Methods for treating myeloproliferative neoplasms

Abstract

Methods for inhibiting or treating myeloproliferative neoplasms by using a bispecific molecule that binds to mutant calreticulin and CD3 are described.

Description

[0001] Cross-reference to related applications This application claims priority to each of U.S. Provisional Patent Application No. 63 / 601,323, filed November 21, 2023, and U.S. Provisional Patent Application No. 63 / 648,951, filed May 17, 2024, the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] This application relates to a bispecific molecule that binds to mutant calreticulin and CD3 for use in methods of treating a subject with myeloproliferative neoplasm or reducing the severity of a subject's myeloproliferative neoplasm.

[0003] Sequence listing reference submitted electronically This application contains a sequence list submitted electronically. The information contained in the electronic sequence list (Sequence Listing JBI6861PCT1 Sequence Listing.xml; size: 78.3KB; creation date: November 18, 2024) is incorporated herein by reference in its entirety. Background Technology

[0004] Myeloproliferative neoplasms (MPNs) are clonal diseases of blood cell production arising in the compartments of hematopoietic stem cells (HSCs), characterized by an overproduction of mature myeloid blood cells. Transformation into secondary acute myeloid leukemia (sAML) is a major cause of death in MPN patients. Current treatment options for MPN patients are limited to symptomatic care. Currently, the only treatment with the potential to cure the disease or prolong survival in MPN is stem cell transplantation (SCT). Transplant-related deaths or severe morbidity occur in more than half of transplant recipients, thus requiring individual patient risk assessment. Therefore, finding novel, disease-modifying treatments for MPN that can prevent its progression to sAML represents an unmet medical need.

[0005] Mutations in JAK2, thrombopoietin receptor (TPOR, also known as myeloproliferative leukemia protein or MPL), and calreticulin (CALR) are phenotypic drivers in the pathogenesis of MPN. CALR mutation (CALRmut) is the second most common mutation in MPN. CALRmut causes an insertion or deletion of the last exon in the gene, resulting in the loss of the KDEL ER-retaining motif and the production of a 36-amino acid positively charged C-terminal neoantigen. Due to the loss of the KDEL motif, CALRmut is not limited to the ER. Specifically, a tetrameric complex formed between two CALRmut and two MPL proteins shuttles across the Golgi apparatus and is presented on the cell membrane, leading to constitutive activation of the downstream kinase JAK2. Cell surface presentation of the CALRmut / MPL complex is essential for constitutive activation and oncogenic transformation of JAK2. In wild-type cells, MPL is present only on the cell surface with mature glycosylation, whereas MPL complexed with CALRmut exists on the cell surface in an immature glycosylated state.

[0006] Because CALRmut is expressed on the surface of MPN, it can be considered a target for immunotherapy. There is a need for treatment improvement in MPN disease targeting CALRmut. This article discloses a bispecific antibody that binds to CALRmut and CD3, and its antigen-binding fragment, for use in treating subjects with myeloproliferative neoplasms or reducing the severity of myeloproliferative neoplasms in subjects. Summary of the Invention

[0007] This article provides a method for inhibiting the growth or proliferation of myeloproliferative neoplasms (MPN) or for treating MPN, comprising administering to a subject in need, such as a human subject in need, a therapeutic dose of 0.6 mg to 400 mg per administration of an anti-mutant calreticulin (CALRmut) / anti-CD3 bispecific antibody, wherein the anti-CALRmut / anti-CD3 bispecific antibody comprises a first antigen-binding domain that specifically binds to CALRmut and a second antigen-binding domain that specifically binds to CD3ε.

[0008] In some embodiments, the first antigen-binding domain comprises the first HCDR1, first HCDR2, and first HCDR3 of the first heavy chain variable region (VH1) of SEQ ID NO:14, and wherein the first antigen-binding domain comprises the first light chain complementarity-determining region (LCDR) 1, first LCDR2, and first LCDR3 of the first light chain variable region (VL1) of SEQ ID NO:16. In some embodiments, the second antigen-binding domain comprises the second HCDR1, second HCDR2, and second HCDR3 of the second heavy chain variable region (VH2) of SEQ ID NO:23, and the second LCDR1, second LCDR2, and second LCDR3 of the second light chain variable region (VL2) of SEQ ID NO:27.

[0009] In some embodiments, the anti-CALRmut / anti-CD3 bispecific antibody used in this invention comprises a heavy chain, a light chain, and a stapled single-chain fragment variable (spFv) chain. The heavy chain associates with the light chain to form an antigen-binding site that specifically binds to the mutant calreticulin (CALRmut); The spFv chain specifically binds to CD3 immune function; and among them... a) The heavy chain comprises: a heavy chain complementarity determination region (CDR) 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 12, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13; The light chain comprises: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9; and The spFv chain comprises: a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 25, a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 22; b) The heavy chains include: heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 68, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 69, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13; The light chain comprises: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9; and The spFv chain comprises: a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 25, a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 75, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 76, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 22; c) The heavy chains include: heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 10, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 67, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13; The light chain comprises: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9; and The spFv chain comprises: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 24, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 25, light chain CDR3 containing the amino acid sequence of SEQ ID NO: 26, heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 73, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 74, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 22; or d) The heavy chains include: heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 70, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 71, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 72; The light chains comprise: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 66, light chain CDR2 containing the amino acid sequence of DAS, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9; and The spFv chain comprises: a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 81, a light chain CDR2 containing the amino acid sequence of YAS, a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 78, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 79, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 80.

[0010] In some embodiments, the heavy chain comprises a heavy chain variable region (VH) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO: 14. In a preferred embodiment, the VH contains the amino acid sequence of SEQ ID NO: 14.

[0011] In some embodiments, the light chain comprises a light chain variable region (VL) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 16. In a preferred embodiment, the VL contains the amino acid sequence of SEQ ID NO: 16.

[0012] In some embodiments, the heavy chain variable region (VH) contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region (VL) contains the amino acid sequence of SEQ ID NO: 16.

[0013] In some embodiments, the bispecific antibody is IgG. In some embodiments, the bispecific antibody comprises an IgG1 isotype Fc region. In some embodiments, the bispecific antibody further comprises L234A, L235A, and D265S substitutions in the Fc region. In some embodiments, the bispecific antibody further comprises a mortar (KiH) substitution. In some embodiments, the bispecific antibody further comprises H435R and Y436F substitutions in the Fc region.

[0014] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the spFv chain comprises the amino acid sequence of SEQ ID NO: 28.

[0015] In some implementations, a bispecific antibody or its bispecific antigen-binding fragment is administered to the subject at a dose of at least 0.6 mg, such as a therapeutic dose of 0.6 mg to 400 mg, 5 mg to 100 mg, or 20 mg to 200 mg.

[0016] In some embodiments, the method further includes administering one or more escalating doses of anti-CALRmut / anti-CD3 bispecific antibody to the subject prior to the administration of the therapeutic dose, wherein none of the escalating doses exceeds the therapeutic dose. In some embodiments, escalating doses, such as 0.6 mg, 1 mg, or 1.2 mg of anti-CALRmut / anti-CD3 bispecific antibody, are administered to the subject 3 to 8 days prior to the initial administration of the therapeutic dose, for example, to reduce injection site reactions and CRS.

[0017] In some embodiments, the bispecific antibody or its bispecific antigen-binding fragment is administered every 1, 2, 3, 4, 5, 6, 7 or 8 weeks, preferably every 3 weeks.

[0018] In some embodiments, this application relates to a method of treating MPN, the method comprising subcutaneously administering, once every three weeks, a therapeutic dose of antiCALRmut / antiCD3 bispecific antibody of 1.2 mg to 400 mg, such as 5 mg to 100 mg or 20 mg to 200 mg, to a human subject in need, each time, wherein the antiCALRmut / antiCD3 bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO:15, a first light chain having the amino acid sequence of SEQ ID NO:17, and a second heavy chain having the amino acid sequence of SEQ ID NO:28, optionally, the method further comprising subcutaneously administering, one week prior to the initial therapeutic dose, an escalating dose of 0.6 mg or 1.2 mg of the antiCALRmut / antiCD3 bispecific antibody to the subject.

[0019] In some embodiments, the MPN is characterized by the presence of a mutant calreticulin (CALR). In some embodiments, the subject requiring treatment has one or more CALR mutations of type 1, type 1-like, type 2, or type 2-like. Other types of mutation patterns, such as those described, for example, by Pietra et al., Leukemia. 2016 Feb; 30(2):431, are also contemplated in some aspects of the invention.

[0020] In some implementations, the subject undergoes a splenectomy. In other implementations, the subject receives an allogeneic graft, such as an allogeneic bone marrow or stem cell graft.

[0021] In some implementations, the subject is ineligible, intolerant, or resistant to JAK inhibitor therapy.

[0022] In some implementations, the subject has already received prior therapy. For example, the subject may have been treated with one or more other lines of treatment, such as treatment with a JAK inhibitor and / or hydroxyurea. Optionally, one or more prior lines of treatment for the subject have failed.

[0023] In some implementations, the MPN is selected from the group consisting of: chronic myeloid leukemia, polycythemia vera, primary myelofibrosis (MF), essential thrombocythemia (ET), chronic neutrophilic leukemia, and chronic eosinophilic leukemia. In some implementations, the subject has a myelodysplastic syndrome selected from ET, pre-fibrotic MF, dominant primary MF, and accelerated blast phase MF.

[0024] In some embodiments, the subject is diagnosed with ET, particularly ET with a high risk of thrombosis or bleeding and who is intolerant to, resistant to, or refractory to hydroxyurea. Preferably, the method induces at least one of the following: 1) normal spleen size at imaging; and 2) a peripheral blood platelet count ≤400 × 10⁻⁶. 9 Cells / L and / or white blood cell count ≤10×10 9 per L.

[0025] In some implementations, the subject is diagnosed with myofibrosis (MF). For example, the subject may have primary MF, such as primary MF with a risk score of intermediate 1 (Int-1), intermediate 2 (Int-2), or high risk (HR) according to the Dynamic International Prognostic Scoring System (DIPSS, Passamonti 2010, Blood. 115:1703-1708), optionally with the percentage of blast cells in the blood or bone marrow not always exceeding 20%. In some implementations, the subject may have post-ET MF, such as post-ET MF with a risk score of Int-1, Int-2, or HR according to the Prognostic Model for Secondary Myofibrosis (PV and ET) (MYSEC-PM, Passamonti 2017, Leukemia. 31:2726-2731), optionally with the percentage of blast cells in the blood or bone marrow not always exceeding 20%. Preferably, the method of this application causes a reduction in the spleen volume of the subject compared with the baseline spleen volume measured before administration of the anti-CALRmut / anti-CD3 bispecific antibody, preferably a reduction of at least 35% in spleen volume compared with the baseline spleen volume measured before administration of the anti-CALRmut / anti-CD3 bispecific antibody.

[0026] In some embodiments, MPN is MF and the method causes at least one of the following: 1) age-adjusted normal cellularity of bone marrow, 2) <5% blast cells, and 3) peripheral blood hemoglobin ≥10 g / dL and neutrophil count ≥1 × 10⁻⁶. 9 CFU / L, and / or platelet count ≥100×10⁻⁶ 9 / L. In some implementations, this method improves MF disease, for example, by exerting a clinically meaningful effect on subject survival outcomes and / or restoration of normal hematopoiesis along with improvement in myelofibrosis through a substantial and persistent reduction in the clonal burden of the disease. See, for example, Pemmaraju et al. 2022, J Clin Oncol.40(26):3032-3036, for disease improvement of MF, the full text of which is incorporated herein by reference.

[0027] In some embodiments, the method reduces the level of soluble CALRmut in the subject's serum, preferably by at least 50% compared to the baseline level measured before administration of the anti-CALRmut / anti-CD3 bispecific antibody.

[0028] In another implementation, the method causes a reduction in CALRmut-positive cells in bone marrow samples.

[0029] In other embodiments, the method results in improved bone marrow architecture, such as reduced bone marrow reticular fibrosis and improved bone marrow cell composition.

[0030] In some embodiments, the method of this application further includes administering at least one first adjunctive therapeutic agent to the subject before administering a therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody, or before administering an escalating dose of the anti-CALRmut / anti-CD3 bispecific antibody. For example, the at least one first adjunctive therapeutic agent may be a glucocorticoid, an antihistamine, an antipyretic, an antiemetic, or any combination thereof.

[0031] In some embodiments, the method of this application further includes administering at least one second adjunct therapy to the subject before and optionally after administering a therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody. For example, the at least one second adjunct therapy may be an H1 antagonist, an H2 antagonist, or a leukotriene inhibitor, or any combination thereof.

[0032] This application also relates to an anti-CALRmut / anti-CD3 bispecific antibody for use in a method for treating MPN, according to any of the foregoing embodiments of this application.

[0033] Other aspects, features, and advantages of the invention will be better understood by reading the following detailed description of the invention and its claims. Attached Figure Description

[0034] The foregoing and other objects, aspects, features and advantages of the exemplary embodiments will become more apparent and better understood by referring to the following description in conjunction with the accompanying drawings.

[0035] Figure 1 The anti-CALRmut×CD3 bispecific antibody was described, in which AAS (L234A, L235A, D265S); CALRmut, mutant calreticulin; CD, differentiation cluster; Fab, antigen-binding fragment; Fc, crystallizable fragment; RF, H435R and Y436F; and spFv, the variable region of the stapled single-strand fragment.

[0036] Figure 2 The antitumor efficacy of the anti-CALRmut / anti-CD3 bispecific antibody (C3CRB73) in SC ELF-153 MPL CALRdel52 xenografts in T-cell humanized NSG mice was depicted. SC humanized NSG mice with ELF-153 MPL CALRdel52 tumors (first arrow indicates T-cell injection) were administered C3CRB73 via intraperitoneal injection (IP) at 1 mg / kg, 5 mg / kg, or 20 mg / kg (second to last arrows indicate administration). Tumor volume was measured twice weekly, and results are expressed as mean tumor volume ± SEM for each group. Tumor volume curves are shown when only two-thirds of the animals in each group were in the study.

[0037] Figure 3A and Figure 3B The antitumor efficacy of C3CRB73 in disseminated OCI-M2 MPLCALRins5 xenografts from humanized T-cell NSG mice was described. Figure 3A It depicts data on animal weight loss, while Figure 3B Survival data were depicted. T-cell humanized NSG mice with OCI-M2 MPL CALRins5 tumors (first arrow indicates T-cell injection) were administered via intravenous injection of the specified dose and antibody (second to last arrows indicate administration). Negative clinical signs and / or ≥20% body weight loss were used as surrogate endpoints for death. Body weight curves are shown when only two-thirds of the animals in each group were in the study.

[0038] Figure 4A and Figure 4B The mutation patterns of CALR mutations in MPN patients are depicted. The wide black bars represent exon 9 of CALR, the narrow bars represent the 3' UTR of the gene, and the thin lines represent introns and intergenic regions. Figure 4A The cDNA sequence shown is the start and end of exon 9. Below the cDNA sequence are the amino acid sequences derived from the three optional reading frames. Figure 4B The three reading frames are shown to produce different peptide compositions, especially regarding the charge of the amino acids. Figure 4C This study describes the spectrum of CALRmut myeloproliferative disorders as defined by CALRmut, degree of fibrosis, blood cell count, and treatment.

[0039] Figure 5 Serum concentration-time curves were plotted after a single IV dose of 0.5 mg / kg C3CRB73 in male cynomolgus monkeys (n=3 / group).

[0040] Figure 6 A to Figure 6 F shows that C3CRB73-mediated T cell activation occurs only in the presence of CALRmut-positive tumor cells. Figure 6 A and Figure 6 B shows that no cytotoxicity was observed in healthy hematopoietic cells. Figure 6 C to Figure 6 F demonstrates C3CRB73-mediated potent T-cell cytotoxicity against ex vivo CALRmut patient-derived CD34+ cells.

[0041] Figure 7 It describes how to incorporate an escalation dosing schedule into an activity schedule when administering one or more escalation doses.

[0042] Figure 8 A schematic overview of the research design is depicted.

[0043] Figure 9 The cohort results of hemoglobin recovery after treatment with compound 1 are depicted. Detailed Implementation

[0044] This disclosure provides implementation schemes for bispecific antibodies that bind to CALRmut and CD3, which are used to treat subjects with myeloproliferative neoplasms (MPN) or reduce their severity.

[0045] As used herein, the terms “a” or “an” mean “at least one / a kind” or “one or more / a combination of” unless the context clearly indicates otherwise.

[0046] As used herein, the term "about" means that the index value is approximate and small variations will not significantly affect the practice of the disclosed embodiments. When numerical limits are used, the "about" index value may vary by + / - 10% and remain within the range of the disclosed embodiments unless the context otherwise indicates. Furthermore, although the value may be preceded by the term "about," this document also provides precise values ​​where the term "about" is not used.

[0047] "Antigen" refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, parts thereof, or combinations thereof) capable of binding by an antigen-binding domain or a T-cell receptor capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens can be genetically expressed, synthetic, or purified from biological samples such as tissue samples, tumor samples, cells or fluids containing other biological components, organisms, protein / antigen subunits, and whole cells or lysates that kill or inactivate them.

[0048] An "antigen-binding fragment" or "antigen-binding domain" refers to the portion of a protein that binds to an antigen. Antigen-binding fragments can be synthetic, enzymatically produced, or genetically engineered polypeptides, and may include portions of immunoglobulins that bind antigens, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd, and Fv fragments; domain antibodies (dAbs) consisting of a VH domain or a VL domain; shark variable IgNAR domains; humped VH domains; VHH domains; minimal recognition units consisting of amino acid residues of the CDRs of antibody mimics (such as FR3-CDR3-FR4 portions, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3); alternative scaffolds for binding antigens; and multispecific proteins containing antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked together via synthetic linkers to form various types of monoclonal antibody designs. In those cases where the VH and VL domains are expressed by separate single chains, the VH / VL domains can be paired intramolecularly or intermolecularly to form monovalent antigen-binding domains, such as single-chain Fvs (scFvs), stapled single-chain Fvs (spFvs), or biantibodies. In some embodiments, the antibody fragment includes a stapled single-chain Fv (or spFv). Antigen-binding fragments can also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which can be monospecific or multispecific to engineer bispecific and multispecific proteins.

[0049] "Antibody" broadly refers to and includes immunoglobulin molecules, specifically including monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), dimer, tetramer, or multimer antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of immunoglobulin molecules containing an antigen-binding site with desired specificity. A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, and their polymers (e.g., IgM). Each HC consists of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be designated into five major classes based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequence of their constant domain, antibody light chains of any vertebrate species can be designated into one of two completely different types, namely κ and λ.

[0050] The term "antibody molecule" also encompasses complete or antigen-binding fragments of domain or single-domain antibodies, which may also be referred to as "sdAb" or "VHH". Domain antibodies include VH or VL that can function as independent antibody fragments. Additionally, domain antibodies include antibodies consisting only of the heavy chain (HCAb). Domain antibodies also include the CH2 domain of IgG as a scaffold, into which a CDR loop is inserted. It can also generally be defined as a polypeptide or protein containing an amino acid sequence consisting of four frame regions interrupted by three complementarity-determining regions. This is represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. sdAbs can be produced in camel species (such as llamas) but can also be synthesized using techniques well known in the art. The amino acid residue numbering of sdAbs or polypeptides follows the general numbering of the VH domain as given by Kabat et al. ("Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, MD, 91, the full text of which is incorporated herein by reference). According to this numbering, sdAb's FR1 contains amino acid residues at positions 1 to 30, sdAb's CDR1 contains amino acid residues at positions 31 to 36, sdAb's FR2 contains amino acids at positions 36 to 49, sdAb's CDR2 contains amino acid residues at positions 50 to 65, sdAb's FR3 contains amino acid residues at positions 66 to 94, sdAb's CDR3 contains amino acid residues at positions 95 to 102, and sdAb's FR4 contains amino acid residues at positions 103 to 113. Domain antibodies are also described in WO2004 / 041862 and WO2016 / 065323, both of which are incorporated herein by reference in their entirety.

[0051] As used herein, the term "bispecific antibody" refers to an antibody that binds to no more than two epitopes or two antigens. A bispecific antibody is characterized by a first variable heavy chain and variable light chain pair that binds specifically to a first epitope (e.g., an epitope on the CALRmut antigen) and a second variable heavy chain and variable light chain pair that binds specifically to a second epitope (e.g., an epitope on T cells, such as CD3). As used herein, "bispecific antibody" encompasses a bispecific antibody containing one or more immunoglobulin (Ig) constant regions and one or more bispecific antigen-binding fragments thereof.

[0052] When used in the context of antibodies or antibody fragments, "immunospecific" means binding to one or more epitopes of the protein of interest via a domain encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene, without preferentially binding to other molecules in a sample containing a mixed molecular population. Typically, the Kd of antibody binding to a homologous antigen is less than about 1 × 10⁻⁸ M, as determined by surface plasmon resonance assays or cell binding assays. Phrases such as "anti-[antigen] antibody" (e.g., anti-CALRmut antibody) are intended to express that the antibody specifically binds to the antigen.

[0053] As used herein, when referring to proteins with different domains or heterologous sequences, the terms "fusion" or "linkage" mean that the protein domains are portions of the same peptide chain linked together by peptide bonds or other covalent bonds. Domains or segments may be directly linked or fused together, or another domain or peptide sequence may be between two domains or sequences, and such sequences will still be considered fused or linked together. In some embodiments, the various domains or proteins provided herein are directly linked or fused together or directly linked or fused with adapter sequences such as glycine / serine, glycine / alanine adapters, or other types of peptide adapters commonly known to link two domains together. If two peptide sequences are directly linked together, they are directly linked; otherwise, if an adapter or other structure is present connecting the two regions, they are indirectly linked. An adapter can be directly linked to two different peptide sequences or domains.

[0054] As used herein, the terms “variable region” and “variable domain” refer to portions of the light and heavy chains of an antibody that include the amino acid sequences of complementarity-determining regions (CDRs, such as CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and framework regions (FRs). According to the methods used in this disclosure, the amino acid positions assigned to CDRs and FRs are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of the CDR (further defined herein) or FR (further defined herein) of the variable region. For example, the heavy chain variable region may include a single inserted residue (i.e., residue 52a according to Kabat) after residue 52 of CDR H2 and an inserted residue (i.e., residues 82a, 82b, 82c, etc. according to Kabat) after residue 82 of the heavy chain FR. The Kabat residue number of a given antibody can be determined by comparing the homologous region of the antibody sequence with the "standard" Kabat number sequence.

[0055] The complementarity-determining region (CDR) is the antibody region that binds to the antigen. There are three CDRs (HCDR1, HCDR2, HCDR3) in VH and three CDRs (LCDR1, LCDR2, LCDR3) in VL. CDRs can be defined using various descriptions, such as Kabat (Wu et al., (1970) J Exp Med 132: 211-50; Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27: 55-77), and AbM (Martin and Thornton, J Bmol Biol 263: 800-15, 1996). The correspondence between various depictions and variable region numbers is described (see, for example, Lefranc et al., (2003) Dev Comp Immunol, 27: 55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-70; International Immunogenetics (IMGT) Database; Web resource, http: / / www_imgt_org). Available programs (such as abYsis for UCL Business PLC) can be used to depict CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” include CDRs as defined by any of the above methods (Kabat, Chothia, IMGT, or AbM).

[0056] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene, cDNA, or RNA encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be said to encode that protein, or other products of that gene or cDNA.

[0057] "Fab" or "Fab fragment" refers to an antibody fragment composed of the VH domain, CH1 domain, VL domain, and CL domain.

[0058] "Fv" or "Fv fragment" refers to an antibody fragment consisting of the VH and VL domains of a single arm of the antibody.

[0059] "Single-chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH), wherein the VL and VH are sequentially linked via a peptide linker and are expressible as a single-chain polypeptide. Unless otherwise specified, as used herein, scFv may have VL and VH variable regions in any order; for example, the scFv may comprise VL-linker-VH or VH-linker-VL relative to the N-terminus and C-terminus of the polypeptide.

[0060] The "studded single-chain Fv" or "spFv" described in WO2021030657A1 refers to an scFv containing one or more disulfide bonds between VH and the linker or between VL and the linker. Typically, an spFv may contain one disulfide bond between VH and the linker, one disulfide bond between VL and the linker, or two disulfide bonds between VH and the linker and between VL and the linker. spFv molecules containing a disulfide bond between VH and VL are excluded from the term "spFv".

[0061] In some embodiments, the antibodies described herein may also contain mutations (e.g., amino acid substitutions, additions, and / or deletions) outside the CDR (i.e., in the frame region (FR). Amino acid substitutions, additions, and / or deletions may be substitutions, additions, and / or deletions of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more). Amino acid substitutions, additions, and / or deletions may be substitutions, additions, and / or deletions of eight or fewer, seven or fewer, six or fewer, five or fewer, four or fewer, three or fewer, or two or fewer single amino acids. In some embodiments, the antibodies described herein may include amino acid substitutions, additions, and / or deletions in the constant region of the antibody (e.g., the Fc region) that result in reduced effector functions, such as reduced complement-dependent cytolysis (CDC), antibody-dependent cell-mediated cytolysis (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or reduced B-cell killing. For many applications of therapeutic antibodies, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions can be detrimental and may pose safety risks by inducing extramechanistic toxicity. Modification of effector functions can be achieved by engineering the Fc region to weaken its binding to FcgR or complement factors. The binding of IgG to activating (FcgRI, FcgRIIa, FcgRIIIa, and FcgRIIIb) and repressive (FcgRIIb) FcgR or the first component (C1q) of complement depends on residues located in the hinge region and CH2 domain. Mutations can be introduced into IgG1, IgG2, and IgG4 to reduce or silence Fc function. Silent mutations may include, but are not limited to, IgG1 AA (F234A, L235A), IgG4 PAA (S228P, F234A, L235A), IgG2 AA (V234A, G237A), IgG1 FEA (L234F, L235E, D265A), or IgG1 FES (L234F / L235E / P331S). In some embodiments, the disclosed antibody or its antigen-binding fragment may contain an IgG1 AA (F234A, L235A) mutation. In some embodiments, the disclosed antibody or its antigen-binding fragment may contain an IgG4 PAA (S228P, F234A, L235A) mutation. In some embodiments, the disclosed antibody or its antigen-binding fragment may contain an IgG2 AA (V234A, G237A) mutation. In some embodiments, the disclosed antibody or its antigen-binding fragment may contain IgG1 FEA (L234F, L235E, D265A) mutations. In some embodiments, the disclosed antibody or its antigen-binding fragment may contain IgG1 FES (L234F / L235E / P331S) mutations.In some embodiments, the disclosed antibody or its antigen-binding fragment may contain IgG1 L234A, L235A, and / or F405L mutations. In some embodiments, the disclosed antibody or its antigen-binding fragment may contain S228P, L234A, L235A, F405L, and / or R409K mutations. In some embodiments, the disclosed antibody or its antigen-binding fragment may contain IgG-AA Fc-L234A, L235A, and F405L.

[0062] The disclosed antibody or its antigen-binding fragment may contain an Fc region having one or more of the following characteristics: (a) reduced effector function compared to the parental Fc; (b) reduced affinity for FcgRI, FcgRIIa, FcgRIIb, FcgRIIIIb and / or FcgRIIIIa; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (f) reduced affinity for FcgRIIb; or (g) reduced affinity for FcgRIIIIa.

[0063] The term "CD3" refers to the human CD3 protein multi-subunit complex. The CD3 protein multi-subunit complex consists of six distinct polypeptide chains. These chains include the CD3γ chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and a CD3ζ chain homodimer (SwissProt 20963), and the complex associates with the T cell receptor α and β chains. Unless otherwise stated, the term "CD3" includes any CD3 variant, isotype, and species homolog that is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding those polypeptides.

[0064] “T cells” and “T lymphocytes” are interchangeable and used synonymously in this document. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+CD8+ T cells, or any other subset of T cells. Also included are “NKT cells,” which refers to a specialized population of T cells that expresses the semi-invariant αβ T cell receptor but also expresses multiple molecular markers commonly associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1- cells, as well as CD4+, CD4-, CD8+, and CD8- cells. The unique feature of the TCR on NKT cells is that it recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. Because NKT cells can produce cytokines that promote inflammation or immune tolerance, they can have protective or destructive effects. Also included are "γ-δ T cells (γδ T cells)," a specialized subset of T cells with unique TCRs on their surface. Unlike most T cells, where the TCR consists of two glycoprotein chains named α-TCR and β-TCR, the TCR in γδ T cells consists of both γ- and δ-chains. γδ T cells can play a role in immune surveillance and immune regulation and have been found to be an important source of IL-17 and induce strong CD8+ cytotoxic T cell responses. Also included are "regulatory T cells" or "Tregs," which are T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4+ T cells, and may also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0065] The term “mutant calreticulin” or “CALRmut” refers to a mutant calreticulin identified and found to be associated with myeloid malignancies; see Klampfl et al. (N Engl J Med 2013; 369:2379-2390, December 19, 2013) and Nangalia et al. (N Engl J Med 2013; 369:2391-2405; EP 14 18 4835.8; PCT / EP2014 / 069638 and U.S. Application No. 14 / 486,973). The terms “CALR” or “CALRwt” are used interchangeably and refer to wild-type calreticulin.

[0066] As used herein, the term "percentage of identity (%)" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence (e.g., an antibody or antigen-binding fragment thereof of this disclosure) that are identical to amino acid (or nucleic acid) residues in a reference sequence (e.g., a wild-type antibody or antigen-binding fragment thereof) after sequence alignment and gap introduction (if necessary) to achieve the maximum percentage of identity (i.e., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignments performed for the purpose of determining the percentage of identity can be performed in various ways within the scope of the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences to be compared. In some implementations, the percentage of amino acid (or nucleic acid) sequence identity of a given candidate sequence with or relative to a given reference sequence is calculated as follows (which can be alternatively expressed as the percentage of a given candidate sequence with or relative to a given reference sequence having or including a certain number of amino acid (or nucleic acid) sequence identity): 100 × (fraction of A / B) Where A is the number of amino acid (or nucleic acid) residues that are identical in the alignment of the candidate sequence and the reference sequence, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percentage of amino acid (or nucleic acid) sequence identity between the candidate sequence and the reference sequence will not be equal to the percentage of amino acid (or nucleic acid) sequence identity between the reference sequence and the candidate sequence.

[0067] In some embodiments, for the purpose of comparing a reference sequence with a candidate sequence, the candidate sequence may exhibit 50% to 100% identity at the full length of the candidate sequence or at selected portions of consecutive amino acid (or nucleic acid) residues. The length of the candidate sequence compared for comparative purposes is at least 30%, for example, at least 40%, for example, at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. The molecule is identical at that position when a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence. Positions can be altered by substitution, deletion, or insertion. Substitution, deletion, or insertion may contain a number of amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more). When describing substitutions, deletions, or insertions of no more than n amino acids, this means that the substitutions, deletions, or insertions contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or n amino acids. The number of substitutions, deletions, or insertions may represent a certain percentage of the total sequence (e.g., 1%, 5%, 10%, 15%, 20%, or more), where the number of substitutions, deletions, or insertions changes the percentage of amino acids in the total sequence to 5%, 10%, 15%, 20%, or more.

[0068] A "polynucleotide" or "nucleic acid" is a synthetic molecule consisting of a chain of nucleotides or other equivalent covalently linked phosphate sugars. cDNA is a typical example of a polynucleotide. Polynucleotides can be DNA or RNA molecules.

[0069] "Vector" refers to an expression vector that can be used in a biological or regenerated biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including clomids, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that have bound recombinant polynucleotides.

[0070] As used herein, the terms “comprising” (and any form of “comprising” such as “comprise”, “comprises”, and “comprised”), “having” (and any form of “having” such as “have” and “has”), “including” (and any form of “including” such as “includes” and “include”), or “containing” (and any form of “containing” such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. Any composition or method that uses the term “comprising” should also be understood to describe such composition as being composed of, consisting of, or substantially composed of the said components or elements.

[0071] As used herein, the terms “individual,” “subject,” or “patient” are used interchangeably to mean any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates such as humans. As used herein, the term “mammal” refers to a rodent (i.e., a mouse, rat, or guinea pig), monkey, cat, dog, cattle, horse, pig, or human. In some embodiments, the mammal is a human.

[0072] As used herein, the phrase "in need of" means that the subject has been identified as requiring a method or treatment. In some embodiments, the identification may be made by any diagnostic means. In any of the methods and treatments described herein, the subject may be in need of it. In some embodiments, the subject is in or is about to travel to an environment where a particular disease, condition, or symptom is prevalent.

[0073] As used in this article, the phrase “integer from X to Y” means any integer including the endpoints. For example, the phrase “integer from X to Y” means 1, 2, 3, 4, or 5.

[0074] Anti-CALRmut×CD3 bispecific antibody In some embodiments, the bispecific antibody comprises one binding arm that binds to CD3ε and another binding arm that binds to CALRmut. In some embodiments, the CD3ε binding arm comprises a stapled single-stranded variable (spFv) chain. In some embodiments, the CD3ε binding arm is described in WO 2021 / 240388, the entire contents of which are incorporated herein by reference. In some embodiments, the CALRmut binding arm is any binding segment disclosed herein.

[0075] In some embodiments, the anti-mutated calreticulin (CALRmut) / anti-CD3 bispecific antibody comprises a first antigen-binding domain that specifically binds to CALRmut and a second antigen-binding domain that specifically binds to CD3ε, wherein the first antigen-binding domain comprises the first HCDR1, first HCDR2, and first HCDR3 of the first heavy chain variable region (VH1) of SEQ ID NO: 14, and wherein the first antigen-binding domain comprises the first light chain complementarity-determining region (LCDR) 1, first LCDR2, and first LCDR3 of the first light chain variable region (VL1) of SEQ ID NO: 16. In some embodiments, the second antigen-binding domain comprises the second HCDR1, second HCDR2, and second HCDR3 of the second heavy chain variable region (VH2) of SEQ ID NO: 23 and the second LCDR1, second LCDR2, and second LCDR3 of the second light chain variable region (VL2) of SEQ ID NO: 27.

[0076] In some implementations, the bispecific antibody comprises a heavy chain, a light chain, and an spFv chain. The heavy chains include: heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 11, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 12, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13. The light chains include: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9; The spFv chain includes: light chain CDR1 containing the amino acid sequence RARQSIGTAIH (SEQ ID NO: 24), light chain CDR2 containing the amino acid sequence YASESIS (SEQ ID NO: 25), light chain CDR3 containing the amino acid sequence QQSGSWPYT (SEQ ID NO: 26), heavy chain CDR1 containing the amino acid sequence GFTFSRYNMN (SEQ ID NO: 20), heavy chain CDR2 containing the amino acid sequence SISTSSNYIY (SEQ ID NO: 21), and heavy chain CDR3 containing the amino acid sequence GWGPFDY (SEQ ID NO: 22). The heavy chain associates with the light chain to form an antigen-binding site that specifically binds to the mutant calreticulin (CALRmut); and The spFv chain specifically binds to CD3 immune cells.

[0077] In some implementations, the bispecific antibody comprises a heavy chain, a light chain, and an spFv chain. The heavy chains include: heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 68, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 69, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13. The light chains include: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9; The spFv chain includes: light chain CDR1 containing the amino acid sequence RARQSIGTAIH (SEQ ID NO: 24), light chain CDR2 containing the amino acid sequence YASESIS (SEQ ID NO: 25), light chain CDR3 containing the amino acid sequence QQSGSWPYT (SEQ ID NO: 26), heavy chain CDR1 containing the amino acid sequence GFTFSRY (SEQ ID NO: 75), heavy chain CDR2 containing the amino acid sequence STSSNY (SEQ ID NO: 76), and heavy chain CDR3 containing the amino acid sequence GWGPFDY (SEQ ID NO: 22). The heavy chain associates with the light chain to form an antigen-binding site that specifically binds to the mutant calreticulin (CALRmut); and The spFv chain specifically binds to CD3 immune cells.

[0078] In some implementations, the bispecific antibody comprises a heavy chain, a light chain, and an spFv chain. The heavy chains include: heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 10, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 67, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13. The light chains include: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9; The spFv chain includes: light chain CDR1 containing the amino acid sequence RARQSIGTAIH (SEQ ID NO: 24), light chain CDR2 containing the amino acid sequence YASESIS (SEQ ID NO: 25), light chain CDR3 containing the amino acid sequence QQSGSWPYT (SEQ ID NO: 26), heavy chain CDR1 containing the amino acid sequence RYNMN (SEQ ID NO: 73), heavy chain CDR2 containing the amino acid sequence SISTSSNYIYYADSVKG (SEQ ID NO: 74), and heavy chain CDR3 containing the amino acid sequence GWGPFDY (SEQ ID NO: 22). The heavy chain associates with the light chain to form an antigen-binding site that specifically binds to the mutant calreticulin (CALRmut); and The spFv chain specifically binds to CD3 immune cells.

[0079] In some implementations, the bispecific antibody comprises a heavy chain, a light chain, and an spFv chain. The heavy chains include: heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 70, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 71, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 72; The light chains include: light chain CDR1 containing the amino acid sequence of SEQ ID NO: 66, light chain CDR2 containing the amino acid sequence of DAS, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 9. The spFv chain includes: light chain CDR1 containing the amino acid sequence QSIGTA (SEQ ID NO: 81), light chain CDR2 containing the amino acid sequence YAS, light chain CDR3 containing the amino acid sequence QQSGSWPYT (SEQ ID NO: 26), heavy chain CDR1 containing the amino acid sequence GFTFSRYN (SEQ ID NO: 78), heavy chain CDR2 containing the amino acid sequence ISTSSNYI (SEQ ID NO: 79), and heavy chain CDR3 containing the amino acid sequence TRGWGPFDY (SEQ ID NO: 80). The heavy chain associates with the light chain to form an antigen-binding site that specifically binds to the mutant calreticulin (CALRmut); and The spFv chain specifically binds to CD3 immune function. In some embodiments, heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 11, heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 12, and heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 7, light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, spFv light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 24, spFv light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 25, spFv light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 26, spFv heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 20, spFv heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 21, and spFv heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 22.

[0080] In some embodiments, heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 68, heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 69, and heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 7, light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, spFv light chain CDR1 consists of SEQ ID NO: 24, spFv light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 25, spFv light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 26, spFv heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 75, spFv heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 76, and spFv heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 22.

[0081] In some embodiments, heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 10, heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 67, and heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 7, light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 8, and light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, spFv light chain CDR1 consists of SEQ ID NO: 24, spFv light chain CDR2 consists of the amino acid sequence of SEQ ID NO: 25, spFv light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 26, spFv heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 73, spFv heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 74, and spFv heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 22.

[0082] In some embodiments, heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 70, heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 71, and heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 72. In some embodiments, light chain CDR1 consists of the amino acid sequence of SEQ ID NO: 66, light chain CDR2 consists of the amino acid sequence of DAS, and light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, spFv light chain CDR1 consists of SEQ ID NO: 81, spFv light chain CDR2 consists of the amino acid sequence of YAS, spFv light chain CDR3 consists of the amino acid sequence of SEQ ID NO: 26, spFv heavy chain CDR1 consists of the amino acid sequence of SEQ ID NO: 78, spFv heavy chain CDR2 consists of the amino acid sequence of SEQ ID NO: 79, and spFv heavy chain CDR3 consists of the amino acid sequence of SEQ ID NO: 80.

[0083] In some embodiments, the heavy chain of the bispecific antibody comprises a VH containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 14. In some embodiments, the heavy chain VH comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the heavy chain VH consists entirely of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the light chain of the bispecific antibody or its bispecific antigen-binding fragment comprises a light chain VL containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 16. In some embodiments, the light chain VL comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the light chain VL consists entirely of the amino acid sequence of SEQ ID NO: 16.

[0084] In some embodiments, the spFv chain of the bispecific antibody or its bispecific antigen-binding fragment comprises spFvVH, wherein the spFvVL contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 23. In some embodiments, the spFvVH contains the amino acid sequence of SEQ ID NO: 23. In some embodiments, the spFvVH is composed of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the spFv chain of the bispecific antibody or its bispecific antigen-binding fragment comprises spFvVL, wherein the spFvVL contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 27. In some embodiments, the spFvVL contains the amino acid sequence of SEQ ID NO: 27. In some embodiments, the spFvVL is composed of the amino acid sequence of SEQ ID NO: 27.

[0085] In some embodiments, the bispecific antibody comprises a heavy chain VH containing the amino acid sequence of SEQ ID NO: 14 and a light chain VL containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the bispecific antibody comprises a heavy chain VH consisting of the amino acid sequence of SEQ ID NO: 14 and a light chain VL consisting of the amino acid sequence of SEQ ID NO: 16.

[0086] In some embodiments, the bispecific antibody comprises: a heavy chain VH containing the amino acid sequence of SEQ ID NO: 14, a light chain VL containing the amino acid sequence of SEQ ID NO: 16, an spFv VH containing the amino acid sequence of SEQ ID NO: 23, and an spFv VL containing the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody comprises: a heavy chain VH consisting of the amino acid sequence of SEQ ID NO: 14, a light chain VL consisting of the amino acid sequence of SEQ ID NO: 16, an spFv VH consisting of the amino acid sequence of SEQ ID NO: 23, and an spFv VL consisting of the amino acid sequence of SEQ ID NO: 27.

[0087] In some embodiments, the bispecific antibody is IgG. In some embodiments, the bispecific antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibody is an IgG1 isotype, or contains an IgG1 isotype Fc region.

[0088] In some embodiments, the bispecific antibody further comprises L234A, L235A, and D265S substitutions in the Fc region. In some embodiments, these substitutions in the Fc region (referred to as AAS substitutions) are known to restrict or eliminate the interaction between the antibody and the Fc receptor.

[0089] In some implementations, the bispecific antibody also includes a mortar and pestle (KiH) substitution in the Fc region. One Fc region contains a T366W substitution (“mortar” Fc region), while the other Fc region contains T366S, L368A, and Y407V substitutions (“mortar” Fc region).

[0090] In some embodiments, the bispecific antibody further comprises H435R and Y436F substitutions in the Fc region. In some embodiments, these substitutions in the Fc region are known to disrupt the binding of protein A in the monomeric and homodimeric "mortar" Fc region.

[0091] In some embodiments, the bispecific antibody comprises a heavy chain containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 15. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain consists entirely of the amino acid sequence of SEQ ID NO: 15.

[0092] In some embodiments, the bispecific antibody comprises a light chain containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 17. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the heavy chain consists entirely of the amino acid sequence of SEQ ID NO: 17.

[0093] In some embodiments, the bispecific antibody comprises an spFv chain containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 28. In some embodiments, the spFv chain comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the spFv chain consists entirely of the amino acid sequence of SEQ ID NO: 28.

[0094] In some embodiments, the bispecific antibody comprises: a heavy chain containing the amino acid sequence of SEQ ID NO: 15, a light chain containing the amino acid sequence of SEQ ID NO: 17, and an spFv chain containing the amino acid sequence of SEQ ID NO: 28, wherein the heavy chain associates with the light chain to form an antigen-binding site that specifically binds to the mutant calreticulin (CALRmut); and wherein the spFv chain specifically binds to CD3.

[0095] In some embodiments, the bispecific antibody comprises: a heavy chain consisting of the amino acid sequence of SEQ ID NO: 15, a light chain consisting of the amino acid sequence of SEQ ID NO: 17, and an spFv chain consisting of the amino acid sequence of SEQ ID NO: 28, wherein the heavy chain and the light chain associate to form an antigen-binding site that specifically binds to the mutant calreticulin (CALRmut); and wherein the spFv chain specifically binds to CD3.

[0096] Vector, host cell and antibody production The antibodies disclosed herein, including but not limited to the bispecific antibodies disclosed herein, can be generated from host cells. A host cell is defined as a carrier comprising the necessary cellular components (e.g., organelles) required for the expression of the polypeptides and constructs described herein from their corresponding nucleic acids. Nucleic acids may be included in a nucleic acid carrier, which can be introduced into the host cell using conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The choice of nucleic acid carrier depends in part on the host cell to be used. Typically, the host cell is of prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0097] Nucleic acid vector construction and host cells Nucleic acid sequences encoding the amino acid sequences of the antibodies disclosed herein (including, but not limited to, the bispecific antibodies disclosed herein) can be prepared using various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the antibodies disclosed herein can be obtained using standard techniques (e.g., gene synthesis). Alternatively, standard techniques in the art (e.g., QuikChange) can be used. ™ Mutagenesis can be used to mutate the nucleic acid molecule encoding the antibody disclosed herein to contain specific amino acid substitutions. The nucleic acid molecule can be synthesized using a nucleotide synthesizer or PCR technology.

[0098] In some implementations, the nucleotide sequence encoding the anti-CALRmut heavy chain includes SEQ ID NO: 18.

[0099] In some implementations, the nucleotide sequence encoding the anti-CALRmut light chain includes SEQ ID NO: 19.

[0100] In some implementations, the nucleotide sequence encoding the anti-CD3 spFv chain includes SEQ ID NO: 29.

[0101] The nucleic acid sequence encoding the disclosed antibody can be inserted into a vector capable of replicating and expressing nucleic acid molecules in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purposes of this disclosure. Each vector may contain various components that can be tuned and optimized for compatibility with a particular host cell. For example, vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding the protein of interest, and a transcription termination sequence.

[0102] In some embodiments, mammalian cells are used as the host cells of this disclosure. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / O, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In other embodiments, Escherichia coli (E. coli) cells are used as the host cells of this disclosure. Examples of E. coli strains include, but are not limited to, Escherichia coli 294 (ATCC). ® 31,446), Escherichia coli λ1776 (ATCC) ® 31,537), Escherichia coli BL21 (DE3) (ATCC) ® BAA-1025) and Escherichia coli RV308 (ATCC) ®31,608). Different host cells possess characteristic and specific mechanisms for the post-translational processing and modification of protein products. Appropriate cell lines or host systems can be selected to ensure the proper modification and processing of the expressed antibody. The aforementioned expression vectors can be introduced into suitable host cells using conventional techniques in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection). Once the vector is introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified to induce promoters, select transformants, or amplify genes encoding the desired sequences. Methods for the expression of therapeutic proteins are known in the art, see, for example, Paulina Balbas and Argelia Lorence (eds.), *Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology*), Humana Press; 2nd edition, 2004 (July 20, 2004); and Vladimir Voynov and Justin A. Caravella (eds.), *Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology)*, Humana Press; 2nd edition, 2012 (June 28, 2012). In some embodiments, the antibodies of this disclosure are isolated from at least one component of a sample from which the antibody is obtained (e.g., a cell culture).

[0103] Pharmaceutical compositions, formulations and kits This disclosure is characterized by pharmaceutical compositions comprising one or more of the bispecific antibodies described herein. In addition to a therapeutically effective amount of antibody, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers or excipients, which may be formulated by methods known to those skilled in the art.

[0104] The acceptable carriers and excipients in the pharmaceutical composition are non-toxic to the recipient at the dose and concentration used. Acceptable carriers and excipients may include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. The pharmaceutical composition may be administered parenterally in the form of an injectable formulation. Sterile solutions or any pharmaceutically acceptable liquid may be used as carriers to formulate pharmaceutical compositions for injection. Pharmaceutically acceptable carriers include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco modified Eagle medium (DMEM), α-modified Eagle medium (α-MEM), F-12 medium). Formulation methods are known in the art; see, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd edition), Taylor & Francis Group, CRC Press (2006).

[0105] In some embodiments, any bispecific antibody disclosed herein may be packaged in a kit. In some embodiments, the kit comprises a polynucleotide encoding any antibody or antigen-binding fragment disclosed herein. In some embodiments, the kit comprises any pharmaceutical composition containing any antibody disclosed herein or an antigen-binding fragment thereof. In some embodiments, the kit comprises one or more of any bispecific antibody disclosed herein, any pharmaceutical composition disclosed herein, the polynucleotide disclosed herein, or any combination thereof.

[0106] Treatment methods and indications In some embodiments, the anti-CALRmut / anti-CD3 bispecific antibody described herein is administered as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the bispecific antibody.

[0107] As used herein, the term "therapeutic effective amount" refers to an amount that effectively induces the desired biological effect in a subject or patient or effectively treats a patient with the condition or disorder described herein, such as a drug dose. It should also be understood that "therapeutic effective amount" can be interpreted as an amount administered at a single dose or by any dose or route, alone or in combination with other therapeutic agents, to achieve the desired therapeutic effect.

[0108] As used herein, the term "not more than" refers to a quantity less than or equal to. This can be an integer quantity. For example, "not more than two substitutions" can mean 0, 1, or 2 substitutions.

[0109] As used herein, the terms “treatment” or “treating” refer to reducing or decreasing the risk of a particular disease or condition or its side effects. This reduction or decrease in risk or side effects is relative to a subject who is not receiving treatment, such as a control, baseline, or known control level or measurement.

[0110] In some embodiments, a method is provided for redirecting T cells to cells expressing mutant calreticulin, the method comprising administering a therapeutically effective amount of any antibody or antigen-binding fragment thereof disclosed herein to a subject in need of such redirection to cells expressing mutant calreticulin.

[0111] Myeloproliferative neoplasms (MPNs) are a rare group of blood cancers in which an abnormal and uncontrolled excess of red blood cells, white blood cells, or platelets is produced in the bone marrow. In some implementations, MPNs are selected from the group consisting of: chronic myeloid leukemia, polycythemia vera, primary myelofibrosis, secondary myelofibrosis, secondary acute myeloid leukemia, essential thrombocythemia, chronic neutrophilic leukemia, and chronic eosinophilic leukemia.

[0112] MPN is characterized by non-cytokine-dependent activation of the Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling pathway. Somatic mutations in JAK2, thrombopoietin receptor (also known as MPL), and CALR (calreticulin) in hematopoietic stem cells are phenotypic drivers in the pathogenesis of MPN. These mutations lead to pathological and constitutive activation of the JAK2-STAT5 pathway, resulting in clonal expansion of myeloid progenitor cells that no longer require cytokine activation to induce cell proliferation and differentiation. CALR is the second most frequently mutated gene in MPN after JAK2. Therefore, in some embodiments, MPN is characterized by the presence of a mutant calreticulin (CALRmut).

[0113] CALR wild-type protein is a major molecular chaperone and Ca2+-binding protein of the endoplasmic reticulum (ER). It plays a major role in protein folding, cell survival regulation, and immune responses. Its protein structure has three domains: (i) an amino domain, which is essential for molecular chaperone function through its lectin binding site and contains the ER signal peptide sequence; (ii) a proline-rich P domain, which binds calcium and contains the molecular chaperone lectin binding site; and (iii) a carboxyl domain, which also plays a role in calcium binding and includes the ER retention signal (KDEL motif).

[0114] All CALR mutations are insertions or deletions that cause a frameshift in the last exon of the gene (exon 9), resulting in the loss of the KDEL motif and the production of a positively charged mutant-specific 36-amino acid C-terminal tail (common to most known CALR mutations) (see [link to relevant documentation]). Figure 4A and Figure 4B Due to the absence of the KDEL motif, CALRmut is not limited to the ER and is transported to the cell membrane via MPL interaction. To date, more than 50 different types of mutations have been detected in CALR, but the deletion of 52 base pairs (CALRdel52; type 1 mutation) and the insertion of 5 base pairs (CALRins5; type 2 mutation) are the most common types, found in more than 80% of CALRmut patients overall. Other mutations include type 1-like or type 2-like mutations, depending on the extent of amino acid deletion. See, for example, Pietra et al., Leukemia. 2016 Feb; 30(2):431, mutational subtypes in CALR mutant myeloproliferative neoplasms, the full text of which is incorporated herein by reference. The spectrum of CALRmut myeloproliferative disorders can be defined by CALRmut, degree of fibrosis and blood cell count and their treatment. Figure 4C ).

[0115] An exemplary C-terminal amino acid residue / sequence of the mutant calreticulin contains the amino acid sequence of SEQ ID NO: 30-65.

[0116] It is envisioned that the antibody provided herein can specifically bind to a fragment or portion of the C-terminus of the mutant calreticulin. Preferably, the antibody provided herein specifically binds to RRKMSPARPRTSCREACLQGWTEA (SEQ ID NO: 2).

[0117] CALRmut lacks a transmembrane domain and requires binding to the extracellular domain of MPL to activate downstream signaling and trigger its oncogenic transformation. The binding of CALRmut to MPL depends on the binding of an immature asparagine-linked glycan at residue N117 and several negatively charged MPL fragments in the ER. This results in the formation of a tight complex and charge-driven interactions, providing CALRmut with specificity for MPL. CALRmut protects the N117-linked glycan of MPL from further processing in the Golgi apparatus. The tetrameric complex formed between two CALRmut and two MPL proteins shuttles across the Golgi apparatus, thereby presenting on the cell membrane and leading to constitutive activation of the downstream kinase JAK2. MPL complexed with CALRmut exists on the cell surface in an immature glycosylated state. Recent genetic and pharmacological screenings have identified the N-glycosylation pathway, more specifically the immature glycosylation of MPL, as essential for CALRmut-driven tumorigenesis. Conversely, in CALR wild-type cells, MPL is expressed on the cell surface in a mature glycosylated state and cannot be bound or stimulated by soluble CALRmut.

[0118] In some embodiments, a method for inhibiting the growth or proliferation of myeloproliferative neoplasms (MPNs) is provided, the method comprising administering to a subject in need a therapeutically effective amount of the bispecific antibody disclosed herein or a bispecific antigen-binding fragment thereof to inhibit the growth or proliferation of MPNs.

[0119] In some implementations, a method for treating MPN is provided, which includes administering any antibody or antigen-binding fragment thereof disclosed herein to a subject in need of treatment for a duration sufficient to treat the cancer.

[0120] In some implementation schemes, the subjects were diagnosed with essential thrombocytosis or myelofibrosis.

[0121] As used in this article, "essential thrombocythemia" or "ET" is an MPN characterized by the proliferation of megakaryocytes in the bone marrow, which causes thrombocytosis, i.e., an elevated platelet concentration in the peripheral circulation. The presence of CALRmut in peripheral blood mononuclear cells (PBMNCs) has been used for diagnosis (WHO Classification of Tumours Editorial Board 2022) and accounts for up to 25% of ET cases (Klampfl 2013, N Engl J Med.369:2379-2390).

[0122] The hydroxyurea-resistant population (resistant to cytoreductive therapy with hydroxyurea) in ET patients already at high vascular risk represents a patient population without a clear standard of care, for whom disease-modifying therapies may offer particular benefit. Indeed, HU resistance in this high vascular risk group is associated with increased disease progression and decreased overall survival, with a cumulative 10-year risk of progression of 13% and a conversion rate to leukemia of 8.5% (Hernandez-Boluda 2011, Br JHaematol.152(1):81-88). In some implementations, the subject is resistant to cytoreductive therapy with hydroxyurea.

[0123] In some embodiments, the method of this application is used to treat ET, particularly ET patients with a high risk of thrombosis or bleeding who are intolerant to, resistant to, or refractory to hydroxyurea. Preferably, the method induces at least one of the following: 1) normal spleen size at imaging; and 2) a peripheral blood platelet count ≤400 × 10⁻⁶. 9 Cells / L and / or white blood cell count ≤10×10 9 per L.

[0124] As used in this article, “primary myelofibrosis” or “PMF” is characterized by the proliferation of megakaryocytes and granulocytes in the bone marrow. This increase in myelofibrosis and osteosclerosis drives extramedullary hematopoiesis, leading to significant hepatosplenomegaly. Splenomegaly is common, progressive, and often substantial. PMF is associated with a greater burden of concomitant genetic mutations and chromosomal abnormalities than ET, which in turn tends to disease progression and leukemic transformation. Symptoms of PMF arise from a hypermetabolic state, fatigue, anorexia, weight loss, and symptoms attributable to splenomegaly, including abdominal discomfort and early satiety. Risk stratification for PMF can be performed at diagnosis using the IPSS (Cervantes 2009, Blood. 113:2895-2901). The Dynamic International Prognostic Scoring System (DIPS) can be used to predict survival during follow-up by incorporating prognostic information from karyotype, platelet count, and transfusion status. DIPS is described in Passamonti 2010, Blood. 115:1703-1708, the relevant content of which is incorporated herein by reference. In some embodiments, the method of this application is used to treat primary myocardial infarction (MF) with a DIPS risk score of intermediate 1 (Int-1), intermediate 2 (Int-2), or high risk (HR). In some embodiments, the percentage of primitive cells in the blood or bone marrow of subjects with primary MF does not always exceed 20%.

[0125] "Post-esophageal myelofibrosis" or "post-ET MF" refers to secondary MF arising from progression of ET. The median time to progression of CALRmut ET is 12.1 years, with a 15-year cumulative incidence of 13.4% (Mora 2018, LeukRes.69:100-102). The PV and ET secondary myelofibrosis prognostic model (MYSEC-PM) has defined four risk categories based on overall survival. MYSEC-PM is described in Passamonti 2017, Leukemia.31:2726-2731, the relevant content of which is incorporated herein by reference. In some embodiments, the methods of this application are used to treat post-ET MF, such as post-ET MF with a MYSEC-PM risk score of Int-1, Int-2, or HR. In some embodiments, the percentage of blast cells in the blood or bone marrow of subjects with post-ET MF does not always exceed 20%.

[0126] In some embodiments, the method of this application is used to treat MF in subjects in need, such as PMF or MF following ET. The method causes a reduction in spleen volume in subjects with MF compared to baseline spleen volume measured before administration of anti-mutant calreticulin (CALRmut) / anti-CD3 bispecific antibody, preferably a reduction of at least 35%, such as 35%, 40%, 45%, and 50%, or any value between these values, compared to baseline spleen volume measured before administration of anti-mutant calreticulin (CALRmut) / anti-CD3 bispecific antibody. In some embodiments, the treatment causes at least one of the following: 1) age-adjusted normal bone marrow cellularity, 2) <5% blast cells, and 3) peripheral blood hemoglobin ≥10 g / dL and neutrophil count ≥1 × 10⁻⁶. 9 CFU / L, and / or platelet count ≥100×10⁻⁶ 9 / L. In some implementations, this method improves MF disease, for example, by exerting a clinically meaningful effect on subject survival outcomes and / or restoration of normal hematopoiesis along with improvement in myelofibrosis through a substantial and persistent reduction in the clonal burden of the disease. See, for example, Pemmaraju et al. 2022, J Clin Oncol.40(26):3032-3036, for disease improvement of MF, the relevant content of which is incorporated herein by reference in its entirety.

[0127] In some embodiments, the method of this application causes a decrease in soluble CALRmut levels in the subject's serum, preferably a decrease of at least 50% compared to baseline levels measured before administration of the anti-mutated calreticulin (CALRmut) / anti-CD3 bispecific antibody, such as 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any value between these values. In some embodiments, the method causes a reduction in CALRmut-positive cells in the subject's bone marrow. In some embodiments, the method causes improved bone marrow architecture, such as a reduction in bone marrow reticular fibrosis, and improved bone marrow cellular composition.

[0128] In some implementations, subjects are ineligible, intolerant, or resistant to JAK inhibitor therapy. Ineligibility indicates that JAKi is contraindicated due to a history of serious infections, such as tuberculosis, progressive multifocal leukoencephalopathy, and skin malignancies known to be associated with or exacerbated by JAKi, or other significant considerations documented by the treating physician. Intolerance indicates hematologic toxicity, such as a platelet count <50 × 10⁻⁶. 9 ≤0.5×10⁶ cells / L and / or ≤0.5×10⁶ neutrophils / L 9 Evidence of resistance and / or refractory after administration of JAK inhibitors will include persistent splenomegaly, lack of symptom improvement, persistent leukocytosis, anemia <10 g / dL, or leukocytosis >11 × 10⁻⁶ cells / L. 9 per L.

[0129] In some embodiments, the subject has already received prior therapy. In some embodiments, the subject is diagnosed with essential thrombocytosis, and prior therapy includes at least two lines of prior cytoreductive therapy, at least one of which must be hydroxyurea. In some embodiments, the subject is diagnosed with myelofibrosis, and prior therapy includes at least one prior JAK inhibitor (JAKi) therapy. In some embodiments, prior therapy includes interferon-alpha (pegylated or standard formulation), anagrelide, or busulfan. In other embodiments, prior therapy includes JAKi, immunomodulatory drug therapy (such as thalidomide), danazol, or other therapies designed to produce disease improvement.

[0130] Example Example 1: Characteristics of CALRmut×CD3 bispecific IgG antibody Figure 1The general molecular architecture of an exemplary bispecific molecule targeting mutant calreticulin (CALRmut) and CD3 (CALRmut×CD3) that can be used in the invention described herein is illustrated. Specifically, C3CRB73 is an IgG1-based bispecific antibody that can simultaneously bind to CD3e (CD3ɛ; Uniprot ID: P07766) on T cells and CALRmut on tumor cells. The antibody has mutations in the fragment crystallizable (Fc) constant region of L234A, L235A, and D265S (i.e., AAS) to eliminate interaction with the Fc receptor and uses a club-and-mortar (KiH) mutation to enhance heterodimerization. The antibody comprises an anti-CD3ɛ spFv (i.e., T366W) fused to the N-terminus of the "clamp" Fc region. The mortar chain (i.e., T366S, L368A, Y407V) is characterized by resistance to CALRmut Fab and contains RF mutations (i.e., H435R, Y436F) to disrupt the binding of the monomeric and homodimeric mortar chain to protein A.

[0131] A. Structure C3CRB73 is generated through the co-expression of the anti-CD3ɛ scFv "pestle" heavy chain (HC) with the anti-CALRmut Fab heavy chain containing "pestle" and RF mutations, and pairs with its homologous light chain (LC). The amino acid sequences of the C3CRB73 heavy and light chains, deduced from the cDNA sequence and confirmed by peptide mapping and mass spectrometry, are shown below, with complementarity-determining regions (CDRs) listed according to AbM, Chothia, Kabat, or IMGT definitions. The CD3 heavy chain sequence includes a pinned CD3'spFv' arm. The CALRmut heavy chain contains heterodimerization mutations (KiH and T366W in the CD3 spFv arm, and T366S, L368A, and Y407V in the CALRmut heavy chain) as well as H435R and Y436F mutations, and also contains an N104S mutation in CDR-H3 of the CALRmut heavy chain.

[0132] Table 1: Amino acid sequence of C3CRB73 CALRmut×CD3 bispecific antibody SEQ IDNO describe sequence 11 CALRmut heavy CDR1 AbM GIDLSNNAIS 12 CALRmut heavy CDR2 AbM VIGNTGDTY 13 CALRmut heavy CDR3 AbM GPPSYSSSVKNI 68 CALRmut Heavy CDR1 Chothia GIDLSNN 69 CALRmut Heavy CDR2 Chothia GNTGD 13 CALRmut Heavy CDR3 Chothia GPPSYSSSVKNI 10 CALRmut heavy CDR1 Kabat NNAIS 67 CALRmut heavy CDR2 Kabat VIGNTGDTYYADSAKG 13 CALRmut heavy CDR3 Kabat GPPSYSSSVKNI 70 CALRmut heavy CDR1 IMGT GIDLSNNA 71 CALRmut re-CDR2 IMGT IGNTGDT 72 CALRmut heavy CDR3 IMGT VRGPPSYSSSVKNI 14 CALRmut VH EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNNAISWVRQAPGKGLEYVGVIGNTGDTYYADSAKGRFTISRDSKTTLYLQMNSLRAEDTAVYFCVRGPPSYSSSVKNIWGQGTLVTVSS 15 CALRmut heavy chain EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNNAISWVRQAPGKGLEYVGVIGNTGDTYYADSAKGRFTISRDSSKTTLYLQMNSLRAEDTAVYFCVRGPPSYSSSVKNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK 7 CALRmut light chain CDR1 AbM QSSQSVYNNNWLS 8 CALRmut light chain CDR2 AbM DASKLES 9 CALRmut light chain CDR3 AbM AGGFTGNVYT 7 CALRmut light chain CDR1 Chothia QSSQSVYNNNWLS 8 CALRmut light chain CDR2 Chothia DASKLES 9 CALRmut light chain CDR3 Chothia AGGFTGNVYT 7 CALRmut light chain CDR1 Kabat QSSQSVYNNNWLS 8 CALRmut light chain CDR2 Kabat DASKLES 9 CALRmut light chain CDR3 Kabat AGGFTGNVYT 66 CALRmut light chain CDR1 IMGT QSSQSVYNNNWLS -- CALRmut light chain CDR2 IMGT DAS 9 CALRmut light chain CDR3 IMGT AGGFTGNVYT 16 CALRmut VL DIQMTQSPSSVSASVGDRVTITCQSSQSVYNNNWLSWLQQKPGKAPKRLIYDASKLESGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCAGGFTGNVYTFGGGTKVEIK 17 CALRmut light chain DIQMTQSPSSVSASVGDRVTITCQSSQSVYNNNWLSWLQQKPGKAPKRLIYDASKLESGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCAGGFTGNVYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20 CD3 spFv Heavy CDR1 AbM GFTFSRYNMN 21 CD3 spFv Heavy CDR2 AbM SISTSSNYIY 22 CD3 spFv Heavy CDR3 AbM GWGPFDY 75 CD3 spFv Heavy CDR1 Chothia GFTFSRY 76 CD3 spFv Heavy CDR2 Chothia STSSNY 22 CD3 spFv Heavy CDR3 Chothia GWGPFDY 73 CD3 spFv Heavy CDR1 Kabat RYNMN 74 CD3 spFv Heavy CDR2 Kabat SISTSSNYIYYADSVKG 22 CD3 spFv Heavy CDR3 Kabat GWGPFDY 78 CD3 spFv Heavy CDR1 IMGT GFTFSRYN 79 CD3 spFv Heavy CDR2 IMGT ISTSSNYI 80 CD3 spFv Heavy CDR3 IMGT TRGWGPFDY 23 CD3 spFv VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYNMNWVRQAPGKGLEWVSSISTSSNYIYYADSVKGRFTFSRDNAKNSLDLQMSGLRAEDTAIYYCTRGWGPFDYWGCGTLVTVSS 24 CD3 spFv Light CDR1 AbM RARQSIGTAIH 25 CD3 spFv Light CDR2 AbM YASESIS 26 CD3 spFv Light CDR3 AbM QQSGSWPYT 24 CD3 spFv Light CDR1 Chothia RARQSIGTAIH 25 CD3 spFv Light CDR2 Chothia YASESIS 26 CD3 spFv Light CDR3 Chothia QQSGSWPYT 24 CD3 spFv Light CDR1 Kabat RARQSIGTAIH 25 CD3 spFv Light CDR2 Kabat YASESIS 26 CD3 spFv Light CDR3 Kabat QQSGSWPYT 81 CD3 spFv Light CDR1 IMGT QSIGTA -- CD3 spFv Light CDR2 IMGT ​ 26 ​ ​ 27 CD3 spFv VL DIQMTQSPSSSLSASVGDRVTITCRARQSIGTAIHWYQQKPGCAPKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSWPYTFGQGTKLEIK 82 CD3 spFv VL-VH DIQMTQSPSSSLSASVGDRVTITCRARQSIGTAIHWYQQKPGCAPKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSWPYTFGQGTKLEIKGGGSGGSGGCPPC GGSGGEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYNMNWVRQAPGKGLEWVSSISTSSNYIYYADSVKGRFTFSRDNAKNSLDLQMSGLRAEDTAIYYCTRGWGPFDYWGCGTLVTVSS 28 CD3 spFv DIQMTQSPSSSLSASVGDRVTITCRARQSIGTAIHWYQQKPGCAPKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSWPYTFGQGTKLEIKGGGSGGSGGCP PCGGSGGEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYNMNWVRQAPGKGLEWVSSISTSSNYIYYADSVKGRFTFSRDNAKNSLDLQMSGLRAEDTAIYYCTRGWGPFDYWGCGTL VTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B. Immunogenicity risk assessment CALRmut is a novel antigen present in malignant MPN clonal cells but not found in normal cells. Therefore, C3CRB73 is not expected to bind to or affect normal cells via its CALRmut target arm. In vivo animal models were not considered relevant to the non-clinical safety test. This is because the CALRmut target is absent in non-clinical species (i.e., mice, rats, dogs, and NHP), lacks cross-reactivity with wild-type CALRmut, and lacks cross-reactivity with the CD3-binding compound (CD3W245) in non-clinical species including NHP. For this reason, the non-clinical safety risk assessment was primarily based on computed, in vitro, and ex vivo data. Specifically, the non-clinical safety assessment consisted of data confirming the specificity of C3CRB73 and the absence of CALRmut in normal human and NHP tissues, as well as an evaluation of the potential effects of soluble CALRmut on C3CRB73 binding, T cell activation, and cytotoxicity.

[0133] Studies assessing the specificity of C3CRB73 included human protein screening arrays (Retrogenix assays), Good Laboratory Practice (GLP)-compliant tissue cross-reactivity studies, and in vitro-based cell function assays. As tested in the Retrogenix human plasma membrane array screening, tumor antigen-negative cell function assays, and GLP-compliant tissue cross-reactivity studies, C3CRB73 is specific for the CALRmut antigen. Importantly, C3CRB73 does not bind to CALR wild-type protein.

[0134] In vitro, C3CRB73-mediated CALRmut selective T cell-mediated cytotoxicity against CALRmut engineered cell lines or CALRmut patient-derived CD34+ cells ( Figure 6 C- Figure 6 F). No cytotoxicity was observed in cells without CALRmut ( Figure 6 A- Figure 6 (B) T cell activation occurs only in the presence of CALRmut-positive tumor cells. When using patient-derived or recombinant CALRmut protein, the presence of soluble CALRmut does not affect C3CRB73 binding or T cell-mediated cytotoxicity against cancer cells expressing MPL and CALRmut or MPL alone. No evidence of off-target binding of C3CRB73 was found in several other target-negative cell lines tested in the presence of soluble CALRmut.

[0135] The potential immunogenicity of antibody sequences was analyzed using T-reg (Treg) adjusted scores from the EpiVax EpiMatrix in silico immunogenicity prediction program (Table 2). The EpiVax program computationally calculates the binding potential with the most common HLA molecules within each “supertype.” This report provides results representing >90% of the world’s population without requiring individual testing for each haplotype. EpiVax scores are calculated by aggregating EpiMatrix scores for all predicted T-cell epitopes contained within a given protein sequence and adjusting for expected T-cell epitope content and protein length. Despite being based on extensive computer simulation analysis, the EpiVax EpiMatrix score has limited experimental capability to predict patient immunogenicity (Table 3). The results in Table 2 indicate that both the anti-CD3 arm v region and the anti-CALRmut v region have acceptable scores. Therefore, C3CRB73 is not considered to pose a significant risk of immunogenicity.

[0136] Table 2: Immunogenicity Assessment describe EpiVax rating CD3 spFv VL -2.39 CD3 spFv VH -42.70 CALRmut VL -34.58 CALRmut VH -10.91 Table 3: Explanation of EpiVax scoring criteria and potential immunogenicity EpiVax rating Immunogenicity standards <-20 ideal -20 to +20 Acceptable >+20 at risk Example 2: In vivo pharmacological and immunogenicity studies of CALRmut×CD3 bispecific antibody A. PK study of C3CRB73 in female cynomolgus monkeys The pharmacokinetic (PK) response of C3CRB73 was investigated in female cynomolgus monkeys. Three animals (n=3) were administered a single intravenous dose of 0.5 mg / kg C3CRB73. Blood samples were collected 30 days after administration. PK parameters were estimated using non-compartmental analysis. The mean Cmax was 15.63 ± 2.02 µg / mL and the mean area under the time-concentration curve (AUCinf) was 104.18 ± 35.82 µg·day / mL.

[0137] B. C3CRB73-induced T cell-mediated efficacy of CALRmut xenografts The antitumor efficacy of C3CRB73 was evaluated in humanized T-cell mice in xenografts of SC human megakaryocytic leukemia ELF-153 MPL CALRdel52 and disseminated acute myeloid leukemia (AML) OCI-M2 MPL CALRins5. For all studies, female NSG mice (Charles River Labs, Lyon, France) were used to provide suitable hosts to reconstruct the human immune system using human donor CD3+ pan-T cells. Prior to T-cell engraftment, 3 × 10⁻⁶ cells were used. 6 Mice were inoculated with 0.5 × 10¹² ELF 153 MPLCALRdel52 tumor cells or with SC. 6Mice were intravenously (IV) injected with OCI-M2 MPL CALRins5 tumor cells. The day of tumor implantation was designated as day 0 of the study.

[0138] Expanded human pan-T cells were intraperitoneally (IP)-inoculated into NSG mice to humanize the immune system. Effector cells were provided by humanized mice expressing CD3 T cells to promote C3CRB73-mediated cytotoxicity against tumor cells expressing human CALRmut. At least 30 minutes prior to C3CRB73 administration, humanized T-cell mice were given Fc-blocking antibody and intravenous immunoglobulin (IVIg) IP to correct for the low Ig environment in NSG mice.

[0139] In the prevention study, NSG mice were randomly assigned to a group of n=10 animals. SCs were implanted with ELF-153 MPLCALRdel52 cells. On day 3 after tumor cell implantation, pan-T cells were humanized. Starting from day 4 after cell implantation, the mice were treated twice a week with 1, 5, or 20 mg / kg C3CRB73 IP for a total of 8 weeks.

[0140] On day 24 post-tumor transplantation, while two-thirds of the control animals treated with the solvent (i.e., phosphate-buffered saline [PBS]) were still in the study, the percentage tumor growth inhibition rate (TGI) of the SC ELF-153 MPL CALRdel52 xenograft was calculated. Statistically significant TGIs were observed with C3CRB73 at 1 mg / kg, 5 mg / kg, and 20 mg / kg, achieving TGIs of 97.4% (p<0.0001), 99.3% (p<0.0001), and 94.9% (p<0.0001), respectively, compared to the solvent-treated controls. Figure 2 In this study, no indication of graft-versus-host disease (GvHD) morbidity due to T-cell humanization was observed.

[0141] In the efficacy study, NSG mice were randomly assigned to groups of n=10 animals and humanized with T cells 6 days after implantation of OCI-M2 MPL CALRins5 tumor cells. Intra-in ...

[0142] Due to significant weight loss and increased tumor burden incidence (hindlimb paralysis), all animals in the solvent (i.e., PBS)-treated control group were removed from the study by day 47. Mice treated with 1, 5, or 20 mg / kg C3CRB73 remained in the study until the end of the experiment (day 63), or were removed from the study due to GvHD caused by human T cell reaction with mouse tissue antigens. C3CRB73 treatment significantly increased lifespan (ILS) compared to solvent-treated control mice, with ILS >50% at all dose levels (p<0.05). Figure 3A and Figure 3B According to the National Cancer Institute's standard definition, an ILS ≥ 25% is considered biologically significant.

[0143] Increased levels of soluble CALRmut protein in plasma have been reported to be associated with disease progression. In the OCI-M2 MPLCALRins5 disseminated model, limited levels of soluble CALRmut (approximately 0.2 ng / mL) were detected in the plasma of animals treated with 1 mg / kg, 5 mg / kg, and 20 mg / kg C3CRB73, while a level of 2.55 ng / mL (corresponding to 12.75 ng / mL in humans) was measured in PBS-treated control mice. These results further confirm the antitumor activity of C3CRB73.

[0144] In both studies, treatment with 1, 5, or 20 mg / kg C3CRB73 did not show any signs of weight loss or other signs of overall toxicity. The first indication of GvHD-related morbidity due to T-cell humanization was observed on day 53.

[0145] B. Summary and Conclusion In vivo, C3CRB73 prevented tumor growth in the ELF-153 MPL CALRdel52 SC xenograft model and significantly increased lifespan in the OCI-M2 MPL CALRins5 disseminated xenograft model.

[0146] Example 3: Pharmacokinetic and Pharmacodynamic Analysis A. Pharmacokinetics in cynomolgus monkeys In non-GLP studies, the pharmacokinetic (PK) characteristics of C3CRB73 were characterized in female cynomolgus monkeys following a single intravenous (IV) dose of 0.5 mg / kg (n=3 / group). Serum samples were collected on days 0.04, 0.25, 1, 2, 4, 7, 10, 14, 18, 23, 28, and 30 post-dose. Total C3CRB73 concentrations were quantified using a qualified research method, namely the Meso Scale Discovery (MSD) electrochemiluminescence immunoassay (ECLIA) with a lower limit of quantification (LLOQ) of 0.08 µg / mL in cynomolgus monkey serum. C3CRB73 was captured and detected using a biotinylated and ruthenium-labeled mouse anti-human IgG monoclonal antibody.

[0147] Figure 5 The serum concentration-time curves of C3CRB73 in monkeys are illustrated in Table 4. Serum PK parameters estimated using non-compartmental analysis (NCA) are shown in Table 4.

[0148] Table 4: PK parameters of C3CRB73 in serum of female cynomolgus monkeys after a single IV dose of 0.5 mg / kg .

[0149] AUC inf Extrapolate to the area under the concentration-time curve to infinity; AUC last Area under the concentration-time curve up to the last sampling point; CL, total clearance; C max Maximum serum concentration; IV, intravenous; values ​​are averages, with standard deviation between parentheses (n=3 / group).

[0150] Furthermore, serum concentration-time data were fitted against a linear 2-compartment PK model for PK parameter estimation. No significant anti-drug antibodies (ADAs) were observed in any animal.

[0151] B. Preliminary effective dose prediction Based on PK parameter estimates from monkeys, assuming a human weight of 70 kg and a cynomolgus monkey weight of 3 kg, allometric scaling was used to predict the human PK of C3CRB73. The scaling factor was 0.85 for clearance-related parameters (total clearance [CL] and distributed clearance [Q]) and 1.0 for distributed volume-related parameters (central compartment volume [Vc] and peripheral compartment volume [Vt]). The model-estimated cynomolgus monkey parameters and predicted human PK parameters are listed in Table 5. Using a 2-compartment model, the estimated T1 / 2 in cynomolgus monkeys was 10.9 days, and the predicted systemic T1 / 2 for C3CRB73 in humans was approximately 17.7 days. Based on internal clinical experience with CD3 redirecting agents, assumptions were made regarding SC bioavailability (i.e., 70%) and absorption rate constant (i.e., 0.3 L / day) in humans.

[0152] Table 5: Model estimated parameters and predicted human PK parameters for C3CRB73 in cynomolgus monkeys CL, Total Clearance; F, Bioavailability; k a Absorption rate constant; PK, pharmacokinetics; Q, distribution and clearance; RSE, relative standard error; V c Volume of the central compartment; V t , volume of the peripheral compartment.

[0153] The initial dose prediction is based on the following three main assumptions.

[0154] The first hypothesis is that human IV PK is predictable based on allometric scaling of body weight from IV bolus data from cynomolgus monkeys.

[0155] Secondly, since neither the anti-CALRmut arm nor the anti-CD3 arm of C3CRB73 cross-reacts with cynomolgus monkeys, target-mediated drug disposal (TMDD) is not included in the current PK model. However, TMDD may be expected in humans, especially at low dose levels.

[0156] Finally, in whole blood from patients, in vitro T cell activation, cytotoxicity, and cytokine release in ELF 153 MPL CALRmut cells at a 1:1 E:T ratio were considered as a systemic factor for predicting FIH initiation dose in CALRmut-positive tumors. The E:T ratio data used for dose prediction were physiologically relevant.

[0157] C. Preliminary recommended scope for Phase 1 human trials A preliminary estimate of the initial FIH dose of C3CRB73 was provided using a MABEL-based approach. The preliminary MABEL concentration was determined to be 0.64 nM from the median EC50 value of cytotoxicity in ELF-153 MPL CALRins5 cells using whole blood from patients; this was the most sensitive and relevant assay based on the current dataset (see U.S. Provisional Application No. 63 / 516,520). A single SC administration of a fixed dose of 0.6 mg (assuming an average human weight of 70 kg) was predicted to result in a maximum serum concentration (Cmax) of C3CRB73 just below the median EC50 value of the most sensitive in vitro assay.

[0158] Example 4: Safety and pharmacokinetics of CALRmutxCD3 bispecific antibody against CALR-mutated myeloproliferative neoplasms The first human study of pharmacokinetics and pharmacodynamics A. Overview of the Plan This clinical protocol is an open-label phase 1 study to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary clinical activity of compound 1 (a clinical batch of C3CRB73, a T-cell redirection bispecific antibody against CALR-mutant myeloproliferative neoplasms), for example, in which adult participants with CALRmut MPN were diagnosed with essential thrombocytosis (ET) or myelofibrosis (MF).

[0159] B. Goals and End Points The following is a list of the objectives and endpoints evaluated in this study, with details consistent with the general objectives listed.

[0160] Table 6: Goals and Endpoints C. Overall Design The study will be conducted in two parts: Part 1 (dose escalation), followed by Part 2 (cohort expansion). Figure 8 Part 1 was designed to determine the expected RP2D and optimal dosing schedule based on preliminary assessments of safety, pharmacokinetic (PK), PD, and efficacy across several dosing regimens. The expected RP2D and optimal dosing schedule determined in Part 1 will be further investigated in Part 2 in participants with specific performance and risk profiles of CALRmut MPN.

[0161] The study of treatment will be based on the following and Figure 8 The dose escalation or cohort expansion strategy outlined herein will be administered subcutaneously (SC) at the dose specified by the sponsor. Study treatment will be administered as an outpatient procedure, but with a minimum hospital stay of 72 hours, to observe each escalation dose of compound 1 and early inflammatory adverse events following the first treatment dose.

[0162] In Part 1, 40 to 60 participants are expected to be recruited into separate disease-specific cohorts (e.g., CALRmut MF and ET), and the total number of participants will depend on the number of dose levels explored to identify the putative RP2D. Initial recruitment efforts will focus solely on CALRmut MF (Int-2 and high-risk), with CALRmut MF (Int-1) participants recruited while reviewing preliminary safety data with SET approval, and CALRmut ET participants recruited eventually. In Part 2, approximately 20 participants will be recruited for each candidate RP2D disease-specific expansion cohort. The total number of participants recruited in the clinical trial is expected to be approximately 100 (DLT evaluable).

[0163] This study will be conducted by the Study Evaluation Team (SET). During the study, safety will be monitored by the SET at regular intervals at each dose escalation step and during Part 2. Delayed toxicities, including long-term cytopenia, will also be monitored in cumulative data across subsequent treatment cycles. SET decisions will be based on a review of all available data, including but not limited to safety data, pharmacokinetic (PK), drug-promoting (PD), and preliminary antitumor activity. The SET may propose changes to dose escalation, dose modification, treatment and procedural schedules, or modifications during the study. For example, as described in Table 7, the target dose escalation level starting from a 0.6 mg subcutaneous injection will not exceed twice the normal dose escalation level.

[0164] Table 7: Maximum Target Dose Increment Target dose series Maximum dose Target dose series Maximum dose 1 0.6mg 5 9.6mg 2 1.2mg 6 19.2mg 3 2.4mg 7 38.4mg 4 4.8mg 8 76.8mg Part 1 Part 1 (dose escalation) is designed to determine the putative RP2D and optimal dosing schedule for further investigation of compound 1 in Part 2. The overall study cohort will include participants with CALRmut ET or MF, placed in a separate dose escalation cohort where treatment options have been exhausted. However, initial recruitment will focus only on CALRmut intermediate-2 (Int-2) and high-risk MF participants. Once preliminary safety data (including the frequency and severity of CRS, coagulopathy, and myelosuppression) are available in this CALRmut MF cohort, SET will review and approve favorable risk / benefit profiles, and may consider including intermediate-1 (Int-1) MF patients who have failed prior JAKi therapy in recruitment.

[0165] This study will explore consecutive, higher doses in the corresponding cohorts, starting with a MABEL-based initial dose of compound 1 in CALRmut MF participants, consistent only with the methods described above. Recruitment of mutCALR ET participants will only be considered after reviewing available clinical data from at least three MF cohorts and a minimum of 12 SET-approved MF participants to inform ET participants of a safe dose escalation approach. The initial starting dose for ET participants will not exceed 2.4 mg.

[0166] SET and the sponsor will review the assessments of safety, efficacy, pharmacokinetic (PK), and PD, and they will agree before any dose escalation. Safety assessments for a given cohort will include a review and discussion of adverse events (AEs) in participants within that cohort. Cohort safety will also be assessed by the proportion of participants in the cohort who experience dose-dependent leukemia (DLT).

[0167] In Part 1, SET and the sponsor will follow a Bayesian Optimal Interval (BOIN) design, which will guide dose escalation if the proportion of participants who have experienced DLT in the DLT evaluable analysis set in the current cohort is >25%.

[0168] Part 1 will explore the initial starting dose of 0.6 mg SC administered every 3 weeks. As dose escalation begins from a MABEL-based starting dose, dose escalation decisions will be guided by a BOIN design with SET approval.

[0169] One or more putative RP2D and optimal dosing schedules can be determined by pooling and evaluating all available PK, PD, target binding, efficacy, safety, and tolerability data in the disease-specific cohort, and further investigated in additional participants in Part 2 (cohort expansion) upon agreement between the SET and the sponsor. The DLT evaluation period begins with the administration of the first dose of Compound 1 (escalation dose or target dose) and covers the period prior to the second administration of the target dose in the dosing schedule. The table below describes the DLT evaluation periods for various dosing scenarios that can be explored in Part 1. Any participant who fails to complete a DLT evaluation period for reasons other than toxicity will be considered non-evaluable for DLT and may be replaced for DLT evaluation. DLT is any adverse event attributable to the study treatment that meets the severity and duration criteria in Table 9 and occurs during the evaluation period summarized in Table 8, unless it can be indisputably attributed to disease or other extraneous causes, such as an accident.

[0170] Table 8: DLT Evaluation Period for Q3W Plan as Needed Table 9: Dosage-limiting toxicity criteria Part 2 The goal of the cohort in Part 2 is to further characterize safety and to provide a preliminary evaluation of the efficacy of the putative RP2D and dosing schedule in specific disease classes. One or more putative RP2Ds and optimal dosing schedules may be selected for Part 2.

[0171] For participants with high-risk cases of ET or MF who are resistant to or intolerant of existing therapies at each dose and schedule, a corresponding cohort of approximately 20 participants can be recruited.

[0172] Based on emerging data, disease categories can be added, excluded, or revised to accommodate other histological studies, such as those determined by SET. The goal of the cohort in Part 2 is to further characterize safety and to provide a preliminary evaluation of the efficacy of the putative RP2D and optimal dosing schedule in specific disease categories.

[0173] Incremental dosage The escalating dose strategy has previously been effective for T-cell redirection bispecific antibodies to mitigate acute cytokine-mediated toxicity associated with the first dose administration.

[0174] In this study, escalation to mitigate the occurrence of cytokine-mediated toxicities can be explored based on an overall evaluation of clinical and supportive data as outlined in SET. Escalation will be guided by experience from early-dose cohorts that have already cleared their DLT phase. Given the SC route of administration and the intended therapeutic range of the molecule, escalation may be initiated prior to clinically observed AEs of these types. Escalation will involve administering one or more doses prior to the subsequent target dose. When reviewing emerging safety data (e.g., DLT) supporting continuous dose escalation using a BOIN design, the escalation dose will be less than or equal to the dose previously safely cleared by SET.

[0175] Figure 7 This demonstrates how to incorporate an escalation dosing schedule into an activity schedule (SoA) when administering one or more escalation doses.

[0176] Research Intervention The study will begin with fixed-dose administration and SC injection as the administration route. Dose escalation will begin with an initial dose of 0.6 mg SC every 21 days (Q3W) and will be guided by a BOIN design and follow dose escalation rules. Following SET approval, alternative dosing schedules (e.g., once weekly, Q2W, Q4W, with or without one or more escalation doses) may be evaluated based on emerging safety and pharmacokinetic data.

[0177] Multiple SC injections may be administered during the administration visit. The dose will be administered at alternating sites on the abdomen (other sites may be acceptable based on the judgment of the treating physician / sponsor's medical monitor). The maximum administration volume at each SC injection site will be 2 mL. In the event of significant discomfort during manual injection, the total dose may be administered at multiple separate sites.

[0178] The observation period begins after SC injection. All participants must be hospitalized for at least 72 hours after each escalation of compound 1 and after administration of the first treatment dose. For the first four target doses, participants should be observed for at least 6 hours after each study treatment administration (SET may review and extend or reduce the observation period as needed).

[0179] D. Research Group Screening for eligible participants will be conducted within 30 days prior to the administration of the study intervention. The inclusion and exclusion criteria for recruiting participants in this study are described below.

[0180] Selection criteria Each potential participant must meet all of the following research recruitment criteria: 1. The applicant must be at least 18 years old at the time of informed consent (or the legal age of majority in the jurisdiction where the research is conducted, whichever is greater).

[0181] 2. Diagnosed with ET that meets the stated risk criteria, as defined by the 2022 WHO criteria (2022 WHO Classification of Cancer Editorial Committee). or MF: Essential thrombocytosis High risk of thrombosis or bleeding is defined as any one of the following: • Age > 60 years old • Platelet count >1500 × 10⁶ at any point during the participant's illness. 9 Units / L • Whether it is a previously documented thrombosis (including transient ischemic attack [TIA]), erythromelalgia, or migraine (severe, recurrent, requiring medication, and considered secondary to MPN) that is considered to be related to the disease, whether it is after diagnosis or within 10 years prior to diagnosis.

[0182] • Previous bleeding or coagulopathy associated with ET • Diabetes or hypertension requiring medication for more than 6 months as well as According to NCCN Guideline Version 1.2023, intolerance, resistance, or refractory to hydroxyurea (HU) is defined as any one of the following: • After 3 months of at least 2 g / day or the maximum tolerated dose (MTD) of HU (2.5 g / day in participants weighing >80 kg), platelet count >600 × 10⁶. 9 Units / L • Platelet count >400 × 10⁻⁶ at any dose of HU 9 cells / L and WBC < 2.5 × 10 9 Units / L (for a period of at least 3 months) • Platelet count >400 × 10⁻⁶ at any dose of HU 9 The blood count / L and hemoglobin level <10 g / dL (for at least 3 months) • Leg ulcers or other unacceptable skin and mucous membrane manifestations are present at any dose of HU. • HU-related fever Myelofibrosis (primary or post-ET) Primary myelofibrosis The Dynamic International Prognostic Scoring System (DIPSS; Passamonti 2010, Blood. 115:1703-1708) classifies intermediate 2 or high risk, where the percentage of blast cells in the blood or bone marrow does not always exceed 20%. SET considers intermediate 1 recruitment based on emerging data.

[0183] ET after MF MYSEC-PM (Passamonti 2017, Leukemia.31:2726-2731) is intermediate 2 or high risk, where the percentage of primitive cells in the blood or bone marrow does not always exceed 20%. SET considers intermediate 1 recruitment based on emerging data.

[0184] as well as Unsuitable for JAK inhibitor (JAKi) therapy or intolerance or It is resistant / difficult to treat.

[0185] Not in compliance with the requirements JAKi is contraindicated due to a history of serious infections, such as tuberculosis, progressive multifocal leukoencephalopathy, and skin malignancies known to be associated with or exacerbated by JAKi, or other important considerations documented by the treating physician.

[0186] intolerance a) Despite recommendations for dose adjustment and discontinuation, hematological toxicity exists – platelet count <50 × 10⁻⁶. 9 ≤0.5×10⁶ cells / L and / or ≤0.5×10⁶ neutrophils / L 9 Units / L; or b) According to the 2017 Common Terminology Standard for Adverse Events (CTCAE) version 5.0, non-hematologic toxicity ≥ grade 3. With resistance / refractory The evidence will include: a) Persistent splenomegaly, or b) Lack of symptom improvement, or c) Persistent leukocytosis, or d) Anemia <10g / dL, or e) Leukocytosis >11×10 9 Units / L 3. ET or MF showed positive CALR driver mutations.

[0187] 4. Has received previous treatment: Essential thrombocytosis At least two prior lines of cytoreductive therapy (unless unavailable or contraindicated), of which at least one must be HU.

[0188] Myelofibrosis Unless the criteria are not met as described in Standard 2, at least one prior JAKi therapy shall be performed.

[0189] 5. The following therapies have been discontinued concurrently: Essential thrombocytosis Interferon-⍺ (pegylated or standard formulation), anagrelide, busulfan. Exception: HU permitted.

[0190] Myelofibrosis JAKi, immunomodulatory drug therapy (such as thalidomide), danazol, or other therapies designed to produce disease improvement.

[0191] 6. ECOG fitness level ≤ 2 (Oken 1982, Am J Clin Oncol. 5:649-655) 7. The following clinical hematological laboratory values ​​were present prior to drug administration: a. Hemoglobin ≥ 8.0 g / dL b. Within 4 weeks of the first dose of the study drug, without granulocyte growth factor assistance, the number of neutrophils was ≥0.75 × 10⁻⁶. 9 Units / L c. Without the aid of thrombopoietin or transfusion, the platelet count is 50 × 10⁻⁶. 9 Units / L and 600×10 9 Between units / L 8. Participants should have the following clinical chemistry laboratory values ​​prior to administration: a.ALT≤3×ULN b. AST≤3×ULN c. Direct bilirubin: ≤1.5×ULN d. Renal function: Glomerular filtration rate estimated or measured according to the CKD-EPI formula ≥40 mL / min 9. A known HIV-positive participant is eligible if they meet all of the following criteria: a. No detectable viral load (i.e., <50 copies / mL) was found during screening. b. During screening, the CD4+ count is >300 cells / mm3. c. No opportunistic infections defined as AIDS within 6 months of screening. d. Receive highly active antiretroviral therapy (HAART). Any changes in HAART due to resistance / progression should occur at least 3 months prior to screening. Changes in HAART due to toxicity are allowed up to 4 weeks prior to screening.

[0192] Note: Exclude HAARTs that may interfere with the study treatment (consult the sponsor to review the drug before recruitment).

[0193] 10. Participants with fertility potential must have a negative high-sensitivity serum (e.g., β-human chorionic gonadotropin [β-hCG]) pregnancy test at screening and within 72 hours of the first dose of study treatment, and must consent to further serum or urine pregnancy tests during the study.

[0194] 11. Participants with fertility potential must use at least one highly effective method of contraception throughout the study and for at least 90 days after the last dose of study treatment.

[0195] Note: If a participant becomes fertile after the start of the study, the participant must adhere to this criterion.

[0196] 12. Participants using oral contraceptives must use an additional barrier method of contraception.

[0197] 13. Participants must agree not to become pregnant, breastfeed, or plan to become pregnant at the time of recruitment to this study or within 90 days after the last dose of study treatment.

[0198] 14. Participants must agree to donate gametes (i.e., eggs or sperm) or freeze gametes for future use without the purpose of assisted reproduction during the study and for a period of 6 months after receiving the last dose of study treatment. Participants should consider preserving gametes before the study treatment, as cancer treatment may impair fertility.

[0199] 15. During the study and for at least 90 days after receiving the last dose of study treatment, participants must wear condoms when engaging in any activity that allows ejaculation into another person.

[0200] If a participant's partner is of fertility potential, the participant must use condoms (with or without spermicide), and the partner must also practice highly effective contraception. Male participants who have had their vas deferens removed must still use condoms (with or without spermicide), but their partners do not need to use birth control pills.

[0201] 16. Participants must agree not to have children at the time of recruitment to this study or within 3 months after the last dose of study treatment.

[0202] 17. Participants must sign an ICF (Initial Clinical Filing Form) indicating that they understand the purpose of the study and the required procedures and are willing to participate in the study.

[0203] 18. Willing and able to comply with the lifestyle restrictions set forth in this plan.

[0204] Exclusion criteria Any potential participant who meets any of the following criteria will be excluded from participation in the study: 1. Known allergy, hypersensitivity or intolerance to the excipients used in the investigational treatment.

[0205] 2. Prior to the planned first dose of study treatment, administer chemotherapy, cytoreductive therapy, targeted therapy, or immunotherapy for 5 half-lives or 2 weeks (whichever is shorter). Exceptions. 3. Any prior treatment with CALRmut targeted therapy. Exception: Participants previously exposed to CALRmut targeted therapy may be considered for recruitment with sponsor approval.

[0206] 4. A concurrent or recently diagnosed or treated malignant tumor was present at the time of participant selection. Exceptions include squamous and basal cell carcinoma of the skin, cervical carcinoma in situ, and any malignant tumor deemed to be cured or have a minimal risk of recurrence within one year of the first dose of study treatment, based on the opinion of both the investigator and the sponsor's medical monitor. Participants who have cured another malignant disease and have shown no signs of recurrence for ≥3 years after the completion of treatment are eligible to enroll in the study.

[0207] 5. Previous solid organ transplantation.

[0208] 6. Any of the following regarding hematopoietic stem cell transplantation: a. Prior treatment with allogeneic stem cell transplantation ≤6 months before the first dose of compound 1, or b. Evidence of graft-versus-host disease (GVHD) requiring immunosuppressive therapy 7. Active autoimmune diseases requiring systemic immunosuppressive drugs (e.g., chronic corticosteroids, methotrexate, or tacrolimus).

[0209] 8. Toxicity from previous anticancer therapies that has not yet regressed to baseline levels or to grade 1 or lower, or to grade ≤2 for alopecia, peripheral neuropathy, and vitiligo.

[0210] 9. Before the first dose of research treatment Within 6 months A clinically significant history of cardiovascular disease, including but not limited to: a. Myocardial infarction b. Severe or unstable angina c. Clinically significant ventricular arrhythmias or unexplained syncope (believed not to be vasovagal or due to dehydration). d. History of severe non-ischemic cardiomyopathy e. Congestive heart failure (New York Heart Association Class III to IV) f. Uncontrolled (persistent) hypertension: systolic blood pressure >159 mm Hg or diastolic blood pressure >99 mm Hg.

[0211] g. Stroke or transient ischemic attack h. Pericarditis or clinically significant pericardial effusion; i. Myocarditis j. Endocarditis k. Acute limb ischemia 10. Clinically significant lung damage, especially requiring supplemental oxygen to maintain adequate oxygenation.

[0212] 11. Evidence of active viral (including chronic Epstein-Barr virus), bacterial, or uncontrolled systemic fungal infection requiring systemic treatment within 7 days prior to the first dose of study treatment.

[0213] 12. Fever (body temperature ≥38.0℃ / 100.4℉) within 48 hours prior to the first dose of study treatment.

[0214] 13. Trauma or major surgery (e.g., requiring general anesthesia) within 28 days prior to the first dose of study treatment. Note: Participants scheduled for surgery under local anesthesia may participate.

[0215] 14. Any serious underlying medical or psychiatric condition (e.g., alcohol or drug abuse), dementia, or altered mental status; or any problem that would impair a participant’s ability to receive or tolerate planned treatment at the study site, to understand informed consent, or any problem that the investigator believes would hinder participation in the study or obscure the study assessment or results specified in the protocol.

[0216] 15. Prohibited drugs that cannot be discontinued, substituted, or temporarily interrupted during the research period.

[0217] 16. Administer the live attenuated vaccine within 4 weeks prior to the first administration of the study treatment.

[0218] 17. Weight <40 kg at screening and / or at the first administration of study treatment.

[0219] 18. Active infectious hepatitis: • Serological positivity for hepatitis B: Defined as a positive hepatitis B surface antigen (HBsAg) test. Real-time polymerase chain reaction (RT-PCR) measurements of hepatitis B virus (HBV) DNA levels must be used to screen for participants whose infection has regressed (i.e., participants who are negative for HBsAg using antibodies against total hepatitis B core antigen [anti-HBc], with or without hepatitis B surface antibody [anti-HBs]). Participants who are positive for RT-PCR will be excluded. Serological findings suggesting HBV vaccination (anti-HBs positivity is the sole serological marker) and participants with a known history of HBV vaccination do not require HBV DNA testing by PCR.

[0220] • Known hepatitis C virus infection or a positive serological test for hepatitis C virus (anti-HCV antibody).

[0221] A positive hepatitis C antibody test result is required at screening or within 3 months prior to starting treatment in the study. Note: Participants with positive hepatitis C antibodies due to remission of the previous disease can only be recruited if a confirmatory negative hepatitis C RNA test is obtained.

[0222] A positive hepatitis C RNA test result was required at screening or within 3 months prior to the first dose of study treatment. Note: Testing is optional, and participants with a negative hepatitis C antibody test do not need to undergo hepatitis C RNA testing.

[0223] • Other known clinically active sources of liver disease.

[0224] 19. History of hemophagocytic lymphohistiocytosis (HLH) / macrophage activation syndrome (MAS).

[0225] 20. History of pneumonia or interstitial lung disease.

[0226] 21. Those without evidence of stable anticoagulation therapy are defined as those who have not received anticoagulation therapy for ≥4 weeks prior to the first administration of study treatment.

[0227] E. Companion therapy Prior to the first dose of study treatment, prophylactic medications such as corticosteroids, antihistamines, and antipyretics will be administered to minimize the risks associated with CRS and sARR. Based on newly emerging data following a review of available data by the sponsor or SET, the prophylactic doses or schedules for subsequent doses may be reduced or omitted. For participants experiencing grade 2 or higher CRS, sARR, or neurotoxicity, pre-treatment corticosteroids will be required for at least one subsequent dose administered to that participant.

[0228] Participants in this study should receive pre-treatment medication as described below (9). Pre-treatment medication may be changed based on emerging safety and other data as determined by SET in Part 1 or Part 2.

[0229] Table 10: Pre-treatment medications F. Research Evaluation Overview Each of the two parts of this study was divided into three phases: screening, treatment, and post-treatment follow-up. All planned assessments, including clinical laboratory tests, must be completed, and results reviewed at each field visit. If multiple assessments are planned at the same time points, the following procedure is recommended: ECG, vital signs, blood draw.

[0230] Treatment decisions will be based on safety and disease assessments conducted at the local laboratory. More frequent study visits and repeated clinical assessments may be conducted if clinically indicated. Sample collection for PK, PD, and biomarker assessments should be kept as close as possible to the designated time. Additional measurements may be performed earlier than the designated time point if necessary. The actual date and time of the assessment will be recorded in the source document and eCRF or laboratory request form. Duplicate or unplanned samples (i.e., PK, PD, biomarkers) may be taken for safety reasons or due to technical issues with the samples. Additional serum or urine pregnancy tests may be performed as determined by the investigator or required by local regulations to determine the absence of pregnancy at any time during participation in this study.

[0231] All PRO assessments in Part 2 must be performed / completed before any testing, procedure, or other counseling to prevent impact on participant responses. For instructions on PRO administration, refer to the PRO Completion Guidelines. Symptom load will be periodically assessed by MPN-SAF-TSS in all participants in Part 2, and in selected participants in Part 1 at the sponsor's discretion.

[0232] Screening period All participants must sign the ICF before proceeding with any study-related procedures. The screening period begins at the time of the first screening assessment and ≤30 days prior to the first dose of study treatment. If the assessment is performed as part of a participant's routine clinical evaluation and is not specifically for this study, it does not need to be repeated after signed informed consent has been obtained, provided that the assessment meets the study requirements and is performed within the specified timeframe prior to the first dose of study treatment. A bone marrow biopsy sample is required at screening to establish the baseline histological status of a potential MPN, but does not confirm recruitment to the study.

[0233] During the screening period, a complete transfusion history for the 12 weeks prior to recruitment will be obtained, including the date, type (e.g., whole blood, platelets, hematocrit), number of transfusion units, and hemoglobin concentration or platelet count at the time of transfusion.

[0234] The presence of a diagnostic CALRmut genetic test is sufficient for recruitment into the trial. Molecular test results from the screening period are not reviewed prior to recruitment; instead, they are used for comparison with later disease assessments. After all screening assessments are completed, the results are reviewed, and eligibility (inclusion / exclusion) criteria are confirmed, and participants proceed to treatment.

[0235] Treatment period The treatment period begins with the first administration of study treatment and continues until the end of treatment (EOT) visit. Participants may be hospitalized for safety monitoring. Vital signs (including but not limited to pulse rate, blood pressure, oxygen saturation, and temperature) will be measured at intervals recorded in the Study Application (SoA) before and after administration of study treatment (Table 9). Potential toxicity to participants will be assessed at each research center visit. Bone marrow biopsy tissue will be collected from participants during the study according to the SoA (Table 9) in Parts 1 and 2 and will be shared with local investigators to allow for central review of bone marrow histopathology and local disease assessment. These samples will be sent to a central laboratory designated by the sponsor.

[0236] Assessments of transfusion events are collected on Day 1 of each cycle and during the EOT visit according to the SoA. Participants may continue to receive study treatment until any treatment discontinuation criteria are met. At the time of discontinuation of study treatment, participants who are clinically able to return for evaluation will complete the EOT visit.

[0237] Post-treatment visit The EOT visit will be completed 30 ± 14 days after the last dose of study treatment or before the start of subsequent anticancer therapy, whichever comes first. In cases where a decision is delayed due to potential toxicities requiring longer follow-up (>30 days), the EOT visit may be completed later. All participants are required to have an EOT visit, including those who discontinue study treatment for any reason other than loss to follow-up, death, or withdrawal of study participation.

[0238] Efficacy evaluation Efficacy assessments will include the procedures outlined in Table 9. These assessments should be performed at each time point throughout the study using the same methods used to assess baseline disease. Investigators will conduct tests that will allow evaluation of the response to therapy against the appropriate disease criteria and according to the schedule outlined in Table 9. The results of these assessments should preferably be available before initiating the next target dose of treatment.

[0239] As determined by the investigators, efficacy evaluation and review of hematological and clinical chemistry results will be conducted at the research center level. Response to treatment will be assessed by the investigators at the research center, and results will be recorded in the eCRF. Efficacy evaluation will continue until disease progression, initiation of subsequent anticancer therapy, withdrawal of study participation consent, or study completion, whichever comes first.

[0240] Hematological and Molecular Response According to Table 9, PB samples (approximately 10 mL of whole blood at each time point) will be collected on day 1 of administration, at 3, 6, 12, and 18 months from day 1, and then annually or when progression is suspected (±1 week) to assess hematological and molecular load responses in all participants. Additional unplanned laboratory monitoring should be performed as clinically indicated to enable the identification and management of new signs or symptoms that may be related to hematological or molecular responses.

[0241] Automated cell counting will be performed locally. Morphological characterization and cell (including platelet) distribution of PB smears or membranes will be evaluated locally. Molecular response assessment includes measuring CALRmut allele load using focused next-generation sequencing assays and will be evaluated by the central laboratory.

[0242] In view of this disclosure, hemoglobin concentrations in human blood can be determined before and during treatment using methods known in the art. See, for example, the Recommended Reference Methods for the Determination of Hemoglobin in Human Blood (ICSH Standard 1995) and the International Standard Specification for Hemoglobin Cyanide (4th Edition). Zwart et al., 7 Clin Pathol 1996; 49:271-274, the contents of which are incorporated herein by reference in their entirety.

[0243] Bone marrow assessment The purpose of repeat bone marrow biopsies is to allow for longitudinal assessment of megakaryocyte morphology and histological grading of fibrosis according to the European Consensus Grading System (Thiele 2005). Bone marrow biopsies were collected from all participants according to the SoA in Table 9, during the screening period and at 3, 6, 12, and 18 months from day 1 of treatment, and then annually or when progression was suspected (±1 week). Additional unplanned bone marrow assessments should be performed as clinically indicated to enable the identification and management of new signs or symptoms that may be related to hematological or molecular responses.

[0244] Biopsies performed during the screening period to assess the morphology, immunohistochemistry (IHC), and fibrosis of core biopsies do not constitute an evaluation of study recruitment eligibility but rather serve as a baseline for subsequent disease response assessment. Bone marrow biopsy samples are required to be provided in the form of formalin-fixed paraffin-embedded (FFPE) blocks to facilitate centralized staining and review. Unstained sections mounted on glass slides may be accepted with prior sponsor approval.

[0245] Spleen volume According to Table 9, all participants should undergo physical examinations at baseline / screening assessment, at 3, 6, 12, and 18 months from day 1, and then annually or when progression is suspected (±1 week). Special attention should be paid to the assessment of splenomegaly and hepatomegaly. In participants who underwent splenectomy, no measurements by imaging were indicated.

[0246] During Parts 1 and 2 of the study, spleen volume will be assessed using computed tomography (CT) or magnetic resonance imaging (MRI). With prior sponsor approval, ultrasound imaging may be used to assess spleen volume.

[0247] The method for assessing spleen volume at the research center should be consistent throughout the study. Typically, the volume segmentation method will be used in conjunction with the resulting total spleen volume calculation. This result needs to be recorded in the radiology report by a radiologist. If no volume method is available at the research center, the spleen volume should be estimated using the following formula by measuring three dimensions via CT or MRI: cephalocaudal (L), maximum dimension in the axial plane (D), and maximum thickness in the axial plane (T) (Prassopoulos 1997, Eur Radiol. 7:246-248).

[0248] Volume (mL) = 30 + (0.58 × L × D × T) If ultrasound imaging is used, the volume can be estimated using the following formula, which includes width (W), thickness (T), maximum length (ML), and head-to-tail length (CCL) (Yetter 2003, AJR Am J Roentgenol.181(6):1615-20).

[0249] Volume (mL) = 0.524 × W × T × (ML + CCL) / 2 In Part 2, spleen assessment should be performed on all participants via physical examination at baseline / screening assessment and during all disease evaluation visits. This will include measurements of organ enlargement below the costal margin.

[0250] Assessment of disease response and disease progression The efficacy of the study analysis will be evaluated according to the modified ELN criteria for essential thrombocythemia (Barosi 2009, Blood.113:4829-4833) and myelofibrosis (Tefferi 2013, Blood.122; 1395-1398) (see Table 11). Efficacy evaluation will be conducted and will continue until the study termination criteria are met.

[0251] Table 11: Response Evaluation Criteria Table 11a. Response criteria for participants with essential thrombocytosis Adapted from Barosi (2009), Blood. 113:4829-4833. Table 11 b. Response criteria for participants with myelofibrosis (Part 1 only) Adapted from Tefferi 2013, Blood. 122;1395-1398 Table 11 c. Response criteria for participants with myelofibrosis (Part 2 only) a. CR allocation does not require cytogenetic and molecular responses.

[0252] b. Immature bone marrow cells consist of primitive cells + premyelocytes + myelocytes + postmyelocytes + nucleated erythrocytes. In patients who have undergone splenectomy, <5% of immature bone marrow cells are permissible.

[0253] c. Increased severity of anemia leading to new transfusion dependence or a sustained decrease in hemoglobin levels of ≥2 g / dL relative to baseline for ≥12 weeks. According to CTCAE version 5.0, an increase in the severity of thrombocytopenia or neutropenia is defined as a decrease of 2 grades in platelet count or absolute neutrophil count relative to baseline. In addition, allocation to CI requires a minimum platelet count ≥25,000 × 10 (9) cells / L and an absolute neutrophil count ≥0.5 × 10 (9) cells / L.

[0254] d. Applicable only to patients with baseline hemoglobin <10 g / dL. In patients who do not meet the strict transfusion dependence criteria at the time of study recruitment but have received a transfusion within the previous month, the pre-transfusion hemoglobin level should be used as the baseline.

[0255] e. In patients who have had their spleen removed, palpable hepatomegaly is replaced with the same measurement strategy.

[0256] f. A splenic or hepatic response must be confirmed by imaging studies (for applicability, baseline splenomegaly or hepatomegaly must be palpable), which requires a ≥35% reduction in spleen volume as assessed by ultrasound (or other imaging modalities). Furthermore, regardless of the contents of the physical examination report, a ≥35% reduction in spleen or liver volume by ultrasound or other imaging modalities constitutes a response.

[0257] g. Disease progression assignment for splenomegaly needs to be confirmed by ultrasound or other imaging modalities showing an increase in spleen volume of ≥25% relative to baseline.

[0258] Adapted from Tefferi 2013, Blood. 122;1395-1398 Security assessment Safety will be monitored by SET. Safety assessment will be measured by adverse events (AEs), clinical laboratory test results, ECG, vital sign measurements, physical examination findings (including basic neurological examination), and ECOG performance status scores at the time points in Table 9. Safety monitoring (clinical and laboratory assessments) may be performed more frequently if clinically indicated, and investigators should evaluate AEs according to standard practice.

[0259] Adverse events AEs will be reported and tracked by the investigator, and will be graded according to NCI-CTCAE version 5.0, except that CRS and ICANS will be graded via ASCTC. Any clinically relevant changes that occur during the study must be documented in the AE section of the eCRF.

[0260] The study will include the following evaluations of safety and tolerability based on the time points provided in the SoA (Table 9).

[0261] Of particular interest are systemic administration-related reactions (sARR), neurotoxicity, and grade 3 cytokine release syndrome (CRS), which will be tracked as an AESI.

[0262] F. Analysis Procedure Pharmacokinetics Serum samples were analyzed by the sponsor or under the sponsor's supervision using a validated, specific, and sensitive immunoassay to determine the concentration of compound 1.

[0263] Individual pharmacokinetic (PK) parameters will be estimated, and descriptive statistics for each dose level will be calculated. PK parameters include, but are not limited to, Cmax, C-valence, Tmax, T1 / 2, AUC0-t, AUC0-τ, apparent total clearance (CL / F), and apparent volume of distribution (V / F); these parameters will be calculated if sufficient data are available for estimation. Furthermore, exploratory population-based PK methods can also be applied to PK analysis. Results of population PK analysis will be reported separately.

[0264] Immunogenicity Anti-compound 1 antibodies will be detected and characterized using a validated assay, either by the sponsor or under the sponsor's supervision. All serum samples collected for the detection of anti-compound 1 antibodies will also be evaluated for serum compound 1 concentrations to enable the interpretation of antibody data. Immune response analysis may be performed on PK samples collected at other time points. Additionally, serum samples should be collected from subjects who discontinued treatment or withdrew from the study at the final visit. These samples will be tested by the sponsor or its designated personnel.

[0265] Antibodies binding to compound 1 will be screened in serum samples, and the titers of confirmed positive samples will be reported. Neutralizing antibodies against compound 1 in ADA-positive samples may be further tested. If deemed necessary, immunoreactivity analysis may be performed on PK samples collected at other time points. Other analyses may be performed to characterize immunogenicity.

[0266] Receptor occupancy (RO) Whole blood samples were collected and analyzed for RO assessment to quantify the binding of compound 1 to CALRmut on the cell surface via flow cytometry. If the RO sample results from Part 1 were comprehensive and meaningful for the study of treatment response assessment, RO samples would be collected in Part 2.

[0267] Soluble CALRmut Serum levels of soluble CALRmut will be measured to explore its relationship with the pharmacokinetics, safety, and efficacy of compound 1. Serum samples will be analyzed using the Meso Scale Discovery (MSD) ECLIA method to determine soluble CALRmut concentrations. Serum samples used for soluble CALRmut assessment will be collected in Parts 1 and 2.

[0268] biomarkers Biomarker samples will be collected to identify PD markers indicating MOA of compound 1 and to evaluate the response / resistance mechanism.

[0269] Samples collected for biomarker evaluation will include bone marrow biopsies and PB samples (Table 12). These samples may be analyzed using techniques including, but not limited to, next-generation sequencing and proteomics. Flow cytometry-based evaluation or time-of-flight cytometry (CYTOOF) of immune cell subsets from PB will also be performed to identify exploratory biomarkers and evaluate hypotheses that treatment may be regulated by T-cell phenotype. Serum fractions isolated from blood will be used to analyze cytokines (including but not limited to IFN-γ, IL-2, IL-6, and IL-10), chemokines, or other secreted proteins. Samples may be used to help address emerging problems and enable the development of safer and more effective therapies. Biological samples collected in this study may be used to develop companion diagnostics. The table below provides an analysis of the planned sample collection during the study.

[0270] Table 12: Biomarkers of pharmacodynamics Immunophenotyping Bispecific antibodies exert their effects by binding to T cells, inducing T cell activation, margination, expansion, and tumor-killing activity. Overstimulation of the TCR and robust co-inhibitory molecular signaling, or the restricted presence of co-stimulatory mechanisms via CD28, can lead to T cell exhaustion and a lack of clinical response to T cell adaptors. Therefore, biomarkers for T cell activation and exhaustion will be evaluated, and the proportions of T cell subsets will be listed to help identify doses and procedures that may optimize therapeutic potential.

[0271] Pro-inflammatory cytokines Activated T cells produce certain pro-inflammatory cytokines to induce cytotoxic and immune responses. Therefore, pro-inflammatory cytokines will be measured to confirm MOA. Furthermore, since chronic inflammation is a hallmark of MPN and increased cytokine release can lead to CRS events, pro-inflammatory cytokine levels will be monitored to ensure safety and implement mitigation strategies if necessary.

[0272] Mutation spectrum analysis The frequency of variant alleles of the mutant CALR in peripheral blood will be monitored to correlate mutational burden with clinical response. Furthermore, many disease-specific comutations or cytogenetic abnormalities are common during disease progression. These mutations can drive disease severity and promote resistance to many therapies. Therefore, it is important to explore whether specific genetic alterations are associated with the antitumor activity of the investigated treatment.

[0273] Immunohistochemistry To evaluate bone marrow response, fibrosis, megakaryocyte morphology, and the presence of mutant CALR+, CD34+, and T cells will be reported intensively in bone marrow biopsies using H&E, special staining agents, and IHC.

[0274] Predictive biomarkers To identify predictors of response, clinical outcomes will be correlated with the mutant CALR load, presence of co-mutations, cytokine expression, and levels of immune cell subsets in tumor samples from whole blood at baseline. Other biomarkers may also be explored if new data become available. These results can serve as markers for predicting clinical response or resistance to guide future research.

[0275] G. Sample quantity determination Part 1: It is expected that 40-60 participants will be recruited in Part 1; the total number of participants recruited in Part 1 will depend on the number of queues explored to identify the putative RP2D and the number of participants recruited in each queue.

[0276] Part 2: In Part 2, up to approximately 20 participants will be recruited for each disease category cohort under each candidate RP2D and the selected schedule.

[0277] The total number of participants recruited in the clinical trial (including Part 1 and Part 2) is expected to be as high as approximately 100.

[0278] Regarding the determination of sample size in Part 2 and the evaluation of clinical activity or AE rate in 20 participants in a cohort, Table 13 provides 95% exact confidence intervals for several potential observed responses or AEs.

[0279] Table 13: Observed response or adverse event rates and 95% confidence intervals (sample size 20) Number of responses or adverse events Observed response rate or adverse event rate 95% confidence interval 2 10% (1%，32%) 3 15% (3%，38%) 4 20% (6%，44%) 5 25% (9%，49%) 6 30% (12%，54%) H. Statistical Analysis Primary endpoint The primary endpoints were the frequency of DLT and the frequency and severity of AE.

[0280] The PK analysis set will be used. All serum compound 1 concentrations below the lower limit of quantification or missing data will be marked as such in the concentration data display or Statistical Analysis System (SAS) dataset. In the summary statistics, serum compound 1 concentrations below the lower limit of quantification will be considered as zero.

[0281] Descriptive statistics will be used to summarize the serum concentration of compound 1 and its pharmacokinetic parameters (PK parameters) at each sampling time point: AUC0-τ, C-valence, and Cmax. Other PK parameters, including but not limited to Tmax, T1 / 2, AUC0-t, CL / F, and V / F (when available), will also be summarized. Mean (standard deviation) serum compound 1 concentration-time curves will be plotted after treatment administration, and individual serum compound 1 concentration-time curves may also be plotted.

[0282] The immunogenicity analysis will be descriptive in nature and will include the number and percentage of participants who produced antibodies against compound 1. A list of participants who were positive for antibodies against compound 1 will be provided. For participants who were positive for antibodies against compound 1, the maximum titer of antibodies against compound 1 will also be summarized. The impact of antibodies against compound 1 on pharmacokinetics, safety, and efficacy may also be evaluated.

[0283] The plan is to conduct analyses to identify MOA (modity of expression) indicative of the investigational treatment or biomarkers predicting efficacy and resistance. The association between biomarkers and clinical response or time-event endpoints will be assessed using appropriate statistical methods (analysis of variance [ANOVA], categorical, or survival models) based on the endpoints. The correlation between baseline expression levels or changes in expression levels and response or time-on-event endpoints will identify responding or resistant subgroups.

[0284] Secondary endpoint In Part 1 (Dose Escalation), the primary biomarkers of response will be hematological, molecular, histological, and splenic responses. Part 2 (Cohort Expansion) will also incorporate measures of symptom burden based on an appropriate Patient-Reported Outcomes (PRO) questionnaire.

[0285] The response rate will be tabulated along with its two-sided 95% exact confidence intervals. Additionally, the number and percentage of participants in each response category will be tabulated. Time to Response (TTR) and Do-Obstruction (DOR) estimates will be provided using the Kaplan-Meier method. These will be applied to the response components and the overall response rate according to ELN 2013.

[0286] The efficacy endpoint is: • Overall Response Rate (ORR): ORR is defined as the proportion of participants who achieve PR and CR according to disease criteria. Investigators will evaluate the response to treatment administered with the study treatment.

[0287] • Complete Response Rate (CRR): The CR rate is defined as the proportion of participants who achieve the optimal CR response based on the disease.

[0288] • Time to Response (TTR): For participants who achieve PR or CR, TTR is defined as the time from the first dose of study treatment to the first response of PR or CR.

[0289] • Duration of Response (DOR): For participants who achieved PR or CR, DOR is defined as the time between the initial recorded date of PR or CR and the date of the first recorded evidence of disease progression or death, whichever comes first. For participants who did not progress or die, data will be reviewed at the last disease evaluation.

[0290] result This study is ongoing. No unexpected safety or tolerability issues were identified from the interim safety and tolerability results. All patients experienced low-grade injection site reactions (ISR). All were grade 1 except for one case of grade 2 ISR. Some patients experienced ISR again at subsequent doses, which improved over time. The peak ISR grade did not increase as the dose increased from 0.6 mg to 4.8 mg (8-fold). Low-grade CRS was observed in some patients. An escalation strategy was implemented in all new cohorts to mitigate ISR and CRS.

[0291] Early efficacy observations guided dose escalation. Cumulative evidence showed benefit from doses increasing from 2.4 mg. Cohort results showed hemoglobin recovery after treatment with compound 1. See, for example, Table 14 and... Figure 9 .

[0292] Table 14. Changes in hemoglobin relative to baseline in subjects treated with various doses of compound 1 .

[0293] Dosage 1: 1.2 mg Dosage 2: 1.8 mg Dosage 3: 2.4 mg (with or without increments) A negative value indicates a decrease in hemoglobin levels relative to baseline. The baseline is the hemoglobin level before administration on day 1 of treatment.

[0294] sequence list

Claims

1. A method for inhibiting the growth or proliferation of myeloproliferative neoplasm (MPN) or treating MPN, the method comprising administering to a subject in need, preferably a human subject in need, a therapeutic dose of 0.6 mg to 400 mg per administration of an anti-mutant calreticulin (CALRmut) / anti-CD3 bispecific antibody, wherein the anti-CALRmut / anti-CD3 bispecific antibody comprises a first antigen-binding domain specifically binding to CALRmut and a second antigen-binding domain specifically binding to CD3ε.

2. The method according to claim 1, wherein the first antigen-binding domain comprises the first HCDR1, first HCDR2 and first HCDR3 of the first heavy chain variable region (VH1) of SEQ ID NO:14, and the first antigen-binding domain further comprises the first light chain complementarity-determining region (LCDR)1, first LCDR2 and first LCDR3 of the first light chain variable region (VL1) of SEQ ID NO:16, and wherein the first HCDR1, first HCDR2, first HCDR3, first LCDR1, first LCDR2 and first LCDR3 are defined by the Kabat, Chothia, IMGT or AbM numbering system.

3. The method of claim 2, wherein the first antigen-binding domain comprises the first HCDR1, the first HCDR2, the first HCDR3, the first LCDR1, the first LCDR2, and the first LCDR3 having the following amino acid sequence: 1) These are SEQ ID NOs: 11, 12, 13, 7, 8, and 9, respectively; 2) These are SEQ ID NOs: 68, 69, 13, 7, 8, and 9, respectively; 3) They are SEQ ID NO: 10, 67, 13, 7, 8 and 9 respectively; or 4) The amino acid sequences of SEQ ID NO: 70, 71, 72 and 66, DAS and SEQ ID NO: 9, respectively.

4. The method according to any one of claims 1 to 3, wherein the first antigen-binding domain comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

16.

5. The method according to any one of the preceding claims, wherein the first antigen-binding domain comprises VH1 of SEQ ID NO:14 and VL1 of SEQ ID NO:

16.

6. The method according to any one of the preceding claims, wherein the first antigen-binding domain comprises Fab.

7. The method according to any one of the preceding claims, wherein the second antigen-binding domain comprises the second HCDR1, second HCDR2 and second HCDR3 of the second heavy chain variable region (VH2) of SEQ ID NO:23 and the second LCDR1, second LCDR2 and second LCDR3 of the second light chain variable region (VL2) of SEQ ID NO:27, wherein the second HCDR1, second HCDR2, second HCDR3, second LCDR1, second LCDR2 and second LCDR3 are defined by the Kabat, Chothia, IMGT or AbM numbering system.

8. The method of claim 7, wherein the second antigen-binding domain comprises the second HCDR1, the second HCDR2, the second HCDR3, the second LCDR1, the second LCDR2, and the second LCDR3 having the following amino acid sequence: 1) These are SEQ ID NOs: 20, 21, 22, 24, 25, and 26, respectively; 2) These are SEQ ID NOs: 75, 76, 22, 24, 25, and 26, respectively; 3) These are SEQ ID NOs: 73, 74, 22, 24, 25, and 26, respectively; or 4) The amino acid sequences of SEQ ID NO: 78, 79, 80 and 81, YAS and SEQ ID NO: 26 are respectively.

9. The method of claim 8, wherein the second antigen-binding domain comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:23, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

27.

10. The method according to claim 9, wherein the second antigen-binding domain comprises VH2 of SEQ ID NO:23 and VL2 of SEQ ID NO:

27.

11. The method according to any one of the preceding claims, wherein the second antigen-binding domain comprises scFv.

12. The method of claim 11, wherein the scFv is an spFv having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

82.

13. The method according to any one of the preceding claims, wherein the first binding domain comprises Fab having a VH1 having the amino acid sequence of SEQ ID NO:14 and a VL1 having the amino acid sequence of SEQ ID NO:16; and wherein the second binding domain comprises spFv having the amino acid sequence of SEQ ID NO:

82.

14. The method according to any one of the preceding claims, wherein the first antigen-binding domain is linked to a first immunoglobulin (Ig) constant region or a fragment of the first Ig constant region, and / or the second antigen-binding domain is linked to a second immunoglobulin (Ig) constant region or a fragment of the second Ig constant region, wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region are IgG1, IgG2 and IgG3 or IgG4 isotypes.

15. The method of claim 14, wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region is an IgG1 isotype.

16. The method according to any one of claims 14 and 15, wherein the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises at least one mutation that causes a reduction in the binding of the anti-CALRmut / anti-CD3 bispecific antibody to FcgR, such as one, two, or three mutations of L234A, L235A, and D265S, wherein the residues are numbered according to the Kabat EU index.

17. The method of claim 16, wherein the FcgR is FcgRI, FcgRIIA, FcgRIIB, or FcgRIII, or any combination thereof.

18. The method according to any one of claims 14 to 17, wherein the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region contain one or more heterodimerization mutations to promote heterodimerization, such as mortar (KiH) substitution.

19. The method of claim 18, wherein one of the first Ig constant region (or a fragment of the first Ig constant region) and the second Ig constant region (or a fragment of the second Ig constant region) comprises the heterodimerization mutation T366W, and the other of the first Ig constant region (or a fragment of the first Ig constant region) and the second Ig constant region (or a fragment of the second Ig constant region) comprises the heterodimerization mutations T366S, L368A, and Y407V.

20. The method according to any one of claims 14 to 19, wherein one of the first Ig constant region (or a fragment of the first Ig constant region) and the second Ig constant region (or a fragment of the second Ig constant region) further comprises the mutations H435R and Y436F.

21. The method according to any one of the preceding claims, wherein the anti-CALRmut / anti-CD3 bispecific antibody comprises a first heavy chain (HC1) having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:15, a first light chain (LC1) having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:17, and a second heavy chain (HC2) having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

28.

22. The method of claim 21, wherein the anti-CALRmut / anti-CD3 bispecific antibody comprises the HC1 having the amino acid sequence of SEQ ID NO:15, the LC1 having the amino acid sequence of SEQ ID NO:17, and the HC2 having the amino acid sequence of SEQ ID NO:

28.

23. The method according to any one of the preceding claims, wherein the therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody is administered subcutaneously, preferably at a therapeutic dose of 20 mg to 200 mg per administration.

24. The method according to any one of the preceding claims, wherein the anti-CALRmut / anti-CD3 bispecific antibody is administered once every 1 to 8 weeks.

25. The method of claim 24, wherein the therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody is administered once every three weeks.

26. The method of claim 24, wherein the therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody is administered once weekly.

27. The method of claim 24, wherein the therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody is administered every two weeks.

28. The method of claim 24, wherein the therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody is administered once every four weeks.

29. The method according to any one of the preceding claims, further comprising administering to the subject one or more escalating doses of the anti-CALRmut / anti-CD3 bispecific antibody prior to administering the therapeutic dose, wherein none of the escalating doses exceeds the therapeutic dose.

30. The method of claim 29, wherein the escalating dose is administered to the subject 3 to 8 days, such as 3, 4, 5, 6, 7, or 8 days, prior to the initial administration of the therapeutic dose.

31. The method of claim 29 or 30, the method comprising administering to the subject a first escalating dose and a second escalating dose of the anti-CALRmut / anti-CD3 bispecific antibody, wherein the first escalating dose is administered before the administration of the second escalating dose, preferably 3 to 8 days prior to the administration of the second escalating dose, such as 3, 4, 5, 6, 7, or 8 days prior to the administration of the second escalating dose, and wherein the second escalating dose is administered before the administration of the initial treatment dose, preferably 3 to 8 days prior to the administration of the initial treatment dose, such as 3, 4, 5, 6, 7, or 8 days prior to the administration of the initial treatment dose.

32. The method of claim 31, wherein the first incremental dose does not exceed the second incremental dose, and the second incremental dose does not exceed the initially applied treatment dose.

33. A method of treating MPN, the method comprising subcutaneously administering, once every three weeks, a therapeutic dose of antiCALRmut / antiCD3 bispecific antibody, such as 20 mg to 200 mg per dose, to a human subject in need, at a dose of 1.2 mg to 400 mg per dose, wherein the antiCALRmut / antiCD3 bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO:15, a first light chain having the amino acid sequence of SEQ ID NO:17, and a second heavy chain having the amino acid sequence of SEQ ID NO:28; optionally, the method further comprises subcutaneously administering, one week prior to the initial administration of the therapeutic dose, the antiCALRmut / antiCD3 bispecific antibody at escalating doses of 0.6 mg or 1.2 mg per dose to the subject.

34. The method according to any one of the preceding claims, wherein the MPN is characterized by the presence of a mutant calreticulin, such as a type 1-like and / or type 2-like CALR mutation.

35. The method according to any one of the preceding claims, wherein the subject is ineligible, intolerant, or resistant to JAK inhibitor therapy.

36. The method according to any one of the preceding claims, wherein the subject undergoes splenectomy.

37. The method according to any one of the preceding claims, wherein the subject receives an allogeneic graft, such as an allogeneic bone marrow or stem cell graft.

38. The method according to any one of the preceding claims, wherein the subject has been administered prior therapy for treating the MPN, such as a JAK inhibitor and / or hydroxyurea, and optionally, one or more prior lines of treatment for the subject have failed.

39. The method according to any one of the preceding claims, wherein the MPN is selected from the group consisting of: chronic myeloid leukemia, polycythemia vera, primary myelofibrosis (MF), essential thrombocythemia (ET), chronic neutrophilic leukemia and chronic eosinophilic leukemia, such as myeloproliferative syndromes selected from ET, pre-fibrotic MF, dominant primary MF and accelerated blast phase MF.

40. The method of claim 39, wherein the subject is diagnosed with ET, particularly having ET with a high risk of thrombosis or bleeding defined as any one of the following: • Age > 60 years old • Platelet count >1500 × 10⁶ at any point during the participant's illness. 9 Units / L • Whether it is a previously documented thrombosis (including transient ischemic attack [TIA]), erythromelalgia, or migraine (severe, recurrent, requiring medication, and considered secondary to MPN) that is considered to be related to the disease, whether it is after diagnosis or within 10 years prior to diagnosis. • Previous bleeding or coagulopathy associated with ET, or • Diabetes or hypertension requiring medication for more than 6 months as well as According to NCCN Guideline Version 1.2023, ET is defined as any one of the following: intolerance to, resistance to, or refractory to hydroxyurea (HU): • After 3 months of at least 2 g / day or the maximum tolerated dose (MTD) of HU (2.5 g / day in participants weighing >80 kg), platelet count >600 × 10⁶. 9 Units / L • Platelet count >400 × 10⁻⁶ at any dose of HU 9 cells / L and WBC < 2.5 × 10 9 Items / L (for a period of at least 3 months), • Platelet count >400 × 10⁻⁶ at any dose of HU 9 The number of cells / L and hemoglobin <10g / dL (for a period of at least 3 months) are required. • At any dose of HU, leg ulcers or other unacceptable skin and mucous membrane manifestations are present, or • HU-related fever.

41. The method of claim 40, wherein the method causes at least one of the following: 1) normal spleen size at imaging; and 2) peripheral blood platelet count ≤ 400 × 10⁻⁶. 9 Cells / L and / or white blood cell count ≤10×10 9 per L.

42. The method of claim 39, wherein the subject is diagnosed with MF, particularly primary MF, such as primary MF with a Dynamic International Prognostic Scoring System (DIPSS) risk score of intermediate 1 (Int-1), intermediate 2 (Int-2), or high risk (HR), optionally with the percentage of blast cells in the blood or bone marrow not always exceeding 20%; or post-ET MF, such as post-ET MF with a PV and ET Secondary Myelofibrosis Prognostic Model (MYSEC-PM) risk score of Int-1, Int-2, or HR, optionally with the percentage of blast cells in the blood or bone marrow not always exceeding 20%.

43. The method of claim 42, wherein the method causes a reduction in the spleen volume of the subject compared to a baseline spleen volume measured before administration of the anti-mutated calreticulin (CALRmut) / anti-CD3 bispecific antibody, preferably a reduction of at least 35% compared to a baseline spleen volume measured before administration of the anti-mutated calreticulin (CALRmut) / anti-CD3 bispecific antibody.

44. The method according to claim 42 or 43, wherein the method results in at least one of the following: 1) age-adjusted normal bone marrow cell composition, 2) <5% blast cells, and 3) peripheral blood hemoglobin ≥10 g / dL and neutrophil count ≥1 × 10⁻⁶ in the subject. 9 CFU / L, and / or platelet count ≥100×10⁻⁶ 9 per L.

45. The method according to any one of the preceding claims, wherein the method reduces the level of soluble CALRmut in the serum of the subject, preferably by at least 50% compared with a baseline level measured before administration of the anti-mutated calreticulin (CALRmut) / anti-CD3 bispecific antibody.

46. ​​The method according to any one of the preceding claims, wherein the method causes a reduction in CALRmut-positive cells in the bone marrow of the subject.

47. The method according to any one of the preceding claims, wherein the method causes an improved bone marrow structure, such as a reduction in bone marrow reticular fibrosis and an improved bone marrow cell composition.

48. The method according to any one of the preceding claims, further comprising administering at least one first additional therapeutic agent to the subject before administering the therapeutic dose of the antiCALRmut / antiCD3 bispecific antibody, or before administering the escalating dose of the antiCALRmut / antiCD3 bispecific antibody.

49. The method of claim 48, wherein the at least one first additional therapeutic agent is a glucocorticoid, an antihistamine, an antipyretic, an antiemetic, or any combination thereof.

50. The method of claim 48, wherein at least one first additional therapeutic agent is dexamethasone.

51. The method of claim 48, wherein at least one first additional therapeutic agent is diphenhydramine or an equivalent.

52. The method of claim 48, wherein at least one first additional therapeutic agent is acetaminophen.

53. The method of claim 48, wherein the at least one first additional therapeutic agent is ondansetron or an equivalent thereof.

54. The method according to any one of the preceding claims, further comprising administering at least one second additional therapeutic agent to the subject before and optionally after administering the therapeutic dose of the anti-CALRmut / anti-CD3 bispecific antibody.

55. The method of claim 54, wherein the at least one second additional therapeutic agent is an H1 antagonist, an H2 antagonist, or a leukotriene inhibitor, or any combination thereof.

56. The method of claim 54, wherein the at least one second additional therapeutic agent is loratadine, cetirizine, or an equivalent thereof.

57. The method of claim 54, wherein the at least one second additional therapeutic agent is ranitidine or an equivalent thereof.

58. The method of claim 54, wherein the at least one second additional therapeutic agent is montelukast or an equivalent thereof.

59. An anti-CALRmut / anti-CD3 bispecific antibody, said anti-CALRmut / anti-CD3 bispecific antibody for use in the method according to any one of the preceding claims.

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