were injected with activated PMEL T cells (5 × 10 6) were treated with adoptive transfer and then, starting on day 6, Fc-IL-4 (20 μg) or PBS control was administered p.t. every other day (or as indicated) (a total of 4 or 8 doses as indicated). One day before this treatment, mice were injected i.p. with anti-CD8 (YTS169.4, BioXcell, 400 μg / mouse), anti-CD4 (YTS177, BioXcell, 400 μg / mouse), anti-NK1.1 (PK136, BioXcell, 400 μg / mouse), anti-IL5 (TRFK5, BioXcell, 1 mg / mouse), anti-Ly6G (NIMP-R14, BioXcell, 400 μg / mouse) or anti-IgG (LTF-2, BioXcell, 400 μg / mouse) and another 2 injections were given during the treatment to deplete the corresponding immune cells. Tumor area and body weight were measured every other day. Tumor area was calculated from caliper measurements of two orthogonal diameters using the formula area = length × width. Mouse survival was monitored for at least 60 days.

[0079] Combination therapy of anti-TIM-3 and Fc-IL-4. Mice (on day 6 after inoculation) bearing established MC38-HER2 tumors with an area of approximately 20 mm 2 were treated with an i.p. injection of anti-TIM3 (RMT3-23, BioXcell, 100 μg / mouse) and then, starting on day 6, Fc-IL-4 (20 μg) or PBS control was administered p.t. every other day (or as indicated) (a total of 4 or 8 doses as indicated). Tumor area and body weight were measured every other day. Tumor area was calculated from caliper measurements of two orthogonal diameters using the formula area = length × width. Mouse survival was monitored for at least 60 days.

[0080] Immunoblot analysis. Cultured PMEL CD8+ T cells (day 7) treated with Fc-IL-4 were harvested and lysed in RIPA lysis buffer (Cell Signaling Technology) containing 1× protease inhibitor cocktail (Cell Signaling Technology). Prior to electrophoresis, whole cell lysates were diluted with 4× protein sample loading buffer (Invitrogen / Thermo Fisher Scientific) containing 1× reducing reagent (Invitrogen / Thermo Fisher Scientific) and incubated at 95 °C for 10 minutes. Electrophoresis of proteins from whole cell lysates was performed using a NuPAGE electrophoresis system (Invitrogen / Thermo Fisher Scientific). The proteins were then transferred to a PVDF membrane (Invitrogen / Thermo Fisher Scientific) at 18 V for 60 minutes using a Trans-Blot SD semidry transfer cell (Bio-Rad). The PVDF membrane was blocked with 5% w / v non-fat dry milk (Sigma-Aldrich) and then incubated overnight at 4 °C with the indicated primary antibodies. After incubation with horseradish peroxidase (HRP)-conjugated antibodies, bands were visualized using SignalFire ECL reagent (Cell Signaling Technology). The primary antibody for Glut1 (number 73015, Cell Signaling Technology) and the secondary antibody for HRP-conjugated anti-rabbit IgG (7074S, 1:3,000) were obtained from Cell Signaling Technology. The HRP-conjugated β-actin monoclonal antibody (BA3R, MA5-15739-HRP, 1:2,000) was obtained from Invitrogen / Thermo Fisher Scientific.

[0081] Statistical analysis. Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software). Data are presented as mean ± s.e.m. unless otherwise stated. Comparison between two groups was done by using two-sided unpaired Student's t-test. Survival data were analyzed using the log-rank test. A statistically significant difference was not considered if the P-value was greater than 0.05.

[0082] Results Considering that the factor IL-4 acts as a lymphocyte growth factor and survival factor, the inventors first attempted to study whether IL-4 could maintain the survival of terminally exhausted CD8+ T cells. To extend the half-life of IL-4, recombinant mouse IL-4 (SEQ ID NO: 20 - mouse IL-4) and mutant IgG1 Fc fusion protein (Fc-IL-4, SEQ ID NO: 22 - mouse Fc-IL-4) with comparable biological activity were designed and obtained (Figure 1a, b). Pharmacokinetic studies revealed that Fc-IL-4 had an increased circulating half-life of 6.31 hours compared to the 0.2-hour circulating half-life of IL-4 (Figure 1c).

[0083] Terminally exhausted CD8+ T cells were sorted from a subset mixture and restimulated with anti-CD3 in the presence or absence of Fc-IL-4 (Figure 2a). Fc-IL-4 significantly increased both the cell viability and cell number of terminally exhausted CD8+ T cells (Figure 2b, c). The apoptosis rate was significantly decreased (Figure 2d). Thus, the inventors demonstrated that Fc-IL-4 could promote the survival of terminally exhausted CD8+ T cells by reducing apoptosis. To further confirm the above results in vivo, activated Thy1.1 PMEL-CD8+ T cells were used when the tumor size was approximately 30 mm 2When reaching [the relevant stage], it was injected into B16F10 melanoma tumor-bearing mice, and these mice were treated with Fc-IL-4 every two days via intratumoral (i.t.) injection (Figure 2e). Two days after the fourth injection of Fc-IL-4, all mice were sacrificed and tumor tissues were collected for TIL analysis. The total number of CD8+ T cells in the tumor increased significantly after Fc-IL-4 treatment. In particular, Fc-IL-4 treatment specifically increased the number of PD-1+TIM-3+CD8+ T cells in the above solid tumors (Figure 2f), which were 7.1-fold and 1.9-fold higher, respectively, in adoptively transferred endogenous CD8+ T cells compared to mice without Fc-IL-4 treatment. In contrast, the numbers of PD-1-TIM-3-CD8+ T cells and PD-1+TIM-3-CD8+ T cells remained unchanged after Fc-IL-4 treatment. Consistent with the ex vivo results, apoptotic PD1+TIM3+CD8+ T cells were significantly inhibited (Figure 2g). Furthermore, the phosphorylation of MLKL in PD1+TIM3+CD8+ T cells that induce T cell necroptosis was also inhibited by Fc-IL-4 (Figure 2h). Western blot results further confirmed that Fc-IL-4 could rescue PD1+TIM3+CD8+ T cells from necroptosis by inhibiting the activation of the necroptosis-related pathway (Figure 2i). In addition, the effector functions of CD8+ T cells, especially PD1+TIM3+CD8+ T cells, were also significantly enhanced with regard to granzyme B and IFNγ expression, as shown in Figure 2j.

[0084] Terminal exhausted T cells can robustly kill tumor cells, and their enrichment has been reported to be positively associated with better outcomes of tumor control. The inventors then sought to examine whether Fc-IL-4 could enhance the antitumor efficacy of ACT against solid tumors. In the established B16F10 mouse melanoma model, treatment with Fc-IL-4 or PMEL-T cells alone moderately suppressed tumor growth but could not eliminate the tumor burden, which gradually increased. Dramatically, combined treatment with Fc-IL-4 and PMEL-T cells could significantly regress tumors, resulting in sustained cure in 80% (4 / 5) of mice bearing B16F10 mouse melanoma (Figures 3a, b). Furthermore, all surviving mice from the combined treatment were given a long-term memory effect and rejected a secondary challenge of the same B16F10 tumor cells (100,000 tumor cells per mouse) two months after treatment (Figure 3c). High tumor burden (size > 40 mm 2 or 100 mm 3) in another mouse melanoma model (YUMM1.7-OVA) with [Fc-IL-4 and co-administration of OT-1 CD8+ T cells showed robust tumor regression, achieving sustained cure in 100% (7 / 7) of mice bearing melanoma tumors, while monotherapy with Fc-IL-4 or ACT achieved only very limited tumor control and cure rates (Figures 3d, e). Similar to the B16F10 melanoma tumor model, all surviving mice from the combination treatment were able to reject a secondary YUMM1.7-OVA challenge (Figure 3f). Next, the inventors extended this combination therapy to CAR-T cell-based ACT treatment in the MC38-HER2 tumor model, a mouse colon adenocarcinoma expressing human epidermal growth factor receptor 2 (HER2). Despite the significantly limited tumor growth inhibition achieved by HER2-CAR-T alone, Fc-IL-4 strongly improved the therapeutic benefit of HER2-CAR-T and ultimately achieved a cure rate of approximately 83% (5 / 6) of tumor-bearing mice in the combination therapy (Figures 3g, h). The anti-tumor immunity induced by Fc-IL-4 and CAR-T was also persistent and durable, as the treated surviving mice were able to reject a secondary MC38-HER2 tumor challenge (Figure 3i). Overall, Fc-IL-4 strongly promoted tumor regression mediated by ACT and was able to induce sustained cure in syngeneic mouse models with pre-established solid tumors.

[0085] Next, to investigate the effect of Fc-IL-4 treatment on the tumor immune microenvironment, the inventors analyzed immune cell infiltration in the B16F10 tumor model. Total CD45+ immune cells were significantly increased by Fc-IL-4, including both innate and adaptive immune cells (Figure 4). In particular, innate immune cells such as eosinophils, NK cells, DCs, and macrophages increased after Fc-IL-4 treatment alone or in combination with ACT, while MDSCs were not affected. Both CD8+ T cells and CD4+ T cells increased after combination treatment with Fc-IL-4 and ACT. Furthermore, Fc-IL-4 significantly enhances the cytolytic ability and multifunctionality of CD8+ T cells in tumors (Figure 4g, h). Considering that Fc-IL-4 induces significant effects on multiple immune cells and that many immune cells such as CD8 T cells, NK cells, and eosinophils have been reported to directly contribute to tumor control, the inventors next attempted to identify the dominant immune cells that determine the potent antitumor efficacy of combination therapy through immune cell depletion studies using the corresponding antibodies (Figure 5a). The inventors found that the antitumor efficacy of coadministration of Fc-IL-4 and ACT remained unchanged after depletion of CD4+ T cells, NK cells, neutrophils, and eosinophils. Nevertheless, the therapeutic benefit achieved by combination therapy was completely abolished after depletion of CD8+ T cells, indicating that CD8+ T cells play an essential role in tumor control in the context of combination therapy with Fc-IL-4 and ACT (Figure 5b, c). Since terminally exhausted CD8+ T cells rather than other CD8+ T cells were the major subset of CD8+ T cells enriched by Fc-IL-4 as shown above, Fc-IL-4 specifically increases the terminally exhausted CD8+ T cell population and effector functions in tumors and is likely to improve the therapeutic benefit of ACT in solid tumors. To further confirm the direct contribution of terminally exhausted CD8+ T cells to tumor control, the inventors used Tcf7, which can recognize the gp33 antigen in mice bearing B16-gp33 tumors and enable selective depletion of precursor terminally exhausted CD8+ T cells by diphtheria toxin (DT) treatment. DTR-GFPTransgenic P14 T cells were transplanted (Figure 5d). The tumor-suppressive effect of the combination of Fc-IL-4 and ACT was not affected after DT-mediated depletion of precursor exhausted T cells (Figures 5e, f), indicating that Fc-IL-4 could act directly on terminally exhausted CD8+ T cells for robust anti-tumor suppression.

[0086] Next, the inventors attempted to clarify the mechanism underlying how Fc-IL-4 extended the survival of terminally exhausted CD8+ T cells. The differentiation and functional changes of CD8+ T cells are closely linked to metabolic reprogramming, which is driven by the metabolically restrictive tumor microenvironment when CD8+ T cells infiltrate the tumor tissue. Considering that Fc-IL-4 conferred a robust survival benefit on terminally exhausted CD8+ T cells, the metabolic changes in this subset were examined after Fc-IL-4 treatment. The inventors first found that Fc-IL-4 could significantly upregulate the expression of glucose transporter-1 (Glut-1) and glucose uptake ability in CD8+ T cells (Figures 6a - c). Furthermore, the extracellular lactate concentration of CD8+ T cells increased after treatment with Fc-IL-4, indicating that Fc-IL-4 was actually involved in the metabolic regulation of CD8+ T cells (Figure 6d). Next, the inventors detected the metabolic activity by seahorse assay and found that Fc-IL-4 significantly increased the glycolytic level of CD8+ T cells in the context of restimulation (Figure 6e), which was shown by basal glycolysis, glycolytic capacity, and glycolytic reserve (Figure 6f). However, Fc-IL-4 did not affect the OXPHOS of T cells. Therefore, Fc-IL-4 regulated the metabolism of CD8+ T cells mainly by enhancing glycolysis. To explore which kinase or factor in the glycolytic pathway was essential for Fc-IL-4 to extend the survival of CD8+ T cells, metabolic inhibitors were examined. The inventors found that FX11, an inhibitor of lactate dehydrogenase A (LDHA), could abrogate the effect of Fc-IL-4 in proliferating terminally exhausted T cells (Figures 6g, h). In summary, Fc-IL-4 may extend the survival of terminally exhausted CD8+ T cells by enhancing glycolysis in the LDHA-dependent pathway.

[0087] In addition to the strong therapeutic improvement of ACT in solid tumors, the inventors have also discovered that Fc-IL-4 can exhibit a synergistic anti-tumor effect with anti-TIM3 (αTIM3) in the MC38-HER-2 tumor model (Figure 7a). αTIM3 alone did not show obvious tumor suppression, but co-administration of Fc-IL-4 and αTIM3 strongly induced tumor regression, and ultimately had a cure rate of 67% (6 / 9) (Figure 7b, c). Surviving mice were able to reject re-challenge with MC38-HER2 tumor cells. This suggests that Fc-IL-4 can act as an adjuvant for immune checkpoint inhibition therapy to induce persistent and long-term anti-tumor immunity (Figure 7d).

[0088] The effect of human Fc-IL-4 (SEQ ID NO: 6 - Fc-IL-4 fusion protein 1) on human T cells was also evaluated, and the inventors found that human Fc-IL-4 (hu.Fc-IL-4) can significantly increase both the cell viability and cell number of human CD8+ T cells by reducing the apoptosis rate (Figure 8a - c). Furthermore, consistent with mouse CD8+ T cells, the cytotoxic effector function of human CD8+ T cells was also significantly increased by hu.Fc-IL-4 (Figure 8d), all of which suggest a promising application of huFc-IL-4 for solid tumors in clinical practice.

[0089] In summary, the inventors have discovered that in multiple established solid mouse tumor models, Fc-IL-4 strongly induces tumor regression and persistent anti-tumor immunity together with ACT or immune checkpoint inhibition (ICB). Preliminary studies have revealed that Fc-IL-4 specifically extends the survival of terminally exhausted CD8+ T cells by increasing glycolysis in the LDHA-dependent pathway. These studies demonstrate a novel therapeutic intervention using Fc-IL-4 to promote the therapeutic benefits of ACT and ICB for more cancer patients, and in addition provide evidence for the anti-tumor characteristics of Fc-IL-4 by extending the survival and lifespan of terminally exhausted CD8+ T cells.

[0090] Array [Chem.] [Chem.] [Chem.] [Chem.]

[0091] References 1. Walz, M., Overbergh, L., Mathieu, C., Kolb, H. and Martin, S. A murine interleukin-4-Ig fusion protein regulates the expression of Th1- and Th2-specific cytokines in the pancreas of NOD mice. Horm. Metab. Res. 34, (2002). 2. Vella, A., Teague, T.K., Ihle, J., Kappler, J. and Marrack, P. Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: Stat6 is probably not required for the effect of IL-4. J. Exp. Med. 186, 325 - 330(1997). 3. Dufort, F.J. et al., Cutting Edge: IL-4-Mediated Protection of Primary B Lymphocytes from Apoptosis via Stat6-Dependent Regulation of Glycolytic Metabolism. J. Immunol. 179, (2007).

Claims

1. A pharmaceutical composition for use in the treatment and / or prevention of disease, i) Interleukin-4 polypeptide (IL-4) fused to a portion, its fragment or manifold, ii) Anti-cancer immunotherapy and Includes, A pharmaceutical composition for use, wherein the portion is a molecule that extends the half-life of IL-4, its fragments or manifolds, and / or stabilizes IL-4, its fragments or manifolds.

2. The pharmaceutical composition for use according to claim 1, wherein the interleukin-4 polypeptide (IL-4), a fragment or variant thereof, is covalently fused to the portion, either directly or indirectly, by a linker essentially consisting of a sequence of Gly and Ser residues.

3. The pharmaceutical composition for use according to claim 2, wherein the linker is selected from the group comprising (GGS)n or (GGGGGS)n, where n is between 1 and 10.

4. The portion is selected from the group comprising polyethylene glycol (PEG), natural polysaccharides and semi-synthetic polysaccharides, such as O-linked oligosaccharides and N-linked oligosaccharides, dextran, hydroxyethyl starch (HES), polysialic acid and hyaluronic acid, albumin, antibodies or fragments thereof, protein polymers, such as homoamino acid polymers, elastin-like polypeptides, XTEN and PAS, to be a pharmaceutical composition for use according to any one of claims 1 to 3.

5. The pharmaceutical composition for use according to claim 4, wherein the antibody fragment is a constant fragment (Fc) domain of an antibody, preferably a silent Fc domain of immunoglobulin G (IgG), and the interleukin-4 polypeptide (IL-4), a fragment or variant thereof is covalently fused to the N-terminus or C-terminus of the IgG Fc domain.

6. The aforementioned disease is selected from the group including solid tumors, humoral tumors, and autoimmune diseases. The pharmaceutical composition for use according to any one of claims 1 to 3.

7. The pharmaceutical composition for use according to claim 6, wherein the solid tumor is selected from the group including lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain tumor, and skin cancer, particularly melanoma, or one or more combinations thereof.

8. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the anti-cancer immunotherapy is selected from the group comprising ACT therapy, immune checkpoint inhibitor therapy, cytokine therapy, cancer vaccine therapy, bispecific antibody therapy, and other cancer immunotherapy, or a combination of one or more of these.

9. The pharmaceutical composition for use according to claim 8, wherein the ACT therapy is selected from the group comprising TCR-T, CAR-T, TIL and NK cell therapy, or a combination of one or more of these.

10. The aforementioned immune checkpoint inhibitor therapy includes an inhibitor selected from the group comprising CTLA-4 inhibitors, TIM3 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors, or a combination of one or more of these, wherein the CTLA-4 inhibitor is selected from the group comprising ipilimumab and tremelimumab, or a combination thereof, or the PD-1 inhibitor is selected from the group comprising nivolumab, pembrolizumab, pidilizumab, dostallimab, and atezolizumab, or a combination thereof. The pharmaceutical composition for use according to claim 8, wherein the PD-L1 inhibitor is selected from the group comprising atezolizumab, avelumab, AMP-224, MEDI-0680, RG-7446, GX-P2, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014, CX-072, and BMS-936559, or a combination thereof, or the TIM3 inhibitor is covolimab.

11. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein the sequence of IL-4 is the human sequence shown in SEQ ID NO: 1, a fragment or variant thereof, which shares at least 85% amino acid sequence identity with SEQ ID NO:

1.

12. A pharmaceutical composition for use according to any one of claims 1 to 3, further comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

13. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein the fused IL-4, its fragments or manifolds increase the efficacy of the anti-cancer immunotherapy by an increase of about 2% or more, about 5% or more, about 20% or more, about 40% or more, about 60% or more, or about 500% or more compared to the efficacy of the anti-cancer therapy in the absence of the partially fused IL-4, its fragments or manifolds.

14. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein a drug useful for cancer treatment or autoimmune disease therapy is administered simultaneously or sequentially with the IL-4, its fragments or manifolds, which are partially fused thereto.

15. A pharmaceutical composition, i) Interleukin-4 polypeptide (IL-4) fused to a portion, its fragment or manifold, ii) Anti-cancer immunotherapy and It includes, and comprises pharmaceutically acceptable carriers, diluents and / or excipients, A pharmaceutical composition in which the aforementioned portion is a molecule that extends the half-life of IL-4, its fragments or manifolds, and / or stabilizes IL-4, its fragments or manifolds.

16. The pharmaceutical composition according to claim 15, wherein the interleukin-4 polypeptide (IL-4), a fragment or manifold thereof, is covalently fused to the portion, either directly or indirectly, by a linker essentially consisting of a sequence of Gly and Ser residues.

17. The pharmaceutical composition according to claim 16, wherein the linker is selected from the group including (GGS)n or (GGGGGS)n, and n is between 1 and 10.

18. The fusion IL-4 comprises or is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22, fragments or variants thereof that share at least 85% amino acid sequence identity with any one of these sequences, or the pharmaceutical composition according to claim 1 or claim 15.

19. The pharmaceutical composition according to claim 1 or 15, wherein the portion is a constant fragment (Fc) domain of immunoglobulin G (IgG), the IgG is selected from the group including human IgG1, IgG2, IgG3, or IgG4, and the interleukin-4 polypeptide (IL-4), its fragment, or variant is covalently fused to the N-terminus or C-terminus of the IgG Fc domain.

20. The pharmaceutical composition according to claim 19, wherein the Fc domain of human IgG comprises IgG1 Fc (SEQ ID NO: 2), IgG2 Fc (SEQ ID NO: 8), IgG3 Fc (SEQ ID NO: 11), and IgG4 Fc (SEQ ID NO: 14), a fragment or variant thereof that shares at least 85% amino acid sequence identity with any one of these sequences, or a combination of one or more of these sequences, or is selected from the group of sequences comprising these.

21. The pharmaceutical composition according to claim 1 or 15, wherein the interleukin-4 polypeptide (IL-4) partially fused, a fragment or variant thereof, and the anti-cancer immunotherapy are administered simultaneously, concurrently, or sequentially to a subject in need thereof.

22. The pharmaceutical composition according to claim 1 or claim 15, further comprising the administration of at least one additional therapeutic agent or therapy, preferably an anticancer agent or anticancer therapy when the disease is cancer.

23. IL-4 fused to a portion of IL-4 that extends the half-life of IL-4 or stabilizes IL-4, wherein the fused IL-4 includes, or is selected from, the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22, or fragments or variants thereof that share at least 85% amino acid sequence identity with any one of these sequences.

24. One or more nucleic acid sequences encoding the fusion IL-4 described in claim 23.

25. A plasmid or vector comprising the nucleic acid sequence described in claim 24.

26. The vector according to claim 25, wherein the vector is selected from the group including retroviral vectors (such as pMSGV), DNA vectors, mouse leukemia virus vectors, SFG vectors, RNA vectors, adenovirus vectors, baculovirus vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, adeno-associated virus vectors (AAV), lentivirus vectors (such as pGAR), or any combination thereof.

27. A prokaryotic host cell or a eukaryotic host cell comprising the plasmid or vector according to claim 25 or claim 26.

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