Methods and Compositions for Cancer Treatment

Abstract

Compositions and methods for treating cancer are provided that involve the use of immunoconjugates in combination with cytotoxic agents or cytotoxic therapies, which exhibit synergistic effects in cancer treatment. [Selection diagram] None

Description

Background Art

[0001] Although an immune response against tumor antigens can be detected (Non-Patent Document 1), malignant cells that cause diseases often cannot induce an immune response that results in rejection. Studies have demonstrated that it is possible to enhance the immunogenicity of tumor cells by introducing immunomodulatory molecules such as cytokines and costimulatory molecules. However, for eradicating residual cancer cells, it may be necessary to target widely scattered micrometastatic tumor deposits that cannot be reached by direct gene transfer. In addition, the expression and stability of the introduced immunomodulatory molecules are often far from sufficient. Immunomodulatory substances such as cytokines produced by cells of the immune system can directly or indirectly activate cells of the adaptive immune response and play an important role in inducing protective anti-tumor immunity. The innate immune system can be triggered by "danger" signals that result in the release of bacterial products or inflammatory cytokines such as interleukin.

[0002] Numerous studies have shown that immunomodulatory substances can be useful for exerting anti-tumor effects in both animal models and cancer patients. However, their use is severely limited due to the short half-life and systemic toxicity associated with the application of immunomodulatory substances. Patent Document 1 describes a chimeric construct containing interferon attached to the C-terminus of an antibody targeting a tumor-associated antigen. However, the fusion protein expressed by such a chimeric construct is typically very unstable in vivo, and its expression yield is typically not high enough for industrial-scale production.

[0003] In addition to immunotherapy, surgery, chemotherapy, hormone therapy, and radiotherapy are also used in the treatment of cancer (see, for example, Non-Patent Document 2). However, surgery may be impossible or unacceptable due to the patient's health condition or advanced stage of the disease, and it frequently occurs that cancer cells cannot be completely removed from the patient after surgery. Radiotherapy is effective only when tumor tissue is more sensitive to "radiation" than normal tissue, and high-dose radiotherapy may often cause serious side effects. Hormone therapy is rarely administered as a single agent and, although it can be effective, it is often used to prevent or delay cancer recurrence after most of the cancer cells have been removed by other treatments. Furthermore, patients may develop drug resistance to chemotherapeutic agents quite rapidly.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, there is still a strong need for new and effective treatment methods for cancer treatment. [[Means for Solving the Problem]]

[0007] The present disclosure provides compositions and methods for treating cancer, including the use of an immunoconjugate in combination with a cytotoxic substance or cytotoxic therapy, and the invention of the present disclosure has shown a significant synergistic effect in cancer treatment.

[0008] In one aspect, the present disclosure provides a composition comprising an immunoconjugate and a cytotoxic substance, wherein the immunoconjugate comprises 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, the first Fc subunit associates with the second Fc subunit to form a dimer, the one or more interleukins are fused to the Fc domain, the cytotoxic substance is capable of inducing immunogenic cell death, and provides a composition.

[0009] In some embodiments, at least one of the one or more interleukins is fused to the amino-terminal amino acid of the Fc domain.

[0010] In some embodiments, the immunoconjugate comprises two or more interleukins.

[0011] In some embodiments, at least two of the two or more interleukins are fused to the amino-terminal amino acid of the Fc domain.

[0012] In some embodiments, at least two of the two or more interleukins are fused to each other via a peptide linker to form an interleukin dimer.

[0013] In some embodiments, at least one of the interleukin dimers is fused to the amino-terminal amino acid of the Fc domain.

[0014] In some embodiments, the two or more interleukins are two or more copies of the same interleukin. In some embodiments, the two or more interleukins are two or more copies of IL10.

[0015] In some embodiments, the immunoconjugate further comprises a targeting moiety fused to the Fc domain, the targeting moiety exhibiting binding specificity for a tumor antigen. In some embodiments, the targeting moiety is fused to the amino-terminal amino acid of the Fc domain. In some embodiments, the tumor antigen is selected from the group consisting of EGFR, HER2 / neu, and FAP. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the IgG is human IgG1.

[0016] In some embodiments, the immunoconjugate is an immunoconjugate comprising a first member and a second member different from the first member, wherein the first member comprises the first Fc subunit, the second member comprises the one or more interleukins fused to the second Fc subunit, and the first Fc subunit associates with the second Fc subunit to form the heterodimer.

[0017] In some embodiments, at least one of the one or more interleukins is fused to the amino-terminal amino acid of the second Fc subunit.

[0018] In some embodiments, in the second member, at least two of the one or more interleukins are fused to each other to form an interleukin dimer, and the interleukin dimer is further fused to the amino-terminal amino acid of the second Fc subunit.

[0019] In some embodiments, the first member further comprises the targeting moiety fused to the first Fc subunit.

[0020] In some embodiments, the immunoconjugate does not contain any targeting moiety.

[0021] In some embodiments, the first Fc subunit is different from the second Fc subunit, and the Fc domain comprises a modification that promotes heterodimerization between the first Fc subunit and the second Fc subunit.

[0022] In some embodiments, the first Fc subunit comprises a first modification, the second Fc subunit comprises a second modification, and the first modification and the second modification are 1) First modification: Y349 and T366, and Second modification: D356, T366, L368, Y407, and F405; 2) First modification: Y349, T366, and F405, and Second modification: D356, T366, L368, and Y407; 3) First modification: Y349, T366, and K409, and Second modification: D356, T366, L368, Y407, and F405; 4) First modification: Y349, T366, F405, K360, and Q347, and Second modification: D356, T366, L368, Y407, and Q347; 5) First modification: Y349, T366, F405, and Q347, and Second modification: D356, T366, L368, Y407, K360, and Q347; 6) First modification: Y349, T366, K409, K360, and Q347, and Second modification: D356, T366, L368, Y407, F405, and Q347; 7) First modification: Y349, T366, K409, and Q347, and Second modification: D356, T366, L368, Y407, F405, K360, and Q347; 8) First modification: T366, K409, and K392, and Second modification: T366, L368, Y407, D399, and F405; 9) First modification: T366 and K409, and Second modification: T366, L368, Y407, and F405; 10) First modification: T366, K409, and Y349, and Second modification: T366, L368, Y407, F405, and E357; 11) First modification: T366, K409, Y349, and S354, and Second modification: T366, L368, Y407, F405, and E357; 12) First modification: T366 and F405, and Second modification: T366, L368, Y407, and K409; 13) First modification: T366, F405, and D399, and Second modification: T366, L368, Y407, K409, and K392; 14) First modification: T366, F405, and Y349, and Second modification: T366, L368, Y407, K409, and E357; 15) First modification: T366, F405, Y349, and S354, and Second modification: T366, L368, Y407, K409, and E357; comprises an amino acid substitution in a position group selected from any of the groups, and the position of the amino acid is determined according to the EU index of the KABAT number.

[0023] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death is selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, platinum-based compounds, and radioactive substances.

[0024] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises an alkylating agent selected from cyclophosphamide.

[0025] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises an antimetabolite selected from the group consisting of capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0026] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises an anthracycline selected from the group consisting of bleomycin, doxorubicin, and mitomycin-C.

[0027] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises a plant alkaloid selected from the group consisting of taxanes, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0028] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises a radioactive substance that emits X-rays and / or gamma rays.

[0029] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises a platinum-based compound selected from the group consisting of carboplatin, cisplatin, and oxaliplatin.

[0030] In another aspect, the present disclosure provides for the use of an immunoconjugate in combination with a cytotoxic therapy in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the immunoconjugate is as defined in the present disclosure and the cytotoxic therapy is capable of inducing immunogenic cell death. The present disclosure provides the use.

[0031] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following forms for carrying out the invention, which illustrate and describe only exemplary embodiments of the present disclosure. As will be recognized, various other embodiments are possible in the present disclosure and various obvious points can be changed in various ways without departing from the present disclosure. Accordingly, the drawings and the specification are to be regarded as illustrative in nature and not restrictive.

[0032] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference to form a part of this specification.

[0033] The novel features of the invention are set forth in detail in the appended claims. The features and advantages of the invention will be better understood by reference to the following forms for carrying out the invention, which describe exemplary embodiments in which the principles of the invention are utilized, and the accompanying drawings (further, the "figures" in this specification ("figure" and "FIG.")). BRIEF DESCRIPTION OF THE DRAWINGS

[0034]

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Best Mode for Carrying Out the Invention

[0035] Before describing the embodiments of the present disclosure, it should be understood that such embodiments are presented by way of example only, and that various alternative forms of the embodiments of the present disclosure described herein can be used in the implementation of the present disclosure. Here, numerous changes, variations, and substitutions will occur to those skilled in the art without departing from the present disclosure.

[0036] 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 this disclosure belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, and suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Here, numerous changes, variations, and substitutions will occur to those skilled in the art without departing from the present disclosure.

[0037] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” generally include plural referents.

[0038] The term “immunoconjugate,” as used herein, generally refers to a proteinaceous molecule formed by the conjugation of one or more antibodies or fragments thereof with one or more second molecules. The second molecules may be the same or different and may include, for example, effector proteins.

[0039] The term “proteinaceous,” as used herein, generally refers to substances or entities that are polypeptides or proteins, related to them, similar to them, or are them. For example, the immunoconjugates of the present disclosure can be heterodimeric proteins or heterodimers containing two or more polypeptides.

[0040] The term "heterodimer", as used herein, generally refers to a molecule (e.g., a proteinaceous molecule) composed of two different members. The two members of the heterodimer may differ in structure, function, activity, and / or composition. For example, the two different members may include polypeptides that differ in the order, number, or type of amino acid residues forming these polypeptides. The two different members of the heterodimer may each independently include one, two, or more units, polypeptide chains, or moieties.

[0041] The term "targeting moiety", as used herein, generally refers to a molecule, complex, or aggregate that specifically, selectively, or preferentially binds to a target molecule, cell, particle, tissue, or aggregate. For example, the targeting moiety can be an antibody, an antigen-binding antibody fragment, a bispecific antibody, or other antibody-based molecule or compound. Other examples of targeting moieties include, but are not limited to, aptamers, avimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides, or proteins. The terms "targeting moiety" and "binding moiety" are used interchangeably herein.

[0042] As used herein, the term "tumor antigen" generally refers to an antigenic substance produced in or by tumor cells that may have the ability to elicit an immune response in a host. For example, a tumor antigen can be a protein, polypeptide, peptide, or fragment thereof that constitutes part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. A tumor antigen peptide may be a peptide generated as a result of the degradation of a tumor antigen in a tumor cell and can induce or activate tumor-specific cytotoxic T lymphocytes when expressed on the cell surface by binding to an HLA molecule. In some embodiments, the term "tumor antigen" refers to a biomolecule (e.g., a protein, carbohydrate, glycoprotein, etc.) that is expressed exclusively or preferentially or differentially on and / or associated with cancer cells, thereby providing a target that is preferential or specific for cancer. For example, preferential expression can be preferential expression compared to any other cell in an organism or preferential expression within a particular region of an organism (e.g., within a particular organ or tissue).

[0043] As used herein, the term "heterodimerization" generally refers to the process of forming a heterodimer between two different members (e.g., two non-identical polypeptides), with or without the formation of a covalent bond between the two different members, by complex formation, association, or aggregation, etc.

[0044] As used herein, the term "covalent bond" generally refers to a chemical bond formed between atoms by the sharing of electrons. For example, a covalent bond may be polar or nonpolar. In some embodiments, the covalent bond is a disulfide bond.

[0045] As used herein, the term "non-covalent affinity" generally refers to non-covalent interactions such as ion pairs, hydrogen bonds, dipole-dipole interactions, charge transfer interactions, π-π interactions, cation-π electron interactions, van der Waals interactions, and dispersion interactions, hydrophobic (lipophilic) interactions, complex formation (e.g., complex formation of transition metal cations), or dimerization or heterodimerization sequences capable of binding to each other by combinations of these interactions.

[0046] As used herein, the term "linker" generally refers to a synthetic amino acid sequence that connects or links two polypeptide sequences, e.g., that links domains of two polypeptides. A linker can connect two amino acid sequences by a peptide bond. In some embodiments, the linker of the present disclosure connects a bioactive moiety to a second moiety in a linear sequence.

[0047] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may include modified amino acids, and may have non-amino acids interrupting them. The term also includes amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation to labeled components. The term may apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term may also include variations of the conventional peptide bond that join the amino acids that make up the polypeptide. For example, "peptide", "polypeptide", and "protein" can be chains of amino acids in which the alpha carbons are linked by peptide bonds. Thus, the terminal amino acid at one end (amino terminus) of the chain has a free amino group, and the terminal amino acid at the other end (carboxy terminus) of the chain may have a free carboxyl group. As used herein, the term "amino terminus" (abbreviated as N-terminus) generally refers to the free alpha amino group on the amino-terminal amino acid of a peptide, or the alpha amino group of an amino acid at any other position within the peptide (when involved in a peptide bond, the amino group). Similarly, the term "carboxy terminus" generally refers to the free carboxyl group on the carboxy-terminal of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides may include, but are not limited to, essentially any polyamino acid, including peptide mimics such as amino acids joined by an ether, as opposed to an amide bond.

[0048] As used herein, the term "amino acid" generally refers to natural and / or non-natural, or synthetic amino acids, including, but not limited to, D- or L-optical isomers, or both, amino acid analogs, and peptide mimics. Standard one-letter or three-letter codes are used to specify the amino acids.

[0049] The term "variant", when used in connection with a proteinaceous molecule (e.g., a polypeptide or a protein), generally refers to a proteinaceous molecule having sequence homology to an unmodified bioactive protein that retains at least a portion of the therapeutic and / or bioactive properties of the bioactive protein. For example, a variant protein may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity compared to a reference bioactive protein. In some embodiments, a "variant" may include a protein that has been intentionally modified, e.g., by site-directed mutagenesis, synthesis of the coding gene, insertion, or a protein that has been incidentally modified by a mutation.

[0050] The terms "conjugated", "linked", "fused", and "fusion" are used interchangeably herein and generally refer to the joining of two or more chemical elements, sequences, or components by means including, for example, chemical bonds or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence when transcription of the sequence is achieved. Generally, "operably linked" means that the DNA sequences being linked are close and in the reading phase or in-frame. "In-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a longer continuous ORF such that the correct reading frame of the original ORF is maintained. Thus, the resulting "fusion polypeptide" is a single protein containing two or more fragments corresponding to the polypeptides encoded by the original ORFs (which segments would not normally join in that manner). A "fusion site" refers to the sequence at which two or more fragments are joined to each other. The fusion site may optionally be a sequence identical to a sequence in two or more of the joined fragments. The fusion site may optionally further include a gap segment that is not identical to any of the sequences of the two or more joined fragments.

[0051] In the context of polypeptides, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide in the direction from the amino terminus to the carboxyl terminus, where adjacent residues in the sequence are proximal in the primary structure of the polypeptide. A "subsequence" is a linear sequence that forms part of a polypeptide and is known to include additional residues in one or both directions.

[0052] The terms "polynucleotide", "nucleic acid", "nucleotide", and "oligonucleotide" are used interchangeably herein and generally refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: the coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis (singular or plural), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can include modified nucleotides such as methylated nucleotides and nucleotide analogs. When present, modifications to the nucleotide structure can be made either before or after polymerization of the polymer. Non-nucleotide components may be interspersed within the nucleotide sequence. A polynucleotide can be further modified, for example, by conjugation with labeling components after polymerization.

[0053] The terms "gene" and "gene fragment" are used interchangeably herein and generally refer to a polynucleotide containing at least one open reading frame capable of encoding a specific protein after transcription and translation. A gene or gene fragment may be genomic DNA or cDNA as long as the polynucleotide contains at least one open reading frame that can span the entire coding region or a segment thereof. A "fusion gene" is a gene composed of at least two heterologous polynucleotides linked together.

[0054] As used herein, the term "antibody" generally refers to a protein comprising one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Immunoglobulin genes can include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. As used herein, a light chain can be classified as either κ or λ. Heavy chains can be classified as γ, μ, α, δ, or ε, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. An antibody can have a structural unit that includes a tetramer. Each tetramer can be composed of two identical pairs of polypeptide chains, and each pair can have one "light" chain (about 25 kD) and one "heavy" chain (about 50 kD to 70 kD). The N-terminus of each chain can define a variable region of about 100 to 110 or more amino acids that is primarily involved in antigen recognition. The terms "light chain variable region" (VL) and "heavy chain variable region" (VH) as used herein generally refer to these regions of the light and heavy chains, respectively. An antibody can exist as a number of well-characterized fragments produced by digestion with various peptidases of native immunoglobulins or produced de novo or expressed de novo. Thus, for example, an antibody can be digested with pepsin under disulfide linkages in the hinge region to produce F(ab)'2, which is a dimer of Fab, itself a light chain joined to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions that cleave the disulfide linkages in the hinge region, whereby the dimer of (Fab')2 can be converted to Fab' monomers. Fab' monomers are essentially Fab with a portion of the hinge region (see Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993) for a more detailed description of other antibody fragments). Although various antibody fragments are defined with respect to digestion of native antibodies, it will be understood by those skilled in the art that such Fab' fragments can be synthesized de novo chemically or by using recombinant DNA methods.Accordingly, as used herein, the term "antibody" can include, but is not limited to, Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nanobody, uniobody, and diabody, and can also include antibody fragments produced by modification of whole antibodies or de novo synthesized using recombinant DNA methods. In some embodiments, antibodies include, but are not limited to, Fab'2, IgG, IgM, IgA, IgE, and single-chain antibodies, such as single-chain Fv (scFv) antibodies in which the variable heavy chain and variable light chain are joined to each other (either directly or via a peptide linker) to form a continuous polypeptide.

[0055] As used herein, the term "antigen-binding site" or "binding moiety" generally refers to the part of an antibody involved in antigen binding. The antigen-binding site can be formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") chain and / or light ("L") chain. Three stretches of high diversity within the V regions of the heavy and light chains are referred to as "hypervariable regions" intervening between more conserved adjacent stretches known as "framework regions" or "FRs". Accordingly, as used herein, the term "FR" generally refers to the amino acid sequences that are naturally found adjacent to each other between the hypervariable regions of immunoglobulins. In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are arranged in three-dimensional space in relation to each other to form an antigen-binding "surface". This surface can mediate the recognition and binding of a target antigen. Each of the three hypervariable regions of the heavy and light chains is referred to as a "complementary determining region" or "CDR" and is characterized, for example, by Kabat et al. Sequences of proteins of immunological interest, 4 th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, Md. (1987).

[0056] The terms "identity", "identical", or "sequence identity", as used herein, generally refer to the sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a program (e.g., Emboss Needle or BestFit) to determine the sequence identity, similarity, or homology between two different amino acid sequences, either the default settings may be used, or an appropriate scoring matrix such as blosum45 or blosum80 may be selected to optimize the identity, similarity, or homology score. In some embodiments, identical polynucleotides hybridize under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity compared to these sequences. Identical polypeptides have at least 80% or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity when optimally aligned with sequences of the same length, or have at least 99% sequence identity.

[0057] The terms "effective amount" or "therapeutically effective amount" refer to the amount of a composition sufficient to achieve the intended use, including, but not limited to, the treatment of a disease. The therapeutically effective amount varies depending on the intended use (e.g., in vitro or in vivo) or the subject and condition being treated, such as the subject's body weight and age, the severity of the condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. This term may apply to the dosage that induces a specific response in target cells, such as target gene transfer, proliferation, and / or apoptosis. The specific dosage varies depending on the particular compound selected, the dosing regimen employed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system employed.

[0058] The terms "treatment", "treating", "alleviating", or "ameliorating" are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic and / or prophylactic benefits. As used herein, a therapeutic benefit generally refers to the eradication of the underlying disorder being treated or a reduction in its severity. A therapeutic benefit is also achieved by the eradication, reduction in severity, or reduction in incidence of one or more physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still suffer from the underlying disorder. For prophylactic benefits, the compositions can be administered to a subject at risk of developing a particular disease or to a subject in which one or more physiological symptoms of the disease have been reported, even if a diagnosis of the disease cannot be made.

[0059] As used herein, the term "therapeutic effect" generally encompasses therapeutic and / or prophylactic benefits as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the manifestation of symptoms of the disease or condition, slowing, halting, or reversing the progression of the disease or condition, or any combination thereof.

[0060] The terms "co-administration", "administering in combination with", "using in combination with", and grammatically equivalent terms, as used herein, generally encompass the application to a subject of two or more agents and therapies such that both agents and / or their metabolites or both therapies are present and / or function in the subject. Co-administration includes simultaneous administration in separate compositions or forms, administration at different times in separate compositions or forms, or administration in a composition in which both agents are present.

[0061] The term "agent", as used herein, generally refers to a biological, pharmaceutical, or chemical compound, or other moiety. Non-limiting examples include simple or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, vitamin derivatives, carbohydrates, toxins, chemotherapeutic compounds, or agents capable of generating and / or emitting radiation.

[0062] As used herein, the term "interleukin" generally refers to a secreted protein or signaling molecule capable of promoting the development or differentiation of T lymphocytes and / or B lymphocytes, and / or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes, as well as monocytes, macrophages, and endothelial cells. As used herein, examples of interleukins (ILs) include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and / or IL-36. As used herein, the term "interleukin" can include full-length interleukins or fragments (e.g., truncated forms) or variants thereof that substantially maintain the biological activity of the corresponding wild-type interleukin (e.g., having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even at least 100% of the biological activity of the corresponding wild-type interleukin). Interleukins can be derived from any mammalian species. In some embodiments, the interleukin is derived from a species selected from the group consisting of humans, horses, cows, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, and non-human primates. In some embodiments, the interleukin may be, for example, a mutant form with an increased or decreased affinity for its receptor.

[0063] As used herein, the term "subject" generally refers to a human or a non-human animal including, but not limited to, cats, dogs, horses, pigs, cows, sheep, goats, rabbits, mice, rats, or monkeys.

[0064] As used herein, the term "EGFR family member" generally refers to a member of the epidermal growth factor receptor family. For example, this can be ErbB-1 (also designated as epidermal growth factor receptor (EGFR)), ErbB-2 (also designated as HER2 in humans and neu in rodents), ErbB-3 (also designated as HER3), and / or ErbB-4 (also designated as HER4). Examples of anti-EGFR family antibodies include, but are not limited to, one or more of the following antibodies: C6.5, C6mL3-9, C6 MH3-B1, C6-B1D2, F5, HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12, EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8, HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8, and HER4.C7, etc. See, for example, U.S. Patent Application Publication No. 2006 / 0099205 and U.S. Patent Application Publication No. 2004 / 0071696, which are incorporated herein by reference in their entirety.

[0065] As used herein, the term "anti-HER2 / neu antibody" generally refers to an antibody that specifically or preferentially binds to the HER2 / neu receptor. For example, an anti-HER2 / neu antibody or an anti-HER2 antibody can be trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0066] As used herein, the term "anti-EGFR antibody" generally refers to an antibody that specifically or preferentially binds to EGFR. In some cases, the anti-EGFR antibody may bind to a mutant form of EGFR (e.g., EGFR variant III, which is the most common extracellular domain mutation of EGFR, also known as EGFRvIII, which results in a deletion of exons 2-7 of the EGFR gene and is characterized by a truncated extracellular domain with ligand-independent constitutive activity). For example, the anti-EGFR antibody can be cetuximab, Mab806, or an antigen-binding fragment thereof.

[0067] As used herein, the term "anti-FAP antibody" generally refers to an antibody that specifically or preferentially binds to FAP. As used herein, the term "FAP" generally refers to fibroblast activation protein (FAP). FAP is present in tumor matrix fibroblasts and plays a role on the cell surface. FAP is a member of the type II serine protease family and is a membrane serine protease having dipeptidyl peptidase activity and collagenase activity. For example, the anti-FAP antibody can be antibody 28H1 or an antigen-binding fragment thereof. In some embodiments, the anti-FAP antibody is antibody 28H1.

[0068] As used herein, the term "member" generally refers to a polypeptide, subunit, or moiety that is one of the components of an immunoconjugate.

[0069] As used herein, the term "Fc domain" generally refers to the Fc portion or Fc fragment of an antibody heavy chain. For example, the Fc domain may refer to the carboxyl-terminal portion of the immunoglobulin heavy chain constant region or an analog or portion thereof capable of binding to an Fc receptor. As is known, each immunoglobulin heavy chain constant region contains four or five domains. The domains are named in order as follows: CH1 - hinge - CH2 - CH3 (-CH4). CH4 is present in IgM and does not have a hinge region. The immunoglobulin heavy chain constant regions useful in the present disclosure may include an immunoglobulin hinge region and may also include the CH3 domain. For example, the immunoglobulin heavy chain constant region may include an immunoglobulin hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the Fc domain according to the present disclosure consists of a hinge - CH2 - CH3 domain.

[0070] As used herein, the term "Fc subunit" generally refers to a component of an Fc domain. For example, an Fc domain is formed by two or more members, and each member may be regarded as one Fc subunit.

[0071] As used herein, the term "complexes with" generally refers to the association (e.g., binding) of one member / subunit of a molecule (e.g., an antibody) with another member / subunit. For example, a light chain may complex with a heavy chain to form a targeting moiety.

[0072] As used herein, the term "binding specificity" generally refers to the ability to specifically bind to (e.g., immunoreact with) a given target (but not bind or substantially bind to non-targets). The targeting moieties of the present disclosure may be monospecific and contain one or more binding sites that specifically bind to the target or multispecific (e.g., bispecific or trispecific) and contain two or more binding sites that specifically bind to the same or different targets.

[0073] As used herein, the terms "associating with" or "associated with" generally refer to one entity being physically associated or in contact with another entity. For example, a first member of an immunoconjugate can "associate" with a second member covalently or non-covalently. In some embodiments, a first member of an immunoconjugate associates with a second member through an interface, which is formed by amino acid residues (i.e., interface residues) from each of the first and second members.

[0074] As used herein, the term "modification" generally refers to any manipulation of a peptide backbone (e.g., amino acid sequence) or any post-translational modification of a polypeptide (e.g., glycosylation). For example, a modification is compared to the sequence of the corresponding wild-type polypeptide. A modification can be a substitution, addition, and / or deletion of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids.

[0075] As used herein, the term "fusion protein" generally refers to a polypeptide comprising or consisting of the amino acid sequence of a polypeptide that is directly or indirectly (e.g., through a linker) fused to the amino acid sequence of a heterologous polypeptide (i.e., a polypeptide that is unrelated to the polypeptide or domain to which the heterologous polypeptide is fused).

[0076] As used herein, the term "C-terminus" generally refers to the carboxy terminus of a polypeptide.

[0077] As used herein, the term "N-terminus" generally refers to the amino terminus of a polypeptide.

[0078] As used herein, the term "immunoglobulin" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include various immunoglobulin variable region genes, along with the constant region genes of κ, λ, α, γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer, consisting of two identical pairs of immunoglobulin chains, with each pair having one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains together are responsible for binding to an antigen, and the constant regions are responsible for antibody effector functions. In addition to antibodies, immunoglobulins can exist in various other forms, including, for example, Fv, Fab, Fab’, and (Fab’)2.

[0079] As used herein, the term "in-frame fusion" generally refers to joining two or more open reading frames (ORFs) to form a continuous longer ORF while maintaining the correct reading frame of the original ORF.

[0080] As used herein, the term "amino acid substitution" generally refers to the replacement of one amino acid at a specific position in a polypeptide with another amino acid.

[0081] As used herein, the term "EU index of Kabat number" generally refers to the index of EU numbers corresponding to the amino acid sequences according to Kabat et al. (1971) Ann. NY Acad, Sci. 190:382 - 391 and Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 - 3242.

[0082] The terms "isolated polynucleotide" and "isolated nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, that are isolated from their natural environment or synthetically produced.

[0083] The term "pharmaceutically acceptable additive" as used herein generally refers to any and all solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, and the like that are compatible with pharmaceutical administration.

[0084] The term "cytotoxic substance" as used herein generally refers to a substance or approach that inhibits or interferes with the function of cells and ultimately causes cell destruction and / or cell death, particularly tumor cell death. Cytotoxic substances can include radioactive isotopes, chemotherapeutic agents, and toxins or fragments thereof such as enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0085] The terms "immunogenic cell death" and "immunogenic apoptosis" are used interchangeably herein and generally refer to forms of cell death that induce an effective anti-tumor immune response, for example, through the activation of dendritic cells (DCs) and the consequent activation of specific T cell responses. Immunogenic cell death may be characterized by the secretion of damage-associated molecular patterns (DAMPs). Examples of DAMPs include calreticulin (CRT), heat shock proteins (HSPs), secreted amphoterin (HMGB1), ATP, and the like. CRT is normally present in the lumen of the endoplasmic reticulum (ER) and can translocate to the surface of dying cells after induction of immunogenic apoptosis, where it functions as an "eat me" signal for professional phagocytes. HSPs include HSP70 and HSP90, which may also translocate to the plasma membrane under stress conditions. HMGB1 is regarded as a late apoptosis marker and appears to require release into the extracellular space for optimal release and presentation of tumor antigens to dendritic cells. ATP can function as a "find-me" signal for monocytes upon secretion, inducing their attraction to apoptotic sites.

[0086] The term "alkylating agent" as used herein generally refers to the oldest and most commonly used class of chemotherapeutic agents, which act by directly damaging DNA and preventing the replication of cancer cells. Alkylating agents are usually cell cycle non-specific and can kill cancer cells at any stage of the cell cycle. An example of an alkylating agent is cyclophosphamide.

[0087] The term "antimetabolite" as used herein generally refers to chemotherapeutic agents that interfere with the growth of DNA and RNA. Antimetabolites are usually cell cycle specific and kill cancer cells at specific stages of cell division. Examples of antimetabolites include capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0088] The term "anthracycline", as used herein, generally refers to a class of agents originally derived from Streptomyces that can be used in cancer chemotherapy. Anthracyclines can interfere with the enzymes necessary for DNA replication. Anthracyclines can be cell cycle non-specific and can be used in the treatment of various cancers. Anthracyclines may include red aromatic polyketides that occur in various forms due to differences in the structure of the aglycone and the attached sugar residues. Examples of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, and mitomycin-C.

[0089] The term "plant alkaloid", as used herein, generally refers to an agent that can inhibit or prevent mitosis or inhibit the production of proteins necessary for cell replication by enzymes. Most plant alkaloids are cell cycle specific, but can cause damage at all stages. Examples of plant alkaloids include taxanes, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0090] The term "radioactive substance", as used herein, generally refers to an agent that can generate and / or emit some form of radiation (e.g., ionizing radiation). Examples of radioactive substances include radioactive isotopes and agents containing radioactive isotopes. A radioactive isotope may be in the form of a radioactive element consisting of atoms with unstable nuclei, which can undergo radioactive decay to a stable form and emit characteristic alpha, beta, or gamma rays.

[0091] The terms "radiation therapy" and "radiotherapy" are used interchangeably herein and generally refer to a form of treatment that uses ionizing radiation (emitted by an X-ray generator or other energy sources such as radioactive isotopes such as gamma, beta, or alpha emitters) to control or kill cancer cells.

[0092] As used herein, the term "fractionated radiotherapy" generally refers to radiotherapy having doses that are divided into multiple fractions (or multiple sessions), where one fraction (or one session) may include the same dose or a different dose than another fraction (or one session).

[0093] As used herein, the term "platinum-based compound" generally refers to an agent (e.g., a chemotherapeutic agent) that includes platinum or a derivative thereof. Examples of platinum-based compounds can include carboplatin, cisplatin, and oxaliplatin.

[0094] A composition comprising an immunoconjugate and a cytotoxic substance In one aspect, the present disclosure provides a composition comprising an immunoconjugate and a cytotoxic agent. The immunoconjugate 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit and may include, The first Fc subunit associates with the second Fc subunit to form a dimer. The one or more interleukins may be fused to the Fc domain. The cytotoxic agent may induce immunogenic cell death.

[0095] For an immunoconjugate according to the present disclosure, at least one of one or more interleukins (e.g., IL10) may be fused (e.g., in-frame) to the amino-terminal amino acid of the Fc domain. The immunoconjugate may comprise two or more interleukins. In some embodiments, at least two of the two or more interleukins are fused to the amino-terminal amino acid of the Fc domain. In some embodiments, one or more interleukins are fused (e.g., in-frame) to the Fc domain via a peptide linker. In some embodiments, at least two of the two or more interleukins are fused (e.g., in-frame) to each other via a peptide linker to form an interleukin dimer. At least one interleukin dimer may be fused to the amino-terminal amino acid of the Fc domain. In some embodiments, the two or more interleukins are two or more copies of the same interleukin. For example, the two or more interleukins are two or more copies of IL10. Thus, in some embodiments, two IL10s are fused in-frame to each other (e.g., via a peptide linker) to form an IL10 dimer, and then the carboxy terminus of the IL10 dimer may be fused (e.g., in-frame, e.g., via a peptide linker) to the amino-terminal amino acid of the Fc domain.

[0096] The linker can be a peptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids. For example, the linker can comprise from 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids), from 1 to 15 amino acids (e.g., from 1 to 11, 12, 13, 14, 15 amino acids), from 1 to 20 amino acids, from 1 to 30 amino acids, or more. In some embodiments, the linker comprises the amino acid sequence defined in SEQ ID NO: 49.

[0097] The immunoconjugate may further comprise a targeting moiety fused to the Fc domain, which may exhibit binding specificity for a tumor antigen. The tumor antigen may be any immunogenic entity or portion thereof that is specifically expressed or present on the surface of the tumor microenvironment or tumor cells. In some embodiments, the tumor antigen is selected from the group consisting of members of the EGFR family (such as EGFR or HER2 / neu) and FAP.

[0098] The targeting moiety may be fused to the amino-terminal amino acid of the Fc domain. In some embodiments, the targeting moiety is fused to the Fc domain by a peptide linker or an immunoglobulin hinge region.

[0099] The targeting moiety may comprise the antigen-binding domain of an antibody. For example, the antigen-binding domain of the antibody may be a Fab portion, a domain antibody, or a ScFv portion. In some embodiments, the antigen-binding domain of the antibody is a Fab portion. The antibody may be selected from the group consisting of anti-EGFR antibodies, anti-HER2 antibodies, and anti-FAP antibodies.

[0100] In some embodiments, the antibody is an anti-EGFR antibody. For example, the anti-EGFR antibody may be cetuximab. In some embodiments, the targeting moiety comprises the heavy chain CDR1 - CDR3 of cetuximab, the light chain CDR1 - CDR3 of cetuximab, the heavy chain variable region of cetuximab, the light chain variable region of cetuximab, and / or the light chain of cetuximab. For example, the targeting moiety may be a Fab portion comprising both the heavy chain variable region and the light chain variable region of cetuximab.

[0101] For example, the targeting moiety may include a heavy chain CDR having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to those included in the corresponding heavy chain CDR1 to CDR3 of cetuximab. Alternatively or additionally, the targeting moiety may include a light chain CDR having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to those included in the corresponding light chain CDR1 to CDR3 of cetuximab. For example, the targeting moiety may include a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding heavy chain variable region of cetuximab. For example, the targeting moiety may include a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding light chain variable region of cetuximab. For example, the targeting moiety may include a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding light chain of cetuximab.

[0102] The heavy chain CDR1 to CDR3 of cetuximab are defined by SEQ ID NO: 60 (CDR1), SEQ ID NO: 61 (CDR2), and SEQ ID NO: 62 (CDR3), respectively. The light chain CDR1 to CDR3 of cetuximab are defined by SEQ ID NO: 56 (CDR1), SEQ ID NO: 57 (CDR2), and SEQ ID NO: 58 (CDR3), respectively. The heavy chain variable region of cetuximab is defined by SEQ ID NO: 63. The light chain variable region of cetuximab is defined by SEQ ID NO: 59.

[0103] In some embodiments, the antibody is an anti-HER2 antibody. For example, the anti-HER2 antibody can be trastuzumab or pertuzumab. In some embodiments, the targeting moiety comprises the heavy chain CDR1-CDR3 of trastuzumab or pertuzumab, the light chain CDR1-CDR3 of trastuzumab or pertuzumab, the heavy chain variable region of trastuzumab or pertuzumab, the light chain variable region of trastuzumab or pertuzumab, and / or the light chain of trastuzumab or pertuzumab. For example, the targeting moiety may be a Fab portion comprising both the heavy chain variable region and the light chain variable region of trastuzumab or pertuzumab.

[0104] For example, the targeting moiety may comprise a heavy chain CDR having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding heavy chain CDR1-CDR3 of trastuzumab or pertuzumab. Alternatively, or additionally, the targeting moiety may comprise a light chain CDR having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding light chain CDR1-CDR3 of trastuzumab or pertuzumab. For example, the targeting moiety may comprise a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding heavy chain variable region of trastuzumab or pertuzumab. For example, the targeting moiety may comprise a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding light chain variable region of trastuzumab or pertuzumab. For example, the targeting moiety may comprise a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding light chain of trastuzumab or pertuzumab.

[0105] The heavy chain CDR1 to CDR3 of trastuzumab are defined by SEQ ID NO: 68 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 70 (CDR3), respectively. The light chain CDR1 to CDR3 of trastuzumab are defined by SEQ ID NO: 64 (CDR1), SEQ ID NO: 65 (CDR2), and SEQ ID NO: 66 (CDR3), respectively. The heavy chain variable region of trastuzumab is defined by SEQ ID NO: 71. The light chain variable region of trastuzumab is defined by SEQ ID NO: 67.

[0106] The heavy chain CDR1 to CDR3 of pertuzumab are defined by SEQ ID NO: 88 (CDR1), SEQ ID NO: 89 (CDR2), and SEQ ID NO: 90 (CDR3), respectively. The light chain CDR1 to CDR3 of pertuzumab are defined by SEQ ID NO: 84 (CDR1), SEQ ID NO: 85 (CDR2), and SEQ ID NO: 86 (CDR3), respectively. The heavy chain variable region of pertuzumab is defined by SEQ ID NO: 91. The light chain variable region of pertuzumab is defined by SEQ ID NO: 87.

[0107] In some embodiments, the antibody is an anti-FAP antibody. For example, the anti-FAP antibody may be 28H1. In some embodiments, the targeting moiety comprises the heavy chain CDR1 to CDR3 of 28H1, the light chain CDR1 to CDR3 of 28H1, the heavy chain variable region of 28H1, the light chain variable region of 28H1, and / or the light chain of 28H1. For example, the targeting moiety may be a Fab portion comprising both the heavy chain variable region and the light chain variable region of 28H1.

[0108] For example, the targeting portion may include a heavy chain CDR having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding heavy chain CDR1 to CDR3 of 28H1. Alternatively, or additionally, the targeting portion may include a light chain CDR having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding light chain CDR1 to CDR3 of 28H1. For example, the targeting portion may include a heavy chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding heavy chain variable region of 28H1. For example, the targeting portion may include a light chain variable region having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding light chain variable region of 28H1. For example, the targeting portion may include a light chain having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that included in the corresponding light chain of 28H1.

[0109] The heavy chain CDR1 to CDR3 of 28H1 are defined by SEQ ID NO: 76 (CDR1), SEQ ID NO: 77 (CDR2), and SEQ ID NO: 78 (CDR3), respectively. The light chain CDR1 to CDR3 of 28H1 are defined by SEQ ID NO: 72 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 74 (CDR3), respectively. The heavy chain variable region of 28H1 is defined by SEQ ID NO: 79. The light chain variable region of 28H1 is defined by SEQ ID NO: 75.

[0110] For an immunoconjugate according to the present disclosure, the Fc domain can be an IgG Fc domain. The IgG can be selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG is human IgG1 and the Fc domain is a human IgG1 Fc domain (wild-type or modified).

[0111] In some embodiments, the immunoconjugate is a proteinaceous homodimer consisting of two identical members. The two identical members can each include one or more interleukins (e.g., IL10) fused to a subunit of the Fc domain (e.g., in-frame, e.g., via a peptide linker). For example, the carboxy terminus of one or more interleukins can be fused to the amino-terminal amino acid of the Fc subunit. In some embodiments, the carboxy terminus of one interleukin (e.g., IL10) is fused in-frame to the amino-terminal amino acid of one of the two Fc subunits to form one member of the homodimer, and two such identical members associate with each other via the interaction between the two Fc subunits to form a homodimer (e.g., as shown in FIG. 8A). In some embodiments, two interleukins (e.g., two IL10s) are fused in-frame to each other (e.g., via a peptide linker) to form an interleukin dimer, and then the carboxy terminus of the interleukin dimer is fused in-frame to the amino-terminal amino acid of one of the two Fc subunits to form one member of the homodimer, and two such identical members associate with each other via the interaction between the two Fc subunits to form a homodimer.

[0112] In some embodiments, the immunoconjugate is a proteinaceous heterodimer comprising a first member and a second member different from the first member, the first member comprising a first Fc subunit, the second member comprising one or more interleukins fused to a second Fc subunit, and the first Fc subunit associating with the second Fc subunit to form a heterodimer.

[0113] In some embodiments, in the second member, at least one of the one or more interleukins is fused to the amino-terminal amino acid of the second Fc subunit.

[0114] In some embodiments, in the second member, at least two of the one or more interleukins are fused to each other to form an interleukin dimer, and the interleukin dimer is further fused to the amino-terminal amino acid of the second Fc subunit. For example, two IL10s are fused in-frame with each other (e.g., via a peptide linker to form an IL10 dimer), and then in-frame fused to the second Fc subunit to form the second member of the proteinaceous heterodimer. For example, the carboxy terminus of the IL10 dimer can be fused to the amino-terminal amino acid of the second Fc subunit (e.g., as shown in FIGS. 8B and 8C).

[0115] For example, the second member of the immunoconjugate can be a fusion protein, and the second Fc subunit can be fused in-frame to the interleukin. In some embodiments, the carboxy terminus of the interleukin(s) is directly or indirectly fused to the amino terminus of the second Fc subunit to form a fusion protein. In some embodiments, the second Fc subunit is fused in-frame to the interleukin(s) via a peptide linker.

[0116] The peptide linker according to the present disclosure may be, for example, a synthetic amino acid sequence that connects or links two polypeptide sequences via a peptide bond. In some embodiments, the linker is a peptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids. For example, the linker may comprise from 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids), from 1 to 15 amino acids (e.g., 1 to 11, 12, 13, 14, 15 amino acids), from 1 to 20 amino acids, from 1 to 30 amino acids, or more. In some embodiments, the linker comprises the amino acid sequence defined in SEQ ID NO: 41. In some embodiments, the linker is resistant or substantially resistant to proteolysis.

[0117] In some embodiments, the first member further comprises a targeting moiety fused to the first Fc subunit. For example, the targeting moiety can be fused to the amino-terminal amino acid of the first Fc subunit. For example, the first member can comprise the Fab portion of an antibody, and the carboxy terminus thereof (e.g., the carboxy terminus of the heavy chain portion such as the CH1 domain or the hinge region) is fused to the amino-terminal amino acid of the first Fc subunit (e.g., as shown in FIG. 8C).

[0118] In some embodiments, the proteinaceous heterodimer does not comprise a targeting moiety. For example, the first member may comprise only the first Fc subunit (e.g., as shown in FIG. 8B).

[0119] In some embodiments, the first Fc subunit is identical to the second Fc subunit (e.g., a subunit of the wild-type human IgG1 Fc domain).

[0120] In some embodiments, the first Fc subunit is different from the second Fc subunit, and the Fc domain contains modifications that promote heterodimerization between the first Fc subunit and the second Fc subunit. For example, the first Fc subunit may contain a first modification, and the second Fc subunit may contain a second modification. For example, the first modification may be in the CH3 domain of the first Fc subunit, and the second modification may be in the CH3 domain of the second Fc subunit. For example, the first modification and / or the second modification are each compared to the sequence of their corresponding wild-type Fc domain.

[0121] For example, the first modification may include an amino acid substitution at position T366 and one or more amino acid substitutions at positions selected from the group consisting of Y349, F405, K409, D399, K360, Q347, K392, and S354, and the positions of the amino acids are determined according to the EU index of the KABAT number.

[0122] In some embodiments, the first modification includes an amino acid substitution at position T366 and one or more amino acid substitutions at positions selected from the group consisting of Y349, F405, K409, D399, K360, Q347, K392, and S354, and the positions of the amino acids are determined according to the EU index of the KABAT number.

[0123] For example, the amino acid substitutions included in the first modification may be selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D, and T366W.

[0124] In some embodiments, the first modification includes 2 to 5 amino acid substitutions.

[0125] In some embodiments, the first modification is 1) Y349 and T366; 2) Y349, T366, and F405; 3) Y349, T366, and K409; 4) Y349, T366, F405, K360, and Q347; 5) Y349, T366, F405, and Q347; 6) Y349, T366, K409, K360, and Q347; 7) Y349, T366, K409, and Q347; 8) T366, K409, and K392; 9) T366 and K409; 10) T366, K409, Y349, and S354; 11) T366 and F405; 12) T366, F405, and D399; and 13) an amino acid substitution of a position group selected from any of the groups of T366, F405, Y349, and S354, wherein the positions of the amino acids are determined according to the EU index of the KABAT number.

[0126] In some embodiments, the first modification is 1) Y349C and T366W; 2) Y349C, T366W, and F405K; 3) Y349C, T366W, and K409E; 4) Y349C, T366W, and K409A; 5) Y349C, T366W, F405K, K360E, and Q347E; 6) Y349C, T366W, F405K, and Q347R; 7) Y349C, T366W, K409A, K360E, and Q347E; 8) Y349C, T366W, K409A, and Q347R; 9) T366W, K409A, and K392D; 10) T366W and K409A; 11) T366W, K409A, and Y349D; 12) T366W, K409A, Y349D, and S354D; 13) T366W and F405K; 14) T366W, F405K, and D399S; 15) T366W, F405K, and Y349D; and 16) a group of amino acid substitutions selected from any of the groups of 16) T366W, F405K, Y349D, and S354D, wherein the positions of the amino acids are determined according to the EU index of the KABAT number.

[0127] In some embodiments, the second modification comprises amino acid substitutions at positions T366, L368, and Y407 and amino acid substitutions at one or more positions selected from the group consisting of D356, D399, E357, F405, K360, K392, K409, and Q347, wherein the positions of the amino acids are determined according to the EU index of the KABAT number.

[0128] In some embodiments, the amino acid substitutions included in the second modification are selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A, and Y407V.

[0129] In some embodiments, the second modification comprises amino acid substitutions at positions 4 to 6.

[0130] In some embodiments, the second modification is 1) D356, T366, L368, Y407, and F405; 2) D356, T366, L368, and Y407; 3) D356, T366, L368, Y407, and Q347; 4) D356, T366, L368, Y407, K360, and Q347; 5) D356, T366, L368, Y407, F405, and Q347; 6) D356, T366, L368, Y407, F405, K360, and Q347; 7) T366, L368, Y407, D399, and F405; 8) T366, L368, Y407, and F405; 9) T366, L368, Y407, F405, and E357; 10) T366, L368, Y407, and K409; 11) T366, L368, Y407, K409, and K392; and 12) an amino acid substitution of a position group selected from any of the groups of T366, L368, Y407, K409, and E357, wherein the positions of the amino acids are determined according to the EU index of the KABAT number.

[0131] In some embodiments, the second modification is 1) D356C, T366S, L368A, Y407V, and F405K; 2) D356C, T366S, L368A, and Y407V; 3) D356C, T366S, L368A, Y407V, and Q347R; 4) D356C, T366S, L368A, Y407V, K360E, and Q347E; 5) D356C, T366S, L368A, Y407V, F405K, and Q347R; 6) D356C, T366S, L368A, Y407V, F405K, K360E, and Q347E; 7) T366S, L368A, Y407V, D399S, and F405K; 8) T366S, L368G, Y407A, and F405K; 9) T366S, L368A, Y407V, F405K, and E357A; 10) T366S, L368A, Y407V, and K409A; 11) T366S, L368A, Y407V, K409A, and K392D; 12) T366S, L368G, Y407A, and K409A; 13) an amino acid substitution group selected from any of the groups of T366S, L368A, Y407V, K409A, and E357A, wherein the positions of the amino acids are determined according to the EU index of the KABAT number.

[0132] In some embodiments, the first Fc subunit comprises a first modification, the second Fc subunit comprises a second modification, and the first modification and the second modification are 1) First modification: Y349 and T366, and Second modification: D356, T366, L368, Y407, and F405; 2) First modification: Y349, T366, and F405, and Second modification: D356, T366, L368, and Y407; 3) First modification: Y349, T366, and K409, and Second modification: D356, T366, L368, Y407, and F405; 4) First modification: Y349, T366, F405, K360, and Q347, and Second modification: D356, T366, L368, Y407, and Q347; 5) First modification: Y349, T366, F405, and Q347, and Second modification: D356, T366, L368, Y407, K360, and Q347; 6) First modification: Y349, T366, K409, K360, and Q347, and Second modification: D356, T366, L368, Y407, F405, and Q347; 7) First modification: Y349, T366, K409, and Q347, and Second modification: D356, T366, L368, Y407, F405, K360, and Q347; 8) First modification: T366, K409, and K392, and Second modification: T366, L368, Y407, D399, and F405; 9) First modification: T366 and K409, and Second modification: T366, L368, Y407, and F405; 10) First modification: T366, K409, and Y349, and Second modification: T366, L368, Y407, F405, and E357; 11) First modification: T366, K409, Y349, and S354, and Second modification: T366, L368, Y407, F405, and E357; 12) First modification: T366 and F405, and Second modification: T366, L368, Y407, and K409; 13) First modification: T366, F405, and D399, and Second modification: T366, L368, Y407, K409, and K392; 14) First modification: T366, F405, and Y349, and Second modification: T366, L368, Y407, K409, and E357; 15) First modification: T366, F405, Y349, and S354, and Second modification: includes amino acid substitutions of a position group selected from any of the groups of T366, L368, Y407, K409, and E357, and the positions of the amino acids are determined according to the EU index of the KABAT number.

[0133] In some embodiments, the first Fc subunit includes the first modification, the second Fc subunit includes the second modification, and the first modification and the second modification are 1) First modification: Y349C and T366W, and Second modification: D356C, T366S, L368A, Y407V, and F405K; 2) First modification: Y349C, T366W, and F405K, and Second modification: D356C, T366S, L368A, and Y407V; 3) First modification: Y349C, T366W, and K409E, and Second modification: D356C, T366S, L368A, Y407V, and F405K; 4) First modification: Y349C, T366W, and K409A, and Second modification: D356C, T366S, L368A, Y407V, and F405K; 5) First modification: Y349C, T366W, F405K, K360E, and Q347E, and Second modification: D356C, T366S, L368A, Y407V, and Q347R; 6) First modification: Y349C, T366W, F405K, and Q347R, and Second modification: D356C, T366S, L368A, Y407V, K360E, and Q347E; 7) First modification: Y349C, T366W, K409A, K360E, and Q347E, and Second modification: D356C, T366S, L368A, Y407V, F405K, and Q347R; 8) First modification: Y349C, T366W, K409A, and Q347R, and Second modification: D356C, T366S, L368A, Y407V, F405K, K360E, and Q347E; 9) First modification: T366W, K409A, and K392D, and Second modification: T366S, L368A, Y407V, D399S, and F405K; 10) First modification: T366W and K409A, and Second modification: T366S, L368G, Y407A, and F405K; 11) First modification: T366W, K409A, and Y349D, and Second modification: T366S, L368A, Y407V, F405K, and E357A; 12) First modification: T366W, K409A, Y349D, and S354D, and Second modification: T366S, L368A, Y407V, F405K, and E357A; 13) First modification: T366W and F405K, and Second modification: T366S, L368A, Y407V, and K409A; 14) First modification: T366W, F405K, and D399S, and Second modification: T366S, L368A, Y407V, K409A, and K392D; 15) First modification: T366W and F405K, and Second modification: T366S, L368G, Y407A, and K409A; 16) First modification: T366W, F405K, and Y349D, and Second modification: T366S, L368A, Y407V, K409A, and E357A; 17) First modification: T366W, F405K, Y349D, and S354D, and Second modification: comprises a group of amino acid substitutions selected from any of the group of T366S, L368A, Y407V, K409A, and E357A, wherein the positions of said amino acids are determined according to the EU index of the KABAT number.

[0134] In some embodiments, the first Fc subunit comprises the first modification, the second Fc subunit comprises the second modification, the first modification comprises the amino acid substitutions T366W and K409A, the second modification comprises the amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the positions of said amino acids are determined according to the EU index of the KABAT number.

[0135] For example, the amino acid sequence of the first Fc subunit may be selected from SEQ ID NO: 39, SEQ ID NO: 43, or SEQ ID NO: 47. The amino acid sequence of the interleukin may be as defined in SEQ ID NO: 50 or SEQ ID NO: 52. The amino acid sequence of the second Fc subunit may be selected from SEQ ID NO: 50.

[0136] In some embodiments, the immunoconjugate of the present disclosure is a proteinaceous heterodimer, and the amino acid sequence of the second member may be selected from SEQ ID NO: 50.

[0137] The cytotoxic substance capable of inducing immunogenic cell death may be selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, platinum-based compounds, and radioactive substances or radiotherapy.

[0138] In some embodiments, the cytotoxic substance or cytotoxic therapy capable of inducing immunogenic cell death comprises an alkylating agent selected from cyclophosphamide.

[0139] In some embodiments, the cytotoxic substance or cytotoxic therapy capable of inducing immunogenic cell death comprises a platinum-based compound selected from the group consisting of carboplatin, cisplatin, and oxaliplatin.

[0140] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises an antimetabolite selected from the group consisting of capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0141] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises an anthracycline selected from the group consisting of bleomycin, doxorubicin, epirubicin, daunorubicin, idarubicin, valrubicin, and mitomycin-C. In some embodiments, the cytotoxic substance or cytotoxic therapy capable of inducing immunogenic cell death comprises doxorubicin.

[0142] In some embodiments, the cytotoxic substance capable of inducing immunogenic cell death comprises a plant alkaloid selected from the group consisting of taxane, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0143] The radioactive substance can be a substance capable of emitting X-rays and / or gamma rays (e.g., a radioactive isotope or containing the same).

[0144] In some embodiments, the radioactive substance can emit X-rays for at least one dose. Each X-ray irradiation can be at a dose of about 50 Gy or less (e.g., about 45 Gy or less, about 40 Gy or less, about 35 Gy or less, about 30 Gy or less, about 25 Gy or less, about 20 Gy or less, about 15 Gy or less, about 14 Gy or less, about 13 Gy or less, about 12 Gy or less, about 11 Gy or less, about 10 Gy or less, about 9 Gy or less, about 8 Gy or less, about 7 Gy or less, about 6 Gy or less, about 5 Gy or less, or about 4 Gy or less). In some embodiments, each X-ray irradiation is at a dose of about 6 Gy to 15 Gy, e.g., 6 to 14 Gy, 6 Gy to 13 Gy, 6 Gy to 12 Gy, 6 Gy to 11 Gy, 6 Gy to 10 Gy, 6 Gy to 9 Gy, 6 Gy to 8 Gy, 6 Gy to 7 Gy, 7 Gy to 15 Gy, 7 Gy to 14 Gy, 7 Gy to 13 Gy, 7 Gy to 12 Gy, 7 Gy to 11 Gy, 7 Gy to 10 Gy, 7 Gy to 9 Gy, or 7 Gy to 8 Gy.

[0145] In some embodiments, the radioactive substance can emit gamma rays for at least one dose. Each gamma-ray irradiation can be at a dose of about 50 Gy or less (e.g., about 45 Gy or less, about 40 Gy or less, about 35 Gy or less, about 30 Gy or less, about 25 Gy or less, about 20 Gy or less, about 15 Gy or less, about 14 Gy or less, about 13 Gy or less, about 12 Gy or less, about 11 Gy or less, about 10 Gy or less, about 9 Gy or less, about 8 Gy or less, about 7 Gy or less, about 6 Gy or less, about 5 Gy or less, or about 4 Gy or less). In some embodiments, each gamma-ray irradiation is at a dose of about 6 Gy to 15 Gy, e.g., 6 Gy to 14 Gy, 6 Gy to 13 Gy, 6 Gy to 12 Gy, 6 Gy to 11 Gy, 6 Gy to 10 Gy, 6 Gy to 9 Gy, 6 Gy to 8 Gy, 6 Gy to 7 Gy, 7 Gy to 15 Gy, 7 Gy to 14 Gy, 7 Gy to 13 Gy, 7 Gy to 12 Gy, 7 Gy to 11 Gy, 7 Gy to 10 Gy, 7 Gy to 9 Gy, or 7 Gy to 8 Gy.

[0146] In some embodiments, the radioactive substance can emit X-rays or gamma rays in an amount of about 2 to 15 times (or divided doses) (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, or more, e.g., 2 to 14 times, 2 to 13 times, 2 to 12 times, 2 to 11 times, 2 to 10 times, 2 to 9 times, 2 to 8 times, 2 to 7 times, 2 to 6 times, 2 to 5 times, 2 to 4 times, 3 to 8 times, 3 to 7 times, or 4 to 10 times).

[0147] The different components of the composition may be independently packaged (e.g., not mixed with each other until administration) or pre-mixed and packaged in the same packaging unit.

[0148] The compositions of the present disclosure may be pharmaceutical compositions and may further contain pharmaceutically acceptable additives. Examples of pharmaceutically acceptable additives include, but are not limited to, inert solid diluents and excipients, diluents, sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers, and adjuvants.

[0149] In some embodiments, the pharmaceutical composition is formulated for oral administration, intravenous administration, intramuscular administration, in-situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration by subcutaneous repository.

[0150] The compositions of the present disclosure may contain a therapeutically effective amount of an active ingredient (e.g., an immunoconjugate and a cytotoxic substance). A therapeutically effective amount is the amount of the composition of the subject that can (at least partially) prevent and / or cure the above-mentioned condition or disorder and / or any complications thereof in a subject suffering from or at risk of developing a condition or disorder (e.g., cancer). The specific amount / concentration of the active ingredient contained may vary depending on the administration method and the needs of the patient, and can be determined, for example, based on volume, viscosity, and / or the patient's weight, etc.

[0151] Methods and uses for cancer treatment In another aspect, the present disclosure provides an immunoconjugate for use in treating cancer in combination with a cytotoxic therapy. The immunoconjugate may comprise 1) one or more interleukins and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer. The one or more interleukins may be fused to the Fc domain. The cytotoxic substance may induce immunogenic cell death.

[0152] In another aspect, the present disclosure provides the use of an immunoconjugate in combination with a cytotoxic therapy in the preparation of a medicament for treating cancer in a subject in need of treatment. The immunoconjugate may comprise 1) one or more interleukins and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer. The one or more interleukins may be fused to the Fc domain. The cytotoxic substance may induce immunogenic cell death.

[0153] In a further aspect, the present disclosure provides a method of treating cancer in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of (a) an immunoconjugate in combination with a therapeutically effective amount of (b) a cytotoxic therapy. The immunoconjugate can comprise 1) one or more interleukins and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer. The one or more interleukins may be fused to the Fc domain. The cytotoxic agent can induce immunogenic cell death.

[0154] The immunoconjugate is as defined in other parts of the present disclosure. For example, the immunoconjugate may be included in the compositions of the present disclosure.

[0155] The cytotoxic therapy capable of inducing immunogenic cell death can be selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, platinum-based compounds, and radioactive substances or radiation therapy.

[0156] In some embodiments, the cytotoxic therapy capable of inducing immunogenic cell death comprises an alkylating agent selected from the group consisting of cyclophosphamide.

[0157] In some embodiments, the cytotoxic therapy capable of inducing immunogenic cell death comprises an antimetabolite selected from the group consisting of capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0158] In some embodiments, the cytotoxic therapy capable of inducing immunogenic cell death comprises an anthracycline selected from the group consisting of bleomycin, doxorubicin, epirubicin, daunorubicin, idarubicin, valrubicin, and mitomycin-C. In some embodiments, the cytotoxic agent or cytotoxic therapy capable of inducing immunogenic cell death comprises doxorubicin.

[0159] In some embodiments, the cytotoxic therapy capable of inducing immunogenic cell death includes plant alkaloids selected from the group consisting of taxane, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0160] In some embodiments, the cytotoxic therapy capable of inducing immunogenic cell death includes platinum-based compounds selected from the group consisting of carboplatin, cisplatin, and oxaliplatin.

[0161] In some embodiments, the cytotoxic therapy capable of inducing immunogenic cell death includes at least one radiotherapy. The radiotherapy may include X-ray irradiation and / or gamma-ray irradiation. The radiotherapy may be fractionated radiotherapy, for example, radiotherapy applied in multiple sessions or as multiple fractional doses.

[0162] Each X-ray irradiation per session or per fractional dose may be at a dose of about 50 Gy or less (e.g., about 45 Gy or less, about 40 Gy or less, about 35 Gy or less, about 30 Gy or less, about 25 Gy or less, about 20 Gy or less, about 15 Gy or less, about 14 Gy or less, about 13 Gy or less, about 12 Gy or less, about 11 Gy or less, about 10 Gy or less, about 9 Gy or less, about 8 Gy or less, about 7 Gy or less, about 6 Gy or less, about 5 Gy or less, or about 4 Gy or less). In some embodiments, each X-ray irradiation per session or per fractional dose is at a dose of about 6 Gy to 15 Gy, for example, 6 Gy to 14 Gy, 6 Gy to 13 Gy, 6 Gy to 12 Gy, 6 Gy to 11 Gy, 6 Gy to 10 Gy, 6 Gy to 9 Gy, 6 Gy to 8 Gy, 6 Gy to 7 Gy, 7 Gy to 15 Gy, 7 Gy to 14 Gy, 7 Gy to 13 Gy, 7 Gy to 12 Gy, 7 Gy to 11 Gy, 7 Gy to 10 Gy, 7 Gy to 9 Gy, or 7 Gy to 8 Gy.

[0163] In some embodiments, a single radiotherapy is used, and the radiation irradiation is X-ray irradiation or gamma-ray irradiation with a dose of about 5 Gy to 15 Gy (for example, 6 Gy to 15 Gy, 7 Gy to 15 Gy, 7 Gy to 10 Gy, or 6 Gy to 12 Gy). In some embodiments, radiotherapy with 4 to 10 sessions or fractionated doses is used, and the radiation irradiation is X-ray irradiation (for example, fractionated X-ray irradiation) or gamma-ray irradiation (for example, fractionated gamma-ray irradiation), and each session includes a dose of about 5 Gy to 15 Gy (for example, 6 Gy to 15 Gy, 7 Gy to 15 Gy, 7 Gy to 10 Gy, or 6 Gy to 12 Gy).

[0164] In some embodiments, a subject may be administered a cytotoxic therapy two or more times (for example, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times or more). For example, a subject may be administered a cytotoxic therapy 2 to 15 times, 2 to 14 times, 2 to 13 times, 2 to 12 times, 2 to 11 times, 2 to 10 times, 2 to 9 times, 2 to 8 times, 3 to 10 times, 4 to 10 times, or 4 to 12 times.

[0165] The immunoconjugate may be administered to the subject after the application of the cytotoxic therapy. In some embodiments, a subject is administered a cytotoxic therapy two or more times, and the immunoconjugate is administered to the subject after the application of the last (or final) session of the cytotoxic therapy.

[0166] For example, a subject may be administered a cytotoxic therapy 2 to 15 times (for example, 2 to 14 times, 2 to 13 times, 2 to 12 times, 2 to 11 times, 2 to 10 times, 2 to 9 times, 2 to 8 times, 3 to 10 times, 4 to 10 times, or 4 to 12 times), and the immunoconjugate may be administered to the subject after the application of the last (or final) session of the cytotoxic therapy.

[0167] The immunoconjugate may be administered to a subject within 10 days (e.g., within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day, or immediately) after the application of the cytotoxic therapy (e.g., after the last administration of the cytotoxic agent). In some embodiments, the immunoconjugate is administered to the subject within 0 to 7 days (e.g., 0 to 6 days, 0 to 5 days, 0 to 4 days, 0 to 3 days, 0 to 2 days, or 0 to 1 day) after the application of the cytotoxic therapy.

[0168] The immunoconjugate may be administered to the subject two or more times (e.g., at least two times, at least three times, at least four or more times).

[0169] The cancer may include solid tumors. For example, the cancer may be selected from the group consisting of B cell lymphoma, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, esophageal cancer, cervical cancer, melanoma, cancer of the uterus or endometrium, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, biliary tract cancer, cancer of the small intestine or appendix, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, liposarcoma, testicular cancer, and malignant fibrous histiocytoma.

[0170] In some embodiments, the cancer is present in a subject's body and is, for example, a cancer or cancer cell within a human or non-human animal (e.g., a mammal). In some embodiments, the mammal is a human. In some embodiments, the mammal is a mouse, rat, cat, dog, rabbit, pig, sheep, horse, cow, goat, shrew, hamster, guinea pig, monkey, or any other mammal. Many such mammals may be subjects known in the art as preclinical models of specific diseases or disorders, including solid tumors and / or other cancers (e.g., Talmadge et al., 2007 Am. J. Pathol. 170:793, Kerbel, 2003, Canc. Biol. Therap. 2(4 Suppl 1):S134, Man et al., 2007 Canc. Met. Rev. 26:737, Cespedes et al., 2006, Clin. TransL Oncol. 8:318).

[0171] The present disclosure also includes the following embodiments.

[0172] 1. A composition comprising an immunoconjugate and a cytotoxic agent, wherein the immunoconjugate comprises 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer, the one or more interleukins are fused to the Fc domain, and the cytotoxic agent is capable of inducing immunogenic cell death. Composition.

[0173] 2. The composition according to embodiment 1, wherein at least one of the one or more interleukins is fused to the amino-terminal amino acid of the Fc domain.

[0174] 3. The composition according to embodiment 1 or 2, wherein the immunoconjugate comprises two or more interleukins.

[0175] 4. The composition according to embodiment 3, wherein at least two of the two or more interleukins are fused to the amino-terminal amino acids of the Fc domain.

[0176] 5. The composition according to any one of embodiments 1 to 4, wherein one or more of the above interleukins are fused to the Fc domain via a peptide linker.

[0177] 6. The composition according to any one of embodiments 3 to 5, wherein at least two of the two or more interleukins are fused to each other via a peptide linker to form an interleukin dimer.

[0178] 7. The composition according to embodiment 6, wherein at least one of the interleukin dimers is fused to the amino-terminal amino acids of the Fc domain.

[0179] 8. The composition according to any one of embodiments 3 to 7, wherein the two or more interleukins are two or more copies of the same interleukin.

[0180] 9. The composition according to embodiment 8, wherein the two or more interleukins are two or more copies of IL10.

[0181] 10. The composition according to any one of embodiments 1 to 9, wherein the immunoconjugate further comprises a targeting moiety fused to the Fc domain, and the targeting moiety exhibits binding specificity for a tumor antigen.

[0182] 11. The composition according to embodiment 10, wherein the targeting moiety is fused to the amino-terminal amino acids of the Fc domain.

[0183] 12. The composition according to embodiment 10 or 11, wherein the targeting moiety is fused to the Fc domain via a peptide linker or an immunoglobulin hinge region.

[0184] 13. The composition according to any one of embodiments 10 to 12, wherein the targeting moiety comprises an antigen-binding domain of an antibody.

[0185] 14. The composition according to embodiment 13, wherein the antigen-binding domain of the antibody is a Fab portion.

[0186] 15. The composition according to any one of embodiments 10 to 14, wherein the tumor antigen is selected from the group consisting of EGFR, HER2 / neu, and FAP.

[0187] 16. The composition according to any one of embodiments 10 to 15, wherein the targeting moiety comprises an antigen-binding domain of an antibody, and the antibody is selected from the group consisting of an anti-EGFR antibody, an anti-HER2 antibody, and an anti-FAP antibody.

[0188] 17. The composition according to embodiment 16, wherein the antibody is an anti-EGFR antibody.

[0189] 18. The composition according to embodiment 17, wherein the anti-EGFR antibody is cetuximab.

[0190] 19. The composition according to embodiment 18, wherein the targeting moiety comprises heavy chain CDR1 to CDR3 of cetuximab.

[0191] 20. The composition according to embodiment 18 or 19, wherein the targeting moiety comprises light chain CDR1 to CDR3 of cetuximab.

[0192] 21. The composition according to any one of embodiments 18 to 20, wherein the targeting moiety comprises the heavy chain variable region of cetuximab.

[0193] 22. The composition according to any one of embodiments 18 to 21, wherein the targeting moiety comprises the light chain variable region of cetuximab.

[0194] 23. The composition according to embodiment 16, wherein the antibody is an anti-HER2 antibody.

[0195] 24. The composition according to embodiment 23, wherein the anti-HER2 antibody is trastuzumab.

[0196] 25. The composition according to embodiment 24, wherein the targeting moiety comprises the heavy chain CDR1-CDR3 of trastuzumab.

[0197] 26. The composition according to embodiment 24 or 25, wherein the targeting moiety comprises the light chain CDR1-CDR3 of trastuzumab.

[0198] 27. The composition according to any one of embodiments 24 to 26, wherein the targeting moiety comprises the heavy chain variable region of trastuzumab.

[0199] 28. The composition according to any one of embodiments 24 to 27, wherein the targeting moiety comprises the light chain variable region of trastuzumab.

[0200] 29. The composition according to embodiment 16, wherein the antibody is an anti-FAP antibody.

[0201] 30. The composition according to embodiment 29, wherein the anti-FAP antibody is 28H1.

[0202] 31. The composition according to embodiment 30, wherein the targeting moiety comprises the heavy chain CDR1-CDR3 of 28H1.

[0203] 32. The composition according to embodiment 30 or 31, wherein the targeting moiety comprises the light chain CDR1-CDR3 of 28H1.

[0204] 33. The composition according to any one of embodiments 30 to 32, wherein the targeting moiety comprises the heavy chain variable region of 28H1.

[0205] 34. The composition according to any one of embodiments 30 to 33, wherein the targeting moiety comprises the light chain variable region of 28H1.

[0206] 35. The composition according to any one of embodiments 1 to 34, wherein the Fc domain is an IgG Fc domain.

[0207] 36. The composition according to embodiment 35, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0208] 37. The composition according to embodiment 36, wherein the IgG is human IgG1.

[0209] 38. The immunoconjugate is an immunoconjugate comprising a first member and a second member different from the first member, the first member comprises the first Fc subunit, the second member comprises the one or more interleukins fused to the second Fc subunit, the first Fc subunit associates with the second Fc subunit to form the heterodimer, The composition according to any one of embodiments 1 to 37.

[0210] 39. The composition according to embodiment 38, wherein in the second member, at least one of the one or more interleukins is fused to the amino-terminal amino acid of the second Fc subunit.

[0211] 40. The composition according to embodiment 39, wherein in the second member, at least two of the one or more interleukins are fused to each other to form an interleukin dimer, and the interleukin dimer is further fused to the amino-terminal amino acid of the second Fc subunit.

[0212] 41. The composition according to any one of embodiments 38 to 40, wherein the first member further comprises the targeting moiety fused to the first Fc subunit.

[0213] 42. The composition according to embodiment 41, wherein in the first member, the targeting moiety is fused to the amino-terminal amino acid of the first Fc subunit.

[0214] 43. The composition according to any one of embodiments 38 to 40, wherein the immunoconjugate does not contain any targeting moiety.

[0215] 44. The composition according to any one of embodiments 1 to 43, wherein the first Fc subunit is different from the second Fc subunit, and the Fc domain contains a modification that promotes heterodimerization between the first Fc subunit and the second Fc subunit.

[0216] 45. The composition according to embodiment 44, wherein the first Fc subunit contains a first modification and the second Fc subunit contains a second modification.

[0217] 46. The composition according to embodiment 45, wherein the first modification includes an amino acid substitution at position T366 and one or more amino acid substitutions selected from the group consisting of Y349, F405, K409, D399, K360, Q347, K392, and S354, and the positions of the amino acids are determined according to the EU index of the KABAT number.

[0218] 47. The composition according to embodiment 46, wherein the amino acid substitutions included in the first modification are selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D, and T366W.

[0219] 48. The composition according to any one of embodiments 45 to 47, wherein the first modification includes 2 to 5 amino acid substitutions.

[0220] 49. The first modification is 1) Y349 and T366; 2) Y349, T366, and F405; 3) Y349, T366, and K409; 4) Y349, T366, F405, K360, and Q347; 5) Y349, T366, F405, and Q347; 6) Y349, T366, K409, K360, and Q347; 7) Y349, T366, K409, and Q347; 8) T366, K409, and K392; 9) T366 and K409; 10) T366, K409, Y349, and S354; 11) T366 and F405; 12) T366, F405, and D399; and 13) A composition according to any one of embodiments 45 to 48, comprising an amino acid substitution of a position group selected from any of the groups of T366, F405, Y349, and S354, wherein the positions of the amino acids are determined according to the EU index of the KABAT number.

[0221] 50. The above first modification is 1) Y349C and T366W; 2) Y349C, T366W, and F405K; 3) Y349C, T366W, and K409E; 4) Y349C, T366W, and K409A; 5) Y349C, T366W, F405K, K360E, and Q347E; 6) Y349C, T366W, F405K, and Q347R; 7) Y349C, T366W, K409A, K360E, and Q347E; 8) Y349C, T366W, K409A, and Q347R; 9) T366W, K409A, and K392D; 10) T366W, and K409A; 11) T366W, K409A, and Y349D; 12) T366W, K409A, Y349D, and S354D; 13) T366W and F405K; 14) T366W, F405K, and D399S; 15) T366W, F405K, and Y349D; and A composition according to any one of embodiments 45 to 49, comprising a group of amino acid substitutions selected from any of the groups of T366W, F405K, Y349D, and S354D, wherein the positions of the amino acids are determined according to the EU index of the KABAT number.

[0222] 51. A composition according to any one of embodiments 45 to 50, wherein the second modification comprises amino acid substitutions at positions T366, L368, and Y407 and amino acid substitutions at one or more positions selected from the group consisting of D356, D399, E357, F405, K360, K392, K409, and Q347, and the positions of the amino acids are determined according to the EU index of the KABAT number.

[0223] 52. A composition according to embodiment 51, wherein the amino acid substitutions comprised in the second modification are selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A, and Y407V.

[0224] 53. A composition according to any one of embodiments 45 to 52, wherein the second modification comprises amino acid substitutions at positions 4 to 6.

[0225] 54. The second modification is 1) D356, T366, L368, Y407, and F405; 2) D356, T366, L368, and Y407; 3) D356, T366, L368, Y407, and Q347; 4) D356, T366, L368, Y407, K360, and Q347; 5) D356, T366, L368, Y407, F405, and Q347; 6) D356, T366, L368, Y407, F405, K360, and Q347; 7) T366, L368, Y407, D399, and F405; 8) T366, L368, Y407, and F405; 9) T366, L368, Y407, F405, and E357; 10) T366, L368, Y407, and K409; 11) T366, L368, Y407, K409, and K392; and 12) a composition according to any one of embodiments 45 to 53, comprising an amino acid substitution of a position group selected from any of the groups of 12) T366, L368, Y407, K409, and E357, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0226] 55. The second modification is 1) D356C, T366S, L368A, Y407V, and F405K; 2) D356C, T366S, L368A, and Y407V; 3) D356C, T366S, L368A, Y407V, and Q347R; 4) D356C, T366S, L368A, Y407V, K360E, and Q347E; 5) D356C, T366S, L368A, Y407V, F405K, and Q347R; 6) D356C, T366S, L368A, Y407V, F405K, K360E, and Q347E; 7) T366S, L368A, Y407V, D399S, and F405K; 8) T366S, L368G, Y407A, and F405K; 9) T366S, L368A, Y407V, F405K, and E357A; 10) T366S, L368A, Y407V, and K409A; 11) T366S, L368A, Y407V, K409A, and K392D; 12) T366S, L368G, Y407A, and K409A; 13) a composition according to any one of embodiments 45 to 54, comprising a group of amino acid substitutions selected from any of the groups of 13) T366S, L368A, Y407V, K409A, and E357A, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0227] 56. The first Fc subunit includes a first modification, the second Fc subunit includes a second modification, and the first modification and the second modification are 1) First modification: Y349 and T366, and Second modification: D356, T366, L368, Y407, and F405; 2) First modification: Y349, T366, and F405, and Second modification: D356, T366, L368, and Y407; 3) First modification: Y349, T366, and K409, and Second modification: D356, T366, L368, Y407, and F405; 4) First modification: Y349, T366, F405, K360, and Q347, and Second modification: D356, T366, L368, Y407, and Q347; 5) First modification: Y349, T366, F405, and Q347, and Second modification: D356, T366, L368, Y407, K360, and Q347; 6) First modification: Y349, T366, K409, K360, and Q347, and Second modification: D356, T366, L368, Y407, F405, and Q347; 7) First modification: Y349, T366, K409, and Q347, and Second modification: D356, T366, L368, Y407, F405, K360, and Q347; 8) First modification: T366, K409, and K392, and Second modification: T366, L368, Y407, D399, and F405; 9) First modification: T366 and K409, and Second modification: T366, L368, Y407, and F405; 10) First modification: T366, K409, and Y349, and Second modification: T366, L368, Y407, F405, and E357; 11) First modification: T366, K409, Y349, and S354, and Second modification: T366, L368, Y407, F405, and E357; 12) First modification: T366 and F405, and Second modification: T366, L368, Y407, and K409; 13) First modification: T366, F405, and D399, and Second modification: T366, L368, Y407, K409, and K392; 14) First modification: T366, F405, and Y349, and Second modification: T366, L368, Y407, K409, and E357; 15) First modification: T366, F405, Y349, and S354, and Second modification: T366, L368, Y407, K409, and E357; comprising an amino acid substitution in a position group selected from any of the groups, wherein the position of the amino acid is determined according to the EU index of the KABAT number, the composition according to any one of embodiments 45 to 55.

[0228] 57. The first Fc subunit comprises a first modification, the second Fc subunit comprises a second modification, and the first modification and the second modification are 1) First modification: Y349C and T366W, and Second modification: D356C, T366S, L368A, Y407V, and F405K; 2) First modification: Y349C, T366W, and F405K, and Second modification: D356C, T366S, L368A, and Y407V; 3) First modification: Y349C, T366W, and K409E, and Second modification: D356C, T366S, L368A, Y407V, and F405K; 4) First modification: Y349C, T366W, and K409A, and Second modification: D356C, T366S, L368A, Y407V, and F405K; 5) First modification: Y349C, T366W, F405K, K360E, and Q347E, and Second modification: D356C, T366S, L368A, Y407V, and Q347R; 6) First modification: Y349C, T366W, F405K, and Q347R, and Second modification: D356C, T366S, L368A, Y407V, K360E, and Q347E; 7) First modification: Y349C, T366W, K409A, K360E, and Q347E, and Second modification: D356C, T366S, L368A, Y407V, F405K, and Q347R; 8) First modification: Y349C, T366W, K409A, and Q347R, and Second modification: D356C, T366S, L368A, Y407V, F405K, K360E, and Q347E; 9) First modification: T366W, K409A, and K392D, and Second modification: T366S, L368A, Y407V, D399S, and F405K; 10) First modification: T366W and K409A, and Second modification: T366S, L368G, Y407A, and F405K; 11) First modification: T366W, K409A, and Y349D, and Second modification: T366S, L368A, Y407V, F405K, and E357A; 12) First modification: T366W, K409A, Y349D, and S354D, and Second modification: T366S, L368A, Y407V, F405K, and E357A; 13) First modification: T366W and F405K, and Second modification: T366S, L368A, Y407V, and K409A; 14) First modification: T366W, F405K, and D399S, and Second modification: T366S, L368A, Y407V, K409A, and K392D; 15) First modification: T366W and F405K, and Second modification: T366S, L368G, Y407A, and K409A; 16) First modification: T366W, F405K, and Y349D, and Second modification: T366S, L368A, Y407V, K409A, and E357A; 17) A group of amino acid substitutions selected from any of the groups of first modification: T366W, F405K, Y349D, and S354D, and second modification: T366S, L368A, Y407V, K409A, and E357A, wherein the positions of the amino acids are determined according to the EU index of the KABAT number, the composition according to any one of embodiments 45 to 56.

[0229] 58. The first Fc subunit contains the first modification, The second Fc subunit contains the second modification, The first modification includes amino acid substitutions T366W and K409A, The second modification includes amino acid substitutions T366S, L368G, Y407A, and F405K, The positions of the amino acids are determined according to the EU index of the KABAT number, the composition according to embodiment 57.

[0230] 59. The composition according to any one of embodiments 1 to 58, wherein the cytotoxic substance capable of inducing immunogenic cell death is selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, platinum-based compounds, and radioactive substances.

[0231] 60. The composition according to embodiment 59, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises an alkylating agent selected from cyclophosphamide.

[0232] 61. The composition according to embodiment 59, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises an antimetabolite selected from the group consisting of capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0233] 62. The composition according to embodiment 59, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises an anthracycline selected from the group consisting of bleomycin, doxorubicin, and mitomycin-C.

[0234] 63. The composition according to embodiment 59, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises a plant alkaloid selected from the group consisting of taxane, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0235] 64. The composition according to embodiment 59, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises a radioactive substance that emits X-rays and / or gamma rays.

[0236] 65. The composition according to embodiment 64, wherein the radioactive substance emits X-rays, and each X-ray irradiation is at a dose of about 50 Gy or less.

[0237] 66. The composition according to embodiment 65, wherein the radioactive substance emits gamma rays, and each gamma-ray irradiation is at a dose of about 50 Gy or less.

[0238] 67. The composition according to embodiment 59, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises a platinum-based compound selected from the group consisting of carboplatin, cisplatin, and oxaliplatin.

[0239] 68. An immunoconjugate used in cancer treatment in combination with cytotoxic therapy, wherein the immunoconjugate is as defined in any one of embodiments 1 to 58, and the cytotoxic therapy is capable of inducing immunogenic cell death.

[0240] 69. The immunoconjugate according to embodiment 68, wherein the cytotoxic therapy that induces immunogenic cell death is selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, platinum-based compounds, and radiation therapy.

[0241] 70. The immunoconjugate according to embodiment 69, wherein the cytotoxic therapy capable of inducing immunogenic cell death includes at least one radiation therapy.

[0242] 71. The immunoconjugate according to embodiment 70, wherein the radiation irradiation includes X-ray irradiation and / or gamma-ray irradiation.

[0243] 72. The immunoconjugate according to embodiment 71, wherein the radiation irradiation includes X-ray irradiation, and each X-ray irradiation is at a dose of about 50 Gy or less.

[0244] 73. The immunoconjugate according to embodiment 71, wherein the radiation irradiation includes gamma-ray irradiation, and each gamma-ray irradiation is at a dose of about 50 Gy or less.

[0245] 74. The immunoconjugate according to any one of embodiments 70 to 73, wherein the radiation therapy is fractionated irradiation therapy.

[0246] 75. The immunoconjugate according to embodiment 74, wherein the fractionated irradiation therapy includes 2 to 15 fractions.

[0247] 76. The immunoconjugate according to embodiment 69, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an alkylating agent selected from cyclophosphamide.

[0248] 77. The immunoconjugate according to embodiment 69, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an antimetabolite selected from the group consisting of capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0249] 78. The immunoconjugate according to embodiment 69, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises an anthracycline selected from the group consisting of bleomycin, doxorubicin, and mitomycin-C.

[0250] 79. The immunoconjugate according to embodiment 69, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises a plant alkaloid selected from the group consisting of taxane, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0251] 80. The immunoconjugate according to embodiment 69, wherein the cytotoxic substance capable of inducing immunogenic cell death comprises a platinum-based compound selected from the group consisting of carboplatin, cisplatin, and oxaliplatin.

[0252] 81. Use of an immunoconjugate in the preparation of a medicament for treating cancer in a subject in need of treatment, in combination with a cytotoxic therapy, wherein the immunoconjugate is as defined in any one of embodiments 1 to 58, and the cytotoxic therapy is capable of inducing immunogenic cell death. Use.

[0253] 82. The use according to embodiment 81, wherein the cytotoxic therapy capable of inducing immunogenic cell death is selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, platinum-based compounds, and radiotherapy.

[0254] 83. The use according to embodiment 82, wherein the cytotoxic therapy capable of inducing immunogenic cell death comprises at least one radiotherapy.

[0255] 84. The use according to embodiment 83, wherein the radiotherapy comprises X-ray irradiation and / or gamma-ray irradiation.

[0256] 85. Use according to embodiment 84, wherein the radiation irradiation includes X-ray irradiation, and each X-ray irradiation is at a dose of about 50 Gy or less.

[0257] 86. Use according to embodiment 84, wherein the radiation irradiation includes gamma-ray irradiation, and each gamma-ray irradiation is at a dose of about 50 Gy or less.

[0258] 87. Use according to any one of embodiments 83 to 86, wherein the radiotherapy is fractionated radiotherapy.

[0259] 88. Use according to embodiment 87, wherein the fractionated radiotherapy includes 2 to 15 fractions.

[0260] 89. Use according to embodiment 82, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an alkylating agent selected from cyclophosphamide.

[0261] 90. Use according to embodiment 82, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an antimetabolite selected from the group consisting of capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0262] 91. Use according to embodiment 82, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an anthracycline selected from the group consisting of bleomycin, doxorubicin, and mitomycin-C.

[0263] 92. Use according to embodiment 82, wherein the cytotoxic substance capable of inducing immunogenic cell death includes a plant alkaloid selected from the group consisting of taxane, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0264] 93. Use according to embodiment 82, wherein the cytotoxic agent capable of inducing immunogenic cell death includes a platinum-based compound selected from the group consisting of carboplatin, cisplatin, and oxaliplatin.

[0265] 94. A method for treating cancer in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of (a) an immunoconjugate in combination with a therapeutically effective amount of (b) a cytotoxic therapy, wherein the immunoconjugate is as defined in any one of Embodiments 1 to 58, and the cytotoxic therapy is capable of inducing immunogenic cell death.

[0266] 95. The method according to Embodiment 94, wherein the immunoconjugate is administered to the subject after the application of the cytotoxic therapy.

[0267] 96. The method according to Embodiment 95, wherein the immunoconjugate is administered to the subject within 10 days after the application of the cytotoxic therapy.

[0268] 97. The method according to Embodiment 95 or 96, wherein the immunoconjugate is administered to the subject within 3 days after the application of the cytotoxic therapy.

[0269] 98. The method according to any one of Embodiments 94 to 97, wherein the immunoconjugate is administered to the subject two or more times.

[0270] 99. The method according to any one of Embodiments 94 to 98, wherein the cytotoxic therapy capable of inducing immunogenic cell death is selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, platinum-based compounds, and radiation therapy.

[0271] 100. The method according to Embodiment 99, wherein the cytotoxic therapy capable of inducing immunogenic cell death comprises at least one radiation therapy.

[0272] 101. The method according to Embodiment 100, wherein the radiation therapy comprises X-ray irradiation and / or gamma-ray irradiation.

[0273] 102. The method according to Embodiment 101, wherein the radiation irradiation comprises X-ray irradiation, and each X-ray irradiation is at a dose of about 50 Gy or less.

[0274] 103. The method according to embodiment 101, wherein the radiation irradiation includes gamma-ray irradiation, and each gamma-ray irradiation is at a dose of about 50 Gy or less.

[0275] 104. The method according to any one of embodiments 100 to 103, wherein the radiation therapy is fractionated radiotherapy.

[0276] 105. The method according to embodiment 104, wherein the fractionated radiotherapy includes 2 to 15 fractions.

[0277] 106. The method according to embodiment 99, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an alkylating agent selected from cyclophosphamide.

[0278] 107. The method according to embodiment 99, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an antimetabolite selected from the group consisting of capecitabine, gemcitabine, pemetrexed, and 5-Fu.

[0279] 108. The method according to embodiment 99, wherein the cytotoxic substance capable of inducing immunogenic cell death includes an anthracycline selected from the group consisting of bleomycin, doxorubicin, and mitomycin-C.

[0280] 109. The method according to embodiment 99, wherein the cytotoxic substance capable of inducing immunogenic cell death includes a plant alkaloid selected from the group consisting of taxane, docetaxel, paclitaxel, vinblastine, vincristine, and vinorelbine.

[0281] 110. The method according to embodiment 99, wherein the cytotoxic substance capable of inducing immunogenic cell death includes a platinum-based compound selected from the group consisting of carboplatin, cisplatin, and oxaliplatin.

[0282] In this specification, various embodiments of the present invention have been shown and described. It will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used.

Example

[0283] The following examples are described to provide a complete disclosure and description of the manufacturing and usage methods of the present invention to those skilled in the art, and are not intended to limit the scope that the inventors consider to be their invention. Also, the following experiments are not intended to represent all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise specified, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or approximately atmospheric pressure. Standard abbreviations such as bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); i.m., intramuscular (into muscle); i.p., intraperitoneal (into the peritoneal cavity); s.c., subcutaneous (under the skin), etc. may be used.

[0284] Example 1 Modification and preparation of nucleic acids 1.1 Modification of Fc Amino acid modifications (such as amino acid substitutions) are made to the interfacial residues of the human IgG1 Fc domain to obtain the following group of modifications (shown in Table 1 below). In the present disclosure, chain A is also referred to as Fc9 or the first Fc subunit, and chain B is also referred to as Fc6 or the second Fc subunit:

[0285]

Table 1

[0286] Subsequently, the formation of heterodimeric proteins containing the modifications of the groups listed in Table 1 above was examined using the ScFv-Fc / Fc system as described in detail below.

[0287] First, the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region was obtained from the database Uniprot (P01857), and the wild-type human IgG1-Fc region amino acid sequence (SEQ ID NO: 30) was obtained. A polynucleotide fragment encoding wild-type human IgG1-Fc was obtained by RT-PCR from total RNA of human PBMCs (designated as SEQ ID NO: 31, the Fc gene fragment). A polynucleotide fragment encoding the mouse kappa III signal peptide (SEQ ID NO: 32) was added to the 5' end of the Fc gene by overlapping PCR and then subcloned into the vector pcDNA4 (Invitrogen, catalog V86220) to obtain a recombinant expression vector for expressing human IgG1-Fc in mammalian cells.

[0288] A nucleic acid molecule encoding the ScFv-Fc fusion protein (SEQ ID NO: 33) was synthesized, where ScFv refers to an anti-HER2 single-chain antibody, and the amino acid sequence of the ScFv-Fc fusion protein is defined in SEQ ID NO: 34. Subsequently, the ScFv-Fc gene fragment was subcloned into the vector pcDNA4 (Invitrogen, catalog V86220) to obtain a recombinant expression vector for expressing the ScFv-Fc fusion protein in mammalian cells.

[0289] In some cases, a polypeptide encoding the variable region of a camelid single-domain antibody (VhH) was fused to the N-terminus of the Fc gene fragment to obtain a fusion gene fragment (defined in SEQ ID NO: 35) encoding the fusion protein VhH-Fc (defined in SEQ ID NO: 36). This was then subcloned into the vector pcDNA4 (Invitrogen, catalog V86220) to obtain a recombinant expression vector for expressing the fusion protein VhH-Fc in mammalian cells.

[0290] Next, the amino acid modifications listed in Table 1 above were introduced into ScFv-Fc (Groups 1 to 17), VhH-Fc (Groups 9 to 12, 14, 15, and 17), and Fc gene fragments (Groups 1 to 8) by overlapping PCR, where Chain A refers to the Fc subunit of ScFv-Fc, and Chain B refers to an independent Fc subunit or the Fc subunit of VhH-Fc. The amino acid-modified gene fragments were each subcloned into the vector pcDNA4 (Invitrogen, Catalog V86220) to obtain recombinant expression vectors for expressing modified ScFv-Fc fusion proteins, modified Fc proteins, and modified VhH-Fc fusion proteins in mammalian cells.

[0291] Next, suspension-cultured HEK293 cells (ATCC CRL-1573 (trademark)) were transformed with the expression vectors constructed with PEI. For each group, the expression vector expressing Chain A (ScFv-Fc fusion protein) and the expression vector expressing Chain B (Fc protein or VhH-Fc fusion protein) were simultaneously transformed at a ratio of 1:1. After culturing for 5 to 6 days, the supernatant of the transient expression product was collected, and the expression product containing the corresponding protein heterodimer was pre-purified using Protein A affinity chromatography. The pre-purified expression products each contained ScFv-Fc / ScFv-Fc homodimer protein, Fc / Fc homodimer protein (or VhH-Fc / VhH-Fc homodimer protein), and ScFv-Fc / Fc heterodimer protein (or ScFv-Fc / VhH-Fc heterodimer protein), which were present in various ratios. Since the molecular weights of these proteins (i.e., homodimers and heterodimers) were different, the corresponding ratios could be determined according to the corresponding band intensities reflected in the non-reducing SDS-PAGE gel. The intensities were quantified, and the results are summarized in Tables 2 to 5 below.

[0292]

Table 2

[0293] [Table 3]

[0294] [Table 4]

[0295] [Table 5]

[0296] As can be seen from the above Tables 2 to 5, all modification groups promoted heterodimer formation very effectively. For illustration, the modification of group 10 (modification of chain A: T366W+K409A; modification of chain B: T366S+L368G+Y407A+F405K) was used in the following examples to generate the immunoconjugates or protein mixtures of the present disclosure.

[0297] 1.2 Preparation of anti-EGFR (cetuximab) The full-length amino acid sequences of the heavy and light chains of cetuximab (also known as Erbitux or Erb, an antibody against the epidermal growth factor receptor EGFR) were obtained, and the corresponding DNA sequences encoding these amino acid sequences were obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). Subsequently, a nucleic acid molecule encoding the light chain of cetuximab (Erb-LC) was synthesized. The amino acid sequence of Erb-LC is defined in SEQ ID NO: 37, and the corresponding polynucleotide sequence encoding it is defined in SEQ ID NO: 38. Subsequently, point mutations (T366W and K409A) were introduced into the polynucleotide sequence encoding the Fc region of the cetuximab heavy chain gene to synthesize a nucleic acid molecule encoding a modified cetuximab heavy chain (referred to herein as erb-Fc9), and the corresponding polypeptide encoded by it was named Erb-Fc9. The amino acid sequence of Erb-Fc9 is defined in SEQ ID NO: 39, and the polynucleotide sequence encoding it is defined in SEQ ID NO: 40.

[0298] 1.3 Preparation of anti-HER2 (trastuzumab) The full-length amino acid sequences of the heavy and light chains of trastuzumab were obtained according to U.S. Patent No. 7,879,325 (which is incorporated herein by reference). Subsequently, the corresponding DNA sequences encoding these amino acid sequences were obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). Next, a nucleic acid molecule encoding the light chain of trastuzumab (T-LC) was synthesized. The amino acid sequence of T-LC is defined in SEQ ID NO: 41, and the corresponding polynucleotide sequence encoding it is defined in SEQ ID NO: 42. Next, point mutations (T366W and K409A) were introduced into the polynucleotide sequence encoding the Fc region of the trastuzumab heavy chain gene, and a nucleic acid molecule encoding the modified trastuzumab heavy chain was synthesized (referred to herein as t-Fc9), and the corresponding polypeptide encoding it was named T-Fc9. The amino acid sequence of T-Fc9 is defined in SEQ ID NO: 43, and the polynucleotide sequence encoding it is defined in SEQ ID NO: 44.

[0299] 1.4 Preparation of anti-FAP (28H1) The full-length amino acid sequences of the heavy and light chains of 28H1 were obtained according to US Patent Application Publication No. 20120128591 (which is incorporated herein by reference). Subsequently, the corresponding DNA sequences encoding these amino acid sequences were obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). Next, a nucleic acid molecule encoding the light chain of 28H1 (28H1-LC) was synthesized. The amino acid sequence of 28H1-LC is defined in SEQ ID NO: 45, and the corresponding polynucleotide sequence encoding it is defined in SEQ ID NO: 46. Next, point mutations (T366W and K409A) were introduced into the polynucleotide sequence encoding the Fc region of the 28H1 heavy chain gene, and a nucleic acid molecule encoding the modified 28H1 heavy chain was synthesized (referred to herein as 28H1-Fc9), and the corresponding polypeptide encoded by it was named 28H1-Fc9. The amino acid sequence of 28H1-Fc9 is defined in SEQ ID NO: 47, and the polynucleotide sequence encoding it is defined in SEQ ID NO: 48.

[0300] 1.5 (IL10) 2 -Fc6 preparation First, the sequence information of human interleukin 10 (IL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Next, the amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) was obtained according to the amino acid sequence of the constant region (P01857) of human immunoglobulin gamma 1 (IgG1) by the protein database Uniprot. Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment. The resulting polypeptide is referred to as Fc6. Next, the linker sequence "(GGGGS)3" (SEQ ID NO: 49) was added to the N-terminus of Fc6 to obtain linker-Fc6. Then, the corresponding DNA sequence encoding it was designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). Next, the linker sequence "(GGGGS)3" (SEQ ID NO: 49) was added between two copies of IL10 to obtain (IL10)2. The polynucleotide sequence encoding (IL10)2 was added to the 5'-end of the polynucleotide sequence encoding linker-Fc6, thereby obtaining and synthesizing the polynucleotide sequence encoding the fusion protein (IL10)2-Fc6. The amino acid sequence of (IL10)2-Fc6 is defined in SEQ ID NO: 50, and the polynucleotide sequence encoding it is defined in SEQ ID NO: 51.

[0301] 1.6 Preparation of IL10-Fc First, the sequence information of human interleukin 10 (IL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Next, the amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) was obtained according to the amino acid sequence of the constant region (P01857) of human immunoglobulin gamma 1 (IgG1) from the protein database Uniprot. Next, the linker sequence "(GGGGS)3" (SEQ ID NO: 49) was added to the N-terminus of IgG1-Fc to obtain linker-Fc. Next, the corresponding DNA sequence encoding it was designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). Next, a polynucleotide encoding the fusion protein IL10-Fc was synthesized by adding the polynucleotide sequence encoding IL10 to the 5'-end of the polynucleotide sequence encoding linker-Fc. The amino acid sequence of IL10-Fc is defined in SEQ ID NO: 52, and the polynucleotide sequence encoding it is defined in SEQ ID NO: 53.

[0302] 1.7 Preparation of Fc9 The amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) was obtained according to the amino acid sequence of the constant region (P01857) of human immunoglobulin gamma 1 (IgG1) from the protein database Uniprot. Thereafter, point mutations (T366W and K409A) were introduced into the IgG1Fc fragment, and the resulting polypeptide was called Fc9. The amino acid sequence of Fc9 is defined in SEQ ID NO: 17, and the polynucleotide sequence encoding it is defined in SEQ ID NO: 54.

[0303] Example 2 Construction of recombinant plasmid The nucleic acid molecules obtained according to Example 1 (encoding Erb-Fc9, T-Fc9, 28H1-Fc9, Fc9, T-LC (trastuzumab light chain), Erb-LC (cetuximab light chain), 28H1-LC, (IL10)2-Fc6, and IL10-Fc) were digested with HindIII and EcoRI (Takara), and then subcloned into the vector pcDNA4 / myc-HisA (Invitrogen, V863-20) respectively. The obtained plasmids were verified by sequencing, and the correct recombinant plasmids were named as follows: pcDNA4-Erb-Fc9, pcDNA4-T-Fc9, pcDNA4-28H1-Fc9, pcDNA4-Fc9, pcDNA4-T-LC, pcDNA4-Erb-LC, pcDNA4-28H1-LC, pcDNA4-(IL10)2-Fc6, and pcDNA4-IL10-Fc.

[0304] Example 3 Expression and purification of immunoconjugate Two days before transfection, a suspension of 12 × 600 mL of domesticated HEK293 (ATCC, CRL-1573™) cells was prepared for transient transfection, and the cells were seeded at a density of 0.8 × 10 6 cells / ml. Two days later, a certain amount of the three cell suspensions was centrifuged and then resuspended in 600 mL of Freestyle293 culture medium.

[0305] The recombinant expression vectors obtained according to Example 2 were divided into the following groups: Group 1: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-(IL10)2-Fc6 (200 μg); Group 2: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-(IL10)2-Fc6 (200 μg); Group 3: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-(IL10)2-Fc6 (200 μg); Group 4: pcDNA4-Fc9 (200 μg) + pcDNA4-(IL10)2-Fc6 (200 μg); Group 5: pcDNA4-IL10-Fc (200 μg).

[0306] All proteins were prepared from 293F cells transiently transfected. Briefly, FreeStyle 293F cells (Invitrogen) were grown in 293F medium (Invitrogen), transfected with linearized plasmid DNA and 293Fectin reagent (Invitrogen), and grown in a shaker flask batch with a volume of 80 mL / flask to 100 mL / flask at 37 °C and 5% CO2 for 6 days. All proteins were purified by one-step protein A chromatography. The quality of each protein was determined by SDS-PAGE and SEC-HPLC. Similarly, the expression and purification results of other immunoconjugates of this application were verified and confirmed by SDS-PAGE.

[0307] The immunoconjugates derived from Groups 1 to 5 obtained in this way are named Erb-(IL10)2, Tmab-(IL10)2, 28H1-(IL10)2, Fc9-(IL10)2, and (IL10-Fc)2, respectively.

[0308] Figures 1A to 1F show the successful expression and purification of the immunoconjugates of Erb-(IL10)2, Tmab-(IL10)2, (IL10-Fc)2, and Fc9-(IL10)2. In Figure 1A, Erb-(IL10)2 (reduced) was loaded in lane 1, the marker was loaded in lane 2, and Erb-(IL10)2 (non-reduced) was loaded in lane 3. In Figure 1B, Tmab-(IL10)2 (reduced) was loaded in lane 1, the marker was loaded in lane 2, and Tmab-(IL10)2 (non-reduced) was loaded in lane 3.

[0309] In Figure 1C, (IL10-Fc)2 (original sample) was loaded into lane 1, (IL10-Fc)2 (flow-through) was loaded into lane 2, (IL10-Fc)2 (elution) was loaded into lane 3, a marker was loaded into lane 4, standard positive control BSA was loaded into lane 5, blank buffer was loaded into lane 6, lane 7 was blank, and (IL10-Fc)2 (elution, non-reduced) was loaded into lane 8.

[0310] In Figure 1D, Fc9-(IL10)2 (original sample) was loaded into lane 1, Fc9-(IL10)2 (flow-through) was loaded into lane 2, Fc9-(IL10)2 (elution) was loaded into lane 3, a marker was loaded into lane 4, standard positive control BSA was loaded into lane 5, blank buffer was loaded into lane 6, lane 7 was blank, and Fc9-(IL10)2 (elution, non-reduced) was loaded into lane 8.

[0311] Figure 1E shows the results of SEC-HPLC, indicating that the proportion of undesirable oligomers in the expression product of (IL10-Fc)2 was about 27%.

[0312] Figure 1F shows the results of SEC-HPLC, indicating that the proportion of undesirable oligomers in the expression product of Fc9-(IL10)2 was about 3.3%.

[0313] From these results, it can be seen that the production of the immunoconjugates of the present disclosure was successful. Interestingly, the expression product of Fc9-(IL10)2 has far fewer undesirable oligomers compared to the expression product of (IL10-Fc)2.

[0314] Example 4 Investigation of radiation therapy dose and tumor control 4.1 Animals and cell culture Female C57BL / 6 mice, aged 6 to 8 weeks, were obtained from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China) and raised under specific pathogen-free conditions. All animals were used in accordance with the approval of the local ethics committee. This study was approved according to the recommendations of the Guidelines for the Care and Use of Laboratory Animals (1998, Ministry of Health, People's Republic of China). Human EGFR and K b The B16-EGFR-SIY melanoma cell line expressing the binding peptide antigen SIYRYYGL (SIY, SEQ ID NO: 55) was generated in-house and grown in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin (Gibco Invitrogen).

[0315] 4.2 Tumor growth B16-EGFR-SIY melanoma cells (5×10 5 ) were subcutaneously (s.c.) inoculated into the ventral part of the mice and allowed to grow for approximately 10 days. Tumor volume was recorded with two perpendicular diameters (length and width), and calculated as V = ab 2 / 2 (where a and b are the longest and shortest diameters, respectively). According to the tumor size, the mice were randomly assigned to groups. Each C57 / BL6 mouse weighing approximately 18 g to 20 g was anesthetized by intraperitoneal (i.p.) injection of approximately 150 μl / 20 g of 1% (w / v) sodium pentobarbital. Each anesthetized mouse placed in the lateral position on the flat horizontal plane of the block was protected with a lead shield with 10 mm × 10 mm holes. Then, local radiation was performed once through the holes (electron beam irradiation, 3 Gy / min, Medical Linear Accelerators, SIEMENS Primus, Germany) in the First Affiliated Hospital of Soochow University, Department of Radiation Oncology, and the tumor volume was measured twice a week. Thereby, a B16-EGFR-SIY control tumor model was obtained.

[0316] 4.3 Radiation therapy dose and tumor control The B16-EGFR-SIY control tumors were treated with local single-fraction radiotherapy (RT) at 5 Gy, 10 Gy, 20 Gy, and 30 Gy. Additionally, a B16-EGFR-SIY control tumor model without further local single-dose irradiation was set as a control.

[0317] After 21 days of RT treatment, the B16-EGFR-SIY control tumor model grew from 123.4 ± 43.7 mm 3 to 2797.1 ± 917.8 mm 3 in volume, which was decelerated by local single-fraction irradiation RT at 5 Gy to 30 Gy (Figure 2). Figure 2 reflects the relationship between single-fraction irradiation of RT and tumor volume. With the increase in the single-dose of RT, the inhibitory effect of RT on tumors was enhanced. 30 Gy mediated almost complete disappearance of the tumors, while 5 Gy had little effect. These results suggested that different radiation regimens had direct effects that were not equivalent regarding the growth inhibition of irradiated tumors.

[0318] Example 5 Synergistic effect of RT and the immunoconjugate of the present disclosure Administration of Erb-(IL10)2 or isotype control (human IgG1) obtained in Example 3 was started 3 days after RT and administered i.p. at a dose of 1 mg / kg every 3 to 4 days for a total of 3 times (unless otherwise specified).

[0319] C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, locally irradiated on day 10, and then 200 μl of Erb-(IL10)2 (1 mg / kg) or Fc9-(IL10)2 (1 mg / kg) or isotype control was intraperitoneally injected on day 13, and this was performed a total of 3 times every 3 to 4 days. The efficacy of Erb-(IL10)2, RT; Fc9-(IL10)2, RT, and the combination of Erb-(IL10)2 and RT; the combination of Fc9-(IL10)2 and RT was determined, and the results are shown in FIGS. 3A to 3C. Data were analyzed using GraphPad Prism version 5.0 software (San Diego, CA). Results are expressed as mean ± SEM. Unpaired Student's t-test was used to compare two groups. All reported p-values were two-sided, and the statistical significance level was * p < 0.05, ** p < 0.01, and *** p < 0.001 was set. Also, a P-value less than 0.05 was considered statistically significant.

[0320] Figures 3A and 3C reflect the relationship between tumor volume and the number of days after tumor challenge by single-dose irradiation of RT at 10 Gy and 5 Gy, respectively. Also, in Figure 3B, the horizontal coordinate is the number of days after tumor challenge, and the vertical coordinate is the percentage of mouse survival. RT 5 Gy had a slight effect on tumor growth compared to the isotype control group, while RT 10 Gy, Erb-(IL10)2 delayed tumor progression (Figures 3A and 3C). Treatment with the combination of RT 10 Gy + Erb-(IL10)2 effectively controlled tumor growth (P = 0.0349, RT 10 Gy vs RT 10 Gy + Erb-(IL10)2 showed tumor volumes of 1597.6 ± 474.7 mm3 vs 329.4 ± 156.5 mm3 on day 34) (Figure 3A). In tumors treated with the combination of RT 10 Gy and Erb-(IL10)2, 5 / 5 mice did not reach a volume of 2000 mm3 compared to 1 / 5 mice in the isotype control group (Figure 3A). Reduction to 5 Gy of single-dose radiation showed no significant synergistic effect in RT 5 Gy + Erb-(IL10)2 (P = 0.7088, Erb-(IL10)2 1940.2 ± 539.7 mm3 vs RT 5 Gy + Erb-(IL10)2 1654.3 ± 504.4 mm3 on day 34) (Figure 3C). Furthermore, as shown in Figure 3B, the combination of RT 10 Gy and Erb-(IL10)2 significantly improved the survival rate.

[0321] These data indicate that Erb-(IL10)2 significantly improves the effect of RT 10 Gy and the combination of RT 10 Gy and Erb-(IL10)2 extends the survival period of mice with B16-EGFR-SIY tumors.

[0322] Administration of Fc9-(IL10)2 or isotype control (human IgG1) obtained in Example 3 was started 3 days after RT and administered i.p. a total of 3 times at a dose of 1 mg / kg every 3 to 4 days (unless otherwise specified).

[0323] C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, locally irradiated on day 10, and then intraperitoneally injected with 0.32 mg / kg of Fc9-(IL10)2 or 0.5 mg / kg of isotype control on day 13, and this was performed a total of 3 times every 3 to 4 days. The efficacy of Fc9-(IL10)2, RT; Fc9-(IL10)2, RT, and the combination of Fc9-(IL10)2 and RT; the combination of Fc9-(IL10)2 and RT was determined, and the results are shown in Fig. 3D. Data were analyzed using GraphPad Prism version 5.0 software (San Diego, California). Results are presented as mean ± SEM.

[0324] Fig. 3D reflects the relationship between tumor volume and the number of days after tumor challenge with a single dose of 10 Gy irradiation RT. Treatment with the combination of RT 10 Gy + Fc9-(IL10)2 effectively controlled tumor growth (Fig. 3D). The data indicate that Fc9-(IL10)2 exhibits an effective synergistic effect with RT 10 Gy.

[0325] Example 6 Determination of the administration schedule of RT and the immunoconjugate of the present disclosure To determine the impact of the dosing schedule on the synergistic effect of RT treatment combined with the immunoconjugate of the present disclosure, Erb-(IL10)2 treatment was provided at different days after RT treatment. Erb-(IL10)2 was intraperitoneally injected into mice 3 days before administration of RT 10 Gy (day 10), simultaneously with RT 10 Gy (day 13), or 3 days after RT 10 Gy (day 16), respectively.

[0326] The results are shown in Fig. 4, where the horizontal axis is the number of days after tumor challenge and the vertical axis is the volume of the tumor. As can be seen from Fig. 4, all combinations had a significant effect on the rate of tumor growth from day 10 to day 30.

[0327] The effect on tumor growth was equivalent in all treatment groups (P = 0.5218, RT 10 Gy d10 + Erb-(IL10)2 d13 vs RT 10 Gy d10 + Erb-(IL10)2 d10 = 853.0 ± 476.2 mm at day 30)3 vs. 1318.8 ± 506.7 mm 3 ; P = 0.3115, on day 30, RT 10 Gy d10 + Erb-(IL10)2 d13 vs. RT 10 Gy d10 + Erb-(IL10)2 d16 = 853.0 ± 476.2 mm 3 vs. 2109.4 ± 1061.2 mm 3 ), surprisingly, administration of Erb-(IL10)2 approximately 3 days after RT exerted the most potent synergistic effect compared to co - administration of Erb-(IL10)2 with RT or delaying the administration of Erb-(IL10)2 until day 16.

[0328] Example 7 The synergistic effect is CD8 + T cell-dependent Next, the mechanism underlying the synergistic effect of the combination of RT and Erb-(IL10)2 was investigated. First, the functions of effector T cells and NK cells in mediating the anti - tumor effect after combination therapy were investigated.

[0329] Mice treated with RT and Erb-(IL10)2 were further given i.p. injections of anti - CD4 mAb (clone TIB207, 200 μg / mouse), anti - CD8 mAb (clone TIB210, 200 μg / mouse), or anti - NK mAb (clone PK136, 200 μg / mouse) starting on day 10 and once a week for a total of 2 applications. As shown in Figures 5A and 5C, depletion of CD4 + T cells or NK cells did not significantly affect the effect of combination therapy on tumor growth (P = 0.2872, on day 27, RT 10 Gy + Erb-(IL10)2 1 mg / kg + anti - CD4 mAb vs. RT 10 Gy + Erb-(IL10)2 1 mg / kg = 771.4 ± 172.5 mm 3 vs. 474.8 ± 190.7 mm 3 ; P = 0.1651, on day 27, RT 10 Gy + Erb-(IL10)2 1 mg / kg + anti - NK mAb vs. RT 10 Gy + Erb-(IL10)2 1 mg / kg = 940.1 ± 219.7 mm 3 vs. 474.8 ± 190.7 mm 3)。However, as shown in Figure 5B, CD8 + T cell depletion dramatically reduced the therapeutic effect and led to rapid tumor growth (P = 0.0376, RT 10 Gy + Erb-(IL10) 21 mg / kg + anti-CD8 mAb vs RT 10 Gy + Erb-(IL10) 21 mg / kg on day 27 = 1896.1 ± 499.6 mm 3 vs 474.8 ± 190.7 mm 3 )。Therefore, these results demonstrate that CD8 + T cells are essential for the antitumor effect of the combination therapy.

[0330] Example 8 The combination therapy mediated an abscopal effect on distant tumors and improved tumor-specific CD8 + T cell function in the tumor mass 8.1 Abscopal effect On day 0, 5 × 10 5 B16-EGFR-SIY cells (primary tumor) were s.c. injected into the right flank and the same number of cells (secondary tumor) were s.c. injected into the left flank of C57BL / 6 mice. As shown, the primary tumor was locally treated with RT 10 Gy on day 10, and then Erb-(IL10)2 at 1 mg / kg was i.p. injected on day 13, which was performed three times every 3 to 4 days. The volumes of the primary and secondary tumors were measured and monitored.

[0331] To evaluate the abscopal effect (indirect effect) of the combination of RT and RT + Erb-(IL10)2, B16-EGFR-SIY cells were s.c. injected into both flanks of C57BL / 6 mice, and the primary tumor on the right flank was irradiated with radiation to determine the direct therapeutic effect, while the secondary tumor on the left flank was not irradiated and was used to measure the potential and indirect systemic effect. These treatments led to a significant growth delay of the primary tumor, similar to that shown in Figure 3A.

[0332] The effect on secondary tumor growth is shown in Fig. 6A. Three experiments were conducted using 5 mice per group. The effect of i.p. administration of Erb-(IL10)2 alone was similar to that seen in the primary tumor. However, a single RT treatment of 10 Gy caused a significant growth delay in the primary tumor, but in the secondary tumor, no effect was observed (Fig. 6A).

[0333] In contrast, the combination of Erb-(IL10)2 and RT resulted in a significant further growth delay in secondary tumors compared to the results obtained with Erb-(IL10)2 administration alone (P = 0.0009, on day 26 Erb-(IL10)2 vs RT 10Gy + Erb-(IL10)2 = 970.2 ± 119.6 mm 3 vs 253.9 ± 71.6 mm 3 ). In summary, these results indicate that a single local radiation exposure (10 Gy) has a low potential to cause an abscopal effect, but the combination of RT 10 Gy and Erb-(IL10)2 can induce a significant anti-tumor effect on distant tumors.

[0334] 8.2 Enhancement of antigen-specific T cell responses To investigate the effect of combination therapy on enhancing the tumor antigen-specific T cell response, SIY peptide was used to identify SIY-reactive CD8 + T cells.

[0335] Enzyme-linked immunosorbent spot (ELISPOT) assay was used to quantify the cells secreting interferon gamma (IFNγ) using a 96-well plate. An ELISPOT kit was used according to the manufacturer's instructions (BD Biosciences catalog number 551083). As described, the tumor-draining lymph node (DLN) was removed to obtain a single cell suspension. The 96-well ELISPOT plate was pre-coated with 5 μg / ml of purified anti-mouse IFN-γ (BD Biosciences catalog number 51-2525kc) at 4 °C overnight. 5 × 10 5Individual lymph node cells were co-cultured in the presence of 10 μg / ml of SIY polypeptide (SL-9, GL Biochem catalog number 057787) or ovalbumin (OVA) polypeptide (InvivoGen catalog number vac-sin), and the latter was used as a negative control for antigen specificity. After 72 hours of incubation, the cells were removed, 2 μg / ml of biotinylated anti-mouse IFN-γ (BD Biosciences catalog number 51-1818kz) was added, and the plates were incubated at room temperature for 2 hours. Spots were visualized using streptavidin-HRP (BD Biosciences catalog number 557630) and AEC substrate (BD Biosciences catalog number 551951), and then image analysis and spot counting were performed. Each condition was tested in replicates and their mean values were shown. As described above, the tumors received RT at 10 Gy and were administered Erb-(IL10)2 at 1 mg / kg i.p. Three days after the second administration of Erb-(IL10)2, the mice were sacrificed, tumor DLNs were removed to obtain single cell suspensions, and ELISPOT assays were performed.

[0336] Three days after the second administration of Erb-(IL10)2 (i.e., day 19), lymphocytes derived from tumor DLNs were isolated from mice inoculated with B16-EGFR-SIY melanoma cells. As shown in Figure 6B, in the ELISPOT assay, lymph node cells in the tumor inflow region of mice in each group were activated in vitro with SIY or OVA peptides, and then the number of IFN-γ secreting cells was measured. SIY-specific IFN-γ-producing CD8 in the DLNs of mice treated with the combination of RT and Erb-(IL10)2 +The number of T cells was significantly increased compared to those treated with RT or Erb-(IL10)2 alone (P = 0.0026, isotype control vs RT + Erb-(IL10)2; P = 0.0034, Erb-(IL10)2 vs RT + Erb-(IL10)2; P = 0.0064, RT vs RT + Erb-(IL10)2). Overall, these findings demonstrate that the combination therapy of RT and Erb-(IL10)2 enhances the tumor control effect by improving the systemic activation of tumor-specific T cells in tumor DLN and enhancing the function of tumor antigen-specific T cells in the tumor microenvironment.

[0337] 8.3 Induction of activated T cells after treatment with combination therapy FTY720 (SIGMA, catalog number SML0700-5MG) was used to test the activation of T cells in the tumor microenvironment. FTY720 is a lipophilic immunomodulatory sphingosine-1-phosphate analog that induces severe peripheral lymphopenia in mice by preventing lymphocytes from exiting lymphoid organs through receptor internalization induced by agonists and maintains lymphopenia.

[0338] B16-EGFR-SIY cells were s.c. inoculated into both flanks of mice. Starting from the 10th day after B16-EGFR-SIY melanoma cell transplantation, the mice in the radiotherapy + Erb-(IL10)2 group received the first i.p. injection. Then 3 days later, after the first administration of depletion antibody or FTY720, lymphocyte suspensions were prepared from the peripheral blood of those mice and labeled with APC anti-mCD4 (clone GK1.5, BioLegend), FITC anti-mCD8a (clone 53-6.7, BioLegend), FITC-anti-mCD3 (clone 17A2, BioLegend), or APC-anti-mNK1.1 (clone PK136, BioLegend). The tumor volumes of both flanks were measured and monitored. Samples were analyzed with a Life Attune Flow Cytometer (Life) and the data were analyzed. Representative data are shown in Figures 6C - 6F based on the results obtained from three experiments performed with 5 - 6 mice per group.

[0339] CD4 in peripheral blood + and CD8 + When the distribution of lymphocytes of and is evaluated 3 days after the first administration of FTY720, CD8 + T cells and CD4 + The relative numbers of T cells were found to decrease to approximately 0.5% and 1.5%, respectively, as shown in Figure 6C.

[0340] However, as shown in Figure 6D, it was observed that the antitumor effect of Erb-(IL10)2 alone at 1 mg / kg was not reduced by FTY720 treatment. In the combination therapy (i.e., RT + Erb-(IL10)2), FTY720 significantly abrogated the antitumor effect against secondary tumors (P = 0.0081, on day 31, RT 10 Gy + Erb-(IL10)2 1 mg / kg vs. RT 10 Gy + Erb-(IL10)2 1 mg / kg + FTY720 = 392.5 ± 33.9 mm 3 vs. 1052.4 ± 197.5 mm 3 )(shown in Figure 6F), and had only a slight effect on the primary tumor (P = 0.0665, on day 31, RT 10 Gy + Erb-(IL10)2 1 mg / kg vs. RT 10 Gy + Erb-(IL10)2 1 mg / kg + FTY720 = 298.1 ± 73.4 mm 3 vs. 537.9 ± 90.4 mm 3 )(shown in Figure 6E). Therefore, the combination of RT and the immunoconjugate of the present disclosure activated T cells in the tumor microenvironment.

[0341] Similar results were obtained for Tmab-(IL10)2, 28H1-(IL10)2, Fc9-(IL10)2, and (IL10-Fc)2 in the above experiments.

[0342] Example 9 Effect of cytotoxic chemotherapy combined with the immunoconjugate of the present disclosure 9.1 Effect of the immunoconjugate according to the present disclosure combined with doxorubicin The B16-EGFR-SIY control tumor model was obtained as described in Example 4 above. To compare the effect of Erb-(IL10)2 combined with doxorubicin, mice were divided into several groups of 5 mice per group: isotype control group, treated with 1 mg / kg of human IgG1 (20 μg / mouse); Erb-(IL10)2 group, treated with 1 mg / kg (20 μg / mouse) of Erb-(IL10)2; doxorubicin group, treated with 5 mg / kg (100 μg / mouse) of doxorubicin; Erb-(IL10)2 + doxorubicin group, treated with 1 mg / kg of Erb-(IL10)2 and 5 mg / kg of doxorubicin. C57BL / 6 mice were inoculated s.c. with B16-EGFR-SIY cells on day 0, Er-(IL10)2 was injected i.p. on days 7, 10, and 14, respectively, and doxorubicin was injected via the tail vein on day 7.

[0343] The results are shown in Figure 7A. It can be seen that a synergistic effect was observed in the combination of Erb-(IL10)2 and doxorubicin in controlling tumor growth.

[0344] To compare Fc9-(IL10)2 combined with doxorubicin, mice were divided into the following several groups of 5 mice per group: isotype control group, treated with 0.5 mg / kg of human IgG1 (10 μg / mouse); Fc9-(IL10)2 group, treated with 0.5 mg / kg (10 μg / mouse) of Fc9-(IL10)2; doxorubicin group, treated with 5 mg / kg (100 μg / mouse) of doxorubicin; Fc9-(IL10)2 + doxorubicin group, treated with 0.5 mg / kg of Fc9-(IL10)2 and 5 mg / kg of doxorubicin. C57BL / 6 mice were inoculated s.c. with B16-EGFR-SIY cells on day 0, Fc9-(IL10)2 was injected i.p. on days 7, 10, and 14, respectively, and doxorubicin was injected via the tail vein on day 7.

[0345] The results are shown in Figure 7B. It can be seen that a synergistic effect was observed in the combination of Fc9-(IL10)2 and doxorubicin in controlling tumor growth.

[0346] 9.2 Effect of the immunoconjugate according to the present disclosure combined with oxaliplatin The B16-EGFR-SIY control tumor model was obtained as described in Example 4 above. To compare the effects of Erb-(IL10)2 combined with oxaliplatin, mice were divided into several groups of 5 mice per group: isotype control group, treated with 0.5 mg / kg of human IgG1 (10 μg / mouse); Erb-(IL10)2 group, treated with 0.5 mg / kg (10 μg / mouse) of Erb-(IL10)2; oxaliplatin group, treated with 15 mg / kg (300 μg / mouse) of oxaliplatin; Erb-(IL10)2 + oxaliplatin group, treated with 0.5 mg / kg of Erb-(IL10)2 + 15 mg / kg of oxaliplatin. C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, Erb-(IL10)2 was intraperitoneally injected on days 7, 10, and 14, and oxaliplatin was injected via the tail vein on day 7.

[0347] The results are shown in Figure 7C. Due to the high toxicity of oxaliplatin, all mice in the oxaliplatin group died after 14 days (see Figure 7I). It can be seen that a synergistic effect was observed in the combination of Erb-(IL10)2 and oxaliplatin for the control of tumor growth.

[0348] To compare the effects of Fc9-(IL10)2 combined with oxaliplatin, mice were divided into several groups of 5 mice per group: isotype control group, treated with 0.5 mg / kg of human IgG1 (10 μg / mouse); Fc9-(IL10)2 group, treated with 0.5 mg / kg (10 μg / mouse) of Fc9-(IL10)2; oxaliplatin group, treated with 15 mg / kg (300 μg / mouse) of oxaliplatin; Fc9-(IL10)2 + oxaliplatin group, treated with 0.5 mg / kg of Fc9-(IL10)2 + 15 mg / kg (300 μg / mouse) of oxaliplatin. C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, Fc9-(IL10)2 was intraperitoneally injected on days 7, 10, and 14, and oxaliplatin was injected via the tail vein on day 7.

[0349] The results are shown in Fig. 7D. Because the toxicity of oxaliplatin is high, all the mice in the oxaliplatin group and the Fc9-(IL10)2 + oxaliplatin group died after 14 days (see Fig. 7J). Therefore, the synergistic effect cannot be compared among the groups.

[0350] 9.3 Effect of the immunoconjugate according to the present disclosure combined with CTX (cyclophosphamide) The B16-EGFR-SIY control tumor model was obtained as described in Example 4 above. To compare the effect of Erb-(IL10)2 combined with CTX, mice were divided into several groups containing 5 mice per group: PBS group, treated with PBS buffer; Erb-(IL10)2 group, treated with 0.5 mg / kg (10 μg / mouse) of Erb-(IL10)2; CTX group, treated with 100 mg / kg (2 mg / mouse) of CTX; Erb-(IL10)2 + CTX group, treated with 0.5 mg / kg of Erb-(IL10)2 and 100 mg / kg of CTX. C57BL / 6 mice were s.c. inoculated with B16-EGFR-SIY cells on day 0, Erb-(IL10)2 was i.p. injected on days 7, 10, and 14, respectively, and CTX was injected via the tail vein on day 7.

[0351] The results are shown in Fig. 7E. It can be seen that a synergistic effect was observed in the combination of Erb-(IL10)2 and CTX in the control of tumor growth.

[0352] To compare the effects of Fc9-(IL10)2 combined with CTX, mice were divided into several groups of 5 mice per group: isotype control group, treated with 1 mg / kg of human IgG1 (20 μg / mouse); Fc9-(IL10)2 group, treated with 0.65 mg / kg (13 μg / mouse) of Fc9-(IL10)2; CTX group, treated with 100 mg / kg (2 mg / mouse) of CTX; Fc9-(IL10)2 + CTX group, treated with 0.65 mg / kg of Fc9-(IL10)2 and 100 mg / kg of CTX. C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, and Fc9-(IL10)2 was intraperitoneally injected on days 7, 10, and 14, respectively, and CTX was injected via the tail vein on day 7.

[0353] The results are shown in Figure 7F. It can be seen that a synergistic effect was observed in the control of tumor growth in the combination of Fc9-(IL10)2 and CTX.

[0354] 9.4 Effect of the immunoconjugate according to the present disclosure combined with vinorelbine The B16-EGFR-SIY control tumor model was obtained as described in Example 4 above. To compare the effects of Erb-(IL10)2 combined with vinorelbine, mice were divided into several groups of 5 mice per group: isotype control group, treated with 0.5 mg / kg of human IgG1 (10 μg / mouse); Erb-(IL10)2 group, treated with 0.5 mg / kg (10 μg / mouse) of Erb-(IL10)2; vinorelbine group, treated with 5 mg / kg (100 μg / mouse) of vinorelbine; Erb-(IL10)2 + vinorelbine group, treated with 0.5 mg / kg of Erb-(IL10)2 + 5 mg / kg of vinorelbine. C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, and Erb-(IL10)2 was intraperitoneally injected on days 7, 10, and 14, respectively, and vinorelbine was injected via the tail vein on day 7.

[0355] The results are shown in Figure 7G. It can be seen that a synergistic effect was observed in the control of tumor growth in the combination of Erb-(IL10)2 and vinorelbine.

[0356] To compare the effects of Fc9-(IL10)2 combined with vinorelbine, mice were divided into several groups of 5 mice per group: an isotype control group, treated with 0.5 mg / kg of human IgG1 (10 μg / mouse); an Fc9-(IL10)2 group, treated with 0.5 mg / kg (10 μg / mouse) of Fc9-(IL10)2; a vinorelbine group, treated with 5 mg / kg (100 μg / mouse) of vinorelbine; an Fc9-(IL10)2 + vinorelbine group, treated with 0.5 mg / kg of Fc9-(IL10)2 + 5 mg / kg of vinorelbine. C57BL / 6 mice were s.c. inoculated with B16-EGFR-SIY cells on day 0, Fc9-(IL10)2 was i.p. injected on days 7, 10, and 14, and vinorelbine was injected via the tail vein on day 7.

[0357] The results are shown in Fig. 7H. It can be seen that a synergistic effect was observed in the combination of Fc9-(IL10)2 and vinorelbine for the control of tumor growth.

[0358] Fig. 7I shows the effects on survival rates of Erb-(IL10)2, oxaliplatin, and the combination of Erb-(IL10)2 and oxaliplatin.

[0359] Fig. 7J shows the effects on survival rates of Fc9-(IL10)2, oxaliplatin, and the combination of Fc9-(IL10)2 and oxaliplatin.

[0360] In the above experiments, similar results were obtained for Tmab-(IL10)2, 28H1-(IL10)2, and (IL10-Fc)2. In addition, it was found that the immunoconjugates of the present disclosure have a (selective) synergistic effect when administered in combination with various other chemotherapeutic agents.

[0361] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within this specification. Although the present invention has been described with reference to the foregoing specification, the description of the embodiments and examples herein is not intended to be construed in a limiting sense. Here, those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the present invention. Further, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions defined herein that depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be utilized in practicing the present invention. Accordingly, the present invention is intended to cover any such alternatives, modifications, variations, or equivalents. The following embodiments define the scope of the present invention, and it is intended that the methods and structures within the scope of these embodiments and their equivalents be covered thereby.

Claims

A pharmaceutical composition for treating cancer in combination with electron beam irradiation, containing an immunoconjugate, wherein the immunoconjugate is 1) an IL10 dimer, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit derived from human IgG1, the first Fc subunit associates with the second Fc subunit to form a dimer, the IL10 dimer is fused to the amino acid at the amino terminus of the first Fc subunit or the amino acid at the amino terminus of the second Fc subunit, the IL10 dimer is one in which two IL10s are linked via a peptide linker, the first Fc subunit contains a first modification, the second Fc subunit contains a second modification, the first modification is T366W and K409A, the second modification is T366S, L368G, Y407A, and F405K, the positions of the amino acids are determined according to the EU index of the Kabat number, pharmaceutical composition. Claim 2 The pharmaceutical composition according to claim 1, wherein the immunoconjugate further comprises a targeting moiety fused to the Fc domain, and the targeting moiety exhibits binding specificity for a tumor antigen. Claim 3 The pharmaceutical composition according to claim 2, wherein the IL10 dimer is fused to one of the amino acid at the amino terminus of the first Fc subunit or the amino acid at the amino terminus of the second Fc subunit, and the targeting moiety is fused to the amino acid at the amino terminus of the other first Fc subunit or the amino acid at the amino terminus of the second Fc subunit. Claim 4 The pharmaceutical composition according to claim 2, wherein the tumor antigen is selected from the group consisting of EGFR, HER2 / neu, and FAP. Claim 5 The pharmaceutical composition according to claim 1, wherein the immunoconjugate does not contain any targeting moiety.

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