Bispecific antibodies against CHI3L1 and PD1 that exhibit enhanced T cell-mediated cytotoxic effects against tumor cells.

Abstract

To provide more effective immunotherapy against individual immune checkpoint inhibitor molecules such as PD-1.SOLUTION: The present invention provides a humanized bispecific antibody that simultaneously detects and neutralizes both CHI3L1 and the immune checkpoint inhibitor PD-1, wherein these antibodies exhibit enhanced synergistic cytotoxic effects compared to the effects of individual CHI3L1 and PD-1 antibodies, alone or in combination. A pharmaceutical composition comprising the bispecific antibody, and a therapeutic agent for cancer comprising the bispecific antibody, are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Embodiments of the present invention relate to bispecific antibodies that simultaneously target both CHI3L1 and the immune checkpoint molecule PD-1. These bispecific antibodies, either alone or in combination, exhibit a greater and more amplified synergistic cytotoxic effect than the effects of individual CHI3L1 and PD-1 antibodies. Statements relating to federally funded research or development This invention was developed with the support of NIH CADET Grant UH2 HL123876, awarded by the National Institutes of Health. The government reserves certain rights to this invention. [Background technology]

[0002] Currently, immunotherapy against individual immune checkpoint inhibitor (ICPI) molecules is effectively applied to patients with various malignancies, including lung cancer and gliablastoma. Examples include antibodies against molecules such as programmed death receptor 1 (PD-1). However, only some patients respond to these therapies. Furthermore, the responses observed are often not sustained. Consequently, extensive efforts are being made worldwide to develop methods to increase the effectiveness of ICPI immunotherapy using antibodies against immune checkpoint molecules, including PD-1. [Overview of the project] [Problems that the invention aims to solve]

[0003] Therefore, more effective immunotherapies are needed for individual immune checkpoint inhibitor molecules such as PD-1. [Means for solving the problem]

[0004] Previous studies have demonstrated that chitinase 3-like-1 (CHI3L1) plays a crucial role in the pathogenesis of various cancers. Furthermore, we have previously demonstrated that a combination of anti-CHI3L1 and anti-PD-1 antibodies produces a synergistic effect in cancer treatment. Based on these findings, we hypothesized that simultaneous targeting of CHI3L1 and PD-1 may have additional synergistic and / or additive antitumor effects. To investigate these possibilities, we developed bispecific antibodies that react simultaneously with CHI3L1 and PD-1.

[0005] Embodiments of the present invention provide a humanized bispecific antibody that simultaneously detects and neutralizes both CHI3L1 and the immune checkpoint inhibitor PD-1. The bispecific antibody comprises an antigen-binding moiety of an anti-human PD-1 antibody and an antigen-binding moiety of an anti-human CHI3L1 antibody.

[0006] In some embodiments, the bispecific antibody comprises an anti-human PD-1 single-chain variable fragment (ScFv-PD1) conjugated to the backbone of an anti-human CHI3L1 antibody. ScFv-PD1 can be conjugated to either the CHI3L1 antibody heavy chain (CHI3L1-HC-PD1) or the CHI3L1 antibody light chain (CHI3L1-LC-PD1).

[0007] In an alternative embodiment, the bispecific antibody comprises an anti-human CHI3L1 single-chain variable fragment (ScFv-CHI3L1) conjugated to the backbone of an anti-human PD-1 antibody. ScFv-CHI3L1 can be conjugated to either the PD-1 antibody heavy chain (PD-1-HC-CHI3L1) or the PD-1 antibody light chain (PD-1-LC-CHI3L1).

[0008] In one embodiment, the antigen-binding portion of the anti-human CHI3L1 antibody includes the following complementarity-determining regions (CDRs): (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 4; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; (c) light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (d) heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; (e) heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2; and (f) heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3. In one embodiment, the antigen-binding portion of the anti-human CHI3L1 antibody includes a heavy chain sequence having the amino acid sequence of SEQ ID NO: 13. In one embodiment, the antigen-binding portion of the anti-human CHI3L1 antibody includes a light chain sequence having the amino acid sequence of SEQ ID NO: 14.

[0009] In one embodiment, the antigen-binding portion of the anti-human PD-1 antibody includes the amino acid sequence of SEQ ID NO: 35.

[0010] As described herein, the bispecific antibodies of the present invention exhibited remarkable antitumor effects. These bispecific antibodies, alone or in combination, showed an enhanced synergistic cytotoxic effect compared to the effects of individual CHI3L1 and PD-1 antibodies. The bispecific antibodies of the present invention (i) increase the adhesion of Jurkat T cells to U87 cells; (ii) increase the ability of Jurkat T cells to induce cytotoxic / apoptotic responses in U87 cells; (iii) increase the accumulation of granzymes and perforins in Jurkat T cells co-cultured with U87 cells; and / or (iv) increase the ability of Jurkat T cells to induce lactate dehydrogenase (LDH) release and cytotoxic responses in U87 cells.

[0011] Embodiments of the present invention also provide pharmaceutical compositions comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises a chemotherapeutic agent.

[0012] Embodiments of the present invention also provide a method for treating cancer in a subject by administering a therapeutically effective amount of the bispecific antibody or pharmaceutical composition of the present invention. In one embodiment, the cancer is a malignant cancer. In one embodiment, the cancer is a primary cancer or a metastatic cancer. In one embodiment, the cancer is one of the following: prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, gliablastoma, melanoma, malignant melanoma, or lung cancer. In one embodiment, the subject is a subject determined to have elevated levels of CHI3L1. In one embodiment, the elevated CHI3L1 level is circulating CHI3L1. In one embodiment, the cancer expresses PD-L1.

[0013] The bispecific antibodies of the present invention target CHI3L1 and PD-1 and, either alone or in combination, have an antitumor cytotoxic effect that surpasses the effects of anti-CHI3L1 and anti-PD-1 antibodies.

[0014] Other embodiments are also described and enumerated herein.

[0015] For illustrative purposes, specific embodiments of the present invention are shown in the drawings below. Similar figures in the drawings indicate similar elements throughout. However, it should be understood that the present invention is not limited to the exact arrangements, dimensions, and fixtures shown. [Brief explanation of the drawing]

[0016] [Figure 1] This figure provides a schematic diagram of the CHI3L1×PD1 bispecific antibody structure. The platform used to generate the bispecific antibody is illustrated in Figure 1A: CHI3L1-LC×ScFv-HC-PD1-ScFv-LC-PD1 (CHI3L1-LC-PD1) and Figure 1B: CHI3L1-HC×ScFv-HC-PD1-ScFv-LC-PD1 (CHI3L1-HC-PD1). [Figure 2]This figure shows the binding affinity of the CHI3L1×PD1 bispecific antibody. The affinity of the CHI3L1-LC-PD1 antibody was evaluated by a competitive ELISA assay: Figure 2A shows the evaluation against recombinant human (rh)CHI3L1; Figure 2B shows the evaluation against rhPD1; and Figure 2C shows the evaluation against a mixture of rhCHI3L1 and rhPD1. There was no difference in binding affinity to rhCHI3L1 or rhPD1 between the CHI3L1-LC-PD1 antibody and the CHI3L1-HC-PD1 antibody. [Figure 3] This figure shows that the bispecific CHI3L1×PD1 antibody significantly increases the adhesion of Jurkat T cells to U87 gliablastoma cells in a co-culture system. Jurkat T cells were activated by anti-human CD3 / CD28 treatment (5 μg / ml each, incubated in 5% CO2 and air at 37°C for 2 hours) and then co-cultured with U87 neurogliablastoma cells. Culture was carried out with isotype control antibodies, as well as antibodies specific to PD-1, CHI3L1, CHI3L1+PD-1, and an antibody bispecific to CHI3L1×PD-1. In Figure 3A, CellBrite cytoplasmic membrane dye was used for fluorescent labeling of U87 (red) and Jurkat T cells (green). Figure 3B shows phase-contrast images taken 6 hours after incubation with IgG control antibody and the indicated antibody (5 mg / ml each). Isotype, IgG control antibody; PD1, α-human PD1 antibody; CHI3L1, α-human CHI3L1 antibody; CHI3L1+PD1, with α-CHI3L1 antibody + α-PD1 antibody; CHI3L1×PD1, bispecific CHI3L1-PD1 antibody. Figure 3C shows the number of Jurkat T cells attached to each U87 cell evaluated via counted fluorescence microscopy (20x magnification of the original; this evaluation included 10 randomly selected regions). Values ​​are mean ± SEM. *p<0.05, **p<0.01, by t-test. [Figure 4]This figure shows that treatment with a CHI3L1×PD1 bispecific antibody increased the U87 gliablastoma cell death response in co-culture of U87-Jurkat T cells. Jurkat T cells were activated by anti-CD3 / CD28 treatment (5 μg / ml each, incubated in 5% CO2 and air at 37°C for 2 hours) and then co-cultured with U87 gliablastoma cells. Culturing was performed with isotype control antibodies, as well as antibodies specific to PD-1, CHI3L1, CHI3L1+PD-1, and antibodies bispecific to CHI3L1×PD-1. In Figures 4A-B, CellBrite cytoplasmic membrane dye was used for fluorescent labeling of live cells (green), and propidium iodide staining was used for dead cells (red). Six hours after incubation, cells were treated with vehicle only (Figure 4A) and with IgG2b isotype control or indicated antibody (5 mg / ml each) (Figure 4B), and fluorescence images were acquired. Figure 4C shows TUNEL staining and images acquired under a bright-field microscope. Figure 4D shows the quantification of TUNEL-positive apoptotic U87 cells. TUNEL-positive apoptotic cells were counted under a light microscope (20x magnification of the original) and expressed as a percentage of the total number of cells evaluated (10 microscope fields were randomly selected and used for this evaluation). Values ​​are mean ± SEM. *p<0.05, **p<0.01, t-test. [Figure 5]This figure shows that treatment with a bispecific CHI3L1×PD1 antibody increased granzyme accumulation in Jurkat T cells in co-culture with U87 gliablastoma cells. Jurkat T cells were activated by anti-human CD3 / CD28 treatment (5 μg / ml each, 2-hour incubation, 5% CO2 and air, 37°C) and then co-cultured with U87 gliablastoma cells. Cells were cultured with isotype control antibodies, as well as antibodies specific to PD-1, CHI3L1, CHI3L1+PD-1, and an antibody bispecific to CHI3L1×PD-1. Fluorescence images were acquired 6 hours after incubation with IgG2b isotype control and the indicated antibody (5 mg / ml each) (Figure 5A). Dual immunohistochemical staining of cells was performed using α-granzyme and α-phalloidin antibodies. Figure 5B shows the quantification of granzyme+ cells. The number of granzyme+ cells was counted under a fluorescence microscope (20x magnification of the original; 10 randomly selected regions were included in this assessment). Values ​​are mean ± SEM. *p<0.05, by t-test. [Figure 6]This figure shows that treatment with a CHI3L1×PD1 bispecific antibody increased perforin accumulation in Jurkat T cells in co-culture with U87 gliablastoma cells. Jurkat T cells were activated by anti-human α-CD3 / α-CD28 treatment (5 μg / ml each, incubated in 5% CO2 and air at 37°C for 2 hours). They were cultured with isotype control antibodies, as well as antibodies specific to PD-1, CHI3L1, CHI3L1+PD-1, and an antibody bispecific to HI3L1×PD-1. Images were acquired 6 hours after incubation with IgG2b isotype control and indicated antibodies (5 mg / ml each) (Figure 6A). Dual immunohistochemical staining of cells was performed using anti-perforin and anti-phalloidin antibodies. Figure 6B shows the quantification of perforin-positive (+) cells. The average number of perforin-+ cells per microscopic field of view was counted (20x the original magnification). Isotype, IgG2b control antibody; PD1, anti-human PD1 antibody; CHI3L1, anti-human CHI3L1 antibody; CHI3L1+PD1, anti-CHI3L1 antibody + anti-PD1 antibody together; CHI3L1×PD1, bispecific CHI3L1×PD1 antibody. 20x magnification of the original. Values ​​are mean ± SEM. *p<0.05, **p<0.01, by t-test. [Figure 7]This figure shows that treatment with a bispecific CHI3L1×PD1 antibody increased LDH release from U87 cells in a Jurkat-U87 coculture. Jurkat T cells were activated by anti-human CD3 / CD28 treatment (5 μg / ml each, 2-hour incubation, 5% CO2 and air, 37°C). They were cultured with isotype control antibodies, as well as antibodies specific to PD-1, CHI3L1, CHI3L1+PD-1, and an antibody bispecific to CHI3L1×PD-1. After 6 hours of incubation with IgG control and indicated antibodies (5 mg / ml each), LDH activity was measured using an assay kit (Pierce LDH cytotoxicity assay kit). ns, not significant, *p<0.05, **p<0.01, ***p<0.001, t-test. The LDH released by U87 cells in co-culture was compared to the levels of total LDH in U87 cells alone (-ve control), U87 cells treated with lysis buffer (+ve control), and LDH released by Jurkat cells treated with lysis buffer (Jurkat). [Figure 8]This figure shows that treatment with bispecific CHI3L1×PD1 antibody induced a synergistic CTL-mediated tumor cell death response and tumor cell PTEN expression. (Column A) Representative demonstration and quantification of apoptotic tumor cell death using the in situ cell detection kit - fluorescein dUTP. TUNEL(+) cells stain green. (Columns B-D) Representative demonstration and quantification of Jurkat T cell expression of CD8 (Column B), perforin (Column C), and granzyme (Column D). Tumor cells are green, and positively stained Jurkat cells are yellow-orange. (Column E) Representative demonstration and quantification of tumor cell PTEN. Tumor cells are green, and PTEN is yellow-orange. (Row F) Quantification of the evaluations in columns A-E. The percentages of TUNEL+ tumor cells (column A), Jurkat cells expressing CD8 (column B), perforin (column C), granzyme (column D), and tumor cells expressing PTEN (column E) are illustrated. These assessments were performed using a fluorescence microscope (20x magnification of the original). Ten randomly selected fields were evaluated in these quantifications. Values ​​in panel F are the mean ± SEM of the four assessments of interest. **P<0.01. ***P<0.001. Scale bar = 10 μm, applicable to all subpanels A-E. [Modes for carrying out the invention]

[0017] To gain a substantial understanding of the present invention, it should be understood that specific aspects, styles, embodiments, variations, and features of the present invention are described below in detail at various levels.

[0018] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implicitly understood from the context, the following terms and phrases have the meanings provided below. The definitions are provided to assist in describing specific embodiments and that the scope of the invention is not limited solely to the claims. Therefore, this specification is not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. In the event of any obvious difference between the use of a term in the art and the definition provided herein, the definition provided herein shall prevail.

[0019] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. For example, a reference to “cell” includes a combination of two or more cells, etc.

[0020] As used herein, the term "or" means "and / or". When used in phrases such as "A and / or B", the term "and / or" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, when used in phrases such as "A, B, and / or C", the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0021] The abbreviation "e.g." originates from the Latin "exempli gratia" and is used herein to indicate non-restrictive examples. Therefore, the abbreviation "e.g." is synonymous with the term "for example."

[0022] Where used herein, the terms “approximately” or “about” with respect to a value or parameter are generally interpreted to include numbers that fall within the range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of a possible value, unless otherwise specified or as is evident from the context (except when such a number is less than 0% or greater than 100%). Where used herein, a reference to a value or parameter using “approximately” or “about” includes (and describes) embodiments directed toward that value or parameter. For example, a statement referring to “about X” includes a statement of “X”.

[0023] As used herein, the term "includes" means that, in addition to the defined elements presented, other elements may also be present. The use of "includes" indicates inclusion, not limitation.

[0024] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not described in the description of the embodiments.

[0025] As used herein, the term “essentially consisting of” refers to elements necessary for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic and novel or functional features (one or multiple) of the embodiments of the invention.

[0026] The terms "statistically significant" or "significantly significant" refer to statistical significance, which generally means a difference of two standard deviations (2SD) or more.

[0027] As used herein, the terms “therapeutably effective amount,” “effective amount,” or “effective dose” mean an amount that provides therapeutic or aesthetic benefit in the treatment, prevention, or management of a tumor or malignant tumor, for example, an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of the tumor or malignant tumor. It is understood that many methods for determining an effective amount for a given use are known in the art. For example, dosage determination Pharmacological methods for determination may be used in therapeutic situations. In the context of therapeutic or prophylactic application, the amount of composition administered to the subject depends on the type and severity of the disease, as well as individual characteristics such as general health, age, sex, weight, and tolerance to the drug. It also depends on the degree, severity, and type of the disease. Those skilled in the art can determine the appropriate dosage according to these and other factors. The composition may also be administered in combination with one or more further therapeutic compounds.

[0028] As used herein, the terms “to treat,” “to treat,” “to treat,” or “to improve” mean, when used in relation to a disease, disorder, or medical condition, a therapeutic treatment for a condition in which the subject is to reverse, reduce, alleviate, inhibit, slow down, or halt the progression or severity of symptoms or conditions. The term “to treat” includes reducing or reducing at least one adverse effect or symptom of a condition. A treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Or, a treatment is “effective” if the progression of a condition is reduced or interrupted. That is, “treatment” includes not only improvement of symptoms or markers but also halting or at least slowing the progression or worsening of symptoms that would be expected without treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduced degree of deficit, stable (i.e., non-worsening) state of a tumor or malignant tumor, delayed or slowed tumor growth and / or metastasis, and extended lifespan, compared to what would be expected without treatment.

[0029] As used herein, the term “administer” means to place the bispecific antibodies disclosed herein into a subject by a method or route that results in at least partial delivery of the drug at the desired site. Pharmaceutical compositions comprising the compounds disclosed herein may be administered by any suitable route that results in effective treatment in a subject.

[0030] As used herein, the term “long-term” administration means that a therapeutic agent or drug is administered for at least 12 weeks. This includes the administration of the therapeutic agent or drug in such a manner that it is effective over a period of at least 12 weeks or for a period of at least 12 weeks, and does not necessarily mean that the administration itself takes place over 12 weeks, for example, when a sustained-release composition or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. Often, long-term administration is at least 4, 5, 6, 7, 8, 9 months or longer, or at least 1, 2, 3, 5, 7, 10 years or longer.

[0031] The compositions intended herein may be administered by any convenient method, including aerosol inhalation, injection, oral ingestion, blood transfusion, implantation, or transplantation. In preferred embodiments, the compositions are administered parenterally. As used herein, “parenteral administration” and “administered parenterally” refer to, but are not limited to, methods of administration other than enteral and topical administration, usually by injection, and include intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. In one embodiment, the compositions intended herein are administered to a target by direct injection into a tumor, lymph node, or site of infection.

[0032] As used herein, the term “cancer” generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and invade nearby tissues. Cancer cells can also spread to other parts of the body via the blood and lymphatic systems. There are several main types of cancer. Carcinomas are cancers that occur in the skin or in tissues that fill or cover internal organs. Sarcomas are cancers that occur in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemias are cancers that occur in hematopoietic tissues such as bone marrow and involve numerous abnormalities. These are cancers that produce blood cells that enter the bloodstream. Lymphoma and multiple myeloma are cancers that originate from cells of the immune system. Central nervous system cancers are cancers that originate from the tissues of the brain and spinal cord.

[0033] In some embodiments of any of the embodiments, the cancer is a primary cancer. In some embodiments of any of the embodiments, the cancer is a malignant cancer. As used herein, the term “malignant” means a cancer in which a group of tumor cells exhibit one or more of the following: uncontrolled proliferation (i.e., division beyond the normal limit), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., dispersal to other parts of the body via the lymph or blood). As used herein, the term “metastatic” means the dispersal of cancer from one part of the body to another. A tumor formed by dispersed cells is called a “metastatic tumor” or “metastasis.” A metastatic tumor contains cells that resemble the original (primary) tumor.

[0034] As used herein, the terms “benign” or “non-malignant” refer to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limiting and typically do not invade or metastasize.

[0035] "Cancer cells" or "tumor cells" refer to cancerous growths or individual cells in tissue. A tumor generally refers to a swelling or lesion formed by the abnormal proliferation of cells, and can be benign, premalignant, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not. The terms cancer(cells) and tumor(cells) are used interchangeably when referring to cancer cells that form tumors.

[0036] A subject with cancer or a tumor is a subject that has objectively measurable cancer cells present in the subject's body. This definition includes malignant, actively proliferating cancers, as well as potentially dormant tumors or micrometastases. Cancers that migrate from their original location and spread to other vital organs can ultimately lead to the subject's death through functional deterioration of the affected organ. Hematopoietic cancers, such as leukemia, can overwhelm the normal hematopoietic compartment in the subject through competition, thereby causing hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately death.

[0037] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (e.g., gastrointestinal cancer); gliablastoma (GBM); hepatocellular carcinoma; hepatocellular carcinoma; carcinoma in situ; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); lymphoma, e.g., Hodgkin lymphoma and non-Hodgkin lymphoma; melanoma; myeloma Neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular tumor; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; vulvar cancer; and other cancers and sarcomas; and B-cell lymphoma (e.g., low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; moderate / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small unsevered cell NHL; bulky disease NHL; mantle cell lymphoma; Examples include AIDS-associated lymphoma; and Valdenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hair cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis, edema (such as that associated with brain tumors), and Meigs syndrome, all of which are associated with phakomatoses.

[0038] "Cancer cells" are cancerous, precancerous, or transformed cells that exhibit spontaneous or induced phenotypic changes, whether in vivo, ex vivo, or in tissue culture, without necessarily involving the uptake of new genetic material. Transformation can occur from infection with transforming viruses and the incorporation of new genomic nucleic acids, or from the uptake of exogenous nucleic acids, but can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is associated, for example, with morphological changes, cell immortalization, abnormal growth control, lesion formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in a suitable animal host such as a nude mouse.

[0039] A subject may be someone who has been previously diagnosed with, is suffering from, or has been identified as having, a condition requiring treatment (e.g., cancer) or one or more complications associated with such a condition, but who, in some cases, does not yet require treatment for the condition or one or more complications associated with such a condition. Alternatively, a subject may be someone who has never been previously diagnosed with a condition requiring treatment or one or more complications associated with such a condition. For example, a subject may be someone who exhibits one or more risk factors for a condition, or one or more complications associated with a condition, or a subject who does not exhibit any risk factors. A “subject requiring treatment” for a particular condition may be someone who has that condition, has been diagnosed with that condition, or is at risk of developing that condition. As further described herein, in some embodiments, a subject is someone who has been determined to have elevated levels of CHI3L1. In some embodiments, CHI3L1 is circulating CHI3L1. In some embodiments, a subject has cancer that expresses PD-L1.

[0040] The terms “decrease,” “decrease,” “decrease,” or “inhibit” are all used herein to mean a decrease of a statistically significant amount. In some embodiments, “decrease,” “decrease,” or “decrease” or “inhibit” typically mean a decrease of at least 10% compared to a reference level (e.g., the absence of a given treatment or drug), and may include decreases of, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “decrease” or “inhibit” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction can preferably be lowered to a level that is acceptable as being within the normal range for individuals without a given disorder.

[0041] The terms “increased,” “increased,” “enhance,” or “activated” are all used herein to mean an increase of a statically significant amount. In some embodiments, the terms “increased,” “increased,” “enhance,” or “activated” can mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% and 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times or more compared to a reference level, or any increase between 2 times and 10 times or more. And, "increase" here refers to a statistically significant increase at this level.

[0042] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a set of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms “protein” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, and the term “peptide” is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologues, paralogs, fragments, and other aforementioned equivalents, variants, fragments, and analogs.

[0043] In the various embodiments described herein, it is intended to further include variants (naturally occurring or not), alleles, homologs, conservedly modified variants, and / or conserved substitutional variants of any of the particular polypeptides described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences that modify a single amino acid or a small number of amino acids in the encoded sequence are “conservedly modified variants,” and that such modifications result in the substitution of an amino acid with a chemically similar amino acid, thereby preserving the desired activity of the polypeptide. Such conservedly modified variants are added to, and not excluded from, polymorphic variants, interspecific homologs, and alleles consistent with the present disclosure.

[0044] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be a functional fragment of an amino acid sequence described herein. As used herein, “functional fragment” is a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide as determined by the assays described herein below. The functional fragment may include conservative substitutions of the sequences disclosed herein.

[0045] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutations in the native nucleotide sequence. As used herein, “variant” is a polypeptide that is substantially homologous to the native or reference polypeptide but has a different amino acid sequence from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide includes the sequence encoding the variant protein or a fragment thereof that, compared to the native or reference DNA sequence, contains one or more additions, deletions, or substitutions of nucleotides but retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0046] As used herein, the terms “nucleic acid” or “nucleic acid sequence” refer to any molecule, preferably a polymer molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogues. A nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid that does not originate from any double-stranded DNA. In one embodiment, the nucleic acid may be DNA. In another embodiment, nucleic acid This can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0047] In some embodiments of any of the features, the polypeptides, nucleic acids, or cells described herein can be manipulated. As used herein, “manipulated” means a form that has been manipulated by human hands. For example, a polypeptide is considered “manipulated” if at least one form of it, for example, its sequence, has been manipulated by human hands to be different from its naturally occurring form. As is common practice and as will be understood by those skilled in the art, the offspring of a manipulated cell are typically still referred to as “manipulated,” even though the actual manipulation was performed on the preceding entity.

[0048] In some embodiments, the nucleic acid encoding the polypeptide described herein (e.g., an antibody or antibody reagent) is contained by a vector. In some embodiments described herein, a nucleic acid sequence encoding a given polypeptide described herein, or any module thereof, is operably ligated to a vector. Examples of vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions.

[0049] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterogeneous to the cell. An expression vector may contain further elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e.g., in human cells for expression and in a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins, and, if applicable, the secretion of proteins, and, where applicable, but not limited to, transcription, transcriptional processing, translation, and protein folding, modification, and processing. “Expression products” include RNA transcribed from genes and polypeptides obtained by translation of mRNA transcribed from genes. The term “gene” means a nucleic acid sequence (DNA) that is transcribed to RNA in vitro or in vivo when operably ligated to a suitable regulatory sequence. A gene may or may not include regions before and after the coding region, such as the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0050] The terms “isolated” or “partially purified,” as used herein, refer to a nucleic acid or polypeptide isolated from at least one other component (e.g., a nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide found in its natural source and / or present with the nucleic acid or polypeptide when expressed by a cell, or, in the case of a secreted polypeptide, secreted. Chemically synthesized nucleic acids or polypeptides or those synthesized using in vitro transcription / translation are considered “isolated.” The terms “purified” or “substantially purified” refer to an isolated nucleic acid or polypeptide that is at least 95% by weight of the nucleic acid or polypeptide of interest, for example, including at least 96%, at least 97%, at least 98%, at least 99%, or more. In some embodiments, the antibodies, their antigen-binding moieties, or chimeric antigen receptors (CARs) described herein are isolated. In some embodiments, the antibodies, antibody reagents, their antigen-binding moieties, or CARs described herein are purified.

[0051] As used herein, “manipulated” refers to a state in which something has been controlled by human hands. For example, an antibody, antibody reagent, its antigen-binding portion, CAR, or bispecific antibody is considered "manipulated" if its sequence has been altered by human hands to differ from that of a naturally occurring antibody. It is common practice, and as those skilled in the art will understand, that the offspring and copies of a manipulated polynucleotide and / or polypeptide are typically still referred to as "manipulated" even though the actual manipulation was performed on the preceding entity.

[0052] As used herein, “epitope” can be formed on a polypeptide from adjacent amino acids or on a polypeptide from adjacent non-adjacent amino acids by tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. Epitopes typically contain at least three, more commonly at least five, about nine, or about eight to ten amino acids in their intrinsic spatial conformation. “Epitope” contains a structural unit that is typically bound by an immunoglobulin VH / VL pair. Epitopes define the minimal binding site of an antibody and thus represent the target of the antibody's specificity. In the case of a single-domain antibody, the epitope represents a structural unit bound by an isolated variable domain. The terms “antigenic determinant” and “epitope” can also be used interchangeably herein. In certain embodiments, the epitope determinant includes a chemically active surface grouping of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments, may have specific three-dimensional structural properties and / or specific charge properties.

[0053] As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule comprising an immunoglobulin molecule with an immunoactive portion, i.e., an antigen-binding site that binds immunospecifically to an antigen. The term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as their full-length antibodies and antigen-binding sites in various forms, including, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-transplant antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies (dAb), diabodies, multispecific antibodies, bispecific antibodies, anti-idiotype antibodies, bispecific antibodies, their functionally active epitope-binding sites, and / or bifunctional hybrid antibodies.

[0054] Each heavy chain consists of a variable region (hereinafter abbreviated as HCVR or VH) and a steady region. The heavy chain steady region consists of three domains CH1, CH2, and CH3. Each light chain consists of a variable region (hereinafter abbreviated as LCVR or VL) and a steady region. The light chain steady region consists of a CL domain. The VH and VL regions are further divided into hypervariable regions called complementarity-determining regions (CDRs), with conserved regions called framework regions (FRs) scattered between them. Thus, each VH and VL region consists of three CDRs and four FRs, aligned from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0055] As used herein, the term “CDR” refers to the complementarity-determining region within the antibody variable sequence. Each of the variable regions in the heavy and light chains has three CDRs, referred to as CDR1, CDR2, and CDR3 for each variable region. The precise boundaries of these CDRs are defined differently by different systems. The system described by Kabat et al. (1987) and (1991) provides not only a clear residue numbering system applicable to any variable region of an antibody, but also precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Other boundaries defining CDRs that overlap with CDRs are described by Padlan et al. (1995), Ma This has been described by cCallum et al. (1996) and Chothia et al. (1987 and 1989). Furthermore, other CDR boundary definitions do not need to strictly adhere to any of the above systems, but nevertheless, they may be shortened or extended in light of predictions or experimental findings that a particular residue or group of residues or even the entire CDR does not significantly affect antigen binding, although they overlap with Kabat CDRs. The methods used herein may utilize CDRs defined according to any of these systems, but preferred embodiments use CDRs defined in Kabat.

[0056] The antibody term "antigen-binding moiety" refers to one or more portions of an antibody described herein, which still possess binding affinity as defined above. It has been shown that a portion of a complete antibody can perform the antigen-binding function of the antibody. According to the antibody term "antigen-binding moiety," examples of binding moieties include: (i) the Fab moiety, i.e., a monovalent moiety consisting of the VL, VH, CL, and CH1 domains; (ii) the F(ab')2 moiety, i.e., a bivalent moiety containing two Fab moieties linked to each other in a hinge region via disulfide crosslinks; (iii) the Fd moiety consisting of the VH and CH1 domains; (iv) the Fv moiety consisting of the FL and VH domains of a single arm of the antibody; and (v) the dAb moiety (dAb, or V) consisting of the VH domain, or VH, CH1, CH2, DH3, or VH, CH2, CH3. L Single-domain antibodies, which contain only the domain, have also been shown to bind specifically to a target epitope. The two domains of the Fv portion, namely VL and VH, are encoded by separate genes, but they can be further linked to each other using a synthetic linker, e.g., a polyG4S amino acid sequence (disclosed as "G4S" as Sequence ID No. 29 in U.S. Patent No. 10,253,111), and recombination methods, which allow the VL and VH regions to be combined and prepared as a single protein chain forming a monovalent molecule (known as single-stranded Fv (ScFv)). The antibody term "antigen-binding portion" is also intended to include such single-stranded antibodies. Other forms of single-stranded antibodies, such as "diabodies," are also included herein. Diabody is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short for the two domains to combine on the same chain, thereby forcing the domains to pair with complementary domains on different chains to form two antigen-binding sites. Immunoglobulin constant domains refer to heavy chain or light chain constant domains. The amino acid sequences of human IgG heavy chain and light chain constant domains are known in the art.

[0057] As used herein, the term “antibody reagent” refers to a polypeptide comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binding to a given antigen. An antibody reagent may comprise an antibody or a polypeptide comprising the antigen-binding domain of an antibody. In some embodiments, an antibody reagent may comprise a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody may comprise two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody reagent” encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments, as well as complete antibodies).

[0058] Antibodies can have the structural features of IgA, IgG, IgE, IgD, IgM (and their subtypes and combinations). Antibodies can originate from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates), and can be primate-like antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.

[0059] Furthermore, the antibodies, antigen-binding moieties, or CARs described herein are the same as the antibodies described above. Alternatively, the antibody moiety may be part of a larger immunoadhesion molecule formed by covalent or non-covalent bonding of one or more further proteins or peptides. Relating to such immunoadhesion molecules are the use of streptavidin core regions for preparing tetrameric scFv molecules, and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidinyls, such as hexahistidinyl tags (disclosed as SEQ ID NO: 30 in U.S. Patent No. 10,253,111, “hexahistidinyl tags”) for generating divalent and biotinylated scFv molecules.

[0060] In some embodiments, the antibodies, antibody reagents, their antigen-binding moieties, or CARs described herein may be immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-transplant antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies, diabodies, multispecific antibodies, bispecific antibodies, anti-idiotype antibodies, bispecific antibodies, and their functionally active epitope-binding moieties.

[0061] In some embodiments, the antibody or its antigen-binding portion is a fully human antibody. In some embodiments, the antibody and its antigen-binding portion are a humanized antibody or antibody reagent. In some embodiments, the antibody and its antigen-binding portion are a fully humanized antibody or antibody reagent. In some embodiments, the antibody or its antigen-binding portion is a chimeric antibody or antibody reagent. In some embodiments, the antibody and its antigen-binding portion are a recombinant polypeptide. In some embodiments, the CAR includes an extracellular domain that binds to CHI3L1, and the extracellular domain includes a humanized antibody or a chimeric antibody or its antigen-binding portion.

[0062] The term "human antibody" refers to an antibody whose variable and constant regions correspond to or are derived from the immunoglobulin sequences of human germ cell lines, as described, for example, by Kabat et al. (1991). However, human antibodies may contain amino acid residues not encoded by the immunoglobulin sequences of human germ cells (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo), for example, in the CDR, particularly CDR3. Recombinant human antibodies described herein may have a variable region and may also contain a constant region derived from the immunoglobulin sequences of human germ cells. See Kabat et al. (1991). However, according to certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic due to a human Ig sequence is used, somatic in vivo mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibody are related to or derived from the VH and VL sequences of the human germline, but are sequences that do not naturally exist in vivo within the human antibody germline repertoire. According to certain embodiments, this type of recombinant antibody is the result of selective mutagenesis or reverse mutagenesis, or both. Preferably, the mutagenesis results in a greater affinity for the target and / or a smaller affinity for the non-target structure than that of the parental antibody. The preparation of humanized antibodies from the sequences and information provided herein can be carried out by those skilled in the art without excessive experimentation. In one approach, there are four common steps used to humanize monoclonal antibodies; see, for example, U.S. Patents 5,585,089, 6,835,823, and 6,824,989. These include (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light chain and heavy chain variable domains; (2) designing the humanized antibody, i.e., determining which antibody framework regions to use during the humanization process; (3) the actual humanization method / technology; and (4) transfection and expression of the humanized antibody.

[0063] In some embodiments, the bispecific antibody, antibody reagent, and antigen conjugate described herein are used. A portion, and / or CAR, may be a variant of the sequence described herein, for example, a conserved substitution variant of an antibody polypeptide. In some embodiments, the variant is a conservedly modified variant. Conservative substitution variants can be obtained, for example, by mutation of a native nucleotide sequence. As used herein, “variant” is a polypeptide that is substantially homologous to a native or reference polypeptide but has a different amino acid sequence from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide includes one or more additions, deletions, or substitutions of nucleotides compared to the native or reference DNA sequence, but comprises a sequence encoding the variant protein or a portion thereof that retains activity, for example, antigen-specific binding activity to the relevant target polypeptide, for example, CHI3L1 or PD-1. A wide variety of PCR-based site-directed mutagenesis approaches are also known in the art and can be applied by those skilled in the art.

[0064] Typically, the CDR regions in humanized antibodies and human antibody variants are substantially identical, and more often than not, to the corresponding CDR regions in the mouse or human antibodies from which they are derived. In some embodiments, it is possible to create one or more conserved amino acid substitutions of CDR residues without significantly affecting the binding affinity of the resulting humanized immunoglobulin or human antibody variant. In some embodiments, substitutions of the CDR region can increase binding affinity.

[0065] The term "chimeric antibody" refers to an antibody that contains sequences for the variable regions of the heavy and light chains of one species, as well as constant region sequences from another species, such as an antibody with mouse heavy and light chain variable regions linked to a human constant region. Humanized antibodies have variable region framework residues substantially from a human antibody (called an acceptor antibody) and complementarity-determining regions substantially from a non-human antibody, such as a mouse antibody (called a donor immunoglobulin). The constant region(s), if present, is substantially or entirely derived from human immunoglobulin. Human variable domains are usually selected from human antibodies whose framework sequences show a high degree of sequence identity with the (mouse) variable region domain from which the CDR was induced. The heavy and light chain variable region framework residues may be substantially similar to regions of the same or different human antibody sequences. Human antibody sequences may be sequences of naturally occurring human antibodies or consensus sequences of several human antibodies.

[0066] Furthermore, techniques developed to produce "chimeric antibodies" can be used by splicing genes from mice or other species, antibody molecules with appropriate antigen specificity, and genes from human antibody molecules with appropriate biological activity. The variable segment of the chimeric antibody is typically linked to at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, such as immortalized B cells, using well-known procedures. Antibodies can contain both light chain and heavy chain constant regions. The heavy chain constant region can include the CH1, hinge, CH2, CH3, and sometimes CH4 regions. For therapeutic purposes, the CH2 domain may be deleted or omitted.

[0067] Furthermore, as described herein, recombinant humanized antibodies may be further optimized for therapeutic use in humans to reduce potential immunogenicity while maintaining functional activity. In this regard, functional activity means a polypeptide capable of exhibiting one or more known functional activities related to the recombinant antibody, its antigen-binding moiety, or CAR described herein. Such functional activities include binding to cancer cells and / or anticancer activity. Furthermore, a polypeptide having functional activity means that the polypeptide exhibits activity similar to, but not necessarily identical to, the activity of the reference antibody, its antigen-binding moiety, or CAR described herein, whether dose-dependent or not, for example, biological activity. This includes mature forms measured by specific assays, such as scientific assays. Where dose-dependency exists, it does not need to be identical to that of the reference antibody, its antigen-binding moiety, or CAR, but rather substantially similar in dose-dependency at a given activity compared to the reference antibody, its antigen-binding moiety, or CAR described herein (i.e., the candidate polypeptide exhibits greater activity than the antibodies, antigen-binding moieties, and / or CARs described herein, or exhibits about 1 / 25th, 1 / 10th, or 1 / 3th of that activity).

[0068] In some embodiments, the antibody reagents described herein (e.g., antibodies or CARs) are not naturally occurring biomolecules. For example, mouse antibodies produced against human-derived antigens do not occur naturally without human intervention and manipulation, e.g., manufacturing processes carried out by humans. Chimeric antibodies are also not naturally occurring biomolecules, for example, in that they contain sequences obtained from multiple species and assembled into recombinant molecules. In certain embodiments, the human antibody reagents described herein are not naturally occurring biomolecules; for example, fully human antibodies directed against human antigens undergo negative selection in nature and are not found naturally in the human body.

[0069] In some embodiments, the antibody, antibody reagent, its antigen-binding portion, and / or CAR is an isolated polypeptide. In some embodiments, the antibody, antibody reagent, its antigen-binding portion, and / or CAR is a purified polypeptide. In some embodiments, the antibody, antibody reagent, its antigen-binding portion, and / or CAR is an engineered polypeptide.

[0070] "Binding activity" is a measure of the strength of binding between an antigen-binding molecule (such as an antibody or its antigen-binding portion described herein) and a related antigen. Binding activity is related to both the affinity of the antigen determinant for its antigen-binding site on the antigen-binding molecule and the number of appropriate binding sites present on the antigen-binding molecule. Typically, an antigen-binding protein (e.g., an antibody or a portion of an antibody described herein) has a dissociation constant (K -5 ~10 -12 mol / L or less, e.g., 10 -7 ~10 -12 mol / L or less, or 10 -8 10 -8 ~10 -12 mol / L) (i.e., an association constant (K A ) of 10 7 ~10 12 L / mol or 10 8 ~10 12 L / mol) for binding to its cognate or specific antigen. Any K -4 value greater than 10 4 M<000002,0>or any K A value lower than 10 -10 M (0.1 nM) to 10 -5 M (10000 nM). The stronger the interaction, the lower its K D ​​​​​​​​​​The affinity becomes lower. For example, the binding sites on antibodies or parts thereof described herein bind to the desired antigen with affinity less than 500 nM, e.g., less than 200 nM, or less than 10 nM, e.g., less than 500 pM. The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any suitable method known in itself, including, for example, scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay, different variants thereof known in the art, and other techniques referred to herein.

[0071] Therefore, as used herein, “selectively bind” or “specifically bind” means that the peptide described herein (e.g., an antibody, CAR, bispecific antibody or a part thereof) binds K D 10 -5 M (10000 nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less refers to the ability to bind to targets such as antigens present on the cell surface of cancer cells. Specific binding is, for example, determined by the affinity and binding activity of polypeptide agents, as well as the concentration of polypeptide agents. This can be influenced by the degree. Those skilled in the art can determine the appropriate conditions for the polypeptide agents described herein to selectively bind to a target using any suitable method, such as titration of the polypeptide agent in a suitable cell binding assay. A polypeptide specifically bound to a target is not substituted by dissimilar competitors. In certain embodiments, an antibody, its antigen-binding moiety, CAR, or bispecific antibody is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0072] In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is 10-5 M (10000 nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or a dissociation constant less than M (K D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is approximately 10 -5 M~10 -6 The dissociation constant of M (K D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is approximately 10 -6 M~10 -7 The dissociation constant of M (K D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is approximately 10 -7 M~10 -8 The dissociation constant of M (K D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is approximately 10 -8 M~10 -9 The dissociation constant of M (K D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is approximately 10 -9 M~10 -10 The dissociation constant of M (K D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is approximately 10 -10 M~10 -11 The dissociation constant of M (K D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody, its antigen-binding moiety, or CAR described herein is approximately 10 -11 M~10 -12 The dissociation constant of M (KD ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibody described herein, its antigen-binding moiety, or CAR is 10 -12 Dissociation constant less than M (K D ) binds to CHI3L1 and / or PD-1.

[0073] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members or elements of the group as found herein. One or more members of a group may be included in or excluded from a group for convenience and / or patentability reasons. In the event of any such inclusion or exclusion, this Specified herein shall be deemed to include the qualified groups and thus satisfy the written description of all Markush groups used in the appended claims.

[0074] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art to which this disclosure pertains. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and is subject to change. Terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention as defined solely by the claims. Definitions of general terms in immunology and molecular biology are found in The Merck Manual of Diagnosis and Therapy, 19th edition (2011); The Encyclopedia of Molecular Cell Biology and Molecular Medicine (1999–2012); Molecular Biology and Biotechnology: A Comprehensive Desk Reference. This information can be found in (1995); Immunology (2006); Janeway's Immunobiology (2014); Lewin's Genes XI (2014); Molecular Cloning: A Laboratory Manual, 4th edition (2012); Basic Methods in Molecular Biology (2012); Laboratory Methods in Enzymology: DNA (2013); Current Protocols in Molecular Biology (CPMB) (2014); Current Protocols in Protein Science (CPPS) (2005); and Current Protocols in Immunology (CPI) (2003), the contents of which are incorporated herein by reference in their entirety.

[0075] Those skilled in the art can easily identify the chemotherapeutic agents to be used. For example, see Physicians' Cancer Chemotherapy Drug Manual (2014); Chapter 85 in Harrison's Principles. See Chapters 28-29 of Internal Medicine, 18th edition (2011); in Abeloffs Clinical Oncology, 5th edition (2013); and The Cancer Chemotherapy Handbook, 4th edition (2003).

[0076] In some embodiments of any aspect, the disclosures described herein do not relate to processes for cloning humans, processes for modifying the genetic identity of human germlines, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that could cause suffering to humans or animals without any substantial medical benefit, nor to animals resulting from such methods.

[0077] Other terms are defined herein within the scope of describing various aspects of the present invention.

[0078] The bispecific antibody of the present invention Immunotherapy using anti-programmed death-1 (PD-1) or anti-PD-1 ligand 1 (PD-L1) antibodies has been approved for the treatment of several cancers due to impressively sustained responses, but overall, only a small percentage of patients currently benefit from PD-1 blockade therapy alone (Topalian et al., 2012; Herbst et al., 2014; Powles et al., 2014; Ansell et al., 2015; Garon et al., 2015; Postow et al., 2015; Robert et al., 2015a,b; Weber et al., 2015; Nghiem et al., 2016; Ribas et al., 2016). Combinations of anti-PD-1 / L1 antibodies with other immunomodulators appear to be more active, but they add significant toxicity (Wolchok et al., 2013; Larkin et al., 2015; Postow et al., 2015).

[0079] In our previous research, we showed that (a) inhibition of CHI3L1 and / or CHI3L1 signaling, and (b) inhibition of at least one immune checkpoint protein such as PD-1, provide a synergistic effect in the treatment of cancer, e.g., lung cancer. See, for example, U.S. Patent Publication No. 2019 / 0062457. When the anti-CHI3L1 antibody FRG was administered in combination with an anti-PD-1 antibody, a synergistic effect was observed, and this combination showed improved efficacy in reducing B16F10 metastasis, thereby suggesting that the combination of CHI3L1 inhibition and checkpoint protein inhibition provides a synergistic effect in the treatment of cancer. See Example 2 of U.S. Patent Publication No. 2019 / 0062457. Specific binding to both CHI3L1 polypeptide and PD-1 polypeptide, and simultaneous detection of both CHI3L1 and the immune checkpoint inhibitor PD-1. It was hypothesized that bispecific antibodies that neutralize the cancer could show improved synergistic effects in cancer treatment.

[0080] This specification describes bispecific antibodies, antibody reagents, their antigen-binding fragments, or chimeric antigen receptors (CARs) that specifically bind to both CHI3L1 polypeptide and PD-1 polypeptide, and simultaneously detect and neutralize both CHI3L1 and the immune checkpoint inhibitor PD-1. Such bispecific antibodies, their antigen-binding moieties, etc., can enable, for example, the diagnosis, prognosis, and / or treatment of cancer. In some embodiments, the techniques described herein relate to chimeric antigen receptors (CARs) and CAR-T therapies for cancer. In some embodiments, the techniques described herein relate to monoclonal antibody therapies for cancer. In some embodiments, the techniques described herein relate to antibody-drug conjugates for the treatment of cancer.

[0081] This specification describes bispecific anti-CHI3L1 antibodies and anti-PD-1 antibodies, antibody reagents, and methods and compositions related to their antigen-binding fragments, exhibiting excellent properties ex vivo and in vivo, such as high sensitivity, high specificity, high binding affinity, and neutralizing activity. Methods of treatment, such as treatment of cancer, by administering the compounds described herein are also provided.

[0082] The bispecific antibody of the present invention comprises an antigen-binding moiety of an anti-human PD-1 antibody and an antigen-binding moiety of an anti-human CHI3L1 antibody. In some embodiments, the bispecific antibody comprises an anti-human PD-1 single-chain variable fragment (ScFv-PD1) bound to the backbone of the anti-human CHI3L1 antibody. In alternative embodiments, the bispecific antibody comprises an anti-human CHI3L1 single-chain variable fragment (ScFv-CHI3L1) bound to the backbone of the anti-human PD-1 antibody.

[0083] Those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences that modify a single amino acid or a small proportion of amino acids in the encoded sequence are “conservatively modified variants” in which the modification replaces an amino acid with a chemically similar amino acid and retains the ability to specifically bind to a target antigen (e.g., CHI3L1 and PD-1). Such conservatively modified variants are added to, and not excluded from, polymorphic variants, interspecific homologs, and alleles consistent with the present disclosure.

[0084] An example of a substitution variant is one that does not change the sequence of CDR, for example, V H or V L Examples include conserved amino acid substitutions within a domain. Conservative substitutions in sequences not included in the CDR may be substitutions in wild-type or naturally occurring sequences, such as constant regions of human or mouse frameworks and / or antibody sequences.

[0085] A given amino acid may be substituted with a residue having similar physicochemical properties, for example, by substituting one aliphatic residue with another (e.g., Ile, Val, Leu, or Ala with each other), or by substituting one polar residue with another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activity, e.g., antigen-binding activity and specificity of the native or reference polypeptide, is preserved.

[0086] Amino acids can be classified according to the similarity in the properties of their side chains (Biochemistry, 2nd edition (1975), pp. 73-75): (1) Nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Tr p(W), Met(M); (2) Non-charged: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q); (3) Acidic: Asp(D), Glu(E); (4) Basic: Lys(K), Arg(R), His(H). Alternatively, naturally occurring residues can be grouped based on the properties of their common side chains: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions require exchanging one member of one of these classes with another. Certain conservative substitutions include, for example, substitutions from Ala to Gly or Ser; Arg to Lys; Asn to Gln or H; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0087] The variant amino acid sequence or DNA sequence is preferably identical to the natural sequence or reference sequence by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more. The degree of homology (identity percentage) between the natural sequence and the mutant sequence can be determined, for example, by comparing the two sequences using a freely available computer program commonly used on the World Wide Web for this purpose (e.g., BLASTp or BLASTn with default settings).

[0088] Modification of natural amino acid sequences can be achieved by any of the numerous techniques known to those skilled in the art. Mutations can be introduced to specific loci, for example, by synthesizing oligonucleotides containing mutant sequences adjacent to restriction sites that allow ligation to fragments of the natural sequence. After ligation, the resulting reconstructed sequence encodes analogs having the desired amino acid insertions, substitutions, or deletions. Alternatively, oligonucleotide-directed site-specific mutagenesis can be used to provide modified nucleotide sequences having specific codons modified according to the required substitutions, deletions, or insertions.

[0089] Any cysteine ​​residue that does not contribute to maintaining the proper three-dimensional structure of a polypeptide can generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine ​​bonds (one or more) can be added to a polypeptide to improve its stability or promote oligomerization.

[0090] In certain embodiments in which the antibodies, their antigen-binding moieties, or CARs described herein include at least one CDR that is not identical in sequence to the CHI3L1 and PD-1 CDRs provided herein, the amino acid sequence of that at least one CDR can be selected by methods well known to those skilled in the art. For example, Fujii, 2004, "Antibody affinity maturation by random mutagenesis," Methods in Molecular Biology: Antibody Engineering, Vol. 248, pp. 345-349 (the entire work is incorporated herein by reference), in particular Figure 2 and Chapter 3.3, describe a method for preparing a library of any CDR of interest. Thus, those skilled in the art will know how to prepare the specific CDRs described herein. Alternative CDRs containing conserved substitution variants of DR sequences can be identified, and when present in the antibodies or antigen-binding moieties described herein, they result in antigens or antigen-binding moieties that bind to cancer cell surface antigens. Furthermore, the method described by Fujii et al. allows those skilled in the art to screen light chain sequences that, when combined with known heavy chain fragments, give desired binding behavior, and vice versa.

[0091] In some embodiments, the CAR comprises an extracellular domain containing an anti-CHI3L1 antibody or its antigen-binding moiety that binds to one or more epitopes of the CHI3L1 polypeptide; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain. Exemplary anti-CHI3L1 and anti-PD-1 antibodies and their antigen-binding moieties, as well as more exemplary epitopes, are described elsewhere in this specification.

[0092] As used herein, “chimeric antigen receptor” or “CAR” refers to an artificially constructed hybrid polypeptide comprising an antigen-binding domain (e.g., the antigen-binding portion of an antibody (e.g., scFv)), a transmembrane domain, and a T cell signaling and / or T cell activation domain. CARs have the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restrictive manner by leveraging the antigen-binding properties of a monoclonal antibody. Non-MHC-restrictive antigen recognition gives T cells expressing CARs the ability to recognize antigens independently of antigen treatment, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR). Most commonly, the extracellular binding domain of a CAR consists of a single-stranded variable fragment (scFv) derived from the fusion of variable heavy and light chain regions of a mouse or humanized monoclonal antibody. Alternatively, in various embodiments, the scFv may be a Fab-derived scFv (e.g., instead of an antibody obtained from a Fab library), which is fused to a transmembrane domain and then to an intracellular signaling domain. "First-generation" CARs include those that provide a CD3 zeta (CD3ζ) signal alone upon antigen binding, "second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ). "Third-generation" CARs include those that provide multiple co-stimulatory (e.g., CD28 and CD137) domains and an activation domain (e.g., CD3ζ). In various embodiments, the CAR is selected to have high affinity or binding activity to the antigen. Further studies of CAR can be found, for example, in Maus et al. (2014); Reardon et al. (2014); Hoyos et al. (2012); Byrd et al. (2014); Maher and Wilkie (2009); and Tamada et al. (2012), each of which is incorporated herein by reference in whole.

[0093] In some embodiments of any aspect, the CAR comprises an extracellular binding domain including a humanized CHI3L1-specific or humanized PD-1-specific binding domain; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain. As used herein, the terms “binding domain,” “extracellular domain,” “extracellular binding domain,” “antigen-specific binding domain,” and “extracellular antigen-specific binding domain” are used interchangeably to provide a CAR having the ability to specifically bind to a target antigen of interest, e.g., CHI3L1 and PD-1. The binding domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source.

[0094] In some embodiments, the CAR intended herein may include linker residues between various domains, which may be added, for example, for proper spacing and conformation of the molecule. In certain embodiments, the linker is a variable region linking sequence. The "variable region linking sequence" links the VH domain and the VL domain so that the resulting polypeptide maintains specific binding affinity to the same target molecule as an antibody containing the same light and heavy chain variable regions. This is an amino acid sequence that provides a spacer function that fits the interaction of the subbinding domains. The CARs contemplated herein may include 1, 2, 3, 4, or 5 or more linkers. In certain embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or the length of any intervening amino acid. In some embodiments, the linker is an amino acid length of 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.

[0095] In certain embodiments, the CAR binding domain is followed by one or more “spacer domains,” which refer to regions that move the antigen-binding domain away from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation. The hinge domain can be derived from a natural, synthetic, semi-synthetic, or recombinant source. In certain embodiments, the spacer domain is, but is not limited to, a portion of an immunoglobulin containing one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0096] The binding domain of a CAR is typically followed by one or more "hinge domains," which play a role in positioning the antigen-binding domain away from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation. CARs generally contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domains may contain amino acid sequences from naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions. Exemplary hinge domains suitable for use in CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be modified. In another embodiment, the hinge domain includes a CD8α hinge region.

[0097] The "transmembrane domain" is a part of the CAR that fuses the extracellular binding portion and the intracellular signaling domain, fixing the CAR to the cell membrane of immunoeffector cells. The TM domain can be derived from either native, synthetic, semi-synthetic, or recombinant sources. The TM domain can be derived from the alpha, beta, or zeta chains of T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1 (i.e., it may include at least one or more of their transmembrane regions).

[0098] In some embodiments, the CAR as envisioned herein includes an intracellular signaling domain. “Intracellular signaling domain” means a portion of the CAR that transduces an effective CAR-binding message to a target antigen into the interior of an immunoeffector cell and is involved in effector cell function, e.g., activation, cytokine production, proliferation, and cytotoxic activity, e.g., release of cytotoxic factors to CAR-binding target cells, or other cellular responses induced by antigen binding to an extracellular CAR domain. In some embodiments, the CAR as envisioned herein includes an intracellular signaling domain comprising one or more “costimulatory signaling domains” and “primary signaling domains.”

[0099] Primary signaling domains stimulate or inhibit the primary activation of the TCR complex. Primary signaling domains that act in a stimulating manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary signaling domains particularly used in the invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0100] As used herein, the term “costimulatory signaling domain” or “costimulatory domain” refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, upon binding to an antigen, provide a secondary signal necessary for the efficient activation and function of T lymphocytes. Examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, the CAR includes one or more co-stimulatory signaling domains selected from the group consisting of primary signaling domains CD28, CD137, and CD134, as well as CD3ζ.

[0101] In some embodiments, antibody-drug conjugates are provided. In certain embodiments, the antibody-drug conjugate comprises an antibody, an antibody reagent, or its antigen-binding moiety, as described herein. The drug may be, for example, a chemotherapeutic molecule as described elsewhere herein. In some embodiments, the antibody-drug conjugate comprises a chemotherapeutic agent directly conjugated to and / or bound to the antibody or its antigen-binding moiety. In some embodiments, the binding may be a non-covalent bond, such as a hydrogen bond, electrostatic interaction, or van der Waals interaction, however, the binding may also be a covalent bond. "Conjugated" means a covalent bond between at least two molecules. In some embodiments, the composition may be an antibody-drug conjugate.

[0102] In some embodiments, an antibody, antibody reagent, or its antigen-binding moiety may be bound to and / or conjugated to multiple chemotherapy molecules. In some embodiments, an antibody-drug conjugate may be bound to and / or conjugated to multiple chemotherapy molecules. In some embodiments, the ratio of a given chemotherapy molecule to the antibody or its antigen-binding moiety may be about 1:1 to about 1,000:1, for example, a single antibody reagent molecule may be linked to and conjugated to about 1 to about 1,000 individual chemotherapy molecules.

[0103] In some embodiments, antibodies, or their antigen-binding moieties, and chemotherapeutic agents may be present in the scaffold material. Suitable scaffold materials for use in therapeutic compositions may include, but are not limited to, nanoparticles; matrices; hydrogels; and biomaterials, biocompatible and / or biodegradable scaffold materials, which are known in the art. As used herein, the term “nanoparticles” means about 10 -9 Alternatively, it refers to particles on the order of one billionth to one billionth of a meter. The term "nanoparticles" includes nanospheres, nanorods, nanoshells, and nanoprisms, and these nanoparticles may be part of a nanonetwork.

[0104] The term “nanoparticles” also encompasses liposomes and lipid particles having nanoparticle size. As used herein, the term “matrix” refers to a three-dimensional structure comprising the components of the compositions described herein (e.g., antibodies or their antigen-binding portions). Non-limiting examples of matrix structures include foams; hydrogels; electrospun fibers; gels; fiber mats; sponges; three-dimensional scaffolds; nonwoven mats; woven materials; knitted materials; fiber bundles; and fibers, as well as other material formats. For example, Rockwood et al. (2011), U.S. Patent Application Publications 2011 / 0167602; 2011 / 0009960; 2012 / 0296352; U.S. See Japanese Patent No. 8,172,901. The structure of the matrix can be selected by those skilled in the art depending on the intended use of the composition; for example, an electrospun matrix may have a larger surface area than a foam.

[0105] In some embodiments, the scaffold is a hydrogel. As used herein, the term “hydrogel” refers to a three-dimensional polymer structure that is insoluble in water but can absorb and retain large amounts of water to form a stable, often soft, and flexible structure. In some embodiments, water can penetrate between polymer chains in a polymer network, subsequently causing swelling and hydrogel formation. Generally, hydrogels are highly absorbent. Hydrogels possess many desirable properties for biomedical applications. For example, they can be non-toxic, conform to tissues, and have high permeability to water, ions, and small molecules. Hydrogels are highly absorbent (they can contain more than 99% water) and can be composed of natural (e.g., silk) or synthetic polymers, such as PEG.

[0106] As used herein, “biomaterial” refers to a material that is biocompatible and biodegradable. As used herein, the term “biocompatible” refers to a substance that is not toxic to cells. In some embodiments, a substance is considered “biocompatible” if its addition to cells in vitro results in less than about 20% cell death. In some embodiments, a substance is considered “biocompatible” if its addition to cells in vivo does not induce inflammation and / or other adverse effects in vivo. As used herein, the term “biodegradable” refers to a substance that is broken down under physiological conditions. In some embodiments, a biodegradable substance is a substance that is broken down by a cellular apparatus. In some embodiments, a biodegradable substance is a substance that is broken down by a chemical process.

[0107] As used herein, the terms “nucleic acid” or “nucleic acid sequence” refer to polymer molecules incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogues. Nucleic acids can be single-stranded or double-stranded. Single-stranded nucleic acids may be single-stranded nucleic acids of denatured double-stranded DNA. In some embodiments, nucleic acids may be cDNA, for example, nucleic acids lacking introns.

[0108] Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and preparation by cassette mutagenesis of previously prepared variants or non-variant versions of antibodies. Nucleic acid sequences encoding at least one antibody, portion, or polypeptide described herein may be recombined using vector DNA according to conventional techniques, including blunt or alternating ends for ligation, restriction enzyme digestion to provide suitable ends, fill-in of adherent ends as needed, alkaline phosphatase treatment to avoid undesirable connections, and ligation with a suitable ligase. Using techniques for such operations, monoclonal antibody molecules, antibody reagents, their antigen-binding regions, or nucleic acid sequences encoding CARs can be constructed.

[0109] Nucleic acid molecules such as DNA are said to be "capable of expressing" polypeptides if they contain nucleotide sequences that include transcriptional and translational regulatory information, and such sequences are "operably linked" to nucleotide sequences that encode polypeptides. An operable linkage is a linkage in which the regulatory DNA sequence and the DNA sequence to be expressed are connected in a manner that allows for gene expression as a peptide or antibody portion in a recoverable amount. The exact nature of the regulatory region required for gene expression can vary from organism to organism, as is well known in similar technical fields.

[0110] In some embodiments, the antibodies, antibody reagents, their antigen-binding moieties, or nucleic acids encoding CARs described herein constitute a vector. In some embodiments described herein, the antibodies, antibody reagents, their antigen-binding moieties, or nucleic acid sequences encoding CARs described herein, or any of these modules, are operably linked to a vector. The term “vector,” as used herein, refers to a nucleic acid construct designed for delivery to or transfer between different host cells. As used herein, a vector may be viral or nonviral. The term “vector” encompasses any genetic element that, when bound with appropriate regulatory elements, can replicate and transfer a gene sequence into a cell. Vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

[0111] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not always, heterogeneous to the cell. An expression vector may contain further elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e.g., in human cells for expression and in a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins, and, if applicable, the secretion of proteins, and, where applicable, but not limited to, transcription, transcript processing, translation and protein folding, modification and processing. “Expression products” include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably ligated to an appropriate regulatory sequence. A gene may or may not contain, before and after the coding region, for example, a 5' untranslated (5'UTR) or "leader" sequence and a 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0112] As used herein, the term “viral vector” means a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged in a viral vector particle. Viral vectors may contain, instead of non-essential viral genes, nucleic acids encoding antibodies, their antigen-binding moieties, or CARs as described herein. Vectors and / or particles can be used for the purpose of transferring any of these nucleic acids into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0113] "Recombinant vector" means a vector containing a heterologous nucleic acid sequence, or "transgene," that can be expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and treatments in some embodiments. In some embodiments, the vector is an episome. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in high copy number extra chromosomal DNA in a target, thereby eliminating the potential effects of chromosomal incorporation.

[0114] In any one embodiment of the specification, the Specified herein describes an antibody, an antibody reagent, its antigen-binding portion, or CAR, or a cell containing a nucleic acid encoding such an antibody, an antibody reagent, its antigen-binding portion, or CAR.

[0115] The expression of antibodies, antibody reagents, their antigen-binding moieties, or CARs described herein can occur in either prokaryotic or eukaryotic cells. In a suitable host, in vivo or Examples of in situ host cells include bacterial or eukaryotic hosts, including yeast, insects, fungi, birds, and mammalian cells, or host cells of mammalian, insect, bird, or yeast origin. Mammalian cells or tissues may be derived from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells can be used. Furthermore, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins thus produced can be processed in vivo or purified and processed in vitro to enable the synthesis of antibodies or parts thereof described herein, having specific amino-terminal sequences. In addition, problems associated with the retention of methionine residues derived from the start codon in direct expression of yeast (or bacteria) can be avoided. Any of the yeast gene expression systems incorporating promoters and termination elements from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in a glucose-rich medium, can be used to obtain the recombinant antibodies or their antigen-binding moieties described herein. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, promoter and terminator signals of phosphoglycerate kinase genes can be utilized.

[0116] The production of antibodies or antigen-binding moieties described herein can be achieved in insects, for example, by infecting an insect host with a baculovirus engineered to express a transmembrane polypeptide by a method known to those skilled in the art.

[0117] In some embodiments, the introduced nucleotide sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. For this purpose, any of the broad range of vectors can be used, and are known and available to those skilled in the art. Important factors in selecting a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the desired copy number of the vector in a particular host; and whether it is desirable that the vector can be "shuttle" between host cells of different species.

[0118] Examples of prokaryotic vectors known in the art include plasmids, such as plasmids that can replicate in Escherichia coli (E. coli). Other gene expression elements useful for the expression of antibodies, their antigen-binding portions, or cDNA encoding CARs include, but are not limited to, (a) viral transcription promoters and their enhancer elements, e.g., the SV40 early promoter, Roussarcoma virus LTR, and Moloney's mouse leukemia virus; (b) polyadenylation sites, such as those derived from splice regions and the late region of SV40; and (c) polyadenylation sites, such as those in SV40. Immunoglobulin cDNA genes may be expressed, for example, using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globin intercalated sequences, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements as expression elements.

[0119] In immunoglobulin genes composed of some cDNA and some genomic DNA, the transcription promoter may be human cytomegalovirus, the promoter enhancer may be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions may be native chromosomal immunoglobulin sequences.

[0120] In some embodiments, in the expression of cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, and the transcription promoter enhancer is either or both a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer. The splice region contains introns exceeding 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the synthesized immunoglobulin chain. In other embodiments, cDNA sequences encoding other proteins are combined with the expression elements to achieve protein expression in mammalian cells.

[0121] The gene is assembled into or inserted into an expression vector. Next, recipient cells capable of expressing the chimeric immunoglobulin chain gene product are transfected with an antibody, its antigen-binding portion, or CAR, or with a chimeric H chain or chimeric L chain coding gene alone, or co-transfected with both the chimeric H chain and chimeric L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the incorporated gene, and the expressed immunoglobulin chain or intact antibody or fragment is recovered from the culture.

[0122] In some embodiments, an antibody, its antigen-binding moiety, CAR, or a gene encoding chimeric H and L chains, or parts thereof, is assembled into a separate expression vector, which is then used to co-transfect recipient cells. Each vector may contain two selectable genes, a first selectable gene designed for selection in a bacterial system, and a second selectable gene designed for selection in a eukaryotic system, with each vector having a different gene pair. This strategy first results in a vector that directs the production of the gene in a bacterial system and enables amplification. The genes thus produced and amplified in the bacterial host are then used to co-transfect eukaryotic cells, enabling the selection of co-transfected cells having the desired transfected gene. Non-limiting examples of selectable genes for use in a bacterial system are a gene conferring resistance to ampicillin and a gene conferring resistance to chloramphenicol. Selectable genes for use as eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (referred to as gpt) and the Tn5-derived phosphotransferase gene (referred to as neo). Alternatively, the genes can be assembled into the same expression vector.

[0123] In the transfection of expression vectors and the production of antibodies, antibody reagents, their antigen-binding moieties, or CARs described herein, the recipient cell line may be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin gene and have a mechanism for immunoglobulin glycosylation. For example, in some embodiments, the recipient cells are recombinant Ig-producing myeloma cells SP2 / 0 (ATCC#CRL 8287). SP2 / 0 cells produce only immunoglobulins encoded by the transfected gene. Myeloma cells can be cultured or grown in the peritoneal cavity of mice, and secreted immunoglobulins can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells such as human or non-human B lymphocytes, human or non-human hybridoma cells, or interspecies heterohybridoma cells.

[0124] Expression vectors having chimeric, humanized, or compound human antibody constructs, antibodies, their antigen-binding moieties, and / or CARs as described herein can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, as well as mechanical means such as electroporation, direct microinjection, and microprojectile impact, which are known to those skilled in the art.

[0125] Traditionally, monoclonal antibodies are produced as natural molecules in the mouse hybridoma system. In addition to this technology, the methods and compositions described herein provide recombinant DNA expression of monoclonal antibodies. This enables the production of humanized antibodies, as well as a spectrum of antibody derivatives and fusion proteins, in selected host species. Antibody production in bacteria, yeast, transgenic animals, and chicken eggs is also an alternative to hybridoma-based production systems. The main advantage of transgenic animals is the potential high yield from a renewable source.

[0126] In one embodiment, cells containing an isolated antibody, its antigen-binding moiety, or CAR as described herein are provided. In some embodiments, the isolated antibody, its antigen-binding moiety, or CAR as described herein is expressed on the cell surface. In some embodiments, the cells contain nucleic acids encoding the isolated antibody, its antigen-binding moiety, or CAR as described herein.

[0127] In some embodiments, the cells are immune cells. As used herein, “immune cells” refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cells are T cells; NK cells; NKT cells; lymphocytes such as B cells and T cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0128] In certain embodiments, cells (e.g., immune cells) are transduced with a retroviral vector encoding a CAR, such as a lentiviral vector. For example, immune effector cells are transduced with a vector encoding a CAR containing an anti-CHI3L1 / anti-PD-1 antibody or its antigen-binding moiety that binds to CHI3L1 and PD-1 polypeptides having intracellular signaling domains of CD3ζ, CD28, 4-1BB, Ox40, or any combination thereof. Thus, these transduced cells can induce a CAR-mediated cytotoxic response.

[0129] Retroviruses are a common tool for gene delivery. In certain embodiments, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a “provirus.” The provirus acts as a template for RNA polymerase II, directing the expression of RNA molecules encoding structural proteins and enzymes necessary to produce new viral particles.

[0130] Exemplary retroviruses suitable for use in specific embodiments include, but are not limited to, Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), Gibbon's ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0131] As used herein, the term “lentivirus” refers to a group (or genus) of compound retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV-1 and HIV-2); bisnamaedi virus (VMV) virus; canine arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector skeleton ( That is, HIV cis-active sequence elements are preferred. In certain embodiments, a lentivirus is used to deliver polynucleotides containing CARs to cells.

[0132] Retroviral vectors, more specifically lentiviral vectors, can be used when carrying out certain embodiments of the present invention. Therefore, the terms “retrovirus” or “retroviral vector” as used herein mean “lentivirus” and “lentiviral vector,” respectively.

[0133] CHI3L1 antigen binding portion As used herein, “CHI3L1,” “Chintinase-3-like protein 1,” or “YKL-40” refers to a glycoprotein of approximately 40 kDa secreted by at least macrophages, chondrocytes, neutrophils, synovial cells, and some cancer cells. CHI3L1 is a non-chitinase-active, Th2-promoting cytokine involved in the AKT anti-apoptotic signaling pathway and inducing astrocyte migration. The sequences of CHI3L1 expression products are known in many species, for example, human CHI3L1 (NCBI gene ID 1116) mRNA (NCBI Ref Seq: NM_001276.1 and NCBI Ref Seq: NM_001276.2) and polypeptide (NCBI Examples include Ref Seq:NP_001267.1 and NCBI Ref Seq:NP_001267.2.

[0134] In some embodiments, the CHI3L1 antigen-binding portion of the bispecific antibody of the present invention comprises one or more heavy chain CDRs having the amino acid sequences of SEQ ID NOs. 1-3 disclosed in U.S. Patent No. 10,253,111 and reproduced in Table 1 below, and / or light chain CDRs having the amino acid sequences of SEQ ID NOs. 4-6.

[0135] [Table 1]

[0136] In some embodiments of the model, a bispecific antibody, antibody reagent, its antigen-binding moiety, or CAR that specifically binds to the CHI3L1 polypeptide specifically binds to an epitope selected from SEQ ID NOs. 13-24 disclosed in U.S. Patent No. 10,253,111. In some embodiments of the model, a bispecific antibody, antibody reagent, its antigen-binding moiety, or CAR that specifically binds to the CHI3L1 polypeptide specifically binds to the epitope of SEQ ID NO: 13 disclosed in U.S. Patent No. 10,253,111.

[0137] In some embodiments, the backbone of the anti-human CHI3L1 antibody comprises a conservative substitution to a heavy chain sequence having the amino acid sequence of SEQ ID NO: 36 disclosed in U.S. Patent No. 10,253,111, or a light chain sequence having the amino acid sequence of SEQ ID NO: 38, wherein the conservative substitution is in a sequence not included in the CDR. In alternative embodiments, the backbone of the anti-human CHI3L1 antibody comprises a heavy chain sequence of an FRG antibody having the amino acid sequence of SEQ ID NO: 36 disclosed in U.S. Patent No. 10,253,111, or a light chain sequence of an FRG antibody having the amino acid sequence of SEQ ID NO: 38, provided below as SEQ ID NO: 13 and SEQ ID NO: 13, respectively.

[0138] [Table 2]

[0139] In other alternative embodiments, the CHI3L1 antigen-binding moiety of the bispecific antibody of the present invention comprises one or more heavy chain CDRs having the amino acid sequences of SEQ ID NOs. 1 to 12 disclosed in Table 3, and / or one or more light chain CDRs having the amino acid sequences of SEQ ID NOs. 13 to 20. See, for example, International Publication Application No. 2019060675.

[0140] [Table 3-1]

[0141] [Table 3-2]

[0142] PD-1 antigen binding portion Examples of anti-PD-1 antibodies are disclosed in U.S. Patent No. 10,344,090 (Yuan et al.); No. 10,323,091 (van Dijk et al.); No. 10,316,089 (Baruah et al.); No. 10,280,224 (Wang et al.); No. 10,239,942 (Amirina et al.); No. 10,221,244 (Wong et al.); No. 10,155,037 (Abdiche et al.); U.S. Patent Publication No. 2011 / 0123550 (Shibayama et al.); No. 2016 / 0376367 (Yuan et al.); and No. 2017 / 0210806 (Liu).

[0143] Any antigen-binding moiety of an anti-PD-1 antibody can be used in the bispecific antibodies of the present invention. In some embodiments, bispecific antibodies for detecting and neutralizing CHI3L1 and PD1 include a PD-1 single-strand variable fragment (scFv-PD1) and a linker (shown in bold and underlined), and are provided in Table 4.

[0144] [Table 4]

[0145] Pharmaceutical composition In any one embodiment of the embodiments, the Specified Specified Description includes a composition comprising a bispecific antibody, antibody reagent, its antigen-binding moiety, or CAR described herein, or a nucleic acid encoding an antibody, antibody reagent, its antigen-binding moiety, or CAR described herein, or a cell described herein. In some embodiments, the composition is a pharmaceutical composition. As used herein, the term “pharmaceutical composition” means an activator combined with a pharmaceutically acceptable carrier that is acceptable for use in the pharmaceutical industry. The term “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is commensurate with a reasonable benefit / risk ratio.

[0146] The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed within the composition is well understood in the art and does not need to be limited by formulation. Typically, such compositions are prepared for injection as either a liquid solution or a suspension; however, a solid form suitable for solution or suspension in liquid before use can also be prepared. Preparations may also be emulsified or provided as liposome compositions. The active ingredient can be mixed with excipients that are pharmaceutically acceptable, compatible with the active ingredient, and suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. Furthermore, if desired, the composition may contain small amounts of auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., that increase or maintain the efficacy of the active ingredient. The therapeutic compositions described herein may contain pharmaceutically acceptable salts of the constituents. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with free amino groups of polypeptides) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine. Physiologically acceptable salts are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no substances in addition to the active ingredient and water, or both, such as buffers such as sodium phosphate, physiological saline, or phosphate-buffered physiological saline at a physiological pH value. Furthermore, aqueous carriers may contain more than one buffer salt, as well as salts, such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain a liquid phase in addition to and excluding water. Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, etc. It is a water-oil emulsion. The amount of activator used in the present invention that is effective in treating a specific disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0147] In some embodiments, a composition comprising an antibody, antibody reagent, its antigen-binding moiety, or CAR described herein, or a nucleic acid encoding an antibody, antibody reagent, its antigen-binding moiety, or CAR described herein, may be a lyophilized product.

[0148] In some embodiments, the techniques described herein relate to a syringe or catheter, such as an organ-specific catheter (e.g., a renal catheter, biliary catheter, cardiac catheter, etc.), containing a therapeutically effective amount of the composition described herein.

[0149] In one embodiment, this specification describes a method for inhibiting or killing CHI3L1+ / PD-1+ cells, comprising contacting the cells with an isolated bispecific antibody, antibody reagent, its antigen-binding moiety, or CAR, nucleic acid encoding such polypeptide, a cell containing such polypeptide or nucleic acid, or a composition containing such polypeptide or nucleic acid, as described herein. Inhibiting CHI3L1+ / PD-1+ cells may include inhibiting the metabolic activity, translocation, and / or proliferation of the cells. Assays for measuring metabolic activity, translocation (e.g., migration assays) and proliferation are well known in the art. Similarly, assays for measuring the killing of CHI3L1+ / PD-1+ cells, such as cell viability assays, are well known in the art.

[0150] As used herein, “CHI3L1+ / PD-1+” cells are, for example, healthy cells of the same type, or cells that express elevated levels of CHI3L1+ and PD-1+ compared to the mean levels of CHI3L1+ / PD-1+ found in healthy cells of the same type.

[0151] In some embodiments of the aspects described herein, a subject administered with the composition described herein may be a subject determined to have an elevated level of CHI3L1, or an increased level of CHI3L1 compared to a prior assessment of the level in that subject. In some embodiments of the aspects, the elevated level of CHI3L1 is the level of circulating CHI3L1. In some embodiments of the aspects described herein, a subject administered with the composition described herein may be a subject determined to have cancer cells that are CHI3L1+.

[0152] In some embodiments of any of the embodiments described herein, a method comprising administering the composition described herein may further include a first step of identifying subjects having elevated levels of CHI3L1. In some embodiments of any of the embodiments, the elevated level of CHI3L1 is the level of circulating CHI3L1. In some embodiments of any of the embodiments described herein, a method comprising administering the composition described herein may further include a first step of identifying subjects having cancer cells that are CHI3L1+.

[0153] As used herein, “CHI3L1+” cells are cells that express an increased level of CHI3L1+ compared to, for example, healthy cells of the same type or the average level of CHI3L1 found in healthy cells of the same type. In some embodiments of any of the embodiments, the increased level of CHI3L1 is at least 1.5 times the level found in the reference, and may be, for example, 1.5 times, 2 times, 3 times, 4 times, 5 times or higher than the reference level.

[0154] In one embodiment, the techniques described herein relate to a method comprising administering an antibody, antibody reagent, its antigen-binding moiety, or CAR described herein, or a nucleic acid encoding an antibody, antibody reagent, its antigen-binding moiety, or CAR described herein, to a subject. In some embodiments, the subject requires treatment for cancer and / or malignant tumors. In some embodiments, the subject requires treatment for prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, gliablastoma, melanoma, malignant melanoma, and lung cancer. In some embodiments, the method is a method for treating a subject. In some embodiments, the method is a method for treating cancer in a subject.

[0155] In one embodiment, the techniques described herein are methods comprising administering a bispecific antibody, antibody reagent, its antigen-binding portion, or CAR described herein, or a nucleic acid encoding a bispecific antibody, antibody reagent, its antigen-binding portion, or CAR described herein.

[0156] In one embodiment, this specification describes a method for treating cancer in a target area, the method comprising administering cells described herein, for example, cells containing a bispecific antibody, antibody reagent, its antigen-binding moiety, or CAR described herein. In some embodiments, the cells are immune cells.

[0157] In one embodiment, this specification describes a method for treating cancer in a target subject, the method comprising administering a nucleic acid described herein, or immune cells containing a nucleic acid, to the target subject, the immune cells expressing a polypeptide encoded by the nucleic acid. In some embodiments, the immune cells are T cells. The nucleic acid can be targeted to a specific cell type, for example, by the use of cell type-specific promoters and / or compositions that selectively bind to a desired cell type. For example, conjugation of a nucleic acid to an aptamer can enable targeted delivery. See, for example, McNamara et al. (2006). In alternative embodiments, the nucleic acid can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic ion delivery systems promote the binding of nucleic acid molecules (negatively charged) and increase interactions in negatively charged cell membranes, enabling efficient uptake of the nucleic acid by cells. Cationic lipids, dendrimers, or polymers can bind to nucleic acids or be induced to form vesicles or micelles containing nucleic acids (see, for example, Kim et al. (2008)). The formation of vesicles or micelles further inhibits the degradation of nucleic acids when administered systemically. Methods for preparing and administering cationic-inhibitory nucleic acid complexes are within the capabilities of those skilled in the art. Some non-limiting examples of drug delivery systems useful for systemic delivery of nucleic acids include DOTAP oligofectamine, "solid nucleic acid lipid particles," cardiolipin, polyethyleneimine, Arg-Gly-Asp(RGD) peptide, and polyamidoamine. In some embodiments, nucleic acids form complexes with cyclodextrins for systemic administration. Methods for administering nucleic acids and cyclodextrins and pharmaceutical compositions can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in whole. Targeted delivery of nucleic acids is described, for example, in Ikeda and Taira (2006); Soutschek et al. (2004); and Lorenze et al. (2004), each of which is incorporated herein by reference in whole.For example, nucleic acids can target immune cells by encapsulating inhibitors in liposomes containing ligands for receptors expressed on immune cells, such as TCRs. In some embodiments, the liposomes may contain aptamers specific to immune cells.

[0158] In some embodiments, the methods described herein relate to CAR-T cell therapy. CAR-T cell therapy and related treatments involve adoptive cell transfer of immune cells (e.g., T cells) that express CARs that specifically bind to target cell types (e.g., cancer cells) in order to treat the target. This relates to the following. In some embodiments, the cells administered as part of the treatment may be autologous to the subject. In some embodiments, the cells administered as part of the treatment may not be autologous to the subject. In some embodiments, the cells are engineered to express CARs and / or genetically modified. Further studies of CAR-T therapy can be found, for example, in Maus et al. (2014); Reardon et al., Neuro-Oncology 2014, Vol. 16: pp. 1441-1458; Hoyos et al. (2012); Byrd et al. (2014); Maher and Wilkie (2009); Tamada et al., Clin Cancer Res 2012, Vol. 18: pp. 6436-6445, each of which is incorporated herein by reference in whole.

[0159] Generally, pharmaceutical compositions comprising cells, such as T cells or immune cells, as described herein include all integer values ​​within the range of 10. 2 ~10 10 Cells / kg body weight, preferably 10 5 ~10 6 It may be described that the drug can be administered in doses of cells / kg body weight. The number of cells depends on the final intended use as well as the type of cells contained in the composition. For the uses provided herein, the cells are generally in a volume of 1 liter or less, and may be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically 10 6Greater than cells / mL and generally 10 7 Greater than cells / mL and generally 10 8 cells / mL or more. The number of clinically relevant immune cells is cumulatively 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells and can be distributed among multiple infusions equal to or exceeding that number. In some embodiments of the invention, in particular, since all of the cells injected are redirected to a specific target antigen, a smaller number of cells can be administered in the range of 10 6 / kilogram (10 6 to 10 11 per patient). The cell composition expressing CAR can be administered multiple times at dosages within these ranges. The cells can be allogeneic, syngeneic, xenogeneic, or autologous with respect to the patient being treated. If desired, the treatment can also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) described herein to increase the induction of an immune response. In some embodiments, the dosage can be from about 1×10 5 cells to about 1×10 8 cells per kilogram of body weight. In some embodiments, the dosage can be from about 1×10 6 cells to about 1×10 7 cells per kilogram of body weight. In some embodiments, the dosage can be about 1×10 6 cells per kilogram of body weight. In some embodiments, a single dose of cells can be administered. In some embodiments, the dose of cells can be repeated, for example, once, twice, or more. In some embodiments, the dose of cells can be administered, for example, on a daily, weekly, or monthly basis.

[0160] The dosage range of a drug depends on its potency and encompasses a size sufficient to produce the desired effect, such as slowing tumor growth or reducing tumor size. The dosage should not be so large as to cause unacceptable side effects. Generally, the dosage varies depending on the patient's age, condition, and sex and can be determined by those skilled in the art. In addition, individual physicians can adjust the dosage if complications occur. In some embodiments, the dosage ranges from 0.001 mg / kg body weight to 0.5 mg / kg body weight. In some embodiments, the dosage range is from 5 μg / kg body weight to 100 μg / kg body weight. Alternatively, the dosage range can be adjusted to maintain serum levels between 1 μg / mL and 1000 μg / mL. For systemic administration, the target population may be administered at therapeutic doses, such as 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or higher.

[0161] The above-described doses can be repeated. In some embodiments, the dose is administered once daily or multiple times daily, for example, three times daily, but not limited to these doses. In some embodiments, the above-described doses are administered daily for several weeks or months. The duration of treatment depends on the clinical progress of the subject and their response to treatment.

[0162] In some embodiments, the dose may be approximately 2 mg / kg to approximately 15 mg / kg. In some embodiments, the dose may be approximately 2 mg / kg. In some embodiments, the dose may be approximately 4 mg / kg. In some embodiments, the dose may be approximately 5 mg / kg. In some embodiments, the dose may be approximately 6 mg / kg. In some embodiments, the dose may be approximately 8 mg / kg. In some embodiments, the dose may be approximately 10 mg / kg. In some embodiments, the dose may be approximately 15 mg / kg. In some embodiments, the dose may be approximately 100 mg / kg. 2 ~about 700mg / m 2 This is possible. In some embodiments, the dose is approximately 250 mg / m².2 It can be. In some embodiments, the dosage is about 375 mg / m 2 It can be. In some embodiments, the dosage is about 400 mg / m 2 It can be. In some embodiments, the dosage is about 500 mg / m 2 It can be.

[0163] In some embodiments, the dosage can be administered intravenously. In some embodiments, the intravenous administration can be an infusion that occurs over a period of about 10 minutes to about 3 hours. In some embodiments, the intravenous administration can be an infusion that occurs over a period of about 30 minutes to about 90 minutes.

[0164] In some embodiments, the dosage can be administered about once a week. In some embodiments, the dosage can be administered once a week. In some embodiments, the dosage can be administered once a week for about 12 weeks to about 18 weeks. In some embodiments, the dosage can be administered every about 2 weeks. In some embodiments, the dosage can be administered every about 3 weeks. In some embodiments, the dosage can be administered at about 2 mg / kg to about 15 mg / kg every about 2 weeks. In some embodiments, the dosage can be administered at about 2 mg / kg to about 15 mg / kg every about 3 weeks. In some embodiments, the dosage can be administered intravenously at about 2 mg / kg to about 15 mg / kg every about 2 weeks. In some embodiments, the dosage can be administered intravenously at about 2 mg / kg to about 15 mg / kg every about 3 weeks. In some embodiments, the dosage is about 200 mg / m 2 ~about 400 mg / m 2 can be administered intravenously once a week. In some embodiments, the dosage is about 200 mg / m 2 ~about 400 mg / m 2 can be administered intravenously every about 2 weeks. In some embodiments, the dosage is about 200 mg / m 2 ~about 400 mg / m 2It can be administered intravenously approximately every three weeks. In some embodiments, a total of approximately 2 to 10 doses are administered. In some embodiments, a total of 4 doses are administered. In some embodiments, a total of 5 doses are administered. In some embodiments, a total of 6 doses are administered. In some embodiments, a total of 7 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, administration is carried out over a total of approximately 4 to 12 weeks. In some embodiments, administration is carried out over a total of approximately 6 weeks. In some embodiments, administration is carried out over a total of approximately 8 weeks. In some embodiments, administration is carried out over a total of approximately 12 weeks. In some embodiments, the initial dose may be approximately 1.5 to 2.5 times greater than the next dose.

[0165] In some embodiments, the dose may be about 1 mg to about 2000 mg. In some embodiments, the dose may be about 3 mg. In some embodiments, the dose may be about 10 mg. In some embodiments, the dose may be about 30 mg. In some embodiments, the dose may be about 1000 mg. In some embodiments, the dose may be about 2000 mg. In some embodiments, the dose can be administered by intravenous infusion at a rate of about 3 mg daily. In some embodiments, the dose can be administered by intravenous infusion at a rate of about 10 mg daily. In some embodiments, the dose can be administered by intravenous infusion at a rate of about 30 mg three times a week.

[0166] The therapeutically effective dose is a sufficient amount of the drug to produce a statistically significant and measurable change in tumor size, tumor growth, etc. (Measures of effectiveness are described below in this specification). Such effective doses can be measured in clinical trials as well as in animal studies.

[0167] The drug may be administered intravenously by injection or by gradual infusion over time. Given a suitable formulation for a given route, for example, drugs useful in the methods and compositions described herein may be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, or intracavitarially, and may be delivered by peristaltic means if desired, or by other means known to those skilled in the art. The compounds used herein are preferably administered orally, intravenously, or intramuscularly to patients with cancer. Direct local administration to tumor masses is also particularly intended.

[0168] A therapeutic composition containing at least one drug may, for example, be administered in unit doses. The term “unit dose,” when used in reference to a therapeutic composition, refers to a physically discrete unit appropriate as a single dose for a subject, each unit containing a predetermined amount of the active substance calculated to produce the desired therapeutic effect in relation to the required physiologically acceptable diluent, i.e., a carrier or vehicle.

[0169] The composition is a method adapted to a drug prescription and administered in a therapeutically effective amount. The amount and timing of administration depend on the target being treated, the capacity of the target system utilizing the active ingredient, and the desired degree of therapeutic effect.

[0170] The precise amount of active ingredient required for administration depends on the physician's judgment and is individual to individual. However, the appropriate dosage range for systemic administration is disclosed herein and depends on the route of administration. Suitable regimens also vary, but are typically represented by an initial dose followed by repeated doses at intervals of one hour or more by subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations within the specified range is intended for in vivo treatment.

[0171] In some embodiments, the method further comprises administering the pharmaceutical compositions described herein together with one or more additional chemotherapeutic agents, biological agents, drugs, or treatments as part of a combination therapy. In some such embodiments, the chemotherapeutic agents, biological agents, drugs, or treatments are selected from the group consisting of radiotherapy, surgical procedures, antibody reagents, and / or small molecules.

[0172] In some embodiments of the methods described herein, the method further comprises administering one or more chemotherapeutic agents to a subject to which the pharmaceutical composition described herein is administered. Non-limiting examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carboquan, methuredopa and uredopa; ethyleneimines and methylamelamines, e.g., altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bratacin and bratacinone); and camptothecin (synthetic analogues). Including topotecan; briostatin; calistatin; CC-1065 (including its synthetic analogues adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogues, KW-2189, and CB1-TM1); eryuterobin; pancratistatin; sarcodictin; spongstatin; nitrogen mustard, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine hydrochloride oxy Melphalan, nobembitin, fenestrine, prednimustine, trophosphamide, uracil mustard; nitrothrea, e.g., carmustine, chlorozotosin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., engine antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1); dynemycin, e.g., dynemycin A; bisphosphonates, e.g., clodronate; esperamicin; and neocardinostatin chromophores and related pigments Protein enediin antibiotic chromophore), acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, Idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine; dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., carsterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofsphamide glycosides; aminolevulinic acid; enyluracil;Amsacrin; Bestlovesil; Bisanthren; Edatraxate; Defofamine; Demecolsin; Diadiquan; Elformitin; Erliptinium acetate; Epotilon; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidynin; Maytansinoids, e.g., Maytansine and Ansamitosine; Mitoguazone; Mitoxantrone; Mopidammole; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (Registered Trademark) Polysaccharide Complex (JHS Natural) Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic Acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Beraclin A, Loridine A and Angidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremophore-Free, Albumin-Modified Nanoparticle Formulation of Paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill), and TAXOTERE® Docetaxel (Rhone-Poulenc; Rorer, Antony, France); Chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, e.g., cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE® vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (e.g., irinotecan treatment regimens with 5-FU and leucovorin); Topoisomerase inhibitor RFS2000; Difluoromethicone This may include chloroornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatins, e.g., oxaliplatin treatment regimens (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation, and any pharmaceutically acceptable salts, acids, or derivatives of any one of the above.

[0173] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term also includes radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Intended to include radioactive isotopes of Lu, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, fragments and / or variants thereof.

[0174] As used herein, the terms “chemotherapy” or “chemotherapeutic agent” refer to any chemical agent that has therapeutic utility in the treatment of diseases characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms and cancers, as well as diseases characterized by hyperplastic proliferation. As used herein, chemotherapeutic agents encompass both chemical and biological agents. These agents function to inhibit the cellular activity on which cancer cells depend to maintain their survival. Categories of chemotherapeutic agents include alkylating agents / alkaloids, antimetabolites, hormones or hormone analogs, and other antitumor agents. Most of these drugs, if not all, exhibit direct toxicity to cancer cells and do not require immunostimulation. In one embodiment, a chemotherapeutic agent is a drug used to treat neoplasms such as solid tumors. In one embodiment, a chemotherapeutic agent is a radiomolecule. Those skilled in the art can easily identify the chemotherapeutic agent to be used (e.g., Chapter 86 of Harrison's Principles of Internal Medicine, 14th edition (2001); Chapter 17 of Abeloff's Clinical Oncology, 2nd edition (2000)). The bispecific and multispecific polypeptide agents described herein can be used in conjunction with further chemotherapeutic agents.

[0175] "Radiation therapy" means using directed gamma or beta rays to induce sufficient damage to cells, either to limit their ability to function normally or to completely destroy them. It is understood that there are many methods known in the art to determine the dosage and duration of treatment. Typical treatment is given as a single dose, and typical dosages range from 10 to 200 units (Gray) per day.

[0176] In some embodiments, the methods described herein may further include administering further immunotherapy. As used herein, “immunotherapy” refers to, but is not limited to, a diverse range of therapeutic strategies designed to induce a patient’s own immune system to fight a tumor, including, intravesical BCG immunotherapy for superficial bladder cancer, vaccines that produce specific immune responses such as malignant melanoma and renal cell carcinoma, loading patient-derived dendritic cells with prostatic acid phosphatase peptides to induce a specific immune response against prostate-derived cells, the use of cyplucel-T for prostate cancer, administration of cytokines, growth factors and / or signaling molecules (e.g., interleukins) that stimulate one or more immune cell types, ex vivo expansion and / or stimulation of tumor antigen-specific lymphocytes and / or dendritic cells before reintroduction into the patient, imiquimod, adoptive cell transfer, and / or methods described, for example, International Publication No. 2003 / 063792 and U.S. Patent No. 8,329,660. In some embodiments, immunotherapy stimulates an NK response. In other embodiments, the immunotherapy is a adoptive cell transfer approach, i.e., adoptive immunotherapy. ru.

[0177] In some embodiments, the methods described herein may further include administering to a target a further antibody, an antibody reagent, its antigen-binding moiety, or T cells containing a CAR. In some embodiments, the methods described herein may further include administering to a target a cytokine. Antibody and cytokine-based therapies are known in the art and, in non-limiting examples, may include alemtuzumab; bevacizumab; brentuximab vedotin; cetuximab; gemtuzumab; ibritumomab tiuxetan; ipilimumab; ofatumumab; panchimbumumab; rituximab; tositumomab; trastuzumab; interleukin-2; and interferon-alpha.

[0178] For example, the effectiveness of a given treatment for cancer can be determined by a skilled clinician. However, a treatment is considered "effective" in the terms used herein if, for example, one or all of the signs or symptoms of a tumor change in a beneficial way, or if other clinically recognized symptoms are improved, for example, by at least 10%, or even improved, after treatment with the drugs described herein. Effectiveness can also be measured by whether the individual does not worsen, as is assessed by the need for hospitalization or medical intervention (i.e., cessation of disease progression). Methods for measuring these indicators are known to those skilled in the art and / or are described herein.

[0179] An effective dose for treating a disease means an amount sufficient to produce an effective treatment for the disease when administered to a mammal in need, as the term is defined herein. The effectiveness of a drug can be determined by evaluating physical indicators such as tumor size, tumor volume, tumor density, angiogenesis, and tumor growth rate, for example, in the case of cancer.

[0180] CHI3L1 and PD-1 levels in the subjects In one embodiment, this specification describes a method for detecting, prognosing, and / or diagnosing cancer, the method comprising contacting the sample with a bispecific antibody, antibody reagent or antigen-binding moiety described herein to detect or measure levels of CHI3L1 and / or PD-1 or PD-L1 in a sample obtained from a subject, wherein an increase in the levels of CHI3L1 and PD-1 or PD-L1 relative to a reference level indicates that the subject has cancer and has an increased risk of developing cancer.

[0181] Recent reports have shown that PD-L1 expression as a biomarker for immune checkpoint inhibitors (ICIs) such as PD-1 is moderately sensitive in patients with cancer, and that treatment outcomes are observed in patients with low or absent PD-L1 expression levels (Paz-Ares et al., 2018; Hellmann et al., 2019; Schoenfeld et al., 2020). In other embodiments, a method for detecting, prognosing, and / or diagnosing cancer, the method comprising detecting or measuring the level of CHI3L1 in a sample obtained from a subject by contacting the sample with a bispecific antibody, antibody reagent or its antigen-binding moiety as described herein, wherein an increase in the level of CHI3L1 relative to a reference level indicates that the subject has cancer and is at increased risk of developing cancer.

[0182] In some embodiments of any of the aspects described herein, the subject administered with the composition described herein may be a subject determined to have elevated levels of CHI3L1 and / or PD-1 or PD-L1. In some embodiments, the elevated levels of CHI3L1 and PD-1 or PD-L1 are the levels of circulating CHI3L1 and PD-1 or PD-L1. In some embodiments of any of the aspects described herein The subjects to whom the compositions described herein are administered may be subjects determined to have cancer cells that are CHI3L1+ / PD-1+.

[0183] In some embodiments of any of the embodiments described herein, a method comprising administering the composition described herein may further include a first step of identifying subjects having elevated levels of CHI3L1 and / or PD-1 or PD-L1. In some embodiments, the elevated levels of CHI3L1 and / or PD-1 or PD-L1 are the levels of circulating CHI3L1 and / or PD-1 or PD-L1. In some embodiments of any of the embodiments described herein, a method comprising administering the composition described herein may further include a first step of identifying subjects having cancer cells that are CHI3L1+ / PD-1+ or CHI3L1+ / PD-L1+.

[0184] In one embodiment, the Specified herein describes an assay comprising contacting a test sample obtained from a subject with an antibody, antibody reagent, or its antigen-binding moiety as described herein, and detecting the presence or intensity of a signal indicating the presence or level of CHI3L1 and PD-1 in the sample, wherein an increase in CHI3L1 and PD-1 levels relative to a reference level indicates that the subject has or is at higher risk of developing cancer.

[0185] In one embodiment, this specification describes a method for identifying subjects in need of cancer treatment, the method comprising contacting a test sample obtained from a subject with a bispecific antibody, antibody reagent, or its antigen-binding moiety as described herein, detecting the presence or intensity of a signal indicating the presence or level of CHI3L1 and / or PD-1 or PD-L1 in the sample, and identifying a subject as in need of cancer treatment if the expression level of CHI3L1 and / or PD-1 or PD-L1 is elevated relative to a reference level.

[0186] In one embodiment, the Specified herein describes a method for determining whether a subject is likely to respond to anti-CHI3L1 / anti-PD-1 therapy, such as treatment with an anti-CHI3L1 / anti-PD-1 bispecific antibody, antibody reagent, or its antigen-binding moiety, or with T cells containing bispecific CARs that bind to CHI3L1 and PD-1 or PD-L1, the method comprising contacting a test sample obtained from the subject with the antibody, antibody reagent, or its antigen-binding moiety described herein, detecting the presence or intensity of a signal indicating the presence or level of CHI3L1 and PD-1 or PD-L1 in the sample; determining that the subject is likely to respond to treatment with anti-CHI3L1 / anti-PD-1 therapy if the levels of CHI3L1 and PD-1 or PD-L1 increase relative to a reference level; and determining that the subject is unlikely to respond to treatment with anti-CHI3L1 / anti-PD-1 if the levels of CHI3L1 and PD-1 or PD-L1 do not increase relative to a reference level.

[0187] In one embodiment, this specification describes a method for treating cancer, comprising contacting a test sample obtained from a subject with an antibody, antibody reagent, or its antigen-binding moiety as described herein; detecting the presence or intensity of a signal indicating the presence or level of CHI3L1 and PD-1 in the sample; and treating the subject with anti-CHI3L1 / anti-PD-1 bispecific antibody therapy if the levels of CHI3L1 and PD-1 are elevated relative to a reference level. In one embodiment, this specification describes a method for treating cancer, comprising administering a therapeutically effective amount of anti-CHI3L1 / anti-PD-1 bispecific antibody therapy to a subject determined to require treatment for cancer and further determined to have elevated levels of CHI3L1 and PD-1 relative to a reference level, wherein the anti-CHI3L1 / anti-PD-1 therapy comprises an antibody, antibody reagent, its antigen-binding moiety, or a T cell; nucleic acid; cell; or composition containing a bispecific CAR that recognizes CHI3L1 and PD-1 as described herein.

[0188] In some embodiments, the expression level of CHI3L1 can be measured by determining the level of the expression product of the CHI3L1 gene, e.g., CHI3L 1 RNA transcript or CHI3L1 polypeptide, and the expression level of PD-1 can be measured by determining the level of the expression product of the PD-1 gene, e.g., PD-1 RNA transcript or PD-1 polypeptide. Such molecules can be isolated, induced, or amplified from biological samples, e.g., biological fluids. In some embodiments, a detectable signal is generated by an antibody or its antigen-binding moiety in the presence of a CHI3L1 molecule or a PD-1 molecule. In some embodiments, the antibody or its antigen-binding moiety can be detectably labeled or generate a detectable signal. In some embodiments, the level of CHI3L1 or PD-1 is determined using a method selected from the group consisting of Western blotting; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunoassay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistochemical staining; radioimmunometric assay; immunofluorescence assay; mass spectrometry; FACS; and immunoelectrophoresis assay. In some embodiments, the antibody or its antigen-binding moiety is detectably labeled or generates a detectable signal. In some embodiments, the expression level of CHI3L1 or PD-1 is normalized to the expression level of one or more reference genes or reference proteins. In some embodiments, the reference level of CHI3L1 or PD-1 is the expression level of CHI3L1 or PD-1 in a previous sample obtained from the subject.

[0189] In some embodiments, the level of CHI3L1 or PD-1 may be the level of the CHI3L1 or PD-1 polypeptide. Detection of the CHI3L1 or PD-1 polypeptide can be carried out according to any method known in the art. Immunological methods for detecting the CHI3L1 or PD-1 polypeptide according to this technique include, but are not limited to, antibody techniques such as immunohistochemistry, immunocytochemistry, flow cytometry, fluorescence-activated cell sorting (FACS), immunoblotting, radioimmunoassay, Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and induction techniques using antibody reagents described herein.

[0190] Immunochemical methods require the use of antibody reagents specific to the target molecule (e.g., an antigen, or in embodiments described herein, CHI3L1 or PD-1 polypeptide). In some embodiments, the assays, methods, and / or systems described herein may include anti-CHI3L1 or anti-PD-1 antibody reagents. In some embodiments, the antibody reagent may be detectably labeled. In some embodiments, the antibody reagent may be attached to a solid support (e.g., bound to a solid support). In some embodiments, the solid support may include particles (including, but not limited to, agarose or latex beads or particles or magnetic particles), beads, nanoparticles, polymers, substrates, slides, coverslips, plates, dishes, wells, membranes, and / or diffraction gratings. The solid support may include, but not limited to, many different materials, including polymers, plastics, resins, polysaccharides, silicon or silica-based materials, carbon, metals, inorganic glass, and membranes.

[0191] In one embodiment, the assays, methods, and / or systems described herein may include ELISA. In an exemplary embodiment, a first antibody reagent may be immobilized on a solid support (typically a polystyrene microtiter plate). The solid support can be brought into contact with a sample obtained from a subject, and the antibody reagent binds to ("captures") an antigen for which it is specific (e.g., CHI3L1 or PD-1). The solid support can then be brought into contact with a second labeled antibody reagent (e.g., a detection antibody reagent). The detection antibody reagent may, for example, contain a detectable signal and be covalently linked to an enzyme, or by itself, by a secondary antibody linked to an enzyme via bioconjugation. It can be detected. The presence of a signal indicates that both the first antibody reagent and the second "detection" antibody reagent immobilized on the support have bound to the antigen; that is, the presence of a signal indicates the presence of CHI3L1 or PD-1 molecules. Between each step, the plate is typically washed with a mild surfactant solution to remove any proteins or antibodies that are not specifically bound. After the final washing step, the plate is colored by adding an enzyme substrate to produce a visible signal, indicating the amount of CHI3L1 or PD-1 polypeptide in the sample. Older ELISAs utilize chromogenic substrates, while newer assays use fluorescent substrates with much higher sensitivity. There are other different forms of ELISA that are well known to those skilled in the art.

[0192] In one embodiment, the assays, systems, and methods described herein may include lateral flow immunoassays (LFIA), also known as immunochromatographic assays, or strip tests for measuring or determining the level of CHI3L1 or PD-1 polypeptides in a sample. LFIA is a simple device intended to detect the presence (or absence) of CHI3L1 or PD-1 in a sample. Many LFIA tests are currently used for medical diagnostics, whether for home testing, point-of-care testing, or laboratory use. An LFIA test is a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After applying the sample to a test strip, it encounters a colored antibody reagent that mixes with the sample, and if it binds to a portion of the sample, it passes through a line or zone pre-treated with a second antibody reagent to encounter the substrate. Depending on the level of CHI3L1 or PD-1 present in the sample, the colored antibody reagent may bind at the test line or zone. An LFIA is essentially an immunoassay adapted to operate along a single axis to fit a test strip format or dipstick format. Strip tests are extremely versatile and can be easily modified by those skilled in the art to detect a vast range of antigens from liquid samples such as urine, blood, and water samples. Strip tests are also known as dipstick tests, a name derived from the literal action of "dipping" the test strip into the liquid sample being tested. LFIA strip tests are easy to use, require minimal training, and can be easily incorporated as components of point-of-care (POCT) diagnostics used in the field. LFIA tests can be operated as either competitive assays or sandwich assays. Sandwich LFIA is similar to sandwich ELISA. The sample first encounters colored particles labeled with a target-specific antibody reagent (e.g., CHI3L1 or PD-1 specific antibody reagent). The test line also contains an antibody reagent (e.g., CHI3L1 or PD-1 specific antibody reagent). The test line is indicated as a colored band for positive samples.In some embodiments, lateral flow immunoassays may be bi-antibody sandwich assays, competitive assays, quantitative assays, or variations thereof. There are many variations of lateral flow technology. It is also possible to apply multiple capture zones to create multiple tests.

[0193] A typical test strip consists of the following components: (1) a sample application area containing an absorbent pad (i.e., matrix or material) to which the test sample is applied; (2) a conjugate or reagent pad - which contains one or more target-specific antibody reagents that can be conjugated to colored particles (usually colloidal gold particles or latex microspheres); (3) a reaction membrane - typically a test result area containing a hydrophobic nitrocellulose or cellulose acetate membrane with the antibody reagent immobilized in lines across the membrane as a capture zone or test line (a control zone may also be present, containing antibodies specific to the antibody reagent conjugated to particles or microspheres); and (4) an optional wick or waste reservoir - consisting of an additional absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it. The components of the strip are usually immobilized in an inert backing material and come in a simple dipstick format, or in a plastic case with a sample port and reaction window indicating the capture and control zones. It can be provided within the testing. While not strictly necessary, most tests incorporate a secondary line containing antibodies to recover free latex / gold to confirm that the test worked correctly.

[0194] The use of “dipsticks” or LFIA test strips and other solid supports has been described in the art in the context of immunoassays for many antigen biomarkers. U.S. Patents Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. Patent Applications Nos. 10 / 278,676; 09 / 579,673 and 10 / 717,082, which are incorporated herein by whole reference, are non-limiting examples of such lateral flow testing apparatuses. Three U.S. patents (U.S. Patent No. 4,444,880 issued by H. Tom; U.S. Patent No. 4,305,924 issued by RNPiasio; and U.S. Patent No. 4,135,884 issued by JTShen) describe the use of “dipstick” technology for detecting soluble antigens via immunochemical assays. The apparatus and methods of these three patents broadly describe a first component immobilized on a solid surface on a “dipstick” that is exposed to a solution containing a soluble antigen bound to the component immobilized on the “dipstick” prior to detection of a component-antigen complex on the stick. Modifying the teachings of these “dipstick” technology as required for the detection of CHI3L1 or PD-1 polypeptides is within the scope of the art. In some embodiments, the dipstick (or LFIA) may be suitable for use with urine samples. In some embodiments, the dipstick may be suitable for use with blood samples.

[0195] Immunochemistry is a family of techniques based on the use of specific antibodies, which are used to specifically target molecules inside or on the surface of cells. In some embodiments, immunohistochemistry ("IHC") and immunocytochemistry ("ICC") techniques can be used to detect or measure levels of CHI3L1 or PD-1 polypeptides. IHC is the application of immunochemistry to tissue sections, while ICC is the application of immunochemistry to cells or tissue imprints after they have received specific cytological preparations, such as liquid-based preparations. In some cases, signal amplification can be incorporated into specific protocols, with a labeled secondary antibody following the application of an antibody reagent specific to platelets or leukocytes. Typically, for immunohistochemistry, tissue obtained from the subject, fixed with a suitable fixative such as alcohol, acetone, and paraformaldehyde, is sectioned and reacted with an antibody. Conventional methods for immunohistochemistry are described in Buchwalow and Bocker (eds.), "Immunohistochemistry: Basics and Methods," Springer (2010); and Lin and Prichard, "Handbook of Practical Immunohistochemistry," Springer (2011), which are incorporated herein by reference in their entirety. In some embodiments, immunocytochemistry may be utilized when tissues or cells obtained from a subject are fixed with a suitable fixative such as alcohol, acetone, and paraformaldehyde, and then antibodies are reacted with them. Methods for immunocytological staining of human samples are known to those skilled in the art and are described, for example, in "Immunocytochemistry: A Practical Guide for Biomedical Research" (2009), which are incorporated herein by reference in their entirety.

[0196] In some embodiments, one or more antibody reagents described herein may include detectable labels and / or have the ability to generate a detectable signal (e.g., by catalyzing a reaction that converts a compound into a detectable product). Detectable labels may include, for example, light-absorbing dyes, fluorescent dyes, or radioactive labels. Methods for detecting detectable labels, methods for detecting them, and methods for incorporating them into antibody reagents are described herein. This is well known in the field of technology.

[0197] In some embodiments, the detectable label may include a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, for example, by fluorescence, chemifusion, or chemiluminescence, or by any other suitable means. The detectable labels used in the methods described herein may be primary labels (where the label includes a directly detectable portion or a portion that generates a directly detectable portion) or secondary labels (where the detectable label binds to another portion to generate a detectable signal, as is common in immunological labeling with secondary and tertiary antibodies, for example). The detectable label may be linked to an antibody reagent by covalent or non-covalent means. Alternatively, the detectable label may be linked by directly labeling a molecule that achieves binding to the antibody reagent via, for example, a ligand-receptor pair sequence or other such specific recognition molecule. Detectable labels may include, but are not limited to, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0198] In other embodiments, the detection antibody is labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of an appropriate wavelength, its presence can be detected by fluorescence. In some embodiments, the detectable label may be a fluorescent dye molecule or a fluorophore, and is not limited to, fluorescein, phycoerythrin, phycocyanin, o-phthalaldehyde, fluoresamine, Cy3(trademark), Cy5(trademark), allophycocyanin, Texas Red, peridenine chlorophyll, cyanine, tandem conjugate, e.g., phycoerythrin-Cy(trademark), green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green(trademark), rhodamine and derivatives (e.g., Texas Red and tetralodimine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes(trademark), 6-carboxyphyoresene (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofluoro This includes ceine (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluoroceine (JOE or J), N,N,N',N'-tetramethyl-6 carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g., Cy3, Cy5, and Cy7 dyes; coumarins, e.g., umbelliferone; benzimide dyes, e.g., Hoechst 33258; phenanthoridine dyes, e.g., Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g., cyanine dyes, e.g., Cy3, Cy5, etc.; bodipy dyes and quinoline dyes.

[0199] In some embodiments, the detectable labels are not limited to, 3 H, 125 I, 35 S, 14 C, 32 P, and 33 It may be a radioactive label containing P.

[0200] In some embodiments, the detectable label may be an enzyme, but is not limited to, horseradish peroxidase and alkaline phosphatase. The enzyme label may, for example, produce a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes intended for use to detectably label antibody reagents include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, and glucomirror. It contains ze and acetylcholinesterase.

[0201] In some embodiments, detectable labels are chemiluminescent labels, but are not limited to, lucigenin, luminol, luciferin, isoluminol, seromatic acridinium esters, imidazole, acridinium salts, and oxalate esters.

[0202] In some embodiments, the detectable label may be a spectral colorimetric label comprising, but not limited to, colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0203] In some embodiments, antibodies may also be labeled with detectable tags such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems, such as the biotin-streptavidin system, can also be used. In this system, antibodies that are immunoreactive (i.e., specific) to the biomarker of interest are biotinylated. The amount of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromaten substrate. Such streptavidin-peroxidase detection kits are commercially available, for example, from DAKO;Carpinteria, CA.

[0204] Antibody reagents also contain fluorescent metals, for example, 152 These metals can be detectably labeled with Eu or other lanthanides. These metals can be conjugated to antibody reagents using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0205] The assays and methods described herein may relate to determining whether a subject has elevated levels of CHI3L1 and PD-1 relative to a reference level. In some embodiments, the reference levels of CHI3L1 and PD-1 may be, for example, the levels of CHI3L1 and PD-1 in a healthy subject who does not have or has not been diagnosed with cancer. In some embodiments, the reference levels may be levels in samples of similar cell type, sample type, and sample processing, as the sample to which the levels of CHI3L1 and PD-1 are to be determined, and / or levels obtained from subjects of the same age, sex, and other demographic parameters, as the subject to which the levels of CHI3L1 and PD-1 are to be determined. In some embodiments, the test sample and the control reference sample are of the same type, i.e., obtained from the same biological source and containing the same composition, e.g., the same number and type of cells and / or type of sample material. Thus, in some embodiments, elevated levels of CHI3L1 and PD-1 may change as demographic factors such as age, sex, genotype, environmental factors, and individual medical history change. In some embodiments, the reference level may include, for example, the levels of CHI3L1 and PD-1 (e.g., CHI3L1 and PD-1 polypeptides) in the same type of sample taken from a subject that does not show any signs or symptoms of cancer. In some embodiments, the reference expression level of CHI3L1 and PD-1 may be the expression level of CHI3L1 and PD-1 in a previous sample obtained from the subject. This allows for direct analysis of changes in levels within that individual.

[0206] In some embodiments, the levels of CHI3L1 and PD-1 may be elevated relative to the reference level if the levels are at least 1.25 times the reference level, for example, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, or more. In some embodiments, the expression levels of CHI3L1 and PD-1 may be normalized relative to the expression level of one or more reference genes or reference proteins. Yes, it is possible. In some embodiments, the expression levels of CHI3L1 and PD-1 can be normalized against a reference value.

[0207] In some embodiments, the expression levels of 20 or fewer other genes are determined. In some embodiments, the expression levels of 10 or fewer other genes are determined.

[0208] The terms “sample” or “test sample” as used herein refer to a sample taken from or isolated from a living organism, for example, a urine sample from a subject. Exemplary biological samples include, but are not limited to, biological fluid samples; serum; plasma; urine; saliva; and / or tumor samples. The term also includes mixtures of the above samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments, a test sample may include cells derived from the subject. As used herein, the term “biological fluid” refers to any fluid obtained from a biological source, and is not limited to, blood, urine, and separatory fluids.

[0209] Samples can be obtained by removing a sample from the subject, but this can also be achieved by using a previously isolated sample (e.g., isolated at a previous point in time and by the same or a different person). Furthermore, test samples may be newly recovered or previously collected samples.

[0210] In some embodiments, the test sample may be an untreated test sample. As used herein, the term “untreated test sample” means a test sample that has not undergone any prior pretreatment of the sample, except for dilution and / or suspension in solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample may be a frozen test sample, e.g., frozen tissue. The frozen sample may be thawed before using the methods, assays and systems described herein. After thawing, the frozen sample may be centrifuged and then subjected to the methods, assays and systems described herein. In some embodiments, the test sample is a clarified test sample prepared, for example, by centrifugation and recovery of the supernatant containing the clarified test sample. In some embodiments, the test sample may be a pretreated test sample, e.g., supernatant or filtrate, obtained from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combination thereof. In some embodiments, the test sample may be treated with chemical and / or biological reagents. Chemical and / or biological reagents can be used to protect and / or maintain the stability of samples, such as biomolecules (e.g., nucleic acids and proteins), during processing. One exemplary reagent is a protease inhibitor, which is commonly used to protect or maintain the stability of proteins during processing. Those skilled in the art will be familiar with suitable methods and processes for the pretreatment of biological samples required for determining CHI3L1 levels, as described herein.

[0211] In some embodiments, the methods, assays, and systems described herein may further include a step of obtaining a test sample from a subject. In some embodiments, the subject may be a human subject.

[0212] In some embodiments, the methods, assays, and systems described herein may include generating a report based on CHI3L1 and PD-1 levels. In some embodiments, the report may show the raw values ​​of CHI3L1 and PD-1 in the test sample (plus, optionally, the levels of CHI3L1 and PD-1 in the reference sample), or show a percentage or multiple increase in the levels of CHI3L1 and PD-1 compared to the reference level, and / or whether the subject is at risk of having or not having cancer. It provides a signal indicating that.

[0213] As used herein, “risk of having” means an increase and / or rise in CHI3L1 and PD-1 levels, for example, by 2, 2.5, 3, 4, or more, and at least twice the likelihood of having a particular condition compared to subjects who did not have that risk.

[0214] In some embodiments, the assay or method may further include the step of administering an anti-CHI3L1 / anti-PD-1 therapy. In some embodiments, the anti-CHI3L1 / anti-PD-1 therapy comprises an isolated bispecific antibody, an antibody reagent, its antigen-binding moiety, or CAR or CAR T cells; nucleic acids; cells; or compositions described herein.

[0215] In any one aspect of the embodiments, this specification describes antibodies, antibody reagents, or antigen-binding moieties thereof that are conjugated or bound to a detectable label.

[0216] In any one embodiment of the embodiments, a solid support comprising a bispecific antibody, an antibody reagent, or an antigen-binding fragment thereof as described herein is described. In some embodiments of any of the embodiments, the bispecific antibody, the antibody reagent, or the antigen-binding fragment thereof is detectably labeled. In some embodiments of any of the embodiments, the solid support comprises particles, beads, polymers, or substrates.

[0217] In any one embodiment of the embodiments, the Specified Specified Molecular Complex comprises at least one bispecific antibody, antibody reagent, antigen-binding fragment thereof, or CAR conjugated to a CHI3L1 polypeptide and a PD-1 polypeptide.

[0218] In one embodiment, this specification describes a kit comprising a composition described herein, for example, a bispecific antibody, an antibody reagent, its antigen-binding portion, or a CAR described herein. The kit is any product (e.g., a package or container) comprising at least one reagent, for example, a bispecific antibody, which is promoted, distributed, or sold as a unit for carrying out the method described herein. In some embodiments of any of the embodiments, the bispecific antibody, antibody reagent, or its antigen-binding fragment described herein is immobilized on a solid support. In some embodiments of any of the embodiments, the solid support comprises particles, beads, polymers, or substrates. In some embodiments of any of the embodiments, the antibody, antibody reagent, or its antigen-binding fragment is detectably labeled.

[0219] The kits described herein may optionally include additional components useful for carrying out the methods described herein. For example, the kit may include a fluid (e.g., a buffer) suitable for a composition containing the bispecific antibody described herein, its antigen-binding moiety, or CAR, and instructions describing the performance of the methods described herein. The kit may further include a device and / or reagents for delivering the compositions described herein. In addition, the kit may include an instruction leaflet and / or provide information regarding the relevance of the results obtained.

[0220] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the exact forms disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but as will be understood by those skilled in the art, various equivalent modifications are possible within the scope of the present disclosure. For example, method steps or functions are shown in a given order, but alternative embodiments can perform the functions in a different order or the functions can be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied, as appropriate, to other procedures or methods. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, as needed, using the above references and the compositions, functions, and concepts of the application, to provide still other embodiments of the disclosure. Further, considering biological functional equivalency, some changes can be made to the protein structure without affecting the type or amount of biological or chemical action. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0221] Any particular element of any of the foregoing embodiments can be combined or substituted in other embodiments with respect to that element. Further, the advantages associated with particular embodiments of the present disclosure are described in the context of these embodiments, but other embodiments can also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages in order to fall within the scope of the present disclosure.

[0222] The techniques described herein are further illustrated by the following examples, which should not be construed as being more limiting. In the practice or testing of the present disclosure, methods and materials similar or equivalent to those described herein can be used, but appropriate methods and materials are described below.

Examples

[0223] Here, to generally describe the present invention, the present invention will be more easily understood by referring to the following examples, which are included only for the purpose of exemplifying specific aspects and embodiments of the present invention and are not intended to limit the present invention.

Example

[0224] Generation and Characterization of CHI3L1×PD1 Bispecific Antibody (FRG×PD1-ScFv) To generate a bispecific antibody that detects and neutralizes CHI3L1 and PD1, the inventors used the framework of an anti-human CHI3L1 antibody (referred to as "FRG") recently developed in the inventors' laboratory and described in U.S. Patent No. 10,253,111. The PD1 single-chain variable fragment (scFv-PD1) was generated based on sequence information obtained from public domains with minor modifications. As illustrated in FIG. 1, scFv-PD1 was conjugated to either the light chain or the heavy chain of the CHI3L1 antibody using a linker. The amino acid sequences of scFv-PD1 and the linker are shown in Table 2. The construct of the bivalent CHI3L1×PD1 antibody was confirmed by DNA sequence analysis.

[0225] The CHI3L1×PD1 construct was individually transfected into HEK-293T adherent cells. The binding affinity of the bispecific antibody for CHI3L1 and PD-1 was evaluated by competitive ELISA and compared with the binding of the individual antibody moieties.

[0226] As shown in FIG. 2, the secreted bispecific antibody in the supernatant was able to detect both recombinant human (rh) CHI3L1 and rhPD1. These studies demonstrated that the bispecific antibody and the individual antibody moieties had comparable affinities (KD≒1×10 -9 M) for rhCHI3L1 and rhPD1 and similar limits of detection (LOD; 2 ng / ml). A protein A column was used for further antibody purification.

[0227] In summary, the inventors have successfully generated and characterized bispecific antibodies that react with high affinity to both rhCHI3L1 and rhPD1 (see Figures 1 and 2). As illustrated in Figure 1, these bispecific antibodies were developed using two different approaches. These were generated. Using these platforms, CHI3L1-LC-PD1 and CHI3L1-HC-PD bispecific (bivalent) antibodies were produced, each detecting both human CHI3L1 and human PD1. The affinity of the CHI3L1-LC-PD1 antibody for both rhCHI3L1 and rhPD1 (evaluated by competitive ELISA assay) was K D ≈ 1 × 10 -9 It is estimated to be M, with a limit of detection (LOD) of 2 ng / ml. There was no significant difference in binding affinity to rhCHI3L1 or rhPD1 between the CHI3L1-LC-PD1 antibody and the CHI3L1-HC-PD1 antibody (Figure 2 and data not shown). [Examples]

[0228] Characterization of the T cell-U87 binding and antitumor cytotoxic activity of bispecific CHI3L1×PD1 antibodies. The ability of Jurkat cells to bind to U87 cells and the antitumor cytotoxic activity of a bispecific CHI3L1×PD1 antibody were evaluated using an in vitro co-culture system containing U87 gliablastoma cells (ATCC #HTB-14) and Jurkat T cells (ATCC #TIB152). Human gliablastoma (U87) cells were grown in complete DMEM medium. Jurkat (T cells) were activated by stimulating them with α-CD3 / α-CD28 antibody (5 μg / ml) for 2 hours in RPMI complete medium under 5% CO2 and air. Jurkat cells were then centrifuged, washed, and resuspended in fresh complete RPMI medium. U87 and Jurkat cells were cultured in complete RPMI medium in a 1:6 ratio.

[0229] The reactions in these co-cultures were evaluated under IgG isotype control, anti-PD1 alone, anti-CHI3L1 alone, a combination of anti-CHI3L1 and anti-PD-1 (5 μg / ml each), or in the presence of the bispecific antibodies described above. Overall, there were five treatment groups: (i) isotype IgG control, (ii) α-PD1, (iii) α-CHI3L1, (iv) α-CHI3L1 + α-PD1, and (v) bispecificity-CHI3L1xPD1. Co-cultured cells were incubated in 5% CO2 and air for 6–12 hours. T cell-U87 cell binding was evaluated by microscopy, and cell death was assessed using TUNEL staining and the LDH-releasing cytotoxicity assay described below.

[0230] Quantification of Jurkat cell adhesion to A.U87 cells Jurkat T cells were activated with anti-(α)-human CD3 and α-CD28 antibodies (5 μg / ml, incubated at 37°C for 2 hours in 5% CO2 and air, respectively) and co-cultured with U87 gliablastoma cells and the above-mentioned isotype controls or other antibodies. CellBrite cytoplasmic membrane dye was used for fluorescent labeling of U87 (red) and Jurkat T cells (green). The number of Jurkat T cells per U87 cell was counted using a fluorescence microscope (20x magnification of the original) and averaged across 10 randomly selected microscope fields.

[0231] As shown in Figure 3, treatment with the bispecific CHI3L1×PD1 antibody significantly increased Jurkat T cell adhesion to U87 cells. Importantly, the effect of the bispecific antibody was significantly more pronounced than the effects of treatment with α-CHI3L1 or α-PD1 individually, or with a combination of α-CHI3L1 and α-PD1.

[0232] B. Apoptosis U87 cell death quantification using TUNEL assay We performed dUTP nick-end labeling mediated by terminal deoxynucleotidyltransferases in two different methods for in-situ evaluation of cell death: (i) Cells co-cultured in different chambers were fixed, permalized, and stained with propidium fluorescein iodide (red dye) and Cyto (green dye). Cells that fluoresced red were dead cells, and those that fluoresced green were living cells. The number of living and dead U87 cells was counted using a fluorescence microscope (20 times the original magnification) and randomly selected. The values ​​were averaged across 10 microscope fields.

[0233] (ii) Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling for in-situ assessment of cell death. In selected studies, TUNEL-positive staining was assessed using bright-field microscopy. Dead or apoptotic cells were stained blue, and living cells were stained with nuclear first red. The number of living and dead U87 cells was counted using fluorescence microscopy and conventional microscopy (20x the original magnification) and averaged across 10 randomly selected microscopic fields.

[0234] As shown in Figure 4, treatment with the bispecific CHI3L1×PD1 antibody significantly increased the ability of Jurkat T cells to induce a cytotoxic / apoptotic response in U87 cells. Importantly, the effect of the bispecific antibody was significantly more pronounced than the effects of treatment with α-CHI3L1 or α-PD1 individually, or with a combination of α-CHI3L1 and α-PD1.

[0235] Quantitative analysis of C. granzyme and accumulation of perforin Granzyme and perforin are major cytotoxic enzymes secreted by various activated cytotoxic T cells, including Jurkat cells. Expression levels of these cytotoxic enzymes were measured in co-cultured cells using double-labeled immunohistochemistry with antibodies against granzyme (Figure 5) or perforin (Figure 6) (red) and phalloidin actin filaments (green). The number of granzyme-+ or perforin-+ cells was counted using a fluorescence microscope (20x magnification) and averaged across 10 randomly selected microscope fields.

[0236] As shown in Figures 5 and 6, treatment with bispecific CHI3L1×PD1 antibodies significantly increased the accumulation of granzymes and perforins in Jurkat T cells co-cultured with U87 cells. Importantly, the effect of the bispecific antibodies was significantly more pronounced than the effects of treatment with α-CHI3L1 or α-PD1 individually, or with a combination of α-CHI3L1 and α-PD1.

[0237] These results suggest that the bispecific CHI3L1×PD1 antibody synergistically enhances the cytotoxic effect on T cells.

[0238] D.LDH-releasing cytotoxicity assay Lactate dehydrogenase (LDH) is released into the culture medium after membrane integrity is lost due to cytotoxic damage. Thus, LDH release has been used as an indicator of cell death. This was evaluated using a coupled two-step reaction. In the first step of the reaction, LDH is released by oxidizing lactate to pyruvate, thereby converting NADH and H2. + NAD to + It catalyzes the reduction of H. In the second step, diaphorase catalyzes the reduction of the newly formed NADH and H. + This catalyzes the reduction of tetrazolium salts (INT) to dark-colored formazans that strongly absorb at 490-520 nm.

[0239] Cytotoxicity levels were determined using a commercially available assay kit (Pierce LDH Cytotoxicity Assay Kit) and the protocol provided by the manufacturer. In these experiments, cells were co-cultured in and out of the presence of the antibodies mentioned above. LDH in the culture medium was evaluated after overnight incubation. These values ​​were compared to the following controls: (a) a complete medium control without cells to determine the background LDH activity present in the serum used for medium supplementation; (b) serum-free medium; (c) LDH activity control (water); and (d) the maximum LDH activity released by cells treated with lysis buffer. In these assays, cytotoxicity % was calculated as follows.

[0240]

Number

[0241] As shown in Figure 7, treatment with the bispecific CHI3L1×PD1 antibody significantly increased the ability to induce LDH release and cytotoxic responses of Jurkat T cells in U87 cells. Importantly, the effect of the bispecific antibody was significantly more prominent than that of individual treatment with α-CHI3L1 or α-PD1, or treatment with the combination of α-CHI3L1 and α-PD1.

[0242] The synergistic effect of the bispecific CHI3L1×PD1 antibody is further illustrated in Figure 8. The antitumor effect of the FRG×PD-1 bispecific antibody was evaluated in a co-culture system containing Jurkat cells and A375 human melanoma cells. Jurkat cells were activated by pretreatment with anti-CD3 and anti-CD28 (1 μg / mL each, incubated in 5% CO2 and air at 37°C for 2 hours). Jurkat cells were then co-cultured with A357 human melanoma cells for 24 hours. These co-cultures were performed in the presence of the following antibodies: isotype control antibody (5 μg / mL), anti-PD-1 or anti-CHI3L1 (FRG) alone (5 μg / mL) or in combination (2.5 μg / mL each), and bispecific FRG×PD-1 antibody (5 μg / mL). Column A provides representative demonstration and quantification of apoptotic tumor cell death using the in situ cell detection kit - fluorescein dUTP. TUNEL(+) cells are stained green. Columns B–D provide representative demonstrations and quantifications of Jurkat T cell expression for CD8 (Column B), perforin (Column C), and granzyme (Column D). Tumor cells are green, and stain-positive Jurkat cells are yellow-orange. Column E provides representative demonstrations and quantifications of PTEN in tumor cells. Tumor cells are green, and PTEN is yellow-orange. Column F provides quantifications of the assessments in columns A–E. Examples include % TUNEL+ tumor cells (Column A), % Jurkat cells expressing CD8 (Column B), perforin (Column C), and granzyme (Column D), and % tumor cells expressing PTEN (Column E). These assessments were performed using a fluorescence microscope (20x magnification of the original). Ten randomly selected fields were evaluated in these quantifications. The data in Figure 8 demonstrate that treatment with a bispecific CHI3L1×PD1 antibody induces a synergistic CTL-mediated tumor cell death response and tumor cell PTEN expression.

[0243] In summary, the newly developed bispecific CHI3L1×PD1 antibody increased T cell and U87 cell binding and enhanced cytotoxic effects against U87 tumor cells compared to α-CHI3L1 and α-PD1 antibody therapy alone or in combination. These results suggest that the bispecific CHI3L1×PD1 antibody of the present invention is a more effective treatment for tumors in which CHI3L1 and its receptor, as well as PD1 and its ligands (PD-L1, PD-L2), are dysregulated than α-CHI3L1 or α-PD1 antibody alone or in combination.

[0244] The above specification is considered sufficient to enable those skilled in the art to carry out the present aspects and embodiments. The present aspects and embodiments are not limited in scope by the provided examples, for the examples are intended as single examples of one aspect, and other functionally equivalent embodiments are within the scope of the disclosure. In addition to those shown and described herein, various modifications will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objectives described herein are not necessarily limited to each embodiment. Those skilled in the art can recognize or confirm many equivalents to the particular embodiments described herein by mere everyday experimentation. Such equivalents are intended to be covered by the following claims.

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Claims

1. A bispecific antibody for detecting and neutralizing CHI3L1 and PD-1, comprising an antigen-binding moiety of an anti-human programmed death receptor 1 (PD-1) antibody and an antigen-binding moiety of an anti-human chitinase 3-like-1 (CHI3L1) antibody, The antigen-binding portion of the anti-human CHI3L1 antibody comprises the following complementarity-determining regions (CDRs): (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 4; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; (c) light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (d) heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; (e) heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2; and (f) heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

3. The antigen-binding portion of the anti-human PD-1 antibody contains the amino acid sequence of SEQ ID NO:

35. The bispecific antibody exhibits superior efficacy compared to anti-human PD-1 antibody and anti-human CHI3L1 antibody alone or in combination.

2. The bispecific antibody according to claim 1, comprising an anti-human PD-1 single-chain variable fragment (ScFv-PD1) conjugated to the backbone of an anti-human CHI3L1 antibody.

3. The bispecific antibody according to claim 1, comprising an anti-human CHI3L1 single-strand variable fragment (ScFv-CHI3L1) conjugated to the backbone of an anti-human PD-1 antibody.

4. The bispecific antibody according to claim 2, wherein the ScFv-PD1 is bound to the heavy chain of the CHI3L1 antibody (CHI3L1-HC-PD1).

5. The bispecific antibody according to claim 2, wherein the ScFv-PD1 is bound to the light chain of the CHI3L1 antibody (CHI3L1-LC-PD1).

6. The bispecific antibody according to claim 4, wherein the heavy chain of the CHI3L1 antibody has the amino acid sequence of SEQ ID NO:

13.

7. The bispecific antibody according to claim 5, wherein the light chain of the CHI3L1 antibody has the amino acid sequence of SEQ ID NO:

14.

8. A bispecific antibody according to any one of claims 1 to 7, which increases the attachment of Jurkat T cells to U87 cells.

9. The bispecific antibody according to claim 8, which increases the ability of Jurkat T cells to induce cytotoxic / apoptotic responses in U87 cells.

10. The bispecific antibody according to claim 8, which increases the accumulation of granzyme and perforin in Jurkat T cells co-cultured with U87 cells.

11. The bispecific antibody according to claim 8, which enhances the ability of Jurkat T cells to induce lactate dehydrogenase (LDH) release and cytotoxic responses in U87 cells.

12. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 11, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, further comprising a chemotherapeutic agent.

14. A pharmaceutical composition according to claim 12 or 13 for the treatment of cancer.

15. A cancer treatment agent comprising a bispecific antibody according to any one of claims 1 to 11.

16. The cancer treatment agent according to claim 15, wherein the cancer is a malignant cancer.

17. The cancer treatment agent according to claim 15, wherein the cancer is a primary cancer or a metastatic cancer.

18. The cancer treatment agent according to claim 15, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, gliablastoma, melanoma, malignant melanoma, and lung cancer.

19. A cancer treatment agent according to any one of claims 15 to 18, administered to a subject determined to have elevated levels of CHI3L1.

20. The cancer treatment agent according to claim 19, wherein the level of CHI3L1 is circulating CHI3L1.

21. A cancer treatment agent according to any one of claims 15 to 20, wherein the cancer expresses PD-L1.

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