Compositions and methods for treating 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 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. The present disclosure describes fusion proteins and their use in treating subjects with cancer.

Description

Background Art

[0001] The past decade has witnessed a transformation in the treatment of relapsed and refractory B malignancies. The use of CD19-targeted cell therapies has provided high clinical response rates in some of these otherwise difficult-to-treat patient populations. In addition, some patients have achieved durable clinical remissions that appear to be curative. However, these results have been tempered by the finding that approximately half of patients who have an initial response to cell therapy relapse, and most relapse quickly, typically within six months. Therefore, there is a need for additional therapeutic agents for the treatment of cancer. 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 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 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 the following: a hyaluronic acid binding motif, a PAS polypeptide, a proline / alanine random coil polypeptide, and albumin or a 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 an 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 NO: 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 mutant thereof.

[0006] In some embodiments, the fusion protein comprises an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical 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, the 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 extension polypeptide. In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding a fusion protein as described herein. In some embodiments, the present disclosure provides a host cell comprising a nucleic acid encoding a fusion protein as described herein. In some embodiments, the present disclosure provides a method of producing a fusion protein as described herein, the method comprising culturing a host cell comprising a nucleic acid encoding a fusion protein as described herein. In some embodiments, the present disclosure provides a method of treating a subject having a tumor, comprising administering an effective amount of a fusion protein as described herein to the subject.

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

[0009] Other features, objects and advantages of the present invention are apparent in the following detailed description. However, it should be understood that the detailed description, although indicating embodiments of the present invention, is given by way of example only and is not limiting. According to the detailed description, various changes and modifications within the scope of the present invention will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The numbers in the figures are for illustrative purposes only and are not limiting.

[0011] Figure 1 The isoelectric point distributions of protein preparations CTE1, CTE2 and CTE3 are shown. CTE1 is a mixture of sialylated and non-sialylated proteins, CTE2 is predominantly non-sialylated and CTE3 is predominantly sialylated.

[0012] Figure 2 Shown are the isoelectric point distributions of protein preparations CTE1 after various treatments to remove sialylation (eg, sialidase).

[0013] Figure 3Shown are the binding affinity curves of protein preparations CTE1 (red circles), CTE2 (green squares) and CTE3 (blue circles) to CD19 negative / CD20 positive JeKo-1 cells (JeKo-19KO) as measured by flow cytometry.

[0014] Figure 4 Shown are in vitro binding affinity curves of protein preparations CTE1 (green triangles), CTE2 (red circles) and CTE3 (blue squares) to purified biotinylated human albumin (left panel) or biotinylated human CD20 membrane preparations (right panel), as measured by ELISA.

[0015] Figure 5 Shown are the killing curves of protein preparations CTE1 (red circles), CTE2 (blue squares), and CTE3 (pink triangles) against CD19-negative / CD20-positive JeKo-1 cells (JeKo-19KO) when administered together with CAR-19 T cells.

[0016] Figure 6 It is shown that wild-type JeKo-1 cells are primarily positive for CD19 and CD20 expression as measured by flow cytometry.

[0017] 7A to 7C Shown are CD19 and CD20 expression levels of JeKo-1 B cell lymphoma cells treated with CAR-19 T cells over 13 days, as measured by flow cytometry. Fig. 7A , Figure 7B and Figure 7C Experiments corresponding to CAR-19T:JeKo-1 cell ratios of 1:1 (A), 0.3:1 (B), or 0.1:1 (C)

[0018] Fig. 8A Shown are the CD19 and CD20 expression levels 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 when CAR-19 T cells, fusion protein (middle), or both (bottom) were subsequently added to the cells.

[0019] Fig. 9 Shown is the time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with CAR-19 T cells plus CTE1, CAR-19 T cells alone, CAR-20 T cells alone, or left untreated.

[0020] Fig.10Shown is the time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with CAR-19 T cells plus CTE1, CAR-19 T cells plus CTE2, CAR-19 T cells alone, or left untreated.

[0021] Fig.11 Shown is the time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with different concentrations of CAR-19 T cells plus CTE2, CAR-19 T cells alone, or left untreated.

[0022] Fig.12 Shows Fig.11 The average body weights of mice are reported in .

[0023] Fig.13 Shows Fig.11 The mean luminescence of mice reported in . The right panel is an expanded view of 0.016, 0.08, 0.4 and 2 mg / kg fusion protein.

[0024] Fig.14 Shows Fig.11 The survival probability of mice reported in .

[0025] Fig.15 Shown is the time course of protein concentrations of fusion proteins CTE1, CTE2, and CTE3 in the serum of NSG mice for estimating the half-life of the proteins in the in vivo circulation.

[0026] Fig.16 Shown are the binding affinity curves of various fusion protein constructs to CD20 positive / CD19 negative JeKo-1 cells (JeKo-19KO), as measured by ELISA.

[0027] Fig.17 Shown are the killing curves of various fusion protein constructs against CD19-negative / CD20-positive JeKo-1 cells (JeKo-19KO) when administered together with CAR-19 T cells.

[0028] Fig.18 Binding and killing curves for additional fusion proteins are shown.

[0029] Fig.19A Shown is the binding affinity curve of CTE3 fusion protein to CD19 negative / CD20 positive JeKo-1 cells (JeKo-19KO), as measured by flow cytometry. Fig.19B Shown are the binding affinity curves of CTE3 fusion proteins to wild-type JeKo-1 cells, as measured by flow cytometry. Fig.19CShown are the binding affinity curves of the fusion proteins to wild-type Ramos Hodgkin Lymphoma cells, as measured by flow cytometry.

[0030] Fig. 20 Shown is the killing curve of CTE3 fusion protein preparation against CD19-negative / CD20-positive JeKo-1 cells (JeKo-19KO) when administered together with CAR-19T cells.

[0031] Fig.21A Binding of CTE3 to 293T cells transfected with cynomolgus macaque CD20 is shown. Fig.21B Binding of CTE3 to 293T cells transfected with human CD20 is shown.

[0032] Fig.22A The effect of human serum on CTE3-mediated CAR19 cytotoxicity is shown. Increased CTE3 levels are shown on the x-axis. HS = human serum. Fig. 22B Shown is the effect of human serum albumin (HSA) on CTE3-mediated CAR19 cytotoxicity. Increased CTE3 levels are shown on the x-axis.

[0033] Fig.23A CD79b x CD20 bispecific CTE binding to biotinylated CD20 is shown. Fig. 23B CTE#650 binding to biotinylated CD79b is shown. Fig.23C Binding of monospecific and bispecific anti-CD20 and anti-CD79b proteins to 293T-CD20 cells is shown. Fig.23D Binding of monospecific and bispecific anti-CD20 and anti-CD79b proteins to 293T-CD79b cells is shown. Fig.23E The cytotoxic capacity of anti-CD19 CAR T cells plus monospecific and bispecific anti-CD20 and anti-CD79b proteins against JeKo-1 CD19KO cells is shown.

[0034] Fig.24A Shown is the binding of CTE3 to biotinylated CD20 using SPR. Fig. 24B Shown is the binding of CTE3 to biotinylated CD19 using SPR. Fig.24C Shown is the binding of CTE3 to biotinylated HSA using SPR.

[0035] Fig.25A Shown are the cytotoxicity curves of JeKo-1 CD19 KO cells after one round of CAR19 stimulation. Fig.25B Shown are the cytotoxicity curves of JeKo-1 CD19 KO cells after 3 rounds of CAR19 stimulation. DETAILED DESCRIPTION

[0036] definition

[0037] In order to make the present invention easier to understand, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0038] Administration: As used herein, the term "administering" refers to administering a composition to a subject or system. It can be administered to an animal subject (e.g., a human) by any appropriate route. For example, in some embodiments, administration can be bronchial (including by bronchial instillation), buccal, enteral, intercutaneous, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, transtracheal (including by intratracheal instillation), transdermal, vaginal, and vitreous. In some embodiments, administration can be intratumoral or peritumoral administration. In some embodiments, administration can involve intermittent administration. In some embodiments, administration can involve continuous administration (e.g., perfusion) over at least one selected period of time.

[0039] 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 treatment method that involves the use of lymphocytes with anti-tumor activity, expansion of these cells to large numbers in vitro, and infusion into a host carrying cancer. In some embodiments, ACT is a treatment method that involves the use of lymphocytes with anti-tumor activity, infusion into a host carrying cancer, and in vivo expansion.

[0040] Agent: The term "agent" as used herein may refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, carbohydrates, lipids, small molecules, metals, or combinations thereof. It will be clear from the context that, in some embodiments, an agent may be or include a cell or organism, or a fraction, extract, or component thereof. In some embodiments, an agent is or includes a natural product, because it is found in nature and / or obtained from nature. In some embodiments, an agent is or includes one or more artificial entities, because it is designed, engineered, and / or produced by artificial means and / or does not exist in nature. In some embodiments, an agent may be used in an isolated or pure form; in some embodiments, an agent may be used in a crude form. In some embodiments, potential agents are provided as a collection or library, for example, which may be screened to identify or characterize active agents therein. Some specific embodiments of agents that may be used according to the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics, etc. In some embodiments, an agent is or includes 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 polymer portion. In some embodiments, the agent lacks or is substantially free of any polymer portion.

[0041] Amelioration: As used herein, "amelioration" means preventing, reducing and / or alleviating a subject's condition, or improving a subject's condition. Amelioration includes but does not require complete recovery or complete prevention of a disease, disorder or condition.

[0042] 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 H 2N–C(H)(R)–COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a d-amino acid; in some embodiments, the amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty 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 substitutes. Amino acids, including carboxyl and / or amino terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. An amino acid may contain one or more post-translational modifications, such as association with one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and / or an amino acid residue of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be apparent from the context in which it is used.

[0043] Antibody: As used herein, the term "antibody" refers to a typical immunoglobulin sequence element sufficient to confer specific binding to a particular target antigen. In addition, the term "antibody" as used herein may refer to any construct or format known or developed in the art in appropriate embodiments (unless otherwise specified or clear from the context) for utilizing antibody structural and functional characteristics in alternative presentations. For example, in some embodiments, the form of an antibody used in accordance with the present disclosure is selected from, but not limited to, complete IgG, IgE and IgM, bispecific or multispecific antibodies (e.g. etc.), bi- or multi-paratopic antibodies, single-chain Fv, polypeptide-Fc fusions, Fab, camelid antibodies, masked antibodies (e.g. ), small modular immunopharmaceuticals (“SMIPsTM”), single-chain or tandem diabodies VHH, Mini-antibodies, Ankyrin repeat protein or DART, TCR-like antibodies, MicroProteins and As is known in the art, naturally occurring intact IgG antibodies are tetramers 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) that associate with each other into a structure commonly referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each about 110 amino acids in length): an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH1, CH2, and carboxyl-terminal CH3 (located at the base of the Y stem). A short region called a "switch" connects the heavy chain variable region and the constant region. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides in the intact antibody to each other. Each light chain consists of two domains: an amino-terminal variable (VL) domain, followed by a carboxyl-terminal constant (CL) domain, separated from each other by another "switch". The complete antibody tetramer is composed of two heavy chain-light chain dimers, in which the heavy chain and the light chain are interconnected by a single disulfide bond; the other two disulfide bonds interconnect the heavy chain hinge region so that the dimers are interconnected and form a tetramer. Naturally produced antibodies are also glycosylated, usually on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold", which is formed by stacking two β sheets (e.g., 3, 4 or 5 chain sheets) in a compressed antiparallel β barrel. Each variable domain contains three hypervariable loops (called "complementarity determining regions" (CDR1, CDR2 and CDR3)) and four "framework" regions (FR1, FR2, FR3 and FR4) that are invariant to some extent. When the natural antibody is folded, the FR region forms a β sheet that provides a structural framework for the domain, and the CDR loop regions from the heavy and light chains are gathered together in three-dimensional space to produce a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also binds to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As known in the art, the affinity and / or other binding properties of the Fc region of an Fc receptor can be regulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or used according to the present disclosure include glycosylated Fc domains, including such glycosylated Fc domains with modifications or engineering. For the purposes of the present disclosure, in certain embodiments, any polypeptide or polypeptide complex including sufficient immunoglobulin domain sequences found in natural antibodies may be referred to and / or used as an "antibody", whether such polypeptides are naturally produced (e.g., produced by an organism reacting with an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, antibodies are polyclonal; in some embodiments, antibodies are monoclonal.In some embodiments, the antibody has a constant region sequence that is unique to mouse, rabbit, primate or human antibodies. In some embodiments, the antibody sequence elements are completely human, or are humanized, primatized, chimeric, etc., as known in the art. In some embodiments, the antibody may lack the covalent modification (e.g., attachment of polysaccharides) that will be present when it is naturally produced. In some embodiments, the antibody may contain covalent modifications (e.g., attachment of polysaccharides, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.) or other side groups (e.g., polyethylene glycol, etc.)). In some embodiments, a protein scaffold produced to bind to a target antigen can be used as an antigen binding fragment. In some embodiments, a protein binding molecule can be generated in a computer based on the amino acid sequence and / or resolved structure of the 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).

[0044] Antibody-dependent cellular toxicity: As used herein, the term "antibody-dependent cellular toxicity" or "ADCC" refers to the phenomenon that target cells bound by antibodies are killed by immune effector cells. Without wishing to be bound by any particular theory, ADCC is generally understood to involve effector cells carrying Fc receptors (FcRs) that can recognize and subsequently kill antibody-coated target cells (e.g., cells expressing a specific antigen to which the antibody binds on its surface). 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.

[0045] Antibody fragment: As used herein, "antibody fragment" includes a portion of an intact antibody, such as the antigen binding region or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed by antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments (composed of the variable regions of the heavy and light chains), recombinant single-chain polypeptide molecules ("scFv proteins") in which the light and heavy chain variable regions are connected by peptide linkers, recombinant single-domain antibodies composed of antibody heavy chain variable regions (e.g., VHH), and the minimum recognition unit composed of amino acid residues that mimic hypervariable regions (e.g., hypervariable regions of the heavy chain variable region (VH), hypervariable regions of the light chain variable region (VL), one or more CDR domains within VH, and / or one or more CDR domains within VL). In many embodiments, an antibody fragment contains enough parent antibody sequence to be a fragment that binds to the same antigen as the parent antibody; 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 The antigen binding fragment of an antibody can be produced by any means. For example, the antigen binding fragment of an antibody can be produced enzymatically or chemically by fragmentation of a complete antibody and / or it can be produced recombinantly from a gene encoding a partial antibody sequence. Alternatively or additionally, the antigen binding fragment of an antibody can be produced in whole or in part synthetically. The antigen binding fragment of an antibody can optionally include a single-chain antibody fragment. Alternatively or additionally, the antigen binding fragment of an antibody can include multiple chains connected together, for example, by disulfide bonds. The antigen binding fragment of an antibody can optionally include a multimolecular complex. Functional antibody fragments typically include at least about 50 amino acids and more typically include at least about 200 amino acids.

[0046] Antigen: The term "antigen" as used herein refers to an agent that triggers 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 triggers a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, an antigen triggers a cellular response (e.g., involving T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen is bound to an antibody and may or may not induce a specific physiological response in an organism. Typically, an antigen may be or include any chemical entity, such as a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, in addition to a biopolymer (e.g., in addition to a nucleic acid or amino acid polymer)), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a polysaccharide. It will be understood by those of ordinary skill in the art that, in general, an antigen may be provided in an isolated or pure form, or may alternatively be provided in a crude form (e.g., together with other materials, such as in an extract, such as a cell extract or other relatively crude preparation containing an antigen source), or may be present on or in a cell. In some embodiments, the antigen is a recombinant antigen.

[0047] Approximately or approximately: As used herein, the term "approximately" or "approximately" 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 "approximately" refers to a range of values ​​that fall 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 than or less than), unless otherwise stated or otherwise apparent from the context (except when this number exceeds 100% of the possible value).

[0048] Binding: It should be understood that as used herein, the term "binding" generally refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of contexts, including when studying the interacting entities or moieties in isolation or in the context of a more complex system (e.g., when covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0049] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "cancer" are used interchangeably herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include precancerous (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 name just a few non-limiting examples, in some embodiments, the teachings of the present disclosure are applied to one or more cancers, such as, for example, hematopoietic cancers, including leukemias, lymphomas (Hodgkin's and non-Hodgkin's), myelomas, and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the oral cavity, pharynx, larynx, and lung, liver cancer, genitourinary cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer and endometrial cancer and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and cancers of the nervous system, benign lesions such as papilloma, and the like.

[0050] Chimeric Antigen Receptor (CAR): As used herein, "chimeric antigen receptor" or "CAR" refers to a recombinant cell surface receptor that is engineered to be expressed on an immune effector cell and to specifically target the cell and / or bind an antigen.

[0051] 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' cap formation and / or 3' end formation, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0052] Fusion protein: As used herein, the term "fusion protein" generally refers to a polypeptide comprising at least two segments, each segment showing a high degree of amino acid identity with the following peptide portions: (1) existing in nature, and / or (2) representing a functional domain of a polypeptide. Generally, a polypeptide containing at least two such segments is considered a fusion protein if the two segments are parts of the following: (1) not included in the same peptide in nature, and / or (2) have not been previously linked to each other in a single polypeptide, and / or (3) have been linked to each other by artificial means.

[0053] 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, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via phosphodiester linkages. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. For example, in some embodiments, nucleic acids are, comprise, or consist of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and are considered to be 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 bonds. In some embodiments, the nucleic acid is, comprises or consists of one or more natural 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, pyrrolopyrimidine, 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-oxyadenosine, 8-oxyguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, embedded 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 sugars in natural nucleic acids. In some embodiments, nucleic acid has a nucleotide sequence encoding a functional gene product, such as an RNA or protein. In some embodiments, nucleic acid includes one or more introns. In some embodiments, nucleic acid is prepared by separation from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, propagation in a recombinant cell or system, and one or more of 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; in some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element, the at least one element encoding a polypeptide, or the complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0054] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to substances that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0055] Polypeptide: As used herein, a "polypeptide" is generally a string of at least two amino acids connected to each other by peptide bonds. In some embodiments, a polypeptide may contain at least 3-5 amino acids, each amino acid connected to the other amino acids by at least one peptide bond. In some embodiments, a polypeptide may be longer than 5 amino acids, each amino acid connected to the other amino acids by at least one peptide bond. One of ordinary skill in the art will understand that polypeptides sometimes include "non-natural" amino acids or other entities that are still capable of being optionally incorporated into a polypeptide chain.

[0056] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids interconnected by peptide bonds). Proteins may contain moieties other than amino acids (e.g., they may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. One of ordinary skill in the art will recognize that a "protein" may be a complete polypeptide chain (with or without a signal sequence) produced by a cell, or may be a portion thereof. One of ordinary skill in the art will recognize that a protein may sometimes contain more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means. A polypeptide 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, and the like. In some embodiments, a protein may contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0057] Subject: "Subject" refers to a mammal (e.g., a human). In some embodiments, the subject suffers from a disease, disorder, or condition of interest. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person with one or more characteristics that are characteristic of a 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 to whom a diagnosis and / or therapy is administered and / or has been administered.

[0058] Suffering from: An individual "suffering from" a disease, disorder, or condition (eg, cancer) has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, or condition.

[0059] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease, disorder and / or condition when administered to a population suffering from or susceptible to a disease, condition and / or illness according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, condition and / or illness, stabilizes one or more characteristics thereof and / or delays its onset. It should be understood by those of ordinary skill in the art that the term "therapeutically effective amount" does not actually require successful treatment to be achieved in a particular subject. Instead, a therapeutically effective amount may be an amount that provides a specific desired pharmacological response in a large number of subjects when administered to a patient in need of such treatment. For example, in some embodiments, a "therapeutically effective amount" refers to an amount that will block, stabilize, weaken or reverse the cancer support process occurring in the individual, or will enhance or increase the cancer suppression process in the individual when administered to an individual in need in the context of the present therapy. In the context of cancer treatment, a "therapeutically effective amount" refers to an amount that will prevent, stabilize, inhibit or reduce the further development of cancer in an individual when administered to a subject diagnosed with cancer. Particularly preferred "therapeutically effective amounts" of the compositions described herein reverse (in therapeutic treatment) the development of malignancies such as pancreatic cancer or help achieve or prolong remission of malignancies. The therapeutically effective amount administered to an individual to treat cancer in the individual may be the same or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the methods of treatment described herein should not be interpreted as, limited to, or otherwise limited to a "cure" of cancer; rather, the methods of treatment involve using the compositions described to "treat" cancer, i.e., produce a desired or beneficial change in the health of an individual suffering from cancer. Such benefits have been recognized by skilled healthcare providers 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 vital functions (e.g., improvement in the function of cancerous tissues or organs), reduction or inhibition of further metastasis, reduction in opportunistic infections, increase in survivability, reduction in pain, improvement in motor function, improvement in cognitive function, improvement in sense of energy (vitality, reduction in discomfort), improvement in sense of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. Additionally, regression of a particular tumor in an individual (e.g., as a result of treatment as described herein) can also be assessed by collecting cancer cell samples from a tumor site such as pancreatic cancer (e.g., during treatment) and testing the cancer cells for levels of metabolic and signaling markers to monitor the state of the cancer cells, thereby verifying at the molecular level the regression of the cancer cells to a less malignant phenotype.For example, tumor regression induced by using the methods of the present invention will be indicated by finding a decrease in one or more pro-angiogenic markers, an increase in anti-angiogenic markers, a metabolic pathway that exhibits abnormal activity in an individual diagnosed with cancer, an intercellular signaling pathway, or a normalization of an intracellular signaling pathway (i.e., a change to a state found in a normal individual not suffering from cancer). It will be understood by one of ordinary skill in the art that in some embodiments, a therapeutically effective amount can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount can be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0060] Treatment: As used herein, the term "treatment" (also referred to as "treat" or "treating") refers to any administration of a substance that partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment may be directed to subjects who do not show signs of the relevant disease, disorder, and / or condition, and / or subjects who only show early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be directed to subjects who show one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0061] Vector: As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid associated with it. In some embodiments, a vector is capable of extrachromosomal replication and / or expression of a nucleic acid to which it is attached in a host cell, such as a eukaryotic and / or prokaryotic cell. A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector."

[0062] Overview

[0063] The promise of cancer cell therapy has become clear with the introduction of autologous T cells transduced with chimeric antigen receptors (CARs) targeting the B-cell malignancy antigen CD19. Anti-CD19 CAR-T cells (CAR-19s) induce 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 approaching 10 years cancer-free (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).

[0064] However, the cost of CAR-19 therapy remains high, and the necessary infrastructure limits the environment in which CARs can be produced and administered (Lin et al., J Clin Oncol. 2019; 37: 2105–19). Preparative apheresis, consolidation, lymphocyte clearance, and toxicity limit the use of CAR-T therapy in frail patients. The side effects of CAR-19 treatment can be significant, including grade 3+ cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), which can lead to prolonged hospital stay and additional care costs (Brudno et al., Blood Rev. 2019; 34: 45–55). More than half of the patients who respond subsequently relapse, usually within a few months after 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).

[0065] Antigen loss recurrence is a phenomenon driven by natural selection, that is, CAR-19T cells can exert strong selection pressure on malignant B cell populations, thereby driving the selection of clones in which target protein CD19 is lost or reduced. Antigen loss can occur via mutation events that prevent the expression of CD19 extracellular domain (ECD) or ECD intraregions and / or via 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 in the treatment mode of cancer therapy (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 escape from CAR-19 therapy have been described and include lineage switching to myeloid leukemia and trogocytosis, a process in which CD19 is physically stripped from lymphoma cells by CAR (Perna, Translational Cancer Research [Internet]. AME Publishing Company; 2016 [cited January 12, 2022]; 5 (available from tcr.amegroups.com / article / view / 9016); Miao et al., Frontiers in Immunology. 2021; 12: 1862).

[0066] The fusion proteins described herein provide solutions to one or more of these problems. In some embodiments, the fusion proteins described herein can be expressed by cells transduced with lentivirus, retrovirus or other gene therapy vectors, and / or can be expressed by in vitro mammalian cell culture and then purified to produce injectable biologics. In some embodiments, the fusion protein of the present disclosure comprises three functional domains: a modified CD19ECD, an anti-CD20 binding domain, and an anti-albumin binding domain. In some such embodiments, the fusion protein of the present disclosure binds to CD20 and displays CD19 ECD. As described herein, such fusion proteins can (i) increase the CD19 antigen density on target tumor cells, regardless of their CD19 expression level, (ii) effectively trigger CD19 negative tumor cell death in the presence of CAR-19T cells in vitro, (iii) prevent antigen loss recurrence to escape CAR-19 therapy, (iv) prevent CD19 negative tumor expansion, for example at relatively low doses, and / or (v) have a significant effect on survival. As described herein, the fusion proteins of the present disclosure can be expressed by transfected mammalian cells, can be efficiently purified, and exhibit favorable biophysical properties, indicating that such fusion proteins may be suitable for scale-up and production.

[0067] Fusion Protein

[0068] 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 target 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 target polypeptide. In some embodiments, the fusion protein described herein comprises or consists of: at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 50 and 52, but lacking the amino acid sequence of the disclosed half-life extending polypeptide in each 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 50, but lacks the disclosed half-life extension polypeptide in each sequence and lacks the disclosed C-terminal hexa-histidine 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 SEQ ID NO.55.

[0069] 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 target 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 target 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 NO: 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 includes (amino to carboxyl terminus): a signal sequence; an anti-CD20 VHH; a first linker (GSGGGGSGGGGS); a polypeptide of interest; a second linker (SRGGGGSGGGGSGGGGS); a half-life extending polypeptide; and a hexahistidine tag. 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, 6, 10, 14, 18, 22, 26, 30, 34, and 50, and lacks a 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 includes (amino to carboxyl terminus): anti-CD20 VHH; a first linker (GSGGGGSGGGGS); a polypeptide of interest; a second linker (SRGGGGSGGGGSGGGGS); and a half-life extending polypeptide.

[0070] In addition to those sequences described herein, those skilled in the art will recognize many amino acid sequences suitable for use as linkers. In some embodiments, the first and / or second linkers comprise a series of naturally occurring amino acids. In some embodiments, the linker is derived from a naturally occurring multi-domain protein. In some embodiments, the linker is flexible. In some embodiments, a computational tool familiar to those skilled in the art, such as JPred (Dorzdetskiy et al., Nucl Acids Res. 2015; 43: W389-94) is used to design a flexible linker. In some embodiments, the linker adopts a preferred configuration. In some embodiments, the linker comprises a GS linker, wherein the linker comprises the formula [G x S y ] z wherein x, y, and z are positive integers. In some embodiments, the linker comprises a polyglycine linker, wherein the linker comprises the amino acid sequence G x , wherein x is a positive integer. In some embodiments, the linker comprises a linker rich in glycine and serine, wherein glycine and serine together constitute 50% or more of the linker amino acid sequence. Many embodiments and applications of specific linker sequences in fusion proteins and linker design methods 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.

[0071] Antigen Binding Peptides

[0072] In some embodiments, the fusion proteins of the present disclosure comprise an antigen binding polypeptide that binds to a tumor antigen, such as 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, such as an anti-CD20 single domain antibody described herein.

[0073] In some embodiments, the fusion protein of the present disclosure comprises a first antigen-binding polypeptide that binds to CD20, and a second antigen-binding polypeptide that binds to 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, such as an anti-CD20 single domain antibody described herein.

[0074] Single domain antibodies

[0075] Single-domain antibodies are antibodies whose complementary determining regions are part of single-domain polypeptides. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally free of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single-domain antibodies may be any known in the art, or any future single domain antibodies. Single-domain antibodies may be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, cattle. According to one aspect of the present disclosure, the single-domain antibodies used herein are referred to as single-domain antibodies of heavy chain antibodies free of light chains. Such single-domain antibodies are disclosed in, for example, WO 94 / 04678. Variable domains derived from naturally free of light chain heavy chain antibodies (e.g., single domain antibodies) are referred to herein as "VHH" or "nanoantibodies". Such VHH may be derived from antibodies produced in camelid species, such as camels, dromedaries, llamas, llamas, alpacas, and guanacos. Species other than Camelidae may produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the present disclosure.

[0076] The amino acid residues of the VHH domains from camelids are numbered according to the general numbering of VH domains given in Kabat et al., "Sequence of proteins of immunological interest", US Public Health Services, NIH (Bethesda, MD), publication number 91-3242 (1991); see also Riechmann et al., J. Immunol. Methods 231: 25-38 (1999). According to this numbering, FR1 comprises the amino acid residues at positions 1-30, CDR1 comprises the amino acid residues at positions 31-35, FR2 comprises the amino acid residues at positions 36-49, CDR2 comprises the amino acid residues at positions 50-65, FR3 comprises the amino acid residues at positions 66-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

[0077] However, it should be noted (as is well known in the art for VH and VHH domains) that the total number of amino acid residues in each CDR may be different 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 means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0078] Alternative methods for numbering the amino acid residues of a VH domain are known in the art and can also be applied in a similar manner to a VHH domain. Unless otherwise indicated, in the present disclosure, claims and drawings, CDRs are defined according to IMGT numbering (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38: D301-D307 (2010).

[0079] Anti-CD20 VHH

[0080] In some embodiments, the fusion protein of the present 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.

[0081] 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.

[0082] In some embodiments, the anti-CD20 VHH comprises or consists of at least one CDR (e.g., CDR1, CDR2, and / or CDR3) depicted in any one of SEQ ID NOs: 37-44 and 53. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 37. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 38. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 39. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 40. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 41. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 42. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 43. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 44. In some embodiments, the anti-CD20 VHH comprises CDR1, CDR2, and CDR3 depicted in SEQ ID NO: 53.

[0083] 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) depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted 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 the CDR1 depicted 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 the CDR2 depicted 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 the CDR3 depicted in SEQ ID NO:53.

[0084] Antibodies or fragments can be produced by any method known in the art for synthesizing antibodies (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; WO 92 / 22324; WO 98 / 46645). Chimeric antibodies can be produced using, e.g., the method described in Morrison, 1985, Science 229: 1202, and humanized antibodies can be produced by, e.g., the method described in U.S. Pat. No. 6,180,370.

[0085] Target peptide

[0086] In some embodiments, the fusion protein of the present disclosure comprises a target polypeptide. In some embodiments, the target polypeptide is an antigen target for a cell therapy agent, such as a CAR-T cell, an antibody, or an antibody drug conjugate as described in WO2017 / 075537, WO2017 / 075533, WO2018156802, and WO2018156791.

[0087] In some embodiments, the target polypeptide comprises or consists of all or part of a tumor-associated antigen (TAA) or a 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 multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens generated by 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, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-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-associated protein, TAAL6, TAG72, TLP, MUC16, IL13Rα2, FRα, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, Nectin-4, AGS-16, guanylate cyclase C, MUC-1, CFC1B, integrin α3 chain (belonging to laminin receptor chain a3b1), and TPS.

[0088] In some embodiments, the target polypeptide comprises or consists of all or part of a tumor antigen selected from the group consisting of CD19, CD20, CD22, CD30, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLEC12A, ROR1, phosphatidylinositol glycan 3 (GPC3), mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1, and MAGE A3.

[0089] In some embodiments, the polypeptide of interest comprises or consists of all or part of the following B cell-specific markers: such as 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).

[0090] CD19 is a 95 kDa type I transmembrane glycoprotein used as a biomarker for B cell development (Wang et al., Exp. Hematol. Oncol. 1:36 (2012)). The expression of CD19 in lymphomas and leukemias makes 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 unique and effective expression of CD19 in CAR-T cell therapy, treatment methods have been described that involve using antibody-CD19 fusions or CD19 variants engineered to directly bind to tumor biomarkers to bind CD19. - Tumor "transformation" to CD19 + Tumors (see, for example, WO2017 / 075537 and WO2017 / 075533).

[0091] It is assumed that the extracellular region of CD19 contains 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)). Homology modeling supports this ( et al., Nucleic Acids Res. 33:244–248 (2005)). However, a later published structure indicated that CD19 does not contain a C2-like immunoglobulin domain (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.

[0092] In some embodiments, the target polypeptide comprises all or part of the amino acid sequence of SEQ ID NO:47 or consists thereof. 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 with 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 contiguous amino acids of SEQ ID NO:47. In some embodiments, the target polypeptide comprises or consists of all or part of the extracellular domain (ECD) of CD19. For example, in some embodiments, the target polypeptide comprises or consists of a fragment of SEQ ID NO:47 (e.g., 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 part 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 with the amino acid sequence of SEQ ID NO: 48. In some embodiments, the target polypeptide comprises or consists of all or part of a CD19 variant or a fragment thereof. In some embodiments, the target polypeptide comprises or consists of all or part of an amino acid 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 with the amino acid sequence of SEQ ID NO: 48. In some embodiments, the target polypeptide comprises or consists of all or part 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, which comprises one or more amino acid substitutions as described herein. In some embodiments, the CD19 variant is or comprises a CD19ECD (e.g., SEQ ID NO: 48 or SEQ ID NO: 56) or a portion thereof, which comprises 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 an 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 an amino acid sequence of SEQ ID NO: 46.

[0093] Half-life extension

[0094] In some embodiments, the fusion proteins of the present disclosure include an agent that increases the half-life of the fusion protein, for example relative to a fusion protein without the agent. In some embodiments, such an agent is a polypeptide, referred to herein as a "half-life extending polypeptide".

[0095] In some embodiments, the half-life extension 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 extension polypeptide is an 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 extension polypeptide is a polypeptide that binds serum proteins. In some embodiments, the half-life extension polypeptide is a serum albumin binder (e.g., BSA, HSA or MSA binder).

[0096] In some embodiments, the serum albumin binder is an albumin binding peptide. Peptides bound to albumin are described in WO200145746, WO2002076489, WO2008068280, WO2009127691, WO2011095545, and U.S. Patent Publication Nos. 20040001827, 20080187517, and 20130316952. Those skilled in the art are familiar with methods for directly connecting proteins and antibodies to albumin or albumin domains, such as, for example, in Patterson et al., Bioconjugate Chem. 2016, 27, 10, 2271–2275; Bern et al., Sci. Trans. Med., October 14, 2020, Vol. 12, No. 565.

[0097] In some embodiments, the half-life extension polypeptide is or comprises a hyaluronic acid binding domain. Hyaluronic acid (HA), also known as hyaluronic acid, is a glycosaminoglycan that is present in connective tissue and other tissues and is abundant in synovial fluid, skin and vitreous. HA binds to a large number of naturally occurring hyaluronic acid binding proteins (HABPs). Some HABPs contain an HA binding domain, called a connection module, through which HABP binds to HA (Kohda, CJ et al., Cell 86 (1996) 767–775.). Some HAPBs contain a linear 9–11 residue HA binding motif, which contains a plurality of basic amino acids called B-X7-B motifs (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).

[0098] 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 accounts for more than about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100% of the total amino acid sequence of the half-life extending polypeptide. In general, the PAS polypeptide is characterized in that it adopts a random coil conformation under physiological conditions, as described in U.S. Patent Publication No. 20100292130. PAS polypeptides that can be used as half-life extending polypeptides in the fusion protein of the present disclosure are further described in WO 2008 / 155134, U.S. Patent Publication No. 20100292130, U.S. Patent No. 8,563,521 and / or U.S. Patent No. 9,260,494.

[0099] In some embodiments, the half-life extension polypeptide is composed only of proline and alanine, or is mainly composed of proline and alanine but may have up to 1%, 2%, 3%, 5% or 10% of other amino acid residues. In the presence of other amino acids, they may all be the same, or a variety of different amino acids may be present. Examples of polypeptides that are mainly or completely composed 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.

[0100] In some embodiments, the half-life extending polypeptide is characterized in that the sum of glycine, alanine, serine, threonine, glutamate and proline residues accounts for more than about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or 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 4 of these 6 different amino acids. Such polypeptides can be said to be composed mainly 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).

[0101] 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 an amino acid sequence disclosed or described in U.S. Publication No. 20070269422, the entire contents of which are incorporated herein by reference. In some embodiments, the anti-albumin VHH comprises or consists of an 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.

[0102] Tumor

[0103] The present disclosure provides techniques that can be used to treat any tumor. In some embodiments, the tumor is or includes a hematological malignancy, including but not limited to acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Langerhans cell histiocytosis, multiple myeloma, or a myeloproliferative neoplasm.

[0104] 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 cancer, rectal cancer, gastric cancer, or esophageal cancer.

[0105] In some specific embodiments, tumor is or includes advanced tumor and / or refractory tumor.In some embodiments, when certain pathologies are observed in tumor (for example, in tissue samples obtained from tumor, such as biopsy samples) and / or when cancer patients with such tumors are generally considered to be non-candidates for conventional chemotherapy, tumor is characterized as advanced stage.In some embodiments, tumor is characterized as advanced stage pathology and may include tumor size, expression changes of genetic markers, invasion of adjacent organs and / or lymph nodes by tumor cells.In some embodiments, when patients with such tumors have drug resistance to one or more known treatments (for example, one or more conventional chemotherapy regimens) and / or when specific patients show drug resistance (for example, lack of responsiveness) to one or more such known treatments, tumor is characterized as refractory.

[0106] Cell therapy

[0107] In some embodiments, the fusion proteins described herein can be administered to a subject as a cell therapy. For example, a nucleotide sequence encoding a fusion protein described herein can be introduced into a cell to be administered to a subject as a cell therapy. In some embodiments, the cell therapy can be produced by immune cells, such as cells that can be used or capable of being used in adoptive cell therapy. In some embodiments, the cell therapy is produced by a cell type selected from the group consisting of: TIL, T cells, CD8 + cells, CD4 + Cells, NK cells, γ-δ T cells, regulatory T 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 left the bloodstream and migrated to the tumor. Lymphocytes can be divided into three groups, including B cells, T cells, and natural killer cells. As used herein, "T cells" refers to CD3 + cells, including CD4 + Helper cells, CD8 + Cytotoxic T cells and delta-gamma T cells.

[0108] In certain embodiments, a cell therapy agent is produced by genetically modifying (e.g., transforming) cells, such as immune cells, with nucleic acids encoding fusion proteins as described herein. In some embodiments, such nucleic acids are contained in a recombinant expression vector. The recombinant expression vector may include any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and may contain natural, non-natural or altered nucleotides. The recombinant expression vector may include naturally occurring or non-naturally occurring internucleotide linkages, or two types of linkages.

[0109] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for breeding and amplification or for expression or for both, such as plasmids and viruses. For example, vectors can be selected from 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.). Phage 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).

[0110] In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, retroviral vectors, alpha viruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes viruses, and fowl pox virus vectors, and preferably have natural or engineered capabilities for transforming immune cells (e.g., T cells).

[0111] In ex vivo applications such as cell therapy, gamma-retroviral vectors derived from murine leukemia virus (MLV) were first developed and are still in use. Lentiviral vectors based on human immunodeficiency virus (HIV) are widely used. The general strategy for designing lentiviral vectors is based on the deletion and alteration of native viral sequences to prevent the production of replication-competent viruses. Therefore, the lentiviral components are divided into three or four different plasmid constructs with the aim of preventing the possibility of complete recombination into fully replication-competent lentivirus (RCL). The viral vector genome contains at least a transgene expression cassette, a long terminal repeat (LTR), and a packaging signal. In most cases, three additional plasmids provide the factors required for viral production and packaging (e.g., gag, pol, env). The promoter-enhancer region from the 3'LTR is also deleted, preventing transcription from this region and subsequent viral replication (called self-inactivating vector; SIN). The basic steps of ex vivo cell transformation or transduction include cell isolation and culture of the desired cell type for selection, amplification, and differentiation before or after transduction of the cells with the viral vector. In the case of hematopoietic cells, most of these steps are performed in a closed system using disposable blood collection and processing bags. For CAR T cell therapy, patient blood cells are harvested, and the desired T cell population is selected and grown to the desired level. It is then transduced with a viral vector carrying the desired gene cassette, and the CAR T cells are subsequently expanded to the billion cell level. Lentiviral vectors have been shown to efficiently transduce T cells and are therefore the preferred vector for introducing CAR into patient target cells. The expanded cells are then reintroduced into the patient.

[0112] In certain in vivo applications, a nucleic acid encoding a fusion protein described herein or a vector comprising 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 described herein can be provided as described in, for example, Nawaz et al., Blood Cancer Journal, Vol. 11, Article No. 119 (June 23, 2021); Carbonaro-Sarracino et al., Molecular Therapy: Methods & Clinical Development, Vol. 16, March 2020; Cantore and Naldini Haemophilia, Vol. 27, No. S3, pp. 122-125; Gouze-Decaris et al., Arthritis Res. 2001; 3 (Suppl. 1): p. 34; Breuer et al., Scientific Reports, Vol. 10, Article No. 4544 (2020), Naldini et al., SCIENCE, April 12, 1996, Vol. 272, No. 5259, pp. 263-267. Viral vector particles can be used to deliver nucleic acids directly in vivo. Examples of such viral vector particles include lentivirus, retrovirus, AAV, HVS, vaccinia virus, and many other viral types. The viral vector particles can be modified to optimize delivery, such as delivery to specific cell types, including immune cells. See, for example, 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 Vol. 334, June 10, 2021, pp. 106-113; Jang et al., Molecular Therapy Vol. 19, No. 8, August 2011, pp. 1407-1415.

[0113] Recombinant expression vectors can be prepared using standard recombinant DNA techniques as described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 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. Circular or linear expression vector constructs can be prepared to include a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0114] The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc.; providing prototrophic complementation in auxotrophic hosts; etc. Suitable marker genes for recombinant expression vectors include, for example, neomycin / G418 resistance genes, puromycin resistance genes, hygromycin resistance genes, histamine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0115] The carriers useful in the context of the present disclosure may be "naked" nucleic acid carriers (i.e., carriers with little or no protein, sugar and / or lipid encapsulating it), or carriers complexed with other molecules. Other molecules that may be suitably combined with the carrier 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.

[0116] 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 a variety of art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, gene guns, 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 nutritional polypeptides, anti-immunosuppressive polypeptides, and half-life extension polypeptides.

[0117] Protein therapeutics

[0118] In some embodiments, the fusion proteins described herein can be produced and administered to a subject as a protein therapeutic, rather than being produced by or with a cell therapeutic described herein. Such polypeptides can be included in a composition, such as a pharmaceutical composition, and used as a protein therapeutic. For example, a protein therapeutic comprising a fusion protein described herein can be administered in combination with a cell therapeutic targeting CD19, such as a CAR-T cell or ADC.

[0119] A variety of methods for preparing polypeptides are known in the art and can be used to prepare polypeptides to be included in protein therapeutics. For example, polypeptides can be recombinantly produced by utilizing a host cell system engineered to express a nucleic acid encoding a polypeptide. Recombinant expression of a gene can include constructing an expression vector containing a polynucleotide encoding a polypeptide. Once the polynucleotide is obtained, a vector for producing the polypeptide can be produced by recombinant DNA technology using techniques known in the art. Expression vectors containing a polypeptide coding sequence and appropriate transcription and translation control signals can be constructed using known methods. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0120] 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.

[0121] A variety of host-expression vector systems are available (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems can be used to produce polypeptides and, if desired, subsequently purify. Such host expression systems include microorganisms, such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing polypeptide coding sequences; yeast (e.g., Saccharomyces and Pichia pastoris) transformed with recombinant yeast expression vectors containing polypeptide coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing polypeptide coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing polypeptide coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0 and 3T3 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., late adenovirus promoters; vaccinia virus 7.5K promoters).

[0122] For bacterial systems, a variety of expression vectors can be used, including but not limited to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12: 1791); pIN vector (Inouye and Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 24: 5503-5509); etc. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).

[0123] For expression in mammalian host cells, viral-based expression systems can be used (see, e.g., Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Expression efficiency can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544).

[0124] In addition, host cell strains that regulate the expression of inserted sequences or modify and process gene products in a desired ad hoc manner can be selected. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of expressed polypeptides. Such cells include, for example, established mammalian cell lines and insect cell lines, animal cells, fungal cells, and yeast cells. Mammalian host cells include, for example, the BALB / c mouse myeloma cell line (NSO / 1, ECACC No. 85110503); human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands); SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cell subclones 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 CCL10); 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 carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).

[0125] For long-term, high-yield production of recombinant proteins, host cells are engineered to stably express polypeptides. Host cells can be transformed with DNA controlled by appropriate expression control elements known in the art, including promoters, enhancers, sequences, transcription terminators, polyadenylation sites, and selectable markers. Methods commonly known in the art of recombinant DNA technology can be used to select the desired recombinant clones.

[0126] Once the protein described herein has been produced by recombinant expression, it can be purified by any purification method known in the art, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, and sieving column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. For example, antibodies can be separated and purified by appropriately selecting and combining affinity columns (such as protein A columns) with chromatographic columns, filtration, ultrafiltration, salting out, and dialysis procedures (see Antibodies: A Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). In addition, as described herein, polypeptides can be fused to heterologous polypeptide sequences to facilitate purification. Alternatively or additionally, polypeptides or fusion proteins can be prepared partially or completely by chemical synthesis.

[0127] Virus delivery

[0128] In some embodiments, nucleic acid encoding fusion protein as described herein can be introduced into cells and / or administered to subjects as viral vectors. In some embodiments, such viral vectors can be used to introduce fusion protein into cancer cells (e.g., tumor cells). The introduction of such fusion protein can increase the susceptibility to the subject's immune system and / or one or more additional therapeutic agents (see, e.g., WO2017 / 075533).

[0129] Vector design

[0130] The nucleotide sequence encoding fusion protein as herein described can be introduced into various types of vectors. For example, nucleic acid can be cloned into plasmid, phagemid, phage derivative, animal virus and clay. Other vectors can include expression vector, replication vector, probe generation vector, sequencing vector and viral vector. In other examples, the vector can be a foamy virus (FV) vector, a retroviral vector made of foamy virus. Viral vector design and technology are well known in the art, such as Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2001) and other virology and molecular biology manuals.

[0131] Viral transduction

[0132] Viruses are very efficient in delivering nucleic acids to specific cell types, while generally avoiding detection by infected host immune systems. These features make some viruses become attractive candidate vectors 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, slow viruses, poxviruses, herpes simplex virus type 1, herpes viruses, oncogenic viruses (such as murine leukemia viruses), etc. Typically, suitable vectors contain a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers in at least one organism (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0133] 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 hexadimethrine bromide) used to increase the efficiency of lentiviral or retroviral transduction.

[0134] For example, retrovirus provides a platform for gene delivery system. Retrovirus is an enveloped virus belonging to the family Retroviridae. Once entering the host cell, the virus will be replicated by using viral reverse transcriptase to transcribe its RNA into DNA. Retroviral DNA is replicated as a part of the host genome and is referred to as provirus. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of the subject in vivo. Many retroviral systems are known in the art (see, for example, U.S. Patent Nos. 5,994,136, 6,165,782 and 6,428,953).

[0135] Retroviruses include alpha retroviruses (e.g., avian leukosis virus), beta retroviruses (e.g., mouse mammary tumor virus), delta retroviruses (e.g., bovine leukemia virus and human T-lymphotropic virus), epsilon retroviruses (e.g., walleye dermatosarcoma virus), and lentiviruses. In some embodiments, the retrovirus is a lentivirus, a genus of viruses in the family Retroviridae, characterized, for example, by a long latency period. The lentivirus is the only retrovirus that can infect non-dividing cells; it can deliver a large amount of genetic information to the DNA of the host cell and can therefore be used as an efficient gene delivery vector. In some examples, the lentivirus can be, but is not limited to, human immunodeficiency virus (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIA), and visna virus. Vectors derived from lentiviruses provide a method for achieving significant levels of gene transfer in vivo.

[0136] In some embodiments, the vector is an adenovirus vector. Adenovirus is a large class of viruses containing double-stranded DNA. It replicates the host cell's DNA while using the host's cellular machinery to synthesize viral RNA DNA and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, to accommodate large transgenes, and to encode proteins without integrating into the host cell genome.

[0137] In some embodiments, AAVP vectors are used. AAVP vectors are hybrids of prokaryotic-eukaryotic vectors, which are chimeras of genetic cis elements of recombinant adeno-associated viruses (AAV) and bacteriophages. AAVP combines selected elements of bacteriophage and AAV vector systems to provide a vector that is easy to produce in bacteria and can show little or no packaging restrictions, while allowing infection of mammalian cells and integration into host chromosomes. Vectors containing many appropriate elements are commercially available and can be further modified to include necessary sequences by standard methods. In addition, AAVP does not require helper viruses or trans-acting factors. In addition, since there is no AAV capsid formation, the natural tropism of AAV to mammalian cells is eliminated. Other methods and details are found in U.S. Patents 8,470,528 and Hajitou A. et al., Cell, 125:358-398.

[0138] In some embodiments, human papillomavirus (HPV) pseudovirus is used. DNA plasmids can be packaged into papillomavirus L1 and L2 capsid proteins to generate pseudovirions that can effectively deliver DNA. Encapsulation protects DNA from nucleases and provides targeted delivery with a high level of stability. HPV pseudoviruses can alleviate many safety issues associated with the use of viral vectors. Other methods and examples are found in Hung, C. et al., Plos One, 7:7 (e40983); 2012, U.S. Pat. No. 8,394,411 and Kines, R. et al. Int J of Cancer, 2015.

[0139] In some embodiments, an oncolytic virus is used. Oncolytic virus therapy can selectively replicate the virus in cancer cells, and can then spread within the tumor, for example, without affecting normal tissue. Alternatively, oncolytic viruses can preferentially infect and kill cells without causing damage to normal tissue. Oncolytic viruses can also effectively induce immune responses to themselves and to infected tumor cells. Generally, oncolytic viruses are divided into two categories: (I) viruses that preferentially replicate naturally in cancer cells and are non-pathogenic to humans. Exemplary (I) class oncolytic viruses include autonomous small viruses, myxoma viruses (poxviruses), Newcastle disease (Newcastledisease) viruses (NDV; paramyxoviruses), reoviruses, and Seneca Valley (Seneca Valley) viruses (picoRNA viruses). The second category (II) includes viruses that have been genetically manipulated for use as vaccine vectors, including measles viruses (paramyxoviruses), polioviruses (picoRNA viruses), and vaccinia viruses (poxviruses). In addition, oncolytic viruses may include viruses that have been genetically engineered to have mutations / deletions in genes required for replication in normal cells rather than cancer cells, including adenovirus, herpes simplex virus, and vesicular stomatitis virus. Oncolytic viruses can be used as a viral transduction method due to their low probability of genetic resistance, as they can target multiple pathways and replicate in a tumor-selective manner. Due to in situ viral amplification, the viral dose in the tumor increases over time (compared to small molecule therapies that decrease over time), and safety features (i.e., drug and immune sensitivity) can be built in.

[0140] Preparation and administration

[0141] Certain embodiments of the present disclosure include methods of administering to a subject a cell therapeutic agent (or a population thereof) described herein, a protein therapeutic agent described herein, a composition comprising a cell therapeutic agent, and / or a composition comprising a protein therapeutic agent, for example, in an amount effective to treat the subject. In some embodiments, the method is effective to treat cancer in the subject.

[0142] In some embodiments, the cell therapeutic comprises autologous cells that are administered to the same subject from which the immune cells were obtained. Alternatively, the immune cells are obtained from a subject and transformed, e.g., transduced, with an expression construct as described herein to obtain a cell therapeutic that is allogeneically transferred into another subject.

[0143] In some embodiments, the cellular therapeutic is autologous to the subject, and the subject can be naive, immune, diseased, or in another state prior to isolating the immune cells from the subject.

[0144] In some embodiments, additional steps can be performed before applying to a subject. For example, a cell therapy agent can be amplified in vitro after an immune cell contacts an expression construct as described herein (e.g., transduction or transfection) but before applying to a subject. Before applying to a subject, in vitro amplification can be performed for 1 day or longer, such as 2 days or longer, 3 days or longer, 4 days or longer, 6 days or longer, or 8 days or longer. Alternatively or additionally, before applying to a subject, in vitro amplification can be performed for 21 days or shorter, such as 18 days or shorter, 16 days or shorter, 14 days or shorter, 10 days or shorter, 7 days or shorter, or 5 days or shorter. For example, before applying to a subject, in vitro amplification can be performed for 1-7 days, 2-10 days, 3-5 days, or 8-14 days.

[0145] In some embodiments, during in vitro expansion, the cell therapeutic agent can be stimulated with an antigen (e.g., a TCR antigen). Antigen-specific expansion can optionally be supplemented with 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 cell therapeutic agent can be directly administered to the subject or can be frozen for future use, i.e., for subsequent administration to the subject.

[0146] In some embodiments, the cell therapy is treated with interleukin-2 (IL-2) ex vivo before infusion into the cancer patient, and the cancer patient is treated with IL-2 after infusion. In addition, in some embodiments, the cancer patient may undergo preparative lymphocyte clearance (temporary elimination of the immune system) before administering the cell therapy. The combination of IL-2 treatment and preparative lymphocyte clearance can enhance the persistence of the cell therapy.

[0147] In some embodiments, the cell therapeutic agent is transduced or transfected with a nucleic acid encoding a cytokine, which can be engineered to provide constitutive, regulatable or time-controlled expression of the cytokine. Suitable cytokines include, for example, cytokines that enhance the survival of T lymphocytes during contraction, which can promote the formation and survival of memory T lymphocytes.

[0148] In certain embodiments, the cell therapy agent is administered before, substantially simultaneously with, or after the administration of another therapeutic agent such as a cancer therapeutic agent. The cancer therapeutic agent can be, for example, a chemotherapeutic agent, a biological agent, or radiotherapy. In some embodiments, the subject receiving the cell therapy agent is not administered a treatment sufficient to cause immune cell depletion, such as lymphocyte removal chemotherapy or radiotherapy.

[0149] Cell therapy agents described herein can be formed into compositions, such as cell therapy agents and pharmaceutically acceptable carriers. In certain embodiments, the composition is a pharmaceutical composition comprising at least one cell therapy agent described herein and a pharmaceutically acceptable carrier, diluent and / or excipient. Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients and diluents, are well known and readily available to those skilled in the art. Preferably, the pharmaceutically acceptable carrier is chemically inert to active agents such as cell therapy agents, and does not cause any harmful side effects or toxicity under the conditions of use.

[0150] The composition may be formulated for administration by any suitable route, such as, for example, intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural and / or subcutaneous administration routes. Preferably, the composition is formulated for parenteral administration routes.

[0151] Compositions suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injection solutions, which may contain, for example, antioxidants, buffers, bacteriostats and solutes that make the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilizers, thickeners, stabilizers and preservatives.

[0152] The dosage applied to a subject, particularly a person, will vary with the specific embodiment, the composition used, the method of administration, and the specific part and subject treated. However, the dosage should be sufficient to provide a therapeutic response. Clinicians in the art can determine the therapeutically effective amount of the composition to be applied to a person or other subject to treat or prevent a specific medical condition. The precise amount of the composition required for effective treatment will depend on many factors, such as the specific activity of the cell therapy agent, and the route of administration, the amount of one or more available antigens on tumor cells (e.g., as a result of tumor volume or tumor load degree) and / or normal cells, and many considerations specific to the subject well known to those skilled in the art. In some embodiments, the appropriate dosage of the cell therapy agent for one or more specific cancer indications can be defined in a dose escalation clinical trial.

[0153] Any suitable number of cell therapy cells may be administered to a subject. Although a single cell therapy cell as described herein is capable of expansion and providing therapeutic benefit, in some embodiments, administration of 10 2or more, for example 10 3 or more, 10 4 or more, 10 5 or more, or 10 8 or more cell therapy cells. Alternatively or additionally, administering 10 12 or less, e.g. 10 11 or less, 10 9 or less, 10 7 or less, or 10 5 or fewer cell therapy cells described herein. In some embodiments, administration of 10 2 -10 5 , 10 4 -10 7 , 10 3 -10 9 , or 10 5 -10 10 The cell therapy cells described herein.

[0154] The dosage of the cell therapy agent described herein can be administered to a mammal once or in a series of sub-doses over an appropriate period of time, such as daily, half-weekly, weekly, biweekly, half-monthly, bimonthly, half-yearly or annually, as required. The dosage unit containing an effective amount of the cell therapy agent can be administered in a single daily dose, or the total daily dose can be administered in two, three, four or more doses per day as required.

[0155] The polypeptides described herein can be incorporated into pharmaceutical compositions (e.g., used as protein therapeutic agents). Pharmaceutical compositions comprising polypeptides can be prepared 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 injectable preparations comprising sterile solutions or suspensions in water or another pharmaceutically acceptable liquid. For example, pharmaceutical compositions can be prepared in the following manner: the polypeptides are appropriately combined with pharmaceutically acceptable vehicles or media, such as sterile water and saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, adhesives, and then mixed in the unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation is such that a suitable dose is provided within a specified range.

[0156] Sterile compositions for injection can be prepared according to conventional pharmaceutical practice using distilled water for injection as a vehicle. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used as aqueous solutions for injection, optionally in combination with suitable solubilizers, for example alcohols such as ethanol and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80TM, HCO-50, etc.

[0157] Non-limiting examples of oily liquids include sesame oil and soybean oil, and they can be combined with benzyl benzoate or benzyl alcohol as solubilizers. Other items that can be included are buffers such as phosphate buffers or sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The prepared injection can be packaged in a suitable ampoule.

[0158] Protein half-life is affected by the degree of sialylation, which is the post-translational covalent addition of terminal sialic acid to glycosylated proteins (Hossler et al., Glycobiology. 2009; 19: 936-49). Proteins with a higher degree of sialylation may have a longer half-life 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.

[0159] The administration route may be parenteral administration, such as administration by injection, nasal administration, pulmonary administration or transdermal administration. It may be systemically or locally administered by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.

[0160] Suitable administration methods 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, the dosage can be selected from the range of 0.001 to 100000 mg / kg body weight, but the disclosure is not limited to such ranges. The dosage and method of administration can vary according to the subject's weight, age, condition, etc., and can be appropriately selected by those skilled in the art as needed.

[0161] In some embodiments, a pharmaceutical composition containing a fusion protein is administered in combination with a cell therapy agent such as a CAR-T cell or in combination with an ADC targeting CD19, as described herein. In some embodiments, the pharmaceutical composition comprises a fusion protein as described herein and a cell therapy agent as described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered simultaneously, concomitantly, or sequentially with a cell therapy agent as described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days before administering a cell therapy agent as described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 week, 2 weeks, 3 weeks, or 4 weeks before administering a cell therapy agent as described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 month, 2 months, 3 months, 4 months, or 5 months before administering a cell therapy agent as described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administering a cell therapy agent as described herein. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1 week, 2 weeks, 3 weeks, or 4 weeks after the cell therapy agent described herein is administered. In some embodiments, the pharmaceutical composition containing the fusion protein is administered about 1 month, 2 months, 3 months, 4 months, or 5 months after the cell therapy agent described herein is administered.

[0162] All publications, patent applications, patents and other references mentioned herein, including GenBank accession numbers, are incorporated herein by reference in their entirety. In addition, materials, methods and examples are only illustrative and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein are identical with the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used for practice or test of the present invention, suitable methods and materials are described herein.

[0163] The present disclosure is further illustrated by the following examples. These examples are for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way.

[0164] CAR-T therapy relapse

[0165] Typically, CAR-T therapy includes administering T cells expressing a chimeric antigen receptor (CAR) that binds to a target antigen. In some embodiments, the CAR-T target antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA) as described herein.

[0166] In some embodiments, the present disclosure is based in part on the recognition that certain individuals who are being treated with CAR-T therapy for cancer and then relapse (e.g., stop showing one or more beneficial responses to CAR-T therapy, as described herein) can be "rescued" from relapse by administering the fusion proteins described herein. In some embodiments, the present disclosure provides compositions and methods comprising fusion proteins for treating subjects who exhibit cancer relapse during or after CAR-T therapy.

[0167] In some embodiments, the disclosure is based in part on the recognition that some individuals who are being treated for cancer with CAR-T therapy will have a suboptimal response to therapy and may therefore relapse, and therefore receive treatment to prevent recurrence. As a non-limiting example, suboptimal response can be improved by increasing the density of target antigens on tumor cells. In some embodiments, increasing the density of target antigens on tumor cells can be achieved by binding the fusion protein of the present invention to tumor cells. In some embodiments, the disclosure provides compositions and methods comprising fusion proteins for treating subjects who are expected to have or have a suboptimal response to CAR T cell therapy, for example, patients who have achieved stable disease, partial response, excellent partial response, or complete response without reaching a minimal residual disease negative state. (See, for example, www.cibmtr.org / manuals / fim / 1 / en / topic / multiple-myeloma-response-crit eria).

[0168] Subject identification

[0169] In some embodiments, the subject is identified and / or selected to administer a fusion protein as described herein. In some embodiments, the subject may be identified and / or selected for treatment based on the diagnosis of refractory or drug-resistant cancer. In some embodiments, the subject may be identified and / or selected for treatment based on a prescription for receiving ACT therapy. In some embodiments, the subject may be identified and / or selected for treatment based on evidence of ACT therapy recurrence. In some embodiments, the subject may be identified and / or selected for treatment based on one or more measured or observed signs of cancer recurrence (e.g., non-beneficial reactions, loss or downregulation of the target antigen of the cells used in ACT, or progressive disease). In some embodiments, the subject is administered fusion protein. In some embodiments, after administering fusion protein therapy, the subject exhibits a positive clinical response to ACT therapy, such as an improvement based on one or more clinical and / or objective criteria (e.g., a reduction in tumor load, tumor size, and / or tumor stage).

[0170] The methods described herein may include preparing and / or providing a report, such as in an electronic, web-based or paper form. The report may include one or more outputs from the methods described herein, such as tumor load, tumor size and / or tumor stage, disease stability, loss or downregulation of target antigens. In some embodiments, a report is generated, such as in paper or electronic form, identifying the presence or absence of one or more tumor antigens of a cancer patient, and optionally, a recommended course of cancer therapy. In some embodiments, the report includes an identifier of the cancer patient. In one embodiment, the report is in a web-based form.

[0171] In some embodiments, additionally or alternatively, the report includes information about prognosis, drug resistance, or potential or suggested treatment options. The report may include information about the possible effectiveness of the treatment option, the acceptability of the treatment option, or the desirability of applying the treatment option to the cancer patient, such as information identified in the report. For example, the report may include information or suggestions about administering a cancer therapy, such as administering a preselected dose to the patient or administering it with a preselected treatment regimen, such as in combination with one or more alternative cancer therapies. The report may be delivered within 7, 14, 21, 30, or 45 days after performing the methods described herein, for example, to an entity described herein. In some embodiments, the report is a personalized cancer treatment report.

[0172] In some embodiments, each time a cancer subject is tested using the methods described herein, a report is generated for record. The cancer subject may be re-evaluated at regular intervals, such as monthly, bimonthly, six-monthly, or annually, or more frequently or less frequently, to monitor the subject's responsiveness to cancer therapy and / or improvement in one or more cancer symptoms, such as those described herein. In some embodiments, the report may record at least the treatment history of the cancer subject.

[0173] In one embodiment, the method further comprises providing the report to another party. The other party can 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.

[0174] example

[0175] Example 1. Construction and expression of CTE1 and CTE2 fusion proteins

[0176] Genetic fusion protein constructs comprising a secretory signal peptide, anti-CD20 VHH, a portion of the CD19 ECD, and an anti-albumin llama VHH were cloned into a mammalian cell expression vector and transiently transfected into Expi293 cells or CHO cells. The cell culture was clarified by centrifugation, and the fusion protein was purified from the supernatant. Two fusion proteins were produced. CTE1 (having the amino acid sequence of SEQ ID NO:2) was expressed in Expi293 cells. CTE2 (having the amino acid sequence of SEQ ID NO:2 and lacking a C-terminal hexa-histidine tag (e.g., SEQ ID NO.55) was expressed in CHO cells.

[0177] The CTE1 fusion protein was purified by His-NTA column chromatography and analyzed by reducing and non-reducing PAGE. Superdex 75 purification was used to separate the main peak and remove any endotoxin, and SEC-HPLC was used to separate the monomeric protein from the small aggregates present. The CTE2 fusion protein was purified by protein A affinity chromatography, cation exchange chromatography and SEC-HPLC purification. As determined by SEC-HPLC and PAGE, the CTE2 fusion protein was purified to homogeneity.

[0178] Example 2. Biophysical characteristics of CTE1 and CTE2 fusion proteins

[0179] Capillary isoelectric focusing (cIEF) analysis was used to evaluate the sialylation of CTE1 and CTE2 fusion proteins. In cIEF separations, a continuous pH gradient formed by applying a voltage across a capillary filled with carrier ampholytes separates proteins by their isoelectric points (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 relative to total charge, which in turn is affected by the presence or absence of sialic acid relative to the fusion protein isoforms. In short, proteins can be evaluated as the percentage of basic and acidic peaks, where the acidic peaks represent sialylated proteins.

[0180] Three protein preparations were evaluated for 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 found to have sialylated and unsialylated forms. CTE3 was prepared by separation using an anion exchange column (AEX) and collecting the eluate.

[0181] Sialylation was assessed using imaging capillary isoelectric focusing (icIEF), a high-resolution technique that separates species primarily based on the intrinsic net charge of the molecule’s pI. icIEF takes into account both surface exposed and internal amino acids without losing resolution due to hydrophobic interactions. icIEF separation is performed by focusing the isoforms in a zwitterionic pH gradient via an applied electric field. The focusing step is followed by a detection step, which in conventional capillary isoelectric focusing (cIEF) requires chemical or pressure mobilization via a fixed UV detector. icIEF lacks the mobilization step, and detection is achieved by continuously scanning the entire capillary. Due to the elimination of the mobilization step, charge variation profiles can be recorded with the highest possible resolution within an effective run time of 15–20 min. Protein preparations CTE1, CTE2, and CTE3 showed different icIEF profiles, such as 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.

[0182] Since the appearance of basic and acidic peaks may be influenced by other features besides sialylation, sialidase treatment was performed to demonstrate that the presence of the acidic peak was due to sialic acid modification within the protein preparation. Several CTE1 preparations in different formulation buffers (FB) were evaluated + / - sialidase. Figure 2 Coded as CTE-Test and CTE-Test2.

[0183] This experiment showed that sialidase removed all of the more acidic peaks (pH 5.1–pH 7.9) and slightly reduced the size of one basic peak (pH 8.55).

[0184] Example 3. Fusion protein binding to CD20 and albumin

[0185] The binding of CTE1 and CTE2 fusion proteins (as described in Example 1) and CTE3 (AEX preparation) to CD19-positive and CD19-negative cells was evaluated. Briefly, the JeKo-1 mantle cell lymphoma cell line was edited with CRISPR / Cas9 to eliminate CD19 expression, thereby generating the JeKo-19KO cell line. JeKo-19KO cells express CD20 but not CD19. The cells were incubated with CTE proteins, washed to remove unbound proteins, and then incubated with anti-CD19 antibody FMC63 labeled with phycoerythrin (FMC63-PE). The cells were then washed again and fixed with PBS containing 2% formalin for analysis by flow cytometry.

[0186] like Figure 3As shown in , CTE1 (red circles), CTE2 (hollow green squares) and CTE3 (hollow blue circles) proteins bind equivalently to CD19-negative / CD20-positive JeKo-19KO cells, as detected with anti-CD19-PE antibody (FMC63), i.e., regardless of the degree of sialylation. The half-maximal effective concentrations calculated from these binding data are reported in Table 1.

[0187] Table 1:

[0188] Test molecules <![CDATA[EC 50 (ng / ml)]]> <![CDATA[EC 50 (nM)]]> CTE1 7.5 0.13 CTE2 3.2 0.06 CTE3 11.5 0.2

[0189] Fusion protein is assessed for binding to CD20 and albumin. In brief, two ELISA formats are implemented using anti-CD19 antibody FMC63 to capture fusion protein. ELISA plates (Thermo Fisher) are coated with 1 μg / ml FMC63 overnight at 4 ° C, and blocked with 200 μl / well Tris buffered saline (TBS) containing 0.3% skim milk for 1 hour at room temperature. The fusion protein is added to TBS containing 1% BSA, the concentration is gradually reduced, and incubated for 1 hour. The plate is washed 3 times with TBS. Then 0.5 μg / ml biotinylated human albumin (Novus Biologics) or 0.5 μg / ml biotinylated human CD20-"nanodisc" (AcroBiosystems) is added to TBS containing 1% BSA and maintained for 1 hour, the plate is washed again, and then incubated with streptavidin-HRP and TMB peroxidase substrate (Thermo Fisher) to detect binding protein.

[0190] like Figure 4 As shown in , CTE1 (green triangles), CTE2 (red circles) and CTE3 (blue squares) proteins bind equivalently to anti-CD19 coated plates as detected with biotinylated albumin (left) or biotinylated CD20 membrane preparations (right), i.e. regardless of the degree of sialylation. The half maximal effective concentrations calculated from these binding data are reported in Table 2.

[0191] Table 2:

[0192] Test molecules <![CDATA[EC 50 ng / ml(biotinylated albumin)]]> <![CDATA[EC 50 ng / ml (biotinylated CD20)]]> CTE2 13.5 22.3 CTE1 24.6 22.0 CTE3 15.1 18.6

[0193] Example 4. Fusion protein mediates CAR-19 T cell targeting and cytotoxicity

[0194] The CAR-19 T cell targeting and cytotoxicity of CTE1 and CTE2 fusion proteins (as described in Example 1) were evaluated. Briefly, 96-well round-bottom plates were incubated with 1×10 450 μL of JeKo-1 CD19KO target cells carrying the luciferase gene were inoculated at a density of 50 cells / well. The test protein was diluted in 50 μL RPMI / FBS and added to the cells. The CAR-19T cells were thawed and washed once with RPMI / FBS, collected by centrifugation at 550RCF for 10 minutes and added to the wells in a volume of 50 μL to obtain a defined CAR-19T: target (E: T) cell ratio. The plate was incubated at 37 ° C for 48 hours. The plate was centrifuged at 550RCF for 5 minutes, the pellet was rinsed with PBS, and centrifuged again. Then, 20 μL of 1× lysis buffer (Promega) was added to the pellet, and the lysate was transferred to a 96-well opaque tissue culture plate (Fisher Scientific). The plate was read in a photometer with a syringe to distribute the substrate (Promega). The killing percentage was calculated based on the average luminescence loss of the experimental conditions relative to the control conditions of only target cells plus CAR-19T cells.

[0195] like Figure 5 As shown in, CAR-19T cells have equivalent cytotoxic activity in the presence of target JeKo-19KO cells and CTE1 (red circles), CTE2 (blue squares) and CTE3 (open triangles) proteins, regardless of the degree of sialylation. The half-maximal inhibitory concentration calculated from these cytotoxicity data is reported in Table 3.

[0196] Table 3:

[0197] CTE1 CTE2 CTE3 Cytotoxicity IC50 (ng / ml) 0.012 0.018 0.020

[0198] These results show that CTE fusion protein can redirect CAR-19T cells to CD19 bound to CD20 cell surface protein and displayed on the protein. Fusion proteins that bind to CD20 and display the CD19 extracellular domain can increase the apparent density of CD19 on wild-type lymphoma cells that naturally express two antigens. Using the "Bang Beads" method (Bangs Laboratories, Fishers, IN USA), when CTE3 is added to wild-type JeKo-1 mantle cell lymphoma cells or wild-type Ramos Hodgkin's lymphoma cells, the number of apparent CD19 molecules on the surface of lymphoma cells is shown to increase in a dose-dependent manner. It was found that the total number measured was approximately equal to the number of CD19 receptors plus the number of CD20 receptors measured by flow cytometry staining. The experiment was performed using the manufacturer's instructions.

[0199] method

[0200] Bead staining (Bangs Lab QSC beads catalog number: 815B5ML; anti-mouse IgG, lot number 15515, access code: 22020217-1): Manually shake the bottle to ensure a homogenous suspension. Do not vortex. Blank population: Do not stain. Stain each marker population (1-4) separately. Add one drop of QSC beads to 50 μL FACS buffer and tap gently to mix. Add 30 μL FMC63-PE (3×10 6 cells) for CD19 standard curve beads, add 60 μL a-CD20-PE (a total of 3×10 6 10 cells) and gently tap to mix immediately. Incubate at 4°C in the dark for 30 minutes. Add 1 ml FACS buffer and centrifuge at 2500 g for 5 minutes. Wash twice with 1 ml FACS buffer and centrifuge at 2500 g for 5 minutes; resuspend in 500 μL PBS.

[0201] Cell staining: In a 96-well FACS plate, 50 μL of Fc-blocked cells (approximately 5×10 5 50 μL of 3x serial dilutions of 606 protein (CTE3, SEQ ID NO. 55, sialylated) were incubated at 4°C for 30 minutes. The cells were washed twice with 200 μL of FACS buffer. The cells were resuspended in 50 μL of FACS buffer, 30 μL of FMC63-PE were added, and incubated at 4°C for 30 minutes. For controls: a. Unstained cells, b. FMC63-PE stained cells (30 μL / test for 3×10 6 cells), c. Anti-CD20-PE stained cells (60 μL anti-CD20-PE, for 3×10 6 Cells were collected and gently tapped to mix immediately. Cells were incubated at 4°C in the dark for 30 minutes. Cells were washed 3 times with 200 μL FACS buffer and centrifuged at 500 g for 2 minutes. Cells were resuspended in 200 μL PBS containing 1% paraformaldehyde to fix cells for 30 minutes. FACS analysis: Beads and cells were run on the same day with the same settings.

[0202] Beads were analyzed on an Accuri C6 plus machine: the flow rate was set to 100-200 events / second (medium flow control), and 1000 events were collected for each bead population. The dyed beads were combined and run in a single tube (5×1000 events). Using the FSC / SSC dot plot, FSC-H / FSC-A was created to obtain a single peak and SSC-A / FL2 (PE channel). A histogram / FL2 (PE channel) was created and a half-height / full-width gate was applied to each bead population (M1, M2, M3, M4, M5). The geometric mean or median of the FL2 (PE channel) of each bead population was recorded for entry into the QuickCal spreadsheet (download QuickCal v 3.0 using the access code). The median of the FL2 (PE channel) was entered to obtain a standard curve.

[0203] Run cells with the same settings as the bead analysis on the Accuri C6 plus, and collect 5000 events for each test. Using the FSC / SSC dot plot, create FSC-H / FSC-A to obtain a single peak and SSC-A / FL2 (PE channel). Create a histogram / FL2 (PE channel) and apply the half-height / full-width gate to the cell population. Record the geometric mean or median of the FL2 (PE channel) of the cell population for input into the QuickCal spreadsheet. Enter the median of the FL2 (PE channel) and obtain the ABC (antibody binding capacity) value. Subtract the ABC value of the unstained cell population or isotype control from the ABC value of the cell. If it is assumed that the monovalent antibody is bound to the surface receptor, the ABC value = # surface receptor. The results show that in the range between 10ng / ml and 10μg / ml CTE3, the apparent number of CD19 molecules on the JeKo-1 cell surface is significantly increased in a dose-dependent manner. The results showed that in the range between 10ng / ml and 10μg / ml CTE3, the apparent number of CD19 molecules on the surface of Ramos cells increased significantly in a dose-dependent manner. The results showed that the effect of increasing the number of CD19 molecules on target lymphoma cells can enhance CAR-19-mediated toxicity for up to 24 hours at different E:T ratios. The results showed enhanced cytotoxicity over the E:T ratio range including 0.1:1, 0.3:1, 1:1, 3:1 and 5:1.

[0204] It is concluded that CTE3 increases the apparent CD19 density on lymphoma cells to a sufficient extent to increase CAR-19-mediated cytotoxicity.

[0205] Example 5. Treatment of CD19 antigen escape using fusion protein

[0206] The ability of CTE1 fusion protein (as described in Example 1) to regulate CD19 antigen escape of target cells was evaluated. To generate a CD19 antigen escape model, wild-type CD19-positive / CD20-positive JeKo-1 cells carrying the luciferase gene (JeKo-luc) were cultured at 1×10 4 100 μl of RPMI / FBS per well of a 96-well round bottom plate was plated. 100 μl of RPMI / FBS containing CAR-19 T cells was added to give a JeKo-luc:CAR-19 T cell ratio of 1:1, 0.3:1, or 0.1:1, and the co-cultures were incubated for up to 13 days. Samples were evaluated for CD19 by flow cytometry on days 1, 6, 11, and 13. - CD20 + Accumulation of populations (FMC63-PE, Millipore; anti-CD20-APC, BD Pharmingen).

[0207] Once the CD19-negative population appeared, JeKo-luc cells were plated at 1×10 4 The cells were re-plated in 50 μl RPMI / FBS. Then, 1×10 5 50μl RPMI / FBS of CAR-19T cells was added to make the ratio of JeKo-luc:CAR-19T be 10: 1, with or without fusion protein in 50μl RPMI / FBS. In a set of control wells, the fusion protein was added to the JeKo-luc cells alone without adding CAR-19T cells. In each case, 150μl of co-culture was incubated for 48 hours. The cells were then stained with HIB-CD19-PE (BioLegend) and anti-CD20-APC (BD Pharmingen) and analyzed by flow cytometry for JeKo-1 cells that were not incubated with protein or incubated with anti-CD20-APC and anti-ROR1-PE (BioLegend) antibodies for samples treated with protein. 7AAD was used to gate out dead cells.

[0208] Wild-type JeKo-1 cells have a very high proliferation index and are used as a model for antigen escape from CAR-T cell therapy. The phenotype of these cells under CAR-T pressure was first characterized. JeKo-1 cells are CD19-positive and CD20-positive, and the vast majority are double positive, such as Figure 6 as shown in .

[0209] Figure 6Wild-type JeKo-1 cells are shown to express CD19 (X-axis) and CD20 (Y-axis), so that nearly all cells (90%) are located in the upper right quadrant, indicating double positivity.

[0210] Using titration of the effector:target ratio (E:T, here CAR-T:JeKo-1), where the number of CAR-T cells was varied relative to a fixed number of JeKo-1 cells, and further by varying the culture duration, a clear antigen escape model was created.

[0211] Figure 7 shows the development of the JeKo-1 antigen escape model after in vitro treatment with CAR-19T cells. The locations of the CAR-19 cells and the target JeKo-1 cells are indicated in the flow cytometry profiles, shown on the left side of the figure. The names of the CD19-positive and CD20-positive cell populations are shown on the right side of the figure. A) From day 1 to day 13, the E:T ratio was 1:1. B) From day 1 to day 13, the E:T ratio was 0.3:1. C) From day 1 to day 13, the E:T ratio was 0.1:1.

[0212] As shown in Figure 7, JeKo-1 cells can escape the cytotoxicity of CAR-19 by losing CD19 expression at the cell population level. Fig. 7A , as shown on days 6 and 13, this occurs even when the E:T ratio is 1:1. Figure 7B (Day 6 and Day 13) and Figure 7C As shown in Figure 2, the effect was more pronounced at 0.3:1 and 0.1:1 E:T ratios, with the effect being particularly pronounced on day 13. Notably, the 0.1:1 E:T ratio was ineffective at the cytotoxicity readout level in the luciferase assay, but the selection pressure imposed by the CAR induced a predominantly double-positive (CD19 and CD20 positive) population ( Figure 7C , day 1) to mixed groups ( Figure 7C , day 6) to a population dominated by CD19-negative and CD20-positive cells ( Figure 7C , day 13) transformation.

[0213] Since escape occurred within this range of E:T ratios and incubation days, a second study was performed to evaluate the effect of adding CD19-anti-CD20 fusion protein.

[0214] Fig. 8A Shown are CD19 (x-axis) and CD20 (y-axis) expression levels after 13 days of incubation of CAR-19 cells with JeKo-1 cells (E:T 1:1), as measured by flow cytometry. The expression of CD19 and CD20 was monitored by FACS. Figure 8BResults are shown when only CTE1 protein, only CAR-19 T cells, or both CTE1 and CAR-19 T cells were added to the culture.

[0215] In this experiment, the addition of CAR-19T cells and CTE1, but not CAR-19T cells alone or CTE1 protein alone, was sufficient to kill all target JeKo-1 cells after escape.

[0216] These results suggest that antigen escape, particularly that expressed by CD19, can be induced in vitro and can be reversed using CTE1 targeting CD20.

[0217] Example 6. In vivo treatment of cancer in mice using fusion proteins

[0218] CTE1 and CTE2 fusion proteins (as described in Example 1) were evaluated for tumor therapy in a mouse model. Briefly, female NOD-scid IL2Rγ 空 (NSG) mice (Jackson Labs) were acclimated in a vivarium for at least 3 days before the start of the study. All animals were socially group housed in static, sterile, biocontainment disposable cages prefilled with corncob bedding. Food (LabDiet) and acidified water were provided ad libitum. On study day 1, mice were injected intravenously with 0.1 mL / animal of 2.5×10 6 JeKo-19KO cells. On study day 3, all animals were imaged and then randomized to give equivalent mean tumor burden in each of 6 different cohorts of n=8 mice / cohort. The next day, study day 4, mice in different cohorts were given fusion protein at doses ranging from 0 to 5 mg / kg, depending on the study, followed by 1×10 7 The fusion protein was then administered three times a week for a total of 14 injections. The control cohort consisted of mice treated with CAR-19 T cells alone, without protein injections, and untreated mice that received no treatment.

[0219] Animals were subjected to cage-side health checks and clinical observations at least once daily. Clinical observations were performed more frequently if animals in the study exhibited abnormal clinical signs. Body weights were recorded prior to tumor induction, CAR-19 T cell administration, and three times weekly thereafter unless otherwise indicated by the animal's health status. The most recently collected body weight was used to calculate protein and D-luciferin doses. Imaging was performed on day 3 to randomize animals into cohorts with similar mean baseline luminescence. Following infusion of CAR-19 cells and CTE protein, whole-body imaging was performed twice weekly in all animals 10-15 minutes after subcutaneous administration of 0.2 mL PBS containing 15 mg / mL luciferin. Isoflurane was administered during the described procedures. Above 1×10 10 The value of total flux (measured in lumens) reaches a maximum and is considered cause for humane euthanasia.

[0220] Several studies were performed using the JeKo-19KO cell line, CTE protein, and CAR-19T cells. Animals were injected with JeKo-19KO lymphoma cells and allowed to engraft for 4 days before starting treatment with CAR-19T cells and CTE1 protein. CAR-19T cells were injected once, and CTE1 protein was given 3 times per week. Tumor burden was quantified using luciferase-mediated luminescence.

[0221] In the first study, the in vivo efficacy of CTE1 protein was tested. 6 The JeKo-CD19-KO tumor cell dose was intravenously injected into NSG mice. CAR-19 T cells (1×10 7 ) and CTE1. CTE1 was then administered 3 times a week, but was stopped on day 32 to monitor lymphoma recurrence. Fig. 9 As shown in Figure 2, mice treated with anti-CD19 CAR T cells and CTE1 were cured and no relapse was observed by day 45. Both CAR-19 and CAR-CD20 cohorts without CTE1 were included; as expected, CAR-19 treated animals developed lymphomas and 4 of 5 animals died by day 45, as did the untransduced T cell control cohort (UTD). CD20 CAR worked well except for one animal that developed lethal systemic lymphoma.

[0222] These results show that mice treated with CAR-19 T cells plus CTE1 protein were protected from lethal lymphoma. In addition, even at the lowest dose used, 0.56 mg / kg, durable responses were achieved after drug withdrawal.

[0223] In the second study, CTE1 and CTE2 protein preparations were screened for activity at a 2 mg / kg protein dose compared to a CAR-19 T cell-only cohort and an untreated cohort. Fig.10 As shown in , three times weekly administration of CTE1 and CTE2 proteins inhibited lymphoma development until day 31, when both untreated (NA) mice and CAR-19-treated mice began to succumb to disease.

[0224] These results suggest that at a dose of 2 mg / kg, CTE1 and CTE2 proteins have similar effects on lymphoma growth in vivo.

[0225] In the third study, two parameters were further investigated. First, a dose titration of CTE2 was performed in four cohorts: 2 mg / kg, 0.4 mg / kg, 0.08 mg / kg, and 0.016 mg / kg. Second, the animals were dosed with protein until day 31, at which time dosing was stopped to evaluate the animals for signs of lymphoma recurrence. Nine days later, on day 42, there were no signs of luminescence in animals treated with CAR-19 T cells plus 2 mg / kg CTE2, and only minimal signals were observed in two of the eight animals treated with CAR-19 T cells plus 0.4 mg / kg CTE2, as Fig.11 Lower doses of CTE2 were less effective in preventing relapse after discontinuation of treatment.

[0226] These results showed 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 and 0.08 mg. / kg cohorts.

[0227] Body weight is a quantitative means of tracking animal health and is often used in conjunction with clinical examinations to ensure humane euthanasia in cancer models. Mice treated with CAR-19 plus CTE2 protein continued to gain weight throughout the protein administration period (until day 31), while control animals began to lose weight around day 25, as shown in Figure 2. Fig.12 Even after cessation of CTE2 protein administration, animals in the treatment cohort maintained body weight by the end of the study (Day 42), and animals in the highest dose treatment cohort (receiving 2 mg / kg of CTE2 until Day 31) continued to gain weight, with the last recorded body weight on Day 39.

[0228] These results show that even after CTE2 dosing was stopped at day 31, animals in the three highest dose cohorts remained healthy enough to gain weight, whereas the negative control groups (CAR-19 only and 'no treatment') steadily lost weight after day 21.

[0229] Fig.13Luminous intensity for each cohort is shown, showing a rapid increase in the untreated group and a slightly delayed but still rapid increase in the CAR-19 treated group. Line terminations for the untreated and CAR-19 treated groups correspond to the loss of all animals in these cohorts. Since the signal intensity in the control cohort is very high, it is impossible to discern differences between the CTE2 dosing cohorts unless those groups are removed. In the absence of those groups, a clear dose response is evident, as there is a complete lack of luminescent signal in the 2mg / kgCTE2 cohort as of day 42. Since the last protein dose was given on day 31, this indicates that these animals have eliminated CD19-negative lymphoma.

[0230] These results showed complete protection from lymphoma as measured by luminescence in all animals treated with 2 mg / kg CTE2 (8 / 8 animals showed no signal), most animals treated with 0.4 mg / kg CTE2 (6 / 8 animals showed no signal), and half of the animals treated with 0.08 mg / kg CTE2 (4 / 8 animals showed no signal). The lack of luminescence signal and continued increase in body weight indicated that some mice were disease-free, an indication supported by the Kaplan-Meier survival curves of the different cohorts, as shown in Figure 2. Fig.14 as shown in .

[0231] Survival data exemplified that only 1 of 32 treated animals (3%) died of lymphoma during the study; the animal was in the lowest dose cohort of 0.016 mg. / kg. In contrast, 14 / 16 (87.5%) control animals died of lymphoma during the study.

[0232] In vivo protein efficacy varies with the pharmacokinetic (PK) properties of the injected fusion protein. The PK properties of CTE1, CTE2, and CTE3 were evaluated in mouse serum after a single injection. Fig.15 As shown in . The half-life of the fusion protein in mice depends on albumin-mediated recycling via FcRN binding, as there is no antigen target - the anti-CD20 domain does not bind to mouse CD20, and the human CD19 ECD has no measurable binding properties in solution, but the anti-albumin domain can bind to mouse albumin. CTE1 protein was evaluated in the serum of Balb / c and NSG mice after a single IV injection and showed similar half-lives of 21 hours and 28 hours, respectively. The half-life of CTE2 (hyposialylated) in Balb / c mice was shorter than expected at 7.6 hours, and CTE3 (hypersialylated) had the longest half-life of 36 hours.

[0233] Protein half-life is also affected by the degree of sialylation, which is the post-translational covalent addition of terminal sialic acid to glycosylated proteins. Proteins with a higher degree of sialylation have a longer half-life in vivo. Table 4 summarizes the half-life of the fusion proteins in Balb / c mice and NSG mice and the icIEF profiles of the fusion proteins. The icIEF data showed that the CTE1 protein contained a mixture of acidic and basic peaks, the CTE2 protein was almost completely basic, indicating the lowest degree of sialylation, and the CTE3 was almost completely acidic, indicating a high degree of sialylation. These results explain the different PK properties of the three protein preparations.

[0234] Table 4

[0235]

[0236] Example 7. Production and testing of additional fusion proteins

[0237] The ability of other fusion proteins to bind and kill CD19-CD20+Jeko-1 cells was evaluated. First, the ability of anti-CD20 VHHs (SEQ ID NO:37-42) with different CDR2 sequences within SEQ ID 6 to bind CD20 on CD19-CD20+Jeko-1 cells was evaluated using FACS. The following table depicts the changes made to the CDR2 sequence of SEQ ID NO:37. As shown in Table 5, VHHs including the CDR2 sequences shown exhibited different levels of binding, from complete loss of activity to almost no loss of activity, as well as changes in mean fluorescence intensity (MFI).

[0238] Table 5:

[0239]

[0240]

[0241] Substitution of Thr-Tyr (TY) at the N-terminus of CDR2 of SEQ ID NO: 37 with Ser-Trp (SW) significantly affected binding. Surprisingly, highly non-conservative substitution of the Ser-Pro (SP) sequence at the C-terminus of CDR2 of SEQ ID NO: 37 with Trp-Ile (WI) did not affect binding. In addition, mutations (AA) within CDR3 of the anti-CD20 VHH of SEQ ID NO: 37 N PTYGSDWNAEN to AA D PTYGSDWNAEN) had only a modest effect on binding to CD20.

[0242] A series of fusion protein constructs were generated, which included a secretion signal peptide, anti-CD20 VHH, a GGGGSGGGGSGGGGSGGGGS linker, a portion of the CD19 ECD, SRGGGGSGGGGSGGGGS linker, anti-albumin VHH and a hexahistidine tag. The amino acid sequences of the constructs are provided in Table 6:

[0243] Table 6:

[0244] Construct number SEQ ID NO: 526(CTE1) 6 631 10 632 14 633 18 634 22 635 26 636 30 637 34

[0245] The fusion protein was purified and then assayed for binding and killing of CD20 positive Jeko-1 CD19KO cells. The binding is summarized in Table 7 below:

[0246] Table 7.

[0247]

[0248] The direct cell binding results of the constructs to CD20 on JeKo-19KO cells, measured by binding of the anti-CD19 mAb FMC63, fully confirmed the preliminary FACS results. Constructs #631 and #634, with four and three AA changes, respectively (relative to SEQ ID NO:37), did not bind to the cells at all. Construct #633, with two AA changes relative to SEQ ID NO:37, was almost equivalent to construct #526 (CTE1). Construct #635, with conservative changes at the C-terminus (Pro to Thr) (relative to SEQ ID NO:37), clearly lost fivefold binding; and construct #636 (CDR3 Asn mutated to Asp relative to SEQ ID NO:37) retained full activity. Importantly, combining the mutations in CDR3 with the two AA substitutions in construct #633 produced a construct (#637) that was almost equivalent to construct #526. The binding curves are illustrated in Fig.16 middle.

[0249] The killing of JeKo-19KO cells by the fusion protein in the presence of CAR-19 was evaluated. The data are summarized below Fig.17 and Table 8.

[0250] Table 8:

[0251]

[0252] Fig.17 and Table 8 show that the cytotoxicity data are consistent with the binding data.

[0253] Example 8. Evaluation of Additional Fusion Proteins

[0254] Additional fusion protein construct #607 was generated, which contained a different anti-CD20 VHH. The amino acid sequences of the constructs are provided in Table 9:

[0255] Table 9:

[0256] Construct number SEQ ID NO: 526(CTE1) 6 606(CTE2) 2, but lacking the C-terminal hexa-histidine tag (e.g., SEQ ID NO.55) 607 50

[0257] Constructs #526(CTE1) and #607 bound to cell surface CD20 almost equivalently, at about 0.4 nM for construct #526(CTE1); and at about 0.8 nM for construct #607. Fig.18 , left). However, construct #607 was about one-tenth as effective in killing JeKo-19KO cells ( Fig.18 , right).

[0258] Table 10:

[0259]

[0260] Example 9 Biophysical Characterization and Optimization of Biologics

[0261] Determine the biophysical properties of a biopharmaceutical as described herein. Use standard techniques known in the art to assess fusion proteins as described herein to evaluate the characteristics of a biopharmaceutical for therapy. Assess the stability of a fusion protein (e.g., as described in the present embodiment) (e.g., stability in a composition buffer and / or a biological fluid (e.g., blood or saliva)).

[0262] The fusion protein described in this embodiment is formulated into a preparation that exhibits an optimal melting temperature (Tm), enhanced stability, reduced aggregation and / or degradation level, increased protease resistance level, increased antioxidant level, enhanced biodistribution and / or improved PK / PD properties in vivo. Such features are evaluated using standard methods and assays known in the art, such as differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism (CD), temperature scanning viscometer, analytical ultracentrifugation (AUC), size exclusion chromatography (SEC, SEC-MALS), dynamic light scattering (DLS), light resistance, zeta potential, capillary electrophoresis CE (e.g., CZE, MECC, gel CE, 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., phosphates, acetates, 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 / chelators (e.g., ethylenediaminetetraacetic acid, EDTA), and / or preservatives (e.g., benzyl alcohol).

[0263] Example 10. Fusion protein mediation of CAR-19 T cell targeting and cytotoxicity.

[0264] This example further demonstrates that the fusion protein as described herein mediates CAR-19T cell targeting in a concentration-dependent manner. In addition, this example further demonstrates that the fusion protein as described herein mediates CAR-19T cell cytotoxicity. These characteristics are particularly described in Su et al. Oncoimmunology 2022, Vol. 11, No. 1, e2111904 (13 pages), the entire contents of which are incorporated by reference.

[0265] The CAR-19 T cell targeting and cytotoxicity of CTE3 fusion proteins (as described in Example 1) were evaluated. Briefly, 96-well round-bottom plates were incubated with 1×10 450 μL of JeKo-1 CD19KO target cells carrying the luciferase gene were inoculated at a density of 50 cells / well. The test protein was diluted in 50 μL RPMI / FBS and added to the cells. The CAR-19T cells were thawed and washed once with RPMI / FBS, collected by centrifugation at 550RCF for 10 minutes and added to the wells with a volume of 50 μL to obtain a defined CAR-19T: target (E: T) cell ratio. The plate was incubated at 37 ° C for 48 hours. The plate was centrifuged at 550RCF for 5 minutes, the pellet was rinsed with PBS, and centrifuged again. Then, 20 μL of 1× lysis buffer (Promega) was added to the pellet, and the lysate was transferred to a 96-well opaque tissue culture plate (Fisher Scientific). The plate was read in a photometer with a syringe to distribute the substrate (Promega). The killing percentage was calculated based on the average luminescence loss of the experimental conditions relative to the control conditions of only target cells plus CAR-19T cells. 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's lymphoma cells was determined using the "Bang Beads" method (Bangs Laboratories, Fishers, IN USA) according to the manufacturer's instructions.

[0266] CTE fusion protein can redirect CAR-19 T cells to CD19 bound to and displayed on the CD20 cell surface protein. Fusion proteins that bind to CD20 and display the extracellular domain of CD19 can increase the apparent density of CD19 on wild-type lymphoma cells that naturally express both antigens. FIG. 19A to FIG. 19C As shown in , when CTE3 fusion protein was added to JeKo-1 CD19KO target cells, wild-type JeKo-1 mantle cell lymphoma cells, or wild-type Ramos Hodgkin's lymphoma cells, the number of apparent CD19 molecules on the lymphoma cell surface increased in a concentration-dependent manner. The total number of CD19 receptors or CD20 receptors on the test cells measured after treatment with 10 μg / mL CTE3 was found to be approximately equal to the number of CD19 receptors plus the number of CD20 receptors.

[0267] CAR-19T cells have cytotoxic activity in the presence of target JeKo-19KO cells and CTE protein. Fig. 20As shown in the (left figure), after 18 hours, the CAR-19T cells had an average of at least 60% cytotoxic activity after incubation with JeKo-1 cells treated with 0.01, 0.1, 1 or 10 μg / mL CTE protein (E: T3: 1). CAR-19T cells had an average of at least 50% cytotoxic activity after incubation with JeKo-1 cells treated with 0.001 μg / mL CTE protein (E: T 3: 1). After 18 hours, the CAR-19T cells had an average of at least 40% cytotoxic activity after incubation with JeKo-1 cells treated with 0.01, 0.1, 1 or 10 μg / mL CTE protein (E: T 1: 1). CAR-19T cells had an average of at least 20% cytotoxic activity after incubation with JeKo-1 cells treated with 0.001 μg / mL CTE protein (E: T 1: 1). After 18 hours, CAR-19T cells had an average of about 30% cytotoxic activity after incubation with JeKo-1 cells treated with 0.01, 0.1, 1 or 10 μg / mL CTE protein (E:T 0.3:1). CAR-19T cells had an average of at least 10% cytotoxic activity after incubation with JeKo-1 cells treated with 0.001 μg / mL CTE protein (E:T 0.3:1). After 48 hours, CAR-19T cells showed increased cytotoxic activity after incubation with JeKo-1 cells.

[0268] These results further demonstrate that the fusion protein increases the apparent CD19 density on lymphoma cells to a degree sufficient to increase CAR-19-mediated cytotoxicity.

[0269] Example 11: CTE3 binds to human and cynomolgus monkey CD20 expressed on 293T cells

[0270] This example further demonstrates that the fusion proteins described herein bind to human and cynomolgus monkey CD20.

[0271] Human 293T cells were grown to a cell density of about 80%. Cells were transfected with cDNA to express human CD20 (GenScript OHu00965D) or "cynomolgus monkey" CD20 (A157V mutation of human CD20 cDNA) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). The A157V mutation is the only amino acid difference with human CD20 in the extracellular domain. Cells were removed by Accutase approximately 48 hours after transfection and harvested by centrifugation at 500 x g for 2 minutes at 4°C. The cell pellets were resuspended in FACS buffer (PBS containing 1% BSA and 0.1% sodium azide). Then, 10 cells were added to each well. 65 μl of Fc blocking agent (BD) per cell was added to the cell suspension and incubated at room temperature for 10 minutes. The cells were then centrifuged as above, and the cells were resuspended in FACS buffer and 50 μl (approximately 5×10 5 Cells). Then, 50 μl of CTE3 fusion protein was added to the cells, starting from 10 μg / ml (final concentration) and then serially diluted 3 times in FACS buffer. The plate was incubated at 4°C for 30 minutes and then centrifuged as described 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 FACS buffer) and incubated at 4°C for 30 minutes. The cells were washed twice as above and then fixed with 150 μl 2% PFA. Samples were analyzed on a BD Accuri C6 flow cytometer using FlowJo software.

[0272] like Fig.21A and Fig.21B As shown in , the EC50 of CTE3 binding to human and non-human primate (cynomolgus macaque) CD20 is about 6 ng / ml. These results indicate that CTE3 has cross-species specificity between humans and non-human primates (cynomolgus macaques).

[0273] Example 12: CTE3 is active as a CAR T cell engager in the presence of human serum or human serum albumin

[0274] This example further demonstrates that CTE3 is still highly cytotoxic to cells expressing CD20 at pM concentrations.

[0275] CTE3 contains an albumin binding domain. The ability to kill CD20-expressing cells in the presence of human serum or human serum albumin (HSA) was evaluated. The cytotoxicity assay used JeKo-1 CD19KO-luc cells expressing luciferase to determine the activity of CTE3 in the presence of 50% human serum or 30 or 60 mg / ml HSA. JeKo-1 CD19KO-luc cells were cultured at 1×10 4Cells were plated in RPMI medium (96 round bottom plates) containing 50% human serum or 10% FBS (control conditions) or 10% FBS RPMI medium without human serum albumin and containing 30 or 60 mg / l human serum albumin, 50 μl / well. Starting from 100 ng / ml, CTE3 was titrated in 4-fold serial dilutions in the same serum / HSA dilution as above. CTE3 dilutions (25 μl) were distributed to the corresponding wells. CAR19 T cells in RPMI medium containing 50% human serum or 10% FBS or in 10% FBS RPMI medium without human serum albumin and containing 30 or 60 mg / ml human serum albumin at a ratio of 2: 1 or 5: 1 (CAR T cells to target cells) were added in a volume of 25 μl / well. The plate was incubated at 37 ° C for 48 hours. After washing and pelleting the cells, lysis buffer (Glomax Multi Detection System) was added, the lysate was transferred to a 96 white opaque plate, and luciferase levels were measured using a GloMax plate reader with an automatic injector (Promega). Data were graphed using GraphPad Prism software.

[0276] Table 11:

[0277] CTE3 IC50 ng / ml IC50 pM In 50% HS 0.46 8.3 In 10% FBS 0.11 2

[0278] like Fig.22A As shown in Figure 2, the IC values ​​of samples containing 10% FBS were 50 Compared with human serum, the IC 50 The IC50 values ​​for the cytotoxicity assay were determined and are shown in Table 11. The potency of CTE3 in 50% human serum (IC 50 about 8.3 pM) was lower than the potency in 10% FBS (IC 50 In control wells containing CAR19 cells but without CTE3, a decrease in cell growth in the presence of 50% human serum was observed compared to 10% FBS.

[0279] Table 12:

[0280] CTE3 <![CDATA[IC 50 (ng / ml)]]> <![CDATA[IC 50 pM]]> In 10% FBS 0.09 1.62 In 30mg / ml HSA 0.35 6.3 In 60 mg / ml HSA 0.18 3.2

[0281] like Fig. 22B As shown in Figure 2, the inclusion of 30 mg / ml or 60 mg / ml HSA reduced the ability of CTE3 to induce cytotoxicity in JeKo-1 CD19 KO-luc cells compared to control samples containing 10% FBS. Determination of the IC of the cytotoxicity assay 50 The values ​​are shown in Table 12. In the presence of HSA, IC50 The levels of cytotoxicity in the samples were reduced 2-4 fold and the maximum cytotoxicity level was slightly reduced compared to the samples assayed in 10% FBS alone.

[0282] These results indicate that, although activity is reduced in the presence of 50% human serum or physiological levels of HSA, CTE3 is still highly cytotoxic to CD20-expressing cells at pM concentrations.

[0283] Example 13: CD20 x CD79b Bispecific Anti-CD19 CAR T Engagement Protein Antigen Binding

[0284] The bispecific CD79b x CD20 CAR19 engagement protein was generated by assembling anti-CD79b scFv with anti-CD20 VHH and CD19 ECD in CTE3. The construct was designed to place the anti-CD79b scFv at the N-terminus (#650) or C-terminus (#651) of the anti-CD20 VHH-CD19 ECD core sequence. The plasmid was transfected into 293T cells using Lipofectamine 2000, and the supernatant was harvested and titrated. The binding of #650 and #651 proteins to biotinylated CD20 or CD79b was determined in an ELISA format.

[0285] 96-well plates were coated with 1.0 μg / ml FMC63 / 0.1M carbonate, pH 9.5 overnight at 4°C. The plates were blocked with 200 μl / well Tris-buffered saline (TBS) containing 0.3% skim milk at room temperature for 1 hour. The plates were then washed three times with wash buffer (1×TBST: 0.1M Tris, 0.5M NaCl, 0.05% Tween 20). Next, 100 μl of #650 and #651 supernatants were serially diluted 3 times in TBS / 1% BSA, pH 7.4 (dilution buffer) starting from 5 μg / ml. The samples were incubated at room temperature for 1 hour, and the plates were then washed as above. Next, 100 μl of dilution buffer containing 0.5 μg / ml biotinylated CD20 nanodiscs (ACROBiosystems) or biotinylated CD79b (homemade) was added to each well and incubated for 1 hour at 37°C. After washing the plate again, 100 μl of 1:2000 diluted HRP-streptavidin (in dilution buffer) was added to each well and incubated at 37°C in the dark for 1 hour. For detection, 100 μl of 1-Step Ultra TMB-ELISA was added to each well. When color 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 Fig.23A and Fig. 23B middle.

[0286] like Fig.23A As shown in , placing the CD79b scFv at the N- or C-terminal position of the anti-CD20 VHH did not significantly affect binding to biotinylated CD20. Fig. 23B As shown in , binding to biotinylated CD79b does appear to be affected if the anti-CD79b scFv domain is located at the C-terminus of the molecule.

[0287] The ability to bind to cell surface CD20 or CD79b was examined by flow cytometry. Controls for binding to CD20 (#606 anti-CD20 VHH-CD19 ECD-anti-albumin) and CD79b (#645 anti-CD79b scFv-CD19 ECD) were included in this assay. Fig.23C and Fig.23D As shown in Table 13, #650 and #651 bind to both antigens. Table 13 shows the EC binding to 293T-CD20 cells. 50 Table 14 shows the EC binding to 293T-CD79b cells. 50 value.

[0288] Table 13:

[0289] 293T-CD20 binding 606 645 650 651 EC50(ng / mL) 6.70 - 34.45 24.46 EC50(nM) 0.121 0.000 0.487 0.345 MW(Da) 55560 56800 70700 70800

[0290] EC binding of CTE#650 and #651 to CD20 expressed on the cell surface 50 Similar, but slightly lower than #606.

[0291] 293T-CD20 binding 606 645 650 651 EC50(ng / mL) - 425.80 200.9 269.4 EC50(nM) 0.000 7.496 2.842 3.805 MW(Da) 55560 56800 70700 70800

[0292] On cells expressing CD79b, both #650 and #651 bound 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 present on the cell surface.

[0293] like Fig.23E As shown in , the cytotoxic activity of CD20 x CD79b CTE #650 and #651 against CD20+ / CD79b+ JeKo-1CD19 KO-luc cells was tested. CTE3 was replaced with #606 protein or #645, #650 or #651 supernatant. Table 14 shows the IC values ​​for cytotoxicity against JeKo-1CD19 KO cells. 50 value.

[0294] Table 14:

[0295] <![CDATA[IC 50 ng / ml]]> <![CDATA[IC 50 pM]]> MW kDa #606 0.078 1.4 55.6 #645 10.45 184.0 56.8 #650 0.069 0.99 70.8 #651 0.037 0.52 70.8

[0296] The monospecific anti-CD20 CTE (#606) and the bispecific anti-CD20 / anti-CD79b CTE (#650 / #651) can be used at an IC of approximately 1 pM. 50 The killing effect of anti-CD79b monospecific CTE was low.

[0297] Example 14: Surface Plasmon Resonance (SPR) Results of CTE3 Binding to Anti-CD19 FMC63 and HSA

[0298] SPR measurements were performed on a Biacore 8K (Cytiva) at 25°C using 1×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 in which the biotinylated substrate binds almost irreversibly to the sensor chip surface. This coupling allows all immobilized molecules to be in the same orientation and high substrate density can be achieved.

[0299] 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 seconds, flow rate 10 μL / min. Anti-CD19 and HSA were diluted into running buffer (1.5 μg / mL and 4.0 μg / mL, respectively) and injected onto FC2 in separate experiments until the appropriate immobilization level (i.e., 1000-1500 response units (RU)) was reached. The remaining streptavidin was inactivated by injecting biotin (0.2 μg / ml, contact time 100 seconds, flow rate 10 μL / min) on all FCs.

[0300] 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 onto the immobilized target protein: association time 180 sec, dissociation time 900 sec, flow rate 30 μL / min. Chip regeneration was then 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 for HSA binding was 30 sec twice, regeneration time for anti-CD19 binding was 30 sec once and flow rate 10 μL / min. In all experiments, free biotin immobilized on FC1 was used as reference for blank subtraction.

[0301] like Fig. 24B and Fig.24C As shown in Figure , the association and dissociation curves indicate that CTE3 has good affinity for anti-CD19 and HSA, respectively. For anti-CD19, the equilibrium dissociation constant (K D ) is 8.13 nM, and the half-life (t 1 / 2 ) was 15.03 min (Table 15). In a similar manner, the K D It was determined to be 65.1nM, t 1 / 2 It is 1.78 minutes (Table 15).

[0302] Table 15

[0303]

[0304] Example 15: Surface Plasmon Resonance (SPR) Results of CTE3 Binding to CD20

[0305] 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 in which the biotinylated substrate binds almost irreversibly to the sensor chip surface. This coupling allows all immobilized molecules to be in the same orientation and high substrate density can be achieved.

[0306] 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 sec, flow rate 10 μL / min. Biotinylated CD20 was diluted into running buffer (0.2 μg / mL) and injected onto FC2 until the appropriate immobilization level (i.e., 1000-1500 response units (RU)) was reached. Remaining streptavidin was inactivated by injecting biotin (0.2 μg / ml, contact time 100 sec, flow rate 10 μL / min) on all FCs.

[0307] 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 onto the immobilized target protein: association 180 seconds, dissociation 2400 seconds, 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 resulting association and dissociation curves were consistent with a 1:1 binding model fit. Table 16 shows the measured kinetic parameters.

[0308] like Fig.24A As shown in Figure , the association and dissociation curves indicate that CTE3 has a good affinity for CD20 (K D is 2.93nM and t 1 / 2 The measured kinetic parameters for anti-CD20 are detailed in Table 16.

[0309] Table 16

[0310]

[0311] Example 16: CTE3 is more cytotoxic than CD20 x CD3 BiTE to cells expressing CD20

[0312] This example further demonstrates that CTE3 mediates improved CAR-19 T cell cytotoxicity. Specifically, this example demonstrates that CTE3 is more cytotoxic than CD20 xCD3 BiTE to cells expressing CD20 after repeated stimulation of anti-CD19 CAR T cells.

[0313] Raji cells (about 10 × 10 6 ) were treated with 0.5 μg / ml silk fungus C for 1 hour at 37°C to generate B cells for stimulation. The cells were washed twice with RPMI+10% FBS (R10) and then incubated at 1×10 6 Resuspend at 10 cells / ml. CAR19 cells were stimulated and restimulated with MMC-treated Raji cells every four days by co-culturing at a ratio of 2 CAR19 cells: 1 Raji cell. The cell density was maintained at approximately 0.5 × 10 6Cells / ml. Every four days, just before restimulation, cells were sampled and stained with anti-CD3-FITC and anti-Flag-APC to determine CAR19 amplification; flow cytometry and Bang Beads were used to determine the number of cells. After determining the CAR19 cell concentration, a CTE3 or bispecific anti-CD20 x anti-CD3 (BPS Biosciences, catalog number: 100836-2) bridging cytotoxicity assay was established for Jeko-1 CD19 KO cells using restimulated CAR19 cells. The results were read 48 hours after the assay.

[0314] like Fig.25A As shown in Figure , the ability of CTE3 or anti-CD20 x anti-CD3 bispecific antibodies to kill Jeko-1 CD19 KO cells using CAR19 cells that had undergone one round of Raji cell stimulation was evaluated. 50 is 2.8 pM, and the IC of the bispecific antibody 50 is 35.7pM. Fig.25B As shown in Figure 2, the potency of CAR19 cells that have undergone 3 rounds of stimulation decreased. 50 (7.7 pM) is still lower than the IC of the bispecific 50 (124.1pM).

[0315] These results suggest that CTE3 is more cytotoxic than a commercial anti-CD20 x anti-CD3 bispecific antibody when CAR19 cells are used in an assay that has been repeatedly stimulated with B cells. Therefore, CTE3 may be more effective in preventing relapse in CAR19-treated patients.

[0316] Example 17: Batch Analysis of CTE3 Drug

[0317] This example further demonstrates that compositions comprising fusion proteins as described herein can be formulated into preparations that exhibit enhanced stability, reduced levels of aggregation and / or degradation, increased levels of protease resistance, increased levels of antioxidant activity, enhanced biodistribution, and / or improved PK / PD properties in vivo.

[0318] A batch of CTE3 was produced and analyzed for various drug product properties. Table 17 shows the details and release testing of the CTE3 batch.

[0319] Table 17:

[0320]

[0321]

[0322]

[0323] The stability of Test Batch 1B was evaluated after 1 month and 3 months. Table 18 shows the real-time stability data for Test Batch 1B.

[0324] Table 18

[0325]

[0326]

[0327] As shown in Table 18, after 1 month and 3 months, the compositions comprising CTE3 exhibited similar properties as when initially tested.

[0328] In another example, the stability of the composition comprising CTE3 was evaluated at time points exceeding 3 months. Table 19 describes the stability storage conditions and testing time points. The composition comprising CTE3 remained stable for at least 3 months.

[0329] Table 19:

[0330]

[0331] Equivalent

[0332] Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but is set forth as follows:

[0333] Sequence Listing

[0334] SEQ ID NO:1

[0335] ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGCAACTGCAACTGGAGTACATAGCCAGGTGCAATTACAAGAGTCTGGTGGAGGCCTAGCACAAGCGGGCGGTTCCCTGAGACTCAGCTGCGCTGCTAGCGGACGTACATTTTCCATGGGCTGGTTTCGTCAAGCTCCTGGCAAGGAACGCGAGTTTGTGGCTGCTATTACCTACTCTGGAGGCTCCCCTTACTACGCGAGTTCTGTTCGCGGCCGGTTTACAATCTCCCGGGATAATGCTAAGAATACAGTTTATCTACAAATGAACTCCCTGAAACCCGAGGATACCGCTGTTTATTACTGTGCCGCTAACCCAACCTACGGCTCTGATTGGAACGCTGAGAACTGGGGCCAGGGCACACAGGTGACTGTTAGTTCCGGTGGCGGTGGCAGCGGTGGCGGAGGATCTGGAGGCGGTGGAAGTGGAGGTGGAGGTTCTCCCGAGGAGCCCCTGGTTGTGAAAGTCGAGGAAGGCGACACCGCCGCCCTGTGGTGCCTCAAGGGTACCTCTGATGGACCCACGCAACAATTGACATGGTCTCGTGAATCCCCTCTGAAGCCTTTCCTCAAGTACAGCCTGGGAGTCCCCGGCCTTGGAGTACATGTCAGGCCAGACGCGATTAGTGTTGTCATCCGCAATGTTAGTCAGCAGATGGGCGGCTTTTACTTGTGTCAACCCGGCCCTCCTTCCGAGAAGGCATGGCAGCCTGGCTGGACCGTGAATGTTGAAGGCAGCGGCGAGCTGTTCCGATGGAACGTCAGTGACCTTGGCGGCCTGGGCTGCGGACTGAAGAACAGATCCAGCGAAGGACCAAGTAGCCCTAGCGGCAAGTTGATGTCTCCAAAATTATATGTTTGGGCAAAGGATAGGCCCGAGATTTGGGAGGGGGAACCCCCATGCCTGCCTCCACGAGACAGCCTCAATCAAAGCCTGAGTCGAGATCTGACCGTTGCTCCTGGTTCAACACTGTGGCTGTCATGCGGCGTACCACCGGATTCTGTTTCTCGCGGGCCCCTGTCTTGGACCCACGTCCATCCTAAGGGTCCCAAGTCCCTCCTGAGCCTGGAGCTCAAGGACGACCGGCCAGCACGCGATATGTGGGTTATGGGCACAAGCCTCATGCTGCCCCGAGCAACCGCGCAAGACGCCGGCAAATGGTACTGCCATAGGGGTAACCTTACCATGAGTTTTCACTTGGAGATCACAGCTAGGCCTTCTCGAGGTGGCGGTGGCTCCGGAGGCGGCGGATCAGGAGGTGGCGGCAGTGAAGTCCAACTTGTGGAGTCTGGCGGAGGCTTGGTCCAACCTGGCAATTCTCTGCGCTTGTCCTGCGCTGCTAGCGGGTTCACTTTTTCATCTTTCGGAATGTCTTGGGTTAGACAGGCTCCAGGCAAGGGACTGGAATGGGTTTCGTCCATTAGTGGCAGCGGGTCTGACACTCTTTACGCGGACAGTGTCAAGGGCAGGTTCACGATATCAAGAGACAACGCAAAAACCACACTCTACCTCCAAATGAACTCTTTGAGACCTGAAGATACAGCAGTGTATTATTGCACAATTGGTGGCTCTCTGTCCCGCTCGTCCCAAGGGACACTCGTAACGGTCAGCTCACATCATCACCATCACCAT

[0336] SEQ ID NO:2

[0337] (Sequence: [Signal sequence]-[VHH (CDR1, CDR2, and CDR3 in bold)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Hexa-histidine tag])

[0338] [MGWSCIILFLVATATGVHS]- [QVQLQESGGGLAQAGGSLRL SCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKV EEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLP RATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSG GGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-

[0339] [HHHHHH]

[0340] SEQ ID NO:3

[0341] CAGGTGCAATTACAAGAGTCTGGTGGAGGCCTAGCACAAGCGGGCGGTTCCCTGAGACTCAGCTGCGCTGCTAGCGGACGTACATTTTCCATGGGCTGGTTTCGTCAAGCTCCTGGCAAGGAACGCGAGTTTGTGGCTGCTATTACCTACTCTGGAGGCTCCCCTTACTACGCGAGTTCTGTTCGCGGCCGGTTTACAATCTCCCGGGATAATGCTAAGAATACAGTTTATCTACAAATGAACTCCCTGAAACCCGAGGATACCGCTGTTTATTACTGTGCCGCTAACCCAACCTACGGCTCTGATTGGAACGCTGAGAACTGGGGCCAGGGCACACAGGTGACTGTTAGTTCCGGTGGCGGTGGCAGCGGTGGCGGAGGATCTGGAGGCGGTGGAAGTGGAGGTGGAGGTTCTCCCGAGGAGCCCCTGGTTGTGAAAGTCGAGGAAGGCGACACCGCCGCCCTGTGGTGCCTCAAGGGTACCTCTGATGGACCCACGCAACAATTGACATGGTCTCGTGAATCCCCTCTGAAGCCTTTCCTCAAGTACAGCCTGGGAGTCCCCGGCCTTGGAGTACATGTCAGGCCAGACGCGATTAGTGTTGTCATCCGCAATGTTAGTCAGCAGATGGGCGGCTTTTACTTGTGTCAACCCGGCCCTCCTTCCGAGAAGGCATGGCAGCCTGGCTGGACCGTGAATGTTGAAGGCAGCGGCGAGCTGTTCCGATGGAACGTCAGTGACCTTGGCGGCCTGGGCTGCGGACTGAAGAACAGATCCAGCGAAGGACCAAGTAGCCCTAGCGGCAAGTTGATGTCTCCAAAATTATATGTTTGGGCAAAGGATAGGCCCGAGATTTGGGAGGGGGAACCCCCATGCCTGCCTCCACGAGACAGCCTCAATCAAAGCCTGAGTCGAGATCTGACCGTTGCTCCTGGTTCAACACTGTGGCTGTCATGCGGCGTACCACCGGATTCTGTTTCTCGCGGGCCCCTGTCTTGGACCCACGTCCATCCTAAGGGTCCCAAGTCCCTCCTGAGCCTGGAGCTCAAGGACGACCGGCCAGCACGCGATATGTGGGTTATGGGCACAAGCCTCATGCTGCCCCGAGCAACCGCGCAAGACGCCGGCAAATGGTACTGCCATAGGGGTAACCTTACCATGAGTTTTCACTTGGAGATCACAGCTAGGCCTTCTCGAGGTGGCGGTGGCTCCGGAGGCGGCGGATCAGGAGGTGGCGGCAGTGAAGTCCAACTTGTGGAGTCTGGCGGAGGCTTGGTCCAACCTGGCAATTCTCTGCGCTTGTCCTGCGCTGCTAGCGGGTTCACTTTTTCATCTTTCGGAATGTCTTGGGTTAGACAGGCTCCAGGCAAGGGACTGGAATGGGTTTCGTCCATTAGTGGCAGCGGGTCTGACACTCTTTACGCGGACAGTGTCAAGGGCAGGTTCACGATATCAAGAGACAACGCAAAAACCACACTCTACCTCCAAATGAACTCTTTGAGACCTGAAGATACAGCAGTGTATTATTGCACAATTGGTGGCTCTCTGTCCCGCTCGTCCCAAGGGACACTCGTAACGGTCAGCTCA

[0342] SEQ ID NO:4

[0343] (Sequence: [VHH (CDR1, CDR2, and CDR3 in bold)] - [First linker] - [Target polypeptide] - [Second linker] - [Half-life extended polypeptide])

[0344] [QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGK EREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQ QLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]

[0345] SEQ ID NO:5

[0346]

[0347] SEQ ID NO:6

[0348] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0349] [MEFGLSWVFLVALFRGVQC]- [ QVQLQESGGGLAQAGGSLR LSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVK VEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWA KDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSG GGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH]

[0350] SEQ ID NO:7

[0351]

[0352] SEQ ID NO:8

[0353] (Sequence: [VHH (CDR1, CDR2, and CDR3 in bold)] - [First linker] - [Target polypeptide] - [Second linker] - [Half-life extended polypeptide])

[0354] [ QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGK EREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQ QLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS]

[0355] SEQ ID NO:9

[0356]

[0357] SEQ ID NO:10

[0358] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0359] [MEFGLSWVFLVALFRGVQC]- [ QVQLQESGGGLAQAGGSLR LSCAASGRTFSMGWFRQAPGKEREFVAAISWSGGWIYYASSVR GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVV KVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYV WAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGG GSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYL QMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-

[0360] [HHHHHH]

[0361] SEQ ID NO:11

[0362]

[0363] SEQ ID NO:12

[0364] (Order: [VHH (CDR1, CDR2, and CDR3 in bold)] - [first linker] - [target polypeptide] - [second linker] - [half-life extended polypeptide])

[0365] [ QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGK EREFVAAISWSGGWIYYASSVRGRFTISRDNAKNTVYLQMNSLK PEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQ QLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS]

[0366] SEQ ID NO:13

[0367]

[0368] SEQ ID NO:14

[0369] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0370] [MEFGLSWVFLVALFRGVQC]- [ QCQVQLQESGGGLAQAGGS LRLSCAASGRTFSMGWFRQAPGKEREFVAAISWSGGSPYYASSV RGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLV VKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYV WAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGG GSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYL QMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-

[0371] [HHHHHH]

[0372] SEQ ID NO:15

[0373]

[0374] SEQ ID NO:16

[0375] (In the order: [VHH (CDR1, CDR2, and CDR3 are in bold)] - [first linker] - [target polypeptide] - [second linker] - [half-life extended polypeptide])

[0376] [ QCQVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAISWSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-

[0377] [PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP][SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]

[0378] SEQ ID NO:17

[0379]

[0380] SEQ ID NO:18

[0381] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0382] [MEFGLSWVFLVALFRGVQC]- [ QVQLQESGGGLAQAGGSLR LSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGWIYYASSVRG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVK VEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWA KDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSG GGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMN SLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH]

[0383] SEQ ID NO:19

[0384]

[0385] SEQ ID NO:20

[0386] (Order: [VHH (CDR1, CDR2, and CDR3 in bold)] - [first linker] - [target polypeptide] - [second linker] - [half-life extended polypeptide])

[0387] [ QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGK EREFVAAITYSGGWIYYASSVRGRFTISRDNAKNTVYLQMNSLK PEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQ QLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS]

[0388] SEQ ID NO:21

[0389]

[0390] SEQ ID NO:22

[0391] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0392] [MEFGLSWVFLVALFRGVQC]-[QVQLQESGGGLAQAGGSLR LSCAASGRTFSMGWFRQAPGKEREFVAAISWSGGSTYYASSVRG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVK VEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWA KDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSG GGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMN SLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH]

[0393] SEQ ID NO:23

[0394]

[0395] SEQ ID NO:24

[0396] (Order: [VHH (CDR1, CDR2, and CDR3 in bold)] - [first linker] - [target polypeptide] - [second linker] - [half-life extended polypeptide])

[0397] [QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGK EREFVAAISWSGGSTYYASSVRGRFTISRDNAKNTVYLQMNSLK PEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQ QLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS]

[0398] SEQ ID NO:25

[0399]

[0400] SEQ ID NO:26

[0401] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0402] [MEFGLSWVFLVALFRGVQC]-[QCQVQLQESGGGLAQAGGS LRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSTYYASSV RGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLV VKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYV WAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGG GSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYL QMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH]

[0403] SEQ ID NO:27

[0404]

[0405] SEQ ID NO:28

[0406] (In the order: [VHH (CDR1, CDR2, and CDR3 in bold)] - [first linker] - [target polypeptide] - [second linker] - [half-life extended polypeptide])

[0407] [QCQVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSTYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]

[0408] SEQ ID NO:29

[0409]

[0410] SEQ ID NO:30

[0411] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0412] [MEFGLSWVFLVALFRGVQC]-[QVQLQESGGGLAQAGGSLR LSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVK VEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWA KDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSG GGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMN SLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH]

[0413] SEQ ID NO:31

[0414]

[0415] SEQ ID NO:32

[0416] (Order: [VHH (CDR1, CDR2, and CDR3 in bold)] - [First linker] - [Target polypeptide] - [Second linker] - [Half-life extended polypeptide])

[0417] [QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGK EREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCAADPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQ QLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS]

[0418] SEQ ID NO:33

[0419]

[0420] SEQ ID NO:34

[0421] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0422] [MEFGLSWVFLVALFRGVQC]-[QVQLQESGGGLAQAGGSLR LSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGWIYYASSVRG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVK VEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWA KDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSG GGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMN SLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH]

[0423] SEQ ID NO:35

[0424]

[0425] SEQ ID NO:36

[0426] (Sequence: [VHH (CDR1, CDR2, and CDR3 in bold)] - [First linker] - [Target polypeptide] - [Second linker] - [Half-life extended polypeptide])

[0427] [QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGK EREFVAAITYSGGWIYYASSVRGRFTISRDNAKNTVYLQMNSLK PEDTAVYYCAADPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQ QLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPG NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS SQGTLVTVSS]

[0428] SEQ ID NO:37 (CDR1, CDR2, and CDR3 in bold)

[0429] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS

[0430] SEQ ID NO:38 (CDR1, CDR2, and CDR3 in bold)

[0431] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAISWSGGWIYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS

[0432] SEQ ID NO:39 (bold indicates CDR1, CDR2, CDR3)

[0433] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAISWSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKP EDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS

[0434] SEQ ID NO:40 (bold indicates CDR1, CDR2, CDR3)

[0435] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGWIYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS

[0436] SEQ ID NO:41 (bold indicates CDR1, CDR2, CDR3)

[0437] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAISWSGGSTYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS

[0438] SEQ ID NO:42 (bold indicates CDR1, CDR2, CDR3)

[0439] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSTYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS

[0440] SEQ ID NO:43 (bold indicates CDR1, CDR2, CDR3)

[0441] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPTYGSDWNAENWGQGTQVTVSS

[0442] SEQ ID NO:44 (bold indicates CDR1, CDR2, CDR3)

[0443] QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGWIYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPTYGSDWNAENWGQGTQVTVSS

[0444] SEQ ID NO:45 anti - albumin VHH

[0445] EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAP GKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS

[0446] SEQ ID NO:46

[0447] PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP

[0448] SEQ ID NO:47

[0449] MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR

[0450] SEQ ID NO:48

[0451] PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKA WQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP

[0452] SEQ ID NO:49

[0453]

[0454] SEQ ID NO:50

[0455] (The order is: [Signal sequence]-[VHH (bold indicates CDR1, CDR2, CDR3)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Histidine tag])

[0456] [MEFGLSWVFLVALFRGVQC]-[EVQLVESGGGLVQPGGSLRL SCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSL KDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLV VKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYV WAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGG GSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH]

[0457] SEQ ID NO:51

[0458]

[0459] SEQ ID NO:52

[0460] (Order: [VHH (CDR1, CDR2, and CDR3 are in bold)] - [First linker] - [Target polypeptide] - [Second linker] - [Half-life extended polypeptide])

[0461] [EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]

[0462] SEQ ID NO:53 (CDR1, CDR2, and CDR3 are in bold)

[0463] EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS

[0464] SEQ ID NO:54

[0465] ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGCAACTGCAACTGGAGTACATAGCCAGGTGCAATTACAAGAGTCTGGTGGAGGCCTAGCACAAGCGGGCGGTTCCCTGAGACTCAGCTGCGCTGCTAGCGGACGTACATTTTCCATGGGCTGGTTTCGTCAAGCTCCTGGCAAGGAACGCGAGTTTGTGGCTGCTATTACCTACTCTGGAGGCTCCCCTTACTACGCGAGTTCTGTTCGCGGCCGGTTTACAATCTCCCGGGATAATGCTAAGAATACAGTTTATCTACAAATGAACTCCCTGAAACCCGAGGATACCGCTGTTTATTACTGTGCCGCTAACCCAACCTACGGCTCTGATTGGAACGCTGAGAACTGGGGCCAGGGCACACAGGTGACTGTTAGTTCCGGTGGCGGTGGCAGCGGTGGCGGAGGATCTGGAGGCGGTGGAAGTGGAGGTGGAGGTTCTCCCGAGGAGCCCCTGGTTGTGAAAGTCGAGGAAGGCGACACCGCCGCCCTGTGGTGCCTCAAGGGTACCTCTGATGGACCCACGCAACAATTGACATGGTCTCGTGAATCCCCTCTGAAGCCTTTCCTCAAGTACAGCCTGGGAGTCCCCGGCCTTGGAGTACATGTCAGGCCAGACGCGATTAGTGTTGTCATCCGCAATGTTAGTCAGCAGATGGGCGGCTTTTACTTGTGTCAACCCGGCCCTCCTTCCGAGAAGGCATGGCAGCCTGGCTGGACCGTGAATGTTGAAGGCAGCGGCGAGCTGTTCCGATGGAACGTCAGTGACCTTGGCGGCCTGGGCTGCGGACTGAAGAACAGATCCAGCGAAGGACCAAGTAGCCCTAGCGGCAAGTTGATGTCTCCAAAATTATATGTTTGGGCAAAGGATAGGCCCGAGATTTGGGAGGGGGAACCCCCATGCCTGCCTCCACGAGACAGCCTCAATCAAAGCCTGAGTCGAGATCTGACCGTTGCTCCTGGTTCAACACTGTGGCTGTCATGCGGCGTACCACCGGATTCTGTTTCTCGCGGGCCCCTGTCTTGGACCCACGTCCATCCTAAGGGTCCCAAGTCCCTCCTGAGCCTGGAGCTCAAGGACGACCGGCCAGCACGCGATATGTGGGTTATGGGCACAAGCCTCATGCTGCCCCGAGCAACCGCGCAAGACGCCGGCAAATGGTACTGCCATAGGGGTAACCTTACCATGAGTTTTCACTTGGAGATCACAGCTAGGCCTTCTCGAGGTGGCGGTGGCTCCGGAGGCGGCGGATCAGGAGGTGGCGGCAGTGAAGTCCAACTTGTGGAGTCTGGCGGAGGCTTGGTCCAACCTGGCAATTCTCTGCGCTTGTCCTGCGCTGCTAGCGGGTTCACTTTTTCATCTTTCGGAATGTCTTGGGTTAGACAGGCTCCAGGCAAGGGACTGGAATGGGTTTCGTCCATTAGTGGCAGCGGGTCTGACACTCTTTACGCGGACAGTGTCAAGGGCAGGTTCACGATATCAAGAGACAACGCAAAAACCACACTCTACCTCCAAATGAACTCTTTGAGACCTGAAGATACAGCAGTGTATTATTGCACAATTGGTGGCTCTCTGTCCCGCTCGTCCCAAGGGACACTCGTAACGGTCAGCTCA

[0466] SEQ ID NO:55

[0467] (Sequence: [Signal sequence]-[VHH (CDR1, CDR2, and CDR3 are in bold)]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide])

[0468] [MGWSCIILFLVATATGVHS]- [QVQLQESGGGLAQAGGSLRL SCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKV EEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWA KDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSG GGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQ APGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]

[0469] SEQ ID NO:56

[0470] PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKSE

Claims

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

2. The fusion protein of claim 1, wherein the half-life extending polypeptide is any one of the following: a hyaluronic acid binding motif, a PAS polypeptide, a proline / alanine random coil polypeptide, and an antibody or an antigen-binding fragment thereof.

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

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

5. The fusion protein of claim 4, wherein the anti-albumin antibody or antigen-binding fragment thereof comprises an anti-albumin VHH.

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

45.

7. The fusion protein of any one of claims 1 to 6, wherein the tumor antigen is CD20, and wherein the antibody or antigen-binding fragment thereof is an anti-CD20 antibody or antigen-binding fragment thereof.

8. The fusion protein of claim 7, wherein the anti-CD20 antibody or antigen-binding fragment thereof comprises an anti-CD20 VHH.

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

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

11. The fusion protein of any one of claims 1-10, wherein the target polypeptide is a tumor antigen.

12. The fusion protein of any one of claims 1-11, wherein the target polypeptide is a B cell antigen.

13. The fusion protein of any one of claims 1 to 12, wherein 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.

14. The fusion protein of any one of claims 1 to 13, wherein the target polypeptide is CD19 or a fragment or mutant thereof.

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

47.

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

47.

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

47.

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

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

46.

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

46.

21. The fusion protein of any one of claims 1-20, comprising a first linker between the antibody or antigen-binding fragment thereof and the target polypeptide.

22. The fusion protein of any one of claims 1-21, comprising a second linker between the polypeptide of interest and the half-life extending polypeptide.

23. The fusion protein of claim 21 or 22, wherein the first linker or the second linker comprises an amino acid sequence of any one of (i) GS connector, (ii) a polyglycine linker, (iii) a glycine- and serine-rich linker, or (iv) Flexible joints.

24. The fusion protein of claim 21, wherein the first linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS.

25. The fusion protein of claim 22, wherein the second linker comprises the amino acid sequence SRGGGGSGGGGSGGGGS.

26. A fusion protein as described in any one of claims 1-25, wherein the fusion protein comprises an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequence of any one of SEQ ID NO:2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 50, 52, and 55.

27. The fusion protein of any one of claims 1-26, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 50, 52, and 55.

28. The fusion protein of any one of claims 1-27, wherein the fusion protein consists of the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 50, 52, and 55.

29. The fusion protein of any one of claims 1-26, wherein the fusion protein comprises an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2, 6, 14, 18, 26, 30, 34, and 50, and lacks a C-terminal hexahistidine tag.

30. The fusion protein of any one of claims 1-26, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 2, 6, 14, 18, 26, 30, 34, and 50, and lacks a C-terminal hexahistidine tag.

31. The fusion protein of any one of claims 1-26, wherein the fusion protein consists of the amino acid sequence of any one of SEQ ID NOs: 2, 6, 14, 18, 26, 30, 34 and 50, and lacks a C-terminal hexahistidine tag.

32. The fusion protein of any one of claims 1-31, comprising a second antibody or antigen-binding fragment thereof that binds to a second tumor antigen.

33. A fusion protein as described in 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 of any one of claims 1-33.

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

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

37. A host cell comprising the nucleic acid of claim 34 or 35 or the vector of claim 28.

38. A method for producing a fusion protein, comprising culturing the host cell of claim 34 under conditions suitable for expressing the fusion protein.

39. A method for treating a subject having a tumor, comprising administering to the subject an effective amount of the fusion protein of any one of claims 1-33, thereby treating the subject.

40. The method of claim 39, wherein the tumor expresses the tumor antigen.

41. The method of claim 39 or 40, wherein the tumor does not express CD19.

42. The method of any one of claims 39-41, wherein upon administration, the fusion protein binds to the tumor antigen.

43. The method of any one of claims 39-42, further comprising administering to the subject an antibody, an antibody drug conjugate, or a cell therapy agent (e.g., a CAR-T cell) that specifically recognizes the target polypeptide.

44. The method of claim 43, wherein the antibody, antibody drug conjugate, or cell therapy agent (e.g., CAR-T cell) binds to the fusion protein after administration to the subject.

45. The method of claim 44, wherein binding of the antibody, the antibody drug conjugate, or the cell therapy agent (e.g., CAR-T cells) to the fusion protein induces killing of the tumor.

46. ​​An antibody or antigen-binding fragment thereof, comprising a VHH or a fragment thereof having an amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44 and 53.

47. An antibody or antigen-binding fragment thereof, wherein the VHH comprises at least one CDR (eg, CDR1, CDR2, and / or CDR3) depicted in any one of SEQ ID NOs 37, 39, 40, 42-44, and 53.

48. A nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 46 or 47. A vector comprising the nucleic acid sequence of claim 48 .

50. A host cell comprising the nucleic acid sequence of claim 48 or the vector of claim 49.

51. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell of claim 50 under conditions suitable for expressing the antibody or the antigen-binding fragment thereof or the fusion protein.

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