Bispecific antibodies against chi3l1 and PD1 with enhanced t cell-mediated cytotoxic effects on tumor cells

Abstract

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

Description

Technical Field

[0001] Embodiments of the present invention relate to bispecific antibodies that simultaneously target both CHI3L1 and the immune checkpoint molecule PD-1. These bispecific antibodies, alone or in combination, exhibit an increased synergistic cytotoxic effect that is greater than the effects of the individual CHI3L1 and PD-1 antibodies. Statement regarding federally funded research or development This invention was developed with NIH CADET grant UH2 HL123876 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0002] Currently, immunotherapies against individual immune checkpoint inhibitor (ICPI) molecules are effectively applied to patients with various malignancies, including lung cancer and glioblastoma. Examples include antibodies against moieties such as programmed death receptor 1 (PD-1). However, only some patients respond to these treatments. In addition, the responses seen are often not sustained. As a result, intensive efforts have been made worldwide to develop methods to increase the effectiveness of ICPI immunotherapies using antibodies against immune checkpoint molecules, including PD-1.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Therefore, there is a need for more effective immunotherapies against individual immune checkpoint inhibitor molecules such as PD-1.

Means for Solving the Problems

[0004] Previous studies have demonstrated that chitinase 3-like-1 (CHI3L1) plays an important role in the etiology of various cancers. Furthermore, the inventors have previously demonstrated that the combination of anti-CHI3L1 antibody and anti-PD-1 antibody results in a synergistic effect in the treatment of cancer. Based on these findings, the inventors hypothesized that the simultaneous targeting of CHI3L1 and PD-1 may have additional synergistic and / or additive antitumor effects. To investigate these possibilities, a bispecific antibody that reacts with both CHI3L1 and PD-1 was developed.

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

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

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

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

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

[0010] As described herein, the bispecific antibodies of the present invention have a significant anti-tumor effect. These bispecific antibodies, alone or in combination, exhibited an increased synergistic cytotoxic effect as compared to the effects of the individual CHI3L1 and PD-1 antibodies. The bispecific antibodies of the present invention (i) increase the attachment of Jurkat T cells to U87 cells; (ii) increase the ability of Jurkat T cells to induce a cytotoxic / apoptotic response in U87 cells; (iii) increase the accumulation of granzyme and perforin in Jurkat T cells co-cultured with U87 cells, and / or (iv) increase the ability of Jurkat T cells to induce lactate dehydrogenase (LDH) release and a cytotoxic response in U87 cells.

[0011] 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.

[0012] Embodiments of the present invention also provide a method of treating cancer in a subject by administering a therapeutically effective amount of a bispecific antibody or pharmaceutical composition of the present invention. In one embodiment, the cancer is a malignant cancer. In one embodiment, the cancer is a primary cancer or a metastatic cancer. In one embodiment, the cancer is one of 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 elevated levels of CHI3L1. In one embodiment, the elevated CHI3L1 level is circulating CHI3L1. In one embodiment, the cancer expresses PD-L1.

[0013] The bispecific 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, either alone or in combination.

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

[0015] For illustrative purposes, certain embodiments of the present invention are shown in the drawings described below. Like numbers in the drawings indicate like elements throughout. However, it should be understood that the present invention is not limited to the exact arrangements, dimensions, and instruments shown.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

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

[0018] Definitions For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implicit from the context, the following terms and phrases include the meanings provided below. The definitions are provided to assist in explaining specific embodiments and the scope of the present invention is limited only by the claims. Since it is to be determined, it is not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an obvious difference between the use of terms in the art and the definitions provided in this specification, the definitions provided in this specification shall prevail.

[0019] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes combinations of two or more cells and the like.

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

[0021] The abbreviation "e.g." is derived from the Latin exempli gratia and is used in this specification to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0022] As used herein, the term "substantially" or "about" with respect to a value or parameter generally means within 5%, 10%, 15%, or 20% in either direction (greater or less) of the number, unless otherwise specified or apparent from the context (except where such numbers are less than 0% or greater than 100% of the possible values). As used herein, a reference to a value or parameter of "substantially" or "about" includes (and describes) embodiments directed to that value or parameter. For example, a description that refers to "about X" includes a description of "X".

[0023] As used herein, the term "comprising" means that other elements may also be present in addition to the recited defined elements, and the use of "comprising" indicates inclusion rather than limitation.

[0024] The term "consisting of" refers to the compositions, methods, and each of their components described herein, and excludes any element not described in that description of the embodiment.

[0025] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic and novel or functional characteristics (singly or plurally) of that embodiment of the invention.

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

[0027] As used herein, the phrases "therapeutically effective amount", "effective amount" or "effective dosage amount" 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 provides a statistically significant reduction in at least one symptom, sign or marker of a tumor or malignancy. It is understood that many methods are known in the art for determining an effective amount for a given use. For example, dosing determination Pharmacological methods for determination can be used in a therapeutic situation. In the context of therapeutic or prophylactic application, the amount of the composition administered to a subject depends on the type and severity of the disease, as well as individual characteristics such as general health, age, gender, weight, and drug tolerance. It also depends on the degree, severity, and type of the disease. A person skilled in the art can determine an appropriate dosage according to these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds.

[0028] As used herein, the terms "treating", "treatment", "treat", or "ameliorate", when used with respect to a disease, disorder, or medical condition, refer to a therapeutic treatment for a condition in which a subject causes the progression or severity of symptoms or conditions to regress, reduce, alleviate, inhibit, decelerate, or stop. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. When one or more symptoms or clinical markers are reduced, the treatment is generally "effective". Alternatively, when the progression of a condition is reduced or interrupted, the treatment is "effective". That is, "treatment" includes not only improvement of symptoms or markers, but also stopping or at least decelerating the progression or worsening of symptoms expected if treatment is not performed. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms (singly or plurally), decrease in the degree of deficit, stable (i.e., non-worsening) state of a tumor or malignancy, delay or deceleration of tumor growth and / or metastasis, and extension of lifespan, compared to what is expected if treatment is not performed.

[0029] As used herein, the term "administering" refers to placing a bispecific antibody disclosed herein within a subject by a method or route that results in at least partial delivery of the drug at the desired site. A pharmaceutical composition containing a compound disclosed herein can be administered by any suitable route that provides an effective treatment in a subject.

[0030] As used herein, the term "long-term" administration means that a therapeutic agent or drug is administered for at least 12 weeks. This includes that the therapeutic agent or drug is administered to be effective over a period of at least 12 weeks or for a period of at least 12 weeks. For example, when a sustained-release composition or a long-acting therapeutic agent or drug is used, it does not necessarily mean that the administration itself is carried out for 12 weeks. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or at least 1, 2, 3, 5, 7, 10 years or more.

[0031] Administration of the compositions contemplated herein can be carried out by any convenient method including aerosol inhalation, injection, oral ingestion, transfusion, implantation or transplantation. In a preferred embodiment, the composition is administered parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, 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.

[0032] As used herein, the term "cancer" generally relates to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body via the blood and lymphatic systems. There are mainly several types of cancer. A carcinoma is a cancer that occurs in the skin or in tissues that line or cover internal organs. A sarcoma is a cancer that arises from bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissues. Leukemia is a cancer that originates in hematopoietic tissues such as the bone marrow and has a large number of abnormalities Cancer is a disease that produces abnormal blood cells and releases them into the bloodstream. Lymphoma and multiple myeloma are cancers that originate from cells of the immune system. Central nervous system cancers are cancers that originate from the tissues of the brain and spinal cord.

[0033] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, 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 beyond normal limits), invasion (i.e., penetration and destruction of adjacent tissues), and metastasis (i.e., spread to other parts of the body via lymph or blood). As used herein, the term "metastasize" refers to the spread of cancer from one part of the body to another. A tumor formed by the dispersed cells is called a "metastatic tumor" or a "metastasis". Metastatic tumors contain cells similar to those of the original (primary) tumor.

[0034] As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow larger but does not spread to other parts of the body. Benign tumors are self-limiting and typically do not invade or metastasize.

[0035] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by abnormal growth of cells and can be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not necessarily form tumors. For cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

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

[0037] Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, leukemias, basal cell cancers, biliary tract cancers; bladder cancers; bone cancers; brain and CNS 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 (e.g., gastrointestinal cancers); glioblastoma (GBM); hepatocellular carcinomas; hepatocellular cancers; intraepithelial neoplasms; kidney cancers or renal 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, e.g., 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 cancers; stomach cancers; testicular tumors; thyroid cancers; uterine cancers or endometrial cancers; urinary system cancers; vulvar cancers; and other carcinomas and sarcomas; and B cell lymphomas (e.g., low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth, edema (such as that associated with brain tumors), and Meigs syndrome associated with phakomatoses.

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

[0039] A subject has been previously diagnosed with, or is suffering from or identified as having, a condition (e.g., cancer) that requires treatment or one or more complications associated with such a condition, although in some cases the subject may not yet need treatment for the condition or one or more complications associated with that condition. Alternatively, a subject may be one who has not been previously diagnosed with a condition that requires treatment or one or more complications associated with such a condition. For example, a subject may be one who exhibits one or more risk factors for a condition, or one or more complications associated with a condition, or a subject who exhibits no risk factors. A "subject in need of treatment" for a particular condition can be a subject who has the condition, has been diagnosed as having the condition, or is at risk of developing the condition. As further described herein, in some embodiments, the subject is a subject determined to have an elevated level of CHI3L1. In some embodiments, CHI3L1 is circulating CHI3L1. In some embodiments, the subject has a cancer that expresses PD-L1.

[0040] The terms "decrease", "reduce", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce", "reduction" or "decrease" or "inhibit" typically means a decrease of at least 10% compared to a reference level (e.g., the absence of a given treatment or agent), e.g., 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, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The reduction can preferably be lowered to a level that is considered within the normal range for an individual without a given disorder.

[0041] The terms "increased", "increase", "augment", or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "increased", "increase", "augment", or "activate" mean an increase of at least 10% compared to a reference level, e.g., 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 an increase including up to and including 100%, or any increase between 10 - 100%, 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 or more compared to a reference level, or any increase between 2-fold - 10-fold or more. In the context of a marker or symptom Here, "increasing" is a statistically significant increase at such levels.

[0042] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues linked to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids that include modified amino acids (e.g., phosphorylated, glycosylated, glycocylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used with respect to relatively large polypeptides, while the term "peptide" is often used with respect to small polypeptides, although their use in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, as well as other equivalents, variants, fragments, and analogs as described above.

[0043] In various embodiments described herein, it is further contemplated to include any variant (naturally occurring or not) of a particular polypeptide described, alleles, homologs, conservatively modified variants, and / or conservative substitution variants. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small number of amino acids in the encoded sequence are "conservatively modified variants," and that such modifications result in substitution of chemically similar amino acids and retain the desired activity of the polypeptide. Such conservatively modified variants are added to and not excluded from polymorphic variants, interspecies homologs, and alleles that are consistent with the present disclosure.

[0044] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) can be a functional fragment 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.

[0045] In some embodiments, the polypeptides described herein can be variants of the sequences described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained, for example, by mutation of a native nucleotide sequence. As referred to 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 that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes sequences that contain one or more additions, deletions, or substitutions of nucleotides as compared to the native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0046] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid 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.

[0047] In some embodiments of any of the aspects, the polypeptides, nucleic acids, or cells described herein can be manipulated. As used herein, "manipulated" refers to a manner that is manipulated by human hands. For example, a polypeptide is considered "manipulated" if at least one aspect of the polypeptide, such as its sequence, is manipulated by human hands so as to be different from its naturally occurring aspect. It is a common practice and, as understood by those skilled in the art, the progeny of a manipulated cell are typically still referred to as "manipulated" even though the actual manipulation was performed on an entity that preceded them.

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

[0049] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptide from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. An expression vector can contain additional elements. For example, an expression vector can have two replication systems and thus be capable of maintaining itself in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and protein, and, if applicable, the secretion of the protein, and includes, but is not limited to, for example, transcription, transcriptional processing, translation, and protein folding, modification and processing. "Expression product" includes RNA transcribed from a gene and polypeptide obtained by translation of mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene can or can not include regions before and after the coding region, for example, 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "trailer" sequence, and intervening sequences (introns) between individual coding segments (exons).

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

[0051] As used herein, "engineered" refers to a manner that is manipulated by human hands. For example an antibody, antibody reagent, antigen-binding portion thereof, CAR or bispecific antibody is considered "engineered" if its sequence of the antibody, antibody reagent, antigen-binding portion thereof, CAR or bispecific antibody is manipulated by human hands such that it is different from the sequence of a naturally occurring antibody. As is common practice and understood by those of skill in the art, progeny and copies of engineered polynucleotides and / or polypeptides are typically still referred to as "engineered" even though the actual manipulation was performed on an entity that preceded them.

[0052] As used herein, an "epitope" can be formed on a polypeptide from adjacent amino acids, or on a polypeptide from non-adjacent amino acids that are adjacent by the tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are typically lost when treated with a denaturing solvent. An epitope typically contains at least 3, more usually at least 5, about 9, or about 8 - 10 amino acids in its native spatial conformation. An "epitope" includes a structural unit that is normally bound by an immunoglobulin VH / VL pair. An epitope defines the minimal binding site of an antibody and thus represents the target of antibody specificity. In the case of a single domain antibody, the epitope represents a structural unit bound by the isolated variable domain. The terms "antigenic determinant" and "epitope" can also be used interchangeably herein. In certain embodiments, an epitope determinant includes a chemically active surface grouping of a molecule such as an amino acid, sugar side chain, phosphoryl, or sulfonyl, and in certain embodiments can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0053] As used herein, the term "antibody" refers to an immunoglobulin molecule and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that immunospecifically bind to an antigen. The term also refers to antibodies composed of various forms including two immunoglobulin heavy chains and two immunoglobulin light chains, and their full-length antibodies and antigen-binding portions, e.g., immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab’, F(ab’)2, Fv, disulfide-bonded Fv, scFv, single domain antibodies (dAb), diabodies, multispecific antibodies, bispecific antibodies, anti-idiotype antibodies, bispecific antibodies, their functionally active epitope-binding portions, and / or bifunctional hybrid antibodies.

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

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

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

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

[0058] Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be derived from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates), and can be primatized antibodies. Antibodies can also include diabodies, humanized antibodies, chimeric antibodies, and the like.

[0059] Furthermore, the antibodies, antigen-binding portions thereof, or CARs described herein can be part of a larger immune adhesion molecule formed by covalent or non-covalent binding of the above antibodies or antibody portions to one or more additional proteins or peptides. Examples of such immune adhesion molecules include the use of a streptavidin core region to prepare a tetrameric scFv molecule, as well as the use of cysteine residues, marker peptides, and C-terminal polyhistidinyl, e.g., a hexahistidinyl tag (the "hexahistidinyl tag" disclosed as SEQ ID NO: 30 in U.S. Patent No. 10,253,111), to generate divalent and biotinylated scFv molecules.

[0060] ​In some embodiments, the antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein can be immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fabs, Fab’, F(ab’)2, Fvs, disulfide-bonded Fvs, scFvs, single-domain antibodies, diabodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, bispecific antibodies, and functionally active epitope-binding portions thereof.

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

[0062] The term "human antibody" refers to an antibody whose variable and constant regions correspond to or are derived from immunoglobulin sequences of the human germ line, as described, for example, by Kabat et al. (1991). However, a human antibody can include amino acid residues not encoded by the immunoglobulin sequences of the human germ line (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by somatic mutations in vivo), for example, in the CDRs, particularly CDR3. The recombinant human antibodies described herein can have variable regions and can also include constant regions derived from immunoglobulin sequences of the human germ line. See Kabat et al. (1991). However, according to certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if transgenic animals that are due to human Ig sequences are used, somatic in vivo mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibody are related to or derived from the VH and VL sequences of the human germ line but are sequences that do not naturally occur in vivo within the human antibody germ line repertoire. According to certain embodiments, this type of recombinant antibody is the result of selective mutagenesis or back-mutation, or both. Preferably, the mutagenesis results in an affinity for a target that is greater than that of the parental antibody and / or an affinity for a non-target structure that is less than that of the parental antibody. Generating humanized antibodies from the sequences and information provided herein can be carried out by one of ordinary skill in the art without undue experimentation. In one approach, there are four general steps used to humanize a monoclonal antibody. See, for example, U.S. Patent Nos. 5,585,089, 6,835,823, and 6,824,989. These 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 during the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody.

[0063] In some embodiments, the bispecific antibodies, antibody reagents, their antigen-binding portions, and / or CARs described herein can be variants of the sequences described herein, e.g., conservative substitution variants of antibody polypeptides. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained, for example, by mutation of a 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 that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes one or more additions, deletions, or substitutions of nucleotides as compared to the native or reference DNA sequence, but encodes a variant protein or a portion thereof that retains an activity, e.g., antigen-specific binding activity to a relevant target polypeptide, e.g., CHI3L1 or PD-1. A variety of PCR-based site-directed mutagenesis approaches are also known in the art and can be applied by those skilled in the art.

[0064] Typically, the CDR regions in humanized antibodies and human antibody variants are substantially identical, and more typically identical, to the corresponding CDR regions in the mouse or human antibodies from which they are derived. In some embodiments, it is possible to make one or more conservative amino acid substitutions of CDR residues without significantly affecting the binding affinity of the resulting humanized immunoglobulin or human antibody variant. In some embodiments, the substitutions in the CDR regions can increase the binding affinity.

[0065] The term "chimeric antibody" refers to an antibody that includes sequences for the variable regions of the heavy and light chains from one species, and constant region sequences from another species, such as an antibody having mouse heavy and light chain variable regions linked to human constant regions. A humanized antibody has substantially the variable region framework residues from a human antibody (referred to as the acceptor antibody), and complementarity determining regions from a substantially non-human antibody, such as a mouse antibody (referred to as the donor immunoglobulin). Also, the constant region(s), when present, are substantially or completely of human immunoglobulin origin. The human variable domain is usually selected from a human antibody whose framework sequence shows a high degree of sequence identity with the (mouse) variable region domain from which the CDRs were 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 sequence can be that of a naturally occurring human antibody or a consensus sequence of several human antibodies.

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

[0067] Furthermore, as described herein, recombinant humanized antibodies can be further optimized to reduce potential immunogenicity while maintaining functional activity for treatment in humans. In this regard, functional activity means a polypeptide that can exhibit one or more known functional activities 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. Furthermore, a polypeptide having functional activity means that the polypeptide exhibits an activity similar to, but not necessarily identical to, the activity of the reference antibody, antigen-binding portion thereof, or CAR described herein, and includes the mature form measured in a specific assay such as a biological assay, regardless of the presence or absence of dose-dependence. When dose-dependence exists, it need not be identical to that of the reference antibody, antigen-binding portion thereof, or CAR, but rather is substantially similar to the dose-dependence in a given activity as compared to the reference antibody, antigen-binding portion thereof, or CAR described herein (i.e., the candidate polypeptide exhibits greater activity or activity less than or equal to about 1 / 25, about 1 / 10, or about 1 / 3 of the antibody, antigen-binding portion, and / or CAR described herein). In some embodiments, the antibody reagents (e.g., antibodies or CARs) described herein are not naturally occurring biomolecules. For example, a mouse antibody raised against a human-derived antigen does not occur naturally without human intervention and manipulation, e.g., manufacturing processes performed by humans. Chimeric antibodies are also not naturally occurring biomolecules, e.g., in that they are obtained from multiple species and contain sequences assembled into a recombinant molecule. In certain embodiments, the human antibody reagents described herein are not naturally occurring biomolecules, e.g., fully human antibodies directed against human antigens are subject to negative selection in nature and are not naturally found in the human body.

[0068]

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

[0070] "Binding activity" is a measure of the strength of binding between an antigen-binding molecule (such as an antibody or antigen-binding portion thereof described herein) and a related antigen. Binding activity is related to both the affinity of the epitope for its antigen-binding site on the antigen-binding molecule and the number of appropriate binding sites present on the antigen-binding molecule. Typically, an antigen-binding protein (e.g., an antibody or portion of an antibody described herein) has a dissociation constant (10 -5 ~10 -12 mol / liter or less of K D , e.g., 10 -7 ~10 -12 mol / liter or less, or 10 -8 10 -8 ~10 -12 mol / liter) (i.e., 10 5 ~10 12 liter / mol or more of an association constant (K A ), e.g., 10 7 ~10 12 liter / mol or 10 8 ~10 12 liter / mol) for binding to their cognate or specific antigen. Any K -4 value greater than 10 D mol / liter (or any K 4 value lower than 10 -1 M) is generally considered to indicate non-specific binding. K A for biologically relevant (e.g., specific) interactions is typically in the range of 10 D M (0.1 nM) to 10 -10 M (10000 nM). The stronger the interaction, the lower the K -5 M (10000 nM). The stronger the interaction, the lower the K DIt becomes low. For example, the binding site on the antibody or a part thereof described in this specification binds to the desired antigen with an affinity of less than 500 nM, such as less than 200 nM, or less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or epitope can be determined by, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, different variants thereof that are known per se in the art, and any other appropriate methods mentioned herein that are known per se.

[0071] Accordingly, as used herein, "selectively binds" or "specifically binds" means that the peptide (e.g., antibody, CAR, bispecific antibody, or a part thereof) described herein has a K D 10 -5 M (10000 nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less, and refers to the ability to bind to a target such as an antigen present on the cell surface of a cancer cell. Specific binding can be affected by, for example, the affinity and binding activity of the polypeptide agent, as well as the concentration of the polypeptide agent. One of ordinary skill in the art can determine the appropriate conditions for the polypeptide agent described herein to selectively bind to a target using any appropriate method such as titration of the polypeptide agent in an appropriate cell-binding assay. A polypeptide specifically bound to a target is not displaced by a non-similar competitor. In certain embodiments, an antibody, an antigen-binding portion thereof, a CAR, or a bispecific antibody is said to specifically bind to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, the bispecific antibody, an antigen-binding portion thereof, or a CAR described herein has a 10

[0072] In some embodiments, the bispecific antibody, an antigen-binding portion thereof, or a CAR described herein has a 10-5 Less than M(10000 nM), for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or a dissociation constant (K D ) less than that binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -5 ) of about 10 -6 M to 10 D ) that binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -6 ) of about 10 -7 M to 10 D ) that binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -7 ) of about 10 -8 M to 10 D ) that binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -8 ) of about 10 -9 M to 10 D ) that binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -9 ) of about 10 -10 M to 10 D ) that binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -10 ) of about 10 -11 M to 10 D ) that binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -11 ) of about 10 -12 M to 10D ) binds to CHI3L1 and PD-1. In some embodiments, the bispecific antibodies, antigen-binding portions thereof, or CARs described herein have a dissociation constant (K -12 ) of less than 10 D M and bind to CHI3L1 and / or PD-1.

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

[0074] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It is to be understood that the invention is not limited to the specific methods, protocols, and reagents, etc. described herein, as these may themselves vary. The terms used herein are for the purpose of describing only particular embodiments and are not intended to limit the scope of the invention, which is defined only by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition (2011); The Encyclopedia of Molecular Cell Biology and Molecular Medicine (1999 - 2012); Molecular Biology and Biotechnology: A Comprehensive Desk Reference (1995); Immunology (2006); Janeway’s Immunobiology (2014); Lewin’s Genes XI (2014); Molecular Cloning: A Laboratory Manual, 4th Edition (2012); Basic Methods in Molecular Biology (2012); Laboratory Methods in Enzymology: DNA (2013); Current Protocols in Molecular Biology (CPMB) (2014); Current Protocols in Protein Science (CPPS) (2005); and Current Protocols in Immunology (CPI) (2003), the contents of which are hereby incorporated by reference in their entirety.

[0075] One of ordinary skill in the art can readily identify the chemotherapeutic agents to be used. For example, see Physicians’ Cancer Chemotherapy Drug Manual (2014); Chapter 85 in Harrison’s Principles of Internal Medicine, 18th Edition (2011); Chapters 28-29, in Abeloffs Clinical Oncology, 5th Edition (2013); The Cancer Chemotherapy Handbook, 4th Edition (2003).

[0076] In some embodiments of any of the aspects, the disclosure described herein is not directed to processes of cloning a human, processes of modifying the genetic identity of the human germ line, uses of a human embryo for industrial or commercial purposes, or processes of modifying the genetic identity of an animal that are likely to cause suffering to the animal without any substantial medical benefit to a human or an animal, and also animals resulting from such methods.

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

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

[0079] In previous studies by the inventors, it has been shown that (a) inhibition of CHI3L1 and / or CHI3L1 signaling, and (b) inhibition of at least one immune checkpoint protein such as PD-1 provide a synergistic effect in the treatment of cancer, such as lung cancer. See, for example, U.S. Application Publication No. 2019 / 0062457. When the anti-CHI3L1 antibody FRG was administered in combination with an anti-PD-1 antibody, a synergistic effect was observed, and this combination showed improved efficacy in reducing B16F10 metastasis, thereby suggesting that the combination of CHI3L1 inhibition and inhibition of checkpoint proteins provides a synergistic effect in the treatment of cancer. See Example 2 of U.S. Application Publication No. 2019 / 0062457. A bispecific antibody that specifically binds to both the CHI3L1 polypeptide and the PD-1 polypeptide and simultaneously detects and neutralizes is hypothesized to be able to exhibit an improved synergistic effect in the treatment of cancer.

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

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

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

[0083] One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence are “conservatively modified variants” where the alteration results in substitution of an amino acid with a chemically similar amino acid and which maintain the ability to specifically bind to a target antigen (e.g., CHI3L1 and PD-1). Such conservatively modified variants are included within the scope of the present disclosure in addition to polymorphic variants, interspecies homologs, and alleles and are not excluded.

[0084] Examples of substitution variants include, without altering the sequence of the CDR, for example, conservative substitutions of amino acids within the V H or V L domain. Conservative substitutions in sequences not included in the CDR may be substitutions to the wild-type or naturally occurring sequence, for example, substitutions to the framework and / or constant regions of a human or murine antibody sequence.

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

[0086] Amino acids can be classified according to the similarity in the nature of their side chains (Biochemistry, 2nd Edition (1975) pages 73-75): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Tr (1) Hydrophobic: Phe(F), Trp(W), Met(M); (2) Non-charged 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, the naturally occurring residues can be grouped based on the nature of their common side chains: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions require the exchange of one member of one of these classes for another class. Certain conservative substitutions include, for example, substitution of Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Ala or Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg, Gln or Glu; Met for Leu, Tyr or Ile; Phe for Met, Leu or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, Ile or Leu.

[0087] The variant amino acid sequence or DNA sequence is 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 the native or reference sequence. The degree of homology (percent identity) between the native sequence and the mutant sequence can be determined, for example, for this purpose, by comparing the two sequences using freely available computer programs commonly used on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0088] Modification of a natural amino acid sequence can be achieved by any of a number of techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides that contain a mutant sequence adjacent to a restriction site that enables ligation to a fragment of the natural sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis method can be used to provide a modified nucleotide sequence having specific codons modified according to the required substitution, deletion, or insertion.

[0089] Any cysteine residues not involved in maintaining the proper conformation of the polypeptide can generally also be replaced with serine in order to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or promote oligomerization.

[0090] In certain embodiments where the antibodies, antigen-binding portions thereof, or CARs described herein comprise 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 skilled in the art. For example, Fujii, 2004, "Antibody affinity maturation by random mutagenesis" Methods in Molecular Biology: Antibody Engineering 248:345-349 (entirely incorporated herein by reference), particularly FIGS. 2 and 3.3, describe methods for generating a library of any CDR of interest. Thereby, one skilled in the art can obtain the specific C described herein. Alternative CDRs that include conservative substitution variants of the DR sequences can be identified and, when present in the antibodies or antigen-binding portions thereof described herein, result in an antigen or antigen-binding portion thereof that binds to a cancer cell surface antigen. Also, the methods described by Fujii et al. enable one of ordinary skill in the art to screen for light chain sequences that, when combined with known heavy chain fragments, give the desired binding behavior, and vice versa.

[0091] In some embodiments, the CAR comprises an extracellular domain that includes 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, as well as exemplary epitopes, are described elsewhere herein.

[0092] As used herein, "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide comprising an antigen-binding domain (e.g., the antigen-binding portion of an antibody (e.g., scFv)), a transmembrane domain, and a T cell signaling and / or T cell activation domain. A CAR has the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner by utilizing the antigen-binding properties of a monoclonal antibody. Non-MHC-restricted antigen recognition confers upon T cells expressing the CAR the ability to recognize antigens that are independent of antigen processing, thus bypassing a major mechanism of tumor escape. Further, when expressed in T cells, the CAR preferably does not dimerize with the endogenous T cell receptor (TCR) alpha and beta chains. Most commonly, the extracellular binding domain of a CAR is composed of a single-chain variable fragment (scFv) derived from fusing the variable heavy and light chain regions of a mouse or humanized monoclonal antibody. Alternatively, in various embodiments, the scFv can use an scFv derived from a Fab (e.g., instead of an antibody obtained from a Fab library), which is fused to a transmembrane domain and then to an intracellular signaling domain. "First-generation" CARs include those that provide only the CD3 zeta (CD3ζ) signal upon antigen binding, and "second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ). "Third-generation" CARs include those that provide multiple co-stimulatory (e.g., CD28 and CD137) domains and an activation domain (e.g., CD3ζ). In various embodiments, the CAR is selected to have high affinity or binding activity 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); Tamada et al. (2012), each of which is incorporated herein by reference in its entirety.

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

[0094] In some embodiments, the CAR contemplated herein can include linker residues between the various domains and can be added, for example, for proper spacing and conformation of the molecule. In certain embodiments, the linker is a variable region linker sequence. A "variable region linker sequence" connects the VH domain and the VL domain such that the resulting polypeptide retains the specific binding affinity for the same target molecule as an antibody that includes the same light and heavy chain variable regions. It is an amino acid sequence that provides a spacer function compatible with the interaction of the two sub-binding domains. The CAR contemplated herein can include 1, 2, 3, 4, or 5 or more linkers. In certain embodiments, the length of the linker is from about 1 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids, or any intervening amino acid length. 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.

[0095] In certain embodiments, one or more "spacer domains" follow the binding domain of the CAR, which refers to a region that moves the antigen-binding domain away from the effector cell surface and enables appropriate cell / cell contact, antigen binding, and activation. The hinge domain can be derived from any of natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is part of an immunoglobulin that includes, but is not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0096] Following the binding domain of the CAR, generally one or more "hinge domains" follow, which serve to position the antigen-binding domain away from the effector cell surface and enable appropriate cell / cell contact, antigen binding, and activation. The CAR generally includes one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from any of natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in 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 modified. In another embodiment, the hinge domain includes the CD8α hinge region.

[0097] The "transmembrane domain" is part of the CAR that fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAR to the cell membrane of immune effector cells. The TM domain can be derived from any of natural, synthetic, semi-synthetic, or recombinant sources. The TM domain can be derived from the alpha, beta, or zeta 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 (i.e., can include at least its transmembrane region(s)).

[0098] In some embodiments, the CARs contemplated herein include an intracellular signaling domain. The "intracellular signaling domain" refers to the part of the CAR that transduces the message of effective CAR binding to the target antigen inside the immune effector cell and is involved in effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, e.g., the release of cytotoxic factors to CAR-bound target cells, or other cellular responses induced 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 a "primary signaling domain".

[0099] The primary signaling domain either positively or negatively regulates the primary activation of the TCR complex. A primary signaling domain that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs that include the primary signaling domain particularly used in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Examples of ITAMs that include the primary signaling domain particularly used in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0100] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor, and when bound to an antigen, provides a second signal necessary for efficient activation and function of T lymphocytes. Examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In one embodiment, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of the primary signaling domains of CD28, CD137, and CD134, and CD3ζ.

[0101] In some embodiments, an antibody-drug conjugate is provided. In certain embodiments, the antibody-drug conjugate comprises an antibody, an antibody reagent, or an 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 conjugated directly to and / or bound to the antibody or an antigen-binding portion thereof. In some embodiments, the binding can be a non-covalent bond, such as a hydrogen bond, an electrostatic interaction, or a van der Waals interaction, however, the binding can also be a covalent bond. "Conjugated" means a covalent bond between at least two molecules. In some embodiments, the composition can be an antibody-drug conjugate.

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

[0103] In some embodiments, an antibody, or antigen-binding portion thereof, and a chemotherapeutic agent can be present in a scaffold material. Scaffold materials suitable for use in 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 particles on the order of about 10 -9 to one billionth to several billionths of a meter of 1 meter. The term "nanoparticle" includes nanospheres, nanorods, nanoshells, and nanoplisms, and these nanoparticles can be part of a nanonetwork.

[0104] The term "nanoparticle" also encompasses liposomes and lipid particles having the size of nanoparticles. As used herein, the term "matrix" refers to a three-dimensional structure that includes components of the compositions described herein (e.g., an antibody or antigen-binding portion thereof). Non-limiting examples of matrix structures include foams; hydrogels; electrospun fibers; gels; fiber mats; sponges; three-dimensional scaffolds; non-woven mats; woven materials; knit materials; fiber bundles; and fibers, as well as other material formats. For example, Rockwood et al. (2011), US Patent Application Publication Nos. 2011 / 0167602; 2011 / 0009960; 2012 / 0296352; each of which is incorporated herein by reference in its entirety; US See U.S. Patent No. 8,172,901. The structure of the matrix can be selected by one of ordinary skill in the art according to the intended use of the composition. For example, an electrospun matrix can have a larger surface area than a foam.

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

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

[0107] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to a polymeric molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be a single-stranded nucleic acid of denatured double-stranded DNA. In some embodiments, the nucleic acid can be cDNA, e.g., a nucleic acid lacking introns.

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

[0109] A nucleic acid molecule, such as DNA, contains nucleotide sequences that include transcriptional and translational regulatory information, and is said to be "capable of expressing" a polypeptide when such sequences are "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequence and the DNA sequence being expressed are connected in such a way as to permit gene expression in recoverable amounts as a peptide or antibody portion. The exact nature of the regulatory regions required for gene expression may vary depending on the organism, as is well known in the art for similar technical fields.

[0110] In some embodiments, the antibodies, antibody reagents, antigen-binding portions thereof, or nucleic acids encoding a CAR described herein are comprised by a vector. In some aspects of the embodiments described herein, the antibodies, antibody reagents, antigen-binding portions thereof, or nucleic acid sequences encoding a CAR, or any module thereof described herein, are operably linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery 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 when combined with appropriate control elements and transfer a gene sequence into a cell. Vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

[0111] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. An expression vector can contain additional elements. For example, an expression vector can have two replication systems and thus be capable of maintaining itself in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and protein and, optionally, the secretion of the protein, and includes, where applicable, but is not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA to RNA) in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene can or cannot include, before and after the coding region, for example, 5' untranslated (5'UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0112] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. A viral vector can contain, instead of non-essential viral genes, a nucleic acid encoding an antibody, an antigen-binding portion thereof, or a CAR described herein. The vector and / or particle can be used for the purpose of transferring any nucleic acid into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0113] "Recombinant vector" means a vector containing a heterologous nucleic acid sequence capable of being expressed in vivo or a "transgene". It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest at a high copy number in extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0114] In one aspect of any of the embodiments, described herein are cells comprising an antibody, antibody reagent, antigen-binding portion thereof, or CAR described herein, or a nucleic acid encoding such an antibody, antibody reagent, antigen-binding portion thereof, or CAR.

[0115] Expression of the antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include in vivo or A bacterial or eukaryotic host containing yeast, insect, fungal, avian, and mammalian cells, or a host cell of mammalian, insect, avian, or yeast origin, can be used in any of the in-situ methods. Mammalian cells or tissues can be from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells can be used. Additionally, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins produced in this way can be processed in vivo or purified and processed in vitro, enabling the synthesis of the antibodies or portions thereof described herein that have a specific amino-terminal sequence. Furthermore, problems associated with retention of the methionine residue derived from the start codon in direct expression in yeast (or bacteria) can be avoided. Any of a series of yeast gene expression systems incorporating promoters and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large quantities when yeast is grown in a glucose-rich medium can be used to obtain the recombinant antibodies or antigen-binding portions thereof 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.

[0116] Production of the antibodies or antigen-binding portions thereof described herein can be achieved in insects. For example, by infecting an insect host with a baculovirus engineered to express a transmembrane polypeptide by methods known to those skilled in the art.

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

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

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

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

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

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

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

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

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

[0126] In one aspect, provided are isolated antibodies, antigen-binding portions thereof, or cells comprising a CAR, as described herein. In some embodiments, the isolated antibodies, antigen-binding portions thereof, or CARs described herein are expressed on the cell surface. In some embodiments, the cell comprises a nucleic acid encoding the isolated antibody, antigen-binding portion thereof, or CAR, as described herein.

[0127] In some embodiments, the cell is an immune cell. 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; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cell is a T cell; an NK cell; an NKT cell; a lymphocyte such as a B cell and a T cell; and a myeloid cell such as a monocyte, macrophage, eosinophil, mast cell, basophil, and granulocyte.

[0128] In certain embodiments, cells (e.g., immune cells) are transduced with a retroviral vector encoding a CAR, such as a lentiviral vector. For example, immune effector cells are transduced with a vector encoding a CAR comprising an anti-CHI3L1 / anti-PD-1 antibody or 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 induce a CAR-mediated cytotoxic response.

[0129] Retroviruses are common tools for gene delivery. In certain embodiments, 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 reverse transcribes its genomic RNA into a linear double-stranded DNA copy, which is then covalently integrated into the host genome. When the virus is integrated into the host genome, it is called a "provirus." The provirus functions as a template for RNA polymerase II and directs the expression of RNA molecules encoding the structural proteins and enzymes necessary to produce new virus particles.

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

[0131] As used herein, the term "retrovirus" refers to a group (or genus) of complex retroviruses. Exemplary retroviruses 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., an HIV cis-acting sequence element) is preferred. In certain embodiments, a retrovirus is used to deliver a polynucleotide comprising a CAR to a cell. That is, an HIV cis-acting sequence element) is preferred. In certain embodiments, a retrovirus is used to deliver a polynucleotide comprising a CAR to a cell.

[0132] Retroviral vectors, and more particularly lentiviral vectors, can be used in practicing certain embodiments of the present invention. Thus, as used herein, the terms "retrovirus" or "retroviral vector" each mean to include "lentivirus" and "lentiviral vector", respectively.

[0133] CHI3L1 antigen-binding portion As used herein, "CHI3L1", "chitinase-3-like protein 1", or "YKL-40" refers to a ~40 kDa glycoprotein secreted by at least macrophages, chondrocytes, neutrophils, synoviocytes, and some cancer cells. CHI3L1 has no chitinase activity, is a Th2-promoting cytokine, is associated with the AKT anti-apoptosis signaling pathway, and induces the migration of astrocytes. The sequences of CHI3L1 expression products are known in many species, for example, human CHI3L1 (NCBI gene ID number 1116) mRNA (NCBI Ref Seq: NM_001276.1 and NCBI Ref Seq: NM_001276.2) and polypeptide (NCBI Ref Seq: NP_001267.1 and NCBI Ref Seq: NP_001267.2).

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

[0135] [Table 1]

[0136] In some embodiments of any of the aspects, 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 of the aspects, 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.

[0137] In some embodiments, the framework of the anti-human CHI3L1 antibody includes conservative substitutions with respect 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 SEQ ID NO: 38 disclosed in U.S. Patent No. 10,253,111, and the conservative substitutions are in sequences not included in the CDRs. In alternative embodiments, the framework of the anti-human CHI3L1 antibody includes 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 SEQ ID NO: 38 disclosed in U.S. Patent No. 10,253,111, provided below as SEQ ID NO: 13 and SEQ ID NO, respectively.

[0138] [Table 2]

[0139] In other alternative embodiments, the CHI3L1 antigen-binding portion of the bispecific antibody of the present invention comprises one or more of the heavy-chain CDRs having the amino acid sequences of SEQ ID NOs: 1-12 disclosed in Table 3, and / or one or more of the light-chain CDRs having the amino acid sequences of SEQ ID NOs: 13-20. See, for example, International Patent Application Publication No. WO 2019 / 060675.

[0140]

Table 3-1

[0141]

Table 3-2

[0142] PD-1 antigen-binding portion Examples of anti-PD-1 antibodies are disclosed in U.S. Patent 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.); U.S. Patent Application Publication Nos. 2011 / 0123550 (Shibayama et al.); 2016 / 0376367 (Yuan et al.); 2017 / 0210806 (Liu).

[0143] The antigen-binding portion of any anti-PD-1 antibody can be used in the bispecific antibody of the present invention. In some embodiments, the bispecific antibody that detects and neutralizes CHI3L1 and PD1 comprises a PD-1 single-chain variable fragment (scFv-PD1) and a linker (shown in bold and underlined) and is provided in Table 4.

[0144]

Table 4

[0145] Pharmaceutical composition In one aspect of any of the embodiments, this specification describes a bispecific antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR described herein, or a nucleic acid encoding an antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR described herein, or a composition comprising a cell 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, within the scope of sound medical judgment, to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

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

[0147] In some embodiments, the compositions containing the antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein, or nucleic acids encoding the antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein can be lyophilized products.

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

[0149] In one aspect, methods of inhibiting or killing CHI3L1+ / PD-1+ cells are described herein, which include contacting the cells with the isolated bispecific antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein, nucleic acids encoding such polypeptides, cells containing such polypeptides or nucleic acids, or compositions containing such polypeptides or nucleic acids. 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.

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

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

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

[0153] As used herein, a "CHI3L1+" cell is, for example, a cell that expresses increased levels of CHI3L1+ compared to the average level of CHI3L1 found in normal cells of the same type or normal cells of the same type. In some embodiments of any of the aspects, the increased level of CHI3L1 is at least 1.5 times the level found in the reference, and can be, for example, 1.5 - fold, 2 - fold, 3 - fold, 4 - fold, 5 - fold or higher than the reference level.

[0154] In one aspect, the technology described herein relates to a method comprising administering to a subject an antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR described herein, or a nucleic acid encoding an antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR 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 cancer in a subject.

[0155] In one aspect, the technology described herein relates to a method comprising administering to a subject a bispecific antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR described herein, or a nucleic acid encoding a bispecific antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR described herein.

[0156] In one aspect, described herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a cell described herein, such as a cell comprising a bispecific antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR described herein. In some embodiments, the cell is an immune cell.

[0157] In one embodiment, the present specification describes a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a nucleic acid described herein, or an immune cell comprising the nucleic acid, wherein the immune cells of the subject are caused to express a polypeptide encoded by the nucleic acid. In some embodiments, the immune cells are T cells. The nucleic acid can be targeted to a specific cell type, for example, by use of a cell type-specific promoter and / or composition that selectively binds to the desired cell type. For example, conjugation of the nucleic acid to an aptamer can enable targeted delivery. See, for example, McNamara et al. (2006). In alternative embodiments, the nucleic acid can be delivered using a drug delivery system such as a nanoparticle, dendrimer, polymer, liposome, or cationic delivery system. A positively charged cationic delivery system facilitates binding of the nucleic acid molecule (negatively charged), increases interactions at the negatively charged cell membrane, and enables efficient uptake of the nucleic acid by the cell. 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)). Formation of vesicles or micelles further prevents degradation of the nucleic acid when administered systemically. Methods of making and administering cationic-inhibitory nucleic acid complexes are within the capabilities of those skilled in the art. Some non-limiting examples of drug delivery systems useful for systemic delivery of nucleic acids include DOTAP oligoFectamine, "solid nucleic acid lipid particles", cardiolipin, polyethyleneimine, Arg-Gly-Asp (RGD) peptide, and polyamidoamine. In some embodiments, the nucleic acid forms a complex with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of the nucleic acid and cyclodextrin can be found in U.S. Patent No. 7,427,605, which is hereby incorporated by reference in its entirety. Targeted delivery of nucleic acids is described, for example, in Ikeda and Taira (2006); Soutschek et al. (2004); Lorenze et al. (2004), each of which is hereby incorporated by reference in its entirety.As an example, nucleic acids can target immune cells by encapsulating an inhibitor in liposomes that express ligands for receptors on immune cells, such as TCR. In some embodiments, the liposomes can contain aptamers specific for immune cells.

[0158] In some embodiments, the methods described herein relate to CAR-T cell therapy. CAR-T cells and related therapies involve adoptive transfer of immune cells (e.g., T cells) that express a CAR that specifically binds to a target 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 are not autologous to the subject. In some embodiments, the cells are engineered to express a CAR and / or genetically modified. 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); Tamada et al., Clin Cancer Res 2012, 18:6436-6445, each of which is incorporated herein by reference in its entirety.

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

[0160] The dosage range of the agent depends on the efficacy and includes a magnitude sufficient to produce the desired effect, such as a deceleration of tumor growth or a reduction in tumor size. The dosage should not be so large as to cause unacceptable side effects. Generally, the dosage varies according to the patient's age, condition, and gender and can be determined by those skilled in the art. Also, in the event of complications, an individual physician can adjust the dosage. In some embodiments, the dosage ranges from 0.001 mg / kg body weight to 0.5 mg / kg body weight. In some embodiments, the dosage range is from 5 μg / kg body weight to 100 μg / kg body weight. Alternatively, the dosage range can be adjusted to maintain the serum level between 1 μg / mL and 1000 μg / mL. For systemic administration, the subject can be administered at a therapeutic dose, such as 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg or more.

[0161] The administration of the above dosages can be repeated. In some embodiments, the dosage is administered once a day or multiple times a day, for example, but not limited to, 3 times a day. In some embodiments, the above dosages are administered daily for several weeks or months. The treatment period depends on the clinical progression of the subject and the responsiveness to the treatment.

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

[0163] 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.

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

[0165] In some embodiments, the dose can be from about 1 mg to about 2000 mg. In some embodiments, the dose can be about 3 mg. In some embodiments, the dose can be about 10 mg. In some embodiments, the dose can be about 30 mg. In some embodiments, the dose can be about 1000 mg. In some embodiments, the dose can be about 2000 mg. In some embodiments, the dose can be administered intravenously at about 3 mg daily. In some embodiments, the dose can be administered intravenously at about 10 mg daily. In some embodiments, the dose can be administered intravenously at about 30 mg three times a week.

[0166] A therapeutically effective amount is an amount of the agent that is statistically significant in terms of tumor size, tumor growth, etc. and sufficient to produce a measurable change (the measures of effectiveness are described below herein). Such an effective amount can be measured in clinical trials as well as in animal experiments.

[0167] The agent can be administered intravenously by injection or by gradually infusing over time. When the appropriate formulation for a given route is provided, for example, agents useful in the methods and compositions described herein can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and, if desired, by peristaltic means or by other means known to those skilled in the art. The compounds used herein are preferably administered orally, intravenously or intramuscularly to patients having cancer. Direct local administration to the tumor mass is also specifically contemplated.

[0168] A therapeutic composition containing at least one agent can, for example, usually be administered in unit dosage. The term "unit dosage", when used with respect to a therapeutic composition, refers to a physically discrete unit suitable as a single dosage for a subject, each unit containing a predetermined quantity of the active substance calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0169] The composition is in a manner compatible with the dosage regimen and is administered in a therapeutically effective amount. The amount and timing of administration depend on the subject being treated, the capacity of the subject's system to utilize the active ingredient, and the degree of the desired therapeutic effect.

[0170] The exact amount of the active ingredient required for administration depends on the judgment of the physician and is specific to each individual. However, dosage ranges suitable for systemic administration are disclosed herein and depend on the route of administration. Dosage regimens suitable for administration also vary, but are typified by an initial administration followed by repeated administrations at intervals of more than one hour by injection or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain the concentration in the blood within the range specified for in vivo treatment is contemplated.

[0171] In some embodiments, the method further comprises administering the pharmaceutical composition described herein, as part of a combination therapy, with one or more additional chemotherapeutic agents, biological agents, drugs, or treatments. In some such embodiments, the chemotherapeutic agent, biological agent, drug, or treatment is selected from the group consisting of radiation therapy, surgery, antibody reagents, and / or small molecules.

[0172] In some embodiments of the methods described herein, the method further comprises administering one or more chemotherapeutic agents to a subject to whom the pharmaceutical composition described herein is administered. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bradycidin and bradycidinone); camptothecin (including the synthetic analogue topotecan); bryostatin; calicheamicin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (especially 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, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride Melphalan, Novembicin, Phestrolin, Prednimustine, Trofosfamide, Uracil Mustard; Nitrosureas, for example, Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, and Ranimustine; Antibiotics, for example, enediyne antibiotics (for example, Calicheamicin, especially Calicheamicin gamma 1I and Calicheamicin omega I1; Dynemicin, for example, Dynemicin A; Bisphosphonates, for example, Clodronate; Esperamicin; as well as Neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), Acrasinomycin, Actinomycin, Autramycin, Azaserine, Bleomycin, Cactinomycin, Carabicin, Caminomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5-oxo-L-norleucine, ADRIAMYCIN® Doxorubicin (for example, Morpholino-Doxorubicin, Cyanomorpholino-Doxorubicin, 2-Pyrrolino-Doxorubicin and Deoxydoxorubicin), Epirubicin, Esorubicin, Idarubicin, Marcellomycin, Mitomycin, for example, Mitomycin C, Mycophenolic Acid, Nogalamycin, Olivomycin, Peplomycin, Potfiromycin, Puromycin, Keramycin, Rhodomycin, Streptozocin, Streptonigrin, Tubercidin, Ubenimex, Dinostatin, Zorubicin; Antimetabolites, for example, Methotrexate and 5-Fluorouracil (5-FU); Folic acid analogs, for example, Denopterin, Methotrexate, Pteropterin, Trimethoprim; Purine analogs, for example, Fludarabine, 6-Mercaptopurine, Thiamiprine, Thioguanine; Pyrimidine analogs, for example, Ancytabine, Azacitidine, 6-Azauridine, Carmofur, Cytarabine; Dideoxyuridine, Doxifluridine, Enocitabine, Floxuridine; Androgens, for example, Calusterone, Drostanolone Propionate, Epithiostanol, Mepitiostane, Testolactone; Antiadrenal, for example, Aminoglutethimide, Mitotane, Trilostane; Folic acid supplements, for example, Folic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; deferoxamine; demeclocycline; diacquone; elfomycin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, for example, maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichloroethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitocin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, for example, TAXOL (registered trademark) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE (registered trademark) Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill), and TAXOTERE (registered trademark) docetaxel (Rhone-Poulenc; Rorer, Antony, France); Chlorambucil; GEMZAR (registered trademark) Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, such as Cisplatin, Oxaliplatin and Carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE (registered trademark) Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (e.g., treatment regimen of irinotecan with 5-FU and Leucovorin); Topoisomerase inhibitor RFS2000; Difluorometh Thiolornithine (DMFO); Retinoids, such as Retinoic acid; Capecitabine; Combretastatin; Leucovorin (LV); Oxaliplatin, such as Oxaliplatin treatment regimen (FOLFOX); Lapatinib (Tykerb (registered trademark)); Inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., Erlotinib (Tarceva (registered trademark)) and VEGF-A that reduce cell proliferation, and any one of the above pharmaceutically acceptable salts, acids or derivatives may be included.

[0173] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells 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 small molecule toxins or enzyme active toxins of bacterial, fungal, plant or animal origin, fragments and / or variants thereof, etc.

[0174] As used herein, the term "chemotherapy" or "chemotherapeutic agent" refers to any chemical agent having therapeutic utility in the treatment of diseases characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms and cancers, as well as diseases characterized by hyperplastic growth. Chemotherapeutic agents used herein include both chemical agents and biological agents. These agents function to inhibit cell activities on which cancer cells depend to maintain survival. Categories of chemotherapeutic agents include alkylating agents / alkaloid agents, antimetabolites, hormones or hormone analogs, and other antitumor agents. Most of these drugs are directly toxic to cancer cells, if not all, and do not require immune stimulation. In one embodiment, the chemotherapeutic agent is a drug used in treating neoplasms such as solid tumors. In one embodiment, the chemotherapeutic agent is a radioactive molecule. Those skilled in the art can readily identify the chemotherapeutic agent to be used (e.g., Chapter 86 of Harrison’s Principles of Internal Medicine, 14th Edition (2001); Chapter 17 of Abeloff’s Clinical Oncology, 2nd Edition (2000)). The bispecific and multispecific polypeptide agents described herein can be used together with additional chemotherapeutic agents.

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

[0176] In some embodiments, the methods described herein can further include administering to the subject additional immunotherapies. As used herein, "immunotherapy" refers to a diverse set of treatment strategies designed to induce the patient's own immune system to fight the tumor, including but not limited to intravesical BCG immunotherapy for superficial bladder cancer, vaccines that generate specific immune responses such as for melanoma and renal cell carcinoma, loading patient-derived dendritic cells with prostate acid phosphatase peptides to induce specific immune responses against prostate-derived cells, use of sipuleucel-T for prostate cancer, administration of cytokines, growth factors, and / or signaling molecules (e.g., interleukins) that stimulate one or more immune cell types, ex vivo expansion and / or stimulation of 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 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 approach, i.e., adoptive immunotherapy. is.

[0177] In some embodiments, the methods described herein can further include administering to the subject additional antibodies, antibody reagents, antigen-binding portions thereof, or T cells comprising a CAR. In some embodiments, the methods described herein can further include administering a cytokine to the subject. Antibody- and cytokine-based therapies are known in the art and can include, as non-limiting examples, alemtuzumab; bevacizumab; brentuximab vedotin; cetuximab; gemtuzumab; ibritumomab tiuxetan; ipilimumab; ofatumumab; panitumumab; rituximab; tositumomab; trastuzumab; interleukin-2, and interferon-alpha.

[0178] For example, the effectiveness of a given treatment for cancer can be determined by a skilled clinician. However, a treatment is considered an "effective treatment" as used herein if, for example, any one or all of the signs or symptoms of the tumor change in a beneficial way, or if other clinically recognized symptoms are improved, for example, by at least 10% after treatment with the agents described herein. Effectiveness can also be measured by the individual not worsening, as evaluated 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.

[0179] An effective amount for the treatment of a disease means an amount sufficient to provide an effective treatment for that disease when administered to a mammal in need thereof, as the term is defined herein. The effectiveness of an agent can be determined, for example, for cancer, by evaluating physical metrics such as tumor size, tumor volume, tumor density, angiogenesis, tumor growth rate, and the like.

[0180] CHI3L1 and PD-1 levels in a subject In one aspect, described herein are methods for detecting, prognosticating, and / or diagnosing cancer, the methods 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 levels of CHI3L1 and PD-1 or PD-L1 relative to a reference level indicates that the subject has cancer and has an increased risk of developing cancer.

[0181] Recent reports have shown that the sensitivity of PD-L1 expression as a biomarker for immune checkpoint inhibitors (ICIs) such as PD-1 in patients with cancer is moderate, and treatment outcomes are 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 of detecting, predicting the prognosis of, 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, an antibody reagent, or an antigen-binding portion thereof as described herein, wherein an increase in the CHI3L1 level relative to a reference level indicates that the subject has cancer and has an increased risk of developing cancer.

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

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

[0184] In one aspect, described herein is an assay comprising contacting a test sample obtained from a subject with an antibody, an antibody reagent, or an 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 subject has or is at a higher risk of developing cancer.

[0185] In one aspect, described herein is a method of identifying a subject in need of treatment for cancer, the method comprising contacting a test sample obtained from the subject with a bispecific 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 / or PD-1 or PD-L1 in the sample; and identifying the subject as in need of treatment for cancer if the expression level of CHI3L1 and / or PD-1 or PD-L1 is increased relative to a reference level.

[0186] In one aspect, the present specification describes a method for determining whether a subject is likely to respond to treatment with an anti-CHI3L1 / anti-PD-1 therapy, such as an anti-CHI3L1 / anti-PD-1 bispecific antibody, an antibody reagent, or an antigen-binding portion thereof, or a bispecific CAR that binds to CHI3L1 and PD-1 or PD-L1 and is included in T cells. The method includes contacting a test sample obtained from the subject with an antibody, an antibody reagent, or an antigen-binding portion thereof described herein, detecting the presence or intensity of a signal indicating the presence or level of CHI3L1 and PD-1 or PD-L1 in the sample; determining that the subject is likely to respond to treatment with an anti-CHI3L1 / anti-PD-1 therapy when the levels of CHI3L1 and PD-1 or PD-L1 are increased relative to a reference level; and determining that the subject is likely to have a low response to treatment with anti-CHI3L1 / anti-PD-1 when the levels of CHI3L1 and PD-1 or PD-L1 are not increased relative to the reference level.

[0187] In one aspect, the present specification describes a method for treating cancer, including 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 indicating 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 therapy when the levels of CHI3L1 and PD-1 are increased relative to a reference level. In one aspect, the present specification describes a method for treating cancer, including administering a therapeutically effective amount of an anti-CHI3L1 / anti-PD-1 bispecific antibody therapy to a subject determined to require treatment for cancer and further determined to have increased levels of CHI3L1 and PD-1 relative to a reference level. The anti-CHI3L1 / anti-PD-1 therapy includes an antibody, an antibody reagent, an antigen-binding portion thereof, or T cells including a bispecific CAR that recognizes CHI3L1 and PD-1; a nucleic acid; a cell; or a composition as described herein.

[0188] In some embodiments, the expression level of CHI3L1 can be measured by determining the level of the expression product of the CHI3L1 gene, such as the level of 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, such as the level of PD-1 RNA transcript or PD-1 polypeptide. Such molecules can be isolated, derived, or amplified from a biological sample, such as a biological fluid. In some embodiments, a detectable signal is generated by an antibody or an antigen-binding portion thereof 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 can generate a detectable signal. In some embodiments, the level of CHI3L1 or PD-1 is determined using a method selected from the group consisting of Western blot; 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 can generate a detectable signal. In some embodiments, the expression level of CHI3L1 or PD-1 is normalized to the expression level of one or more reference genes or reference proteins. In some embodiments, the reference level of CHI3L1 or PD-1 is the expression level of CHI3L1 or PD-1 in a previous sample obtained from the subject.

[0189] In some embodiments, the level of CHI3L1 or PD-1 can be the level of the CHI3L1 or PD-1 polypeptide. Detection of the CHI3L1 or PD-1 polypeptide can follow 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 induction techniques using the antibody reagents described herein.

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

[0191] In one embodiment, the assays, methods, and / or systems described herein can include ELISA. In an exemplary embodiment, the first antibody reagent can be immobilized on a solid support (usually a polystyrene microtiter plate). The solid support can be contacted with a sample obtained from a subject, and the antibody reagent binds (``captures'') to an antigen to which it is specific (e.g., CHI3L1 or PD-1). Next, the solid support can be contacted with a second labeled antibody reagent (e.g., a detection antibody reagent). The detection antibody reagent can include, for example, a detectable signal and can be covalently linked to an enzyme or can itself be detected by a secondary antibody linked to an enzyme via bioconjugation. The presence of the 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 the signal indicates the presence of CHI3L1 or PD-1 molecules. Between each step, the plate is typically washed with a mild surfactant solution to remove any non-specifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzyme substrate to generate a visible signal, indicating the amount of CHI3L1 or PD-1 polypeptide in the sample. Older ELISAs utilize chromogenic substrates, while newer assays use fluorescent substrates with very high sensitivity. There are other different forms of ELISA well known to those skilled in the art. The presence of the 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 the signal indicates the presence of CHI3L1 or PD-1 molecules. Between each step, the plate is typically washed with a mild surfactant solution to remove any non-specifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzyme substrate to generate a visible signal, indicating the amount of CHI3L1 or PD-1 polypeptide in the sample. Older ELISAs utilize chromogenic substrates, while newer assays use fluorescent substrates with very high sensitivity. There are other different forms of ELISA well known to those skilled in the art.

[0192] In one embodiment, the assays, systems, and methods described herein can include a lateral flow immunoassay (LFIA), also known as an immunochromatography assay, or a strip test for measuring or determining the level of CHI3L1 or PD-1 polypeptide in a sample. The LFIA is a simple device intended to detect the presence (or absence) of CHI3L1 or PD-1 in a sample. Currently, there are many LFIA tests used for medical diagnosis for either at-home testing, point-of-care testing, or laboratory use. The LFIA test is in the form of an immunoassay where the test sample flows along a solid substrate via capillary action. After applying the sample to the test strip, it encounters a colored antibody reagent that mixes with the sample and, if it binds to a portion of the sample, passes through a substrate that encounters a line or zone pretreated with a second antibody reagent. Depending on the level of CHI3L1 or PD-1 present in the sample, the colored antibody reagent can become bound at the test line or zone. The LFIA is essentially an immunoassay adapted to operate along a single axis to fit into a test strip format or a dipstick format. The strip test is extremely versatile and can be easily modified by those skilled in the art to detect a vast range of antigens from liquid samples such as urine, blood, water samples, etc. The strip test is also known as a dipstick test and gets its name from the literal action of "dipping" the test strip into the liquid sample being tested. The LFIA strip test is easy to use, requires minimal training, and can be easily included as a component of point-of-care testing (POCT) diagnosis for use in the field. The LFIA test can be operated as either a competitive assay or a sandwich assay. The sandwich LFIA is similar to a sandwich ELISA. The sample first encounters colored particles labeled with an antibody reagent specific to the target (e.g., a CHI3L1- or PD-1-specific antibody reagent). The test line also contains an antibody reagent (e.g., a CHI3L1- or PD-1-specific antibody reagent). The test line shows up as a colored band for a positive sample.In some embodiments, the lateral flow immunoassay can be a sandwich immunoassay, a competitive immunoassay, a quantitative immunoassay, or variations thereof. There are many variations of lateral flow technology. It is also possible to apply multiple capture zones to create multiplex tests.

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

[0194] The use of "dipsticks" or LFIA test strips and other solid supports has been described in the art in the context of immunoassays for many antigen biomarkers. U.S. Patent Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, which are hereby incorporated by reference in their entirety; U.S. Patent Application Nos. 10 / 278,676; 09 / 579,673; and 10 / 717,082 are non-limiting examples of such lateral flow test devices. Three U.S. Patents (U.S. Patent No. 4,444,880 issued to H. Tom; U.S. Patent No. 4,305,924 issued to R. N. Piasio; and U.S. Patent No. 4,135,884 issued to J. T. Shen) describe the use of "dipstick" technology for detecting soluble antigens via immunoassays. The devices and methods of these three patents broadly describe a first component immobilized on a solid surface on the "dipstick" that is exposed to a solution containing soluble antigen that binds to a component immobilized on the "dipstick" prior to detection of the component - antigen complex on the stick. It is within the scope of those skilled in the art to modify the teachings of these "dipstick" technologies as necessary for the detection of CHI3L1 or PD-1 polypeptides. In some embodiments, the dipstick (or LFIA) may be suitable for use with urine samples. In some embodiments, the dipstick may be suitable for use with blood samples.

[0195] Immunochemistry is a family of techniques based on the use of specific antibodies, which are used to specifically target molecules inside or on the surface of cells. In some embodiments, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used to detect or measure 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 receiving specific cytological preparations, such as liquid-based preparations. In some cases, signal amplification can be incorporated into specific protocols, and secondary antibodies containing labels follow the application of antibody reagents specific for platelets or white blood cells. Typically, for immunohistochemistry, tissue obtained from a subject and fixed with an appropriate fixative such as alcohol, acetone, and paraformaldehyde is sectioned and reacted with an antibody. Conventional methods for immunohistochemistry are described in Buchwalow and Bocker (eds.) “Immunohistochemistry: Basics and Methods”, Springer (2010): Lin and Prichard “Handbook of Practical Immunohistochemistry”, Springer (2011), which are hereby incorporated by reference in their entirety. In some embodiments, immunocytochemistry can generally be utilized when tissue or cells obtained from a subject are fixed with an appropriate fixative such as alcohol, acetone, and paraformaldehyde and then reacted with an antibody. Methods for immunocytological staining of human samples are known to those of skill in the art and are described, for example, in Immunocytochemistry: A Practical Guide for Biomedical Research (2009), which is hereby incorporated by reference in its entirety.

[0196] In some embodiments, one or more of the antibody reagents described herein can include a detectable label and / or can include 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, light-absorbing 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.

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

[0198] In other embodiments, the detection antibody is labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of an appropriate wavelength, its presence can be detected by fluorescence. In some embodiments, the detectable label can be a fluorescent dye molecule, or fluorophore, including but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthalaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanin, Texas Red, peridinin chlorophyll, cyanine, tandem conjugate, e.g., phycoerythrin-Cy™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas Red and tetrarhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfluorescein (commonly known by the abbreviations 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, e.g., Cy3, Cy5 and Cy7 dyes; coumarin, e.g., umbelliferone; benzimidazole dyes, e.g., Hoechst 33258; phenanthridine dyes, e.g., Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g., cyanine dyes, e.g., Cy3, Cy5, etc.; BODIPY dyes and quinoline dyes are included.

[0199] In some embodiments, the detectable label can be, including but not limited to, 3 H, 125 I, 35 S, 14 C, 32 P, and 33 a radiolabel containing P.

[0200] In some embodiments, detectable labels can be, but are not limited to, enzymes including horseradish peroxidase and alkaline phosphatase. Enzyme labels can generate, for example, chemiluminescent signals, color signals, or fluorescent signals. Enzymes intended to be used for detectably labeling antibody reagents include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

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

[0202] In some embodiments, detectable labels can be, but are not limited to, spectral colorimetric labels including colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0203] 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, such as the biotin-streptavidin system, can also be used. In this system, the antibody that is immunoreactive (i.e., specific) with the biomarker of interest is biotinylated. The amount of biotinylated antibody bound to the biomarker is determined using streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, for example, from DAKO; Carpinteria, CA.

[0204] The antibody reagent can also be detectably labeled using a luminescent metal, such as 152 Eu, or other members of the lanthanide series. These metals can be conjugated to the antibody reagent using a metal chelating group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0205] The assays and methods described herein may be related to determining whether a subject has increased levels of CHI3L1 and PD-1 relative to a reference level. In some embodiments, the reference levels of CHI3L1 and PD-1 may be, for example, the levels of CHI3L1 and PD-1 in healthy subjects who do not have, or have not been diagnosed with, cancer. In some embodiments, the reference level is the level in a sample of a similar cell type, sample type, sample processing, and / or of the same age, sex, and other demographic parameters as the subject for whom the levels of CHI3L1 and PD-1 are to be determined. In some embodiments, the test sample and the control reference sample are of the same type, i.e., obtained from the same biological source and containing the same composition, e.g., the same number and type of cells and / or type of sample material. Thus, in some embodiments, increased levels of CHI3L1 and PD-1 may vary as demographic factors such as age, sex, genotype, environmental factors, and individual medical history change. In some embodiments, the reference level can include, for example, the levels of CHI3L1 and PD-1 (e.g., CHI3L1 and PD-1 polypeptides) in the same type of sample taken from a subject showing no signs or symptoms of any cancer. In some embodiments, the reference expression levels of CHI3L1 and PD-1 can be the expression levels of CHI3L1 and PD-1 in previous samples obtained from the subject. Thereby, changes in levels in that individual can be directly analyzed.

[0206] In some embodiments, when the levels of CHI3L1 and PD-1 are at least 1.25-fold, for example, 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 more of the reference level, the levels of CHI3L1 and PD-1 may 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. This can be done. In some embodiments, the expression levels of CHI3L1 and PD-1 can be normalized relative to a reference value.

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

[0208] As used herein, the term "sample" or "test sample" refers to a sample taken or isolated from a biological organism, e.g., a urine sample from a subject. Exemplary biological samples include, but are not limited to, biological fluid samples; serum; plasma; urine; saliva; and / or tumor samples. The term also includes mixtures of the above samples. Also, the term "test sample" includes untreated or pretreated (or pre-processed) biological samples. In some embodiments, the test sample can contain cells derived from a subject. As used herein, the term "biological fluid" refers to any fluid obtained from a biological source and includes, but is not limited to, blood, urine, and fractions thereof.

[0209] A sample can be obtained by removing the sample from a subject, but can also be achieved by using a previously isolated sample (e.g., isolated at a previous time point and isolated by the same or another person). Further, the test sample can be newly collected or a previously collected sample.

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

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

[0212] In some embodiments, the methods, assays, and systems described herein can include generating a report based on the levels of CHI3L1 and PD-1. In some embodiments, the report can indicate 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 the percentage or fold increase in the levels of CHI3L1 and PD-1 compared to a reference level, and / or provide a signal indicating whether the subject has or does not have a risk of having cancer. As used herein, "has a risk of" refers to having at least a two-fold higher likelihood of having a particular condition compared to a subject that did not have an increase and / or elevation in the levels of CHI3L1 and PD-1, e.g., 2-fold, or 2.5-fold, or 3-fold, or 4-fold, or more.

[0213] In some embodiments, the assay or method can further include administering an anti-CHI3L1 / anti-PD-1 therapy. In some embodiments, the anti-CHI3L1 / anti-PD-1 therapy includes an isolated bispecific antibody, an antibody reagent, an antigen-binding portion thereof, or a CAR or CAR T cell; a nucleic acid; a cell; or a composition described herein.

[0214] In one aspect of any of the embodiments, described herein is an antibody, antibody reagent, or antigen-binding portion thereof described herein conjugated or bound to a detectable label.

[0215] In one aspect of any of the embodiments, described herein is a solid support comprising a bispecific antibody, antibody reagent, antigen-binding fragment thereof described herein. In some embodiments of any of the aspects, the bispecific antibody, antibody reagent, or antigen-binding fragment thereof is detectably labeled. In some embodiments of any of the aspects, the solid support includes a particle, bead, polymer, or substrate.

[0216]

[0217] ​ In one aspect of any of the embodiments, described herein is a molecular complex comprising at least one bispecific antibody, antibody reagent, antigen-binding fragment thereof, or CAR described herein, which binds to a CHI3L1 polypeptide and a PD-1 polypeptide.

[0218] In one aspect, described herein is a kit comprising a composition described herein, for example, a composition comprising a bispecific antibody, antibody reagent, antigen-binding portion thereof, or CAR described herein. The kit is at least one reagent, for example, any product (such as a package or container) comprising a bispecific antibody, and the product is promoted, distributed, or sold as a unit for performing the methods described herein. In some embodiments of any of the aspects, the bispecific antibody, antibody reagent, antigen-binding fragment thereof described herein is immobilized on a solid support. In some embodiments of any of the aspects, the solid support comprises particles, beads, polymers, or substrates. In some embodiments of any of the aspects, the antibody, antibody reagent, or antigen-binding fragment thereof is detectably labeled.

[0219] The kit described herein can optionally include additional components useful for performing the methods described herein. As an example, the kit can include a fluid (such as a buffer) suitable for a composition comprising a bispecific antibody, antigen-binding portion thereof, or CAR described herein, instructions for performing the methods described herein, and the like. The kit can further include a device and / or reagent for delivery of the composition described herein. Additionally, the kit can include a handling leaflet and / or can provide information regarding the relevance of the results obtained.

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

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

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

Examples

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

Example

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

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

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

[0227] In summary, the inventors successfully generated and characterized bispecific antibodies that react with high affinity to both rhCHI3L1 and rhPD1 (see Figures 1 and 2). As illustrated in Figure 1, two different approaches were used to generate these bispecific antibodies generated. Using these platforms, CHI3L1-LC-PD1 and CHI3L1-HC-PD bispecific (bivalent) antibodies were generated, each of which detected both human CHI3L1 and human PD1. The affinity of the CHI3L1-LC-PD1 antibody for both rhCHI3l1 and rhPD1 (evaluated by competitive ELISA assay) was K D ≈1×10 -9 M, and the limit of detection (LOD) was 2 ng / ml. There was no significant difference in the binding affinity for rhCHI3L1 or rhPD1 between the CHI3L1-LC-PD1 antibody and the CHI3L1-HC-PD1 antibody (Figure 2 and data not shown).

Example

[0228] Characterization of the T cell-U87 binding and anti-tumor cell cytotoxic activity of the bispecific CHI3L1×PD1 antibody An in vitro co-culture system containing U87 glioblastoma cells (ATCC #HTB-14) and Jurkat T cells (ATCC #TIB152) was used to evaluate the ability of Jurkat cells to bind to U87 cells and the anti-tumor cell cytotoxic activity of the bispecific CHI3L1×PD1 antibody. Human glioblastoma (U87) cells were grown in complete DMEM medium. Jurkat (T cells) were activated by stimulation with α-CD3 / α-CD28 antibody (5 μg / ml) for 2 hours in RPMI complete medium with 5% CO2 and air. Next, the Jurkat cells were 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.

[0229] The reactions in these co-cultures were evaluated in the presence of IgG isotype control, anti-PD1 alone, anti-CHI3L1 alone, the combination of anti-CHI3l1 and anti-PD-1 (5 μg / ml each) or the bispecific antibody described above. Overall, there were five treatment groups: (i) isotype IgG control, (ii) α-PD1, (iii) α-CHI3L1, (iv) α-CHI3L1 + α-PD1, and (v) bispecific-CHI3L1xPD1. The co-cultured cells were incubated for 6 - 12 hours in 5% CO2 and air. T cell - U87 cell binding was evaluated microscopically and cell death was evaluated using TUNEL staining and the LDH release cytotoxicity assay described below.

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

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

[0232] B. TUNEL assay quantification of apoptotic U87 cell death Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling for in situ evaluation of cell death was performed in two different ways: (i) Cells co-cultured in different chambers were fixed, permeabilized, and stained with fluorescein iodide propidium (red dye) and Cyto (green dye). Cells emitting red fluorescence are dead cells, and green ones are live cells. The number of live U87 cells and dead U87 cells was counted using a fluorescence microscope (20 times the original magnification) and averaged over 10 randomly selected microscopic fields.

[0233] (ii) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling for in situ evaluation of cell death. In the selected studies, TUNEL positive staining was evaluated using a bright-field microscope. Dead cells or apoptotic cells were stained blue, and live cells were stained with nuclear fast red. The number of live U87 cells and dead U87 cells was counted using a fluorescence microscope and a normal microscope (20 times the original magnification) and averaged over 10 randomly selected microscopic fields.

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

[0235] C. Granzyme quantification and perforin accumulation Granzyme and perforin are major cytotoxic enzymes secreted by various activated cytotoxic T cells, including Jurkat cells. The expression levels of these cytotoxic enzymes were measured in co-cultured cells 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 (20 times the original magnification) and averaged over 10 randomly selected microscopic fields.

[0236] As shown in FIGS. 5 and 6, treatment with the bispecific CHI3L1×PD1 antibody significantly increased 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 the effect of individual treatment with α-CHI3L1 or α-PD1, or treatment with a combination of α-CHI3L1 and α-PD1.

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

[0238] D. LDH release cytotoxicity assay Lactate dehydrogenase (LDH) is released into the medium after the integrity of the membrane is lost due to cytotoxic damage. Thus, LDH release was used as an indicator of cell death. This was evaluated using a coupled two-step reaction. In the first step of the reaction, LDH catalyzes the oxidation of lactate to pyruvate, thereby reducing NAD to NADH and H + to NAD + In the second step, diaphorase catalyzes the reduction of the newly formed NADH and H + to a darkly colored formazan that strongly absorbs at 490 - 520 nm of the tetrazolium salt (INT).

[0239] The cytotoxicity level % was determined using a commercially available assay kit (Pierce LDH cytotoxicity assay kit) using the protocol provided by the manufacturer. In these experiments, cells were co-cultured in the presence and absence of the above antibodies. LDH in the medium was evaluated after overnight incubation. These values were compared to the following controls: (a) cell-free complete medium control to determine the LDH background activity present in the serum used for medium supplementation; (b) serum-free medium; (c) LDH activity control (water); and (d) maximum LDH activity released by cells treated with lysis buffer. In these assays, the cytotoxicity % was calculated as follows.

[0240] [Number]

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

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

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

[0244] The foregoing specification is considered to be sufficient to enable one of ordinary skill in the art to practice the present embodiments. The present embodiments are not limited in scope by the examples provided, which are intended as illustrations of one aspect only, and other functionally equivalent embodiments are within the scope of the present disclosure. Various modifications will become apparent to those of ordinary skill in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objectives described herein are not necessarily included in each embodiment. One of ordinary skill in the art can recognize or confirm many equivalents to the specific embodiments described herein using only routine experimentation. Such equivalents are intended to be included in the scope of the following claims.

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Claims

**Claim 1** A bispecific antibody that detects and neutralizes CHI3L1 and PD-1, comprising an antigen-binding portion of an anti-human programmed death receptor 1 (PD-1) antibody and an antigen-binding portion of an anti-human chitinase 3-like-1 (CHI3L1) antibody. **Claim 2** The bispecific antibody according to claim 1, comprising an anti-human PD-1 single-chain variable fragment (ScFv-PD1) bound to the backbone of an anti-human CHI3L1 antibody. **Claim 3** The bispecific antibody according to claim 1, comprising an anti-human CHI3L1 single-chain variable fragment (ScFv-CHI3L1) bound to the backbone of an anti-human PD-1 antibody. **Claim 4** The bispecific antibody according to claim 2, wherein the ScFv-PD1 is bound to the heavy chain of the CHI3L1 antibody (CHI3L1-HC-PD1). **Claim 5** The bispecific antibody according to claim 2, wherein the ScFv-PD1 is bound to the light chain of the CHI3L1 antibody (CHI3L1-LC-PD1). **Claim 6** The bispecific antibody according to any one of claims 1 to 5, wherein the antigen-binding portion of the anti-human CHI3L1 antibody comprises the following complementarity-determining regions (CDRs): (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 4; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; (c) light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (d) heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; (e) heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2; and (f) heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

3. **Claim 7** The bispecific antibody according to claim 4, wherein the heavy chain of the CHI3L1 antibody has the amino acid sequence of SEQ ID NO:

13. **Claim 8** The bispecific antibody according to claim 5, wherein the light chain of the CHI3L1 antibody has the amino acid sequence of SEQ ID NO:

14. **Claim 9** The bispecific antibody according to any one of claims 1 to 8, wherein the antigen-binding portion of the anti-human PD-1 antibody comprises the amino acid sequence of SEQ ID NO:

35. **Claim 10** The bispecific antibody according to any one of claims 1 to 9, which increases the adhesion of Jurkat T cells to U87 cells. **Claim 11** The bispecific antibody according to claim 10, which increases the ability of Jurkat T cells to induce a cytotoxic / apoptotic response in U87 cells. **Claim 12** The bispecific antibody according to claim 10, which increases the accumulation of granzyme and perforin in Jurkat T cells co-cultured with U87 cells.

13. The bispecific antibody according to claim 10, which increases the ability of Jurkat T cells to induce the release of lactate dehydrogenase (LDH) and the cytotoxic response in U87 cells.

14. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, further comprising a chemotherapeutic agent.

16. The pharmaceutical composition according to claim 14 or 15 for the treatment of cancer.

17. A therapeutic agent for cancer, comprising the bispecific antibody according to any one of claims 1 to 13.

18. The therapeutic agent for cancer according to claim 17, wherein the cancer is a malignant cancer.

19. The therapeutic agent for cancer according to claim 17, wherein the cancer is a primary cancer or a metastatic cancer.

20. The therapeutic agent for cancer according to claim 17, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignant melanoma, and lung cancer.

21. The therapeutic agent for cancer according to any one of claims 17 to 20, which is administered to a subject determined to have an elevated level of CHI3L1.

22. The therapeutic agent for cancer according to claim 21, wherein the level of CHI3L1 is circulating CHI3L1.

23. The therapeutic agent for cancer according to any one of claims 17 to 22, wherein the cancer expresses PD-L1.

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