Compositions and methods for the treatment of cancer

Abstract

The present disclosure provides a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to a tumor antigen and a target polypeptide. In one aspect, the present disclosure provides a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to a tumor antigen; a target polypeptide; and a half-life extending polypeptide. In some embodiments, the fusion protein comprises at least a first linker. In some embodiments, the fusion protein comprises at least a first linker and a second linker. Fusion proteins and their uses in treating a subject with cancer are described.

Description

[Technical field]

[0001] Over the past decade, changes in the treatment of relapsed and refractory B-cell malignancies have been occurring. The use of cell therapy agents targeting CD19 can result in high clinical response rates in some otherwise hard-to-treat patient populations. In addition, some patients achieve durable clinical remissions that appear to be curative. However, these results are tempered by the finding that nearly half of all patients with an initial response to cell therapy will relapse, with the majority of these relapses rapidly, often within 6 months. Thus, additional therapeutic agents for the treatment of cancer are needed. Summary of the Invention

[0002] In some embodiments, the present disclosure provides a fusion protein comprising (i) an antibody or antigen-binding fragment thereof that binds to a tumor antigen, and (ii) a targeting polypeptide. In one aspect, the present disclosure provides a fusion protein comprising (i) an antibody or antigen-binding fragment thereof that binds to a tumor antigen, (ii) a targeting polypeptide, and (iii) a half-life extending polypeptide. In some embodiments, the fusion protein comprises at least a first linker. In some embodiments, the fusion protein comprises at least a first linker and a second linker.

[0003] In some embodiments, the half-life extending polypeptide is any one of a hyaluronan binding motif, a PAS polypeptide, a proline / alanine random coil polypeptide, and an albumin protein or fragment (e.g., HSA), a polypeptide that binds albumin, and an antibody or antigen-binding fragment thereof. In some embodiments, the half-life extending polypeptide is an anti-albumin antibody or antigen-binding fragment thereof.

[0004] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-CD20 antibody or antigen-binding fragment thereof. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises an anti-CD20 VHH. In some embodiments, the anti-CD20 VHH comprises the amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53.

[0005] In some embodiments, the target polypeptide is a B cell antigen. In some embodiments, the target polypeptide is any one of CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, CD180, ROR1, BCMA, HLA-DR10, CD1, CD5, CD21, CD25, CD27, CD30, CD38, CD78, CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2, CD79a, and CD79b. In some embodiments, the target polypeptide is CD19 or a fragment or variant thereof.

[0006] In some embodiments, the fusion protein comprises an amino acid sequence having at least about 90%, at least about 95%, or about 100% identity to the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 50, 52, and 55.

[0007] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of a fusion protein comprising (i) an antibody or antigen-binding fragment thereof that binds to a tumor antigen, and (ii) a target polypeptide. In one aspect, the present disclosure provides a fusion protein comprising (i) an antibody or antigen-binding fragment thereof that binds to a tumor antigen, (ii) a target polypeptide, and (iii) a half-life extending polypeptide. In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding a fusion protein described herein. In some embodiments, the present disclosure provides a host cell comprising a nucleic acid encoding a fusion protein described herein. In some embodiments, the present disclosure provides a method of producing a fusion protein described herein, comprising culturing a host cell comprising a nucleic acid encoding a fusion protein described herein. In some embodiments, the present disclosure provides a method of treating a subject having a tumor, comprising administering to the subject an effective amount of a fusion protein described herein.

[0008] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a VHH having an amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53, or a fragment thereof. In some embodiments, the present disclosure provides a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising a VHH having an amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53, or a fragment thereof. In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising a VHH having an amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53, or a fragment thereof. In some embodiments, the present disclosure provides a host cell comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising a VHH having an amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53, or a fragment thereof. In some embodiments, the disclosure provides a method for producing an antibody or antigen-binding fragment thereof, the method comprising culturing a host cell containing a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising a VHH having an amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53, or a fragment thereof.

[0009] Other features, objects and advantages of the present invention will become apparent from the following detailed description. It should be understood that the detailed description, while illustrating embodiments of the present invention, is provided by way of illustration and not by way of limitation. Various changes and modifications falling within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0010] The drawing figures are for illustrative purposes only and not for limitation. [Brief description of the drawings]

[0011] [Figure 1] The isoelectric distribution of protein preparations CTE1, CTE2, and CTE3 is shown. CTE1 is a mixture of sialylated and non-sialylated protein, CTE2 is primarily non-sialylated, and CTE3 is primarily sialylated. [Diagram 2] 1 shows the isoelectric distribution of protein preparation CTE1 after various treatments (eg, sialidase) to remove sialylation. [Diagram 3] Binding affinity curves of protein preparations CTE1 (red circle), CTE2 (green square), and CTE3 (blue circle) to CD19 negative / CD20 positive JeKo-1 cells (JeKo-19KO) measured by flow cytometry are shown. [Figure 4] Binding affinity curves of in vitro protein preparations CTE1 (green triangles), CTE2 (red circles), and CTE3 (blue squares) to either purified biotinylated human albumin (left panel) or a biotinylated human CD20 membrane preparation (right panel) as measured by ELISA. [Diagram 5] Shown are killing curves of protein preparations CTE1 (red circle), CTE2 (blue square), and CTE3 (pink triangle) against CD19 negative / CD20 positive JeKo-1 cells (JeKo-19KO) when administered with CAR-19 T cells. [Figure 6] Wild-type JeKo-1 cells are shown to be predominantly positive for both CD19 and CD20 expression as determined by flow cytometry. [Figure 7A] 1 shows CD19 and CD20 expression levels measured by flow cytometry for JeKo-1 B cell lymphoma cells treated with CAR-19 T cells for 13 days, corresponding to an experiment with a 1:1 ratio of CAR-19 T cells:JeKo-1 cells. [Figure 7B] 1 shows CD19 and CD20 expression levels measured by flow cytometry for JeKo-1 B cell lymphoma cells treated with CAR-19 T cells for 13 days, corresponding to an experiment with a CAR-19 T cell:JeKo-1 cell ratio of 0.3:1. [Figure 7C]1 shows CD19 and CD20 expression levels measured by flow cytometry for JeKo-1 B cell lymphoma cells treated with CAR-19 T cells for 13 days, corresponding to an experiment with a CAR-19 T cell:JeKo-1 cell ratio of 0.1:1. [Figure 8A] Shows the expression levels of CD19 and CD20 of CAR-19 T cells incubated with JeKo-1 cells at a 1:1 ratio for 13 days as measured by flow cytometry. [Figure 8B] The corresponding flow cytometry profiles are shown after adding either CAR-19 T cells, the fusion protein (middle panel), or both (bottom panel) to the cells. [Figure 9] Shown is a time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with either CAR-19 T cells+CTE1, CAR-19 T cells alone, CAR-20 T cells alone, or left untreated. [Figure 10] Shown is a time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with either CAR-19 T cells+CTE1, CAR-19 T cells+CTE2, CAR-19 T cells only, or left untreated. [Figure 11] Shown is a time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with various concentrations of CAR-19 T cells+CTE2, CAR-19 T cells alone, or left untreated. [Figure 12] The average body weight of the mice reported in FIG. [Figure 13] The average light output of the mice reported in Figure 11 is shown. The graph on the right shows an expanded plot of 0.016, 0.08, 0.4, and 2 mg / kg fusion protein. [Figure 14] The survival probability of the mice reported in FIG. [Figure 15]FIG. 1 shows the time course of protein concentration of fusion proteins CTE1, CTE2, and CTE3 in NSG mouse serum, which was used to estimate protein half-life in the in vivo circulation. [Figure 16] Binding affinity curves of various fusion protein constructs to CD20 positive / CD19 negative JeKo-1 cells (JeKo-19KO) as measured by ELISA. [Figure 17] 1 shows the killing curves of various fusion protein constructs against CD19 negative / CD20 positive JeKo-1 cells (JeKo-19KO) when administered with CAR-19 T cells. [Figure 18] Binding and killing curves of additional fusion proteins are shown. [Figure 19A] Binding affinity curves of CTE3 fusion proteins to CD19-negative / CD20-positive JeKo-1 cells (JeKo-19KO) as measured by flow cytometry. [Figure 19B] Binding affinity curves of CTE3 fusion proteins to wild-type JeKo-1 cells as measured by flow cytometry. [Figure 19C] Binding affinity curves of fusion proteins to wild-type Ramos Hodgkin's lymphoma cells as measured by flow cytometry. [Figure 20] 1 shows the killing curve of CTE3 fusion protein preparations against CD19 negative / CD20 positive JeKo-1 cells (JeKo-19KO) when administered with CAR-19 T cells. [Figure 21A] Binding of CTE3 to cynomolgus CD20-transfected 293T cells is shown. [Figure 21B] Binding of CTE3 to human CD20-transfected 293T cells is shown. [Figure 22A] Figure 1 shows the effect of human serum on CTE3-mediated CAR19 cytotoxicity. Increasing levels of CTE3 are shown on the x-axis. HS = human serum. [Figure 22B]Figure 1 shows the effect of human serum albumin (HSA) on CTE3-mediated CAR19 cytotoxicity. Increasing levels of CTE3 are shown on the x-axis. [Figure 23A] 1 shows that both CD79b×CD20 bispecific CTEs bind to biotinylated CD20. [Figure 23B] Shows that CTE#650 binds to biotinylated CD79b. [Figure 23C] Binding of monospecific and bispecific anti-CD20 and anti-CD79b proteins to 293T-CD20 cells is shown. [Figure 23D] Binding of monospecific and bispecific anti-CD20 and anti-CD79b proteins to 293T-CD79b cells is shown. [Figure 23E] Figure 1 shows the cytotoxic potential of anti-CD19 CAR T cells plus monospecific and bispecific anti-CD20 and anti-CD79b proteins against JeKo-1 CD19KO cells. [Figure 24A] 1 shows the binding of CTE3 to biotinylated CD20 using SPR. [Figure 24B] 1 shows the binding of CTE3 to biotinylated CD19 using SPR. [Figure 24C] 1 shows the binding of CTE3 to biotinylated HSA using SPR. [Figure 25A] Cytotoxicity curves of JeKo-1 CD19 KO cells after one CAR19 stimulation are shown. [Figure 25B] Cytotoxicity curves of JeKo-1 CD19 KO cells after three rounds of CAR19 stimulation are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] definition In order to facilitate understanding of the present invention, certain terms are first defined below. Further definitions of these and other terms are set forth throughout the specification.

[0013] Administration: As used herein, the term "administration" refers to administration of a composition to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route. For example, in some embodiments, administration can be bronchial (including by bronchial instillation), oral mucosal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, intravaginal, and intravitreal. In some embodiments, administration can be intratumoral or peritumoral. In some embodiments, administration can include intermittent administration. In some embodiments, administration can include continuous administration (e.g., perfusion) for at least a selected period of time.

[0014] Adoptive Cell Therapy: As used herein, "adoptive cell therapy" or "ACT" involves the transfer of immune cells with anti-tumor activity into a cancer patient. In some embodiments, ACT is a therapeutic approach that involves the use of lymphocytes with anti-tumor activity, their mass expansion in vitro, and their transfer into a cancer-bearing host. In some embodiments, ACT is a therapeutic approach that involves the use of lymphocytes with anti-tumor activity, their transfer into a cancer-bearing host, and their mass expansion in vivo.

[0015] Agent: As used herein, the term "agent" may refer to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. As will be clear from the context, in some embodiments, an agent may include a cell or organism, or a fraction, extract, or component thereof. In some embodiments, an agent is or includes a natural product that occurs in nature and / or is obtained from nature. In some embodiments, an agent is or includes one or more entities that are artificial in that they are designed, engineered, and / or created through the action of the hand of man, and / or do not occur in nature. In some embodiments, an agent may be utilized in an isolated or pure form, and in some embodiments, an agent may be utilized in a crude form. In some embodiments, candidate agents are provided as a collection or library, and can be screened, for example, to identify or characterize active agents therein. Some specific embodiments of agents that may be utilized in accordance with the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptidomimetics, and the like. In some embodiments, the agent is or comprises a polymer. In some embodiments, the agent is not a polymer and / or is substantially free of any polymer. In some embodiments, the agent contains at least one polymeric moiety. In some embodiments, the agent is free of or substantially free of any polymeric moiety.

[0016] Amelioration: As used herein, "amelioration" refers to the prevention, reduction and / or palliative of a condition, or improvement in a subject's condition. Amelioration includes, but does not require, complete recovery or complete prevention of a disease, disorder or condition.

[0017] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a d-amino acid, and in some embodiments, an amino acid is an l-amino acid. "Standard amino acid" refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including those at the carboxy and / or amino terminal ends of a peptide, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting the activity of the amino acid. The amino acids may participate in disulfide bonds. The amino acids may include one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to free amino acids and / or amino acid residues of peptides. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which the term is used.

[0018] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. Furthermore, the term "antibody," as used herein, may, in appropriate embodiments (unless otherwise stated or clear from the context), refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations. For example, in some embodiments, antibodies utilized in accordance with the present disclosure may include, but are not limited to, intact IgG, IgE and IgM, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), biparatopic or multiparatopic antibodies, single chain Fv, polypeptide-Fc fusions, Fab, camelid antibodies, masked antibodies (e.g., Probodies®), small modular immunopharmaceuticals ("SMIPs™"), single chain or tandem diabodies (T and Ab®), VHHs, Anticalins®, and the like. The format is selected from: IgG, Nanobodies, Minibodies, BiTEs, Ankyrin Repeat Proteins or DARPINs, Avimers, DART, TCR-like antibodies, Adnectins, Affilins, Trans-bodies, Affibodies, TrimerX, Microproteins, Fynomers, Centyrins, and KALBITOR. As is known in the art, naturally produced intact IgG antibodies are tetrameric entities of approximately 150 kDa, consisting of two identical heavy chain polypeptides (each about 50 kDa) and two identical light chain polypeptides (each about 25 kDa), which associate with each other in a structure commonly referred to as "Y-shaped". Each heavy chain consists of at least four domains (each approximately 110 amino acids long): an amino-terminal variable (VH) domain (located at the tip of the Y-shape) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 domain (located at the bottom of the stem of the Y).A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. In intact antibodies, disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other. Each light chain consists of two domains, an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch". An intact antibody tetramer is composed of two heavy-light chain dimers, with the heavy and light chains linked to each other by one disulfide bond and two other disulfide bonds connecting the heavy chain hinge regions to each other, thus connecting the dimers to each other to form a tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., three-, four-, or five-stranded sheets) held together by a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form a beta sheet that confers a structural framework to the domain, and the CDR loop regions of both the heavy and light chains come together in three-dimensional space to form a single hypervariable antigen-binding site located at the tip of the Y-shaped structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding attributes of the Fc region for the Fc receptor can be modulated via glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized in accordance with the present disclosure comprise a glycosylated Fc domain, including modified or engineered Fc domains, such as glycosylated Fc domains.For the purposes of this disclosure, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in a natural antibody may be referred to and / or used as an "antibody," regardless of whether such polypeptide is naturally produced (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, an antibody is polyclonal, and in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are fully human, or humanized, primatized, chimeric, etc., as known in the art. In some embodiments, an antibody may lack covalent modifications (e.g., attachment of glycans) that it would have if it were naturally produced. In some embodiments, an antibody may contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.). In some embodiments, protein scaffolds generated to bind to target antigens can be used as antigen-binding fragments. In some embodiments, protein binding molecules can be generated in silico based on the amino acid sequence and / or degradation structure of a protein of interest (e.g., Cao et al. Design of protein binding proteins from target structure alone. Nature, 2022; DOI: 10.1038 / s41586-022-04654-9).

[0019] Antibody-dependent cellular cytotoxicity: As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to the phenomenon in which antibody-bound target cells are killed by immune effector cells. Without wishing to be bound by any particular theory, it is understood that ADCC typically involves Fc receptor (FcR)-bearing effector cells recognizing and subsequently killing antibody-coated target cells (e.g., cells expressing on their surface a specific antigen to which the antibody binds). Effector cells that mediate ADCC may include immune cells, including, but not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0020] Antibody fragment: As used herein, "antibody fragment" includes a portion of an intact antibody, such as, for example, an antigen-binding region or a variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments (comprising variable regions of heavy and light chains), recombinant single-chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("scFv protein"), recombinant single-domain antibodies (e.g., VHH) consisting of the variable region of an antibody heavy chain, and a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region (e.g., a hypervariable region of a heavy chain variable region (VH), a hypervariable region of a light chain variable region (VL), one or more CDR domains in VH, and / or one or more CDR domains in VL). In many embodiments, an antibody fragment is a fragment that contains sufficient sequence of its parent antibody to bind to the same antigen as the parent antibody, and in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, heavy chain variable regions, and isolated complementarity determining region (CDR) regions. Antigen-binding fragments of antibodies may be produced by any means. For example, antigen-binding fragments of antibodies may be produced enzymatically or chemically by fragmentation of an intact antibody and / or may be produced recombinantly from a gene encoding a partial antibody sequence. Alternatively or additionally, antigen-binding fragments of antibodies may be produced synthetically in whole or in part. Antigen-binding fragments of antibodies may optionally include single chain antibodies. Alternatively or additionally, an antigen-binding fragment of an antibody may comprise multiple chains linked together, for example, by disulfide bonds. An antigen-binding fragment of an antibody may optionally comprise a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids, more typically at least about 200 amino acids.

[0021] Antigen: The term "antigen" as used herein refers to an agent that elicits an immune response; and / or an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody or antibody fragment. In some embodiments, an antigen elicits a humoral response (e.g., including the production of antigen-specific antibodies), and in some embodiments, an antigen elicits a cellular response (e.g., involving a T cell's receptor specifically interacting with the antigen). In some embodiments, an antigen binds to an antibody and may or may not induce a specific physiological response in an organism. In general, an antigen can be or include any chemical entity, such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, other than a biological polymer (e.g., other than a nucleic acid or amino acid polymer)). In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a glycan. Those of skill in the art will generally understand that antigens may be provided in isolated or pure form, or may be provided in crude form (e.g., together with other substances, e.g., an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source), or may be present on or in a cell. In some embodiments, the antigen is a recombinant antigen.

[0022] Approximately or about: As used herein, the term "approximately" or "about" refers to a value similar to the stated reference value when applied to one or more values ​​of interest. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater or less), unless otherwise stated or clear from the context (except when the number exceeds 100% of the possible values).

[0023] Binding: The term "binding" as used herein is understood to refer to a non-covalent association between two or more entities. "Direct" binding involves physical contact between the entities or moieties, and indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied alone, or in the context of a more complex system (e.g., when covalently bound or otherwise associated with a carrier entity and / or in a biological system or cell).

[0024] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used interchangeably herein to refer to cells that exhibit relatively abnormal, unregulated, and / or autonomous growth, thereby exhibiting an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. In general, cells of interest for detection or treatment in this application include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The teachings of the present disclosure may be relevant to any and all cancers. To give some non-limiting examples, in some embodiments, the teachings of the present disclosure are applied to one or more cancers, such as hematopoietic cancers including, for example, leukemia, lymphoma (Hodgkin's and non-Hodgkin's lymphoma), myeloma, and myeloproliferative disorders; genitourinary system cancers, such as sarcoma, melanoma, adenocarcinoma, carcinoma of solid tissues, squamous cell carcinoma of the mouth, throat, larynx, and lung, liver cancer, prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer, as well as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancers, benign lesions such as papillomas, etc.

[0025] Chimeric Antigen Receptor (CAR) As used herein, "chimeric antigen receptor" or "CAR" refers to a recombinant cell surface receptor that is designed to be expressed on immune effector cells and specifically targets the cells and / or binds to an antigen.

[0026] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0027] Fusion Protein: As used herein, the term "fusion protein" generally refers to a polypeptide that includes at least two segments, each of which (1) is naturally occurring and / or (2) exhibits a high degree of amino acid identity to a peptide portion that represents a functional domain of the polypeptide. Typically, a polypeptide that contains at least two such segments is considered a fusion protein if the two segments are (1) portions that are not naturally contained in the same peptide, and / or (2) portions that have not previously been linked to each other in a polypeptide, and / or (3) portions that have been linked to each other through the action of the hand of man.

[0028] Nucleic acid: As used herein, "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is any compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As is clear from the context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., nucleotide and / or nucleoside), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain that includes individual nucleic acid residues. In some embodiments, "nucleic acid" is or includes RNA, and in some embodiments, "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, includes, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, includes, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," as known in the art, and peptide nucleic acids having peptide bonds rather than phosphodiester bonds in the backbone are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is single stranded, and in some embodiments, the nucleic acid is double stranded.In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide, or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0029] Pharmaceutically acceptable: The term "pharmacologically acceptable," as used herein, refers to a material that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0030] Polypeptide: As used herein, a "polypeptide" is, generally speaking, a string of at least two amino acids bound together by peptide bonds. In some embodiments, a polypeptide may contain at least 3-5 amino acids, each bound together by at least one peptide bond. In some embodiments, a polypeptide may be longer than 5 amino acids, each bound together by at least one peptide bond. One of skill in the art will appreciate that a polypeptide may sometimes contain "non-natural" amino acids or other entities, which may still be optionally incorporated into the polypeptide chain.

[0031] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be otherwise processed or modified. One of skill in the art will understand that a "protein" may be an entire polypeptide chain (with or without a signal sequence) as produced by a cell, or a portion thereof. One of skill in the art will understand that a protein may include more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, sialylation, acetylation, amidation, methylation, phosphorylation, etc. In some embodiments, a protein may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0032] Subject: "Subject" means a mammal (e.g., a human). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who possesses one or more characteristics characteristic of susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom and / or upon whom a diagnosis and / or treatment is to be performed.

[0033] Suffering from: An individual who is "suffering from" a disease, disorder, or condition (e.g., cancer) has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, or condition.

[0034] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease, disorder, and / or condition when administered according to a therapeutic administration regimen to a population suffering from or susceptible to the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that results in a reduction in the incidence and / or severity of, stabilization of, and / or delay in the onset of, one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not in fact require the achievement of successful treatment in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, results in a particular desired pharmacological response in a significant number of subjects. For example, in some embodiments, "therapeutically effective amount" in the context of the therapeutic method of the present invention refers to an amount that, when administered to an individual in need thereof, blocks, stabilizes, attenuates, or reverses a cancer-supportive process occurring in the individual, or enhances or increases a cancer-supportive process in the individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, prevents, stabilizes, inhibits, or reduces further development of cancer in the individual. Particularly preferred "therapeutically effective amounts" of the compositions described herein serve to reverse the development of a malignant tumor, such as pancreatic cancer (in the case of a therapeutic treatment), or to achieve or prolong the remission of a malignant tumor. The therapeutically effective amount administered to an individual to treat cancer in the individual may be the same as or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer treatments, the treatments described herein are not intended to be construed as, limited to, or otherwise limited to a "cure" of cancer; rather, these treatments relate to the use of the compositions described to "treat" cancer, i.e., to effect a desired or beneficial change in the health of an individual with cancer.Such benefits are recognized by skilled medical practitioners in the field of oncology and include, but are not limited to, stabilization of the patient's condition, reduction in tumor size (tumor regression), improvement in function (e.g., improved function of cancerous tissue or organ), reduction or inhibition of further metastasis, reduction in opportunistic infections, increased survival rate, reduction in pain, improvement in motor function, improvement in cognitive function, improvement in sense of energy (vitality, reduced fatigue), improvement in sense of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as a result of treatment described herein) may be assessed by taking samples of cancer cells from the site of the tumor, such as pancreatic adenocarcinoma (e.g., over the course of treatment), and testing the cancer cells for levels of metabolic and signaling markers to monitor the state of the cancer cells and confirm at the molecular level that the cancer cells have regressed to a less aggressive phenotype. For example, tumor regression induced by employing the methods of the present invention is indicated by finding a decrease in one or more pro-angiogenic markers, an increase in anti-angiogenic markers, or a normalization of a metabolic pathway, intercellular signaling pathway, or intracellular signaling pathway that exhibits aberrant activity in an individual diagnosed with cancer (i.e., a change to a state seen in a normal individual not afflicted with cancer). One of skill in the art will appreciate that in some embodiments, a therapeutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0035] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance that results in partial or complete alleviation, amelioration, reduction, inhibition, prevention, delay of onset, lessening of the severity, and / or reduction in the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment may be for subjects who do not exhibit symptoms of the relevant disease, disorder, and / or condition and / or subjects who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be for subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be for subjects who have been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be for subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0036] Vector: As used herein, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, a vector is capable of extrachromosomal replication and / or expression of a nucleic acid to which it is linked in a host cell, such as a eukaryotic cell and / or a prokaryotic cell. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors."

[0037] Detailed Description of Specific Embodiments overview The promise of cell therapy for cancer has been highlighted by the introduction of autologous T cells transduced with chimeric antigen receptors (CARs) targeting the B cell malignant tumor antigen CD19. Anti-CD19 CAR-T cells (CAR-19) induced high response rates and durable remissions in patients with relapsed and refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) (Ruella et al., Curr Hematol Malig Rep. 2016;11:368-84). Long-term follow-up studies have demonstrated the potential for cure, with some patients remaining cancer-free for nearly a decade (Chong et al., New England Journal of Medicine. Massachusetts Medical Society; 2021; 384: 673-4; Neelapu et al., Blood. 2021; 138: 93; Cappell et al., J Clin Oncol. 2020; 38: 3805-15; Schuster et al., Biology of Blood and Marrow Transplantation. 2019; 25: S20-1; Grupp et al., Biology of Blood and Marrow Transplantation. Elsevier; 2019; 25: S126-7).

[0038] However, the cost of CAR-19 therapy remains high, and the necessary infrastructure also limits the settings in which CARs can be made and administered. (Lin et al., J Clin Oncol. 2019;37:2105-19). Preparative apheresis, consolidation, lymphodepletion, and toxicity may limit the use of CAR-T therapy in frail patients. Side effects from CAR-19 treatment can be significant, including grade 3+ cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), leading to prolonged hospitalization and additional treatment costs (Brudno et al., Blood Rev. 2019;34:45-55). More than half of responding patients subsequently relapse from treatment, often within months of treatment (Maude et al., New England Journal of Medicine. Massachusetts Medical Society; 2018; 378: 439-48; Neelapu et al., New England Journal of Medicine. Massachusetts Medical Society; 2017; 377: 2531-44). Many patients relapse due to loss or downregulation of the CD19 protein on leukemia or lymphoma cells (Shah et al., Nat Rev Clin Oncol. 2019; 16: 372-85).

[0039] Relapse due to antigen loss is a phenomenon driven by natural selection; that is, CAR-19 T cells exert strong selective pressure on malignant B cell populations, which may result in the selection of clones with lost or reduced target protein, CD19. Antigen loss can occur via mutational events that prevent expression of the CD19 extracellular domain (ECD) or regions within the ECD and / or transcriptional downregulation of expression (Plaks et al., Blood. 2021; 138: 1081-5; Majzner et al., Cancer Discov. American Association for Cancer Research; 2018; 8: 1219-26). Such escape mechanisms are common across therapeutic modalities for cancer treatment (Donk et al., Blood Cancer Discov. American Association for Cancer Research Journals; 2021; 2: 302-18; Ruella et al., Comput Struct Biotechnol J. 2016; 14: 357-62). Other mechanisms of evasion of CAR-19 therapy have also been described, including lineage switching to myeloid cell leukemia and nibbling, a process in which CD19 is physically stripped from lymphoma cells by the CAR (Perna, Translational Cancer Research [Internet]. AME Publishing Company; 2016 [cited January 12, 2022]; 5 (available at tcr.amegroups.com / article / view / 9016); Miao et al., Frontiers in Immunology. 2021; 12:1862).

[0040] The fusion proteins described herein provide a solution to one or more of these problems. In some embodiments, the fusion proteins described herein can be expressed by cells transduced with lentiviral, retroviral or other gene therapy vectors and / or expressed by mammalian cell culture in vitro and then purified to generate an injectable biologic. In some embodiments, the fusion proteins of the present disclosure comprise three functional domains: a modified CD19 ECD, an anti-CD20 binding domain, and an anti-albumin binding domain. In some such embodiments, the fusion proteins of the present disclosure bind to CD20 and present a CD19 ECD. As described herein, such fusion proteins can (i) increase CD19 antigen density on target tumor cells regardless of their CD19 expression level, (ii) potently induce CD19-negative tumor cell death in vitro in the presence of CAR-19 T cells, (iii) prevent relapse due to antigen loss that evades CAR-19 therapy, (iv) prevent CD19-negative tumor expansion, e.g., at relatively low doses, and / or (v) significantly impact survival. As described herein, fusion proteins of the present disclosure can be expressed by transfected mammalian cells, can be efficiently purified, and can exhibit favorable biophysical properties indicating that such fusion proteins may be suitable for scale-up and production.

[0041] Fusion proteins In some embodiments, the present disclosure provides a fusion protein comprising or consisting of (i) a first antigen-binding polypeptide that binds to a first tumor antigen and (ii) a targeting polypeptide. In some embodiments, the present disclosure provides a fusion protein comprising or consisting of (i) a first antigen-binding polypeptide that binds to a first tumor antigen, (ii) a second antigen-binding polypeptide that binds to a second tumor antigen, and (iii) a targeting polypeptide. In some embodiments, the fusion protein described herein comprises or consists of an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 50, and 52, but lacking the half-life extending polypeptide of each of the present disclosures of the sequence. In some embodiments, the fusion proteins described herein comprise or consist of an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, and 502, but lacking the half-life extending polypeptide of the present disclosure of each of the sequences and lacking the C-terminal hexahistidine tag of the present disclosure. In some embodiments, the fusion proteins described herein comprise or consist of an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55.

[0042] In some embodiments, the present disclosure provides a fusion protein comprising or consisting of (i) an antigen-binding polypeptide that binds to a tumor antigen, (ii) a targeting polypeptide, and (iii) a half-life extending polypeptide. In some embodiments, the present disclosure provides a fusion protein comprising or consisting of (i) a first antigen-binding polypeptide that binds to a first tumor antigen, (ii) a second antigen-binding polypeptide that binds to a second tumor antigen, (iii) a targeting polypeptide, and (iv) a half-life extending polypeptide. In some embodiments, the fusion protein described herein comprises or consists of an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 50, and 52. As shown in the sequence listing provided herein, each of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, and 50 comprises (from amino terminus to carboxyl terminus) a signal sequence, an anti-CD20 VHH, a first linker (GSGGGGSGGGGS), a targeting polypeptide, a second linker (SRGGGGSGGGGSGGGGS), a half-life extending polypeptide, and a hexahistidine tag. In some embodiments, the fusion proteins described herein comprise or consist of an amino acid sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, and 50, and lacks the C-terminal hexahistidine tag. As shown in the sequence listing provided herein, each of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, and 52 comprises (from amino terminus to carboxyl terminus) an anti-CD20 VHH, a first linker (GSGGGGSGGGGS), a targeting polypeptide, a second linker (SRGGGGSGGGGSGGGGS), and a half-life extending polypeptide.

[0043] In addition to those sequences described herein, one of skill in the art will recognize many amino acid sequences suitable for use as linkers. In some embodiments, the first and / or second linker(s) comprise a naturally occurring sequence of amino acids. In some embodiments, the linker is derived from a naturally occurring multidomain protein. In some embodiments, the linker is flexible. In some embodiments, flexible linkers are designed using computational tools well known to those of skill in the art, such as JPred (Dorzdetskiy et al., Nucl Acids Res. 2015;43:W389-94). In some embodiments, the linker adopts a preferred conformation. In some embodiments, the linker comprises a GS linker, the linker being of the formula [G x S y ] z wherein x, y, and z are positive integers. In some embodiments, the linker comprises a polyglycine linker, the linker comprising the amino acid sequence G x In some embodiments, the linker comprises a glycine and serine-rich linker, and glycine and serine together account for 50% or more of the linker amino acid sequence. Many embodiments and applications of specific linker sequences in fusion proteins and methods of linker design are described in Chen et al., Adv Drug Deliv Rev. 2013; 65: 1357-69; and Liu et al., Bioinformatics. 2015; 31: 3700-2, the contents of each of which are incorporated herein by reference.

[0044] Antigen-binding polypeptides In some embodiments, the fusion protein of the present disclosure comprises an antigen-binding polypeptide that binds to a tumor antigen, e.g., a tumor antigen described herein. In some embodiments, the antigen-binding polypeptide binds to CD20. In some embodiments, the antigen-binding polypeptide is an anti-CD20 antibody or a CD20-binding fragment thereof. In some embodiments, the antigen-binding polypeptide is or comprises an anti-CD20 scFv, Fv, or other multi-domain binding fragment. In some embodiments, the antigen-binding polypeptide is or comprises an anti-CD20 single domain antibody, e.g., an anti-CD20 single domain antibody described herein.

[0045] In some embodiments, a fusion protein of the disclosure comprises a first antigen-binding polypeptide that binds CD20 and a second antigen-binding polypeptide that binds a second tumor antigen described herein (e.g., CD79b, HER-2 / neu, c-met, EGFR, Ga733\EpCAM, CD21, ROR1, CLL-1 / CLEC12A, HLA-DR10, CD1, CD5, CD21, CD25, CD27, CD30, CD38, CD78, CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2, or BCMA). In some embodiments, the first antigen-binding polypeptide is an anti-CD20 antibody or a CD20-binding fragment thereof. In some embodiments, the first antigen-binding polypeptide is or comprises an anti-CD20 scFv, Fv, or other multi-domain binding fragment. In some embodiments, the first antigen-binding polypeptide is or comprises an anti-CD20 single domain antibody, eg, an anti-CD20 single domain antibody described herein.

[0046] Single Domain Antibodies A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that do not naturally have light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any in the art or any future single domain antibody. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, cow. According to one aspect of the present disclosure, a single domain antibody as used herein is a single domain antibody known as a heavy chain antibody without a light chain. Such single domain antibodies are disclosed, for example, in WO94 / 04678. Variable domains derived from heavy chain antibodies that do not naturally have light chains (e.g., single domain antibodies) are referred to herein as "VHH" or "nanobody". Such VHHs may be derived from antibodies produced in Camelidae species, such as camel, dromedary, llama, vicuna, alpaca and guanaco. Other non-Camelidae species can also produce heavy chain antibodies that do not naturally have light chains, and such VHHs are also within the scope of the present disclosure.

[0047] The amino acid residues of VHH domains from Camelidae are numbered according to the general numbering system for VH domains as set out by Kabat et al., "Sequence of proteins of immunological interest", US Public Health Services, NIH (Bethesda, MD), Publication No 91-3242 (1991), see also Riechmann et al., J. Immunol. Methods 231:25-38 (1999). According to this numbering system, FR1 comprises amino acid residues at positions 1-30, CDR1 comprises amino acid residues at positions 31-35, FR2 comprises amino acid residues at positions 36-49, CDR2 comprises amino acid residues at positions 50-65, FR3 comprises amino acid residues at positions 66-94, CDR3 comprises amino acid residues at positions 95-102, and FR4 comprises amino acid residues at positions 103-113.

[0048] However, it should be noted (for VH and VHH domains, as is well known in the art) that the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This generally means that the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

[0049] Alternative methods for numbering the amino acid residues of VH domains can be applied in a similar manner to VHH domains and are known in the art. In this disclosure, claims and figures, CDRs are defined according to the IMGT numbering system unless otherwise indicated (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010)).

[0050] anti-CD20 VHH In some embodiments, a fusion protein of the disclosure comprises a CD20 binding polypeptide that is or comprises an anti-CD20 VHH. In some embodiments, the anti-CD20 VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 37-44 and 53, or a CD20 binding fragment thereof.

[0051] In some embodiments, the anti-CD20 VHH comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 37-44 and 53, or a CD20-binding portion thereof.

[0052] In some embodiments, the anti-CD20 VHH comprises or consists of at least one CDR (e.g., CDR1, CDR2, and / or CDR3) set forth in any one of SEQ ID NOs: 37-44 and 53. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 37. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 38. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 39. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 40. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 41. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 42. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 43. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 44. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 53.

[0053] In some embodiments, the anti-CD20 VHH comprises or consists of at least one CDR that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a CDR (e.g., CDR1, CDR2, and / or CDR3) set forth in any one of SEQ ID NOs: 37-44 and 53. In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:37, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:37, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:37. In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:38, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:38, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:38.In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:39, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:39, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:39. In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:40, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:40, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:40. In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:41, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:41, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:41.In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:42, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:42, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:42. In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO: 43, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO: 43, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO: 43. In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:44, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:44, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:44.In some embodiments, the anti-CD20 VHH comprises a CDR1 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1 set forth in SEQ ID NO:53, a CDR2 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR2 set forth in SEQ ID NO:53, and a CDR3 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR3 set forth in SEQ ID NO:53.

[0054] The antibodies or fragments can be produced by any method of synthesizing antibodies known in the art (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO92 / 22324; WO98 / 46645). Chimeric antibodies can be produced, for example, using the methods described in Morrison, 1985, Science 229:1202, and humanized antibodies can be produced, for example, by the methods described in U.S. Patent No. 6,180,370.

[0055] Target Polypeptide In some embodiments, the fusion protein of the present disclosure comprises a targeting polypeptide, which in some embodiments is an antigen target for a cellular therapy, such as a CAR-T cell, an antibody, or an antibody drug conjugate, as described in WO2017 / 075537, WO2017 / 075533, WO2018156802, and WO2018156791.

[0056] In some embodiments, the target polypeptide comprises or consists of all or part of a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.Other tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, MUC1 6, IL13Rα2, FRα, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, nectin 4, AGS-16, guanaryl cyclase C, MUC-1, CFC1B, integrin alpha 3 chain (a3b1, laminin receptor chain), and TPS.

[0057] In some embodiments, the target polypeptide comprises or consists of all or a portion of a tumor antigen selected from CD19, CD20, CD22, CD30, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLEC12A, ROR1, glypican 3 (GPC3), mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1, and MAGE A3.

[0058] In some embodiments, the target polypeptide comprises or consists of all or a portion of a B cell specific marker, e.g., CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, CD180, ROR1, BCMA, HLA-DR10, CD1, CD5, CD21, CD25, CD27, CD30, CD38, CD78, CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2, CD79a, and CD79b (see, e.g., LeBien et al., Blood 112:1570-1580 (2008)).

[0059] CD19 is a 95 kDa type I transmembrane glycoprotein that is used as a biomarker for B cell development (Wang et al., Exp. Hematol. Oncol. 1:36 (2012)). CD19 expression in lymphomas and leukemias has made it an effective therapeutic target, especially for chimeric antigen receptor (CAR) T cell therapy (Maude et al., Blood 125:4017-4024 (2015)). Based on the inherent efficacy of CD19 in CAR-T cell therapy, therapeutic approaches have been described that involve "converting" CD19- tumors to CD19+ tumors using antibody-CD19 fusions or CD19 variants engineered to directly bind tumor biomarkers. (See, e.g., WO2017 / 075537 and WO2017 / 075533).

[0060] The extracellular region of CD19 has been hypothesized to contain two C2-like immunoglobulin domains (see, e.g., Wang et al., Exp. Hematol. Oncol. 1:36 (2012); Tedder et al., Nat. Rev. Rheumatol. 5:572-577 (2009)). This is supported by homology modeling (Soding et al., Nucleic Acids Res. 33:244-248 (2005)). However, later published structures have shown that CD19 does not contain C2-like immunoglobulin domains (Teplyakov et al., Proteins 86:495-500 (2018)). The amino acid sequence of wild-type human CD19 is provided herein as SEQ ID NO:47.

[0061] In some embodiments, the target polypeptide comprises or consists of all or a portion of the amino acid sequence of SEQ ID NO: 47. In some embodiments, the target polypeptide comprises or consists of an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or about 100% identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the target polypeptide comprises or consists of about 200, 210, 220, 230, 240, 250, 260, 270, or 280 consecutive amino acids of SEQ ID NO: 47. In some embodiments, the target polypeptide comprises or consists of all or a portion of the CD19 extracellular domain (ECD). For example, in some embodiments, the target polypeptide comprises or consists of a fragment of SEQ ID NO: 47, such as a fragment comprising about amino acid 20 to about amino acid 278 of SEQ ID NO: 47. In some embodiments, the target polypeptide comprises or consists of all or a portion of the amino acids of SEQ ID NO: 48. In some embodiments, the target polypeptide comprises or consists of an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or about 100% identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the target polypeptide comprises or consists of all or a portion of a CD19 variant or a fragment thereof. In some embodiments, the target polypeptide comprises or consists of all or a portion of the amino acids of SEQ ID NO: 56. In some embodiments, the target polypeptide comprises or consists of an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or about 100% identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the target polypeptide comprises or consists of all or a portion of a CD19 variant or a fragment thereof. In some embodiments, the CD19 variant is or comprises a full-length CD19 polypeptide (e.g., SEQ ID NO: 47) or a portion thereof, comprising one or more amino acid substitutions described herein.In some embodiments, the CD19 variant is or comprises a CD19 ECD (e.g., SEQ ID NO: 48 or SEQ ID NO: 56) or a portion thereof, comprising one or more amino acid substitutions. In some embodiments, the CD19 variant is or comprises a CD19 ECD or a portion thereof described in WO2019 / 118918, the entire contents of which are incorporated herein by reference. In some embodiments, the CD19 variant comprises or consists of the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CD19 variant comprises or consists of an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or about 100% identity to the amino acid sequence of SEQ ID NO: 46.

[0062] Half-life extension In some embodiments, a fusion protein of the present disclosure includes an agent that increases the half-life of the fusion protein, e.g., compared to the fusion protein without the agent, in some embodiments, such an agent is a polypeptide referred to herein as a "half-life extending polypeptide."

[0063] In some embodiments, the half-life extending polypeptide is a transferrin polypeptide or a portion thereof. Transferrin is recycled by binding to the transferrin receptor (see, e.g., Widera et al., Adv. Drug Deliv. Rev. 55:1439-66 (2003)). In some embodiments, the half-life extending polypeptide is albumin (e.g., bovine serum albumin (BSA), human serum albumin (HSA), or mouse serum albumin (MSA)) or a fragment thereof. In some embodiments, the half-life extending polypeptide is a polypeptide that binds to a serum protein. In some embodiments, the half-life extending polypeptide is a serum albumin binder (e.g., a BSA, HSA, or MSA binder).

[0064] In some embodiments, the serum albumin binder is an albumin binding peptide. Albumin binding peptides are described in WO200145746, WO2002076489, WO2008068280, WO2009127691, WO2011095545, and US Patent Publication Nos. 20040001827, 20080187517, and 20130316952. Those skilled in the art are familiar with methods for directly linking proteins and antibodies to albumin or albumin domains, for example, as described in Patterson et al., Bioconjugate Chem. 2016, 27, 10, 2271-2275; Bern et al., Sci.Trans.Med., 14 Oct 2020 · Vol 12, Issue 565.

[0065] In some embodiments, the half-life extending polypeptide is or comprises a hyaluronan binding domain. Hyaluronan (HA), also known as hyaluronic acid, is a glycosaminoglycan present in connective and other tissues, and is abundant in synovial fluid, skin, and vitreous. HA binds to a number of naturally occurring hyaluronan binding proteins (HABPs). Some HABPs contain and bind HA through a HA binding domain called the link module (Kohda, CJ et al., Cell 86 (1996) 767-775). Some HABPs contain a linear HA binding motif of 9-11 residues containing multiple basic amino acids, called the B-X7-B motif (Yang B., et al. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J., 13:286-296, 1994).

[0066] In some embodiments, the half-life extending polypeptide is a PAS polypeptide. As used herein, a "PAS polypeptide" is a polypeptide characterized in that the sum of proline, alanine, and serine residues constitutes more than about 80%, or more than about 85%, or more than about 90%, or more than about 95%, or more than about 96%, or more than about 97%, or more than about 98%, or more than about 99%, or 100% of the total amino acid sequence of the half-life extending polypeptide. Generally, PAS polypeptides are characterized in that they adopt a random coil conformation under physiological conditions, as described in U.S. Patent Publication No. 20100292130. PAS polypeptides that may be used as half-life extending polypeptides of the fusion proteins of the present disclosure are further described in WO2008 / 155134, U.S. Patent Publication No. 20100292130, U.S. Patent No. 8,563,521, and / or U.S. Patent No. 9,260,494.

[0067] In some embodiments, the half-life extending polypeptide consists exclusively of proline and alanine, or consists primarily of proline and alanine, but may have up to 1%, 2%, 3%, 5%, or 10% of other amino acid residues. If other amino acids are present, they may all be the same, or multiple different amino acids may be present. Examples of polypeptides that are composed primarily or entirely of proline and alanine and adopt a random coil conformation under physiological conditions, referred to as proline / alanine random coil polypeptides, are described in U.S. Patent Publication No. 20130072420, U.S. Patent No. 9,221,882, and / or U.S. Patent No. 10,081,657.

[0068] In some embodiments, the half-life extending polypeptide is a polypeptide characterized in that the total of glycine, alanine, serine, threonine, glutamic acid, and proline residues make up more than about 80%, or more than about 85%, or more than about 90%, or more than about 95%, or more than about 96%, or more than about 97%, or more than about 98%, or more than about 99%, or 100% of the total amino acid sequence of the half-life extending polypeptide, and the half-life extending polypeptide comprises at least four of these six different amino acids. Such polypeptides may be referred to as being composed primarily of amino acids selected from G, A, S, T, E, and P (see, e.g., U.S. Patent Publication Nos. 20030228309; 9,926,351; 9,976,166; and 10,961,287).

[0069] In some embodiments, the half-life extending polypeptide is an antibody or a fragment thereof. In some embodiments, the half-life extending polypeptide is an anti-albumin antibody or an albumin binding fragment thereof. In some embodiments, the half-life extending polypeptide is or comprises an anti-albumin single domain antibody. In some embodiments, the half-life extending polypeptide is or comprises an anti-albumin VHH. In some embodiments, the anti-albumin VHH comprises or consists of the amino acid sequence disclosed or described in US Publication No. 20070269422, the entire contents of which are incorporated herein by reference. In some embodiments, the anti-albumin VHH comprises or consists of the amino acid sequence of SEQ ID NO: 45 or an albumin binding portion thereof. In some embodiments, the anti-albumin VHH comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45, or an albumin binding portion thereof.

[0070] tumor The present disclosure provides techniques useful for the treatment of any tumor. In some embodiments, the tumor is or comprises a hematological malignancy, including, but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Langerhans cell histiocytosis, multiple myeloma, or myeloproliferative neoplasm.

[0071] In some embodiments, the tumor is or comprises a solid tumor, including, but not limited to, breast cancer, squamous cell carcinoma, colon cancer, head and neck cancer, ovarian cancer, lung cancer, mesothelioma, genitourinary system cancer, rectal cancer, gastric cancer, or esophageal cancer.

[0072] In some particular embodiments, the tumor is or comprises an aggressive tumor and / or a refractory tumor. In some embodiments, a tumor is characterized as aggressive if a particular pathology is observed in the tumor (e.g., a tissue sample, such as a biopsy sample, taken from the tumor) and / or if a cancer patient having such a tumor is not typically considered a candidate for conventional chemotherapy. In some embodiments, the pathology that characterizes a tumor as aggressive may include tumor size, changes in expression of genetic markers, invasion of adjacent organs and / or lymph nodes by tumor cells. In some embodiments, a tumor is characterized as refractory if a patient having such a tumor is resistant to one or more known therapeutic modalities (e.g., one or more conventional chemotherapy regimens) and / or if a particular patient has shown resistance (e.g., lack of response) to one or more such known therapeutic modalities.

[0073] Cell Therapy In some embodiments, the fusion proteins described herein can be administered to a subject as a cellular therapy. For example, a nucleotide sequence encoding the fusion proteins described herein can be introduced into a cell for administration to a subject as a cellular therapy. In some embodiments, the cellular therapy can be made from immune cells, e.g., cells that are useful or usable for adoptive cell therapy. In some embodiments, the cellular therapy can be made from immune cells, e.g., cells that are useful or usable for adoptive cell therapy. In some embodiments, the cellular therapy can be made from immune cells, e.g., cells that are useful or usable for adoptive cell therapy. + cells, CD4 + The tumor-infiltrating lymphocytes are made from a cell type selected from the group consisting of CD3+ T cells, NK-cells, gamma delta T cells, regulatory cells, iNKT cells, monocytes, macrophages, IPSC-derived cells, or peripheral blood mononuclear cells. As used herein, "tumor infiltrating lymphocytes" or TILs refer to white blood cells that have migrated into the bloodstream and into tumors. Lymphocytes can be classified into three groups, including B cells, T cells, and natural killer cells. As used herein, "T cells" refers to cells that are CD3+ T cells or TILs ... + Refers to cells, CD4 + Helper cells, CD8 + Includes cytotoxic T cells and delta gamma T cells.

[0074] In certain embodiments, cellular therapeutic agents are produced by genetically modifying (e.g., transforming) cells, e.g., immune cells, with a nucleic acid encoding a fusion protein described herein. In some embodiments, such a nucleic acid is contained in a recombinant expression vector. Recombinant expression vectors can contain any type of nucleotide, including, but not limited to, DNA and RNA, can be single-stranded or double-stranded, can be synthetic, can be derived in part from natural sources, and can contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring internucleotide linkages, or can contain both types of linkages.

[0075] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and propagation or expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0076] In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, retroviral vectors, alphaviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes viruses, and fowlpox virus vectors, preferably with native or engineered ability to transform immune cells (e.g., T cells).

[0077] For ex vivo applications such as cell therapy, gammaretroviral vectors derived from murine leukemia virus (MLV) were first developed and are still used. Lentiviral vectors based on human immunodeficiency virus (HIV) are also widely used. A common strategy in designing lentiviral vectors is based on deleting and modifying native viral sequences to prevent the generation of replication-competent viruses. Thus, lentiviral components are separated into three or four different plasmid constructs with the aim of preventing the possibility of complete recombination into fully replication-competent lentiviruses (RCLs). The viral vector genome minimally contains a transgene expression cassette, long terminal repeats (LTRs), and a packaging signal. In most cases, three additional plasmids provide factors necessary for virus production and packaging (e.g., gag, pol, env). The promoter-enhancer region of the 3'LTR is deleted to prevent transcription from this region and subsequent viral replication (called self-inactivating vectors; SINs). The basic steps of ex vivo cell transformation or transduction include cell isolation and culture of the desired cell type, allowing selection, expansion, and differentiation either before or after transduction of the cells with a viral vector. For hematopoietic cells, most of these steps are performed in a closed system using single-use blood collection and processing bags. In CAR T cell therapy, the patient's blood cells are collected, the desired T cell population is selected and grown to the required level. They are then transduced with a viral vector carrying the desired gene cassette, and the CAR T cells are subsequently expanded to the billion level. Lentiviral vectors have been shown to efficiently transduce T cells and are therefore the preferred vector for introducing CARs into the patient's target cells. The expanded cells are then reintroduced into the patient.

[0078] In certain in vivo applications, a nucleic acid encoding a fusion protein described herein, or a vector containing a nucleic acid encoding a fusion protein described herein, is administered to an individual in need thereof. For example, a recombinant expression vector comprising a nucleic acid encoding a fusion protein as described herein can be provided as described, for example, in Nawaz et al., Blood Cancer Journal volume 11, Article number: 119 (23 June 2021) Carbonaro-Sarracino et al., Molecular Therapy: Methods & Clinical Development Vol. 16 March 2020; Cantore and Naldini Haemophilia, Volume 27, Issue S3 p. 122-125; Gouze-Decaris, et al., Arthritis Res. 2001; 3 (Suppl 1): P34; Breuer et al., Scientific Reports volume 10, Article number: 4544 (2020), Naldini et al., SCIENCE · 12 Apr 1996 · Vol 272, Issue 5259 · pp. 263-267. Viral vector particles can be used to directly deliver nucleic acid in vivo. Examples of such viral vector particles include lentivirus, retrovirus, AAV, HVS, vaccinia and many other virus types. Viral vector particles can be modified to optimize delivery to specific cell types, including, for example, immune cells.See, e.g., Yang et al., PNAS August 1, 2006 | 103(31)11479-11484; Schaffer et al., Annu Rev Biomed Eng. 2008; 10:169-194; Lee et al., J. of Controlled Release Volume 334, 10 June 2021, Pages 106-113; Jang et al., Molecular Therapy Volume 19, Issue 8, August 2011, Pages 1407-1415.

[0079] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Expression vector constructs can be circular or linear and prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

[0080] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation to confer prototrophy in auxotrophic hosts, etc. Suitable marker genes for recombinant expression vectors include, for example, the neomycin / G418 resistance gene, the puromycin resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0081] Vectors useful in the context of the present disclosure can be "naked" nucleic acid vectors (i.e., vectors with little or no protein, sugar, and / or lipids enveloping the vector) or vectors complexed with other molecules. Other molecules that may be suitably combined with vectors include, but are not limited to, viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties, such as ligands, receptors, or antibodies that target cellular molecules.

[0082] Vector DNA can be introduced into cells, such as immune cells (e.g., T cells), via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" and "transduction" are intended to refer to various techniques for introducing foreign nucleic acids (e.g., DNA) into cells that are recognized in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, gene gun, or electroporation. The cell lines used to produce viral vector particles can themselves be modified to introduce useful features, including, but not limited to, non-self-shielding polypeptides, cell-type nutrient polypeptides, anti-immunosuppressive polypeptides, and half-life extension polypeptides.

[0083] Protein Therapeutics In some embodiments, the fusion proteins described herein can be produced and used as protein therapeutics instead of or in addition to being produced by the cellular therapeutics described herein. Such polypeptides can be included in compositions, e.g., pharmaceutical compositions, and used as protein therapeutics. For example, protein therapeutics including the fusion proteins described herein can be administered in combination with cellular therapeutics, e.g., CAR-T cells or ADCs, that target CD19.

[0084] A variety of methods for making polypeptides are known in the art and can be used to make polypeptides for inclusion in protein therapeutics. For example, a polypeptide can be produced recombinantly by utilizing a host cell system engineered to express a nucleic acid encoding the polypeptide. Recombinant expression of a gene can include the construction of an expression vector containing a polynucleotide encoding the polypeptide. Once a polynucleotide is obtained, a vector for producing the polypeptide can be made by recombinant DNA technology using techniques known in the art. Known methods can be used to construct an expression vector containing a polypeptide coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination.

[0085] The expression vector can be transferred to a host cell by conventional techniques and the transfected cells can then be cultured by conventional techniques to produce the polypeptide.

[0086] A variety of host-expression vector systems can be used (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems can be used to produce a polypeptide, which can then be purified as desired. Such host expression systems include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the polypeptide coding sequence; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the polypeptide coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the polypeptide coding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0087] In bacterial systems, many expression vectors may be used, including, but not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791); pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509), etc. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).

[0088] For expression in mammalian host cells, viral-based expression systems can be utilized (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). The efficiency of expression can be enhanced by including appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544).

[0089] In addition, a host cell line can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed polypeptide. Such cells include, for example, established mammalian and insect cell lines, animal cells, fungal cells, and yeast cells. Mammalian host cells include, for example, BALB / c mouse myeloma cell lines (NS0 / 1, ECACC No: 85110503); human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell lines (e.g., HT1080); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); Mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1) 587); Human cervical cancer cells (HELA, ATCC CCL 2); Dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocytes (Hep G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC) CCL51); TRI cells (Mather et al.,Annals NYAcad. Sci., 383:44-68, 1982); MRC5 cells; FS4 cells; and a human hepatoma line (HepG2).

[0090] For long-term, high-yield production of recombinant proteins, host cells are engineered to stably express the polypeptide. Host cells can be transformed with DNA controlled by suitable expression control elements known in the art, including promoters, enhancers, sequences, transcription terminators, polyadenylation sites, and selection markers. Desired recombinant clones can be selected using methods commonly known in the art of recombinant DNA technology.

[0091] Once the proteins described herein are produced by recombinant expression, they can be purified by any purification method known in the art, such as chromatography (e.g., ion exchange, affinity, and size column chromatography), centrifugation, differential solubility, or any other standard protein purification method. For example, antibodies can be isolated and purified by appropriately selecting affinity columns, such as protein A columns, in combination with chromatography columns, filtration, ultrafiltration, salting out, and dialysis methods (see Antibodies: A Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). Furthermore, as described herein, the polypeptides can be fused to heterologous polypeptide sequences to facilitate purification. Alternatively or additionally, the polypeptides or fusion proteins can be partially or completely prepared by chemical synthesis.

[0092] Viral delivery In some embodiments, a nucleic acid encoding a fusion protein described herein can be introduced into a cell and / or administered to a subject as a viral vector. In some embodiments, such a viral vector can be used to introduce the fusion protein into a cancer cell (e.g., a tumor cell). The introduction of such a fusion protein can increase the subject's immune system and / or its susceptibility to one or more additional therapeutic agents (see, e.g., WO2017 / 075533).

[0093] Vector Design The nucleic acid sequence encoding the fusion protein described herein can be introduced into many kinds of vectors. For example, the nucleic acid can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Other vectors can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and viral vectors. As another example, the vector can be a foamy virus (FV) vector, which is a type of retroviral vector made from a spuma virus. The design and technology of viral vectors are well known in the art, as described in Sambrook et al (Molecular Cloning: A Laboratory Manual, 2001) and other virology and molecular biology manuals.

[0094] Viral transduction Viruses are highly efficient in nucleic acid delivery to certain cell types and often evade detection by the infected host immune system. These characteristics make certain viruses attractive candidates as vehicles for introducing cell therapy targets into cancer cells, such as solid tumor cells. Many virus-based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, poxviruses, herpes simplex virus 1, herpes viruses, oncoviruses (e.g., murine leukemia viruses), and the like. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0095] Lentiviral and retroviral transduction can be enhanced by the addition of LentiBOOST (Mayflower Bioscience SBPLV10112) or TransDux (System Biosciences LV850 A-1) or polybrene (SantaCruz sc-134220; Millipore TR-1003-G; Sigma 107689), a cationic polymer (also known as hexamethrine bromide) used to increase the efficiency of lentiviral or retroviral transduction.

[0096] For example, retroviruses provide a platform for gene delivery systems. Retroviruses are enveloped viruses that belong to the Retroviridae family of viruses. Once inside a host cell, the virus replicates by transcribing RNA into DNA using viral reverse transcriptase. Retroviral DNA replicates as part of the host genome and is called a provirus. Using techniques known in the art, a selected gene can be inserted into a vector and packaged into retroviral particles. The recombinant virus can then be isolated and delivered in vivo to the cells of a subject. Many retroviral systems are known in the art (see, for example, U.S. Pat. Nos. 5,994,136, 6,165,782, and 6,428,953).

[0097] Retroviruses include the alpharetrovirus (e.g., avian leukemia virus), betaretrovirus (e.g., mouse mammary tumor virus), deltaretrovirus (e.g., bovine leukemia virus and human T-lymphotropic virus), epsilonretrovirus (e.g., walleye dermal sarcoma virus), and lentivirus. In some embodiments, the retrovirus is a lentivirus, a genus of viruses in the Retroviridae family, characterized by, for example, a long incubation period. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells and can deliver large amounts of genetic information to the DNA of host cells, allowing them to be used as efficient gene delivery vectors. In some examples, lentiviruses can be, but are not limited to, human immunodeficiency viruses (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia (EIA), and visna virus. Lentivirus-derived vectors provide a means to achieve significant levels of in vivo gene transfer.

[0098] In some embodiments, the vector is an adenovirus vector. Adenovirus is a large family of viruses that contain double-stranded DNA. Adenovirus replicates the DNA of host cells and uses the host cell machinery to synthesize viral RNA DNA and protein. It is known in the art that adenovirus acts on both replicating and non-replicating cells, can accommodate large transgenes, and can code proteins without being integrated into the genome of host cells.

[0099] In some embodiments, AAVP vectors are used. AAVP vectors are hybrid prokaryotic-eukaryotic vectors, chimeras of recombinant adeno-associated virus (AAV) genetic cis elements and phage. AAVP combines selected elements from both phage and AAV vector systems, resulting in vectors that are easy to generate in bacteria, exhibit little or no packaging limitations, and allow for mammalian cell infection and integration into host chromosomes. Vectors containing many of the appropriate elements are commercially available and can be further modified by standard methods to include the required sequences. Among other things, AAVP does not require helper viruses or trans-acting factors. In addition, the lack of AAV encapsidation eliminates the natural tropism of AAV for mammalian cells. Other methods and details can be found in U.S. Pat. No. 8,470,528 and Hajitou A. et al., Cell, 125:358-398.

[0100] In some embodiments, human papillomavirus (HPV) pseudoviruses are used. DNA plasmids can be packaged into papillomavirus L1 and L2 capsid proteins to generate pseudovirions that can efficiently deliver DNA. Encapsulation can protect DNA from nucleases, resulting in a high level of stability in targeted delivery. Many of the safety concerns associated with the use of viral vectors can be alleviated by HPV pseudoviruses. Other methods and examples can be found in Hung, C., et al., Plos One, 7:7(e40983); 2012, US Patent 8,394,411, and Kines, R., et al Int J of Cancer, 2015.

[0101] In some embodiments, oncolytic viruses are used. Oncolytic virus therapy can selectively replicate the virus in cancer cells, which can then spread within the tumor, for example, without affecting normal tissues. Alternatively, oncolytic viruses preferentially infect and kill cells without damaging normal tissues. Oncolytic viruses can also effectively induce immune responses against themselves, as well as against the infected tumor cells. Typically, oncolytic viruses belong to two classes: (I) viruses that naturally replicate preferentially in cancer cells and are non-pathogenic in humans. Exemplary class (I) oncolytic viruses include autonomous parvoviruses, myxoma viruses (poxviruses), Newcastle disease virus (NDV; paramyxoviruses), reoviruses, and Seneca Valley viruses (picornaviruses). The second class (II) includes viruses that have been genetically engineered for use as vaccine vectors, including measles virus (paramyxoviruses), polioviruses (picornaviruses), and vaccinia virus (poxviruses). Additionally, oncolytic viruses can include those genetically engineered to have mutations / deletions in genes required in normal cells but not in cancer cells, including adenovirus, herpes simplex virus, and vesicular stomatitis virus. Oncolytic viruses can be used as a viral transduction method since they can target multiple pathways and replicate in a tumor-selective manner, resulting in a low probability of genetic resistance. The viral load in the tumor can increase over time due to in situ viral amplification (compared to small molecule therapies that decrease over time), and safety features can be incorporated (i.e., drug and immune susceptibility).

[0102] Formulation and Administration Certain embodiments of the disclosure include methods of administering to a subject a cellular therapy (or a population thereof) described herein, a protein therapeutic described herein, a composition comprising a cellular therapy, and / or a composition comprising a protein therapeutic, e.g., in an amount effective to treat the subject. In some embodiments, the method effectively treats cancer in the subject.

[0103] In some embodiments, the cellular therapy comprises autologous cells that are administered to the same subject from which the immune cells were obtained, or alternatively, immune cells are obtained from a subject and transformed, e.g., transduced, with an expression construct described herein to provide a cellular therapy for allogeneic transfer into another subject.

[0104] In some embodiments, the cellular therapy is autologous to the subject, which may be in an immunologically naive, immunized, diseased, or otherwise state prior to isolation of immune cells from the subject.

[0105] In some embodiments, additional steps may be performed prior to administration to a subject. For example, the cellular therapeutic agent may be expanded in vitro after contacting (e.g., transducing or transfecting) immune cells with an expression construct described herein and prior to administration to a subject. The in vitro expansion may be performed for 1 or more days, e.g., 2 or more days, 3 or more days, 4 or more days, 6 or more days, or 8 or more days, prior to administration to a subject. Alternatively or additionally, the in vitro expansion may be performed for 21 days or less, e.g., 18 days or less, 16 days or less, 14 days or less, 10 days or less, 7 days or less, or 5 days or less, prior to administration to a subject. For example, the in vitro expansion may be performed for 1-7 days, 2-10 days, 3-5 days, or 8-14 days prior to administration to a subject.

[0106] In some embodiments, during in vitro expansion, the cellular therapy may be stimulated with an antigen (e.g., a TCR antigen). Antigen-specific expansion can optionally be assisted by expansion under conditions that non-specifically stimulate lymphocyte proliferation, such as, for example, anti-CD3 antibodies, anti-Tac antibodies, anti-CD28 antibodies, or phytohemagglutinin (PHA). The expanded cellular therapy can be administered directly to a subject or frozen for future use, i.e., for later administration to a subject.

[0107] In some embodiments, the cellular therapy is infused into the cancer patient after ex vivo treatment with interleukin-2 (IL-2), and the cancer patient is treated with IL-2 after infusion. Additionally, in some embodiments, the cancer patient may undergo conditioning lymphodepletion (temporary ablation of the immune system) prior to administration of the cellular therapy. The combination of IL-2 treatment and conditioning lymphodepletion can increase the persistence of the cellular therapy.

[0108] In some embodiments, the cellular therapeutic agent is transduced or transfected with a nucleic acid encoding a cytokine, which can be engineered to provide for constitutive, regulatable, or transiently controlled expression of the cytokine. Suitable cytokines include, for example, cytokines that act to enhance survival of T lymphocytes during contraction and can promote the formation and survival of memory T lymphocytes.

[0109] In certain embodiments, the cellular therapy is administered prior to, about simultaneously with, or after administration of another therapeutic agent, such as a cancer therapeutic agent. The cancer therapeutic agent may be, for example, a chemotherapeutic agent, a biologic, or radiation therapy. In some embodiments, the subject receiving the cellular therapy has not received sufficient therapy to result in the depletion of immune cells, such as lymphodepleting chemotherapy or radiation therapy.

[0110] The cellular therapeutic agents described herein can be formed as compositions, e.g., compositions of cellular therapeutic agents and pharma- ceutically acceptable carriers. In certain embodiments, the compositions are pharmaceutical compositions comprising at least one cellular therapeutic agent described herein and a pharma- ceutically acceptable carrier, diluent, and / or excipient. The pharma- ceutically acceptable carriers described herein, e.g., vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available. Preferably, the pharma- ceutically acceptable carrier is chemically inert to the active agent(s), e.g., cellular therapeutic agent, and does not induce any adverse side effects or toxicity under the conditions of use.

[0111] The composition can be formulated for administration by any suitable route, such as, for example, intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and / or subcutaneous routes of administration. Preferably, the composition is formulated for parenteral administration routes.

[0112] Compositions suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes, such as those which render the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0113] The dosage administered to a subject, particularly a human, may vary with the particular embodiment, composition employed, method of administration, and the particular site and subject being treated. However, the dose must be sufficient to produce a therapeutic response. A clinician skilled in the art can determine the therapeutically effective amount of the composition administered to a human or other subject to treat or prevent a particular medical condition. The exact amount of the composition required to be therapeutically effective will depend on a number of factors, such as the specific activity and route of administration of the cellular therapy agent, the amount of antigen(s) available on tumor cells (e.g., due to tumor size or extent of tumor burden) and / or on normal cells, in addition to many subject-specific considerations, but are within the skill of the art. In some embodiments, the appropriate dose of the cellular therapy agent for a particular cancer indication(s) may be defined in a dose escalation clinical trial.

[0114] Any suitable number of cell therapy cells may be administered to a subject. Although a single cell therapy cell as described herein can be expanded to provide therapeutic benefit, in some embodiments, up to 10 2 More than, for example, 10 3 That's it, 10 4 That's it, 10 5 More than or equal to 10 8 Alternatively or additionally, 10 or more cellular therapy cells are administered. 12 For example, 10 11 Below, 10 9 Below, 10 7 Less than or equal to 10 5 Cell therapy cells as described herein below are administered to the subject. In some embodiments, 2 ~10 5 , 10 4 ~10 7 , 10 3 ~10 9 , or 10 5 ~10 10 The cell therapy cells described herein are administered.

[0115] The dose of the cellular therapy described herein can be administered to a mammal once, or in a series of subdoses over a suitable period of time, e.g., daily, twice weekly, weekly, biweekly, twice monthly, bimonthly, twice yearly, or yearly, as needed. A dosage unit containing an effective amount of the cellular therapy can be administered in a single daily dose, or the total daily dosage can be administered in two, three, four or more divided doses daily, as needed.

[0116] The polypeptides described herein can be incorporated into pharmaceutical compositions (e.g., for use as protein therapeutics). Pharmaceutical compositions containing the polypeptides can be formulated by methods known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences pp.1447-1676 (Alfonso R. Gennaro, ed., 19th ed.1995)). Pharmaceutical compositions can be administered parenterally in the form of an injectable solution, including a sterilized solution or suspension in water or another pharma- ceutically acceptable liquid. For example, pharmaceutical compositions can be formulated by suitably combining the polypeptide with a pharma- ceutically acceptable vehicle or medium, such as sterile water and saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, binders, and the like, and then mixing in a unit dosage form required by generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation is such that a suitable dosage within the specified range is provided.

[0117] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, using distilled water for injection as vehicle.For example, physiological saline or the isotonic solution containing glucose and other additives such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used as aqueous solution for injection, optionally combined with suitable solubilizers, for example alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80 (trademark), HCO-50, etc.

[0118] Non-limiting examples of oily liquids include sesame oil and soybean oil, which may be mixed with benzyl benzoate or benzyl alcohol as a solubilizer. Other items that may be included are buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injections can be packaged in suitable ampoules.

[0119] Protein half-life can be influenced by the degree of sialylation, the post-translational covalent addition of terminal sialic acids to glycosylated proteins (Hossler et al., Glycobiology. 2009; 19:936-49). More sialylated proteins can have longer half-lives in vivo (Flintegaard et al., Endocrinology. 2010; 151:5326-36; Bork et al., J Pharm Sci. 2009; 98:3499-508). In some embodiments, the fusion proteins described herein are formulated as sialylated protein therapeutics.

[0120] The route of administration may be parenteral, e.g., by injection, intranasal, pulmonary, or transdermal administration. Administration may be systemic or local, e.g., by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0121] A suitable administration means can be selected based on the age and condition of the subject. A single dose of a pharmaceutical composition containing a polypeptide can be selected from the range of 0.001 to 1000 mg / kg body weight. On the other hand, a dose can be selected within the range of 0.001 to 100,000 mg / kg body weight, but the present disclosure is not limited to such a range. The dose and administration method may vary depending on the body weight, age, condition, etc. of the subject, and can be suitably selected by a person skilled in the art as necessary.

[0122] In some embodiments, the pharmaceutical composition containing the fusion protein is administered in combination with a cellular therapy targeting CD19, e.g., CAR-T cells, or in combination with an ADC, as described herein. In some embodiments, the pharmaceutical composition comprises a fusion protein described herein and a cellular therapy described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered simultaneously, together, or sequentially with a cellular therapy described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1, 2, 3, 4, 5, 6, or 7 days prior to administration of a cellular therapy described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1, 2, 3, or 4 weeks prior to administration of a cellular therapy described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1, 2, 3, 4, or 5 months prior to administration of a cellular therapy described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1, 2, 3, 4, 5, 6, or 7 days after administration of a cellular therapy described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1, 2, 3, or 4 weeks after administration of a cellular therapy described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1, 2, 3, 4, or 5 months after administration of a cellular therapy described herein.

[0123] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, including GenBank accession numbers. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to carry out or test the present invention, suitable methods and materials are described below.

[0124] The present disclosure is further illustrated by the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way.

[0125] Relapse of CAR-T therapy Generally, CAR-T therapy involves the administration of T cells expressing a chimeric antigen receptor (CAR) that binds to a target antigen. In some embodiments, the target antigen of the CAR-T is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA) as described herein.

[0126] In some embodiments, the present disclosure is based, in part, on the recognition that certain individuals being treated for cancer with CAR-T therapy who relapse (e.g., fail to exhibit one or more beneficial responses to CAR-T therapy as described herein) may be "rescued" from the relapse by administration of a fusion protein as described herein. In some embodiments, the present disclosure provides compositions and methods comprising the fusion proteins for treating subjects who exhibit a recurrence of cancer during or after CAR-T therapy.

[0127] In some embodiments, the present disclosure is based, in part, on the recognition that certain individuals undergoing treatment for cancer with CAR-T therapy may relapse due to a suboptimal response to the therapy, and are therefore treated to prevent relapse. As a non-limiting example, the suboptimal response may be improved by increasing the density of the target antigen on the tumor cells. In some embodiments, increasing the density of the target antigen on the tumor cells may be achieved by binding the fusion protein of the present invention to the tumor cells. In some embodiments, the present disclosure provides compositions and methods comprising the fusion proteins for treating subjects who are predicted to have or have a suboptimal response to CAR T cell therapy, for example, patients who have achieved a stable disease, partial response, best partial response, or complete remission that does not reach a minimal residual disease negative status (see, e.g., www.cibmtr.org / manuals / fim / 1 / en / topic / multiple-myeloma-response-criteria).

[0128] Identifying the target In some embodiments, subjects are identified and / or selected for administration of the fusion proteins described herein. In some embodiments, subjects may be identified and / or selected for treatment based on a diagnosis of refractory or resistant cancer. In some embodiments, subjects may be identified and / or selected for treatment based on a prescription to receive ACT therapy. In some embodiments, subjects may be identified and / or selected for treatment based on evidence of relapse to ACT therapy. In some embodiments, subjects may be identified and / or selected for treatment based on one or more measured or observed signs of relapse in cancer (e.g., non-beneficial response, loss or downregulation of target antigens of cells used in ACT, or progression). In some embodiments, the fusion protein is administered to the subject. In some embodiments, upon administration of the fusion protein therapy, the subject exhibits a positive clinical response to the ACT therapy, e.g., exhibits improvement based on one or more clinical and / or objective criteria (e.g., exhibits a reduction in tumor burden, tumor size, and / or tumor stage).

[0129] The methods described herein can include generating and / or providing a report, such as in electronic, web-based, or paper form. The report can include one or more results from the methods described herein, such as tumor burden, tumor size, and / or tumor stage, disease stability, loss or downregulation of target antigens. In some embodiments, the report is generated in a form such as paper or electronic and presents the presence or absence of one or more tumor antigens for the cancer patient, and optionally a recommended course of cancer therapy. In some embodiments, the report includes an identifier for the cancer patient. In one embodiment, the report is in a web-based form.

[0130] In some embodiments, the report additionally or alternatively includes information regarding prognosis, resistance, or potential or proposed treatment options. The report may include, for example, information regarding the likely effectiveness of a treatment option, the acceptability of a treatment option, or the suitability of applying a treatment option to a cancer patient identified in the report. For example, the report may include information or recommendations regarding the administration of a cancer therapy to a patient, for example, administering a preselected dosage or administering a preselected treatment regimen, for example, in combination with one or more alternative cancer therapies. The report may be sent, for example, to an entity described herein within 7, 14, 21, 30, or 45 days of carrying out the method described herein. In some embodiments, the report is a personalized cancer treatment report.

[0131] In some embodiments, a report is generated for recording each time a cancer subject is tested using the methods described herein.Cancer subjects can be re-evaluated at intervals such as monthly, bimonthly, six months, or yearly, or more frequently or less frequently, to monitor the subject for responsiveness to such cancer therapy and / or improvement of one or more cancer symptoms, such as those described herein.In some embodiments, a report can record at least the treatment history of the cancer subject.

[0132] In one embodiment, the method further includes providing the report to another party, which may be, for example, a cancer subject, a caregiver, a physician, an oncologist, a hospital, a clinic, a third party payer, an insurance company, or a government agency. EXAMPLES

[0133] Example 1. Construction and expression of CTE1 and CTE2 fusion proteins A gene fusion protein construct containing a secretion signal peptide, an anti-CD20 VHH, a portion of the CD19 ECD, and an anti-albumin llama VHH was cloned into a mammalian cell expression vector and transiently transfected into Expi293 or CHO cells. The cell culture was clarified by centrifugation and the fusion protein was purified from the supernatant. Two fusion proteins were produced. In Expi293 cells, CTE1 (having the amino acid sequence of SEQ ID NO:2) was expressed. In CHO cells, CTE2 (having the amino acid sequence of SEQ ID NO:2 and lacking the C-terminal hexahistidine tag (e.g., SEQ ID NO:55) was expressed.

[0134] The CTE1 fusion protein was purified by His-NTA column chromatography and analyzed by reducing and non-reducing PAGE. Superdex75 purification was used to isolate the main peak and remove any endotoxin, and SEC-HPLC was used to isolate monomeric protein from minor aggregates present. The CTE2 fusion protein was purified by Protein A affinity chromatography, cation exchange chromatography, and SEC-HPLC purification. The CTE2 fusion protein was purified to homogeneity as determined by SEC-HPLC and PAGE.

[0135] Example 2. Biophysical characterization of CTE1 and CTE2 fusion proteins Capillary isoelectric focusing (cIEF) analysis was used to assess the sialylation of CTE1 and CTE2 fusion proteins. In cIEF separation, a continuous pH gradient is created by applying a voltage to a carrier ampholyte-filled capillary, and proteins are separated according to their isoelectric point (pI). The pI of a protein is the pH value at which the total charge of the protein is zero. Thus, cIEF separates protein isoforms with respect to their overall charge, and is therefore influenced by the presence or absence of sialic acid for fusion protein isoforms. Briefly, proteins can be assessed as the percentage of basic and acidic peaks, with the acidic peak representing the sialylated protein.

[0136] Three protein preparations were evaluated for the degree of sialylation: CTE1 and CTE2, as described in Example 1, and CTE3 (SEQ ID NO:55), a 100% sialylated form purified from a second batch of CTE2 that was found to have sialylated and non-sialylated forms. CTE3 was prepared by separation using an anion exchange column (AEX) and collecting the eluate.

[0137] Sialylation was assessed using imaging capillary isoelectric focusing (icIEF), a high-resolution technique that separates species primarily based on the pI-specific net charge of the molecule. icIEF considers surface-exposed and interior amino acids and does not suffer from loss of resolution due to hydrophobic interactions. icIEF separation is performed by focusing isoforms in an amphoteric pH gradient through an applied electric field. Following the focusing step is a detection step, which in conventional capillary isoelectric focusing (cIEF) requires chemical or pressure-induced migration to a fixed UV detector. In icIEF, there is no migration step and detection is achieved by continuously scanning the entire capillary. As the migration step is omitted, charge variant profiles can be recorded with the highest possible resolution within an efficient runtime of 15-20 min. Protein preparations CTE1, CTE2 and CTE3 exhibited distinct icIEF profiles, as shown in Figure 1. Specifically, CTE1 is a mixture of sialylated and non-sialylated proteins, CTE2 is almost entirely non-sialylated proteins, and CTE3 is almost entirely sialylated proteins.

[0138] Since the appearance of basic and acidic peaks may be influenced by other features in addition to sialylation, sialidase treatment was performed to demonstrate that the presence of the acidic peaks was due to sialic acid modifications in the protein preparation. Several CTE1 preparations in different formulation buffers (FB) were evaluated with + / - sialidase and are labeled CTE test and CTE test 2 in Figure 2.

[0139] This experiment shows that sialidase eliminated all of the more acidic peaks (pH 5.1-pH 7.9) and slightly reduced the size of one basic peak (pH 8.55).

[0140] Example 3. Fusion proteins bind to CD20 and albumin CTE1 and CTE2 fusion proteins (described in Example 1) and CTE3 (AEX preparation) were evaluated for binding to CD19-positive and CD19-negative cells. Briefly, JeKo-1 mantle cell lymphoma cell line was subjected to CRISPR / Cas9 editing to remove CD19 expression to generate JeKo-19KO cell line. JeKo-19KO cells express CD20 but not CD19. Cells were incubated with CTE proteins, washed to remove unbound proteins, and then incubated with phycoerythrin-labeled anti-CD19 antibody FMC63 (FMC63-PE). Cells were then washed again, fixed with 2% formalin in PBS, and analyzed by flow cytometry.

[0141] As shown in Figure 3, CTE1 (red circle), CTE2 (open green square) and CTE3 (open blue circle) proteins bound equally, i.e., regardless of the degree of sialylation, to CD19-negative / CD20-positive JeKo-19KO cells as detected with an anti-CD19-PE antibody (FMC63). The half-maximal effective concentrations calculated from these binding data are reported in Table 1. [Table 1]

[0142] Fusion proteins were assessed for binding to CD20 and albumin. Briefly, two ELISA formats were performed using the anti-CD19 antibody FMC63 to capture the fusion proteins. ELISA plates (Thermo Fisher) were coated with 1 μg / ml FMC63 overnight at 4° C. and blocked with 200 μl / well of Tris-buffered saline (TBS) containing 0.3% nonfat milk for 1 h at room temperature. Fusion proteins were added at decreasing concentrations in TBS containing 1% BSA and incubated for 1 h. Plates were washed three times with TBS. Then, either biotinylated human albumin at 0.5 μg / ml (Novus Biologics) or biotinylated human CD20-"nanodiscs" at 0.5 μg / ml (Acro Biosystems) was added in TBS containing 1% BSA for 1 h, and the plates were washed again followed by incubation with streptavidin-HRP and TMB peroxidase substrate (Thermo Fisher) to detect bound protein.

[0143] As shown in Figure 4, CTE1 (green triangles), CTE2 (red circles) and CTE3 (blue squares) proteins bound comparably, i.e., regardless of the degree of sialylation, to anti-CD19 coated plates as detected with biotinylated albumin (left) or biotinylated CD20 membrane preparations (right). The half maximal effective concentrations calculated from these binding data are reported in Table 2. [Table 2]

[0144] Example 4. Fusion proteins mediate targeting and cytotoxicity of CAR-19 T cells CTE1 and CTE2 fusion proteins (described in Example 1) were evaluated for targeting and cytotoxicity of CAR-19 T cells. Briefly, in a 96-well round-bottom plate, 50 μL of JeKo-1 CD19KO target cells carrying the luciferase gene were cultured at 1×10 in RPMI 1640 medium containing 10% FBS without antibiotics (RPMI / FBS).4 Cells / well were seeded. Dilutions of test proteins were made in 50 μL RPMI / FBS and added to the cells. CAR-19 T cells were thawed, washed once with RPMI / FBS, collected by centrifugation at 550 RCF for 10 min, and added to the wells in a volume of 50 μL to obtain a given CAR-19 T:target (E:T) cell ratio. Plates were incubated at 37° C. for 48 hours. Plates were centrifuged at 550 RCF for 5 min, pellets were rinsed with PBS, and centrifuged again. 20 μL of 1× lysis buffer (Promega) was then added to the pellet, and the lysate was transferred to a 96-well opaque tissue culture plate (Fisher Scientific). Plates were read in a luminometer (Promega) equipped with an injector to dispense substrate. Percent killing was calculated based on the average luminescence loss of the experimental condition versus the control condition of target cells + CAR-19 T cells only.

[0145] As shown in Figure 5, CAR-19 T cells had comparable cytotoxic activity in the presence of target JeKo-19KO cells and CTE1 (red circle), CTE2 (blue square) and CTE3 (open triangle) proteins, i.e., regardless of the degree of sialylation. The half-maximal inhibitory concentrations calculated from these cytotoxicity data are reported in Table 3. [Table 3] These results show that CTE fusion proteins can redirect CAR-19 T cells to CD19 that binds and is presented on the CD20 cell surface protein. Fusion proteins that bind to CD20 and present the extracellular domain of CD19 can increase the apparent density of CD19 on wild-type lymphoma cells that naturally express both antigens. Using the "Bang Beads" method (Bangs Laboratories, Fishers, IN USA), we show that the addition of CTE3 to either wild-type JeKo-1 mantle cell lymphoma cells or wild-type Ramos Hodgkin lymphoma cells increases the number of apparent CD19 molecules on the surface of lymphoma cells in a dose-dependent manner. The total number measured is shown to be approximately equal to the number of CD19 receptors plus the number of CD20 receptors measured by flow cytometry staining. Experiments are performed using the manufacturer's instructions.

[0146] method Bead staining (Bangs Lab QSC beads catalog number: 815B5ML; anti-mouse IgG, lot 15515, access code: 22020217-1): Manually shake the bottle to ensure a uniform suspension. Do not vortex. Blank population: Do not stain. Stain each labeled population (1-4) separately. Add 1 drop of QSC beads to 50 µL of FACS buffer and gently tap to mix. Add 30 µL of FMC63-PE (3 × 10) for CD19 standard curve beads. 6 Add 60 μL of a-CD20-PE (3 × 10 6 Add 1 ml of FACS buffer (per cell) and mix immediately by tapping gently. Incubate for 30 minutes in the dark at 4°C. Add 1 ml of FACS buffer and centrifuge at 2500g for 5 minutes. Wash twice with 1 ml of FACS buffer, centrifuge at 2500g for 5 minutes, and resuspend in 500 μL of PBS.

[0147] Cell staining: In a 96-well FACS plate, add 50 µL of Fc-blocked cells (approximately 5 × 10 5Incubate 3x10 cells with 50 μL of 3-fold serially diluted 606 protein (CTE3, SEQ ID NO:55, sialylated) for 30 min at 4°C. Wash cells twice with 200 μL of FACS buffer. Resuspend cells in 50 μL of FACS buffer and add 30 μL of FMC63-PE and incubate for 30 min at 4°C. Controls: a. Unstained cells, b. FMC63-PE stained cells (3x10 6 30 μL / test for cells), c. anti-CD20-PE stained cells (3 × 10 6 Add 60 μL of anti-CD20-PE to the cells by tapping gently and mixing immediately. Incubate the cells for 30 minutes in the dark at 4°C. Wash the cells 3 times with 200 μL of FACS buffer and centrifuge at 500g for 2 minutes. Resuspend the cells in 200 μL of PBS containing 1% paraformaldehyde and fix the cells for 30 minutes. FACS analysis: beads and cells are performed on the same day with the same settings.

[0148] Bead analysis on an Accuri C6 plus instrument: set flow rate to 100-200 events / sec (medium fluidics) and collect 1000 events per bead population. Combine stained beads, can also be done in a single tube (5 x 1000 events). Use an FSC / SSC dot plot to create FSC-H / FSC-A for singlets and SSC-A / FL2 (PE channel). Create histograms / FL2 (PE channel) and apply 1 / 2 height / full width gates on each bead population (M1, M2, M3, M4, M5). Record the geometric mean or median of FL2 (PE channel) for each bead population for entry into a QuickCal spreadsheet (download QuickCal v 3.0 using your access code). Enter the median values ​​for FL2 (PE channel) to obtain a standard curve.

[0149] Run the cells on an Accuri C6 plus with the same settings as for the bead analysis and collect 5000 events / trial. Create FSC-H / FSC-A using FSC / SSC dot plots to obtain singlets and SSC-A / FL2 (PE channel). Create histograms / FL2 (PE channel) and apply a 1 / 2 height / full width gate on the cell population. Record the geometric mean or median of FL2 (PE channel) of the cell population for input into the QuickCal spreadsheet. Enter the median of FL2 (PE channel) to obtain the ABC (antibody binding capacity) value. Subtract either the ABC value of the unstained cell population or the isotype control from the ABC value of the cells. If binding of monovalent antibodies to surface receptors is presumed, then ABC value = number of surface receptors. The results show that the apparent number of CD19 molecules on the surface of JeKo-1 cells increases visibly in a dose-dependent manner in the range of 10 ng / ml to 10 μg / ml of CTE3. Results show that the apparent number of CD19 molecules on the surface of Ramos cells is visibly increased in a dose-dependent manner over the range of 10 ng / ml to 10 μg / ml of CTE3. Results show that increasing the number of CD19 molecules on the target lymphoma cells enhances CAR-19-mediated toxicity for up to 24 hours at various E:T ratios. Results show that cytotoxicity is enhanced within a range of E:T ratios including 0.1:1, 0.3:1, 1:1, 3:1, and 5:1.

[0150] In conclusion, CTE3 increases the apparent CD19 density on lymphoma cells sufficiently to increase CAR-19-mediated cytotoxicity.

[0151] Example 5. Treatment of CD19 antigen escape with fusion proteins CTE1 fusion proteins (described in Example 1) were evaluated for their ability to modulate CD19 antigen escape of target cells. To generate a CD19 antigen escape model, wild-type CD19+ / CD20+ JeKo-1 cells carrying the luciferase gene (JeKo-luc) were seeded at 1×10 cells / well in 96-well round-bottom plates containing 100 μl RPMI / FBS. 4CAR-19 T cells were added in 100 μl of RPMI / FBS at a ratio of 1:1, 0.3:1, or 0.1:1 JeKo-luc cells:CAR-19 T cells, and the co-cultures were incubated for up to 13 days. On days 1, 6, 11, and 13, CD19 - CD20 + Samples were assessed by flow cytometry for population accumulation (FMC63-PE, Millipore; anti-CD20-APC, BD Pharmingen).

[0152] Upon the appearance of a CD19-negative population, JeKo-luc cells were counted by flow cytometry using bead counting according to the manufacturer's protocol (Bang Laboratories, Inc.) and then cultured at 1 × 10 in 50 μl of RPMI / FBS. 4 The cells were then replated with 1×10 cells in 50 μl of RPMI / FBS with or without fusion protein in 50 μl of RPMI / FBS. 5 CAR-19 T cells were added at a JeKo-luc:CAR-19 T ratio of 10:1. In one set of control wells, the fusion protein was added only to JeKo-luc cells without CAR-19 T cells. In both cases, 150 μl of the co-culture was incubated for 48 h. Cells were then stained with HIB-CD19-PE (BioLegend) and anti-CD20-APC (BD Pharmingen) for JeKo-1 cells not incubated with protein, or with anti-CD20-APC and anti-ROR1-PE (BioLegend) antibodies for samples treated with protein, and analyzed by flow cytometry. Dead cells were gated out using 7AAD.

[0153] Wild-type JeKo-1 cells have a very high proliferation index, so they were used as an antigen escape model for CAR-T cell therapy. We first characterized the phenotype of these cells under CAR-T pressure. JeKo-1 cells are CD19- and CD20-positive, with the majority being double-positive, as shown in Figure 6.

[0154] FIG. 6 shows that wild-type JeKo-1 cells express CD19 (X-axis) and CD20 (Y-axis), and therefore nearly all cells (90%) are in the upper right quadrant, indicating double positivity.

[0155] A clear antigen escape model was created by using a titration of effector:target ratio (E:T, here referring to CAR-T:JeKo-1) to vary the number of CAR-T cells against a fixed number of JeKo-1 cells and by varying the length of culture time.

[0156] FIG. 7 shows the evolution of the JeKo-1 antigen escape model by in vitro CAR-19 T cell treatment. The positions of CAR-19 cells and target JeKo-1 cells are shown in the flow cytometry profile and are shown on the left side of the panel. The designation of CD19- and CD20-positive cell populations is shown on the right side of the panel. A) E:T ratio of 1:1 on days 1-13. B) E:T ratio of 0.3:1 on days 1-13. C) E:T ratio of 0.1:1 on days 1-13.

[0157] As can be seen in Figure 7, JeKo-1 cells are able to avoid CAR-19 cytotoxicity due to loss of CD19 expression at the cell population level. This occurs even at an E:T ratio of 1:1, as seen in Figure 7A at days 6 and 13. As seen in Figure 7B (days 6 and 13) and Figure 7C, at E:T ratios of 0.3:1 and 0.1:1, this effect is more pronounced, especially at day 13. Notably, although an E:T ratio of 0.1:1 was ineffective at the level of cytotoxicity as read by luciferase assay, the selection pressure imposed by the CAR induced a transition from a population that was predominantly double positive (CD19 and CD20 positive) (Figure 7C, day 1) to a mixed population (Figure 7C, day 6), with a predominance of CD19 negative CD20 positive cells (Figure 7C, day 13).

[0158] Since escape occurred at this range of E:T ratios and incubation days, a second study was set up to evaluate the effect of adding a CD19-anti-CD20 fusion protein.

[0159] Figure 8A shows the expression levels of CD19 (x-axis) and CD20 (y-axis) of CAR-19 cells incubated with JeKo-1 cells (E:T1:1) for 13 days as measured by flow cytometry. Expression of CD19 and CD20 was monitored by FACS. Figure 8B shows the results when only CTE1 protein, only CAR-19 T cells, or both CTE1 and CAR-19 T cells were added to the culture.

[0160] In this experiment, the addition of CAR-19 T cells and CTE1, but not CAR-19 T cells alone or CTE1 protein alone, was sufficient to kill all target JeKo-1 cells after escape.

[0161] These results demonstrate that antigen escape, particularly from CD19 expression, can be induced in vitro and reversed using CD20-targeted CTE1.

[0162] Example 6. In vivo treatment of cancer in mice with fusion proteins CTE1 and CTE2 fusion proteins (described in Example 1) were evaluated for tumor therapy in a mouse model. Briefly, 6-10 week old female NOD-scid IL2Rgamma mice were null NSG mice were obtained (Jackson Labs) and acclimated in the vivarium for a minimum of 3 days prior to the start of the study. All animals were socially housed in static, sterile, biocontained, disposable cages pre-bedded with corncob bedding. Diet (LabDiet) and acidified water were available ad libitum. On study day 1, mice were dosed with 2.5 × 10 6JeKo-19KO cells were injected intravenously at 0.1 mL / mouse. Then, on study day 3, all animals were imaged and randomized to ensure comparable mean tumor burden in each of 6 different cohorts of n=8 mice / cohort. The following day, study day 4, different cohorts of mice received doses of fusion protein ranging from 0-5 mg / kg depending on the study, followed 3-4 hours later by 1 × 10 7 The mice were then injected with 10 CAR-19 T cells. The fusion protein was then administered three times a week for a total of up to 14 doses. Control cohorts included mice treated with CAR-19 T cells alone, without any protein injections, and untreated mice that received no therapeutic agent.

[0163] Animal health checks were performed at least daily from cageside, and clinical observations were performed more frequently if the test animals showed abnormal clinical signs. Body weights were recorded before tumor induction, before CAR-19 T cell administration, and three times weekly thereafter, unless the animal's health indicated otherwise. The most recently collected body weights were used to calculate protein and D-luciferin doses. Imaging was performed on day 3 to randomize animals into cohorts with similar baseline mean luminescence. After injection of CAR-19 cells and CTE protein, whole-body imaging was performed twice weekly for all animals, 10–15 min after subcutaneous administration of 15 mg / mL luciferin in 0.2 mL PBS. Isoflurane was administered during the procedure. Total flux measured by lumin was 1 × 10 10 Higher values ​​were the maximum and were considered reason for humane euthanasia.

[0164] Several studies were performed using the JeKo-19KO cell line, CTE protein, and CAR-19 T cells. Animals were injected with JeKo-19KO lymphoma cells and allowed to settle for 4 days before starting treatment with CAR-19 T cells and CTE1 protein. CAR-19 T cells were injected once and CTE1 protein was administered three times a week. Tumor burden was quantified using luciferase-mediated luminescence.

[0165] In the first study, the in vivo efficacy of the CTE1 protein was tested. 6 A tumor cell dose of 1 × 10 JeKo-CD19-KO cells was injected intravenously into NSG mice. On day 3, CAR-19 T cells (1 × 10 7 ) and CTE1 were added. CTE1 was then administered three times a week, but was stopped on day 32 to monitor lymphoma recurrence. As shown in Figure 9, mice treated with anti-CD19 CAR T cells and CTE1 were cured and no recurrence was observed until day 45. CAR-19 and CAR-CD20 cohorts without CTE1 were included. As expected, animals treated with CAR-19 developed lymphoma and 4 out of 5 died by day 45. This was also true for the untransduced T cell control cohort (UTD). The CD20 CAR worked well, except for one animal that developed a fatal systemic lymphoma.

[0166] These results demonstrate that mice treated with CAR-19 T cells plus CTE1 protein were protected from lethal lymphoma, and even with the lowest dose of 0.56 mg / kg, durable responses were achieved even after treatment was discontinued.

[0167] In the second study, the activity of CTE1 and CTE2 protein preparations at a protein dose of 2 mg / kg was screened against CAR-19 T cell only and untreated cohorts. As shown in Figure 10, administration of CTE1 and CTE2 proteins three times a week suppressed lymphoma development until day 31, at which point disease had begun in untreated (NA) and CAR-19 treated mice.

[0168] These results indicate that CTE1 and CTE2 proteins had similar effects on lymphoma growth in vivo at a dose of 2 mg / kg.

[0169] In the third study, two parameters were further investigated. First, a dose escalation of CTE2 was performed over four cohorts: 2 mg / kg, 0.4 mg / kg, 0.08 mg / kg, and 0.016 mg / kg. Animals were then administered the protein until day 31, at which point administration was discontinued to evaluate the animals for signs of lymphoma recurrence. As shown in Figure 11, nine days later, at day 42, no signs of luminescence were observed in animals treated with CAR-19 T cells + CTE2 2 mg / kg, and only a slight signal was observed in 2 out of 8 animals treated with CAR-19 T cells + CTE2 0.4 mg / kg. The lower the dose of CTE2, the less effective it was in preventing recurrence after discontinuation of administration.

[0170] These results show that administration of CAR-19 T cells and CTE2 prevented lymphoma recurrence in all mice in the highest dose cohort of 2 mg / kg and in the majority of mice in the 0.4 mg / kg and 0.08 mg / kg cohorts.

[0171] Body weight is a quantitative means of tracking animal health and is typically used in conjunction with clinical testing to ensure humane euthanasia in cancer models. As shown in Figure 12, mice treated with CAR-19+CTE2 protein continued to gain weight over the protein administration period (through day 31), while control animals began to lose weight around day 25. After CTE2 protein administration was discontinued, animals in the treatment cohort maintained their weight until the end of the study (day 42), and the highest dose treatment cohort, which received 2 mg / kg CTE2 through day 31, continued to gain weight (last recorded weight on day 39).

[0172] These results show that animals in the three highest dose cohorts were healthy enough to gain weight even after CTE2 treatment was stopped on day 31, while the negative control groups (CAR-19 only and "no treatment") steadily lost weight from day 21 onwards.

[0173] FIG. 13 shows the lumen intensity for each cohort, with a sharp increase in the untreated group and a slower but still sharp increase in the CAR-19-treated group. The end of the lines for the untreated and CAR-19-treated groups corresponds to the loss of all animals in these cohorts. The signal intensity in the control cohorts was so high that it was not possible to discern differences between the CTE2-treated cohorts unless these groups were excluded. With the exception of these groups, a clear dose response was seen, with the 2 mg / kg CTE2 cohort having no luminescent signal until day 42. As the last protein dose was administered on day 31, this suggests that the CD19-negative lymphoma had been eliminated from these animals.

[0174] These results show complete protection from lymphoma as measured by luminescence yield in all animals treated with CTE2 2 mg / kg (8 / 8 animals showed no signal), most of the animals treated with CTE2 0.4 mg / kg (6 / 8 animals showed no signal), and half of the animals treated with CTE2 0.08 mg / kg (4 / 8 animals showed no signal). The lack of luminescence signal and continued weight gain indicate that some mice are disease-free, which is supported by the Kaplan-Meier survival curves of the different cohorts, as shown in Figure 14.

[0175] Survival data show that only one of 32 treated animals (3%) died from lymphoma during the study period, and this animal was in the lowest dose cohort of 0.016 mg / kg. In contrast, 14 / 16 (87.5%) control animals died from lymphoma during the study period.

[0176] The in vivo efficacy of the proteins depends on the pharmacokinetic (PK) properties of the injected fusion proteins. As shown in Figure 15, the PK properties of CTE1, CTE2 and CTE3 were evaluated in mouse serum after a single injection. The half-life of the fusion proteins in mice is dependent on recirculation by albumin via FcRN binding due to the lack of an antigen target. That is, the anti-CD20 domain does not bind to mouse CD20 and human CD19 ECD has no measurable binding properties in solution, whereas the anti-albumin domain can bind to mouse albumin. The CTE1 protein was evaluated in the serum of both Balb / c and NSG mice after a single intravenous injection and showed similar half-lives of 21 and 28 hours, respectively. The half-life of CTE2 (low sialylation) in Balb / c mice was shorter than expected at 7.6 hours, while CTE3 (high sialylation) had the longest half-life at 36 hours.

[0177] Protein half-life can also be influenced by the degree of sialylation, the post-translational covalent addition of terminal sialic acids to glycosylated proteins. More sialylated proteins have longer half-lives in vivo. Table 4 summarizes the fusion protein half-lives in Balb / c and NSG mice, as well as the icIEF profiles of the fusion proteins. The icIEF data show that the CTE1 protein had a mixture of acidic and basic peaks, whereas the CTE2 protein was almost completely basic with little sialylation, and the CTE3 protein was almost completely acidic with extensive sialylation. These results explain the differences in the PK properties of the three protein preparations. [Table 4]

[0178] Example 7. Generation and testing of additional fusion proteins Additional fusion proteins were evaluated for their ability to bind and kill CD19- CD20+ Jeko-1 cells. First, anti-CD20 VHHs (SEQ ID NOs: 37-42) with different CDR2 sequences within SEQ ID NO: 6 were evaluated for their ability to bind to CD20 on CD19- CD20+ Jeko-1 cells using FACS. The table below shows the changes made to the CDR2 sequence of SEQ ID NO: 37. As shown in Table 5, VHHs containing the indicated CDR2 sequences showed varying levels of binding ranging from complete loss of activity to minimal loss of activity, and also had variations in mean fluorescence intensity (MFI). [Table 5]

[0179] Substitution of Thr-Tyr (TY) at the N-terminus of CDR2 of SEQ ID NO: 37 with Ser-Trp (SW) clearly affected binding. Surprisingly, substitution of the Ser-Pro (SP) sequence at the C-terminus of CDR2 of SEQ ID NO: 37 with the highly non-conserved Trp-Ile (WI) did not affect binding. Furthermore, mutations within CDR3 of anti-CD20 VHH of SEQ ID NO: 37 (AANPTYGSDWNAEN to AADPTYGSDWNAEN) only slightly affected binding to CD20.

[0180] A series of fusion protein constructs were made containing a secretory signal peptide, an anti-CD20 VHH, the linker GGGGSGGGGSGGGGSGGGGS, a portion of the CD19 ECD, the linker SRGGGGSGGGGSGGGGGS, an anti-albumin VHH, and a hexahistidine tag. The amino acid sequences of the constructs are listed in Table 6: [Table 6]

[0181] The fusion proteins were purified and then assayed for both binding to and killing of CD20 positive Jeko-1 CD19KO cells. Binding is summarized in Table 7 below: [Table 7]

[0182] Direct cell binding results of the constructs to CD20 on JeKo-19KO cells, measured by binding of anti-CD19 mAb FMC63, fully confirmed the preliminary FACS results. Constructs #631 and #634, containing four and three AA changes (relative to SEQ ID NO:37), respectively, did not bind to cells at all. Construct #633, containing two AA changes relative to SEQ ID NO:37, was nearly equivalent to construct #526 (CTE1). Construct #635, containing a conservative change (relative to SEQ ID NO:37) (Pro to Thr) at the C-terminus, clearly lost five-fold binding, and construct #636 (containing a CDR3 with an Asn to Asp mutation relative to SEQ ID NO:37) retained full activity. Importantly, combining the CDR3 mutation with the two AA substitutions of construct #633 yielded a construct (#637) nearly equivalent to construct #526. The binding curves are shown in FIG.

[0183] Killing of JeKo-19KO cells by the fusion proteins in the presence of CAR-19 was evaluated, and the data are summarized in Figure 17 and Table 8 below. [Table 8]

[0184] Figure 17 and Table 8 show that the cytotoxicity data is consistent with the binding data.

[0185] Example 8. Evaluation of additional fusion proteins An additional fusion protein construct #607 was generated containing a different anti-CD20 VHH. The amino acid sequences of the constructs are listed in Table 9: [Table 9]

[0186] Constructs #526(CTE1) and #607 bound to cell surface CD20 approximately equally, with construct #526(CTE1) at approximately 0.4 nM and construct #607 at approximately 0.8 nM (Figure 18, left panel). However, construct #607 was approximately 10-fold less potent at killing JeKo-19KO cells (Figure 18, right panel). [Table 10]

[0187] Example 9 Biophysical Characterization and Optimization of Biologics Assay the biologics described herein for biophysical characteristics. Evaluate the fusion proteins described herein using standard techniques known in the art to evaluate the characteristics of therapeutic biologics. Evaluate the fusion proteins (e.g., those described in this Example) for stability (e.g., stability in composition buffer and / or biological fluid (e.g., blood or saliva)).

[0188] The fusion proteins described in this example are formulated into preparations that exhibit optimized melting temperature (Tm), enhanced stability, reduced levels of aggregation and / or degradation, increased levels of resistance to proteases, increased levels of resistance to oxidation, improved biodistribution, and / or improved PK / PD properties in vivo. Such characteristics are assessed using standard methods and assays known in the art, such as differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism (CD), temperature scanning viscosimetry, analytical ultracentrifugation (AUC), size exclusion chromatography (SEC, SEC-MALS), dynamic light scattering (DLS), light obscuration, zeta potential, capillary electrophoresis CE (e.g., CZE, MECC, GelCE, cIEF / iCIEF), gel electrophoresis (e.g., native, SDS-PAGE, IEF), electron microscopy (e.g., TEM, SEM). Formulation parameters that impart these properties include, for example, pH, osmolality, buffers (e.g., phosphate, acetate, and histidine), tonicity agents / stabilizers (e.g., sugars such as sucrose, trehalose, or mannitol; polyols such as sorbitol), bulking agents (e.g., lyoprotectants such as mannitol), surfactants (e.g., polysorbates), antioxidants (e.g., methionine), metal ions / chelating agents (e.g., ethylenediaminetetraacetic acid, EDTA), and / or preservatives (e.g., benzyl alcohol).

[0189] Example 10. Fusion proteins mediate targeting and cytotoxicity of CAR-19 T cells This example further shows that the fusion proteins described herein mediate the targeting of CAR-19 T cells in a concentration-dependent manner.In addition, this example further shows that the fusion proteins described herein mediate the cytotoxicity of CAR-19 T cells.These properties are described, inter alia, in Su et al. Oncoimmunology 2022, Vol.11, No.1, e2111904 (13 pages), the entire contents of which are incorporated by reference.

[0190] CTE3 fusion proteins (described in Example 1) were evaluated for targeting and cytotoxicity of CAR-19 T cells. Briefly, in a 96-well round-bottom plate, 50 μL of JeKo-1 CD19KO target cells carrying the luciferase gene were cultured at 1×10 in RPMI 1640 medium containing 10% FBS without antibiotics (RPMI / FBS). 4 Cells / well were seeded. Dilutions of test proteins were made in 50 μL RPMI / FBS and added to the cells. CAR-19 T cells were thawed, washed once with RPMI / FBS, collected by centrifugation at 550 RCF for 10 min, and added to the wells in a volume of 50 μL to obtain a given CAR-19 T:target (E:T) cell ratio. Plates were incubated at 37° C. for 48 hours. Plates were centrifuged at 550 RCF for 5 min, pellets were rinsed with PBS, and centrifuged again. 20 μL of 1× lysis buffer (Promega) was then added to the pellet, and the lysate was transferred to a 96-well opaque tissue culture plate (Fisher Scientific). Plates were read in a luminometer (Promega) equipped with an injector to dispense substrate. Percent killing was calculated based on the average luminescence loss of the experimental condition versus the control condition of target cells + CAR-19 T cells only. In addition, the number of apparent CD19 molecules on the surface of JeKo-1 CD19KO target cells, wild-type JeKo-1 mantle cell lymphoma cells, or wild-type Ramos Hodgkin lymphoma cells was determined using the “Bang Beads” method (Bangs Laboratories, Fishers, IN USA) according to the manufacturer's instructions.

[0191] CTE fusion proteins bind to the CD20 cell surface protein and can redirect CAR-19 T cells to CD19 presented on the CD20 cell surface protein. Fusion proteins that bind to CD20 and present the extracellular domain of CD19 can increase the apparent density of CD19 on wild-type lymphoma cells that naturally express both antigens. As shown in Figures 19A-19C, when CTE3 fusion proteins were added to JeKo-1 CD19KO target cells, wild-type JeKo-1 mantle cell lymphoma cells, or wild-type Ramos Hodgkin lymphoma cells, the number of apparent CD19 molecules on the surface of lymphoma cells increased in a concentration-dependent manner. The total number of CD19 or CD20 receptors on the test cells measured after treatment with 10 μg / mL of CTE3 was found to be approximately equal to the number of CD19 receptors plus the number of CD20 receptors.

[0192] CAR-19 T cells had cytotoxic activity in the presence of target JeKo-19KO cells and CTE protein. As shown in Figure 20 (left panel), after 18 hours, CAR-19 T cells had an average of at least 60% cytotoxic activity when incubated with JeKo-1 cells (E:T 3:1) treated with 0.01, 0.1, 1, or 10 μg / mL of CTE protein. CAR-19 T cells had an average of at least about 50% cytotoxic activity when incubated with JeKo-1 cells (E:T 3:1) treated with 0.001 μg / mL of CTE protein. After 18 hours, CAR-19 T cells had an average of at least 40% cytotoxic activity when incubated with JeKo-1 cells (E:T 1:1) treated with 0.01, 0.1, 1, or 10 μg / mL of CTE protein. CAR-19 T cells had an average of at least about 20% cytotoxic activity when incubated with JeKo-1 cells treated with 0.001 μg / mL of CTE protein (E:T 1:1). After 18 hours, CAR-19 T cells had an average of about 30% cytotoxic activity when incubated with JeKo-1 cells treated with 0.01, 0.1, 1, or 10 μg / mL of CTE protein (E:T 0.3:1). CAR-19 T cells had an average of at least about 10% cytotoxic activity when incubated with JeKo-1 cells treated with 0.001 μg / mL of CTE protein (E:T 0.3:1). After 48 hours, CAR-19 T cells showed improved cytotoxic activity when incubated with JeKo-1 cells.

[0193] These results further demonstrate that the fusion protein increases the apparent CD19 density on lymphoma cells sufficiently to increase CAR-19-mediated cytotoxicity.

[0194] Example 11: CTE3 binds to human and cynomolgus CD20 expressed on 293T cells This example further demonstrates that the fusion proteins described herein bind to human and cynomolgus CD20.

[0195] Human 293T cells were grown to approximately 80% cell density. cDNA for expression of human CD20 (GenScript OHu00965D) or "cynomolgus" CD20 (A157V mutation of human CD20 cDNA) was transfected into the cells using Lipofectamine 2000, using the manufacturer's instructions (Invitrogen). The A157V mutation is the only amino acid that differs from human CD20 in the extracellular domain. Approximately 48 hours after transfection, cells were removed with Accutase and harvested by centrifugation at 500 x g for 2 min at 4°C. The cell pellet was resuspended in FACS buffer (PBS with 1% BSA and 0.1% sodium azide). Fc block (BD) was then added at 10 6 5 μl per cell was added to the cell suspension and incubated at room temperature for 10 min. The cells were then centrifuged as above, and the cells were resuspended in FACS buffer and 50 μl (approximately 5×10 5 Cells) were used. 50 μl of CTE3 fusion protein, starting at 10 μg / ml (final concentration) and then serially diluted 3-fold in FACS buffer, was then added to the cells. The plate was incubated at 4° C. for 30 minutes and then centrifuged as above. The cell pellet was washed twice with FACS buffer and then resuspended in 50 μl of diluted FMC63-PE (2.5 μl per 50 μl of FACS buffer) and incubated at 4° C. for 30 minutes. The cells were washed twice as above and then fixed with 150 μl of 2% PFA. Samples were analyzed on a BD Accuri C6 flow cytometer using FlowJo software.

[0196] As shown in Figures 21A and 21B, the EC50 of CTE3 binding to both human and non-human primate (cynomolgus monkey) CD20 is approximately 6 ng / ml. These results indicate that CTE3 has cross-species specificity between humans and non-human primates (cynomolgus monkeys).

[0197] Example 12: CTE3 is effective as a CAR T cell engager in the presence of human serum or human serum albumin This example further demonstrates that CTE3 maintains high cytotoxicity against CD20-expressing cells even at pM concentrations.

[0198] CTE3 contains an albumin binding domain. Its ability to kill CD20-expressing cells in the presence of human serum or human serum albumin (HSA) was evaluated. In the cytotoxicity assay, luciferase-expressing JeKo-1 CD19KO-luc cells were used to determine the activity of CTE3 in the presence of 50% human serum or 30 mg / ml or 60 mg / ml of HSA. JeKo-1 CD19KO-luc cells were cultured at 1 × 10 in 50 μl / well of RPMI medium containing 50% human serum or 10% FBS (control condition) or without human serum albumin, 30 mg / ml or 60 mg / ml of human serum albumin (96 round bottom plate). 4 Cells were plated with 100ng / ml of CTE3. CTE3 was titrated in 4-fold serial dilutions in the same serum / HSA dilutions as above, starting at 100ng / ml. CTE3 dilutions (25μl) were dispensed into corresponding wells. CAR19 T cells at a ratio of 2:1 or 5:1 (CAR T cells:target cells) in RPMI medium containing either 50% human serum or 10% FBS or 10% FBS RPMI medium without human serum albumin, containing 30mg / ml or 60mg / ml human serum albumin were added in a volume of 25μl / well. Plates were incubated at 37°C for 48 hours. After washing and pelleting the cells, lysis buffer (Glomax Multi Detection System) was added and the lysate was transferred to a 96 white opaque plate and luciferase levels were measured using a GloMax plate reader (Promega) equipped with an autoinjector. Data were graphed using GraphPad Prism software.

[0199] [Table 11]

[0200] As shown in FIG. 22A, human serum inhibited the IC 50 IC of samples containing 10% FBS 50 The IC50 values ​​for the cytotoxicity assay were determined and are shown in Table 11. CTE3 was inhibited in 10% FBS (IC50 of approximately 2 pM). 50 ) than in 50% human serum (IC of approximately 8.3 pM). 50 In control wells containing CAR19 cells but no CTE3, reduced cell growth was observed in the presence of 50% human serum compared to 10% FBS.

[0201] [Table 12]

[0202] As shown in FIG. 22B, the inclusion of HSA at 30 mg / ml or 60 mg / ml reduced the ability of CTE3 to induce cytotoxicity of JeKo-1 CD19 KO-luc cells compared to control samples containing 10% FBS. IC of Cytotoxicity Assay 50 Values ​​were determined and are shown in Table 12. In the presence of HSA, IC 50 A 2- to 4-fold decrease in cytotoxicity was observed, with a slight reduction in maximal cytotoxicity levels.

[0203] These results indicate that CTE3 maintains high cytotoxicity against CD20-expressing cells at pM concentrations, although activity is reduced in the presence of 50% human serum or physiological levels of HSA.

[0204] Example 13: Antigen binding of CD20xCD79b bispecific anti-CD19 CAR T engager protein

[0205] Bispecific CD79bxCD20 CAR19 engager proteins were generated by assembling anti-CD79b scFv with anti-CD20 VHH and CD19 ECD within CTE3. Constructs were made such that anti-CD79b scFv was placed N-terminal (#650) or C-terminal (#651) to the anti-CD20 VHH-CD19 ECD core sequence. Plasmids were transfected into 293T cells using Lipofectamine 2000 and supernatants were harvested and titrated. #650 and #651 proteins were assayed in an ELISA format for binding to biotinylated CD20 or CD79b.

[0206] A 96-well plate was coated with 1.0 μg / ml FMC63 in 0.1 M carbonate, pH 9.5, overnight at 4° C. The plate was blocked with 200 μl / well of Tris-buffered saline (TBS) containing 0.3% nonfat milk for 1 h at room temperature. The plate was then washed 3 times with wash buffer (1× TBST: 0.1 M Tris, 0.5 M NaCl, 0.05% Tween 20). Next, 100 μl of the supernatants #650 and #651 were serially diluted 3-fold in TBS / 1% BSA, pH 7.4 (dilution buffer) starting at 5 μg / ml. The samples were incubated for 1 h at room temperature and then the plate was washed as above. Next, 100 μl of 0.5 μg / ml biotinylated CD20 nanodiscs (ACROBiosystems) or biotinylated CD79b (in-house) in dilution buffer was added per well and incubated for 1 h at 37° C. After washing the plate again, 100 μl of a 1:2000 dilution of HRP-streptavidin (in dilution buffer) was added per well and incubated for 1 h in the dark at 37° C. For detection, 100 μl of 1-Step Ultra TMB-ELISA was added per well. Once color had developed, 100 μl of stop solution was added and the plate was read at 450 nm. Graphs were generated using Prism software and are shown in Figures 23A and 23B.

[0207] As shown in Figure 23A, placing the CD79b scFv at either the N- or C-terminal position of the anti-CD20 VHH does not significantly affect binding to biotinylated CD20. As shown in Figure 23B, binding to biotinylated CD79b appears to be affected when the anti-CD79b scFv domain is at the C-terminus of the molecule.

[0208] The ability to bind to cell surface CD20 or CD79b was examined by flow cytometry. The assay included a control for binding to CD20 (#606 anti-CD20 VHH-CD19 ECD-anti-albumin) and a control for binding to CD79b (#645 anti-CD79b scFv-CD19 ECD). As shown in Figures 23C and 23D, #650 and #651 bind to both antigens. Table 13 shows the EC of binding to 293T-CD20 cells. 50 Table 14 shows the EC values ​​for binding to 293T-CD79b cells. 50 Indicates the value.

[0209] [Table 13]

[0210] EC20 binding of CTE#650 and #651 to cell surface expressed CD20 50 is similar but a little lower than #606. [Table 14]

[0211] On CD79b expressing cells, both #650 and #651 bind better than the monospecific anti-CD79b CTE #645. These results indicate that the anti-CD20 and anti-CD79b domains can bind to their cognate antigens when presented on the cell surface.

[0212] As shown in Figure 23E, CD20xCD79b CTE #650 and #651 were tested for cytotoxic activity against CD20+ / CD79b+ JeKo-1 CD19 KO-luc cells. CTE3 was replaced with #606 protein or #645, #650 or #651 supernatant. Table 14 shows the IC of cytotoxicity against JeKo-1 CD19 KO cells. 50 Indicates the value.

[0213] [Table 15]

[0214] The monospecific anti-CD20 CTE (#606) and the bispecific anti-CD20 / anti-CD79b CTE (#650 / #651) had an IC of approximately 1 pM. 50 Anti-CD79b monospecific CTE is less effective.

[0215] Example 14: Surface Plasmon Resonance (SPR) Results for Binding of CTE3 to Anti-CD19 FMC63 and HSA SPR measurements were performed on a Biacore 8K (Cytiva) at 25°C using 1x HBS-EP+ running buffer. In separate experiments for each ligand, biotinylated anti-CD19 and biotinylated HSA were immobilized on a streptavidin-coated CM5 Biacore Sensor Chip. The biotin / streptavidin interaction is a high affinity, non-covalent interaction, and the biotinylated substrate binds almost irreversibly to the sensor chip surface. This binding ensures that all immobilized molecules are in the same orientation, resulting in high substrate density.

[0216] The CM5 chip surface was activated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS). Free biotin (0.2 μg / ml) was captured on flow cell (FC) 1: contact time 100 s, flow rate 10 μL / min. Anti-CD19 and HSA were separately diluted in running buffer (1.5 μg / mL and 4.0 μg / mL, respectively) and, in separate experiments, injected on FC2 until the appropriate immobilization level was reached (i.e., 1000-1500 response units (RU)). Residual streptavidin was quenched by injecting biotin on all FCs (0.2 μg / ml, contact time 100 s, flow rate 10 μL / min).

[0217] For binding studies, serial dilutions of CTE3 (3.125, 6.25, 12.5, 25, 50, 100 and 200 nM) were prepared in running buffer and injected over the immobilized target protein: association time 180 s, dissociation time 900 s, flow rate 30 μL / min. Subsequently, chip regeneration was performed: regeneration buffer 10 mM glycine-HCl pH 1.5 for HSA binding, 10 mM glycine-HCl pH 2.0 for anti-CD19 binding, regeneration time 30 s twice for HSA binding, 30 s once for anti-CD19 binding and flow rate 10 μL / min. In all experiments, free biotin immobilized on FC1 was used as a reference for blank subtraction.

[0218] As shown in Figures 24B and 24C, the association and dissociation curves showed that CTE3 has good affinity for anti-CD19 and HSA, respectively. In the case of anti-CD19, the equilibrium dissociation constant (K D The K for HSA was 8.13 nM and the half-life (t) was 15.03 min (Table 15). D The titer was 65.1 nM and the t1 / 2 was 1.78 min (Table 15). [Table 16]

[0219] Example 15: Surface Plasmon Resonance (SPR) Results for Binding of CTE3 to CD20 SPR measurements were performed on a Biacore T200 (Cytiva) at 25°C using running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, 0.05% DDM, 0.01% CHS, pH 7.4). Biotinylated CD20 was immobilized on a streptavidin-coated CM5 Biacore Sensor Chip. The biotin / streptavidin interaction is a high-affinity, non-covalent interaction, and the biotinylated substrate binds almost irreversibly to the sensor chip surface. This binding ensures that all immobilized molecules are in the same orientation, resulting in high substrate density.

[0220] The CM5 chip surface was activated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS). Free biotin (0.2 μg / ml) was captured on flow cell (FC) 1: contact time 100 s, flow rate 10 μL / min. Biotinylated CD20 was diluted in running buffer (0.2 μg / mL) and injected on FC2 until an appropriate immobilization level (i.e., 1000-1500 response units (RU)) was reached. Residual streptavidin was quenched by injecting biotin on all FCs (0.2 μg / ml, contact time 100 s, flow rate 10 μL / min).

[0221] For binding studies, CTE3 was exchanged into running buffer using a desalting column. Serial dilutions of CTE3 (12.5, 25, 50, 100 and 200 nM) were prepared and injected over the immobilized target protein: association 180 s, dissociation 2400 s, flow rate 30 μL / min. No regeneration step was performed. In all experiments, free biotin immobilized on FC1 was used as a reference for blank subtraction. All data analysis was performed using Biacore evaluation software (Cytiva). The obtained association and dissociation curves were fitted using a 1:1 binding model fitting. Table 16 shows the measured kinetic parameters.

[0222] As shown in FIG. 24A, the association and dissociation curves showed that CTE3 has good affinity for CD20 (K D is 2.93nM, t 1 / 2 (The time to antibody titer was 52.23 min.) The kinetic parameters measured for anti-CD20 are detailed in Table 16. [Table 17]

[0223] Example 16: Cytotoxicity of CTE3 against CD20-expressing cells is greater than that of CD20xCD3 BiTE This example further demonstrates that CTE3 mediates improved CAR-19 T cell cytotoxicity. Specifically, this example shows that CTE3 is more cytotoxic against CD20-expressing cells than CD20xCD3 BiTE after repeated stimulation of anti-CD19 CAR T cells.

[0224] Raji cells (approx. 10 x 10 in 1 ml) 6 ) were treated with 0.5 μg / ml mitomycin C for 1 hour at 37° C. to prepare B cells for stimulation. The cells were washed twice with RPMI+10% FBS (R10) and then diluted with 1×10 6CAR19 cells were stimulated by co-culturing at a ratio of 2 CAR19 cells:1 Raji cells and restimulated with MMC-treated Raji every 4 days. The cell density was approximately 0.5 × 10 6 The concentration of CAR19 cells was kept at 1000x1000 cells / ml. Every 4 days, cells were sampled immediately before restimulation and stained with anti-CD3-FITC and anti-Flag-APC to determine CAR19 expansion. Cell numbers were determined using flow cytometry with Bang Beads. Once the CAR19 cell concentration was determined, CTE3 or bispecific anti-CD20 x anti-CD3 (BPS Biosciences, Catalog No: 100836-2) crosslinking cytotoxicity assays against Jeko-1 CD19 KO cells were set up using the restimulated CAR19 cells. Results were read 48 hours after the assay.

[0225] As shown in Figure 25A, the ability of CTE3 or anti-CD20 x anti-CD3 bispecific antibody to kill Jeko-1 CD19 KO cells was evaluated together with CAR19 cells stimulated once with Raji cells. The IC of CTE3 50 The IC of the bispecific antibody was 2.8 pM. 50 The IC of CTE3 is 35.7 pM. As shown in FIG. 25B, the potency decreases in CAR19 cells stimulated three times. 50 is a bispecific IC 50 (124.1 pM) remained lower (7.7 pM).

[0226] These results indicate that CTE3 is more cytotoxic than the commercially available anti-CD20 × anti-CD3 bispecific antibody when CAR19 cells repeatedly stimulated with B cells are used in the assay, and therefore CTE3 may be more effective in preventing relapse in CAR19-treated patients.

[0227] Example 17: Batch analysis of CTE3 drug This example further demonstrates that compositions comprising the fusion proteins described herein can be formulated into preparations that exhibit increased stability, reduced levels of aggregation and / or degradation, increased levels of resistance to proteases, increased levels of resistance to oxidation, improved biodistribution, and / or improved in vivo PK / PD properties.

[0228] Batches of CTE3 were manufactured and analyzed for various drug product properties. Table 17 shows the batch details of CTE3 and the release testing of the batch. [Table 18-1] [Table 18-2]

[0229] Test batch 1B was evaluated for stability after 1 month and 3 months. Table 18 shows the real-time stability data for Test batch 1B. [Table 19]

[0230] As shown in Table 18, the composition containing CTE3 exhibited similar properties after 1 and 3 months as when first tested.

[0231] In further examples, compositions containing CTE3 are evaluated for safety over time periods greater than 3 months. Table 19 describes the stability storage conditions and test time points. Compositions containing CTE3 are stable for at least 3 months. [Table 20]

[0232] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above Description, but rather is as set forth in the following claims. Sequence Listing SEQ ID NO:1 SEQ ID NO:2 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:3 SEQ ID NO:4 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:5 SEQ ID NO:6 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:7 SEQ ID NO:8 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:9 SEQ ID NO:10 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:11 SEQ ID NO:12 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:13 SEQ ID NO:14 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:15 SEQ ID NO:16 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:17 SEQ ID NO:18 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:19 SEQ ID NO:20 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:21 SEQ ID NO:22 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:23 SEQ ID NO:24 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:25 SEQ ID NO:26 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:27 SEQ ID NO:28 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:29 SEQ ID NO:30 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:31 SEQ ID NO:32 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:33 SEQ ID NO:34 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:35 SEQ ID NO:36 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO: 37 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 38 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 39 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 40 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 41 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 42 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 43 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 44 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO: 45 Anti-albumin VHH EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO:46 PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPS GKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP SEQ ID NO:47 MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNR SSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP VLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEED SEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR SEQ ID NO:48 PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPS GKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP SEQ ID NO:49 SEQ ID NO:50 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]-[hexahistidine tag]) [ka] SEQ ID NO:51 SEQ ID NO:52 (In order: [VHH (bold indicates CDR1, CDR2, CDR3)] - [first linker] - [target polypeptide] - [second linker] - [half-life extension polypeptide]) [ka] SEQ ID NO:53 (bold indicates CDR1, CDR2, CDR3) [ka] SEQ ID NO:54 SEQ ID NO:55 (In order: [signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[first linker]-[target polypeptide]-[second linker]-[half-life extension polypeptide]) [ka] SEQ ID NO:56 PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKL YVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKSE

Claims

1. A fusion protein comprising (i) an antibody or antigen-binding fragment thereof that binds to a tumor antigen, (ii) a target polypeptide, and (iii) a half-life extension polypeptide.

2. The fusion protein according to claim 1, wherein the half-life-extending polypeptide is one of a hyaluronan-binding motif, a PAS polypeptide, a proline / alanine random coil polypeptide, and an antibody or its antigen-binding fragment.

3. The fusion protein according to claim 2, wherein the half-life-extending polypeptide is an antibody or an antigen-binding fragment thereof.

4. The fusion protein according to claim 3, wherein the half-life-extending polypeptide is an anti-albumin antibody or its antigen-binding fragment.

5. The fusion protein according to claim 4, wherein the anti-albumin antibody or its antigen-binding fragment comprises anti-albumin VHH.

6. The fusion protein according to claim 5, wherein the anti-albumin VHH comprises an amino acid sequence having at least about 90%, at least about 95%, or about 100% identity with SEQ ID NO:

45.

7. The fusion protein according to claim 1, wherein the tumor antigen is CD20, and the antibody or its antigen-binding fragment is an anti-CD20 antibody or its antigen-binding fragment.

8. The fusion protein according to claim 7, wherein the anti-CD20 antibody or its antigen-binding fragment comprises anti-CD20 VHH.

9. The fusion protein according to claim 8, wherein the anti-CD20 VHH comprises any one amino acid sequence of SEQ ID NOs: 37, 39, 40, 42-44, and 53.

10. The fusion protein according to claim 8, wherein the anti-CD20 VHH comprises the amino acid sequence of CDR1, CDR2, or CDR3 shown in any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53.

11. The fusion protein according to claim 1, wherein the target polypeptide is a tumor antigen.

12. The fusion protein according to claim 1, wherein the target polypeptide is a B cell antigen.

13. The fusion protein according to claim 1, wherein the target polypeptide is one of CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, CD180, ROR1, BCMA, HLA-DR10, CD1, CD5, CD21, CD25, CD27, CD30, CD38, CD78, CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2, CD79a, and CD79b.

14. The fusion protein according to claim 1, wherein the target polypeptide is CD19 or a fragment or variant thereof.

15. The fusion protein according to claim 14, wherein CD19 comprises an amino acid sequence having at least about 95%, at least about 97%, at least about 99%, or about 100% identity with the amino acid sequence of SEQ ID NO:

47.

16. The fusion protein according to claim 14, wherein the target polypeptide comprises approximately 230, 240, 250, 260, or 270 consecutive amino acids of SEQ ID NO:

47.

17. The fusion protein according to claim 14, wherein the target polypeptide comprises amino acids 20 to 278 of SEQ ID NO:

47.

18. The fusion protein according to claim 14, wherein the target polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, or about 100% identity with the amino acid sequence of SEQ ID NO: 48 or 56.

19. The fusion protein according to claim 14, wherein the target polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, or about 100% identity with the amino acid sequence of SEQ ID NO:

46.

20. The fusion protein according to claim 14, wherein the target polypeptide comprises the amino acid sequence of SEQ ID NO:

46.

21. The fusion protein according to claim 1, comprising a first linker between the antibody or its antigen-binding fragment and the target polypeptide.

22. The fusion protein according to claim 1, comprising a second linker between the target polypeptide and the half-life extension polypeptide.

23. The first linker or the second linker has one of the following amino acid sequences: (i) GS Linker, (ii) Polyglycine linker, (iii) Glycine and serine-rich linker, or (iv) Flexible linker A fusion protein according to claim 21 or 22, comprising:

24. The fusion protein according to claim 21, wherein the first linker comprises the amino acid sequence GGGGGSGGGGGSGGGGGSGGGGGS.

25. The fusion protein according to claim 22, wherein the second linker comprises the amino acid sequence SRGGGGGGSGGGGGGGGGS.

26. The fusion protein according to claim 1, wherein the fusion protein comprises an amino acid sequence having at least about 90%, at least about 95%, or about 100% identity with any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 50, 52, and 55.

27. The fusion protein according to claim 1, wherein the fusion protein comprises any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 50, 52, and 55.

28. The fusion protein according to claim 1, wherein the fusion protein consists of one amino acid sequence from sequence numbers 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 50, 52, and 55.

29. The fusion protein according to claim 1, wherein the fusion protein comprises an amino acid sequence having at least about 90%, at least about 95%, or about 100% identity with any one of the amino acid sequences of SEQ ID NOs: 2, 6, 14, 18, 26, 30, 34, and 50, and lacking a C-terminal hexahistidine tag.

30. The fusion protein according to claim 1, wherein the fusion protein comprises one amino acid sequence of sequence numbers 2, 6, 14, 18, 26, 30, 34, and 50, and lacks a C-terminal hexahistidine tag.

31. The fusion protein according to claim 1, wherein the fusion protein consists of one amino acid sequence from sequence numbers 2, 6, 14, 18, 26, 30, 34, and 50, and lacks a C-terminal hexahistidine tag.

32. The fusion protein according to claim 1, comprising a second antibody or antigen-binding fragment thereof that binds to a second tumor antigen.

33. The fusion protein according to claim 32, wherein the second tumor antigen is CD79b, HER-2 / neu, c-met, EGFR, Ga733 / EpCAM, CD21, ROR1, CLL-1 / CLEC12A, or BCMA.

34. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the fusion protein described in claim 1.

35. (i) A nucleic acid comprising any one nucleotide sequence of sequence numbers 1, 3, 5, 7, 13, 15, 17, 19, 25, 27, 29, 31, 33, 35, 49, 51, and 54, or (ii) any one nucleotide sequence of sequence numbers 1, 5, 9, 13, 17, 21, 25, 29, 33, and 49, and lacking the last 18 nucleotides that encode a hexahistidine tag.

36. A vector comprising the nucleic acid described in claim 34 or 35.

37. A host cell comprising the nucleic acid described in claim 34 or 35.

38. A host cell comprising the vector according to claim 36.

39. A method for producing a fusion protein, comprising culturing the host cell described in claim 37 under conditions suitable for the expression of the fusion protein.

40. A method for producing a fusion protein, comprising culturing the host cell described in claim 38 under conditions suitable for the expression of the fusion protein.

41. A method for treating a subject having a tumor, comprising administering to the subject an effective amount of the fusion protein described in claim 1, thereby treating the subject.

42. The method according to claim 41, wherein the tumor expresses the tumor antigen.

43. The method according to claim 41, wherein the tumor does not express CD19.

44. The method according to claim 41, wherein the fusion protein binds to the tumor antigen upon administration.

45. The method according to claim 41, further comprising administering to the subject an antibody, antibody-drug conjugate, or cell therapy drug (e.g., CAR-T cells) that specifically recognizes the target polypeptide.

46. The method according to claim 45, wherein, upon administration to the subject, the antibody, the antibody-drug conjugate, or the cell therapy drug (e.g., CAR-T cells) binds to the fusion protein.

47. The method according to claim 46, wherein the binding of the antibody, the antibody-drug conjugate, or the cell therapy drug (e.g., CAR-T cells) to the fusion protein induces the killing of the tumor.

48. An antibody or its antigen-binding fragment, comprising a VHH having one of the amino acid sequences or fragments thereof from sequence numbers 37, 39, 40, 42-44, and 53.

49. An antibody or its antigen-binding fragment, wherein the VHH comprises at least one CDR (e.g., CDR1, CDR2, and / or CDR3) as shown in any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53.

50. A nucleic acid sequence encoding the antibody or its antigen-binding fragment according to claim 48 or 49.

51. A vector comprising the nucleic acid sequence described in claim 50.

52. A host cell comprising the nucleic acid sequence described in claim 50.

53. A host cell comprising the vector according to claim 51.

54. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell described in claim 52 under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof or the fusion protein.

55. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell described in claim 53 under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof or the fusion protein.

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