Combination therapy with anti-CD26 antibodies and immune checkpoint inhibitors

Abstract

To provide a new combination therapy that exerts a superior antitumor effect as compared to administration of an anti-CD26 antibody alone.SOLUTION: A pharmaceutical composition for cancer treatment is used that contains an immune checkpoint inhibitor and an anti-CD26 antibody or an antigen-binding fragment thereof as an active ingredient. The immune checkpoint inhibitor and the anti-CD26 antibody or its antigen-binding fragment are separately administered concurrently, continuously, or with a time interval.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to the field of cancer treatment. [Background technology]

[0002] Malignant pleural mesothelioma is a refractory malignant tumor originating from the pleural mesothelium, caused by asbestos exposure. The latency period from asbestos exposure to the onset of the disease is approximately 30 years, and the number of patients is expected to increase significantly in Asia and the Middle East, including Japan, China, and India. The prognosis is extremely poor, and while surgical treatment, chemotherapy, and radiation therapy are performed, none of them yield satisfactory results, and the establishment of new treatment methods is desired. In addition, lung cancer is the most common type of cancer in Japan, and the 5-year survival rate is low at around 20%, so the establishment of safe and effective new treatment methods is also needed for lung cancer.

[0003] Anti-CD26 antibodies are known to have anticancer effects (Patent Documents 1 and 2). A first-in-human phase I clinical trial was conducted in France, focusing on malignant mesothelioma. Safety was confirmed with no notable side effects except for infusion reactions (acute infusion reactions), and 10 out of 19 patients with anticancer drug-resistant malignant mesothelioma achieved stable disease (SD) in the modified RESIST evaluation, with 5 of these cases maintaining SD for more than 6 months, and the longest being 399 days, suggesting efficacy (Non-Patent Document 1). In Japan, a phase I / II clinical trial for anticancer drug-resistant malignant mesothelioma was also conducted, with administration to a total of 9 patients (3 patients each in 1-3 cohorts) in phase I and 31 patients in phase II, with the final patient in phase II being administered in 2019. In domestic clinical trials, a high proportion of patients with anticancer drug-resistant malignant mesothelioma achieved partial response (PR) and SD, demonstrating antitumor effects (Non-Patent Document 2). On the other hand, there were no complete responses (CR), and some patients progressed from stable disease (SD) to progressive disease (PD) in a relatively short period. Therefore, developing a treatment method using this antibody that exhibits longer-lasting antitumor effects and provides progression-free survival is an important challenge.

[0004] To date, attempts have been made to enhance the therapeutic effect by combining anti-CD26 antibodies with small molecule compounds (Patent Document 3).

[0005] Immune checkpoint inhibitors (ICIs) are a general term for drugs that inhibit the binding of inhibitory receptors expressed on immune cells, particularly T cells, to their ligands, thereby inhibiting the transmission of inhibitory signals to immune cells and the subsequent suppression of their function. Representative examples that are already approved include anti-CTLA-4 (cytotoxoc T-lymphocyte associated ptotein 4) antibody, anti-PD-1 (Programmed cell death 1) antibody, and anti-PD-L1 (Programmed cell death-ligand 1) antibody. ICIs have shown efficacy in various cancer types, including malignant melanoma, non-small cell lung cancer, renal cell carcinoma, rectal cancer, and Hodgkin lymphoma. Although there are reports of extending the asymptomatic period with monotherapy, achieving complete response (CR) remains difficult, and attempts are being made to supplement their therapeutic effect by combining them with other drugs. For example, in order to enhance the therapeutic effect of non-small cell lung cancer, the combination of anti-PD-1 antibodies and anti-CTLA-4 antibodies has been attempted. However, regarding the therapeutic effect, there are reports of additive effects, and reports of no additive effects, and the results are varied. In all cases, the problem is that toxicity is frequently observed with the combination, and the development of further combination therapies is expected (Non-Patent Literature 3, 4). The same is true for mesothelioma, where the results of combination therapy with anti-PD-1 antibodies and anti-CTLA-4 antibodies are varied (Non-Patent Literature 5). Thus, although various methods of combination therapy with ICIs have been attempted, it is known that it is difficult to even obtain an additive effect. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2002 / 14462 [Patent Document 2] International Publication No. 2007 / 114876 [Patent Document 3] International Publication No. 2017 / 043613 [Non-patent literature]

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0008] The inventors have hitherto focused on CD26 expressed in cancer cells as a therapeutic target molecule for cancer and developed a humanized CD26 antibody. In order to develop a new combination therapy that exhibits an antitumor effect for a longer period and provides a progression-free survival period as compared with administration of the humanized anti-CD26 antibody alone, investigations were conducted.

[0009] For ICI to exert an antitumor effect, immune cells centered on T cells are indispensable, and a cancer-bearing mouse model in which a syngeneic mouse tumor strain is transplanted into a mouse is often used. On the other hand, for an anti-CD26 antibody to exert an antitumor effect, the binding site on the human CD26 molecule is also important (Teruo Inamoto, et al., Clin Cancer Res; 13(14):4191-4200(2007)), and since the function of CD26 in the immune system is also very different between humans and mice (Morimoto C, et al, Immunol Rev.; 161:55-70(1998)), analysis using a human tumor strain and the human immune system is essential for obtaining data on humanized anti-CD26 antibodies. Specifically, CD26 is also a T cell co-stimulatory molecule that transmits an activation signal to human T cells, and a humanized anti-CD26 antibody blocks the binding of caveolin-1, which is a ligand of CD26, to CD26, that is, blocks the transmission of the CD26 co-stimulatory signal to T cells. On the other hand, CD26 of mouse T cells does not function as a co-stimulatory molecule. Also, regarding the expression of CD26, in human T cells, CD26 shows a three-phase pattern of strongly positive, weakly positive, and negative, whereas mouse T cells are uniformly weakly positive. Regarding the expression of CD26 in immune cells other than T cells, in humans, CD26 expression is seen in NKT cells other than T cells, but CD26 is hardly expressed in B cells and NK cells, whereas in mice, B cells also show the same weakly positive as T cells. Thus, since there are significant differences between humans and mice in functions in T cells and expression patterns in immune cells, analysis using the human immune system is indispensable for functional analysis of CD26 in the immune system. For the above reasons, first, we attempted to produce humanized immune mice as a model animal for confirming the combined action of humanized anti-CD26 antibody and ICI. As a result, we succeeded in producing a model mouse excellent in the generation of human T cells.

[0010] Next, the combined effect of a humanized anti-CD26 antibody and an immune checkpoint inhibitor was investigated in a tumor-bearing mouse model. As a result, a stronger synergistic effect was observed compared to each agent alone. This synergistic effect could only be confirmed by creating the aforementioned animal model, and it is thought to reflect that a similar synergistic effect would occur in humans, who, like the animal model, have human CD26 expression in cancer cells and a human immune system (T cells). [Effects of the Invention]

[0011] The method for producing the model mouse of the present invention can be used to evaluate molecules involved in the human immune system in animal models, as human immunized mice. Furthermore, the combination agent of the present invention contributes to the realization of more effective cancer treatment. [Brief explanation of the drawing]

[0012] [Figure 1] NOG mice were irradiated with a near-lethal dose (100 cGy), and the following day, CD34-positive hematopoietic stem cells (1 × 10⁵) isolated and purified from human umbilical cord blood were transplanted into the tail vein of the mice. 50 μl of blood was collected from the tail vein of NOG mice 6, 10, 14, and 18 weeks after transplantation, and the percentage of human blood cells in the mouse blood was analyzed by flow cytometry (n=10). The results are shown as the mean ± standard deviation for each individual. [Figure 2]After transplanting human umbilical cord blood-derived CD34-positive hematopoietic stem cells into the tail vein of NOG mice and confirming the generation and engraftment of human T cells in the mouse blood 13 weeks later, human malignant mesothelioma cell line JMN (1 × 10⁶) was subcutaneously transplanted into the flank of the mice. Tumor formation was observed 4-5 weeks after JMN subcutaneous transplantation. From 5 weeks after transplantation, administration of control human IgG1, humanized anti-CD26 antibody (YS110) alone, mouse anti-human PD-1 mAb alone, and a combination of humanized CD26 antibody and PD-1 antibody was started at 200 μg / dose three times a week and continued until dissection at 9 weeks. Tumor size was measured twice a week. The upper left figure shows representative images of the tumor size in each group 8 weeks after JMN subcutaneous transplantation. The figure on the right shows a graph of the time course of tumor volume (average value for each group) up to the 9th week of dissection, and the figure on the lower left shows a representative example of a tumor recovered subcutaneously from a mouse during the 9th week of dissection. [Figure 3] Human umbilical cord blood-derived CD34-positive hematopoietic stem cells were transplanted into the tail vein of NOG mice, and 13 weeks later, the human malignant mesothelioma cell line JMN (1 × 10⁶) was subcutaneously transplanted into the flank of the mice. Five weeks after JMN subcutaneous transplantation, control human IgG1, humanized anti-CD26 antibody (YS110) alone, mouse anti-human PD-1 mAb alone, and a combination of humanized anti-CD26 antibody and PD-1 antibody were administered at 200 μg / dose three times a week, and administration continued until dissection at 9 weeks. Nine weeks after JMN subcutaneous transplantation, the mice were dissected, and the subcutaneous tumors were collected and weighed. The tumor weights for each individual are shown as mean ± standard deviation. The data are cumulative results from two independent experiments (control human IgG group n=4, YS110 alone group n=3, PD-1 antibody alone group n=3, YS110 and PD-1 antibody combination group n=3). [Figure 4]After transplanting human umbilical cord blood-derived CD34-positive hematopoietic stem cells into the tail vein of NOG mice and confirming the generation and engraftment of human T cells in the mouse blood 14 weeks later, human lung adenocarcinoma cell line HCC4006 (1 × 10⁶) was subcutaneously transplanted into the flank of the mice. Tumor formation was observed 2-3 weeks after HCC4006 subcutaneous transplantation. From 3 weeks later, administration of control human IgG1, humanized anti-CD26 antibody (YS110) alone, mouse anti-human PD-1 mAb alone, and a combination of humanized anti-CD26 antibody and PD-1 antibody was started at 200 μg / dose three times a week and continued until dissection at 6.5 weeks. Tumor size was measured twice a week. The upper left figure shows representative images of the tumor size in each group 5.5 weeks after HCC4006 subcutaneous transplantation. The figure on the right shows a graph of the time course of tumor volume (average value for each group) up to dissection at 6.5 weeks, and the figure on the lower left shows a representative example of a tumor recovered subcutaneously from a mouse dissected at 6.5 weeks. [Figure 5] Fourteen weeks after transplanting human umbilical cord blood-derived CD34-positive hematopoietic stem cells into the tail vein of NOG mice, human lung adenocarcinoma cell line HCC4006 (1 × 10⁶) was subcutaneously transplanted into the flank of the mice. Three weeks after subcutaneous transplantation of HCC4006, administration of control human IgG1, humanized anti-CD26 antibody (YS110) alone, mouse anti-human PD-1 mAb alone, or a combination of humanized anti-CD26 antibody and PD-1 antibody was initiated at 200 μg / dose three times a week and continued until dissection at 6.5 weeks. Six.5 weeks after subcutaneous transplantation of HCC4006, the mice were dissected, and the subcutaneous tumors were collected and weighed. The tumor weights for each individual are shown as mean ± standard deviation. The data represent the cumulative results of two independent experiments (control human IgG group n=4, YS110 monotherapy group n=4, PD-1 antibody monotherapy group n=4, YS110 and PD-1 antibody combination group n=3). [Modes for carrying out the invention]

[0013] (cancer) In this specification, “cancer,” “carcinoma,” and “tumor” are used interchangeably and refer to cells that have undergone malignant transformation to be pathogenic to the host organism. Primary cancer cells (i.e., cells obtained near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. In this specification, the definition of cancer includes not only primary cancer but also all cancers derived from primary cancer, such as metastatic cancer. Cancers that manifest as solid tumors are detectable based on the tumor mass and can be identified, for example, by CAT scan, MR imaging, X-ray, ultrasound, or palpation, and / or detection of the expression of one or more cancer-specific antigens in a sample obtained from the patient. Cancer may also be a hematopoietic malignancy, such as a tumor of blood cells.Cancer as used herein includes, but is not limited to, lung cancer, non-small cell lung cancer, small cell lung cancer, non-Hodgkin lymphoma, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, pediatric cerebellar astrocytoma, pediatric cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary tract cancer, extrahepatic cholangiocarcinoma, intrahepatic cholangiocarcinoma, bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrohistiocytoma, brain cancer, brain tumors, brainstem glioma, cerebellar astrocytoma, glioma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic tract and hypothalamic glioma, head and neck cancer, metastatic squamous epithelial cell carcinoma Cancer, bronchial adenoma / carcinoid, carcinoid tumor, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, neuroblastoma, childhood cancer, Seziary syndrome, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, eye cancer, intraocular melanoma, retinoblastoma, salivary gland cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, oral cancer, tongue cancer, esophageal cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), small intestine cancer, colon cancer, colorectal cancer, rectal cancer, anorectal cancer, anal cancer, germ cell tumor, hepatocellular carcinoma (liver) cancer, Hodgkin's lymphoma, laryngeal cancer, pharyngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, paranasal cancer Cancers of the urinary cavity and nasal cavity, pharyngeal cancer, islet cell tumors (endocrine pancreas), pancreatic cancer, parathyroid cancer, liver cancer, gallbladder cancer, appendiceal cancer, kidney cancer, urethral cancer, transitional cell carcinoma of the renal pelvis and ureter and other cancers of the urinary tract, squamous cell carcinoma, penile cancer, testicular cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, prostate cancer, ovarian cancer, ovarian epithelial carcinoma, low-grade ovarian tumors, ovarian germ cell tumors, gestational trophoblastic tumors, breast cancer, uterine cancer, uterine sarcoma, cervical cancer, endometrial cancer, uterine cancer, vaginal cancer, vulvar cancer, Wilms' tumor, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders This includes hairy cell leukemia, primary central nervous system lymphoma, chronic myeloproliferative disorders, cutaneous T-cell lymphoma, lymphoid neoplasms, Waldenstram's macroglobulinemia, medulloblastoma, skin cancer (non-melanoma), skin cancer (melanoma), pheochromocytoma, Merkel cell carcinoma, mesothelioma, malignant mesothelioma, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, pineoblastoma and pituitary tumors, plasmacytoma, pleuropulmonary blastoma, retinoblastoma, rhabdomyosarcoma, sarcoma, Ewing's tumor family, soft tissue sarcoma, mycosis fungoides, and Kaposi's sarcoma.

[0014] In this specification, cancer or cancer cells refer to mammalian cancer or mammalian cancer cells, preferably human cancer or human cancer cells. Cancer in this specification may also refer to cancer that expresses CD26.

[0015] (antibody) In this specification, “antibody” means an immunoglobulin molecule that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, and other antibodies via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term antibody includes not only complete polyclonal or monoclonal antibodies but also their fragments. It is known that the variable region (particularly CDRs) of an antibody confers binding properties, and it is widely known to those skilled in the art that even antibody fragments that are not complete antibodies can utilize these binding properties. In this specification, “fragment” or “antigen-binding fragment” of an antibody means a protein or peptide containing a part (partial fragment) of an antibody that retains the antibody’s action on the antigen (immunoreactivity / binding ability). Examples of such immunoreactive fragments include F(ab')2, Fab', Fab, Fab3, single-stranded Fv (hereinafter referred to as "scFv"), (tandem) bispecific single-stranded Fv (sc(Fv)2), single-stranded triple body, nanobody, divalent VHH, pentavalent VHH, minibody, (double-stranded) diabody, tandem diabody, bispecific tribody, bispecific bibody, dual affinity retargeting molecule (DART), triabody (or tribody), tetrabody (or [sc(Fv)2]2, or (scFv-SA)4), disulfide-bonded Fv (hereinafter referred to as "dsFv"), compact IgG, heavy chain antibodies, or polymers thereof (Nature Biotechnology, 29(1):5-6 (2011); Maneesh Jain et al., TRENDS in Biotechnology, See 25(7)(2007):307-316; and Christoph stein et al., Antibodies(1):88-123(2012). In this specification, the immunoreactive fragment may be monospecific, bispecific, trispecific, or multispecific.Furthermore, the antibody fragments may contain fragments with lengths of at least approximately 10 amino acids, at least approximately 25 amino acids, at least approximately 50 amino acids, at least approximately 75 amino acids, and at least approximately 100 amino acids.

[0016] The antibody does not need to be of a specific class, but may include any class of antibody, such as IgG, IgA, or IgM (or their subclasses). Immunoglobulins are classified into different classes based on the antibody amino acid sequence of the constant domain of the heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The corresponding constant domains of the heavy chains of different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of each class of immunoglobulin are well known. Preferably, the antibody may be an IgG antibody, particularly an IgG1 antibody or an IgG2 antibody, or it may be a human IgG antibody.

[0017] The antibodies used herein may be monoclonal or polyclonal antibodies, preferably monoclonal antibodies. In this specification, “monoclonal antibody” means an antibody obtained from a population of cells that produce substantially homogeneous antibodies; that is, the individual antibodies in that cell population are identical except for a few possible naturally occurring mutants. Monoclonal antibodies are highly specific, targeting a single antigen site. Furthermore, in contrast to typical polyclonal antibodies, which contain different antigens targeting different determinants (epitopes), each monoclonal antibody targets a single determinant of an antigen. The modifier “monoclonal” refers to the characteristic of antibodies obtained from a substantially homogeneous antibody-producing cell population and should not be interpreted as requiring antibody production by a specific method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries prepared using techniques such as those described in McCafferty et al., Nature, 348:552-554 (1990).

[0018] The antibodies used herein may be chimeric antibodies, humanized antibodies, fully human antibodies, or animalized antibodies adapted to any non-human animal being treated, preferably humanized antibodies. As used herein, a “humanized” antibody is an antibody or its antigen-binding fragment (Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing a minimal sequence derived from a non-human immunoglobulin and a sequence derived from a human. Some humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the receptor complementarity-determining region (CDR) are replaced with CDR-derived residues from a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. The Fv framework region (FR) residues of the human immunoglobulin may be replaced with corresponding non-human-derived residues. Humanized antibodies contain a variable region derived from a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and / or capabilities, but one or more residues in the Fv framework region and / or one or more CDR residues may be substituted with corresponding human residues (i.e., residues derived from the human antibody sequence). Humanized antibodies may contain residues not present in the original imported CDR or framework sequence for further improvement and optimization of antibody performance. Most preferably, humanized antibodies contain at least a portion of the constant region (Fc) or domain (generally human immunoglobulin) of human immunoglobulin. Some humanized antibodies have modified Fc regions, as described in International Publication No. 99 / 58572. Some forms of humanized antibodies have one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs that are modified from the original antibody, and these CDRs are also called one or more CDRs "derived" from one or more CDRs of the original antibody.

[0019] In certain embodiments, the antibody comprises one or more constant regions, e.g., human constant regions. The constant region can be a constant region of the heavy chain and / or a constant region of the light chain. The antibody may comprise a constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identity to a human constant region. The antibody may comprise an Fc region, e.g., a human Fc region. The antibody may comprise an Fc region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identity to a human Fc region.

[0020] As used herein, the antibody specifically binds to a target molecule (CD26 or an immune checkpoint molecule, sometimes referred to herein as an "antigen" throughout). The binding affinity of the antibody used herein for the antigen is such that the dissociation constant (i.e., Kd) is less than 1×10 -5 M, less than 5×10 -5 M, less than 1×10 -6 M, less than 5×10 -7 M, less than 1×10 -7 M, less than 5×10 -8 M, less than 1×10 -8 M, less than 5×10 -9 M, less than 1×10 -9 M, less than 5×10 -10 M, less than 1×10 -10 M, less than 5×10 -11 M or less than 1×10 -11 M. Affinities having a dissociation constant of less than 1×10 -15 M or greater, 5×10 -15 M or greater, 1×10 -14 M or greater, 5×10 -14 M or greater, 1×10 -13 M or greater, 5×10 -13 M or greater, 1×10 -12 M or greater, 5×10 -12 M or greater, 1×10 -11 M or greater, 5×10 -11 M or greater, 1×10 -10 M or greater, or 5×10 -10The affinity may be M or higher. Methods for determining affinity are known in the art. For example, binding affinity may be determined using BIAcore biosensors, KinExA biosensors, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and / or yeast displays. Affinity may also be screened using appropriate bioassays.

[0021] One method for determining the binding affinity of an antibody to an antigen is to measure the affinity of the antibody's monofunctional Fab fragment. To obtain a monofunctional Fab fragment, the antibody, for example, IgG, can be cleaved with papain or expressed by recombinant technology. The affinity of the Fab fragment of a monoclonal antibody can be determined by a surface plasmon resonance (SPR) system (BIAcore 3000®, BIAcore, Piscaway, NJ). The SA tip (streptavidin) is used according to the supplier's instructions. The biotinylated antigen can be diluted in HBS-EP (100mM HEPES pH 7.4, 150mM NaCl, 3mM EDTA, 0.005% P20) and injected onto the tip at a concentration of 0.005 mg / mL. Two ranges of antigen density are achieved using variable flow times across individual tip channels: 10–20 response units (RU) for detailed kinetic testing, and 500–600 RU for concentration. A mixture of Pierce elution buffer and 4M NaCl (2:1) efficiently removes bound Fab while maintaining CD26 activity on the tip for more than 200 injections. HBS-EP buffer can be used as the running buffer for all BIAcore assays. Step dilutions of purified Fab samples (0.1–10 × estimated KD) are injected at 100 μL / min for 2 minutes, with dissociation times up to 30 minutes usually acceptable. Fab protein concentrations can be determined by ELISA and / or SDS-PAGE electrophoresis using standard Fab at known concentrations (determined by amino acid analysis). The reaction rates of binding (kon) and dissociation (koff) can be obtained simultaneously by fitting the data to a 1:1 Langmuir binding model using the BIAevaluation program (Lofas & Johnsson, 1990). The equilibrium dissociation constant (KD) value is calculated as koff / kon.

[0022] Antibodies described herein may be modified, and examples of such modifications include functionally equivalent antibodies and variants with enhanced or reduced activity that do not significantly affect the properties of the antibody. Modification of antibodies is a routine procedure in the art and does not need to be described in detail herein. Examples of modifications include conservative substitution of amino acid residues, insertion, addition or deletion of one or more amino acids that do not significantly impair functional activity, or the use of chemical analogs. Insertion or addition of amino acid sequences includes the addition of amino acids to the amino terminus and / or carboxyl terminus, and insertion of one or more amino acid residues into a sequence. Examples of terminal insertions include antibodies having an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include the fusion of an enzyme or antibody to the N-terminus or C-terminus of the antibody that prolongs the serum half-life of the antibody.

[0023] In substitutional mutants, at least one amino acid residue in the antibody sequence is removed and a different residue is inserted in its place. While CDRs are the most effective sites for substitutional mutagenesis, changes to FRs are also intended. Conservative substitutions are shown in Table 1. If such substitutions result in changes to biological activity, they are designated as “exemplary substitutions” in Table 1, or more substantial changes, such as those described below regarding amino acid classes, may be introduced to screen the products.

[0024] [Table 1]

[0025] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that significantly alter the effect of the modification on (a) the structure of the antibody backbone in the substitution region, such as sheet or helix conformation, (b) the molecular charge or hydrophobicity at the target site, or (c) the maintenance of side chain volume. Residues present in nature are classified into the following groups based on their general side chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basicity: Asn, Gln, His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0026] Non-conservative substitutions are made by exchanging one member of one class with another. More conservative substitutions include exchanging one member of one class with another member of the same class. Amino acid substitutions in variable regions can alter binding affinity and / or specificity. For example, 1 to 5 or fewer conservative amino acid substitutions are made within the CDR domain. For example, 1 to 3 or fewer conservative amino acid substitutions are made within the CDR3 domain.

[0027] By substituting any cysteine ​​residue that does not contribute to maintaining the proper three-dimensional structure of the antibody (usually with serine), the oxidative stability of the molecule can be improved and abnormal cross-linking can be prevented. Conversely, especially when the antibody is an antibody fragment such as an Fv fragment, the stability of the antibody can be improved by adding cysteine ​​bonds to the antibody.

[0028] The antibodies described herein include glycosylated and non-glycosylated antibodies, as well as antibodies with other post-translational modifications such as glycosylation, acetylation, and phosphorylation with different sugars. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The absence of fucose in the Fc region of antibodies has been reported to affect antibody-dependent cell-mediated cytotoxicity (ADCC).

[0029] (anti-CD26 antibody) In one embodiment, the anti-CD26 antibody of the present invention includes both a heavy chain variable region containing an amino acid sequence having at least about 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14, and a light chain variable region containing an amino acid sequence having at least about 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7.

[0030] Table 2 shows the amino acid sequences of the humanized VL mutants X376 (SEQ ID NO: 1), X377 (SEQ ID NO: 2), X378 (SEQ ID NO: 3), X379 (SEQ ID NO: 4), X380 (SEQ ID NO: 5), X381 (SEQ ID NO: 6), and X394 (SEQ ID NO: 7). The schemes with Kabat numbers and SEQ ID NOs correspond to the light chain variable region.

[0031] [Table 2]

[0032] Table 3 shows the amino acid sequences of the humanized VH variants X384 (SEQ ID NO: 8), X385 (SEQ ID NO: 9), X386 (SEQ ID NO: 10), X387 (SEQ ID NO: 11), X388 (SEQ ID NO: 12), X399 (SEQ ID NO: 13), and X420 (SEQ ID NO: 14). Both the SEQ ID NO sequence and the Kabat number scheme are shown. The Kabat number scheme includes 82a, 82b, and 82c.

[0033] [Table 3]

[0034] For example, the anti-CD26 antibody has a heavy chain variable region described in SEQ ID NO: 8 and a light chain variable region described in SEQ ID NO: 4. Alternatively, the anti-CD26 antibody has a heavy chain containing SEQ ID NO: 17 or a fragment thereof, preferably a heavy chain containing SEQ ID NO: 17. In one embodiment, the heavy chain of the anti-CD26 antibody has a sequence obtained by removing the signal sequence from SEQ ID NO: 17. In one embodiment, the heavy chain of the anti-CD26 antibody includes the variable region of SEQ ID NO: 17.

[0035] Heavy chain (SEQ ID NO: 17) (Underlined part is the signal sequence) MEWSWVFLFFLSVTTGVHS EVQLVESGAGVKQPGGTLRLTCTASGFSLTTYGVHWVRQAPGKGLEWVGVIWGDGRTDYDAAFMSRVTISKDTSKSTVYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0036] For example, the anti-CD26 antibody has a light chain containing SEQ ID NO: 18 or a fragment thereof, preferably a light chain containing SEQ ID NO: 18. In one embodiment, the light chain of the anti-CD26 antibody has a sequence obtained by removing the signal sequence from SEQ ID NO: 18. In one embodiment, the light chain of the anti-CD26 antibody contains the variable region of SEQ ID NO: 18.

[0037] Light chain (SEQ ID NO: 18) (Underlined part is the signal sequence) MSVPTQVLGLLLLWLTDARCDILLTQSPSSLSATPGERATITCRASQGIRNNLNWYQQKPGQAPRLLIYYSSNLQSGVPSRFSGSGSGTDFTLTISRLQPEDVAAYYCQQSIKLPFTFGGSGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0038] In this specification, "YS110," which is a humanized anti-CD26 antibody, refers to an antibody in which the heavy chain consists of the amino acid sequence described in SEQ ID NO: 17 and the light chain consists of the amino acid sequence described in SEQ ID NO: 18.

[0039] (Immune checkpoint inhibitors) An immune checkpoint inhibitor is a drug that inhibits the function of immune checkpoint molecules by binding to them. The functions of immune checkpoint molecules include suppressing the immune response against oneself to maintain immune homeostasis and suppressing excessive immune responses. Examples of immune checkpoint molecules include PD-1 or its ligand (PD-L1, PD-L2), CTLA-4 or its ligand (B7(CD80 / 86)), TIM-3 or its ligand (Galectin-9), BTLA or its ligand (HVEM), LAG-3, TCR or its ligand (MHC-II). Immune checkpoint inhibitors may be antibodies or their antigen-binding fragments, aptamers, or small molecule compounds, as long as they can bind to these immune checkpoint molecules and inhibit their function, but antibodies or their antigen-binding fragments are preferred. Antibodies that bind to immune checkpoint molecules include, for example, anti-CTLA-4 antibodies (ipilimumab), anti-PD-1 antibodies (pembrolizumab, nivolumab), and anti-PD-L1 antibodies (atezolizumab, durvalumab), which are commercially available as drugs for the treatment of cancer in humans.

[0040] As used herein, “treatment” is an approach to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms, the reduction of the scope of the disease, a stabilization (i.e., non-worsening) state of the disease, a delay or slowing of disease progression, recovery or remission of the disease state, and remission (partial or complete), whether detectable or undetectable. “Treatment” may also mean extending life expectancy compared to the expected life expectancy if no treatment is received.

[0041] An "effective dose" is an amount sufficient to achieve beneficial or desired clinical outcomes, including clinical results. An effective dose may be administered in one or more doses. For the purposes of this invention, an effective dose of the anti-CD26 antibody or immune checkpoint inhibitor described herein is an amount sufficient to delay the progression of a condition associated with tumor growth or to shrink the tumor. The effective dose may vary or depend on, among other factors, the patient's medical history and the type (and / or amount) of anti-CD26 antibody or immune checkpoint inhibitor used.

[0042] The pharmaceutical compositions described herein may, as necessary, contain pharmaceutically acceptable additives. “pharmaceutically acceptable additives” include any material that, when combined with the active ingredient, allows the active ingredient to maintain its biological activity, is non-reactive with the subject’s immune system upon delivery, and is non-toxic to the subject. Examples include, but are not limited to, all standard pharmaceutical carriers such as phosphate-buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for spray or parenteral administration are phosphate-buffered saline or physiological saline (0.9%). Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington’s Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990 and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

[0043] In one embodiment, the antibody is expressed in host cells of any organism or derived from any organism, including but not limited to bacteria, yeast, plants, insects, and mammals. Specific cell types include, but are not limited to, Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, Escherichia coli, Bacillus subtilis, SF9 cells, HEK-293 cells, Neurospora, BHK cells, CHO cells, COS cells, HeLa cells, fibroblasts, Schwanno cell lines, immortalized mammalian bone marrow and lymphoid cell lines, Jurkat cells, mast cells and other endocrine and exocrine cells, and nerve cells.

[0044] Various protein expression systems, vectors, and cell culture media useful for antibody production are known to those skilled in the art. See, for example, International Publication Nos. 03 / 054172, 04 / 009823, and 03 / 064630 (the full texts of which are incorporated herein by reference). In one embodiment, a glutamine synthase (GS) expression system is used for the expression of the antibody.

[0045] The target animals for treatment include mammals such as humans, cattle, horses, dogs, cats, pigs, and sheep, with humans being preferred.

[0046] The pharmaceutical composition of the present invention may be any oral or parenteral formulation, as long as it can be administered to a patient. Examples of compositions for parenteral administration include injections and nasal sprays. Preferably, it is an injection. Examples of dosage forms of the pharmaceutical composition of the present invention include liquid formulations and lyophilized formulations. When the pharmaceutical composition of the present invention is used as an injection, additives such as solubilizers such as propylene glycol and ethylenediamine, buffers such as phosphates, isotonic agents such as sodium chloride and glycerin, stabilizers such as sulfites, preservatives such as phenol, and analgesics such as lidocaine may be added as needed (see "Dictionary of Pharmaceutical Additives" Yakuji Nippo Co., Ltd. and "Handbook of Pharmaceutical Excipients Fifth Edition" APhA Publications). When the pharmaceutical composition of the present invention is used as an injection, examples of storage containers include ampoules, vials, pre-filled syringes, pen-type syringe cartridges, and infusion bags.

[0047] The combination agent of the present invention may contain all or some of the two or more drugs used in combination in a single formulation, or it may be provided as a formulation containing each drug individually for the purpose of combination therapy. Alternatively, the combination agent of the present invention may be provided as a kit or set for combination therapy, comprising formulations containing each drug individually.

[0048] In another embodiment, the present invention relates to a method for treating cancer, comprising administering an anti-CD26 antibody and an immune checkpoint inhibitor to a patient having cancer. In yet another embodiment, the present invention relates to a pharmaceutical composition containing an anti-CD26 antibody and an immune checkpoint inhibitor for use in treating cancer. Alternatively, the present invention relates to the use of an anti-CD26 antibody and an immune checkpoint inhibitor for the manufacture of a cancer treatment agent. In the cancer treatment described herein, the order in which the anti-CD26 antibody and the immune checkpoint inhibitor are administered to the cancer patient is not limited to this order; either may be administered first, i.e., the immune checkpoint inhibitor may be administered after the anti-CD26 antibody, or the anti-CD26 antibody may be administered after the immune checkpoint inhibitor, or both may be administered simultaneously or sequentially.

[0049] The pharmaceutical compositions of the present invention may be administered to mammals by injection (e.g., systemically, intravenously, intraperitoneally, subcutaneously, intramuscularly, intraportally, or into cancerous tissue), or by other methods that ensure delivery into the bloodstream in an effective form (e.g., infusion).

[0050] The methods described herein (including treatment methods) may be administered by a single direct injection at one or more sites at one or more time points. Administration may also be performed at multiple sites almost simultaneously. The frequency of administration will be determined and adjusted during the course of treatment based on the desired outcome. In some cases, a sustained-release formulation of the pharmaceutical composition of the present invention may be appropriate. Various formulations and devices for achieving sustained release are well known in the art.

[0051] The effective dose and schedule for administering the pharmaceutical composition of the present invention are determined empirically, and such determination methods are within the common technical knowledge of the art. Those skilled in the art will understand that the dose of the pharmaceutical composition of the present invention to be administered will vary depending, for example, on the mammal to which the pharmaceutical composition of the present invention is administered, the route of administration, the specific type of drug used, and other drugs administered to the mammal. A typical daily dose of the pharmaceutical composition of the present invention may range from about 1 μg / kg body weight to 100 mg / kg body weight or more, depending on the factors described above. Generally, any of the following doses may be used: at least about 50 mg / kg body weight; at least about 10 mg / kg body weight; at least about 3 mg / kg body weight; at least about 1 mg / kg body weight; at least about 750 μg / kg body weight; at least about 500 μg / kg body weight; at least about 250 μg / kg body weight; at least about 100 μg / kg body weight; at least about 50 μg / kg body weight; at least about 10 μg / kg body weight; or at least about 1 μg / kg body weight, or higher doses.

[0052] Examples are shown below to illustrate the present invention in more detail, but the present invention is not limited thereto. All references cited throughout this application are incorporated herein by reference.

[0053] (Materials and Methods) 1) Cell Human malignant mesothelioma cell line JMN and human lung adenocarcinoma cell line HCC4006 were cultured in RPMI1640 medium supplemented with 10% FBS at 37°C under 5% CO2 conditions. Human umbilical cord blood CD34-positive hematopoietic stem cells were purchased from RIKEN BioResource Center.

[0054] 2) Mouse NOD / Shi-scid,IL-2RγKO Jic(NOD.Cg-Prkdc scid Il2rg tm1SugThe / ShiJic) mice (hereinafter referred to as NOG mice) were purchased from In-Vivo Science Inc. The mice were raised in a specific pathogen-free (SPF) facility at Juntendo University.

[0055] 3) Antibodies and reagents The following human antigen-specific antibodies were used for flow cytometry. BUV395-labeled anti-CD56 mAb (clone NCAM16.2) and PE-labeled anti-CD26 mAb (clone M-A261) were purchased from BD Biosciences. Brilliant Violet 421-labeled anti-CD4 mAb (clone RPA-T4), Brilliant Violet 510-labeled anti-CD14 mAb (clone M5E2), FITC-labeled anti-CD8 mAb (clone HIT8a), PerCP / Cy5.5-labeled anti-CD20 mAb (clone 2H7), APC-labeled anti-CD45 mAb (clone HI30), APC / Fire 750-labeled anti-CD3 mAb (clone SK7), and Brilliant Violet 605-labeled anti-mouse CD45 mAb (clone 30-F11), as well as Human TruStain FcX and TruStain FcX (anti-mouse CD16 / 32) for blocking nonspecific antibody binding, were purchased from BioLegend. Additionally, Brilliant Stain Buffer plus, which suppresses nonspecific binding between Brilliant Violet molecules, was purchased from BD Biosciences.

[0056] (Consideration for ethical aspects) Regarding the research using human umbilical cord blood CD34-positive hematopoietic stem cells, the inventor submitted a research plan and other documents for this research to the Department of Medicine, Graduate School of Medicine, Juntendo University, where the head of the department is located, and it was approved by the Ethics Review Committee (Juntendo Medical Ethics No. 2017167, 2020280). Animal experiments were conducted based on the so-called 3Rs, and the experimental plan was submitted to the Experimental Animal Committee of the Faculty of Medicine, Juntendo University, reviewed, and approved (Approval Numbers: 2020270, 2021056).

[0057] (Example 1) Creation of human immunized mice Since the presence of an immune system, particularly T cells, is essential for ICI to exert its antitumor effect, experiments investigating the combined effects of humanized anti-CD26 antibodies and ICIs require the use of human immunized mice. To develop new combination therapies that take advantage of the fewer side effects of humanized anti-CD26 antibodies, we created human malignant mesothelioma cell lines and human lung cancer cell lines as cancer-bearing models using human immunized mice.

[0058] (1) Creating mice NOG mice were irradiated with a low dose (100 cGy), and the following day, human umbilical cord blood CD34-positive hematopoietic stem cells (1 x 10⁶) were administered. 5 Cells were transferred into the mouse tail vein. Blood samples were collected from the mouse tail vein at various time points to confirm the engraftment of human immune cells.

[0059] To investigate the combined effects of humanized anti-CD26 antibody and PD-1 antibody, we created human immunized mice in which human immune cells were engrafted. Creating these mice required irradiating NOG mice (severely immunodeficient mice) with low-dose radiation and transplanting human hematopoietic stem cells. The time between thawing and transplantation is considered crucial in clinical hematopoietic stem cell transplantation. There are differences in washing buffer, number of washing (centrifugation) cycles, and centrifugation time between the protocol published by RIKEN BioResource Center, the supplier of human umbilical cord blood CD34-positive hematopoietic stem cells, and the protocol published by the Central Institute for Experimental Animals, which developed the NOG mice. Therefore, we investigated protocols that ensure stable engraftment of human immune cells.

[0060] (1-1) Cell preparation a) Central Institute for Laboratory Animals Protocol (https: / / www.ciea.or.jp / laboratory_animal / pdf / NOG_huHSC.pdf) Frozen CD34+ cells (2.5 × 10⁻⁶) 5 After thawing the cells in a 37°C water bath, they were transferred to a 50 mL conical tube. While shaking the tube containing the cells, 1 mL of PBS containing 2% fetal bovine serum (2% FBS-PBS) was slowly added dropwise. After adding another 18 mL of 2% FBS-PBS, the tube was centrifuged at room temperature at 1,200 rpm for 5 minutes. The harvested cells were resuspended in 10 mL of 2% FBS-PBS and centrifuged again under the same conditions. The harvested cells were then resuspended in PBS.

[0061] b) RIKEN BioResource Center protocol (Ishikawa, F., et al., Blood;106:1565-1573(2005)) Frozen CD34+ cells (2.5 × 10⁻⁶) 5 The cells were thawed in a 37°C water bath and then transferred to a 50 mL conical tube. 15 mL of washing solution (PBS) was added dropwise at a rate of 1 drop / 5 seconds. After mixing, an additional 2-3 mL of PBS was added to bring the total volume to 20 mL. The mixture was centrifuged at 4°C and 300 G for 10 minutes. The harvested cells were resuspended in PBS.

[0062] c) Modified protocol by the inventors Frozen CD34+ cells (2.5 × 10⁻⁶) 5 The cells were thawed in a 37°C water bath and then transferred to a 50 mL conical tube. 15 mL of PBS containing 10% fetal bovine serum (10% FBS-PBS) was added dropwise at a rate of 1 drop / 5 seconds. After mixing, an additional 3-4 mL of 10% FBS-PBS was added to bring the total volume to 20 mL. The mixture was centrifuged at 4°C and 1,300 rpm for 5 minutes. The harvested cells were resuspended in PBS.

[0063] (1-2) Introduction of cells into mice In NOG mice irradiated with 100 cGy, 0.2 mL (1 × 10⁶) was injected into the tail vein using a 29G microinjection syringe. 5 A cell suspension of the cells was injected.

[0064] (2) Flow cytometry To confirm the engraftment of human immune cells in mouse bodies, peripheral blood obtained from the tail vein of mice was stained with both human and mouse-specific TruStain FcX, then stained with fluorescently labeled antibodies. After hemolysis and fixation using BD FACS Lysing Solution (BD Biosciences), the blood was washed and measured using BD LSRFortessa (BD Biosciences). The obtained data was then analyzed using FlowJo (BD Biosciences).

[0065] (3) Results In the protocol published by the Central Institute for Experimental Animals, human B cells engrafted stably, but human T cells did not develop even after 20 weeks post-transplantation (results not shown). On the other hand, in the protocol published by RIKEN BioResource Center, the development of human T cells was consistently confirmed, but there were cases where the viability of CD34+ cells after thawing was low. Therefore, after modifying the washing buffer and centrifugation time, the protocol developed by the inventors confirmed the development of human T cells without exception in all mice examined. Up to 10 weeks after transplantation of human hematopoietic stem cells, approximately 90% of the human immune cells in the mouse blood were B cells (CD20 positive). After 10 weeks, the proportion of human CD4 T cells (CD3 positive, CD4 positive) and CD8 T cells (CD3 positive, CD8 positive) gradually increased, and at 14 weeks, approximately 20% of human blood cells were T cells, and at 18 weeks, approximately 30% were T cells (Figure 1). In this model, human NK cells (CD56-positive) accounted for approximately 1-2%, and human monocyte cells (CD14-positive) accounted for approximately 1%.

[0066] (Example 2) Combination study using JMN cell transplanted human immunization model mice Using the mice prepared in Example 1, a human malignant mesothelioma cell JMN model was created, and the combined effect of anti-CD26 antibody and anti-PD-1 antibody was confirmed.

[0067] The malignant mesothelioma cell line JMN proliferates very slowly in vivo, and it takes 5-6 weeks for it to form a tumor after being transplanted subcutaneously into mice. Therefore, the JMN cell line was subcutaneously transplanted 13 weeks after hematopoietic stem cell transplantation, when the number of human T cells in the mouse body had increased.

[0068] Thirteen weeks after transplantation of human hematopoietic stem cells, a cell suspension of JMN cells and Matrigel were mixed in a 1:1 ratio, and 1 × 10⁶ cells were administered per animal. 6 JMN cells were subcutaneously transferred into the flank of each mouse. Five weeks after subcutaneous transfer of JMN, when small tumor formation was confirmed, control human IgG1 (Bio X Cell), humanized anti-CD26 antibody (YS110) (Y's AC Co.,Ltd) alone, mouse anti-human PD-1 mAb (Bio X Cell; clone J116) alone, and a combination of humanized anti-CD26 antibody and PD-1 antibody were administered at 200 μg / dose three times a week, and administration continued until dissection at 9 weeks. Tumor size was measured twice a week, and tumor volume was calculated as (width × length × height) × 1 / 2. Nine weeks after JMN transfer, the mice were dissected, the subcutaneous tumors were collected, and their weight was measured. A portion of the tumor was fixed with 10% formalin for pathological analysis, while the remainder was enzymatically treated with Liberase TL Research Grade (Roche) 0.25 mg / ml, and the tissue was lysed in the presence of DNase I (Roche) to obtain cells from the tumor tissue. For the analysis of tumor-infiltrating lymphocytes, lymphocyte purification was performed using the MagniSort Human CD3 Positive Selection Kit (invitrogen) and EasySep Magnet (STEMCELL). In addition, spleen lymphocyte analysis was performed to compare the properties of tumor-infiltrating lymphocytes with those of other lymphocytes.

[0069] Tumor size was measured twice a week, and the tumor size 9 weeks after JMN transfer was compared to the control antibody group (average 215.5 mm). 3 In comparison with humanized anti-CD26 antibody alone (YS alone) (mean value 176.3 mm), 3 ), PD1 antibody alone (average value 169.6 mm) 3 While tumor growth was suppressed in each group, the tumor size was even smaller in the group treated with both antibodies (YS+PD1) (average 123.6 mm). 3 ), it became clear that a synergistic effect was observed (Figure 2).

[0070] Nine weeks after JMN transfer, mice were dissected, and subcutaneous tumors were collected. Some were used for pathological analysis, while the remainder were used to purify tumor-infiltrating lymphocytes for phenotypic analysis. No significant differences were observed between the control group and the YS alone group, or between the control group and the PD1 alone group, in either tumor volume at 9 weeks of dissection or tumor weight. However, a significant difference (p<0.05) was observed between the control group and the YS+PD1 group (p=0.0167, Fisher's multiple comparison test), suggesting the potential benefits of combining both antibodies (Figure 3).

[0071] (Example 3) Combination study using HCC4006 cell transplanted human immunization model mice Using the mice prepared in Example 1, a human lung adenocarcinoma cell line HCC4006 tumor-bearing model was created, and the combined effect of anti-CD26 antibody and anti-PD-1 antibody was confirmed.

[0072] Fourteen weeks after transplantation of human hematopoietic stem cells, a cell suspension of HCC4006 cells and Matrigel were mixed in a 1:1 ratio, resulting in 1 × 10⁶ cells per animal. 6Cells were subcutaneously transferred into the flank of each mouse. Three weeks after subcutaneous transfer of HCC4006, when small tumor formation was confirmed, administration of control human IgG1 (Bio X Cell), humanized anti-CD26 antibody (YS110) (Y's AC Co.,Ltd) alone, mouse anti-human PD-1 mAb (Bio X Cell; clone J116) alone, or a combination of humanized anti-CD26 antibody and PD-1 antibody was started at 200 μg / dose three times a week and continued until dissection at 6.5 weeks. Tumor size was measured twice a week, and tumor volume was calculated as (width × length × height) × 1 / 2. Six and a half weeks after HCC4006 transfer, the mice were dissected, and the subcutaneous tumors were collected and weighed. A portion of the tumor was fixed with 10% formalin for pathological analysis, while the remainder was enzymatically treated with Liberase TL Research Grade (Roche) 0.25 mg / ml, and the tissue was lysed in the presence of DNase I (Roche) to obtain cells from the tumor tissue. For the analysis of tumor-infiltrating lymphocytes, lymphocyte purification was performed using the MagniSort Human CD3 Positive Selection Kit and EasySep Magnet. In addition, splenic lymphocyte analysis was performed to compare the properties of tumor-infiltrating lymphocytes with those of other lymphocytes.

[0073] Tumor size was measured twice a week, and the tumor size 6 weeks after HCC4006 transfer was compared to the control antibody group (average 445.2 mm). 3 Compared to humanized anti-CD26 antibody alone (YS alone) (mean value 272.3 mm) 3 ), PD1 antibody alone (average value 236.0 mm) 3 While suppression of tumor growth was observed in each group, the tumor size was even smaller in the group treated with both antibodies (YS+PD1) (average 163.7 mm). 3 ), it became clear that a synergistic effect was observed (Figure 4).

[0074] Six and a half weeks after HCC4006 transfer, mice were dissected, and subcutaneous tumors were collected. Some were used for pathological analysis, while the remainder were used to purify tumor-infiltrating lymphocytes for phenotypic analysis. Both tumor volume at 6.5 weeks before dissection and the collected tumor weight showed suppression of tumor growth in both the YS alone group and the PD1 alone group compared to the control group. However, the group treated with both antibodies (YS + PD1) had an even smaller tumor size, suggesting the potential benefits of combining both antibodies (Figure 5).

Claims

1. A pharmaceutical composition for cancer treatment comprising an immune checkpoint inhibitor and an anti-CD26 antibody or its antigen-binding fragment as active ingredients, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody, or their antigen-binding fragments, the anti-CD26 antibody has an antigen-binding site composed of a heavy chain consisting of the sequence described in SEQ ID NO: 17 and a light chain consisting of the sequence described in SEQ ID NO: 18, and the antigen-binding fragment is selected from Fv, Fab, Fab', or F(ab')2.

2. A cancer treatment pharmaceutical composition for use in combination with an immune checkpoint inhibitor, comprising an anti-CD26 antibody or an antigen-binding fragment thereof as an active ingredient, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody, or an antigen-binding fragment thereof, the anti-CD26 antibody has an antigen-binding site composed of a heavy chain consisting of the sequence described in SEQ ID NO: 17 and a light chain consisting of the sequence described in SEQ ID NO: 18, and the antigen-binding fragment is selected from Fv, Fab, Fab', or F(ab')2.

3. A cancer treatment pharmaceutical composition containing an immune checkpoint inhibitor as an active ingredient for use in combination with an anti-CD26 antibody or an antigen-binding fragment thereof, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody, or an antigen-binding fragment thereof, the anti-CD26 antibody has an antigen-binding site composed of a heavy chain consisting of the sequence described in SEQ ID NO: 17 and a light chain consisting of the sequence described in SEQ ID NO: 18, and the antigen-binding fragment is selected from Fv, Fab, Fab', or F(ab')2.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein an immune checkpoint inhibitor and an anti-CD26 antibody or its antigen-binding fragment are administered simultaneously, consecutively, or separately at intervals.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is a cancer that expresses CD26.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof.

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