Bispecific antibody against CHI3L1 and PD1 with enhanced T cell-mediated cytotoxicity against tumor cells

Abstract

Described herein are bispecific antibodies that simultaneously target CHI3L1 and the immune checkpoint molecule PD-1. These antibodies exhibit enhanced synergistic cytotoxic effects compared to the effects of the individual CHI3L1 and PD-1 antibodies (alone or in combination). Also provided are methods of treating cancer by administering the bispecific antibodies described herein.

Description

Field of the Invention

[0001] Embodiments of the invention relate to bispecific antibodies that simultaneously target CHI3L1 and the immune checkpoint molecule PD-1. These bispecific antibodies exhibit enhanced synergistic cytotoxic effects that are stronger than those of the CHI3L1 and PD-1 antibodies alone (either alone or in combination).

[0002] Statement Regarding Federally Sponsored Research or Development

[0003] This invention was made with government support under NIH CADET grant UH2 HL123876 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the Invention

[0005] Currently, immunotherapies targeting a single immune checkpoint inhibitor (ICPI) molecule are being effectively applied to patients with various malignancies, including lung cancer and glioblastoma. Examples include antibodies directed against portions such as programmed death receptor 1 (PD-1). However, only some patients respond to these treatments. In addition, the observed remissions are generally not durable. Therefore, worldwide efforts are intensifying to develop antibodies targeting immune checkpoint molecules, including PD-1, to enhance the effectiveness of ICPI immunotherapy.

[0006] Accordingly, there is a need for more effective immunotherapies against a single immune checkpoint inhibitor molecule, such as PD-1. Summary of the Invention

[0008] Previous studies have demonstrated that chitinase 3-like protein 1 (CHI3L1) plays a key role in the pathogenesis of various cancers. In addition, we have previously shown that the combination of anti-CHI3L1 antibody and anti-PD-1 antibody provides a synergistic effect in cancer treatment. Based on these findings, we hypothesized that simultaneously targeting CHI3L1 and PD-1 might have additional synergistic and / or additive anti-tumor effects. To address these possibilities, bispecific antibodies that react with both CHI3L1 and PD-1 were developed.

[0009] Embodiments of the invention provide humanized bispecific antibodies that simultaneously recognize and neutralize CHI3L1 and the immune checkpoint inhibitor PD-1. The bispecific antibodies comprise an antigen-binding portion of an anti-human PD-1 antibody and an antigen-binding portion of an anti-human CHI3L1 antibody.

[0010] In some embodiments, the bispecific antibody comprises an anti-human PD-1 single-chain variable fragment (ScFv-PD1) attached to an anti-human CHI3L1 antibody backbone. The ScFv-PD1 can be attached to the CHI3L1 antibody heavy chain (CHI3L1-HC-PD1) or the CHI3L1 antibody light chain (CHI3L1-LC-PD1).

[0011] In alternative embodiments, the bispecific antibody comprises an anti-human CHI3L1 single-chain variable fragment (ScFv-CHI3L1) attached to an anti-human PD-1 antibody backbone. The ScFv-CHI3L1 can be attached to the PD-1 antibody heavy chain (PD-1-HC-CHI3L1) or the PD-1 antibody light chain (PD-1-LC-CHI3L1).

[0012] In one embodiment, the antigen-binding portion of the anti-human CHI3L1 antibody comprises the following complementarity-determining regions (CDRs): (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4; (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2; and (f) a 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 comprises 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 comprises a light chain sequence having the amino acid sequence of SEQ ID NO: 14.

[0013] In one embodiment, the antigen-binding portion of the anti-human PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 35.

[0014] As described herein, the bispecific antibodies of the present invention have significant anti-tumor effects. Compared to the effects of the CHI3L1 and PD-1 antibodies alone (alone or in combination), these bispecific antibodies exhibit enhanced synergistic cytotoxic effects. The bispecific antibodies of the present invention (i) enhance the attachment of Jurkat T cells to U87 cells; (ii) enhance the ability of Jurkat T cells to induce a cytotoxic / apoptotic response in U87 cells; (iii) enhance the accumulation of granzyme and perforin in Jurkat T cells co-cultured with U87 cells; and / or (iv) enhance the ability of Jurkat T cells to induce a cytotoxic response and lactate dehydrogenase (LDH) release in U87 cells.

[0015] Embodiments of the present invention also provide a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises a chemotherapeutic agent.

[0016] Embodiments of the present invention also provide a method of 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 or metastatic cancer. In one embodiment, the cancer is one of the following: prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignant melanoma or lung cancer. In one embodiment, the subject is a subject determined to have an elevated CHI3L1 level. In one embodiment, the elevated CHI3L1 level is circulating CHI3L1. In one embodiment, the cancer expresses PD-L1.

[0017] The bispecific antibody of the present invention targets CHI3L1 and PD-1 and has an anti-tumor cytotoxic effect that exceeds the effects of anti-CHI3L1 and anti-PD-1 antibodies alone or in combination.

[0018] Other embodiments are also described and recited herein.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] For illustrative purposes, certain embodiments of the present invention are shown in the drawings described below. Like numbers in the drawings always represent like elements. However, it should be understood that the present invention is not limited to the exact arrangements, dimensions, and instruments shown. In the figures:

[0021] Figure 1 A schematic diagram of the CHI3L1xPD1 bispecific antibody structure is provided. The platform for generating the bispecific antibody is shown in Figure 1 A: CHI3L1-LCxScFv-HC-PD1-ScFv-LC-PD1 (CHI3L1-LC-PD1) and Figure 1 B: CHI3L1-HCxScFv-HC-PD1-ScFv-LC-PD1 (CHI3L1-HC-PD1).

[0022] Figure 2 The binding affinity of the CHI3L1xPD1 bispecific antibody is shown. The affinity of the CHI3L1-LC-PD1 antibody was evaluated by a competitive ELISA assay: Figure 2 A shows the evaluation against recombinant human (rh) CHI3L1; Figure 2 B shows the evaluation against rhPD1; and Figure 2Panel C shows the evaluation for the rhCHI3L1 and rhPD1 mixture. There was no difference in the binding affinity for rhCHI3L1 or rhPD1 between the CHI3L1-LC-PD1 and CHI3L1-HC-PD1 antibodies.

[0023] Figure 3 It shows that the bispecific CHI3L1xPD1 antibody significantly enhanced the attachment of Jurkat T cells to U87 glioblastoma cells in the co-culture system. Jurkat T cells were activated by anti-human CD3 / CD28 treatment (each at 5 μg / ml, incubated in 5% CO 2 and air at 37 °C for 2 hours) and then co-cultured with U87 glioblastoma cells. Cultures were performed with isotype control antibody and antibodies specific for PD-1, CHI3L1, CHI31L1+PD-1, and bispecific CHI3L1xPD-1. In Figure 3 Panel A, the CellBrite cell plasma membrane dye was used for fluorescent labeling of U87 (red) and Jurkat T cells (green). Figure 3 Panel B shows the phase contrast images, which were captured after incubation with IgG control and the indicated antibodies (5 mg / ml each) for 6 hours. Isotype, IgG control antibody; PD1, α-human PD1 antibody; CHI3L1, α-human CHI3L1 antibody; CHI3L1+PD1, α-CHI3L1 antibody plus α-PD1 antibody together; CHI3L1xPD1, bispecific CHI3L1-PD1 antibody. Figure 3 Panel C shows the number of Jurkat T cells attached to each U87 cell evaluated by counting under a fluorescence microscope (original magnification 20×; this evaluation included 10 randomly selected areas). Values are mean ± SEM. By t-test, *p < 0.05, **p < 0.01.

[0024] Figure 4 It shows that treatment with the CHI3L1xPD1 bispecific antibody enhanced the death response of U87 glioblastoma cells in the U87-Jurkat T cell co-culture. Jurkat T cells were activated by anti-CD3 / CD28 treatment (each at 5 μg / ml, incubated in 5% CO 2 and air at 37 °C for 2 hours) and then co-cultured with U87 glioblastoma cells. Cultures were performed with isotype control antibody and antibodies specific for PD-1, CHI3L1, CHI3lL1+PD-1, and bispecific CHI3L1xPD-1. In Figure 4 Panels A-B, the CellBrite cell plasma membrane dye was used for fluorescent labeling of live cells (green), and propidium iodide staining was used for dead cells (red). After incubation for 6 hours, with vehicle only ( Figure 4A) and IgG2b isotype control or indicated antibodies (5 mg / ml, each)( Figure 4 B) Treat the cells and capture fluorescence images. In Figure 4 C, TUNEL staining and images were captured under a bright-field microscope. Figure 4 D shows the quantification of TUNEL-positive apoptotic U87 cells. TUNEL-positive apoptotic cells were counted under an optical microscope (original magnification 20x) and expressed as a percentage of the total cells evaluated (10 microscopic fields were randomly selected for this evaluation). Values are mean ± SEM. By t-test, *p < 0.05, **p < 0.01.

[0025] Figure 5 It shows that treatment with the bispecific CHI3L1xPD1 antibody enhanced the accumulation of granzyme in Jurkat T cells co-cultured with U87 glioblastoma cells. Jurkat T cells were activated by treatment with anti-human CD3 / CD28 (5 μg / ml each, in 5% CO 2 and air and incubated at 37 °C for 2 hours) and then co-cultured with U87 glioblastoma cells. Cultures were performed with isotype control antibodies and antibodies specific for PD-1, CHI3L1, CHI31L1+PD-1, and bispecific CHI3L1xPD-1. Fluorescence images were captured after incubation with IgG2b isotype control and indicated antibodies (5 mg / ml each) for 6 hours ( Figure 5 A). Double immunohistochemical staining of the cells was performed with α-granzyme and α-phalloidin antibodies. Figure 5 B shows the quantification of granzyme+ cells. The number of granzyme+ cells was counted under a fluorescence microscope (original magnification 20x; 10 randomly selected areas were included in this evaluation). Values are mean ± SEM. By t-test, *p < 0.05.

[0026] Figure 6 It shows that treatment with the CHI3L1xPD1 bispecific antibody enhanced the accumulation of perforin in Jurkat T cells co-cultured with U87 glioblastoma cells. Jurkat T cells were activated by treatment with anti-human α-CD3 / α-CD28 (5 μg / ml each, in 5% CO 2 (5%) and air and incubated at 37 °C for 2 hours). Cultures were performed with isotype control antibodies and antibodies specific for PD-1, CHI3L1, CHI31L1+PD-1, and bispecific CHI3L1xPD-1. Images were captured after incubation with IgG2b isotype control and indicated antibodies (5 mg / ml each) for 6 hours ( Figure 6 A). Double immunohistochemical staining of the cells was performed with anti-perforin and anti-phalloidin antibodies. Figure 6B shows the quantification of perforin-positive (+) cells. The average number of perforin+ cells per microscopic field was calculated (original magnification 20x). Isotype, IgG2b control antibody; PD1, anti-human PD1 antibody; CHI3L1, anti-human CHI3L1 antibody; CHI3L1+PD1, anti-CHI3L1 antibody plus anti-PD1 antibody together; CHI3L1xPD1, bispecific CHI3L1-PD1 antibody. Original magnification 20x. Values are mean ± SEM. By t-test, *p < 0.05, **p < 0.01.

[0027] Figure 7 Bispecific CHI3L1xPD1 antibody treatment was shown to enhance LDH release of U87 cells in Jurkat-U87 co-cultures. Jurkat T cells were activated with anti-human CD3 / CD28 treatment (5 μg / ml each, incubated at 37 °C in 5% CO 2 and air for 2 hours). Cultures were performed with isotype control antibody and antibodies specific for PD-1, CHI3L1, CHI3lL1+PD-1, and bispecific CHI3L1xPD-1. After incubation with IgG control and indicated antibodies (5 mg / ml each) for 6 hours, LDH activity was measured by a kit (Pierce LDH cytotoxicity assay kit). ns, not significant, by t-test, *p < 0.05, **p < 0.01, ***p < 0.001. The LDH released by U87 cells in co-culture was compared with the levels of total LDH in U87 alone (-ve control), LDH released by U87 treated with lysis buffer (+ve control), and LDH released by Jurkat cells treated with lysis buffer (Jurkat).

[0028] Figure 8Shown is that bispecific CHI3L1xPD1 antibody treatment induces 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 In Situ Cell Death Detection Kit-Fluorescein-dUTP. TUNEL(+) cells are stained 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. Shown are the percentages of 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 evaluations were done using a fluorescence microscope (original magnification 20x). In these quantifications, 10 randomly selected fields of view were evaluated. Values in Figure F are the mean + SEM of the 4 evaluations described above. **P < 0.01. ***P < 0.001. Scale bar = 10 μm and applies to all subpanels of A-E. Detailed Description

[0029] It should be understood that, to provide a substantial understanding of the present invention, certain aspects, modes, embodiments, variations, and features of the present invention will be described in detail to varying degrees hereinafter.

[0030] Definitions

[0031] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated, or implied from the context, the following terms and phrases have the meanings provided below. The definitions are provided to assist in describing specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If a term has an obvious difference in meaning between the art and the definition provided herein, the definition provided in the specification shall prevail.

[0032] The singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0033] As used herein, the term "or" means "and / or". The term "and / or" used in a phrase such as "A and / or B" is intended to include A and B; A or B; A alone; and B alone. Similarly, the term "and / or" used in a phrase such as "A, B, and / or C" is intended to cover 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.

[0034] The abbreviation "e.g." (exempli gratia in Latin) is used herein to denote non-limiting examples. Thus, the abbreviation "e.g." is synonymous with "for example".

[0035] As used herein, the terms "about" or "approximately" with respect to a numerical value or parameter are generally considered to include numbers that fall within 5%, 10%, 15%, or 20% in either direction (greater than or less than) of a given number, unless otherwise stated or apparent from the context (except where the number is less than 0% of the possible value or more than 100% of the possible value). As used herein, "about" or "approximately" in reference to a value or parameter includes (and discloses) embodiments involving that value or parameter. For example, a description of "about X" includes a description of "X".

[0036] As used herein, the term "comprising" means that other elements may also be present in addition to the elements defined, and the use of "comprising" indicates inclusion rather than limitation.

[0037] The term "consisting of" refers to the compositions, methods, and their respective components described herein, which exclude any element not recited in the description of that embodiment.

[0038] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. This term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0039] The terms "statistically significant" or "significant" refer to statistical significance, typically a difference of two standard deviations (2SD) or greater.

[0040] As used herein, the phrases "therapeutically effective amount," "effective amount," or "effective dose" refer to an amount that provides a therapeutic or aesthetic benefit in the treatment, prevention, or management of a tumor or malignancy, e.g., an amount that results in a statistically significant reduction of at least one symptom, sign, or marker of the tumor or malignancy. It is understood that there will be many methods known in the art for determining the effective amount for a particular application. For example, pharmacological methods for determining dosage can be applied to the context of treatment. In the context of a therapeutic or prophylactic application, the amount of the composition administered to a subject will depend on the type and severity of the disease and the characteristics of the individual, such as general health, age, sex, weight, and tolerance to the drug. It also depends on the extent, severity, and type of the disease. A person skilled in the art will be able to determine the appropriate dosage based on these and other factors. The composition can also be administered in combination with one or more other therapeutic compounds.

[0041] As used herein, the terms "treat," "treatment," "manage," or "ameliorate" when used in reference to a disease, disorder, or medical condition refer to a therapeutic management of the condition, wherein the aim is to reverse, alleviate, improve, inhibit, slow down, or stop the progression or severity of the symptom or condition. The term "treatment" includes reducing or alleviating at least one adverse reaction or symptom of the condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the condition is reduced or stopped. That is, "treatment" includes not only an improvement in symptoms or markers, but also stopping or at least slowing the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, diminishment of the degree of a defect, a stable (i.e., non-worsening) state of a tumor or malignancy, delay or slowing of tumor growth and / or metastasis, and an increase in lifespan compared to that expected in the absence of treatment.

[0042] As used herein, the term "administer" refers to placing a bispecific antibody as disclosed herein into a subject by a method or route that results in at least partial delivery of the agent at the desired site. A pharmaceutical composition comprising a compound disclosed herein can be administered by any suitable route that produces an effective treatment in a subject.

[0043] As used herein, the term "long-term" administration refers to a period of time during which a therapeutic agent or drug is administered for at least 12 weeks. This includes the situation where the therapeutic agent or drug is administered such that it is effective over a period of at least 12 weeks, and does not necessarily mean that the administration itself lasts for 12 weeks, e.g., if a sustained-release composition or a long-acting therapeutic agent or drug is used. Thus, the subject receives treatment for at least 12 weeks. In many cases, long-term administration lasts for at least 4, 5, 6, 7, 8, 9 months or longer, or for at least 1, 2, 3, 5, 7, or 10 years or longer.

[0044] The administration of the compositions contemplated herein can be effected in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. In a preferred embodiment, the composition is administered parenterally. As used herein, the terms "parenteral administration" and "administered parenterally" refer to a form of administration other than enteral and topical administration, usually by injection, including but not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intratumoral, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0045] As used herein, the term "cancer" generally refers to a class of diseases or disorders in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic systems. There are several major types of cancer. Carcinoma (malignant epithelial tumor) is cancer that originates in the skin or tissues that line or cover internal organs. Sarcoma is cancer that originates in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that begins in hematopoietic tissue (such as the bone marrow) and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma originate in cells of the immune system. Central nervous system cancers originate in the tissues of the brain and spinal cord.

[0046] In some embodiments in any aspect, the cancer is a primary cancer. In some embodiments in any aspect, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibits one or more of uncontrolled growth (i.e., division outside the normal range), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body through the lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a "metastatic tumor" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0047] 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 generally do not invade or metastasize.

[0048] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a mass or lesion formed by abnormal cell growth, which can be benign, pre-cancerous, or malignant. Most cancer cells form tumors, but some (such as leukemia) do not necessarily form tumors. For those cells that form tumors, the terms cancer (cell) and tumor (cell) can be used interchangeably.

[0049] An object with cancer or a tumor is an object in which there are objectively measurable cancer cells present in the object's body. This definition includes malignant, actively proliferating cancers, as well as latent dormant tumors or micrometastases. Cancers that migrate from their original location and implant in other vital organs will ultimately cause the death of the object through the deterioration of the function of the affected organs. Hematopoietic cancers (such as leukemia) are able to outcompete the object's normal hematopoietic compartment, leading to hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), and ultimately death.

[0050] Examples of cancers include, but are not limited to: malignant epithelial tumors, lymphomas, germ cell tumors, sarcomas, leukemias, basal cell carcinomas, cholangiocarcinomas; bladder cancers; bone cancers; brain and central nervous system cancers; breast cancers; peritoneal cancers; cervical cancers; choriocarcinomas; colorectal cancers; connective tissue cancers; digestive system cancers; endometrial cancers; esophageal cancers; eye cancers; head and neck cancers; gastric cancers (including gastrointestinal cancers); glioblastoma multiforme (GBM); liver cancers; hepatocellular carcinomas; intraepithelial neoplasms; kidney cancers; laryngeal cancers; leukemias; liver cancers; lung cancers (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma; melanomas; myelomas; neuroblastomas; oral cancers (e.g., lip, tongue, mouth, and pharynx); ovarian cancers; pancreatic cancers; prostate cancers; retinoblastomas; rhabdomyosarcomas; rectal cancers; respiratory system cancers; salivary gland cancers; sarcomas; skin cancers; squamous cell carcinomas; gastric cancers; testicular cancers; thyroid cancers; uterine or endometrial cancers; urinary system cancers; vulvar cancers; and other carcinomas and sarcomas; and B-cell lymphomas (including low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (e.g., edema associated with brain tumors), and Meigs syndrome.

[0051] "Cancer cells" are cancer cells, precancerous cells, or transformed cells in vivo, in vitro, or in tissue culture that have undergone spontaneous or induced phenotypic changes, not necessarily involving the uptake of new genetic material. Although transformation can be caused by the infection of transforming viruses and the incorporation of new genomic nucleic acids, or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to carcinogens, resulting in mutations in endogenous genes. Transformation / cancer involves, for example, morphological changes, cell immortalization, abnormal growth control, focus formation, anchorage independence, malignancy, loss of contact inhibition and growth density limitation, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in a suitable animal host (such as nude mice).

[0052] The subject can be a subject who has been previously diagnosed with or identified as having a condition (e.g., cancer) that requires treatment or one or more complications associated with such a condition, and (optionally but not necessarily) has undergone treatment for a condition or one or more complications associated with that condition. Alternatively, the subject can be a subject who has not been previously diagnosed with a condition that requires treatment or one or more complications associated with such a condition. For example, the subject can be a subject 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 risk factors. A "subject in need of treatment" for a particular condition can be a subject who has the condition, has been diagnosed with the condition, or is at risk of developing the condition. As further explained herein, in some embodiments, the subject is a subject determined to have elevated CHI3L1 levels. In some embodiments, CHI3L1 is circulating CHI3L1. In some embodiments, the subject has a cancer that expresses PD-L1.

[0053] The terms "reduce", "decrease", "lower", or "inhibit" are each used herein to denote a statistically significant amount of reduction. In some embodiments, "decrease", "lower", or "reduce" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., absence of a given treatment or agent), and can include, for example, a reduction of 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, "lower" or "inhibit" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. For an individual without a given condition, a decrease can preferably be reduced to an acceptable level within the normal range.

[0054] The terms "increased", "increase", "enhanced", or "activated" are each used herein to denote a statistically significant increase in amount. In some embodiments, the terms "increased", "increase", "enhanced", or "activated" can mean an increase of at least 10% compared to a reference level, such as 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, and including an increase of 100% or any increase between 10 - 100% compared to the reference level, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold compared to the reference level, or any increase between 2-fold and 10-fold or more. In the context of a biomarker or symptom, an "increase" is a statistically significant increase in that level.

[0055] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to denote a series of amino acid residues joined to one another by peptide bonds between the α-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, glycosylated, glycated, etc.) and amino acid analogs, regardless of size or function. While "protein" and "polypeptide" are generally used to refer to relatively large polypeptides, and "peptide" is generally used to refer to small polypeptides, there is overlap in the use of these terms in the art. When referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0056] In the various embodiments described herein, variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any particular polypeptide described are contemplated and included. With respect to amino acid sequences, one of ordinary skill in the art will recognize that some individual substitutions, deletions, or additions in a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small number of amino acids in the coding sequence are "conservatively modified variants", where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are complementary to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0057] In some embodiments, the polypeptides (or nucleic acids encoding such polypeptides) described herein can be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of the wild-type reference polypeptide as determined by the assays described herein below. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0058] In some embodiments, the polypeptides described herein can be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. For example, conservatively substituted variants can be obtained by mutation of the native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to the native or reference polypeptide but has an amino acid sequence that is different from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequences encoding variant polypeptides include sequences that contain one or more nucleotide additions, deletions, or substitutions as compared to the native or reference DNA sequence, but that encode a variant protein or a fragment thereof that retains activity. Various PCR-based site-directed mutagenesis methods are known in the art and can be applied by one of ordinary skill in the art.

[0059] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, that includes units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be single-stranded or double-stranded. Single-stranded nucleic acids can be one strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that does not originate from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0060] In some embodiments in any aspect, the polypeptides, nucleic acids, or cells described herein can be engineered. As used herein, "engineered" refers to an aspect that has been manually manipulated. For example, a polypeptide is considered to be "engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manually manipulated to be different from its naturally occurring aspect. As is done and known by those skilled in the art, the progeny of engineered cells are generally still referred to as being "engineered", even if the manipulation was performed on a previous entity.

[0061] In some embodiments, the vector includes a nucleic acid encoding a polypeptide (such as an antibody or antibody reagent) described herein. In some aspects described herein, the nucleic acid sequence encoding a given polypeptide or any module thereof described herein is operably linked to a vector. Vectors can include, but are not limited to: cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virus particles, and the like.

[0062] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence that is linked on the vector to a transcriptional regulatory sequence. The sequence to be expressed is often (but not necessarily) heterologous to the cell. The expression vector can contain other elements. For example, the expression vector can have a replication system such that it can be maintained in two organisms, e.g., for expression in human cells and for cloning and amplification in a prokaryotic host. The term "expression" refers to the cellular processes involved in the production of RNA and protein and, where applicable, the secretion of protein, including (where applicable) but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene, as well as polypeptides obtained by translation of the mRNA transcribed from the gene. The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene can include or exclude regions before and after the coding region, such as 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0063] As used herein, the term "isolated" or "partially purified", with respect to a nucleic acid or polypeptide, refers to a nucleic acid or polypeptide that has been separated from at least one other component (such as nucleic acids or polypeptides) that is present with the nucleic acid or polypeptide in a natural source, and / or that would be present with the nucleic acid or polypeptide when the nucleic acid or polypeptide is expressed by a cell, or in the case of a secreted polypeptide, is secreted. Chemically synthesized nucleic acids or polypeptides or nucleic acids or polypeptides synthesized using in vitro transcription / translation are considered "isolated". The term "purified" or "substantially purified" means that the isolated nucleic acid or polypeptide is at least 95% by weight of the principal nucleic acid or polypeptide, e.g., including at least 96%, at least 97%, at least 98%, at least 99% or more. In some embodiments, an antibody, an antigen-binding portion thereof, or a chimeric antigen receptor (CAR) as described herein is isolated. In some embodiments, an antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR as described herein is purified.

[0064] As used herein, "engineered / engineered modification" refers to aspects that have been artificially manipulated. For example, an antibody, an antibody reagent, an antigen-binding portion thereof, a CAR, or a bispecific antibody can be considered "engineered / engineered modification" when its sequence has been artificially manipulated to be different from its naturally occurring sequence. As is done and known by those skilled in the art, progeny and copies of engineered polynucleotides and / or polypeptides are generally still referred to as "engineered / engineered modification", even if the actual manipulation was performed on a previous entity.

[0065] As used herein, an "epitope" can be formed on a polypeptide by contiguous amino acids or by non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by contiguous amino acids generally remain upon exposure to denaturing solvents, while epitopes formed by tertiary folding generally are lost upon treatment with denaturing solvents. An epitope generally comprises at least 3, more typically at least 5, about 9, or about 8 - 10 amino acids presenting a unique spatial conformation. An "epitope" includes the structural units that are conventionally bound by an immunoglobulin VH / VL pair. An epitope defines the minimal binding site of an antibody and thus represents the specific target of the antibody. In the case of a single domain antibody, the epitope represents the structural unit that the variable domain binds independently. The terms "antigenic determinant" and "epitope" may also be used interchangeably herein. In certain embodiments, an epitope determinant includes a chemically active group of surface molecules (e.g., amino acids, sugar side chains, phosphoryl or sulfonyl groups), and in certain embodiments, an epitope determinant may have specific three-dimensional structural features and / or specific charge characteristics.

[0066] As used herein, the term "antibody" refers to an immunoglobulin molecule and the immunologically active portions of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds an antigen. The term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as to various forms including full-length antibodies and antigen-binding portions thereof, including, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab′, F(ab′)2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb), diabodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, bispecific antibodies, functionally active epitope-binding portions thereof, and / or bifunctional hybrid antibodies.

[0067] Each heavy chain comprises the heavy chain variable region (abbreviated herein as HCVR or VH) and the heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain comprises the light chain variable region (abbreviated herein as LCVR or VL) and the light chain constant region. The light chain constant region is composed of the CL domain. The VH and VL domains can be further divided into hypervariable regions called complementarity determining regions (CDRs) and intervening conserved regions called framework regions (FRs). Thus, each VH and VL region is composed of three CDRs and four FRs, arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those of ordinary skill in the art.

[0068] As used herein, the term "CDR" refers to the complementarity determining regions within the variable sequences of an antibody. Each of the variable regions of the heavy and light chains has three CDRs, which are designated CDR1, CDR2, and CDR3 of the variable region. The exact boundaries of these CDRs are defined differently according to 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 that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described in Padlan et al. (1995), MacCallum et al. (1996), and Chothia et al. (1987) and (1989). There are other CDR boundary definitions that may not strictly follow one of the above systems, but still overlap with the Kabat CDRs, and although according to prediction or experimental results, their specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding, they may be shortened or lengthened. The methods used herein can utilize CDRs defined according to any of these systems, although the preferred embodiments use Kabat-defined CDRs.

[0069] The term "antigen-binding portion" of an antibody refers to one or more portions of an antibody as used herein, which also have the binding affinity as defined above herein. Portions of a full antibody have been shown to be capable of performing the antigen-binding function of the antibody. According to the term "antigen-binding portion" of an antibody, examples of the binding portion include (i) Fab portion, i.e., a monovalent portion, including VL, VH, CL, and CH1 domains; (ii) F(ab′)2 portion, i.e., a divalent portion, including two Fab portions connected to each other by a disulfide bridge in the hinge region; (iii) Fd portion, including VH and CH1 domains; (iv) Fv portion, including the VL and VH domains of a single arm of an antibody; and (v) dAb portion, consisting of a VH domain or consisting of VH, CH1, CH2, DH3, or VH, CH2, CH3 (dAb, or single-domain antibody, only containing the VL domain that has also been shown to specifically bind to the target epitope). Although the two domains of the Fv portion (i.e., VL and VH) are encoded by different genes, they can be linked to each other using a synthetic linker (e.g., a poly-G4S amino acid sequence ('G4S' as disclosed in SEQ ID NO: 29 of U.S. Patent No. 10,253,111) and recombinant methods, such that it is possible to prepare them as a single protein chain, in which the VL and VH domains are combined to form a monovalent molecule (referred to as single-chain Fv (ScFv)). The term "antigen-binding portion" of an antibody also means an antibody containing such a single-chain antibody. Other forms of single-chain antibodies such as "diabodies" are also included herein. Diabodies are divalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to combine the two domains on the same chain, thus forcing the domains to pair with complementary domains on different chains to form two antigen-binding sites. Immunoglobulin constant regions refer to the constant regions of heavy or light chains. The amino acid sequences of human IgG heavy and light chain constant regions are known in the art.

[0070] As used herein, the term "antibody reagent" refers to a polypeptide containing at least one immunoglobulin variable domain or an immunoglobulin variable domain sequence that specifically binds to a given antigen. An antibody reagent can contain an antibody or a polypeptide containing the antigen-binding domain of an antibody. In some embodiments, an antibody reagent can contain a monoclonal antibody or a polypeptide containing the antigen-binding domain of a monoclonal antibody. For example, an antibody can contain a heavy (H) chain variable region (abbreviated as VH herein) and a light (L) chain variable region (abbreviated as VL herein).

[0071] In another example, an antibody contains two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" includes antigen-binding fragments of an antibody (e.g., single-chain antibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and single-domain antibody (dAb) fragments) and full antibodies.

[0072] Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes and combinations). Antibodies can be from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates), as well as primatized antibodies. Antibodies can also include midibodies, humanized antibodies, chimeric antibodies, and the like.

[0073] In addition, an antibody, its antigen-binding portion, or a CAR as described herein can be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Associated with such immunoadhesion molecules is the use of a streptavidin core region to prepare tetrameric scFv molecules, and the use of cysteine residues, tag peptides, and C-terminal polyhistidinyl residues, such as hexahistidinyl tags (‘hexahistidinyl tag’ as disclosed in SEQ ID NO: 30 of U.S. Patent No. 10,253,111), to produce divalent and biotinylated scFv molecules.

[0074] In some embodiments, an antibody, antibody reagent, its antigen-binding portion, or a CAR as described herein can be an immunoglobulin molecule, monoclonal antibody, chimeric antibody, CDR-grafted antibody, humanized antibody, Fab, Fab′, F(ab′)2, Fv, disulfide-linked Fv, scFv, single-domain antibody, diabody, multispecific antibody, dual-specific antibody, anti-idiotypic antibody, bispecific antibody, and its functionally activated epitope-binding portion.

[0075] In some embodiments, the antibody or its antigen-binding portion is a fully human antibody. In some embodiments, the antibody or its antigen-binding portion is a humanized antibody or antibody reagent. In some embodiments, the antibody or its antigen-binding portion is 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, its antigen-binding portion is a recombinant polypeptide. In some embodiments, the CAR comprises an extracellular domain that binds CHI3L1, wherein the extracellular domain comprises a humanized or chimeric antibody or its antigen-binding portion.

[0076] The term "human antibody" refers to an antibody whose variable and constant regions correspond to or are derived from immunoglobulin sequences of the human germline, as described, for example, by Kabat et al. (1991). However, a human antibody may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or in vitro site-specific mutagenesis or by in vivo somatic mutation), such as in the CDRs, specifically in CDR3. A recombinant human antibody as described herein has a variable region and may also contain a constant region derived from human germline immunoglobulin sequences. See, Kabat, et al. (1991). However, according to specific embodiments, such a recombinant human antibody undergoes in vitro mutagenesis (or, if using a transgenic animal with human Ig sequences, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibody, while related to or derived from human germline VH and VL sequences, may not be sequences that naturally occur in the in vivo human antibody repertoire. According to specific embodiments, such a recombinant antibody is the result of either selective mutagenesis or back-mutation or both. Preferably, the mutagenesis results in a stronger affinity for the target and / or a weaker affinity for non-target structures than the parental antibody. One of ordinary skill in the art can practice the generation of humanized antibodies from the sequences and information provided herein without undue experimentation. In one method, there are four general steps for humanizing a monoclonal antibody, see, for example, U.S. Patent Nos. 5,585,089; 6,835,823; 6,824,989. They are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains; (2) designing the humanized antibody, i.e., determining which antibody framework regions to use in the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody.

[0077] In some embodiments, the bispecific antibodies, antibody reagents, antigen-binding portions thereof, and / or CARs as described herein may be variants of the sequences described herein, e.g., conservative substitution variants of the antibody polypeptides. In some embodiments, the variants are conservatively modified variants. For example, conservative substitution variants can be obtained by mutation of the native nucleotide sequence. As used herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequences encoding variant polypeptides include sequences that contain one or more nucleotide additions, deletions, or substitutions compared to the native or reference DNA sequence, but that encode a variant protein or portion thereof that retains activity, e.g., antigen-specific binding activity for a relevant target polypeptide (e.g., CHI3L1 or PD-1). Various PCR-based site-specific mutagenesis methods are also known in the art and can be applied by one of ordinary skill.

[0078] The CDR regions of conventional humanized antibodies and human antibody variants are substantially the same, and more typically, are the same as the CDR regions corresponding to human antibodies of murine or its derivative origin. In some embodiments, it is possible to make one or more conservative amino acid substitutions to the CDR residues without significantly affecting the binding affinity of the resulting humanized immunoglobulin or human antibody variant. In some embodiments, substitutions in the CDR regions can enhance the binding affinity.

[0079] The term "chimeric antibody" refers to an antibody that contains variable region sequences of the heavy and light chains from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions. A humanized antibody has variable region framework residues substantially from a human antibody (referred to as the recipient antibody) and complementarity-determining regions substantially from a non-human antibody (e.g., a murine antibody) (referred to as the donor immunoglobulin). If there are one or more constant regions, they are also substantially or entirely from human immunoglobulins. The human variable domains are typically selected from human antibodies that have a high degree of sequence identity with the (murine) variable region domain from which the CDRs are derived. The heavy and light chain variable region framework residues can be substantially similar to regions of the same or different human antibody sequences. The human antibody sequences can be naturally occurring human antibody sequences or can be consensus sequences of several human antibodies.

[0080] In addition, techniques developed for the production of "chimeric antibodies" can also be used, i.e., splicing together the genes of antibody molecules from mice or other species with appropriate antigen specificities with the genes of human antibody molecules with appropriate biological activities. The variable segments of chimeric antibodies are typically linked to at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. The human constant region DNA sequences can be isolated from a variety of human cells according to known methods, such as immortalized B cells. An antibody can contain light and heavy chain variable regions. The heavy chain constant region can contain CH1, hinge, CH2, CH3, and (sometimes) CH4 regions. For therapeutic purposes, the CH2 domain can be deleted or omitted.

[0081] In addition, as described herein, recombinant humanized antibodies can be further optimized to reduce potential immunogenicity while maintaining functional activity for human therapy. In this regard, functional activity means a polypeptide capable of exhibiting one or more functional activities known to be associated with the recombinant antibodies, antigen-binding portions thereof, or CARs described herein. Such functional activities include binding to cancer cells and / or anti-cancer activity. In addition, a polypeptide having functional activity means that the polypeptide exhibits activity similar (but not necessarily identical) to that of the reference antibody, antigen-binding portion thereof, or CAR described herein, including the mature form, as measured in a particular assay (e.g., a biological assay), with or without dose-dependence. Where dose-dependence does exist, it need not be the same as that of the reference antibody, antigen-binding portion thereof, or CAR, but rather is substantially similar to the dose-dependence in a given activity compared to the reference antibody, antigen-binding portion thereof, or CAR described herein (i.e., the candidate polypeptide will exhibit stronger activity, or activity no more than about 25-fold, about 10-fold, or about 3-fold lower relative to the antibodies, antigen-binding portions, and / or CARs described herein).

[0082] In some embodiments, the antibody reagents (e.g., antibodies or CARs) described herein are non-naturally occurring biomolecules. For example, murine antibodies generated against human-derived antigens would not exist in nature without human intervention and manipulation, such as manufacturing steps performed by a human. Chimeric antibodies are also not naturally occurring biomolecules; for example, they include sequences obtained from multiple species and assembled into a recombinant molecule. In certain specific embodiments, the human antibody reagents described herein are not naturally occurring biomolecules; for example, fully human antibodies against human antigens would be negatively selected in nature and do not exist non-naturally in the human body.

[0083] In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and / or CAR are isolated polypeptides. In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and / or CAR are purified polypeptides. In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and / or CAR are engineered polypeptides.

[0084] "Affinity" is a measure of the binding strength between an antigen-binding molecule (such as an antibody or antigen-binding portion thereof described herein) and a related antigen. Affinity is related to the avidity between the epitope and its antigen-binding site on the antigen-binding molecule, as well as the number of related binding sites present on the antigen-binding molecule. Generally, an antigen-binding protein (e.g., an antibody or portion of an antibody described herein) will bind to its cognate or specific antigen with a dissociation constant (K D of 10 -5 to 10 -12 moles per liter or less, such as 10 -7 to 10-12 moles per liter or less, or 10 -8 to 10 -12 moles per liter (i.e., the association constant (K A ) is 10 5 to 10 12 liters per mole or higher, e.g., 10 7 to 10 12 liters per mole or 10 8 to 10 12 liters per mole). Any K D value greater than 10 -4 moles per liter (or any K A value below 10 4 M -1 ) is generally considered to indicate non-specific binding. The K D of a biological interaction considered to be significant (e.g., specific) is typically between 10 -10 M (0.1 nM) and 10 -5 M (10000 nM). The stronger the interaction, the lower its K D . For example, the binding site of an antibody or a portion thereof described herein will bind to the desired antigen with an affinity lower than 500 nM, e.g., lower than 200 nM, or lower than 10 nM, e.g., lower than 500 pM. Specific binding of an antigen-binding protein to an antigen or an epitope can be determined by any suitable method known per se, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, as well as different variants thereof known per se in the art; and other techniques mentioned herein.

[0085] Thus, as used herein, "selectively binds" or "specifically binds" refers to a peptide (e.g., an antibody, a CAR, a bispecific antibody, or a portion thereof) described herein binding to a target, e.g., an antigen present on the cell surface of a cancer cell, with a KD of 10 -5 M (10000 nM) or lower, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12M or lower. Specific binding can be affected by, for example, the affinity and avidity of the polypeptide agent and the concentration of the polypeptide agent. A person of ordinary skill in the art can use any suitable method, such as titrating the polypeptide agent in a suitable cell binding assay, to determine the appropriate conditions for the polypeptide agent described herein to selectively bind to the target. A polypeptide that specifically binds to a target will not be displaced by non-analogous competitors. In some embodiments, an antibody, an antigen-binding portion thereof, a CAR, or a bispecific antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0086] In certain embodiments, a bispecific antibody, an antigen-binding portion thereof, or a CAR as described herein binds to CHI3L1 and PD-1, and its dissociation constant (K D ) is 10 -5 M (10000 nM) or lower, such as 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or lower. In some embodiments, a bispecific antibody, an antigen-binding portion thereof, or a CAR as described herein binds to CHI3L1 and PD-1, and its dissociation constant (K D ) is about 10 -5 M to 10 -6 M. In some embodiments, a bispecific antibody, an antigen-binding portion thereof, or a CAR as described herein binds to CHI3L1 and PD-1, and its dissociation constant (K D ) is about 10 -6 M to 10 -7 M. In some embodiments, a bispecific antibody, an antigen-binding portion thereof, or a CAR as described herein binds to CHI3L1 and PD-1, and its dissociation constant (K D ) is about 10 -7 M to 10 -8 M. In some embodiments, a bispecific antibody, an antigen-binding portion thereof, or a CAR as described herein binds to CHI3L1 and PD-1, and its dissociation constant (K D ) is about 10 -8 M to 10 -9 M. In some embodiments, a bispecific antibody, an antigen-binding portion thereof, or a CAR as described herein binds to CHI3L1 and PD-1, and its dissociation constant (K D ) is about 10 -9 M to 10 -10M. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs as described herein bind to CHI3L1 and PD-1, and their dissociation constant (K D ) is about 10 -10 M to 10 -11 M. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs as described herein bind to CHI3L1 and PD-1, and their dissociation constant (K D ) is about 10 -11 M to 10 -12 M. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs as described herein bind to CHI3L1 and PD-1, and their dissociation constant (K D ) is less than 10 -12 M.

[0087] Groupings of alternative elements or embodiments of the invention disclosed herein should not be construed as limitations. Each member of a group can be referred to and claimed individually with any other member of that group or in any combination with other elements herein. For convenience and / or patentability reasons, one or more members of a group may be included in (or deleted from) the group. When any such inclusion or deletion occurs, the specification is deemed to include the modified group herein, thereby satisfying the written description of all Markush groups used in the appended claims.

[0088] Unless otherwise noted, the scientific and technical terms used in this application have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein, and thus can be varied. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy (T HE M ERCK M ANUAL OF D IAGNOSIS AND T HERAPY ), 19th ed. (2011); Encyclopedia of Molecular Cell Biology and Molecular Medicine (T HE E NCYCLOPEDIA OF M OLECULAR C ELL B IOLOGY AND M OLECULAR M EDICINE ), (1999 - 2012); Molecular Biology and Biotechnology: A Comprehensive Desk Reference (M OLECULAR B IOLOGY AND B IOTECHNOLOGY : A C OMPREHENSIVE DESK R EFERENCE ),(1995); Immunology (I MMUNOLOGY )(2006); J ANEWAY 's Immunobiology (I MMUNOBIOLOGY )(2014); LEWIN's Genes XI (G ENES XI)(2014); Molecular Cloning: A Laboratory Manual (M OLECULAR C LONING : A L ABORATORY M ANUAL ), 4th ed. (2012); Current Protocols in Molecular Biology (B ASIC M ETHODS IN M OLECULAR B IOLOGY )(2012); Laboratory Methods in Enzymology: DNA (L ABORATORY M ETHODS IN E NZYMOLOGY : DNA)(2013); Current Protocols in Molecular Biology (C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY )(CPMB)(2014); Current Protocols in Protein Science C URRENT P ROTOCOLS IN P ROTEIN S CIENCE (CPPS)(2005); and Current Protocols in Immunology C URRENT P ROTOCOLS IN I MMUNOLOGY (CPI)(2003), the contents of which are hereby incorporated by reference in their entirety.

[0089] One of ordinary skill in the art can readily identify the chemotherapeutic agents used. See, e.g., The Physician's Cancer Chemotherapy Drug Manual (P HYSICIANS′ C ANCER C HEMOTHERAPY D RUG M ANUAL )(2014); Harrison's Principles of Internal Medicine (HARRI SON′S P RINCIPLES OF I NTERNAL M EDICINE ), 18th ed., Chapter 85 (2011); A BELOFFS Clinical Oncology (A BELOFFS C LINICAL O NCOLOGY ), 5th ed., Chapters 28-29 (2013); and The Cancer Chemotherapy Handbook (T HE C ANCER CHEMOTHERAPY H ANDBOOK ), 4th Edition (2003).

[0090] In some embodiments in any respect, the disclosure herein does not relate to processes of cloning humans, processes of modifying the genetic identity of the human germ line, uses of human embryos for industrial or commercial purposes, or processes of modifying the genetic identity of animals that may cause them to suffer pain and that do not provide any substantial medical benefit to humans or animals, and no animals are produced from such processes.

[0091] Other terms are defined herein within the description of the various aspects of the present invention.

[0092] The bispecific antibodies of the present invention

[0093] Due to impressive and durable clinical responses, immunotherapies using anti-programmed death-1 (PD-1) or anti-PD-1 ligand 1 (PD-L1) antibodies have been approved for the treatment of several cancers; however, overall, only a small fraction of patients currently benefit from single-agent PD-1 blockade therapy (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 immune modulators appear to be more active, but they increase significant toxicity (Wolchok et al., 2013; Larkin et al., 2015; Postow et al., 2015).

[0094] In our previous work, we demonstrated that inhibition of (a) CHI3L1 and / or CHI3L1 signal transduction and (b) at least one immune checkpoint protein, such as PD-1, provides synergy in the treatment of cancer (e.g., lung cancer). See, for example, U.S. Patent Publication No. 2019 / 0062457. Synergy was observed when anti-CHI3L1 antibody (FRG) was administered in combination with anti-PD-1 antibody, and the combination showed improved efficacy in reducing B16F10 metastases, thus indicating that the combination of CHI3L1 inhibition and checkpoint protein inhibition provides synergistic efficacy in cancer treatment. See Example 2 of U.S. Patent Publication No. 2019 / 0062457. It is hypothesized that bispecific antibodies that specifically bind CHI3L1 polypeptide and PD-1 polypeptide and simultaneously detect and neutralize CHI3L1 and the immune checkpoint inhibitor PD-1 can demonstrate improved synergistic efficacy in cancer treatment.

[0095] Disclosed herein are bispecific antibodies, antibody reagents, antigen-binding fragments thereof, or chimeric antigen receptors (CARs) that combine CHI3L1 polypeptide and PD-1 polypeptide and simultaneously detect and neutralize CHI3L1 and the immune checkpoint inhibitor PD-1. Such bispecific antibodies, their antigen-binding portions, etc., can permit, 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 cancer treatment.

[0096] Disclosed herein are methods and compositions related to bispecific anti-CHI3L1 and anti-PD-1 antibodies, antibody reagents, and antigen-binding fragments thereof that exhibit excellent properties such as high sensitivity, high specificity, high binding affinity, and neutralizing activity in vitro and ex vivo. Also provided are methods of treatment by administering the compounds described herein, such as treating cancer.

[0097] The bispecific antibodies of the invention comprise an antigen-binding portion of an anti-human PD-1 antibody and an antigen-binding portion of an anti-human CHI3L1 antibody. In some embodiments, the bispecific antibody comprises an anti-human PD-1 single-chain variable fragment (ScFv-PD1) attached to the anti-human CHI3L1 antibody backbone. In alternative embodiments, the bispecific antibody comprises an anti-human CHI3L1 single-chain variable fragment (ScFv-CHI3L1) attached to the anti-human PD-1 antibody backbone.

[0098] Those skilled in the art will recognize that some individual substitutions, deletions, or additions of nucleic acid, peptide, polypeptide, or protein sequences that alter a single amino acid or a small number of amino acids in the coding sequence are "conservative modified variants", wherein the alteration results in the amino acid being replaced by a chemically similar amino acid and retains the ability to specifically bind to a target antigen (e.g., CHI3L1 and PD-1). Such conservative modified variants are complementary to polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure and are not excluded therefrom.

[0099] Examples of substitution variants include conservative substitutions of amino acids, such as in the V H or V L domains without altering the sequence of the CDRs. Conservative substitutions not included in the sequence of the CDRs can be substitutions relative to the wild-type or naturally occurring sequence, such as the human or murine framework and / or the constant region of the antibody sequence.

[0100] A given amino acid can be replaced by a residue having similar physiochemical properties. For example, one aliphatic residue can be substituted for another (such as Ile, Val, Leu, or Ala for each other), or one polar residue can be substituted for another (such as between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions are well known, such as substitution of entire regions having similar hydrophobic properties. Polypeptides containing conservative amino acid substitutions can be tested in any of the assays described herein to confirm the desired activity, such as that the antigen-binding activity and specificity of the native or reference polypeptide are retained.

[0101] Amino acids can be grouped according to similarities in their side-chain properties (Biochemistry (B IOCHEMISTRY ) 2nd ed. (1975) pp. 73-75): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: 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 common side-chain characteristics: (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 would require replacement of a member of one group with a member of another group. Specific conservative substitutions include, for example; Ala becoming Gly or becoming Ser; Arg becoming Lys; Asn becoming Gln or becoming His; Asp becoming Glu; Cys becoming Ser; Gln becoming Asn; Glu becoming Asp; Gly becoming Ala or becoming Pro; His becoming Asn or becoming Gln; Ile becoming Leu or becoming Val; Leu becoming Ile or becoming Val; Lys becoming Arg, becoming Gln or becoming Glu; Met becoming Leu, becoming Tyr or becoming Ile; Phe becoming Met, becoming Leu or becoming Tyr; Ser becoming Thr; Thr becoming Ser; Trp becoming Tyr; Tyr becoming Trp; and / or Phe becoming Val, becoming Ile or becoming Leu.

[0102] Variant amino acid or DNA sequences are preferably 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 identical to a native or reference sequence. The degree of homology (percent identity) between the native and mutant sequences can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0103] Alterations of native amino acid sequences can be accomplished by any of a variety of techniques known to those of skill in the art. For example, mutations can be introduced at specific loci by synthesizing oligonucleotides containing the mutant sequence, which are flanked by restriction sites and capable of ligating to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-directed mutagenesis procedures can be used to provide nucleotide sequences having specific codon changes according to the desired substitution, deletion, or insertion.

[0104] Any cysteine residues that do not participate in maintaining the correct conformation of the polypeptide can also be substituted, usually with serine, to increase the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, one or more cysteine bonds can be added to the polypeptide to increase its stability or promote oligomerization.

[0105] In certain embodiments, where the antibody, antigen-binding portion thereof, or CAR as described herein comprises at least one CDR that is not identical to the sequences of the CHI3L1 and PD-1 CDRs provided herein, the amino acid sequence of the at least one CDR can be selected by methods well known to those of skill in the art. For example, Fujii, 2004, “Antibody affinity maturation by random mutagenesis” in Methods in Molecular Biology: Antibody Engineering 248:345-349 (incorporated herein by reference in its entirety), particularly in Figure 2 and 3.3 describes methods for generating libraries of any CDRs of interest. This allows one of ordinary skill in the art to identify alternative CDRs, including conservative substitution variants of the specific CDR sequences described herein, which, when present in the antibodies or antigen-binding portions thereof described herein, will result in an antigen or antigen-binding portion thereof that will bind to a cancer cell surface antigen. The methods described by Fujii et al. also allow one of ordinary skill in the art to screen light chain sequences that will exhibit the desired binding behavior when combined with known heavy chain fragments, and vice versa.

[0106] In some embodiments, the CAR comprises an extracellular domain comprising an anti-CHI3L1 antibody or antigen-binding portion thereof that binds to one or more epitopes of the CHI3L1 polypeptide; a transmembrane domain, one or more intracellular co-stimulatory signaling domains, and a primary signaling domain. Exemplary anti-CHI3L1 and anti-PD-1 antibodies and antigen-binding portions thereof, and exemplary epitopes are described elsewhere herein.

[0107] As used herein, "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that comprises 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. Using the antigen-binding properties of monoclonal antibodies, CARs are capable of redirecting the specificity and reactivity of T cells to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition enables CAR-expressing T cells to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor escape. In addition, when expressed in T cells, CARs advantageously do not dimerize with the endogenous T cell receptor (TCR) α and β chains. Most commonly, the extracellular binding domain of a CAR comprises a single-chain variable fragment (scFv) that is derived from the fusion of the variable heavy and light chain regions of a murine or humanized monoclonal antibody. Alternatively, an scFv derived from a Fab (rather than from an antibody, e.g., obtained from a Fab library) can be used, which in various embodiments is fused to a transmembrane domain and then to an intracellular signal transduction domain in various embodiments. "First-generation" CARs comprise CARs that provide only CD3ζ (CD3 zeta) signaling upon antigen binding, "second-generation" CARs include CARs that provide co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ), and "third-generation" CARs include CARs that provide multiple co-stimulation (e.g., CD28 and CD137) domains and activation domains (e.g., CD3ζ). In various embodiments, CARs are selected that have high affinity or avidity for the antigen. Further discussion of CARs 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 its entirety.

[0108] In some embodiments in any aspect, the CAR comprises an extracellular binding domain that comprises a humanized CHI3L1-specific or humanized PD-1-specific binding domain; a transmembrane domain; one or more intracellular co-stimulatory signal transduction domains; and a primary signal transduction 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 and provide a CAR that is capable of specifically binding to a target antigen of interest (e.g., CHI3L1 and PD-1). The binding domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0109] In some embodiments, the CARs contemplated herein can include linker residues between different domains, e.g., to add appropriate spacing and conformation for the molecule. In certain embodiments, the linker is a variable region joining sequence. A "variable region joining sequence" is an amino acid sequence that joins the VH and VL domains and provides a spacer function compatible with interaction with both sub-binding domains, such that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody containing the same light and heavy chain variable regions. The CARs contemplated herein can include one, two, three, four, or five or more linkers. In certain embodiments, the linker has a length of about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any amino acid length therebetween. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0110] In certain embodiments, the binding domain of the CAR is followed by one or more "spacer domains", which refers to regions that move the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is part of an immunoglobulin and includes, but is not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can include the naturally occurring immunoglobulin hinge region or an amino acid sequence of an altered immunoglobulin hinge region.

[0111] The binding domain of the CAR is typically followed by one or more "hinge domains" that function to position the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. The CAR typically includes one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can include the naturally occurring immunoglobulin hinge region or an amino acid sequence of an altered immunoglobulin hinge region. Exemplary hinge domains suitable for the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8α, CD4, CD28, and CD7, which can be the wild-type hinge regions from these molecules or can be altered. In another embodiment, the hinge domain comprises the CD8α hinge region.

[0112] "Transmembrane domain" is a part of the CAR that fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. The TM domain can be derived from natural, synthetic, semi-synthetic or recombinant sources. The TM domain can be derived from (i.e., contain at least one or more transmembrane regions of) the α, β or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD1.

[0113] In some embodiments, the CARs contemplated herein include an intracellular signaling domain. "Intracellular signaling domain" refers to the part of the CAR that is involved in transducing the information of effective CAR binding to a target antigen into the interior of an immune effector cell to trigger effector cell functions, such as activation, cytokine production, proliferation and cytotoxic activity, including releasing cytotoxic factors to the target cells bound by the CAR, or other cellular responses caused by antigen binding to the extracellular CAR domain. In some embodiments, the CARs contemplated herein include an intracellular signaling domain that includes one or more "costimulatory signaling domains" and "primary signaling domains".

[0114] The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary signaling domain acting in a stimulatory manner may contain signal transduction motifs, which are referred to as immunoreceptor tyrosine-based activation motifs or ITAMs. Exemplary instances of ITAMs of the primary signaling domains specifically used in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3g, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0115] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. Exemplary instances 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 comprises one or more costimulatory signaling domains selected from CD28, CD137, CD134, and the CD3ζ primary signaling domain.

[0116] In some embodiments, antibody-drug conjugates are provided. In certain embodiments, the antibody-drug conjugate comprises an antibody, antibody reagent, or antigen-binding portion thereof as described herein. The drug can be, for example, a chemotherapeutic molecule as described elsewhere herein. In some embodiments, the antibody-drug conjugate comprises a chemotherapeutic agent directly conjugated and / or bound to the antibody or antigen-binding portion thereof. In some embodiments, the binding can be non-covalent, e.g., by hydrogen bonding, electrostatic, or van der Waals interactions; however, the binding can also be covalent. "Conjugation" refers to the covalent linkage of at least two molecules. In some embodiments, the composition can be an antibody-drug conjugate.

[0117] In some embodiments, the antibody, antibody reagent, or antigen-binding portion thereof can be bound to and / or conjugated to multiple chemotherapeutic molecules. In some embodiments, the antibody-drug conjugate can be bound to and / or conjugated to multiple chemotherapeutic molecules. In some embodiments, the ratio of a given chemotherapeutic molecule to the antibody or antigen-binding portion thereof can be from about 1:1 to about 1,000:1, e.g., a single antibody reagent molecule can be linked to, conjugated to, etc., about 1 to about 1,000 individual chemotherapeutic molecules.

[0118] In some embodiments, the antibody or antigen-binding portion thereof and the chemotherapeutic agent can be present in a scaffold material. Scaffold materials suitable for therapeutic compositions are known in the art and can include, but are not limited to: nanoparticles; matrices; hydrogels; and biomaterials, biocompatible and / or biodegradable scaffold materials. As used herein, the term "nanoparticle" refers to approximately about 10 -9Particles in the range of one meter to one billionth to several billionths of a meter. The term "nanoparticle" includes nanospheres; nanorods; nanoshells; and nanoplates; these nanoparticles can be part of a nanonetwork.

[0119] The term "nanoparticle" also encompasses liposomes and lipid particles having nanoparticle size. As used herein, the term "matrix" refers to a three-dimensional structure that contains the components of the compositions described herein (e.g., an antibody or an antigen-binding portion thereof). Non-limiting examples of matrix structures include foams; hydrogels; electrospun fibers; gels; fiber pads; sponges; three-dimensional scaffolds; nonwoven pads; woven materials; braided materials; fiber bundles; and fibers and other material forms. See, e.g., Rockwood et al. (2011) and U.S. Patent Publication Nos. 2011 / 0167602; 2011 / 0009960; 2012 / 0296352; and U.S. Patent No. 8,172,901, each of which is incorporated herein by reference in its entirety. One of ordinary skill in the art can select the structure of the matrix based on the intended application of the composition. For example, an electrospun matrix can have a larger surface area than a foam.

[0120] In some embodiments, the scaffold is a hydrogel. As used herein, the term "hydrogel" refers to a three-dimensional polymeric structure that is insoluble in water but capable of absorbing and retaining a large amount of water to form a stable, generally soft and flexible structure. In some embodiments, water can penetrate between the polymer chains of the polymer network, subsequently causing swelling and the formation of a hydrogel. Generally, hydrogels are highly water-absorbent. Hydrogels have the properties required for many biomedical applications. For example, they can be made non-toxic and tissue-compatible, and they are highly permeable to water, ions, and small molecules. Hydrogels are highly water-absorbent (they can contain more than 99% water) and can be composed of natural (e.g., silk) or synthetic polymers (e.g., PEG).

[0121] As used herein, "biomaterial" refers to biocompatible and biodegradable materials. The term "biocompatible" as used herein refers to a substance that is non-toxic to cells. In some embodiments, a substance is considered "biocompatible" if adding the substance to cells in vitro results in less than or equal to about 20% cell death. In some embodiments, a substance is considered "biocompatible" if adding the substance to cells in vivo does not induce inflammation and / or other adverse reactions in vivo. As used herein, the term "biodegradable" refers to a substance that degrades under physiological conditions. In some embodiments, a biodegradable substance is a substance that is broken down by cellular mechanisms. In some embodiments, a biodegradable substance is a substance that is broken down by chemical processes.

[0122] As used herein, the terms "nucleic acid" or "nucleic acid sequence" refer to polymeric molecules comprising units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be single-stranded or double-stranded. Single-stranded nucleic acids can be one strand of a denatured double-stranded DNA. In some embodiments, the nucleic acid can be cDNA, e.g., a nucleic acid lacking introns.

[0123] 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: the preparation of early preparation variants or non-variant versions of antibodies by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis. Nucleic acid sequences encoding at least one antibody, portion, or polypeptide described herein can be recombined with vector DNA according to conventional techniques, including blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, filling in sticky ends as appropriate, alkaline phosphatase treatment to avoid unwanted ligation, and ligation with an appropriate ligase. Techniques for such manipulations can be used to construct nucleic acid sequences encoding monoclonal antibody molecules, antibody reagents, their antigen-binding regions, or CARs.

[0124] A nucleic acid molecule is said to be "capable of expressing" a polypeptide if it (such as DNA) contains a nucleotide sequence that includes transcriptional and translational regulatory information and such sequence is "operably linked" to a nucleotide sequence encoding a polypeptide. Operable linkage means that the regulatory DNA sequence and the DNA sequence to be expressed are linked in such a way that the gene is expressed as a peptide or antibody portion in recoverable amounts. The exact nature of the regulatory regions required for gene expression may vary from organism to organism and is well known in the art.

[0125] In some embodiments, the nucleic acids encoding antibodies, antibody reagents, their antigen-binding portions, or CARs as described herein are included in a vector. In some aspects described herein, the nucleic acid sequences encoding antibodies, antibody reagents, their antigen-binding portions, or CARs as described herein are operably linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed to deliver to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that can replicate and transfer a gene sequence to a cell when associated with appropriate control elements. Vectors can include, but are not limited to: cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virus particles, and the like.

[0126] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence that is ligated to a transcriptional regulatory sequence on the vector. The expressed sequence is often (but not necessarily) heterologous to the cell. The expression vector can contain other elements. For example, the expression vector can have a replication system so that it can be maintained in two organisms, such as for expression in human cells and for cloning and amplification in a prokaryotic host. The term "expression" refers to the cellular processes involved in the production of RNA and protein and, where applicable, secretion of the protein, including (where applicable) but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene, as well as polypeptides obtained by translation of the mRNA transcribed from the gene. The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions before and after the coding region, such as 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0127] As used herein, the term "viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and is capable of being packaged into viral vector particles. The viral vector can contain nucleic acids encoding the antibodies, antigen-binding portions thereof, or CARs described herein, in place of non-essential viral genes. The vector and / or particle can be used to transfer any nucleic acid into cells in vitro or in vivo. Many forms of viral vectors are known in the art.

[0128] A "recombinant vector" refers to a vector that contains a heterologous nucleic acid sequence (or "transgene") capable of being expressed in vivo. It should be understood that the vectors described herein, in some embodiments, are combined with other suitable compositions and treatment methods. In some embodiments, the vector is episomal. Use of a suitable episomal vector provides a way to maintain the nucleotide of interest in the subject in extrachromosomal DNA at a high copy number, thereby eliminating the potential effects of chromosomal integration.

[0129] In one aspect of any embodiment, described herein are cells that contain the antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein, or nucleic acids encoding such antibodies, antibody reagents, antigen-binding portions thereof, or CARs.

[0130] Expression of an antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein can occur in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian, and mammalian in vivo or in situ cells, or host cells derived from mammals, insects, birds, or yeast. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, but any other mammalian cells can be used. In addition, for example, in vivo synthesis of ubiquitin transmembrane polypeptide fusion proteins can be accomplished by using, for example, the yeast ubiquitin hydrolase system. The fusion proteins so produced can be processed in vivo or purified and processed in vitro, thereby allowing synthesis of an antibody or portion thereof having a specific amino-terminal sequence as described herein. In addition, problems associated with methionine residues derived from the start codon retained in direct yeast (or bacterial) expression can be avoided. When yeast is grown in a glucose-rich medium, any of a series of yeast gene expression systems can be utilized, in combination with promoter and terminator elements from actively expressed genes encoding for the high-level production of glycolytic enzymes, to obtain recombinant antibodies or antigen-binding portions thereof as described herein. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0131] Production of an antibody or antigen-binding portion thereof as described herein in insects can be achieved. For example, by methods known to those of ordinary skill in the art, insect hosts are infected with baculoviruses engineered to express transmembrane polypeptides.

[0132] In some embodiments, the introduced nucleotide sequence is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a variety of vectors can be used for this purpose and are known and available to those of skill in the art or ordinary skill in the art. Important factors in the selection of a particular plasmid or viral vector include: the ease with which recipient cells containing the vector can be identified and selected from those not containing the vector; the number of vector copies required in a particular host; and whether it is necessary to be able to "shuttle" the vector between host cells of different species.

[0133] Exemplary prokaryotic vectors known in the art include plasmids, such as plasmids capable of replicating in Escherichia coli. Other gene expression elements that can be used to express the cDNA encoding an antibody, its antigen-binding portion, or a CAR include, but are not limited to, (a) viral transcriptional promoters and their enhancer elements, such as the SV40 early promoter, Rous sarcoma virus LTR, and Moloney murine leukemia virus; (b) splice regions and polyadenylation sites, such as those derived from the SV40 late region, and (c) polyadenylation sites, such as SV40. Immunoglobulin cDNA genes can be expressed, for example, using expression elements, the SV40 early promoter and its enhancer, the murine immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit β-globin intervening sequence, the immunoglobulin and rabbit β-globin polyadenylation sites, and the SV40 polyadenylation element.

[0134] For immunoglobulin genes containing partial cDNA and partial genomic DNA, the transcriptional promoter can be the human cytomegalovirus, the promoter enhancer can be the cytomegalovirus and murine / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be the native chromosomal immunoglobulin sequences.

[0135] In some embodiments, to express a cDNA gene in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancer is one or both of the murine immunoglobulin heavy chain enhancer and the viral LTR enhancer, the splice region contains an intron greater than 31 bp, and the polyadenylation and transcriptional termination regions are from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above-described expression elements to effect protein expression in mammalian cells.

[0136] The gene is assembled into or inserted into an expression vector. Then, recipient cells capable of expressing the chimeric immunoglobulin chain gene product are transfected with the antibody, its antigen-binding portion, or a CAR, or the encoding gene of the chimeric H or chimeric L chain alone, or co-transfected with the chimeric H and chimeric L chain genes. The transfected recipient cells are cultured under conditions permitting incorporation of the gene expression, and the expressed immunoglobulin chain or intact antibody or fragment is recovered from the culture.

[0137] In some embodiments, genes encoding an antibody, an antigen-binding portion thereof, a CAR, or a chimeric heavy and light chain or portions thereof are assembled in separate expression vectors and 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, wherein each vector has a different pair of genes. The vectors produced by this strategy first direct the production of the genes in the bacterial system and allow amplification. The genes so produced and amplified in the bacterial host are then used to co-transfect eukaryotic cells and allow selection of the co-transfected cells carrying the desired transfected genes. Non-limiting examples of selectable genes for the bacterial system are genes conferring ampicillin resistance and genes conferring chloramphenicol resistance. Selectable genes for eukaryotic transfectants include the xanthine-guanine phosphoribosyltransferase gene (named gpt) and the phosphotransferase gene from Tn5 (named neo). Alternatively, the genes can be assembled on the same expression vector.

[0138] Recipient cell lines for transfection of expression vectors and production of antibodies, antibody reagents, antigen-binding portions thereof, or CARs as described herein can be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by transfected immunoglobulin genes and have a mechanism for immunoglobulin glycosylation. For example, in some embodiments, the recipient cell is the recombinant Ig-producing myeloma cell line SP2 / 0 (ATCC#CRL 8287). SP2 / 0 cells produce only immunoglobulins encoded by transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, and the secreted immunoglobulins can be obtained from ascites. Other suitable recipient cells include lymphocytes, such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells.

[0139] Expression vectors carrying chimeric, humanized, or composite human antibody constructs, antibodies, antigen-binding portions thereof, 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 polycation application (e.g., diethylaminoethyl (DEAE) dextran), as well as mechanical means such as electroporation, direct microinjection, and particle bombardment well known to those skilled in the art.

[0140] Traditionally, monoclonal antibodies have been produced as natural molecules in murine hybridoma cell lines. In addition to this technology, the methods and compositions described herein provide for recombinant DNA expression of monoclonal antibodies. This allows for the production of humanized antibodies as well as a range of antibody derivatives and fusion proteins in selected host species. Production of antibodies in bacteria, yeast, transgenic animals, and eggs are also alternatives to the hybridoma-based production systems. The main advantage of transgenic animals is the potential for high yields from renewable resources.

[0141] In one aspect, provided are cells comprising the isolated antibodies, antigen-binding portions thereof, or CARs described herein. In some embodiments, the isolated antibodies, antigen-binding portions thereof, or CARs as described herein are expressed on the cell surface. In some embodiments, the cells comprise a nucleic acid encoding the isolated antibodies, antigen-binding portions thereof, or CARs as described herein.

[0142] In some embodiments, the cells are immune cells. As used herein, "immune cell" refers to a cell that plays a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells; natural killer cells; 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.

[0143] In a specific embodiment, the cells (e.g., immune cells) are transduced with a retroviral vector (e.g., a lentiviral vector) encoding a CAR. For example, immune effector cells are transduced with a vector encoding a CAR that comprises an anti-CHI3L1 / anti-PD-1 antibody or an antigen-binding portion thereof that binds to CHI3L1 and PD-1 polypeptides and has an intracellular signaling domain of CD3ζ, CD28, 4-1BB, Ox40, or any combination thereof. Thus, these transduced cells can initiate a CAR-mediated cytotoxic response.

[0144] Retroviruses are commonly used tools for gene delivery. In a specific embodiment, retroviruses are used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) to cells. As used herein, the term "retrovirus" refers to an RNA virus that transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus". The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoding the structural proteins and enzymes required for the production of new virus particles.

[0145] Exemplary retroviruses suitable for particular embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)) and lentiviruses.

[0146] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); caprine 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 backbone (i.e., HIV cis-acting sequence elements) is preferred. In certain embodiments, lentiviruses are used to deliver polynucleotides comprising a CAR to cells.

[0147] Retroviral vectors, and more specifically lentiviral vectors, can be used to practice the specific embodiments of the present invention. Thus, as used herein, the terms "retrovirus" or "retroviral vector" are meant to include "lentivirus" and "lentiviral vector", respectively.

[0148] CHI3L1 antigen-binding portion

[0149] As used herein, "CHI3L1", "chitinase-3-like protein 1", or "YKL-40" refers to a glycoprotein of approximately 40 kDa secreted by at least macrophages, chondrocytes, neutrophils, synoviocytes, and some cancer cells. CHI3L1 does not have chitinase activity, is a Th2-promoting cytokine, is associated with the AKT anti-apoptotic signal transduction pathway, and induces the migration of astrocytes. The sequences of the CHI3L1 expression products are known for many species, e.g., human CHI3L1 (NCBI gene number: 1116) mRNA (NCBI reference sequences: NM_001276.1 and NCBI reference sequence: NM_001276.2) and polypeptide (NCBI reference sequences: NP_001267.1 and NCBI reference sequence NP_001267.2).

[0150] 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 and / or one or more light-chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6, which are disclosed in U.S. Patent No. 10,253,111 and reproduced in Table 1 below.

[0151] Table 1 Sequences of the variable complementarity-determining regions (CDRs) of the FRG antibody

[0152] Heavy-chain CDR

[0153]

[0154] Light-chain CDR

[0155]

[0156] In some embodiments of any aspect, the bispecific antibody, antibody reagent, antigen-binding portion thereof, 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 any aspect, the bispecific antibody, antibody reagent, antigen-binding portion thereof, 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.

[0157] In some embodiments, the framework of the anti-human CHI3L1 antibody comprises conservative substitutions relative to the heavy-chain sequence having the amino acid sequence of SEQ ID NO: 36 or the light-chain sequence having the amino acid sequence of SED ID NO: 38, which are disclosed in U.S. Patent No. 10,253,111, wherein the conservative substitutions in the sequences are not included in the CDRs. In alternative embodiments, the framework of the anti-human CHI3L1 antibody comprises the heavy-chain sequence of the FRG antibody having the amino acid sequence of SEQ ID NO: 36 or the light-chain sequence of the FRG antibody having the amino acid sequence of SED ID NO: 38, which are disclosed in U.S. Patent No. 10,253,111, and the two are provided below as SED ID NO: 13 and SED ID NO: 14, respectively.

[0158] Table 2

[0159] FRG heavy-chain sequence

[0160]

[0161] FRG light-chain sequence

[0162]

[0163] In other alternative 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: 1-12 and / or one or more light chain CDRs having the amino acid sequences of SEQ ID NOs: 13-20, which are disclosed in Table 3. See, for example, International Publication No. WO 2019060675.

[0164] Table 3

[0165] Heavy chain CDR

[0166]

[0167]

[0168]

[0169] Light chain CDR

[0170]

[0171]

[0172] PD-1 antigen-binding portion

[0173] Examples of anti-PD-1 antibodies are disclosed in U.S. Patent Nos. 10,344,090 (Yuan et al.); 10,323,091 (van Dijk et al.); 10,316,089 (Baruah et al.); 10,280,224 (Wang et al.); 10,239,942 (Amirina et al.); 10,221,244 (Wong et al.); 10,155,037 (Abdiche et al.); and U.S. Publication Nos. 2011 / 0123550 (Shibayama et al.); 2016 / 0376367 (Yuan et al.); 2017 / 0210806 (Liu).

[0174] The antigen-binding portion of any anti-PD-1 antibody can be used in the bispecific antibody of the present invention. In some embodiments, Table 4 provides bispecific antibodies that detect and neutralize CHI3L1 and PD1, comprising a PD-1 single-chain variable fragment (scFv-PD1) and a linker (shown in bold and underlined).

[0175] Table 4

[0176]

[0177] Pharmaceutical composition

[0178] In one aspect of any embodiment, described herein are compositions comprising a bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein, or a nucleic acid encoding an antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein, or a cell as described herein. In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier that is acceptable for use in the pharmaceutical industry. The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that, according to reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, and that conform to a reasonable risk / benefit ratio.

[0179] The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed therein is well known in the art and is not limited based on the formulation. Generally, such compositions are prepared as injectable liquid solutions or suspensions, however, solid forms suitable for solutions or suspensions to be placed in a liquid prior to use can also be prepared. The formulation can also be emulsified or in the form of a liposome composition. The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the treatment methods described herein. Suitable excipients include, for example, water, saline, glucose, glycerol, ethanol, and the like, and combinations thereof. Additionally, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, etc., which enhance or maintain the effectiveness of the active ingredient. The therapeutic compositions described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide), which are formed from inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups can also be derived from inorganic bases, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain nothing other than the active ingredient and water, or contain a buffer such as sodium phosphate at physiological pH, saline, or both, for example, phosphate buffered saline. Further, the aqueous carrier can contain more than one buffering salt, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. In addition to water, the liquid composition can contain a liquid phase. Examples of such other liquid phases are glycerol, vegetable oils (such as cottonseed oil), and water-oil emulsions. The amount of the active agent effective for treating a particular disease or disorder used in the present invention will depend on the nature of the disease or disorder and can be determined by standard clinical techniques.

[0180] In some embodiments, a composition comprising an antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein, or a nucleic acid encoding an antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein, can be a lyophilized product.

[0181] In some embodiments, the techniques described herein relate to syringes or catheters (including organ-specific catheters such as renal catheters, bile catheters, cardiac catheters, etc.) that comprise a therapeutically effective amount of a composition as described herein.

[0182] In one aspect, described herein is a method of inhibiting or killing CHI3L1+ / PD-1+ cells, the method comprising contacting the cells with an isolated bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein, a nucleic acid encoding such a polypeptide, a cell comprising such a polypeptide or nucleic acid, or a composition comprising such a polypeptide or nucleic acid. Inhibiting CHI3L1+ / PD-1+ cells can include inhibiting the metabolic activity, metastasis, and / or proliferation of the cells. Assays for measuring metabolic activity, metastasis (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.

[0183] As used herein, a "CHI3L1+ / PD-1+" cell is a cell that expresses increased levels of CHI3L1+ and PD-1+, e.g., as compared to the average level of CHI3L1+ / PD-1+ found in the same type of healthy cells or in healthy cells of the same type.

[0184] In some embodiments of any aspect described herein, the subject to whom the composition as described herein is administered can be a subject determined to have an elevated level of CHI3L1 or an increased level of CHI3L1 as compared to a previous assessment of the level in the subject. In some embodiments of any aspect, the elevated level of CHI3L1 is the level of circulating CHI3L1. In some embodiments of any aspect described herein, the subject to whom the composition as described herein is administered can be a subject determined to have CHI3L1+ cancer cells.

[0185] In some embodiments of any aspect described herein, a method that includes administering a composition as described herein can further include a first step of identifying a subject having an elevated level of CHI3L1. In some embodiments of any aspect, the elevated level of CHI3L1 is the level of circulating CHI3L1. In some embodiments of any aspect described herein, a method that includes administering a composition as described herein can further include a first step of identifying a subject having CHI3L1+ cancer cells.

[0186] As used herein, a "CHI3L1+" cell is a cell that expresses increased CHI3L1+ levels, e.g., compared to the average CHI3L1+ levels found in or compared to the same type of healthy cells. In some embodiments, in any aspect, the increased CHI3L1 level can be at least 1.5-fold the reference level, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more than the reference level.

[0187] In one aspect, the techniques described herein relate to methods comprising administering to a subject an antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein, or a nucleic acid encoding an antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein. In some embodiments, the subject is in need of treatment for cancer and / or malignancy. In some embodiments, the subject is in need of treatment for: prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignant melanoma, and lung cancer. In some embodiments, the method is a method of treating a subject. In some embodiments, the method is a method of treating a subject's cancer.

[0188] In one aspect, the techniques described herein relate to methods comprising administering to a subject a bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein, or a nucleic acid encoding a bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein.

[0189] In one aspect, a method of treating cancer in a subject in need thereof is described, the method comprising administering a cell as described herein, e.g., a cell comprising a bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein. In some embodiments, the cell is an immune cell.

[0190] On the one hand, this document describes a method for treating cancer in a subject in need thereof, the method comprising administering to the subject the nucleic acid described herein or immune cells comprising the nucleic acid, wherein the immune cells of the subject are caused to express the 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 using a cell type-specific promoter and / or a composition that selectively binds to the desired cell type. For example, coupling the nucleic acid to an aptamer can allow for targeted delivery. See, for example, McNamara, et al. (2006). In alternative embodiments, a drug delivery system such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems can be used to deliver the nucleic acid. Positively charged cationic delivery systems facilitate the binding of nucleic acid molecules (negatively charged), and also enhance the interaction at the negatively charged cell membrane to allow for efficient cellular uptake of the nucleic acid. Cationic lipids, dendrimers, or polymers can be conjugated to the nucleic acid, or induced to form vesicles or micelles that encapsulate the nucleic acid (see, for example, Kim, et al. (2008)). When administered systemically, the formation of vesicles or micelles further prevents degradation of the nucleic acid. Methods for preparing and administering cationic-inhibitory nucleic acid complexes are well within the capabilities of those skilled in the art. Some non-limiting examples of drug delivery systems that can be used for systemic delivery of nucleic acids include DOTAP cationic liposomes (Oligofectamine), "solid nucleic acid lipid particles", cardiolipin, polyethyleneimine, Arg-Gly-Asp (RGD) peptides, and polyamidoamine. In some embodiments, the nucleic acid forms a complex with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of nucleic acids and cyclodextrins can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety. Targeted delivery of nucleic acids is described in, for example, Ikeda and Taira (2006); Soutschek et al. (2004); and Lorenze et al. (2004); each of which is incorporated herein by reference in its entirety. By way of example, the nucleic acid can be targeted to immune cells by encapsulating an inhibitor in a liposome that contains a ligand for a receptor expressed on the immune cells, such as a TCR. In some embodiments, the liposome can comprise an aptamer specific for immune cells.

[0191] In some embodiments, the methods described herein relate to CAR-T cell therapy. CAR-T cells and related therapies involve the adoptive cell transfer of immune cells (e.g., T cells) expressing a CAR that specifically binds to a targeted cell type (e.g., cancer cells) to treat a subject. In some embodiments, the cells administered as part of the treatment can be autologous to the subject. In some embodiments, the cells administered as part of the treatment can be non-autologous to the subject. In some embodiments, the cells are engineered and / or genetically modified to express a CAR. Further discussion of CAR-T therapy can be found, for example, in Maus et al. (2014); Reardon et al. Neuro-Oncology 2014 16:1441-1458; Hoyos et al. (2012); Byrd et al. (2014); Maher and Wilkie (2009); and Tamada et al. Clin Cancer Res 2012 18:6436-6445; each of which is incorporated herein by reference in its entirety.

[0192] Generally speaking, a pharmaceutical composition comprising the cells described herein (e.g., T cells or immune cells) can be administered at a dose of 10 2 to 10 10 cells / kg body weight, preferably 10 5 to 10 6 cells / kg body weight, including all integer values within these ranges. The number of cells will depend on the intended final use of the composition, as will the type of cells included. For the uses provided herein, the volume of the cells is typically 1 liter or less, can be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the density of the cells required is typically greater than 10 6 cells / mL and is typically greater than 10 7 cells / mL, typically 10 8 cells / mL or greater. Clinically relevant amounts of immune cells can be administered in multiple infusions that cumulatively equal or exceed 10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 or 10 12 cells. In some aspects of the invention, particularly because all of the infused cells will be redirected to a specific target antigen, a range of 10 6 / kg (10 6 -10 11A lower number of cells. The CAR-expressing cell composition can be administered multiple times at doses within these ranges. For a patient undergoing treatment, the cells can be allogeneic, syngeneic, xenogeneic, or autologous. If desired, the treatment can also include administration of a mitogen (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the induction of an immune response. In some embodiments, the dose can be about 1x10 5 cells per kilogram of body weight to about 1x10 8 cells. In some embodiments, the dose can be about 1x10 6 cells per kilogram of body weight to about 1x10 7 cells. In some embodiments, the dose can be about 1x10 6 cells per kilogram of body weight. In some embodiments, a single dose of cells can be administered. In some embodiments, the cell dose can be repeated, e.g., one, two, or more times. In some embodiments, the dose of cells can be administered, e.g., daily, weekly, or monthly.

[0193] The dose range of the agent depends on potency and includes an amount sufficient to produce the desired effect, e.g., slowing tumor growth or reducing tumor size. The dose should not be so large as to cause unacceptable adverse side effects. Generally, the dose will vary with the age, condition, and sex of the patient and can be determined by one of ordinary skill in the art. In the event of any complications, the dose can also be adjusted by the individual physician. In some embodiments, the dose range is from 0.001 mg / kg body weight to 0.5 mg / kg body weight. In some embodiments, the dose range is from 5 μg / kg body weight to 100 μg / kg body weight. Alternatively, the dose range can be titrated to maintain serum levels between 1 μg / mL and 1000 μg / mL. For systemic administration, a therapeutically effective amount, e.g., 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 more, can be administered to the subject.

[0194] Administration of the doses listed above can be repeated. In some embodiments, the dose is administered once a day or multiple times a day, e.g., but not limited to, three times a day. In some embodiments, the doses listed above are administered daily for several weeks or months. The duration of treatment depends on the clinical progress of the subject and the response to treatment.

[0195] In some embodiments, the dose can be from about 2 mg / kg to about 15 mg / kg. In some embodiments, the dose can be about 2 mg / kg. In some embodiments, the dose can be about 4 mg / kg. In some embodiments, the dose can be about 5 mg / kg. In some embodiments, the dose can be about 6 mg / kg. In some embodiments, the dose can be about 8 mg / kg. In some embodiments, the dose can be about 10 mg / kg. In some embodiments, the dose can be about 15 mg / kg. In some embodiments, the dose can be about 100 mg / m 2 to about 700 mg / m 2 In some embodiments, the dose can be about 250 mg / m 2 In some embodiments, the dose can be about 375 mg / m 2 In some embodiments, the dose can be about 400 mg / m 2 In some embodiments, the dose can be about 500 mg / m 2 .

[0196] In some embodiments, the dose 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.

[0197] In some embodiments, the dose can be administered approximately weekly. In some embodiments, the dose can be administered weekly. In some embodiments, the dose can be administered weekly for about 12 to about 18 weeks. In some embodiments, the dose can be administered approximately every two weeks. In some embodiments, the dose can be administered approximately every three weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered every two weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered every three weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered intravenously every two weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered intravenously every three weeks. In some embodiments, the dose can be about 200 mg / m administered intravenously weekly 2 to about 400 mg / m 2 . In some embodiments, the dose can be about 200 mg / m administered intravenously every two weeks 2 to about 400 mg / m 2 . In some embodiments, the dose can be about 200 mg / m administered intravenously every three weeks 2 to about 400 mg / m 2。In some embodiments, a total of about 2 to 10 doses are administered. In some embodiments, a total of four doses are administered. In some embodiments, a total of five doses are administered. In some embodiments, a total of six doses are administered. In some embodiments, a total of seven doses are administered. In some embodiments, a total of eight doses are administered. In some embodiments, the administration is carried out for a total of about four weeks to about 12 weeks. In some embodiments, the administration is carried out for a total of about six weeks. In some embodiments, the administration is carried out for a total of about eight weeks. In some embodiments, the administration is carried out for a total of about 12 weeks. In some embodiments, the initial dose may be about 1.5 to about 2.5 times higher than the subsequent doses.

[0198] In some embodiments, the dose may be from about 1 mg to about 2,000 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 1,000 mg. In some embodiments, the dose may be about 2,000 mg. In some embodiments, the dose may be administered intravenously by infusion at about 3 mg per day. In some embodiments, the dose may be administered intravenously by infusion at about 10 mg per day. In some embodiments, the dose may be administered intravenously by infusion three times a week at about 30 mg.

[0199] A therapeutically effective amount is the amount of an agent sufficient to produce a statistically significant, measurable change in tumor size, tumor growth, etc. (efficacy measurements are described below). Such effective amounts can be measured in clinical trials and animal studies.

[0200] The agent can be administered intravenously by injection or intravenously by gradual infusion over time. Appropriate formulations for a given route, e.g., agents that can be used in the methods and compositions described herein, can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and if desired, by peristaltic (pump) means, or by other means known to those skilled in the art. The compounds used herein are preferably administered orally, intravenously, or intramuscularly to cancer patients. Administration directly to the tumor mass is also specifically contemplated.

[0201] For example, a therapeutic composition comprising at least one agent can be conventionally administered in unit doses. When used in a therapeutic composition, the term "unit dose" refers to a physically discrete unit suitable as a unit dosage form for a subject, each unit containing a predetermined amount of the active substance, in association with the required physiologically acceptable diluent (i.e., carrier or vehicle), calculated to produce the desired therapeutic effect.

[0202] The composition is administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The dosage and timing depend on the subject to be treated, the subject's body's ability to utilize the active ingredient, and the degree of the desired therapeutic effect.

[0203] The precise amount of the active ingredient to be administered depends on the judgment of the practitioner and varies from individual to individual. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable dosing regimens are also variable but are represented by an initial administration, followed by repeated administrations at intervals of one or more hours by subsequent injections or other administrations. Alternatively, continuous intravenous infusion is contemplated to maintain the concentration in the blood within the range specified for in vivo treatment.

[0204] In some embodiments, the method further comprises co-administering the pharmaceutical composition described herein with one or more additional chemotherapeutic agents, biological agents, drugs, or treatments as part of a combination therapy. In some such embodiments, the chemotherapeutic agent biological product, drug, or treatment is selected from: radiotherapy, surgery, antibody reagents, and / or small molecules.

[0205] In some embodiments of the methods described herein, the method further comprises administering one or more chemotherapeutic agents to a subject to whom the pharmaceutical composition described herein is being administered. Non-limiting examples of chemotherapeutic agents can include: alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, piposulfan; aziridines such as benzodopa, carboquone, memredopa, uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophore, aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,; Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid;Aceglatone; Aldophosphamide glycoside;

[0206] (Aldophosphamide glycoside); Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoid such as Maytansine and Ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; Polysaccharide complexes (JHS Natural Products, Eugene, Oregon); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene (especially T-2 toxin, verrucarin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumburg, IL) and docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan and 5-FU and leucovorin); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Capecitabine; Capecitabine; Leucovorin (LV); Oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); Lapatinib Inhibitors of PKC-α, Raf, H Ras, EGFR (e.g., erlotinib ), and VEGF-A, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0207] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or arrests cell function and / or causes cell destruction. The term is intended to include radioactive isotopes (e.g., radioactive isotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu), chemotherapeutic agents, and toxins, such as enzymatically active toxins or small molecule toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof.

[0208] As used herein, the term "chemotherapy" or "chemotherapeutic agent" refers to any chemical agent that has therapeutic use in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancers as well as diseases characterized by hyperplastic growth. Chemotherapeutic agents used herein include chemical agents and biological agents. The function of these agents is to inhibit the cell activity on which the continued survival of cancer cells depends. The categories of chemotherapeutic agents include alkylating agents / alkaloid agents, antimetabolites, hormones or hormone analogs, and various anti-tumor drugs. Most (if not all) of these drugs are directly toxic to cancer cells and do not require immune stimulation. In one embodiment, a chemotherapeutic agent is an agent used to treat tumors such as solid tumors. In one embodiment, a chemotherapeutic agent is a radioactive molecule. A person skilled in the art can easily determine the chemotherapeutic agent used (e.g., Harrison's Principles of Internal Medicine (Harrison Principles of Internal Medicine)). ARRISON’S P RINCIPLES OF I NTERNAL M EDICINE ), Chapter 86 in 14th edition (2001); BELOFF Clinical Oncology BELOFF’S C LINICAL O NCOLOGY ), Chapter 17 of 2nd edition (2000). The bispecific and multispecific polypeptide agents described herein can be used in conjunction with other chemotherapeutic agents.

[0209] By "radiation therapy" is meant the use of directed gamma or beta radiation to cause sufficient damage to cells to limit their ability to function normally or to destroy the cells completely. It will be appreciated that there are many known methods in the art to determine the dosage and duration of treatment. Typical treatment is a one-time administration, with a typical dosage range of 10 to 200 units (Gray) per day.

[0210] In some embodiments, the methods described herein may further comprise administering additional immunotherapy to a subject. As used herein, "immunotherapy" refers to a variety of therapeutic strategies designed to induce a patient's own immune system to fight tumors and includes, but is not limited to: intravesical BCG immunotherapy for superficial bladder cancer, vaccines that generate a specific immune response (e.g., for malignant melanoma and renal cell carcinoma), treatment of prostate cancer with Sipuleucel-T, wherein dendritic cells from the patient are loaded with prostate acid phosphatase peptides to induce a specific immune response against prostate-derived cells, administration of cytokines, growth factors and / or signal transduction molecules (e.g., interleukins) that stimulate one or more types of immune cells, ex vivo expansion and / or stimulation of lymphocytes and / or dendritic cells specific for tumor antigens prior to reintroduction into the patient, imiquimod, adoptive cell transfer, and / or methods described, for example, in International Publication WO 2003 / 063792 and U.S. Patent No. 8,329,660. In some embodiments, the immunotherapy stimulates an NK response. In other embodiments, the immunotherapy is an adoptive cell transfer method, i.e., adoptive immunotherapy.

[0211] In some embodiments, the methods described herein may further comprise administering additional antibodies, antibody reagents, antigen-binding portions thereof, or T cells comprising a CAR to a subject. In some embodiments, the methods described herein may further comprise administering cytokines to a subject. Antibody- and cytokine-based therapies are known in the art and may include, as non-limiting examples: alemtuzumab; bevacizumab; brentuximab vedotin; cetuximab; gemtuzumab; ibritumomab tiuxetan; ipilimumab; ofatumumab; panitumumab; rituximab; tositumomab; trastuzumab; interleukin-2, and interferon-α.

[0212] The efficacy of a given treatment (e.g., for cancer) can be determined by a clinician of ordinary skill. However, a treatment is considered to be an "effective treatment" as the term is used herein if any one or all signs or symptoms of the tumor, for example, are altered in a beneficial manner, or other clinically acceptable symptoms are improved or even alleviated, e.g., by at least 10% reduction after treatment with an agent described herein. Efficacy can also be measured by the failure of an individual to deteriorate, as assessed by the need for hospitalization or medical intervention (i.e., the progression of the disease stops). Methods for measuring these metrics are known to those of skill in the art and / or are described herein.

[0213] An effective amount for treating a disease refers to an amount that, when administered to a mammal in need thereof, is sufficient to result in an effective treatment of the disease as defined herein for the disease. The efficacy of an agent can be determined by assessing physical indicators such as cancer (e.g., tumor size, tumor mass, tumor density, angiogenesis, tumor growth rate, etc.).

[0214] CHI3L1 and PD-1 levels of the subject

[0215] In one aspect, the present disclosure describes a method for detecting, prognosticating, and / or diagnosing cancer, the method comprising detecting or measuring the levels of CHI3L1 and / or PD-1 or PD-L1 in a sample obtained from a subject by contacting the sample with a bispecific antibody, antibody reagent, or antigen-binding portion thereof described herein, wherein an increase in the CHI3L1 and PD-1 or PD-L1 levels relative to a reference level indicates that the subject has cancer or an increased risk of developing cancer.

[0216] Recent reports have shown that PD-L1 expression has moderate sensitivity as a biomarker for immune checkpoint inhibitors (ICIs) such as PD-1 in cancer patients, and successful treatment outcomes have been observed in patients with low or no PD-L1 expression levels (Paz-Ares, et al., 2018; Hellmann, et al., 2019; Schoenfeld, et al., 2020). Thus, in another aspect, a method for detecting, prognosticating, 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 antigen-binding portion thereof described herein, wherein an increase in the CHI3L1 level relative to a reference level indicates that the subject has cancer or an increased risk of developing cancer.

[0217] In some embodiments of any of the aspects described herein, the subject to whom the composition described herein is administered can 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 can be the levels of circulating CHI3L1 and PD-1 or PD-L1. In some embodiments of any of the aspects described herein, the subject to whom the composition described herein is administered can be a subject determined to have CHI3L1+ / PD-1+ cancer cells.

[0218] In some embodiments of any aspect described herein, a method that includes administering a composition described herein may further include a first step of identifying an object 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 may be circulating levels of CHI3L1 and / or PD-1 or PD-L1. In some embodiments of any aspect described herein, a method that includes administering a composition described herein may further include a first step of identifying an object having cancer cells that are CHI3L1+ / PD-1+ or CHI3L1+ / PD-L1+.

[0219] In one aspect, described herein is an assay that includes contacting a test sample obtained from an object with an antibody, antibody reagent, or antigen-binding portion thereof described herein and detecting the presence or intensity of a signal indicative of the presence or level of CHI3L1 and PD-1 in the sample; wherein an increase in the CHI3L1 and PD-1 levels relative to a reference level indicates that the object has cancer or is at a higher risk of developing cancer.

[0220] In one aspect, described herein is a method of identifying an object in need of treatment for cancer, the method comprising: contacting a sample obtained from the object with a bispecific antibody, antibody reagent, or antigen-binding portion thereof described herein and detecting the presence or intensity of a signal indicative of the presence or level of CHI3L1 and / or PD-1 or PD-L1 in the sample; and identifying the object as being in need of treatment for cancer when the expression level of CHI3L1 and / or PD-1 or PD-L1 is increased relative to a reference level.

[0221] In one aspect, described herein is a method of determining whether an object is likely to respond to a therapy with anti-CHI3L1 / anti-PD-1, such as an anti-CHI3L1 / anti-PD-1 bispecific antibody, antibody reagent, or antigen-binding fragment thereof, or a T cell comprising a bispecific CAR that binds CHI3L1 and PD-1 or PD-L1, the method comprising: contacting a test sample obtained from the object with an antibody, antibody reagent, antigen-binding fragment thereof described herein and detecting the presence or intensity of a signal indicative of the presence or level of CHI3L1 and PD-1 or PD-L1 in the sample; determining that the object is likely to respond to a therapy with anti-CHI3L1 / anti-PD-1 when CHI3L1 and PD-1 or PD-L1 are increased relative to a reference level; and determining that the object is less likely to respond to a therapy with anti-CHI3L1 / anti-PD-1 when CHI3L1 and PD-1 or PD-L1 are not increased relative to a reference level.

[0222] In one aspect, the present disclosure describes a method of treating cancer, comprising: contacting a test sample obtained from a subject with an antibody, an antibody reagent, or an antigen-binding portion thereof described herein; detecting the presence or intensity of a signal indicative of the presence or level of CHI3L1 and PD-1 in the sample; and treating the subject with an anti-CHI3L1 / anti-PD-1 bispecific antibody when the levels of CHI3L1 and PD-1 are increased relative to a reference level. In one aspect, the present disclosure describes a method of treating cancer, comprising: administering a therapeutically effective amount of an anti-CHI3L1 / anti-PD-1 bispecific antibody therapy to a subject determined to be in need of 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, an antibody reagent, an antigen-binding portion thereof, or a T cell comprising a bispecific CAR that recognizes CHI3L1 and PD-1; a nucleic acid; a cell; or a composition as described herein.

[0223] 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., CHI3L1 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, derived, or amplified from a biological sample (e.g., a biological fluid). In some embodiments, the antibody or an antigen-binding portion thereof produces a detectable signal when a CHI3L1 molecule or a PD-1 molecule is present. In some embodiments, the antibody or an antigen-binding portion thereof is detectably labeled or capable of producing 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); immunohistological staining; radioimmunometric assay; immunofluorescence assay; mass spectrometry; FACS; and immunoelectrophoresis assay. In some embodiments, the antibody or an antigen-binding portion thereof is detectably labeled or produces a detectable signal. In some embodiments, the expression level of CHI3L1 or PD-1 is normalized relative 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 prior sample obtained from the subject.

[0224] In some embodiments, the level of CHI3L1 or PD-1 can be the level of the CHI3L1 or PD-1 polypeptide. Detection of the CHI3L1 or PD-1 polypeptide can be according to any method known in the art. Immunological methods for detecting the CHI3L1 or PD-1 polypeptide according to the present technology 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 derivative techniques using the antibody reagents described herein.

[0225] Immunochemical methods require the use of antibody reagents specific for the target molecule (e.g., an antigen, or in the embodiments described herein, the CHI3L1 or PD-1 polypeptide). In some embodiments, the assays, methods, and / or systems described herein can include: anti-CHI3L1 or anti-PD-1 antibody reagents. In some embodiments, the antibody reagents can be detectably labeled. In some embodiments, the antibody reagents can be attached to a solid support (e.g., bound to a solid support). In some embodiments, the solid support can comprise particles (including but not limited to agarose or latex beads or particles or magnetic particles), beads, nanoparticles, polymers, substrates, slides, coverslips, plates, culture dishes, wells, membranes, and / or gratings. The solid support can include many different materials including, but not limited to, polymers, plastics, resins, polysaccharides, silicon- or silica-based materials, carbon, metals, inorganic glass, and membranes.

[0226] In one embodiment, the assays, methods, and / or systems as described herein may include an ELISA. In an exemplary embodiment, a first antibody reagent may be immobilized on a solid support (commonly a polystyrene microtiter plate). The solid support may be contacted with a sample obtained from a subject, and the antibody reagent will bind (“capture”) its specific antigen (e.g., CHI3L1 or PD-1). The solid support may then be contacted with a second labeled antibody reagent (e.g., a detection antibody reagent). The detection antibody reagent may, for example, comprise a detectable signal, be covalently linked to an enzyme, or be itself conjugated to an enzyme via bioconjugation for detection. The presence of a signal indicates that both the first antibody reagent immobilized on the support and the second “detection” antibody reagent have bound to the antigen, i.e., 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 detergent solution to remove any non-specifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzyme substrate to produce a visible signal that indicates the amount of CHI3L1 or PD-1 polypeptide in the sample. Older ELISAs use chromogenic substrates, but newer assays use fluorescent substrates with higher sensitivity. There are also other different forms of ELISA, which are well known to those skilled in the art.

[0227] In one embodiment, the assays, systems, and methods described herein may include a lateral flow immunoassay test (LFIA), also known as an immunochromatographic assay, or strip test, to measure or determine the level of CHI3L1 or PD-1 polypeptides in a sample. An LFIA is a simple device designed to detect the presence (or absence) of CHI3L1 or PD-1 in a sample. There are currently many LFIA tests for medical diagnostics or 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 matrix by capillary action. After the sample is applied to the test strip, it encounters a colored antibody reagent that mixes with the sample and, if bound to a portion of the sample, passes over a line or area on the substrate that has been pretreated with a second antibody reagent. Depending on the level of CHI3L1 or PD-1 present in the sample, the colored antibody reagent may bind to the test line or area. An LFIA is essentially an immunoassay that is adapted to operate along a single axis to accommodate a test strip format or dipstick format. Strip tests are versatile and can be easily modified by those skilled in the art to detect a wide variety of antigens from fluid samples (such as urine, blood, water samples, etc.). Strip tests are also known as dip stick tests, a name derived from the literal action of "dipping" the test strip into the fluid sample to be tested. LFIA strip tests are easy to use, require minimal training, and can be easily used on-site as a component of point-of-care testing (POCT) diagnostics. LFIA tests can be operated as competitive or sandwich assays. Sandwich LFIA is similar to sandwich ELISA. The sample first encounters colored particles labeled with an antibody reagent specific for the target (e.g., a CHI3L1- or PD-1-specific antibody reagent). The test line will also contain an antibody reagent (e.g., a CHI3L1- or PD-1-specific antibody reagent). The test line will appear as a colored band in a positive sample. In some embodiments, the lateral flow immunoassay can be a two-antibody sandwich assay, a competitive assay, a quantitative assay, or a variant thereof. There are many variants of lateral flow technology. Multiple capture regions can also be applied to create multiplex tests.

[0228] A typical test strip consists of the following parts: (1) a sample application area, including an absorbent pad (i.e., matrix or material) onto which the test sample is applied; (2) a conjugate or reagent pad - which contains one or more antibody reagents specific for the target, which may be conjugated to colored particles (usually colloidal gold particles or latex microspheres); (3) a test result area containing a reaction membrane - usually a hydrophobic nitrocellulose or cellulose acetate membrane to which the antibody reagent is immobilized, serving as a capture area or test line (a control area may also be present, which contains an antibody specific for the antibody reagent conjugated to the particles or microspheres); and (4) an optional wicking or waste reservoir - another absorbent pad designed to wick the sample across the reaction membrane and collect it by capillary action. The components of the strip are usually fixed to an inert backing material and can be provided in a simple dipstick form or within a plastic housing, with a sample port and a reaction window showing the capture area and the control area. Although not absolutely necessary, most tests will include a second line which contains an antibody that can absorb free latex / gold to confirm that the test has been carried out correctly.

[0229] In the context of immunoassays for many antigen biomarkers, the use of "dipsticks" or LFIA test strips and other solid supports has been described in the art. U.S. Patent Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. Patent Application Serial Nos. 10 / 278,676; 09 / 579,673 and 10 / 717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Three U.S. patents (U.S. Patent Nos. 4,444,880, issued to H. Tom; 4,305,924, issued to R.N. Piasio; and 4,135,884, issued to J.T. Shen) describe the use of "dipstick" technology to detect soluble antigens by immuno-chemical assays. The devices and methods of these three patents broadly describe a first component immobilized on a solid surface of a "dipstick" which is exposed to a solution containing soluble antigen that binds to the component immobilized on the "dipstick" prior to the component-antigen complex on the detection strip. Modifying the teachings of these "dipstick" technologies to detect CHI3L1 or PD-1 polypeptides is within the skill of the art. In some embodiments, the dipstick (or LFIA) may be applicable to urine samples. In some embodiments, the dipstick may be applicable to blood samples.

[0230] Immunochemistry is a series of techniques based on the use of specific antibodies, where the antibodies 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 the 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 specific cytological preparation (such as liquid-based preparation). In some cases, signal amplification can be incorporated into specific protocols, where, after application of an antibody reagent specific for platelets or white blood cells, a secondary antibody containing a label is used. Generally, for immunohistochemistry, tissue obtained from a subject and fixed with a suitable fixative (such as alcohol, acetone, and paraformaldehyde) is sectioned and reacted with the antibody. Conventional methods of immunohistochemistry are described in Buchwalow and Bocker (eds.) Immunohistochemistry: Basics and Methods Springer (2010); Lin and Prichard Handbook of Practical Immunohistochemistry Springer (2011); which are incorporated herein by reference in their entirety. In some embodiments, immunocytochemistry can be utilized, where, generally, tissue or cells obtained from a subject are fixed with a suitable fixative (such as alcohol, acetone, and paraformaldehyde) and reacted with the antibody. Methods of immunocytological staining of human samples are known to those of skill in the art and are described in, for example, Immunocytochemistry: A Practical Guide for Biomedical Research (I MMUNOCYTOCHEMISTRY : A P RACTICAL G UIDE FOR B IOMEDICAL R ESEARCH )(2009); which are incorporated herein by reference in their entirety.

[0231] In some embodiments, one or more of the antibody reagents described herein may comprise a detectable label and / or comprise the ability to generate a detectable signal (e.g., by catalyzing a reaction that converts a compound into a detectable product). Detectable labels can include, for example, absorbent dyes, fluorescent dyes, or radioactive labels. Detectable labels, methods for detecting them, and methods for incorporating them into antibody reagents are well known in the art.

[0232] In some embodiments, the detectable label can include a label detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means (e.g., fluorescence, chemifluorescence, or chemiluminescence, or any other suitable means). The detectable labels used in the methods described herein can be primary labels (where the label contains a moiety that can be directly detected or gives rise to a moiety that can be directly detected) or secondary labels (where the detectable label binds to another moiety to generate a detectable signal, e.g., as is common in immunolabeling using secondary and tertiary antibodies). The detectable label can be attached to the antibody reagent by covalent or non-covalent means. Alternatively, the detectable label can be attached, e.g., by direct labeling via ligand-receptor binding pair arrangements to a molecule or other such specific recognition molecule that binds to the antibody reagent. The detectable label can include, but is not limited to, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0233] 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, then its presence can be detected based on fluorescence. In some embodiments, the detectable label can be a fluorescent dye molecule or fluorophore, including but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthalaldehyde, fluorescamine, Cy3 TM 、Cy5 TM 、allophycocyanin, Texas Red, peridin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5 TM 、green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC), and Oregon Green TM 、rhodamine and its derivatives (such as Texas Red and tetrarhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes TM, 6 - Carboxyfluorescein (commonly abbreviated as FAM and F), 6 - Carboxy - 2’,4’,7’,4,7 - hexachlorofluorescein (HEX), 6 - Carboxy - 4’,5’ - dichloro - 2’,7’ - dimethoxyfluorescein (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 such as Cy3, Cy5, and Cy7 dyes; coumarins such as umbelliferone; benzylimine dyes such as Hoechst 33258; phenanthridine dyes such as Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes such as Cy3, Cy5 and other cyanine dyes; BODIPY dyes and quinoline dyes.

[0234] In some embodiments, the detectable label can be a radioactive label, including but not limited to 3 H, 125 I, 35 S, 14 C, 32 P, and 33 P.

[0235] In some embodiments, the detectable label can be an enzyme, including but not limited to horseradish peroxidase and alkaline phosphatase. Enzyme labels can generate, for example, chemiluminescent signals, color signals, or fluorescent signals. Enzymes expected to be used for detectably labeling antibody reagents include but are not limited to: malate dehydrogenase, staphylococcal nuclease, δ - V - steroid isomerase, yeast alcohol dehydrogenase, α - glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β - galactosidase, ribonuclease, urease, catalase, glucose - VI - phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0236] In some embodiments, the detectable label is a chemiluminescent label, including but not limited to lucigenin, luminol, fluorescein, isoluminol, thermatic acridinium ester, imidazole, acridinium salts, and oxalate esters.

[0237] In some embodiments, the detectable label can be a spectral colorimetric label, including but not limited to colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, and latex) beads.

[0238] In some embodiments, the antibody can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, such as the biotin-streptavidin system. In this system, the antibody immunoreactive (i.e., specific) with the biomarker of interest is biotinylated. The amount of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromogenic substrate. Such streptavidin-peroxidase detection kits are commercially available, for example, from DAKO; Carpinteria, California.

[0239] The antibody reagent can also be detectably labeled using a fluorescent-emitting metal such as 152 Eu or other metals of the lanthanide series. These metals can be attached to the antibody reagent using a metal chelating group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0240] The assays and methods described herein can involve determining whether an object has increased levels of CHI3L1 and PD-1 relative to a reference level. In some embodiments, the reference levels of CHI3L1 and PD-1 can be the levels of CHI3L1 and PD-1 in healthy objects not suffering from or not diagnosed with (e.g., cancer). In some embodiments, the reference level can be the level in a sample of a similar cell type, sample type, sample processing, and / or obtained from an object with similar age, gender, and other demographic parameters as the object in which the amounts 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, such as the same number and type of cells and / or the same type of sample material. Thus, in some embodiments, the levels of increased CHI3L1 and PD-1 can vary with demographic factors such as age, gender, genotype, environmental factors, and individual medical history. In some embodiments, the reference level can include the levels of CHI3L1 and PD-1 (e.g., CHI3L1 and PD-1 polypeptides) in the same type of sample taken from an object not showing any signs or symptoms of (e.g., cancer). In some embodiments, the reference levels of CHI3L1 and PD-1 can be the expression levels of CHI3L1 and PD-1 in a prior sample obtained from the object. This allows for a direct analysis of any level changes in the individual.

[0241] In some embodiments, if the levels of CHI3L1 and PD-1 are at least 1.25-fold (e.g., at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold or higher) of a reference level, then the levels of CHI3L1 and PD-1 can be increased relative to the reference level. In some embodiments, the expression levels of CHI3L1 and PD-1 can be normalized relative to the expression levels of one or more reference genes or reference proteins. In some embodiments, the expression levels of CHI3L1 and PD-1 can be normalized relative to a reference value.

[0242] In some embodiments, the expression levels of no more than 20 other genes are determined. In some embodiments, the expression levels of no more than 10 other genes are determined.

[0243] As used herein, the term "sample" or "test sample" refers to a sample collected or isolated from an organism, such as 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, etc. The term also includes mixtures of the above samples. The term "test sample" also includes untreated or pre-treated (or pre-processed) biological samples. In some embodiments, a test sample can contain cells from a subject. As used herein, the term "biological fluid" refers to any fluid obtained from a biological source, including but not limited to blood, urine, and body secretions.

[0244] A test sample can be obtained by taking a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g., isolated at a previous time point and by the same person or another person). In addition, a test sample can be a freshly collected or previously collected sample.

[0245] In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase "untreated test sample" refers to a test sample that has not undergone any prior sample pretreatment other than being diluted and / or suspended in a solution. Exemplary methods for processing 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 can be a frozen test sample, such as frozen tissue. Before employing the methods, assays, and systems described herein, the frozen sample can be thawed. After thawing, the frozen sample can be centrifuged before being subjected to the methods, assays, and systems described herein. In some embodiments, the test sample is a clarified test sample, e.g., prepared by centrifugation and collecting the supernatant containing the clarified test sample. In some embodiments, the test sample can be a preprocessed test sample, e.g., a supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combination thereof. In some embodiments, the test sample can be treated with chemical and / or biological reagents. During processing, chemical and / or biological reagents can be used to protect and / or maintain the stability of the sample, including the biomolecules therein (e.g., nucleic acids and proteins). An 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 are well aware of the methods and procedures for preprocessing biological samples suitable for determining CHI3L1 levels as described herein.

[0246] In some embodiments, the methods, assays, and systems described herein can further include the step of obtaining a test sample from a subject. In some embodiments, the subject can be a human subject.

[0247] In some embodiments, the methods, assays, and systems described herein can include creating a report based on the levels of CHI3L1 and PD-1. In some embodiments, the report represents the raw values of CHI3L1 and PD-1 in the test sample (plus, optionally, the levels of CHI3L1 and PD-1 in a reference sample) or, alternatively, it indicates the percentage or fold increase in the levels of CHI3L1 and PD-1 compared to a reference level, and / or provides a signal that the subject has or does not have a risk of having cancer.

[0248] As used herein, "at risk of having a disease" means having at least 2-fold, e.g., 2-fold, or 2.5-fold, or 3-fold, or 4-fold, or greater risk of having a particular disease compared to a subject who does not have elevated and / or increased levels of CHI3L1 and PD-1.

[0249] In some embodiments, the assay or method may further comprise: the step of administering anti-CHI3L1 or anti-PD-1 treatment. In some embodiments, the anti-CHI3L1 / anti-PD-1 therapy comprises an isolated bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR or CAR T cell as described herein; a nucleic acid; a cell; or a composition.

[0250] In one aspect of any of the embodiments, described herein are antibodies, antibody reagents, or antigen-binding portions thereof as described herein conjugated to a detectable label.

[0251] In one aspect of any of the embodiments, described herein are solid supports comprising bispecific antibodies, antibody reagents, antigen-binding fragments thereof as described herein. In some embodiments of any aspect, the bispecific antibody, antibody reagent, or antigen-binding fragment thereof is detectably labeled. In some embodiments of any aspect, the solid support comprises a particle, bead, polymer, or substrate.

[0252] In one aspect of any of the embodiments, described herein are molecular complexes comprising at least one bispecific antibody, antibody reagent, antigen-binding fragment thereof, or CAR as described herein that binds to a CHI3L1 polypeptide and a PD1 polypeptide.

[0253] In one aspect, described herein are kits comprising a composition as described herein, such as a composition comprising a bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR as described herein. A kit is any article (e.g., a package or container) containing at least one reagent (e.g., a bispecific antibody) that is promoted, distributed, or sold as a unit for performing the methods described herein. In some embodiments of any aspect, the bispecific antibody, antibody reagent, or antigen-binding fragment thereof as described herein is immobilized on a solid support. In some embodiments of any aspect, the solid support comprises a particle, bead, polymer, or substrate. In some embodiments of any aspect, the antibody, antibody reagent, or antigen-binding fragment thereof is detectably labeled.

[0254] The kits described herein may optionally comprise additional components for performing the methods described herein. By way of example, the kit may comprise a fluid (e.g., a buffer) suitable for containing a composition comprising a bispecific antibody, antigen-binding portion thereof, or CAR as described herein, instructional materials describing the implementation of the methods described herein, and the like. The kit may also comprise devices and / or reagents for delivering the composition as described herein. Additionally, the kit may include instructions and / or may provide information regarding the relevance of the results obtained.

[0255] The description of the embodiments disclosed herein is not intended to be exhaustive or to limit the scope of the disclosure to the exact forms disclosed. While specific embodiments and examples of the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be recognized by those of ordinary skill in the relevant art. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order or may perform the functions substantially concurrently. The teachings of the disclosure provided herein may be suitably applied to other programs or methods. The various embodiments described herein may be combined to provide further embodiments. If desired, aspects of the disclosure may be modified to employ the components, functions, and concepts of the above references and applications to provide still further embodiments of the disclosure. In addition, for considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the type or amount of biological or chemical action. These and other changes to the disclosure may be made in light of the above detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0256] The specific elements of any of the foregoing embodiments may be combined or replaced with elements in other embodiments. Additionally, while the advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the disclosure.

[0257] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limiting. While methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below.

[0258] Examples

[0259] The foregoing is a general description of the present invention, which will be more readily understood by reference to the following examples, which are only used to illustrate certain aspects and embodiments of the present invention and are not intended to limit the present invention.

[0260] Example 1 Generation and Characterization of the CHI3L1xPD1 Bispecific Antibody (FRGxPD1-ScFv)

[0261] To generate bispecific antibodies that recognize and neutralize CHI3L1 and PD1, we used the framework of an anti-human CHI3L1 antibody (designated "FRG") recently developed in our laboratory and described in U.S. Patent No. 10,253,111. The PD1 single-chain variable fragment (scFv-PD1) was generated with minor modifications based on sequence information obtained from the public domain. As shown in Table 1, ScFv-PD1 was attached to the light or heavy chain of the CHI3L1 antibody via a linker. Table 2 provides the amino acid sequences of ScFv-PD1 and the linker. The construct of the bivalent CHI3L1xPD1 antibody was confirmed by DNA sequence analysis.

[0262] The CHI3L1xPD1 construct was transfected into HEK-293T adherent cells individually. The binding affinities of the bispecific antibody for CHI3L1 and PD-1 were evaluated by competitive ELISA and compared with the binding of the individual antibody moieties.

[0263] As Figure 2 shown, the bispecific antibody secreted in the supernatant was able to detect recombinant human (rh) CHI3L1 and rhPD1. These studies showed that the bispecific antibody and the individual antibody moieties had similar affinity levels (which ) and similar limits of detection (LOD; 2 ng / ml) for rhCHI3L1 and rhPD1. A protein-A column was used to further purify the antibody.

[0264] In summary, we have successfully generated and characterized bispecific antibodies that exhibit high-affinity responses to rhCHI3L1 and rhPD1 (see Figure 1 and Figure 2 ). As Figure 1 shown, two different methods were used to generate these bispecific antibodies. Using these platforms, we generated CHI3L1-LC-PD1 and CHI3L1-HC-PD bispecific (bivalent) antibodies, each of which detected human CHI3L1 and human PD1. The affinity of the CHI3L1-LC-PD1 antibody for rhCHI311 and rhPD1 (evaluated by competitive ELISA assay) was estimated to be K D ≈1x10 -9 M, with a limit of detection (LOD) of 2 ng / ml. There was no significant difference in the binding affinity for rhCHI3L1 or rhPD1 between the CHI3L1-LC-PD1 and CHI3L1-HC-PD1 antibodies ( Figure 2 and data not shown).

[0265] Example 2 Characterization of the T cell-U87 binding and anti-tumor cytotoxic activity of the bispecific CHI3L1xPD1 antibody

[0266] The ability of Jurkat cells to bind to U87 cells and the anti-tumor cytotoxic activity of the bispecific CHI3L1xPD1 antibody were evaluated using an in vitro co-culture system that included U87 glioblastoma (ATCC#HTB-14) and Jurkat T cells (ATCC#TIB152). Human glioblastoma (U87) cells were grown in complete EMEM medium. Jurkat (T cells) were activated by stimulation with α-CD3 / α-CD28 antibody (5 μg / ml) in RPMI complete medium for 2 hours in 5% CO2 and air. The Jurkat cells were then centrifuged, washed and resuspended in fresh complete RPMI medium. U87 and Jurkat cells were cultured at a ratio of 1:6 in complete RPMI medium.

[0267] The responses of these co-cultures were evaluated in the presence of IgG isotype control, anti-PD1 alone, anti-CHI3L1 alone, anti-CHI311 and anti-PD-1 combination (each 5 μg / ml), or the above bispecific antibody. Overall, there were five treatment groups: (i) isotype IgG control, (ii) α-PD1, (iii) α-CHI3L1, (iv) α-CHI3L1 + α-PD1, and (v) bispecific α-CHI3L1xPD1. The 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 evaluated by TUNEL staining and LDH release cytotoxicity assay, as described below.

[0268] A.J URKAT Quantitative analysis of cell attachment to U87 cells

[0269] Jurkat T cells were activated by anti-(α)-human CD3 and α-CD28 antibodies (each 5 μg / ml, incubated at 37 °C in 5% CO 2 and air for 2 hours) and co-cultured with U87 glioblastoma cells using an isotype control or the other antibodies described above. CellBrite cell plasma 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 (original magnification 20x), and the mean was taken in 10 randomly selected microscopic fields.

[0270] As Figure 3 shown, treatment with the bispecific CHI3L1xPD1 antibody prominently enhanced the attachment of Jurkat T cells to U87 cells. Importantly, the effect of the bispecific antibody was significantly more prominent than that of treatment with α-CHI3L1 or α-PD1 alone or with the combination of α-CHI3L1 and α-PD1.

[0271] B. Quantitative analysis of TUNEL assay for apoptotic U87 cell death

[0272] Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling is used for in situ assessment of cell death and there are two different ways to perform it:

[0273] (i) Cells treated with co-culture in different chambers were fixed and permeabilized, and stained with propidium iodide (red dye) and Cyto (green dye). Fluorescent red cells are dead cells and green ones are live cells. The number of live and dead U87 cells was counted using a fluorescence microscope (original magnification 20×) and averaged over 10 randomly selected microscopic fields of view.

[0274] (ii) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling is used for in situ assessment of cell death. In the selected study, TUNEL positive staining was evaluated using a bright-field microscope. Dead or apoptotic cells were stained blue and live cells were stained with nuclear fast red. The number of live and dead U87 cells was counted using fluorescence and ordinary microscopes (original magnification 20×) and averaged over 10 randomly selected microscopic fields of view.

[0275] As Figure 4 shown, treatment with the bispecific CHI3L1xPD1 antibody prominently enhanced 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 prominent than that of treatment with α-CHI3L1 or α-PD1 alone or with the combination of α-CHI3L1 and α-PD1.

[0276] C. Quantification of granzyme and perforin accumulation

[0277] Granzyme and perforin are the major cytotoxic enzymes secreted by various activated cytotoxic T cells including Jurkat cells. The expression levels of these cytotoxic enzymes in co-cultured cells were measured using double-label 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 (original magnification 20×) and averaged over 10 randomly selected microscopic fields of view.

[0278] As Figure 5 and Figure 6As shown, treatment with the bispecific CHI3L1xPD1 antibody prominently enhanced the accumulation of granzyme and perforin in Jurkat T cells co-cultured with U87 cells. Importantly, the effect of the bispecific antibody was significantly more prominent than that of treatment with α-CHI3L1 or α-PD1 alone or with the combination of α-CHI3L1 and α-PD1.

[0279] These results indicate that the bispecific CHI3L1xPD1 antibody enhanced the cytotoxic effect of T cells in a synergistic manner.

[0280] D. LDH release cytotoxicity assay

[0281] Lactate dehydrogenase (LDH) is released into the culture medium after the loss of membrane integrity caused by cytotoxic damage. Therefore, the release of LDH is used as an indicator of cell death. This is evaluated using a coupled two-step reaction. In the first reaction, LDH catalyzes the reduction of NAD+ to NADH and H+ by the oxidation of lactate to pyruvate. + In the second step, diaphorase utilizes the newly formed NADH and H+ + to catalyze the reduction of the tetrazolium salt (INT) to formazan, which has a strong absorption at 490 - 520 nm.

[0282] The cytotoxicity level (%) was determined using a commercial assay kit (Pierce LDH cytotoxicity assay kit) according to the protocol provided by the manufacturer. In these experiments, cells were co-cultured in the presence or absence of the above antibodies. LDH in the culture medium was evaluated after overnight incubation. These values were compared to the following controls: (a) a cell-free complete medium control to determine the background activity of LDH present in the serum used for medium supplementation; (b) serum-free medium; (c) an LDH activity control (water); and (d) the maximum LDH activity released by cells treated with lysis buffer. In these assays, the cytotoxicity % was calculated as described below.

[0283]

[0284] As Figure 7 shown, treatment with the bispecific CHI3L1xPD1 antibody prominently enhanced the ability of Jurkat T cells to induce LDH release and cytotoxic responses in U87 cells. Importantly, the effect of the bispecific antibody was significantly more prominent than that of treatment with α-CHI3L1 or α-PD1 alone or with the combination of α-CHI3L1 and α-PD1.

[0285] Figure 8The synergistic effect of the bispecific CHI3L1xPD1 antibody was further demonstrated. The antitumor effect of the FRGxPD-1 bispecific antibody was evaluated in a co-culture system containing Jurkat cells and A375 human melanoma cells. Jurkat T cells were pre-activated by pretreatment with anti-CD3 and anti-CD28 (1 μg / mL each, incubated for 2 hours at 37 °C in 5% CO 2 and air). Then, Jurkat cells were co-cultured with A357 human melanoma cells for 24 hours. These co-cultures were carried out in the presence of the following antibodies: isotype control antibody (5 μg / mL), anti-PD-1 alone or anti-CHI3L1(FRG) alone (5 μg / mL), or in combination (2.5 μg / mL each), and the bispecific FRGxPD-1 antibody (5 μg / mL). Column A provides a representative demonstration and quantification of tumor cell death by apoptosis using an in situ cell detection kit - fluorescein dUTP. TUNEL(+) cells were stained green. Columns B-D provide a representative demonstration and quantification of the expression of CD8 (Column B), perforin (Column C), and granzyme (Column D) in Jurkat T cells. Tumor cells were green, and stained positive Jurkat cells were yellow-orange. Column E provides a representative demonstration and quantification of PTEN in tumor cells. Tumor cells were green, and PTEN was yellow-orange. Row F provides the quantification evaluated in Columns A-E. The percentages of 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) are illustrated. These evaluations were performed using a fluorescence microscope (original magnification 20×). In these quantifications, 10 randomly selected regions were evaluated. Figure 8 The data in

[0286] showed that treatment with the bispecific CHI3L1xPD1 antibody induced a synergistic CTL-mediated tumor cell death response and tumor cell PTEN expression.

[0287] The above written description is considered sufficient to enable a person skilled in the art to practice the invention and the embodiments. The scope of the invention and the embodiments should not be limited by the examples provided, as these examples are intended to be a single illustration of one aspect, and other functionally equivalent embodiments are also within the scope of the present disclosure. Various modifications other than those shown and described herein will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. The advantages and objectives described herein are not necessarily included in each embodiment. Those skilled in the art should understand or be able to determine many equivalent forms of the specific embodiments described herein using no more than routine experimentation. Such equivalent forms are intended to be covered by the appended claims.

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[0366] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; are hereby expressly incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the methods described in such publications that may be used in conjunction with the techniques described herein. The publications provided are only for their disclosure prior to the filing date of the present application. Nothing in this regard shall be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation of the content of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the date or content of these documents.

Claims

1. A bispecific antibody that detects and neutralizes chitinase 3-like protein 1 (CHI3L1) and programmed death receptor 1 (PD-1), which comprises an antigen-binding portion of an anti-human PD-1 antibody and an antigen-binding portion of an anti-human CHI3L1 antibody, wherein the antigen-binding portion of the anti-human CHI3L1 antibody comprises the following complementarity-determining regions (CDRs): (a) a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4; (b) a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; (c) a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; (d) a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1; (e) a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2; and (f) a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and wherein the antigen-binding portion of the anti-human PD-1 antibody consists of the amino acid sequence of SEQ ID NO: 35, and the bispecific antibody comprises an anti-human PD-1 single-chain variable fragment ScFv-PD1 attached to the anti-human CHI3L1 antibody framework, wherein the ScFv-PD1 is attached to the anti-human CHI3L1 antibody heavy chain or the ScFv-PD1 is attached to the anti-human CHI3L1 antibody light chain.

2. The bispecific antibody according to claim 1, wherein the ScFv-PD1 is attached to the anti-human CHI3L1 antibody heavy chain.

3. The bispecific antibody according to claim 1, wherein the ScFv-PD1 is attached to the anti-human CHI3L1 antibody light chain.

4. The bispecific antibody according to claim 2, wherein the anti-human CHI3L1 antibody heavy chain has the amino acid sequence of SEQ ID NO:

13.

5. The bispecific antibody according to claim 3, wherein the anti-human CHI3L1 antibody light chain has the amino acid sequence of SEQ ID NO:

14.

6. The bispecific antibody according to any one of claims 1-5, wherein the bispecific antibody enhances the attachment of Jurkat T cells to U87 cells.

7. The bispecific antibody according to claim 6, wherein the bispecific antibody enhances the ability of Jurkat T cells to induce a cytotoxic / apoptotic response in U87 cells.

8. The bispecific antibody according to claim 6, wherein the bispecific antibody enhances the accumulation of granzyme and perforin in Jurkat T cells co-cultured with U87 cells.

9. The bispecific antibody according to claim 6, wherein the bispecific antibody enhances the ability of Jurkat T cells to induce a cytotoxic response and lactate dehydrogenase (LDH) release in U87 cells.

10. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1-9 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, which further comprises a chemotherapeutic agent.

12. Use of a bispecific antibody according to any one of claims 1-9 or a pharmaceutical composition according to any one of claims 10-11 in the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the cancer is selected from glioblastoma and melanoma.

13. The use according to claim 12, wherein the cancer is a malignant cancer.

14. The use according to claim 12, wherein the cancer is a primary cancer or a metastatic cancer.

15. The use according to any one of claims 12-14, wherein the subject is determined to have an elevated CHI3L1 level.

16. The use according to claim 15, wherein the CHI3L1 is circulating CHI3L1.

17. The use according to any one of claims 12-14, wherein the cancer expresses PD-L1.

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