Bispecific antibodies targeting CD117 and CD3

Abstract

Provided herein are bispecific antibodies comprising a first antigen-binding region and a second antigen-binding region wherein the first antigen-binding region binds CD117 and comprises a first heavy chain variable region and a first light chain variable region and the second antigen-binding region binds CD3 and comprises a second heavy chain variable region and a second light chain variable region. Also provided herein are compositions comprising the bispecific antibodies and uses of the bispecific antibodies and compositions.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefits of International Application No. PCT / CN2022 / 140683, filed on December 21, 2022, which is incorporated herein by reference. Technical field

[0003] The present invention relates to bispecific antibodies that bind to CD117 and CD3, antigen - binding fragments thereof, and uses of these bispecific antibodies. Background art

[0004] Hematopoietic stem cell transplantation has been proven effective in treating hematological diseases. However, current hematopoietic stem cell transplantation therapies are often associated with severe toxicity due to the use of radiotherapy or chemotherapy, which puts patients at high risk of life - threatening infections and toxicities. Severe infections often occur immediately after hematopoietic stem cell transplantation, and complications caused by chemical toxicity and radiation damage affect multiple organs in the long term. An antibody that can rapidly and transiently deplete hematopoietic stem cells can create the required bone marrow space, thus enabling sufficient hematopoietic stem cell engraftment. A hematopoietic stem cell - depleting antibody with a short in - vivo half - life and high efficiency is ideal for the rapid and effective treatment of patients.

[0005] Antibody - mediated pretreatment of the host bone marrow before hematopoietic stem cell transplantation is promising but not optimal. Among the targets for hematopoietic stem cell depletion, the CD117 receptor is a potential target that is expressed on all hematopoietic stem cells. Czechowicz and his colleagues found that the anti - CD117 antibody ACK2 could block the function of CD117 in mice, resulting in a temporary loss of more than 98% of endogenous hematopoietic stem cells (Czechowicz A, et al., Science. 2007;318(5854):1296 - 1299). Another study by Czechowicz showed that an anti - CD117 antibody - drug conjugate (CD117 - ADC) conjugated with Saporin caused depletion of more than 99% of hematopoietic stem cells in mice (Czechowicz A et al., Nat.Commun. 2019;10(1):617).

[0006] In humans, existing antibodies have poor efficacy. An anti-human CD117 antibody, SR-1, inhibits hematopoietic stem cells in vitro. Humanized SR-1 (AMG191) can deplete hematopoietic stem cells by inhibiting the CD117 signaling pathway (WO 2019 / 113437 A1; Pang W.W. et al., Blood. 2019;133(19):2069-2078). In non-human primates, the circulating time of AMG191 depends on the dose, with a T½ of 1.88 (0.1 mg / kg) to 4.3 days (25 mg / kg). In non-human primates and human bone marrow CD34+ xenograft immunodeficient mice, AMG191 treatment can deplete endogenous hematopoietic stem cells and condition the bone marrow for hematopoietic stem cell transplantation (Kwon H.S. et al., Blood. 2019;133(19):2104-2108). However, the first results of an ongoing clinical trial (trial number: NCT02963064) showed that pretreatment of severe combined immunodeficiency (SCID) patients with the full-length AMG191 antibody targeting CD117 resulted in a low chimerism rate (3-10%) after allogeneic hematopoietic stem cell transplantation (Agarwal R. et al., Biology of Blood and Marrow Transplantation. 2019;25(3):S92). In this study, hematopoietic stem cell transplantation was performed only when the antibody plasma level reached <100 ng / mL, and it might take up to 20 days after treatment to reach this level. Due to the long interval between antibody injection and reaching a level suitable for hematopoietic stem cell transplantation, the synchronous recovery of the endogenous hematopoietic stem cell pool becomes a potential problem with this treatment strategy.

[0007] There is an urgent need for a rapid and low-risk hematopoietic stem cell depletion protocol in current hematopoietic stem cell transplantation technology. Drugs with a short half-life and high hematopoietic stem cell depletion ability are more suitable for safe bone marrow conditioning and rapid hematopoietic stem cell transplantation. Summary of the Invention

[0008] In a first aspect, the present invention provides a bispecific antibody or an antigen-binding fragment thereof, which comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to CD117 and comprises a first heavy chain variable region (VH_A) and a first light chain variable region (VL_A), and the second binding region binds to CD3 and comprises a second heavy chain variable region (VH_B) and a second light chain variable region (VL_B), wherein:

[0009] The VH_A comprises HCDR1, HCDR2, and HCDR3 of VH having an amino acid sequence containing SEQ ID NO: 44;

[0010] The VL_A comprises LCDR1, LCDR2 and LCDR3 of VL containing the amino acid sequence of SEQ ID NO: 47;

[0011] The VH_B comprises HCDR1, HCDR2 and HCDR3 of VH containing the amino acid sequence of SEQ ID NO: 43; and

[0012] The VL_B comprises LCDR1, LCDR2 and LCDR3 of VL containing the amino acid sequence of SEQ ID NO: 46.

[0013] In some embodiments of the bispecific antibody or its antigen-binding fragment,

[0014] The VH_A comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 6, 12 and 18 respectively;

[0015] The VL_A comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 26, 32 and 38 respectively;

[0016] The VH_B comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 5, 11 and 17 respectively; and

[0017] The VL_B comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 25, 31 and 37 respectively.

[0018] In some embodiments of the bispecific antibody or its antigen-binding fragment,

[0019] The VH_A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 44;

[0020] The VL_A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 47;

[0021] The VH_B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 43; and

[0022] The VL_B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 46.

[0023] In some embodiments of the bispecific antibody or antigen-binding fragment thereof,

[0024] the VH_A comprises the amino acid sequence as shown in SEQ ID NO: 44;

[0025] the VL_A comprises the amino acid sequence as shown in SEQ ID NO: 47;

[0026] the VH_B comprises the amino acid sequence as shown in SEQ ID NO: 43; and

[0027] the VL_B comprises the amino acid sequence as shown in SEQ ID NO: 46.

[0028] In some other embodiments of the bispecific antibody or antigen-binding fragment thereof, the antibody comprises three polypeptide chains, wherein:

[0029] the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH_A-CH1; the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL_A-CL-L-VH_B-CH1; and the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL_B-CL; or

[0030] the first polypeptide chain comprises, from the N-terminus to the C-terminus: VH_B-CH1; the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL_B-CL-L-VH_A-CH1; and the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL_A-CL,

[0031] wherein CH1 represents the first domain of the constant region of the immunoglobulin heavy chain; CL each represents the constant region of the immunoglobulin light chain; and L is absent or represents an optional linker.

[0032] In some embodiments, CL in the second polypeptide chain and CL in the third polypeptide chain each independently comprise a constant region derived from a λ light chain or a κ light chain.

[0033] In some embodiments, CL in the second polypeptide chain and CL in the third polypeptide chain are derived from different types of immunoglobulin light chains.

[0034] In some embodiments of the bispecific antibody or antigen-binding fragment thereof, CL in the second polypeptide chain comprises a light chain constant region derived from a λ light chain (such as a human λ light chain), and CL in the third polypeptide chain comprises a light chain constant region derived from a κ light chain (such as a human κ light chain).

[0035] In some other embodiments of the bispecific antibody or antigen-binding fragment thereof, the CL in the second polypeptide chain comprises a light chain constant region derived from a κ light chain (e.g., human κ light chain), and the CL in the third polypeptide chain comprises a light chain constant region derived from a λ light chain (e.g., human λ light chain).

[0036] In some embodiments of the bispecific antibody or antigen-binding fragment thereof, the VL_A comprises a light chain variable region derived from the same type of immunoglobulin light chain as the CL in the same polypeptide chain, and / or the VL_B comprises a light chain variable region derived from the same type of immunoglobulin light chain as the CL in the same polypeptide chain.

[0037] In some embodiments of the bispecific antibody or antigen-binding fragment thereof, each CH1 independently comprises a CH1 derived from immunoglobulin isotype IgG (e.g., human IgG), optionally each CH1 independently comprises a CH1 derived from an IgG subtype selected from the group consisting of IgG1, IgG2, and IgG4 (e.g., human IgG1, IgG2, and IgG4).

[0038] In some embodiments of the bispecific antibody or antigen-binding fragment thereof, the linker comprises the amino acid sequence of (G4S)n, where n is an integer selected from 1-5, such as 1, 2, 3, 4, or 5.

[0039] In some embodiments of the bispecific antibody or antigen-binding fragment thereof,

[0040] the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 57;

[0041] the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 56; and

[0042] the third polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 52.

[0043] In some embodiments of the bispecific antibody or antigen-binding fragment thereof,

[0044] the first polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO: 57;

[0045] The second polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO: 56; and

[0046] The third polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO: 52.

[0047] In some embodiments of the bispecific antibody or antigen-binding fragment thereof, the antibody does not comprise an immunoglobulin Fc region.

[0048] In a second aspect, the present invention provides a nucleotide (e.g., an isolated nucleic acid) encoding the bispecific antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0049] In a third aspect, the present invention provides a vector comprising the nucleic acid of the second aspect of the present invention. In some preferred embodiments, the vector is an expression vector.

[0050] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid of the second aspect of the present invention or the vector of the third aspect of the present invention.

[0051] In a fifth aspect, the present invention provides a method for preparing the bispecific antibody or antigen-binding fragment thereof of the first aspect of the present invention, wherein the method comprises

[0052] a) culturing the host cell of the fourth aspect of the present invention under conditions suitable for producing the bispecific antibody or antigen-binding fragment thereof; and

[0053] b) obtaining the bispecific antibody or antigen-binding fragment thereof from the culture.

[0054] In a sixth aspect, the present invention provides an antibody-drug conjugate (ADC) comprising the bispecific antibody or antigen-binding fragment thereof of the first aspect of the present invention and a cytotoxic moiety conjugated to the bispecific antibody or antigen-binding fragment thereof.

[0055] In a seventh aspect, the present invention provides a composition comprising the bispecific antibody or antigen-binding fragment thereof of the first aspect of the present invention. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier and / or excipient.

[0056] In an eighth aspect, the present invention provides a method for depleting hematopoietic stem cells in a subject in need thereof, the method comprising administering to the subject the bispecific antibody or antigen-binding fragment thereof of the first aspect of the present invention, the nucleic acid of the second aspect of the present invention, the vector of the third aspect of the present invention, the ADC of the sixth aspect of the present invention, or the composition of the seventh aspect of the present invention.

[0057] In a ninth aspect, the present invention provides a method for treating a hematopoietic system disease in a subject in need of treatment, comprising:

[0058] a) administering to the subject a bispecific antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, an ADC according to the sixth aspect of the present invention, or a composition according to the seventh aspect of the present invention; and

[0059] b) transplanting hematopoietic stem cells into the subject.

[0060] In some embodiments of the method, step b) is carried out within 24 hours, 48 hours, 72 hours, 5 days, 7 days, 2 weeks, 3 weeks or 1 month after step a).

[0061] In some embodiments, the hematopoietic system disease is selected from the group consisting of hemoglobinopathies, immunodeficiencies, metabolic disorders, blood system diseases (such as bone marrow failure syndromes, myelodysplastic syndromes, acute lymphocytic and myelocytic leukemias, and chronic lymphocytic and myelocytic leukemias), hematopoietic cell proliferative diseases, transplant rejection, autoimmune diseases, and autoinflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 : Schematic diagram of the structure of the anti-CD117xCD3 bispecific T cell engager PR004384

[0063] Figure 2 : Anti-CD117xCD3 PR004384 specifically binds to CD117-positive hematopoietic cells and CD3-positive T cells

[0064] (A) FACS plots showing the binding of anti-CD117xCD3 PR004384 and the control anti-HELxCD3 bispecific T cell engager to CD117 expressed on TF-1 cells. The RPMI-8226 and U937 cell lines were used as negative controls. (B) Flow cytometry was used to detect the affinity of the anti-CD117xCD3 bispecific T cell engager for TF-1 cells. (C) FACS plots showing the binding of anti-CD117xCD3 PR004384 and the control anti-HELxCD3 bispecific T cell engager to CD3 expressed on T cells and PBMCs. T cells and PBMCs pretreated with an anti-CD3 blocking antibody were used as negative controls.

[0065] Figure 3 : Anti-CD117xCD3 PR004384 promotes the activation of T cells

[0066] (A) FACS plots showing the proliferation of T cells under the specified conditions. Gates show the reduced CellTrace FarRed signal and the percentage of proliferating cells. (B) FACS plots and quantification results of the activation markers CD69 and CD25 expressed on CD4+ and CD8+ T cells when T cells and TF-1 cells are co-cultured together in the presence of the specified reagents. (C) Determination of cytokine production after purified T cells are co-cultured with TF-1 cells under different concentrations of bispecific T cell engager in an in vitro experiment. Supernatants are collected after the TDCC experiment, and the levels of each cytokine in the supernatants are shown in the figure.

[0067] Figure 4 : PR004384 anti-CD117xCD3 PR004384 effectively depletes human CD117-positive hematopoietic cells in vitro

[0068] (A) Percentages of clearance of TF-1 cells (left) and hCD34+ cells (right) mediated by T cells when treated with the anti-CD117xCD3 bispecific T cell engager PR004384 or the control HELxCD3 bispecific T cell engager in vitro. (B) Percentages of viable TF-1 cells when co-treated with the anti-CD117xCD3 bispecific T cell engager PR004384 or the control anti-HELxCD3 bispecific T cell engager and purified mouse T cells.

[0069] Figure 5 : Determination of the in vivo half-life T½ of the anti-CD117xCD3 bispecific T cell engager in serum.

[0070] Blood samples are collected at the specified time points, and the concentration of the bispecific T cell engager in serum is determined by ELISA.

[0071] Figure 6 : Anti-CD117xCD3 PR004384 effectively depletes human CD117-positive hematopoietic cells in humanized mice in vivo

[0072] (A) Treatment protocol for humanized mice. (B) Representative example of FACS plots showing the percentage of human CD34+ CD117+ cells in total human CD45+ bone marrow cells in humanized mice after the specified treatment and at the specified time points. (C) Graph showing the percentage of CD34+CD117+ cells in total human bone marrow cells in humanized mice at the specified time points after the specified treatment. (D) Graph showing the total number of hCD45+ cells in the bone marrow of humanized mice 6 weeks after the specified treatment.

[0073] Figure 7: The anti-CD117 antibody PR004382 binds specifically to CD117 protein and TF-1 cells

[0074] (A) shows the results of the binding of the anti-CD117 antibody PR004382 and human IgG1 control to CD117 protein based on ELISA assay. The EC50 value of the anti-CD117 antibody PR004382 is shown in the figure. (B) shows the results of the binding of the anti-CD117 antibody PR004382 and human IgG1 control to TF-1 cells based on FACS detection. The EC50 value of the anti-CD117 antibody PR004382 is shown in the figure. (C) The association and dissociation curves are used to determine the association constant (K-on) and dissociation constant (K-dis), and thus calculate the affinity (KD value) of the anti-CD117 antibody PR004382 for CD16a.

[0075] Figure 8 : The anti-CD117 antibody PR004382 eliminates CD117-positive cells in in vitro killing assays

[0076] (A) shows the results of the luminescence-based ADCC assay of the anti-CD117 antibody PR004382 and hIgG1 control. The EC50 value of the anti-CD117 antibody PR004382 is shown in the figure. (B) shows the percentage of NK cell-mediated TF-1 cell depletion when treated with different concentrations of the anti-CD117 antibody PR004382 or hIgG1 control antibody in vitro. (C) shows the percentage of NK cell-mediated depletion of human bone marrow CD34+ cells when treated with the anti-CD117 antibody PR004382 or human IgG1 control antibody (1 μg / mL) in vitro. (D) shows the determination of the in vivo half-life T½ value of PR004382 in serum. Blood samples were collected at the indicated time points, and the concentration of the antibody in serum was determined by ELISA.

[0077] Figure 9 : Anti-CD117xCD3 PR004384 depletes primary human leukemia cells in vitro

[0078] Purified leukemia cells obtained from patients with c-KIT-positive acute leukemia (T / myeloid mixed phenotype acute leukemia) were mixed with purified T cells and treated with the indicated concentration of anti-CD117×CD3 PR004384 or control anti-HEL×CD3. The figure shows the percentage of patient leukemia cell death 24 hours after the indicated treatment.

[0079] Figure 10:Anti-CD117×CD3 PR004384 Depletes AML Cells Including Leukemia Initiating Cells

[0080] (A) Treatment protocol for humanized mice engrafted with patient-derived xenograft (PDX) AML cells. (B) FACS plots showing the expression of human CD117 and human CD33 on PDX AML cells. (C) Plot showing the numbers of human CD33-positive and CD117-positive PDX AML cells in the bone marrow of mice engrafted with PDX AML cells after the indicated treatments. (D) Bone marrow cells from the mice treated as indicated were isolated and transplanted into secondary recipient mice. The plot shows the numbers of AML cells in the bone marrow (BM), spleen, and the percentage of AML cells in the peripheral blood (PB) of the secondary recipient mice 6 months after secondary transplantation.

[0081] Figure 11 :Anti-CD117xCD3 PR004384 Binds to Rhesus Monkey Bone Marrow Hematopoietic Stem Cells

[0082] (A) FACS plots showing the expression of CD117 and CD34 on rhesus monkey bone marrow cells after CD34 enrichment. (B) FACS plots showing the binding of the anti-CD117xCD3 bispecific T cell engager PR004384 to rhesus monkey CD117-positive hematopoietic stem cells. Detailed Description of the Invention

[0083] The above and additional features and advantages of the present invention will become more apparent from the following detailed description of the embodiments in conjunction with the accompanying drawings.

[0084] The embodiments described herein with reference to the accompanying drawings are illustrative, explanatory, and for the general understanding of the present invention. The embodiments should not be construed as limiting the scope of the present invention. Throughout the specification, the same or similar elements and elements having the same or similar functions are denoted by the same reference numerals.

[0085] Unless otherwise specified or defined, all terms used have their ordinary meaning in the art, which will be clear to those skilled in the art. For example, reference may be made to standard manuals such as Leuenberger, H.G.W, Nagel, B. and Klbl, H., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd ed.), vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology" (6th ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background art cited above.

[0086] As used herein, unless the context clearly indicates otherwise, the singular forms "a", "and" and "the" include plural referents. Thus, for example, reference to "an antibody" includes plural antibodies, and in some embodiments reference to "an antibody" includes plural antibodies, and so on.

[0087] Unless otherwise specified or defined, the term "comprising" and variations thereof such as "including" and "containing" are to be understood as implying the inclusion of the stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. The term "including" encompasses "comprising" as well as "consisting of", e.g., a composition "including" X may consist only of X, or may include some additional substances, e.g., X+Y.

[0088] The term "about" associated with a numerical value x is optional and means, for example, x ± 10% or x ± 5%.

[0089] As used herein, the term "antibody" refers to an immunoglobulin molecule having the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each heavy and light chain. The variable regions of the heavy and light chains of an antibody contain binding domains that interact with the antigen. The heavy chain constant region contains three domains, such as CH1, CH2, and CH3. The light chain constant region contains one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. Thus, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together constitute the part of the antibody that binds to the antigen.

[0090] The "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of "framework" regions interrupted by three "complementary determining regions" or "CDRs". The framework regions are used to align the CDRs for specific binding to an epitope of the antigen. The CDRs include the amino acid residues of the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxy terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR 1, 2, and 3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; CDR 1, 2, and 3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0091] The assignment of amino acids to each VL and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. Vol. 196: pp. 901-917, 1987; Chothia et al., Nature, Vol. 342: pp. 878-883, 1989); a combination of Chothia Kabat CDRs, where CDR-H1 is a combination of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the contact definition of Martin et al. (world wide webbioinfo.org.uk / abs). Kabat provides a widely used numbering convention (Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are assigned the same number. Although the present disclosure may use CDRs defined according to any of these numbering systems, a preferred embodiment is to use CDRs defined by Chothia.

[0092] Based on the amino acid sequence of the heavy chain constant region of an antibody, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of an antibody can be divided into a λ (lambda) chain or a κ (kappa) chain.

[0093] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications of antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

[0094] In the context of the present invention, the term "bispecific antibody" should be understood as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes within one target. The term "bispecific antibody" as used herein should be understood in its broadest sense and includes full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing antibody epitopes, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

[0095] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Studies have shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody.

[0096] Examples of antigen-binding fragments that are encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, which are monovalent fragments consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) F(ab')2 fragments, which are divalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fab' fragments, which are essentially Fab with a part of the hinge region; (iv) Fd fragments consisting of a VH domain and a CH1 domain; (v) Fd' fragments having a VH domain, a CH1 domain, and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (vii) dAb fragments, which consist of a VH domain; (viii) isolated complementarity-determining regions (CDRs); and (ix) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. In addition, although the two domains (VL and VH) of an Fv fragment are encoded by separate genes, these domains can still be joined using recombinant methods by a synthetic linker such that the domains can be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes "linear antibodies" that contain a pair of tandem Fd segments (VH-CH1-VH-CH1) and modified forms of any of the foregoing fragments, which pair of tandem Fd segments together with a complementary light chain polypeptide form an antigen-binding region, and the modified forms of these fragments retain antigen-binding activity.

[0097] These antigen-binding fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for use in the same manner as intact antibodies.

[0098] As used herein, the terms "bind" or "specifically bind" refer to a non-random binding reaction between two molecules, such as the non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity, represented by the equilibrium dissociation constant (KD) of the dissociation of an antigen from an antibody, is a measure of the strength of binding between an epitope and the antigen-binding site on the antibody: the smaller the value of KD, the stronger the binding between the epitope and the antibody. Alternatively, affinity can also be expressed as an association constant (KA), which is 1 / KD.

[0099] Affinity is a measure of the strength of binding between an antibody and its associated antigen. Affinity is related to both the affinity between the antigenic determinant (epitope) and its antigen-binding site on the antibody and the number of associated binding sites present on the antibody. Typically, an antibody will have a dissociation constant (KD) of 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or bind with a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 (such as at least 10 12 M -1 ). Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), biolayer interferometry (BLI) assays and sandwich competition assays, as well as various variants of these methods known per se in the art.

[0100] The term "epitope" refers to the site on an antigen that binds to an antibody. Epitopes can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by contiguous amino acids (also called linear epitopes) generally remain upon exposure to denaturing solvents, while epitopes formed by tertiary folding (also called conformational epitopes) generally are lost upon treatment with denaturing solvents. Epitopes generally include at least 3, more typically at least 5 or 8 to 10 amino acids in a unique spatial conformation. Epitopes define the minimal binding sites of antibodies and thus are the specific targets of antibodies or their antigen-binding fragments.

[0101] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have the same residues at the same positions in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of the amino acid sequence to SEQ ID NO: Y and is described in detail as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Generally, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0102] In addition, when determining the degree of sequence identity between two amino acid sequences, one of ordinary skill in the art can consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure and having little or essentially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, according to WO 04 / 037999, GB-A-2 357 768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300; and the type and / or combination of such substitutions (preferably) can be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0103] Such conservative substitutions preferably are substitutions in which one amino acid in one of the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0104] Particularly preferred conservative substitutions are as follows: Ala is converted to Gly or Ser; Arg is converted to Lys; Asn is converted to Gln or His; Asp is converted to Glu; Cys is converted to Ser; Gln is converted to Asn; Glu is converted to Asp; Gly is converted to Ala or Pro; His is converted to Asn or Gln; Ile is converted to Leu or Val; Leu is converted to Ile or Val; Lys is converted to Arg, Gln or Glu; Met is converted to Leu, Tyr or Ile; Phe is converted to Met, Leu or Tyr; Ser is converted to Thr; Thr is converted to Ser; Trp is converted to Tyr; Tyr is converted to Trp; and / or Phe is converted to Val, Ile or Leu.

[0105] "CD117" (also known as c-kit or stem cell factor receptor (SCRF)) is a single transmembrane receptor tyrosine kinase that binds to the ligand stem cell factor (SCF). SCF induces the homodimerization of cKIT, thereby activating its tyrosine kinase activity and signaling through the PI3-AKT and MAPK pathways. CD117 is highly expressed on hematopoietic stem cells (HSCs). This expression pattern makes CD117 a potential target for treating a variety of diseases.

[0106] As used herein, the term "CD3" refers to the human CD3 protein complex, which has five peptide chains: the γ chain, δ chain, ε chain, ζ chain, and η chain, and associates with the T cell receptor α chain and β chain to form the TCR-CD3 complex. The term includes any CD3 variants, isomers, and species homologs that are naturally expressed by cells (including T cells) or expressed on cells transfected with genes or cDNAs encoding the above chains.

[0107] As used herein, the term "bispecific T cell engager" or "BiTE" refers to a polypeptide chain molecule having two antigen-binding domains, one of which binds to a T cell antigen and the second of which binds to an antigen present on the surface of a target cell (see P Publication PCTWO05 / 061547; Baeuerle et al., 2008, Drugs of the Future 33:137-147; Bargou et al., 2008, Science 321:974-977, the entire contents of which are incorporated herein by reference). Thus, the BiTEs of the present disclosure have an antigen-binding region that binds CD117 and a second antigen-binding region directed against a T cell antigen.

[0108] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0109] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0110] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other components of the composition and is substantially innocuous to its recipient, and / or such carrier or adjuvant is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0111] As used herein, the term "hematopoietic stem cell" ("HSC") refers to an immature blood cell that has the ability to self-renew and differentiate into mature blood cells containing different lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Such cells may include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are thought to include a subset of cells with the above stem cell characteristics, while in mice, HSCs are CD34−. In addition, HSCs also refer to long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on functional potential and cell surface marker expression. For example, human HSCs are CD34+, CD38−, CD45RA−, CD90+, CD49F+, and Lin− (mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A are all negative). In mice, bone marrow LT-HSCs are CD34−, Sca-1+, c-Kit+, CD135−, Slamf1 / CD150+, CD48−, and Lin− (mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra are all negative), while ST-HSCs are CD34+, Sca-1+, c-Kit+, CD135−, Slamf1 / CD150+, and Lin− (mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra are all negative). In addition, under steady-state conditions, ST-HSCs are less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have a greater self-renewal potential (i.e., they can survive throughout adulthood and can be continuously transplanted through successive recipients), while ST-HSCs have limited self-renewal ability (i.e., they only survive for a limited time and do not have the potential for continuous transplantation). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and can therefore produce differentiated progeny cells more quickly.

[0112] As used herein, terms such as "treatment" (treatment / treating) refer to the administration of an agent or the performance of a procedure in order to obtain an effect. The effect can be a prophylactic effect that completely or partially prevents a disease or its symptoms, and / or can be a therapeutic effect that partially or completely cures a disease and / or its symptoms. As used herein, "treatment" can include treating a disease or disorder (such as cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or disease symptoms in a subject who may be susceptible to the disease but has not been diagnosed as having the disease (for example, including diseases that may be associated with or caused by a primary disease); (b) inhibiting the disease, that is, preventing the development of the disease; and (c) alleviating the disease, that is, causing the regression of the disease. Treatment can refer to any successful marker in the treatment, improvement, or prevention of cancer, including any objective or subjective parameter, such as alleviation; remission; reduction of symptoms or making the patient more tolerant of the disease condition; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. The treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes the administration of an antibody, composition, or conjugate disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms or conditions associated with a disease (such as cancer). The term "therapeutic effect" refers to reducing, eliminating, or preventing a disease, disease symptoms, or side effects of a disease in a subject.

[0113] As used herein, the term "effective amount" refers to an amount sufficient to achieve treatment of a disease when administered to a subject to treat the disease.

[0114] As used herein, the term "subject" refers to any mammalian subject in need of diagnosis, treatment, or therapy. "Mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as laboratory animals, zoo animals, sports animals, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0115] Bispecific antibody

[0116] The present invention provides a bispecific antibody or an antigen-binding fragment thereof, which comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to CD117 and comprises a first heavy-chain variable region (VH_A) and a first light-chain variable region (VL_A), and the second binding region binds to CD3 and comprises a second heavy-chain variable region (VH_B) and a second light-chain variable region (VL_B), wherein:

[0117] The VH_A comprises HCDR1, HCDR2, and HCDR3 of VH having an amino acid sequence containing SEQ ID NO: 44;

[0118] The VL_A comprises LCDR1, LCDR2 and LCDR3 of VL containing the amino acid sequence of SEQ ID NO: 47;

[0119] The VH_B comprises HCDR1, HCDR2 and HCDR3 of VH containing the amino acid sequence of SEQ ID NO: 43; and

[0120] The VL_B comprises LCDR1, LCDR2 and LCDR3 of VL containing the amino acid sequence of SEQ ID NO: 46.

[0121] In some embodiments, the CDRs are determined by Chothia definition. In some other embodiments, the CDRs are determined by Kabat definition. In some embodiments, the CDRs are determined by the combination of Chothia and Kabat definition systems. In some other embodiments, the CDRs are determined by AbM definition.

[0122] In some embodiments of the bispecific antibody or its antigen-binding fragment,

[0123] The VH_A comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 6, 12 and 18 respectively;

[0124] The VL_A comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 26, 32 and 38 respectively;

[0125] The VH_B comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 5, 11 and 17 respectively; and

[0126] The VL_B comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 25, 31 and 37 respectively.

[0127] In some embodiments of the bispecific antibody or its antigen-binding fragment,

[0128] The VH_A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 44;

[0129] The VL_A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 47;

[0130] The VH_B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 43; and

[0131] The VL_B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 46.

[0132] In some embodiments, VH_A comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 44, the functional variant being formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the antibody comprising VH_A containing the functional variant retains the ability to bind CD117. In some embodiments, VL_A comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 47, the functional variant being formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the antibody comprising VL_A containing the functional variant retains the ability to bind CD117. In some embodiments, VH_B comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 43, the functional variant being formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the antibody comprising VH_B containing the functional variant retains the ability to bind CD3. In some embodiments, VL_B comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 46, the functional variant being formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the antibody comprising VL_B containing the functional variant retains the ability to bind CD3.

[0133] The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of the parental polypeptide. For example, the functional variant of SEQ ID NO: 44 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to SEQ ID NO: 44. For example, the functional variant of SEQ ID NO: 47 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to SEQ ID NO: 47. For example, the functional variant of SEQ ID NO: 43 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to SEQ ID NO: 43. For example, the functional variant of SEQ ID NO: 46 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to SEQ ID NO: 46.

[0134] In the case of functional variants, the number of inserted, deleted, and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parental amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and even more preferably 5% to 30%, still more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and most preferably 1 to 2. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0135] In some embodiments, the insertions, deletions, and / or substitutions can be made in the framework (FR) regions, such as at FR1, FR2, FR3, and / or FR4.

[0136] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions in which an amino acid in one of the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0137] Particularly preferred conservative substitutions are as follows: Ala is converted to Gly or Ser; Arg is converted to Lys; Asn is converted to Gln or His; Asp is converted to Glu; Cys is converted to Ser; Gln is converted to Asn; Glu is converted to Asp; Gly is converted to Ala or Pro; His is converted to Asn or Gln; Ile is converted to Leu or Val; Leu is converted to Ile or Val; Lys is converted to Arg, Gln, or Glu; Met is converted to Leu, Tyr, or Ile; Phe is converted to Met, Leu, or Tyr; Ser is converted to Thr; Thr is converted to Ser; Trp is converted to Tyr; Tyr is converted to Trp; and / or Phe is converted to Val, Ile, or Leu.

[0138] In a preferred embodiment, the VH_A comprises the amino acid sequence shown in SEQ ID NO: 44; the VL_A comprises the amino acid sequence shown in SEQ ID NO: 47; the VH_B comprises the amino acid sequence shown in SEQ ID NO: 43; and the VL_B comprises the amino acid sequence shown in SEQ ID NO: 46.

[0139] In some embodiments of the bispecific antibody or its antigen-binding fragment, the antibody does not comprise an immunoglobulin Fc region.

[0140] In some other embodiments, the antibody comprises three polypeptide chains, wherein:

[0141] The first polypeptide chain comprises, from the N-terminus to the C-terminus: VH_A-CH1; the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL_A-CL-L-VH_B-CH1; and the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL_B-CL; or

[0142] The first polypeptide chain comprises, from the N-terminus to the C-terminus: VH_B-CH1; the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL_B-CL-L-VH_A-CH1; and the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL_A-CL;

[0143] Wherein CH1 represents the first domain of the constant region of the immunoglobulin heavy chain; CL each represents the constant region of the immunoglobulin light chain; and L is absent or represents an optional linker.

[0144] In some embodiments, CL in the second polypeptide chain and CL in the third polypeptide chain each independently comprise a constant region derived from a λ light chain or a κ light chain.

[0145] In some embodiments, CL in the second polypeptide chain and CL in the third polypeptide chain are derived from the same type of immunoglobulin light chain (λ light chain or κ light chain). In some other embodiments, CL in the second polypeptide chain and CL in the third polypeptide chain are derived from different types of immunoglobulin light chains.

[0146] When CL in the second polypeptide chain and CL in the third polypeptide chain are derived from different types of immunoglobulin light chains, the bispecific antibody can be purified by affinity chromatography according to the type of CL in the second polypeptide chain, such that the bispecific antibody comprising the second polypeptide chain bound to the first and third polypeptide chains is captured while removing by-products without the second polypeptide chain.

[0147] In some embodiments, the CL in the second polypeptide chain comprises a light chain constant region derived from a λ light chain (e.g., human λ light chain), and the CL in the third polypeptide chain comprises a light chain constant region derived from a κ light chain (e.g., human κ light chain).

[0148] In some other embodiments, the CL in the second polypeptide chain comprises a light chain constant region derived from a κ light chain (e.g., human κ light chain), and the CL in the third polypeptide chain comprises a light chain constant region derived from a λ light chain (e.g., human λ light chain).

[0149] In some embodiments of the bispecific antibody or antigen-binding fragment thereof, the VL_A comprises a light chain variable region derived from the same type of immunoglobulin light chain as the CL in the same polypeptide chain, and / or the VL_B comprises a light chain variable region derived from the same type of immunoglobulin light chain as the CL in the same polypeptide chain.

[0150] In some embodiments of the bispecific antibody or antigen-binding fragment thereof, each CH1 independently comprises a CH1 derived from immunoglobulin isotype IgG (e.g., human IgG), and optionally each CH1 independently comprises a CH1 derived from an IgG subtype selected from the group consisting of IgG1, IgG2, and IgG4 (e.g., human IgG1, IgG2, and IgG4).

[0151] In some embodiments, the linker can be any flexible linker. For example, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1-5, such as 1, 2, 3, 4, or 5. In some embodiments, the linker can comprise an amino acid sequence of GGGGS. In some embodiments, the linker can comprise an amino acid sequence of GGGGSGGGGS. In some embodiments, the linker can comprise an amino acid sequence of GGGGSGGGGSGGGGS. In some embodiments, the linker can comprise an amino acid sequence of GGGGSGGGGSGGGGSGGGGS. In some embodiments, the linker can comprise an amino acid sequence of GGGGSGGGGSGGGGSGGGGSGGGGS.

[0152] In some embodiments of a bispecific antibody comprising three polypeptide chains, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 57; the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 56; and the third polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 52.

[0153] In some embodiments, the first polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 57, the functional variant being formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the antibody comprising the functional variant retains the ability to bind CD117 and / or CD3. In some embodiments, the second polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 56, the functional variant being formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the antibody comprising the functional variant retains the ability to bind CD117 and / or CD3. In some embodiments, the third polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 52, the functional variant being formed by inserting, deleting, and / or substituting one or more amino acids therein, provided that the antibody comprising the functional variant retains the ability to bind CD117 and / or CD3.

[0154] For example, a functional variant of SEQ ID NO: 57 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to SEQ ID NO: 57. For example, a functional variant of SEQ ID NO: 56 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to SEQ ID NO: 56. For example, a functional variant of SEQ ID NO: 52 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to SEQ ID NO: 52.

[0155] In some embodiments, the number of inserted, deleted, and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parental amino acid sequence, more preferably no more than 35%, more preferably from 1% to 33%, and even more preferably from 5% to 30%, still more preferably from 10% to 25%, and even more preferably from 15% to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be from 1 to 50, preferably from 1 to 20, more preferably from 1 to 10, and still more preferably from 1 to 5. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0156] In some embodiments, the insertion, deletion, and / or substitution can be made in the framework (FR) regions, such as at FR1, FR2, FR3, and / or FR4; and / or in the constant regions, such as at CL and / or CH1.

[0157] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are as described above.

[0158] In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 57; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 56; and the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 52.

[0159] In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTE).

[0160] In some embodiments, the antibodies or antigen-binding fragments of the invention are linked to a fluorescent label, a radioactive label or a cytotoxic agent.

[0161] Nucleic acid

[0162] The invention provides nucleic acids (e.g., isolated nucleic acids) encoding the bispecific antibodies or antigen-binding fragments of the invention.

[0163] The term "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, dithiophosphate, selenophosphate, diselenophosphate, phosphoroanilothioate, phoshoraniladate, and aminophosphate.

[0164] For example, the invention provides nucleic acid molecules encoding any of the heavy chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98% or at least 99% identity to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein.

[0165] For example, the invention provides nucleic acid molecules encoding any of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98% or at least 99% identity to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0166] For example, the present invention provides nucleic acid molecules encoding: (i) any one of the heavy chain variable region sequences disclosed herein; and (ii) any one of the light chain variable region sequences disclosed herein. The present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98% or at least 99% identity to the nucleic acids encoding: (i) any one of the heavy chain variable region sequences disclosed herein; and (ii) any one of the light chain variable region sequences disclosed herein.

[0167] For example, the present invention provides nucleic acid molecules encoding a heavy chain variable region sequence comprising the CDR sequences of any one of the heavy chain variable region sequences disclosed herein.

[0168] The present invention also provides nucleic acid molecules encoding a heavy chain variable region sequence comprising CDR sequences having at least 90%, at least 95%, at least 98% or at least 99% identity to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein..

[0169] For example, the present invention provides nucleic acid molecules encoding a light chain variable region sequence comprising the CDR sequences of any one of the light chain variable region sequences disclosed herein..

[0170] The present invention also provides nucleic acid molecules encoding a light chain variable region sequence comprising CDR sequences having at least 90%, at least 95%, at least 98% or at least 99% identity to the CDR sequences of any one of the light chain variable region sequences disclosed herein.

[0171] For example, the present invention provides nucleic acid molecules encoding the following sequences: (i) a heavy chain variable region sequence comprising the CDR sequences of any one of the heavy chain variable region sequences disclosed herein and (ii) a light chain variable region sequence comprising the CDR sequences of any one of the light chain variable region sequences disclosed herein. The present invention also provides nucleic acid molecules encoding the following sequences: (i) a heavy chain variable region sequence comprising CDR sequences having at least 90%, at least 95%, at least 98% or at least 99% identity to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein; and (ii) a light chain variable region sequence comprising CDR sequences having at least 90%, at least 95%, at least 98% or at least 99% identity to the CDR sequences of any one of the light chain variable region sequences disclosed herein..

[0172] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding a bispecific antibody disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding a bispecific antibody disclosed herein.

[0173] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of the cell.

[0174] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent.

[0175] vector

[0176] The present invention provides a vector comprising the nucleic acid of the present invention.

[0177] In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising a variable region of the heavy or light chain of a bispecific antibody. For example, the present invention provides an expression vector comprising any one of the above nucleic acid molecules.

[0178] Any vector is applicable to the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, DNA vector, murine leukemia virus vector, SFG vector, plasmid, RNA vector, adenoviral vector, baculovirus vector, Epstein Barr virus vector, papillomavirus vector, vaccinia virus vector, herpes simplex virus vector, adeno-associated vector (AAV), lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo large TcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0179] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0180] The recombinant expression vector can be prepared using standard recombinant DNA techniques, which are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing Associates and John Wiley & Sons, NY, 1994. Constructs of circular or linear expression vectors can be prepared to contain a replication system that functions in a prokaryotic host cell or a eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0181] For example, the vector can be an adenovirus vector that contains a nucleotide sequence encoding the bispecific antibody disclosed herein. The vector can be administered to a subject and then enter the subject's cells in vivo, thereby integrating the nucleotide sequence encoding the bispecific antibody disclosed herein into the genome of the cells, and subsequently the cells express the bispecific antibody disclosed herein.

[0182] Host cell

[0183] The present invention also provides a host cell comprising the nucleic acid of the present invention or the vector of the present invention.

[0184] Any cell can be used as a host cell for the nucleic acid or vector of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescens and Shigella; Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHOK1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0185] The host cell of the present invention is prepared by introducing in vitro or ex vivo the vector disclosed herein or the nucleic acid disclosed herein. The host cell of the present invention can be administered to a subject, and the host cell expresses the bispecific antibody disclosed herein in vivo.

[0186] The present invention provides a host cell into which any of the above vectors has been introduced. The present invention also provides a method for preparing the bispecific antibody of the present invention, wherein the method comprises a) culturing the host cell according to the fourth aspect of the present invention under conditions suitable for producing the bispecific antibody; and b) obtaining the bispecific antibody from the culture.

[0187] Antibody-drug conjugate

[0188] The present invention also provides immune conjugates and antibody-drug conjugates (ADCs) that comprise the bispecific antibody or antigen-binding fragment thereof of the present invention, and a chemical moiety or effector molecule conjugated to the bispecific antibody or antigen-binding fragment thereof.

[0189] In the context of the present disclosure, a "conjugate" refers to an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are commonly referred to as "immune conjugates". When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an "antibody-drug conjugate" or "ADC".

[0190] In some embodiments, the effector molecule can be a detectable label or an immunotoxin. Specific non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or a modified toxin thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically cause death through hepatotoxicity. However, PE and DT can be modified into forms used as immunotoxins by removing the native targeting components of the toxin (such as domain la of PE and the B chain of DT) and replacing them with different targeting moieties (such as an antibody).

[0191] The terms "conjugated" or "linked" can refer to making two polypeptide molecules into a continuous polypeptide molecule. In one embodiment, the antibody is linked to the effector molecule. In another embodiment, the antibody linked to the effector molecule is further linked to a lipid or other molecule, linked to a protein or peptide, to increase its half-life in vivo. The linkage can be carried out chemically or recombinantly. In one embodiment, the linkage is chemical, wherein the reaction between the antibody moiety and the effector molecule has resulted in the formation of a covalent bond between the two molecules, thereby forming one molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the effector molecule.

[0192] The present invention provides immune conjugates that comprise the antibody or antigen-binding fragment disclosed herein and an effector molecule. In some embodiments, the effector molecule is a toxin, such as, but not limited to, Pseudomonas exotoxin or a variant thereof. In other embodiments, the effector molecule is a detectable label, such as, but not limited to, a fluorophore, an enzyme, or a radioisotope.

[0193] The disclosed antibodies can be conjugated to therapeutic agents or effector molecules. Immunoconjugates include, but are not limited to, molecules in which the therapeutic agent is covalently linked to the antibody. A therapeutic agent is a medicament having a specific biological activity against a specific target molecule or a cell carrying the target molecule. Those skilled in the art will understand that therapeutic agents can include various drugs such as vinblastine, daunomycin, etc.; cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin; encapsulants containing a pharmacological composition (such as liposomes); radioactive agents such as 125 I, 32 P, 14 C, 3 H, and 35 S and other labels; target moieties and ligands.

[0194] The choice of a particular therapeutic agent depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the therapeutic agent can be a cytotoxin for causing the death of a particular target cell such as a tumor cell. Conversely, when a non-lethal biological response is desired, the therapeutic agent can be conjugated to a non-lethal pharmaceutical agent or a liposome containing a non-lethal pharmaceutical agent.

[0195] Any number of ways known to those skilled in the art can be used to link the effector molecule to the antibody of interest. Both covalent linkage methods and non-covalent attachment methods can be used. The method of linking the effector molecule to the antibody varies depending on the chemical structure of the effector. A polypeptide typically contains multiple functional groups; such as carboxylic acid (COOH), free amine (-NH2) or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or link additional reactive functional groups. Derivatization can include linking any of a number of known linker molecules. A linker can be any molecule used to link the antibody to the effector molecule. The linker is capable of forming covalent bonds with both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers or peptide linkers. When the antibody and the effector molecule are polypeptides, the linker can be linked to the constituent amino acids through the side groups of the constituent amino acids (such as through a disulfide bond with cysteine), or to the α-carbon amino and carboxyl groups of the terminal amino acids.

[0196] In some cases, it is desirable to release the effector molecule from the antibody when the immunoconjugate has reached its target site. Thus, in these cases, the immunoconjugate will contain a bond that can be cleaved near the target site.

[0197] Cleavage of the linker can be facilitated by enzymatic activity or conditions that the immunoconjugate undergoes inside the target cell or near the target site to release the effector molecule from the antibody.

[0198] Given the large number of methods reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other reagents to antibodies, those skilled in the art will be able to determine suitable methods for attaching a given reagent to an antibody or other polypeptide.

[0199] The antibodies disclosed herein can be derivatized or conjugated to another molecule, such as another peptide or protein. Generally, the antibody or a portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, an antibody can be functionally linked (by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule, such as a streptavidin core region or a polyhistidine tag.

[0200] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or different types, such as to produce a bispecific antibody). Suitable cross-linking agents include those heterobifunctional cross-linking agents having two distinct reactive groups separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), or those homobifunctional cross-linking agents (such as disuccinimidyl suberate). Such linkers are commercially available.

[0201] Bispecific antibodies can be conjugated to a detectable marker; for example, the detectable marker can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, which include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP).

[0202] Bispecific antibodies or antigen-binding fragments can also be conjugated to enzymes for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen-binding fragment is conjugated to a detectable enzyme, it can be detected by adding an additional reagent, and the enzyme uses this additional reagent to produce a distinguishable reaction product. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. The antibody or antigen-binding fragment can also be conjugated to biotin and detected by indirect measurement through binding to avidin or streptavidin proteins. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0203] Bispecific antibodies can be fused to self-labeling protein tags (such as HaloTag). For example, the protein tag can be cloned at the end of the constant region. HaloTag is a self-labeling protein tag derived from a bacterial enzyme (haloalkane dehalogenase) that is designed to covalently bind to a synthetic ligand. In some cases, the synthetic ligand contains a chloroalkane linker attached to a fluorophore such as a near-infrared fluorophore (Los et al., (2008) ACS Chem Biol. 3(6):373-82).

[0204] Bispecific antibodies can be labeled with magnetic reagents such as gadolinium. Antibodies can also be labeled with lanthanide elements (such as europium and dysprosium) and manganese.

[0205] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Bispecific antibodies can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter gene (such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal-binding domain, an epitope tag). In some embodiments, the label is linked by spacer arms of various lengths to reduce potential steric hindrance.

[0206] Bispecific antibodies can also be labeled with radioactively labeled amino acids. Radioactive labeling can be used for both diagnostic and therapeutic purposes. For example, radioactive labeling can be used to detect the expression of target antigens by X-ray, emission spectroscopy, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radioactive nucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0207] Bispecific antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life or increasing tissue binding.

[0208] Toxins can be used with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin, and their subunits, as well as botulinum toxins A - F. These toxins are readily available from commercial sources (Sigma Chemical Company, St. Louis, MO). Toxins contemplated also include variants of the toxins described herein (see, for example, U.S. Patent Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095).

[0209] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells that express tumor or viral antigens on their surface. Thus, the antibodies of the present disclosure can be directly or via a linker attached to a drug, which will be directly delivered to cells expressing cell surface antigens. This can be used for therapeutic, diagnostic, or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking to single- or double-stranded DNA, and oligonucleotides that form triplexes.

[0210] Alternatively, the molecule linked to the bispecific antibody can be an encapsulation system, such as a nanoparticle, liposome, or micelle, that contains a therapeutic composition, such as a drug, nucleic acid (e.g., antisense nucleic acid), or another therapeutic moiety preferably protected from direct exposure to the circulatory system. Methods for preparing liposomes linked to antibodies are well known to those skilled in the art (see, for example, U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341 - 365, 1985).

[0211] The antibodies described herein may also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such uses include any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels include magnetic beads, fluorescent dyes (such as fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (such as 3H, 125I, 35S, 14C, or 32P), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads.

[0212] Methods for detecting such labels are well known to those of skill in the art. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a light detector to detect the emitted illumination. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product resulting from the action of the enzyme on the substrate, while colorimetric labels are detected by simply visualizing the colored label.

[0213] Composition

[0214] The present invention also provides a composition comprising the bispecific antibody or an antigen-binding fragment thereof of the present invention. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier and / or excipient.

[0215] The bispecific antibody or an antigen-binding fragment or reagent thereof (also referred to herein as "active compound") of the present invention and its derivatives, fragments, analogs, and homologs can be incorporated into a pharmaceutical composition suitable for administration. Such compositions generally comprise a bispecific antibody or an antigen-binding fragment or reagent thereof and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are compatible with the administration of a drug. Suitable carriers are described in the most recent edition of "Remington's Pharmaceutical Sciences", which is a standard reference text in the field and is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous solvents such as fixed oils can also be used. The use of such media and reagents for pharmaceutical active substances is well known in the art. These media or reagents are considered for use in the composition unless any conventional medium or reagent is incompatible with the active compound. Supplementary active compounds can also be incorporated into the composition.

[0216] In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is used to deplete hematopoietic stem cells in a subject and / or treat a hematological disorder of the subject.

[0217] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate; and a reagent for adjusting tonicity, such as sodium chloride or glucose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0218] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injection is possible. It must be stable under the conditions of preparation and storage; and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. Sustained absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0219] A sterile injectable solution can be prepared by incorporating the required amount of the active compound, as needed, with one or a combination of the ingredients listed above into a suitable solvent and then filtering the solution sterilize. Generally, a dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution of the active ingredient plus any additional desired ingredients.

[0220] Oral compositions generally contain an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier as a mouthwash, wherein the compound in the fluid carrier is orally administered and either spat out or swallowed after rinsing. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0221] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressure container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0222] Systemic administration can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, permeants appropriate to the barrier to be permeated are used in the formulation. Such permeants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, pastes, gels, or creams well-known in the art.

[0223] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0224] In one embodiment, the active compounds are prepared with carriers which will protect the compound from rapid elimination from the body, such as controlled release formulations including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals. Liposome suspensions, including liposomes targeted to infected cells with monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. These materials can be prepared by methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0225] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention depends on the unique characteristics of the active compound and the particular therapeutic effect to be achieved and the inherent limitations in the art of compounding such active compounds for the treatment of individuals, and is directly dependent on these factors.

[0226] The pharmaceutical compositions can be contained in a container, package, or dispenser together with instructions for administration.

[0227] The present invention provides a therapeutic composition comprising the bispecific antibody or antigen-binding fragment thereof of the present invention. The therapeutic composition according to the present invention will be administered together with suitable carriers, excipients and other reagents, and these agents are incorporated into the formulation to improve transfer, delivery, tolerance, etc. A large number of suitable formulations can be found in all the formularies known to pharmaceutical chemists: "Remington's Pharmaceutical Sciences" (Mack Publishing Company, Easton, Pennsylvania). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) such as LIPOFECTIN™, DNA conjugates, anhydrous absorbent pastes, water-in-oil and oil-in-water emulsions, Carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0228] The dosage of the bispecific antibody may vary depending on factors such as the age and body size of the subject receiving the administration, the target disease, the condition, and the route of administration. When the antibody of the present invention is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is generally advantageous to administer the antibody of the present invention at a single dose of about 0.1 to about 60 mg / kg body weight, more preferably about 5 to about 60, about 10 to about 50, or about 20 to about 50 mg / kg body weight. The frequency and duration of treatment can be adjusted according to the severity of the condition. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention can be administered at an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, or about 100 mg or about 50 mg. In certain embodiments, a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof can be administered after the initial dose, and the dosage can be approximately the same as or less than the initial dose, with at least a 1-day to 3-day interval; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks between subsequent doses.

[0229] Method of treatment

[0230] The present invention provides a method for depleting hematopoietic stem cells in a subject in need thereof, the method comprising administering to the subject the bispecific antibody of the present invention or the composition of the present invention. In some embodiments, a therapeutically effective amount of the bispecific antibody or the composition is administered to the subject in an amount sufficient to deplete hematopoietic stem cells in the subject.

[0231] The present invention further provides a method for treating a hematopoietic system disease in a subject in need thereof, the method comprising: a) administering to the subject the bispecific antibody of the present invention or the composition of the present invention; and b) transplanting hematopoietic stem cells into the subject. In some embodiments, a therapeutically effective amount of the bispecific antibody or the composition is administered to the subject in an amount sufficient to deplete hematopoietic stem cells in the subject, treat the hematopoietic system disease, or inhibit the signs or symptoms of the hematopoietic system disease in the subject.

[0232] In some embodiments, the hematopoietic system disease is selected from the group consisting of hemoglobinopathies, immunodeficiencies, metabolic disorders, hematological diseases (such as bone marrow failure syndromes, myelodysplastic syndromes, acute lymphocytic and myelocytic leukemias, and chronic lymphocytic and myelocytic leukemias), hematopoietic cell proliferative diseases, transplant rejection, autoimmune diseases, and autoinflammatory diseases.

[0233] In some embodiments, the hematopoietic system diseases are selected from the group of hematopoietic system diseases including hemoglobinopathies, bone marrow failure syndromes, immunodeficiencies, metabolic disorders, myeloid and lymphoid malignancies, and hematopoietic proliferative diseases, such as: mastocytosis, myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute myeloid leukemia with recurrent genetic abnormalities (e.g., molecular changes in CD117, NPM1, CEBPα, FLT3, RUNX1, ASXL1, TP53, IDH1, and IDH2), chromosomal aberrations (e.g., favorable chromosomal changes, including abnormalities in the p13 and q22 bands of chromosome 16 [t(16;16)(p13;q22), inv(16)(p13q22)] and translocations between chromosomes 8 and 21 [t(8;21)], intermediate-risk aberrations including trisomy 8 and translocations between chromosomes 9 and 11 [t(9;11)], unfavorable chromosomal changes including subsets of cases with additional copies of chromosome 8 or 13 [e.g., trisomy 8 (+8)], complete or partial deletions of chromosome 5 or 7, complex changes in many chromosomes, and changes in the q26 band of chromosome 3).AML with myelodysplasia-related changes, AML with therapy-related myeloid neoplasms, AML not otherwise specified (NOS), minimally differentiated myeloid leukemia, M0, myeloid leukemia, M1, with minimal or no maturation, myeloid leukemia, M2, with maturation, promyelocytic leukemia, M3, myelomonocytic leukemia, M4, myelomonocytic leukemia with eosinophilia, M4eo, undifferentiated monocytic leukemia, M5a (monoblast leukemia), differentiated monocytic leukemia, M5b, erythroleukemia, M6, relapsed AML and recurrent AML, acute lymphoblastic leukemia (CLL) positive for c-KIT, mast cell leukemia, megakaryocytic leukemia, chronic myeloid leukemia (including BCR-ABL translocation [t(9;11)], chronic, accelerated, and blast phases of Philadelphia chromosome Ph-positive and Ph-negative CML), chronic lymphocytic leukemia requiring hematopoietic stem cell transplantation, such as in cases of early relapse or refractory cases after purine analogue therapy, or cases carrying p53 mutations, T-lymphoblastic leukemia positive for the CD117 receptor, autoinflammatory diseases, such as familial Mediterranean fever (FMF), cryopyrin-associated periodic syndromes (CAPS), tumor necrosis factor receptor-associated periodic syndromes (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), hyper IgD syndrome (HIDS), systemic autoinflammatory diseases (SAIDS), Blau syndrome, Mejeed syndrome, pyogenic arthritis-pyoderma gangrenosum-acne syndrome (PAPA), periodic fever-aphthous stomatitis-pharyngitis-adenitis syndrome (PFAPA), Behçet's disease, Still's disease, Crohn's disease, and acquired autoinflammatory syndromes, such as Schnitzler syndrome.

[0234] Hematopoietic stem cell transplantation therapy can be administered to subjects in need of treatment to replenish or reconstitute one or more blood cell types. Hematopoietic stem cells typically exhibit pluripotency and are thus capable of differentiating into multiple different blood cell lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). In addition, hematopoietic stem cells have the ability to self-renew and are thus capable of generating daughter cells with the same differentiation potential as the parent cell and also have the ability to be reintroduced into the transplant recipient, where they will home to the hematopoietic stem cell niche and re-establish effective and sustained hematopoiesis.

[0235] Accordingly, hematopoietic stem cells can be administered to a patient having a defect or deficiency in one or more cell types in the hematopoietic lineage, in order to reconstitute in vivo the defective or deficient cell population, thereby treating a disorder associated with a defect or depletion of the endogenous blood cell population. Accordingly, the bispecific antibodies or compositions described herein can be used to treat non-malignant hemoglobinopathies (e.g., mastocytosis, hemoglobinopathies selected from sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome). Additionally or alternatively, the bispecific antibodies or compositions described herein can be used to treat immunodeficiencies, such as congenital immunodeficiencies. Additionally or alternatively, the bispecific antibodies or compositions described herein can be used to treat acquired immunodeficiencies (e.g., acquired immunodeficiencies selected from human immunodeficiency virus infection and acquired immunodeficiency syndrome). The bispecific antibodies or compositions described herein can be used to treat metabolic disorders (e.g., metabolic disorders selected from glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipid storage diseases, and metachromatic leukodystrophy).

[0236] Additionally or alternatively, the bispecific antibodies or compositions described herein can be used to treat malignancies or proliferative diseases, such as hematological cancers, myeloproliferative diseases. In the context of cancer treatment, the bispecific antibodies or compositions described herein can be administered to a subject in order to deplete the endogenous hematopoietic stem cell population prior to hematopoietic stem cell transplantation therapy. In such a case, the transplanted cells are able to home to the microenvironment created by the endogenous cell depletion step and establish effective hematopoietic function. This in turn is able to reconstitute the cell population depleted during the clearance of cancer cells, such as the cell population depleted during systemic chemotherapy. Exemplary hematological cancers that can be treated using the bispecific antibodies or compositions described herein include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma, as well as other cancerous conditions, including neuroblastoma.

[0237] Other diseases that can be treated using the bispecific antibodies or compositions described herein include, but are not limited to, adenosine deaminase deficiency, severe combined immunodeficiency disease, hyperimmunoglobulin M syndrome, Chediak-Higashi syndrome (i.e., Che - Dong syndrome), hereditary lymphocytic histiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, severe thalassemia, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.

[0238] The bispecific antibodies or compositions described herein can be used to induce solid organ transplant tolerance. For example, the bispecific antibodies or compositions described herein can be used to deplete or eliminate a cell population in a target tissue (e.g., deplete hematopoietic stem cells from the bone marrow stem cell niche). After depleting the cells from the target tissue, a population of stem or progenitor cells from an organ donor (e.g., hematopoietic stem cells from an organ donor) can be administered to the transplant recipient, and after implantation of these stem or progenitor cells, a transient or stable mixed chimeric state can be achieved, thereby enabling long-term transplant organ tolerance without further use of immunosuppressants. For example, the bispecific antibodies or compositions described herein can be used to induce transplant tolerance in solid organ transplant recipients (e.g., kidney transplantation, lung transplantation, liver transplantation, and heart transplantation, etc.).

[0239] In addition, the bispecific antibodies or compositions described herein can be directly used to treat cancer, such as cancers characterized by CD117-positive cells. For example, the bispecific antibodies or compositions described herein can be used to treat leukemia, especially in subjects exhibiting CD117-positive leukemia cells. By depleting CD117-positive cancer cells, such as leukemia cells, the bispecific antibodies or compositions described herein can be directly used to treat various cancers. Exemplary cancers that can be treated in this manner include hematological cancers such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma.

[0240] Acute myeloid leukemia (AML) is a cancer of myeloid blood cells, characterized by the rapid proliferation of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML is the most common acute leukemia affecting adults, and its incidence increases with age. The symptoms of AML are caused by the replacement of normal bone marrow by leukemia cells, which leads to a decrease in the number of red blood cells, platelets, and normal white blood cells. As an acute leukemia, AML progresses rapidly and can be fatal within weeks or months if left untreated. In one embodiment, the bispecific antibody or composition described herein is used to treat AML in a human patient in need thereof. In certain embodiments, treatment with the bispecific antibody or composition can deplete AML cells in a subject. In some embodiments, about 50% or more of the AML cells are depleted. In other embodiments, about 60% or more of the AML cells are depleted, or about 70% or more of the AML cells are depleted, or about 80% or more, about 90% or more, or about 95% or more of the AML cells are depleted. In certain embodiments, treatment with the bispecific antibody or composition is a single-dose treatment. In certain embodiments, a single-dose treatment can deplete about 60%, about 70%, about 80%, about 90%, about 95% or more of the AML cells.

[0241] In addition, the bispecific antibody or composition described herein can be used to treat autoimmune diseases. For example, the bispecific antibody or composition can be administered to a subject (such as a human patient) suffering from an autoimmune disease to kill CD117-positive immune cells. CD117-positive immune cells can be autoreactive lymphocytes, such as T cells expressing a T cell receptor capable of specifically binding to a self-antigen and mounting an immune response against the self-antigen. By depleting autoreactive CD117-positive cells, the bispecific antibody or composition described herein can be used to treat autoimmune disorders such as those described below. Additionally or alternatively, the bispecific antibody or composition described herein can treat autoimmune diseases by depleting the endogenous hematopoietic stem cell population prior to hematopoietic stem cell transplantation, in which case the transplanted cells are able to home to the microenvironment created by the endogenous cell depletion step and establish effective hematopoiesis. This, in turn, can reconstitute the cell population depleted during the autoimmune cell clearance process.

[0242] Autoimmune diseases that can be treated with the bispecific antibodies or compositions described herein include, but are not limited to: psoriasis, psoriatic arthritis, type 1 diabetes, rheumatoid arthritis (RA), human systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia totalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Barlow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac disease - dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus, autonomic dysfunction, endometriosis, idiopathic mixed cryoglobulinemia, fibromyalgia - fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain - Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA nephropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Ménière's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff - person syndrome, Takayasu arteritis, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, collagenous colitis, uveitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis.

[0243] In some embodiments of the method, step b) is carried out within 24 hours, 48 hours, 72 hours, 5 days, 7 days, 2 weeks, 3 weeks, or 1 month after step a).

[0244] In some embodiments, the dose administered to a subject may vary depending on the embodiment, the drug used, the method of administration, and the site and subject being treated. However, the dose should be sufficient to provide a therapeutic response. A clinician can determine the effective amount to administer to a human or other subject to treat a medical condition. The exact amount required to achieve a therapeutic effect may depend on numerous factors, such as the activity of the antibody and the route of administration.

[0245] The dose of the bispecific antibody or composition disclosed herein can be administered to a mammal in one dose, or in a series of divided doses over a suitable period of time, for example, administered at frequencies such as daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annually or annually as needed. A dosage unit containing an effective amount of the bispecific antibody or composition can be administered once daily, or the total daily dose can also be administered in two, three, four or more divided doses as needed.

[0246] A suitable administration method can be selected by a doctor. The administration route can be parenteral, for example, administered by injection, intranasally, by pulmonary administration or transdermally. Systemic or local administration can be carried out by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the bispecific antibody or composition is selected for parenteral delivery, inhaled delivery or delivery through the digestive tract, such as oral administration. The dosage and method of administration can vary according to the weight, age, physical condition, etc. of the subject and can be appropriately selected.

[0247] In some embodiments, the method further comprises administering a second therapeutic agent to the subject..

[0248] In certain embodiments, the bispecific antibody or composition disclosed herein is administered before, substantially simultaneously with or after the administration of the second therapeutic agent.

[0249] In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1 / PD-L1 inhibitors, LAG3 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors or glucocorticoids. In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents can include, for example, cytotoxic agents, antimetabolites (such as folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (such as camptothecin derivatives, anthracenediones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (such as taxanes, vinca alkaloids), protein synthesis inhibitors (such as harringtonine, camptothecin derivatives, quinoline alkaloids), alkylating agents (such as alkyl sulfonates, ethyleneimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.), alkaloids, terpenoids and kinase inhibitors.

[0250] Kit

[0251] The present invention provides a pharmaceutical package or kit, which comprises one or more containers filled with one or more ingredients of the compositions described herein, such as the bispecific antibodies disclosed herein. Optionally, associated with the container(s) may be instructions in a form specified by a government agency that regulates the manufacture, use, or sale of drugs or biological products, indicating that the agency has approved the manufacture, use, or sale for human administration.

[0252] In a specific embodiment, the kit comprises a first container containing the bispecific antibody disclosed herein. In a specific embodiment, the kit comprises a first container in the form of a vial containing a lyophilized sterile bispecific antibody powder under vacuum conditions, and the kit further comprises a second container containing a pharmaceutically acceptable liquid.

[0253] In a specific embodiment, the present invention provides an injection device containing the bispecific antibody. In a specific embodiment, the injection device contains the bispecific antibody in a sterile solution. In a specific embodiment, the injection device is a syringe.

[0254] Medical use

[0255] The present invention provides the use of the bispecific antibody or composition disclosed herein in the preparation of a medicament for depleting hematopoietic stem cells in a subject.

[0256] The present invention provides the use of the bispecific antibody or composition disclosed herein in the preparation of a medicament for treating a hematopoietic system disease in a subject.

[0257] The present invention further provides the bispecific antibody or composition disclosed herein for depleting hematopoietic stem cells in a subject.

[0258] The present invention further provides the bispecific antibody or composition disclosed herein for treating a hematopoietic system disease in a subject.

[0259] In some embodiments of the uses disclosed herein, the bispecific antibodies or compositions disclosed herein are used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1 / PD-L1 inhibitors, LAG3 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, or glucocorticoids. In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents can include, for example, cytotoxic agents, antimetabolites (such as folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (such as camptothecin derivatives, anthracenediones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (such as taxanes, vinca alkaloids), protein synthesis inhibitors (such as harringtonine, camptothecin derivatives, quinoline alkaloids), alkylating agents (such as alkyl sulfonates, ethyleneimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.), alkaloids, terpenoids, and kinase inhibitors.

[0260] In some embodiments, the hematopoietic system diseases are selected from the group consisting of hemoglobinopathies, immunodeficiencies, metabolic disorders, hematological diseases (such as bone marrow failure syndromes, myelodysplastic syndromes, acute lymphocytic and myelocytic leukemias, and chronic lymphocytic and myelocytic leukemias), hematopoietic cell proliferative diseases, transplant rejection, autoimmune diseases, and autoinflammatory diseases.

[0261] In some embodiments, the hematopoietic system diseases are selected from the group of hematopoietic system diseases including hemoglobinopathies, bone marrow failure syndromes, immunodeficiencies, metabolic disorders, myeloid and lymphoid malignancies, and hematopoietic proliferative diseases, such as: mastocytosis, myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute myeloid leukemia with recurrent genetic abnormalities (e.g., molecular changes in CD117, NPM1, CEBPα, FLT3, RUNX1, ASXL1, TP53, IDH1, and IDH2), chromosomal aberrations (e.g., favorable chromosomal changes including abnormalities of chromosome 16 at bands p13 and q22 [t(16;16)(p13;q22), inv(16)(p13q22)] and translocations between chromosomes 8 and 21 [t(8;21)], intermediate-risk aberrations including trisomy 8 and translocations of chromosomes 9 and 11 [t(9;11)], and unfavorable chromosomal changes including subsets of cases with additional copies of chromosome 8 or 13 [e.g., trisomy 8 (+8)], complete or partial deletions of chromosome 5 or 7, complex changes in many chromosomes, and changes at band q26 of chromosome 3).AML with myelodysplasia-related changes, treatment-related myeloid neoplasms AML, AML not otherwise specified (NOS), undifferentiated myeloid leukemia, M0 type, myeloid leukemia, M1 type, with little or no maturation, myeloid leukemia, M2 type, with maturation, promyelocytic leukemia, M3 type, myelomonocytic leukemia, M4 type, myelomonocytic leukemia, M4eo type, with eosinophils, undifferentiated monocytic leukemia, M5a type (monoblast leukemia), differentiated monocytic leukemia, M5b type, erythroleukemia, M6 type, recurrent AML and relapsed AML, acute lymphoblastic leukemia (CLL), c-KIT positive, mast cell leukemia, megakaryocytic leukemia, chronic myeloid leukemia (including BCR-ABL translocation [t(9;11)], Philadelphia chromosome Ph positive and Ph negative CML, chronic phase, accelerated phase and blast crisis), chronic lymphocytic leukemia requiring hematopoietic stem cell transplantation, such as early relapse or refractory cases after purine analogue treatment, or cases carrying p53 mutations, T lymphoblastic leukemia, CD117 receptor positive, autoinflammatory diseases, such as familial Mediterranean fever (FMF), cryopyrin-associated periodic syndromes (CAPS), tumor necrosis factor receptor-associated periodic syndromes (TRAPS), IL-1 receptor antagonist deficiency (DIRA), hyper IgD syndrome (HIDS), systemic autoinflammatory diseases (SAIDS), Blau syndrome, Mejeed syndrome, pyogenic arthritis-pyoderma gangrenosum-acne syndrome (PAPA), periodic fever-aphthous stomatitis-pharyngitis-adenitis syndrome (PFAPA), Behçet's disease, Still's disease, Crohn's disease, and acquired autoinflammatory syndromes, such as Schnitzler syndrome.

[0262] Autoimmune diseases that can be treated with the bispecific antibodies or compositions described herein include, but are not limited to: psoriasis, psoriatic arthritis, type 1 diabetes, rheumatoid arthritis (RA), human systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia totalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Barlow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac disease - dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos' disease, discoid lupus, autonomic dysfunction, endometriosis, idiopathic mixed cryoglobulinemia, fibromyalgia - fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain - Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA nephropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Ménière's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff - person syndrome, Takayasu arteritis, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, collagenous colitis, uveitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis..

[0263] Production method

[0264] The antibodies of the present invention can be prepared by any technique known in the art, such as but not limited to any chemical, biological, genetic or enzymatic technique, which can be used alone or in combination. Generally, after knowing the amino acid sequence of the desired sequence, those skilled in the art can easily prepare the antibodies by standard techniques for producing polypeptides. For example, these antibodies can be synthesized using well-known solid-phase methods, preferably using commercially available peptide synthesizers (such as those produced by Applied Biosystems, Foster City, California) and operating according to the manufacturer's instructions. Alternatively, the antibodies of the present invention can be synthesized by recombinant DNA techniques well known in the art. For example, after incorporating the DNA sequence encoding the antibody into an expression vector and introducing these vectors into a suitable eukaryotic or prokaryotic host capable of expressing the desired antibody, the antibody can be obtained as a DNA expression product and subsequently isolated using well-known techniques.

[0265] Examples

[0266] Antibody sequence

[0267] The antibody sequences of the anti-CD117xCD3 bispecific T cell engager PR004384, the anti-HELxCD3 control bispecific T cell engager PR001310, and the recombinant monoclonal antibody PR004382 are shown in Tables 1 to 3 below.

[0268] Table 1. Antibody sequence of PR004384

[0269] PR004384 sequence serial number Chain 1 DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 57 Chain 2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 56 Chain 3 QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGDKAALTLLGAQPEDEAEYFCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 52 <![CDATA[VH that binds to CD117 (CDR with bold table show )]]> <![CDATA[QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSY NMHWVRQAPGQGLEWMGVI YSGNGD TSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR ERDTRFGN WGQGTLVTVSS]]> 44. (Serial numbers of CDRs: 6, 12, and 18) <![CDATA[VL (CDR in combination with CD117 bold table show )]]> <![CDATA[DIVMTQSPDSLAVSLGERATINC RASESVDIYGNSFMH WYQQKPGQPPKLLIY LASNLES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQNNEDPYT FGGGTKVEIK]]> 47 (Serial numbers of CDRs: 26, 32, and 38) <![CDATA[VH that binds to CD3 (CDR is Bold Show Show )]]> <![CDATA[EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTY AMNWVRQASGKGLEWVGRI RSKYNNYA TYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS]]> 43. (Serial numbers of CDRs: 5, 11, and 17) <![CDATA[VL that binds to CD3 (CDR in Bold Table Show )]]> <![CDATA[QAVVTQEPSLTVSPGGTVTLTC RSSTGAVTTSNYAN WVQQKPGQAPRGLIG GTNKRAP WTPARFSGSLLGDKAALTLLGAQPEDEAEYFC ALWYSNLWV FGGGTKLTVL]]> 46. (Serial numbers of CDRs: 25, 31, and 37)

[0270] Table 2. Antibody sequence of PR001310

[0271] PR001310 Sequence Serial number: Chain 1 DIVLTQSPAIMSASPGEKVTMTCSASSSVNYMYWYQQKSGTSPKRWIYDTSKLASGVPVRFSGSGSGTSYSLTISSMETEDAAEYYCQQWGRNPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 55 Chain 2. EVQLQQSGAELMKPGASVKISCKASGYTFSDYWIEWVKQRPGHGLEWIGEILPGSGSTNYHERFKGKATFTADTSSSTAYMQLNSLTSEDSGVYYCLHGNYDFDGWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 54 Chain 3 QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGDKAALTLLGAQPEDEAEYFCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 52

[0272] Table 3. Antibody sequence of PR004382

[0273] PR004382 Sequence Sequence number: Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 50 Light chain DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 53

[0274] Cell culture

[0275] Human erythroleukemia TF-1 cells were cultured in complete medium A (RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Life Technologies), 100 U / mL penicillin (Gibco), 100 μg / mL streptomycin sulfate (Gibco), 1 mM sodium pyruvate (Life Technologies), and 5 ng / mL human granulocyte macrophage colony-stimulating factor (GM-CSF) (Peprotech)). TF-1 cells were maintained at a cell concentration of 2-9×10 5 cells / mL in a humidified incubator with 5% CO2 at 37 °C.

[0276] Primary human CD34+ cells from bone marrow cells were purchased from STEMCELL Technologies. Cells were seeded at a density of 5×10 4 cells / mL and expanded in serum-free StemSpan SFEM II medium supplemented with StemSpan CD34⁺ expansion supplement, UM729 (1 μM), and SR-1 (0.5 μM) (STEMCELLS Technologies, Vancouver, BC, Canada) for up to 7 days. After 3 or 4 days of culture, the cells were diluted to a concentration of 1×10 5 cells / mL. For experiments with human bone marrow CD34⁺ cells, complete medium B (serum-free StemSpan SFEM II medium supplemented with 100 ng / mL stem cell factor (SCF), 100 ng / mL thrombopoietin (TPO), 100 ng / mL Flt3 ligand (Peprotech®, NJ, USA), 1 μM UM729, and 0.5 μM SR-1) was used.

[0277] Human multiple myeloma RPMI-8226 cells and acute myeloid leukemia U937 cells were cultured and expanded in RPMI-1640 medium supplemented with FBS (10%), 2 mM L-glutamine, and 100 U / mL penicillin in a humidified incubator with 5% CO2 at 37 °C.

[0278] Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (Sanquin, Netherlands) by Ficoll density centrifugation (GE Healthcare, Illinois, USA). Human CD3-positive T lymphocytes were purified from cryopreserved human PBMCs using RosetteSep™ Human T Cell Enrichment Cocktail (STEMCELL Technologies) according to the manufacturer's instructions. The purified T cells were seeded in 24-well plates at a concentration of 1×10 6 cells / mL in complete medium C (RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin-sulfate, and 20 UI / mL human interleukin-2 (hIL-2; PeproTech)) and cultured overnight. For the isolation of human NK cells, RosetteSep™ Human NK Cell Enrichment Cocktail (STEMCELLS Technologies) was used according to the manufacturer's instructions. The purity of all samples (>90%) was detected by BD LSRII-Fortessa (BD Biosciences), and data analysis was performed using FlowJo VX (BD Biosciences). Subsequently, the density of NK cells was adjusted to 1×10 6 cells / mL and placed in 24-well plates containing complete medium C.

[0279] Binding Detection of Flow Cytometry-Based Bispecific T Cell Engagers

[0280] TF-1 cells, human PBMCs, or purified human CD3-positive T cells (2 - 5x10 5The cells were washed in FACS wash buffer (FWB), resuspended in 50 μL of FWB solution containing the bispecific T cell engager or antibody solution at the indicated concentration, and incubated on ice for 2 hours. For the negative control, human CD3-positive T cells were pre-incubated with mouse anti-human CD3 antibody (BD, 10 μg / mL) at 4 °C for 30 minutes. The cells were washed twice and then incubated with Alexa Fluor 647-labeled goat anti-human F(ab')2 fragment (Jackson ImmunoResearch Laboratories, PA, USA) for 1 hour at room temperature (RT) for the detection of the bispecific T cell engager. Subsequently, they were washed twice with FWB and fixed in paraformaldehyde (1%) and calcium chloride (1 mM) solution. RPMI-8226 and U937 cell lines that do not express CD117 were used as negative controls. The expression of CD117 was measured using a BD LSRII-Fortessa flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (LLC, CA, USA).

[0281] Flow cytometry-based PR004382 binding assay

[0282] The recombinant monoclonal antibody PR004382 was serially diluted 5-fold with FACS buffer (PBS containing 2% FBS). TF-1 cells were washed with FWB and then resuspended in a 96-well V-bottom plate at a density of 1×10 5 cells / well, and 100 μL of the diluted antibody solution was added to each well and incubated at 4 °C for 60 minutes. After the cells were washed twice, they were incubated with the secondary antibody (AlexaFluor® 647-labeled affinity-purified F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific, 1:1000 dilution, Jackson ImmunoResearch, catalog number 109 - 606 - 008) at 4 °C for 60 minutes, and then washed twice with FWB and fixed. The fluorescence values were read on an ACEA NovoCyte flow cytometer, the data were analyzed using FlowJo software (LLC, CA, USA), and the data were processed and plotted using GraphPad Prism software.

[0283] ELISA-based PR004382 binding assay

[0284] Coat a 96-well plate with CD117 protein (SinoBiological, cat# 11996-H08H) at a concentration of 1 μg / ml and a volume of 100 μl / well, and incubate overnight at 4°C. Wash the plate 3 times with 200 μL / well of washing buffer (0.05% BSA, 1X PBS, 0.05% Tween); then incubate with 200 μL / well of blocking buffer (2% BSA, 1X PBS, 0.05% Tween) for 2 hours at room temperature. Serial dilute the antibody to be tested 5-fold with the washing buffer. Add the diluted antibody to the plate at a volume of 100 μL / well, and then incubate for 1 hour at room temperature. Wash the plate 3 times, and then incubate with the secondary antibody (goat anti-human IgG (Fc specific)-peroxidase antibody, Sigma, cat# A0170-1ML) for 30 minutes at room temperature. After washing 3 times, add 100 μl / well of 3,3',5,5'-tetramethylbenzidine (TMB), incubate for 15 minutes at room temperature, and then add the ELISA stop solution. Record the absorbance at 450 nm using a microplate reader (Molecular Devices, SpectraMax). Process and plot the data using GraphPad Prism software.

[0285] Flow cytometry staining

[0286] For mouse whole blood samples (20 - 50 μL), incubate the red blood cells in 2 mL of lysis buffer (ACK lysis buffer) containing 150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA on ice for 2 minutes. For staining of surface antigens of blood cell types, after washing the cells with FWB, incubate with 20 μL of a monoclonal antibody mixture (see Table 4: Monoclonal antibodies) at 4°C for 20 minutes. After staining, wash the cells with FWB and fix them on ice with a fixative containing 1% PFA and 1 mM CaCl2. To exclude apoptotic and dead cells from the analysis, add Annexin V antibody (BD Biosciences) and 2.5 mM CaCl2 to the staining solution. Detect the signals using a BD LSRII-Fortessa flow cytometry system (BD Biosciences) and FACS Diva software, and perform data analysis using FlowJo VX software (BD Biosciences).

[0287] Table 4: Monoclonal antibodies

[0288] Reagent Catalog number Company Alexa Fluor® 700 anti-mouse CD45 antibody 103127 BioLegend Europe BV PerCp-eF710 anti-mouse CD3 antibody 46-0037-42 eBioscienc. BV421 anti-human CD117 313216 BioLegend Europe BV FITC anti-human CD45 antibody 304006 BioLegend Europe BV APC anti-human CD34 antibody 343607 BioLegend Europe BV BV711 anti-human CD69 antibody 563836 BioLegend Europe BV PE-CF594 anti-human CD3 antibody 562280 BioLegend Europe BV BV650 anti-human CD4 antibody 563875 BioLegend Europe BV BV786 anti-human CD8 antibody 563823 BioLegend Europe BV SB600 anti-human CD25 antibody 63-0259-42 eBioscienc. PerCP-Cy™5.5 Annexin V 561431 BD Pharmingen

[0289] T cell-dependent cytotoxicity (TDCC) assay

[0290] According to the manufacturer's protocol, target cells (TF-1 cells or human bone marrow CD34⁺ cells) were labeled with CellTrace™ FarRed / CFSE fluorescent dyes (ThermoFisher Scientific). The labeled cells (1x10 6 cells / mL, 50 µL / well) were seeded in a 96-well round-bottom plate with 100 μL of human CD3-positive T cells at the specified effector-to-target cell (E:T) ratio, using complete medium A or B (see above). 50 μL of complete medium containing the specified concentration of bispecific T cell engager was added to each well and incubated at 37 °C and 5% CO2 for 6 or 24 hours. After washing twice, the cells were stained with Annexin-V. The cells were analyzed by flow cytometry, and at least 10,000 events were collected within the target cell gate. To measure specific target cell lysis, the CellTrace TM -labeled target cells were gated, and Annexin-V positivity was detected. Then, the percentage of specific lysis was calculated using the following formula: Cytotoxicity (%) = [(% of dead target cells) - (% of spontaneous death)] × 100 / [100 - (% of spontaneous death)].

[0291] Cytokine analysis

[0292] To investigate cytokine production by T cells after treatment with bispecific T cell engager in an in vitro setting, supernatants were collected from the TDCC assay and centrifuged to remove residual debris or intact cells. All samples were stored at -20 °C for up to one month. After thawing the samples on ice, they were centrifuged at 1,300 x g for 5 minutes at room temperature. Samples were serially diluted two-fold in a low-protein binding plate (Corning™ 96-Well Nonbinding Surface [NBS™] Microplates) according to the manufacturer's instructions (Ella automated Immunoassay system, Biotechne). Subsequently, the samples were diluted 1:1 in wash buffer according to the manufacturer's instructions for the supernatants. The diluted samples were loaded into the ELLA Simple Plex kit and analyzed using the ELLA Simple Plex system (Biotechne).

[0293] T cell activation assay

[0294] To determine the activation status of T cells, cells were collected 24 hours after the TDCC assay. Cells were stained with fluorescent antibodies anti-CD4-BV650, anti-CD8-BV786, anti-CD69-BV711 (Biolegend Europe BV) and anti-CD25-SB600 (eBioscience). Target cells labeled with CellTraceTM were excluded during analysis. The expression of CD25 and CD69 in Annexin-V negative CD4⁺ and CD8⁺ T cells was analyzed using FlowJo VX software (BD Biosciences).

[0295] T cell proliferation assay

[0296] To analyze the proliferation of T cells, 0.5×10 5 TF-1 target cells were placed together with purified T cells labeled with FarRed (effector to target cell ratio E:T = 10:1) in complete medium A containing the specified concentration of bispecific T cell engager (see above) and incubated at 37°C for 5 days. Cells were collected and stained with Annexin-V. Samples were examined on a BD LSRII - Fortessa ((BD Biosciences) to obtain data. The proliferation of T cells (Annexin-V negative / CellTrace TM positive) was analyzed using FlowJo VX software ((BD Biosciences).

[0297] Cross-activity assay of bispecific T cell engager on mouse T cells

[0298] Mouse CD3-positive T cells were purified from the spleens of C57BL6 / J mice by negative selection using magnetic beads (EasySep, Stemcell Technologies) and cultured overnight in complete medium C. To determine the cross-activity of the bispecific T cell engager, TF-1 target cells were labeled with CellTrace™ according to the manufacturer's protocol. Then, the labeled TF-1 target cells (1x10 6cells / mL, 50 µL) were seeded in complete medium A in 96-well round-bottom plates, and 100 μl of CD3-positive T cells (effector-to-target cell ratio E:T = 4:1) were added. Bispecific T cell engager (100 ng / mL) in complete medium A was added to each well, and the plates were incubated in an incubator at 37°C and 5% CO2 for 24 hours. To analyze specific target cell lysis induced by mouse T cells, cells were collected and stained with Annexin-V and anti-mouse CD3-PerCp-eF710. TF-1 cells (CellTrace TM positive cells) were detected using flow cytometry with the same gating strategy as used for TDCC detection.

[0299] Pharmacokinetics (PK) of bispecific T cell engager

[0300] Female C57BL6 / J mice (8 to 10 weeks old) (Charles River France) were used, and the anti-CD117xCD3 bispecific T cell engager diluted in sterile saline (0.9%) was intraperitoneally injected at a dose of 1.0 mg / kg. At the designated time points after injection, blood samples (20 - 50 µL) were collected from the tail vein and collected into Microvette CB300 capillary blood collection tubes containing a coagulation activator ((Sarstedt)). After centrifuging at 10000 g for 5 minutes at room temperature to separate the serum, the serum samples were aliquoted and stored at -20°C. Within 4 weeks after separation, the concentration of the anti-CD117xCD3 bispecific T cell engager in the serum was determined by ELISA. In the ELISA assay, 100 µL of human recombinant CD117 protein isoform 2 (P10721 - 2), extracellular domain (Met 1 - Thr 516) with a C-terminal polyhistidine (His) tag (Bio-Connect BV) was dissolved in the coating buffer (carbonate / bicarbonate buffer) at a concentration of 2 µg / mL and used to coat a 96-well multi-array plate overnight at 4°C. The plate was washed three times with 200 µL of PBST (PBS + Tween-20 [0.05%]), and then blocked with PBST containing 5% bovine serum albumin for 2 hours at room temperature. After thawing the samples on ice, they were centrifuged at 13000×g for 5 minutes at room temperature. The samples were diluted with the dilution buffer (PBST BSA [1%]) at a ratio of 1:100 to 1:2000, 50 µL was added to each well, and incubated overnight at 4°C. To detect the bispecific T cell engager, after washing with PBS - T, 100 µL of an antibody specific for the F(ab')2 fragment of goat anti-human IgG conjugated to HRP (Jackson ImmunoResearch Laboratories, PA, USA) (dissolved in the blocking buffer at a ratio of 1:10000) was added and incubated on an oscillator for 1 hour at room temperature. After washing, 50 µL of 3,3',5,5'-tetramethylbenzidine (TMB) solution was added to each well and allowed to act for at least 15 minutes. Then, 50 µL of 2N sulfuric acid (stop solution) was added. The signal was detected at 450 / 650 nm using a Versamax microplate reader (Molecular Devices). The sample concentration range of the calibration curve was 20 to 0.039 ng / mL of CD117 protein, which was prepared by adding the target to a pooled blank serum sample from untreated mice.

[0301] Pharmacokinetics of PR004382

[0302] B6.Cg-Fcgrttm1Dcr Tg (CAG-FCGRT)276Dcr / DcrJ mice, 8 to 10 weeks old, purchased from The Jackson Laboratory, were administered PR004382 (diluted in sterile 0.9% saline at a dose of 1 mg / kg) by intraperitoneal injection (IP). At different time points (1 day, 2 days, 3 days, 5 days, 7 days after injection), blood samples (20 - 50 µL) were collected from the tail vein using Microvette CB300 capillary blood collection tubes (Sarstedt) containing a coagulant. Serum samples were obtained by centrifuging the blood at 10,000 g for 5 minutes at room temperature, then aliquoted and stored at -20°C. Samples were analyzed by ELISA using the same protocol as for the analysis of bispecific T cell engagers, except that an HRP-conjugated antibody specific for the Fcγ fragment of goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used.

[0303] Treatment of humanized NSG mice with anti-CD117×CD3 bispecific T cell engager

[0304] Female mice aged 16 to 24 weeks transplanted with human CD34+ hematopoietic stem cells (hu-CD34 NSG, 005557) were purchased from The Jackson Laboratory. Blood samples for peripheral blood analysis were collected from the tail vein one day before treatment. Human blood cells (hCD45+ cells) in peripheral blood were determined by flow cytometry using fluorescein isothiocyanate (FITC)-labeled anti-human CD45 antibody as described below. Mice with a human CD45+ cell content greater than 70% in peripheral blood were used. The bispecific T cell engager was diluted to a final concentration of 1.0 mg / kg with sterile saline (0.9%) and injected intraperitoneally every 12 hours for a total of 6 injections. At 16 hours and 4.5 days after treatment, the mice were sacrificed by cervical dislocation, and bone marrow cells and peripheral blood cells from the femurs and tibias were collected. The cells were stained according to the flow cytometry surface staining protocol (see above). Hematopoietic stem cells and progenitors in the bone marrow were determined by staining the cells with a cocktail of antibodies containing: FITC-hCD45, AF680-mCD45, APC-hCD34, BV421-CD117 (all from Biolegend Europe BV) and PerCp 5.5-labeled Annexin-V (BD Pharmingen). Data were acquired by a BD LSRII-Fortessa flow cytometer (BD Biosciences).

[0305] Antibody-dependent cell-mediated cytotoxicity (ADCC) assay

[0306] TF-1 cells and Jurkat-NFAT reporter cells expressing CD16A were separately suspended in buffer (RMI1640 + 4% FBS) at cell densities of 6×10 5 / mL and 2×10 6 / mL, respectively. Then 50 μL of TF-1 cells (3×10 4 cells) and 50 μL of Jurkat-CD16a-NFAT cells (1×10 5Add cells (number of cells) to the culture plate (ViewPlateTM-96 TC, PerkinElmer, cat# 6005181). Dilute the antibody to be detected serially 3-fold with buffer, and then add 50 μL of the diluted antibody to the culture plate. Place the culture plate in an incubator containing 5% CO2 and incubate at 37°C for 6 hours. Before reading the luminescence value using an Envision microplate reader (Perkinelmer), add 50 μL / well of Bright-Glo luciferase assay reagent. Process and plot the data using GraphPad Prism software.

[0307] Determination of the affinity between PR004382 and the Cd16a receptor by the Otet biosensor

[0308] Dilute the antibody to be detected to 200 nM in PBS. Insert the HIS1K sensor (Pall ForteBio, cat#18-5120) into the sensor tray of the Octet Red 96e (Pall ForteBio) and pre-treat it in PBS buffer for 10 minutes. Load the human CD16a (V176) protein (NovoProtein, cat# C441) onto the HIS1K sensor to make the loading signal level greater than 0.2 nm, and then immerse the sensor into the well containing the antibody to be detected. Record and analyze the binding signal using the 1:1 global fitting model with ForteBio data analysis software.

[0309] Detection of NK cell-mediated cytotoxicity

[0310] According to the manufacturer's protocol, TF-1 cells and human bone marrow CD34+ cells were labeled with CellTrace™ FarRed / CFSE fluorescent dyes (ThermoFisher Scientific). In a U-bottom 96-well plate, target cells and human NK cells were co-cultured at an effector-to-target (E:T) ratio of 1:1 in complete medium A (TF-1 cells) or complete medium B (human bone marrow CD34+ cells). PR004382 and IgGκ1 isotype control antibody (human IgGκ1, κ isotype control antibody, CBMAB-0402ZL, purchased from Creative Biolabs) were diluted in complete medium A or complete medium B and then added to the cells at a final concentration range of 1 ng / mL to 1 μg / mL (100 μL / well). Cells were harvested after 24 hours and stained with Annexin V. Live target cells were gated for CellTrace TM positive cells, and cell death was detected by Annexin V uptake. At least 10,000 events were collected within the gate of live target cells. Cytotoxicity was calculated using the following formula: Cytotoxicity (%) = [(% of dead target cells) - (% of spontaneous death rate)] × 100 / [100 - (% of spontaneous death rate)].

[0311] Statistics

[0312] All statistical analyses were performed using Graphpad Prism 9 software (GraphPad, La Jolla, CA). Four-parameter variable slope nonlinear regression was used to fit the dose-response curves and perform pharmacokinetic analyses. Data were presented as mean ± standard error and compared using two-tailed independent sample t-tests, unless otherwise specified in the figure legend. Each in vitro experiment was repeated at least three times. The number of asterisks indicates the significance level of the adjusted p-value: indicates p < 0.05, indicates p < 0.01, indicates p < 0.001, and indicates p < 0.0001.

[0313] Ethical statement

[0314] Animal experiments were conducted in accordance with project license number 2010607 issued by the animal welfare institution Instantie voor Dierenwelzijn (IvD). All animal experiments were carried out in accordance with the relevant laws of the Centrale Commissie Dierproeven (CCD) of the Netherlands. All mice were housed in a certified barrier facility at the Erasmus University Medical Center.

[0315] Example 1. Binding specificity of PR004384 (anti-CD117xCD3 bispecific T cell engager)

[0316] Figure 1 shows a schematic diagram of the structure of the anti-CD117×CD3 bispecific T cell engager PR004384. To study the activity of the anti-CD117×CD3 bispecific T cell engager PR004384, an in vitro binding experiment was performed using the human erythroleukemia cell line TF-1, which is known to express the CD117 receptor, and two hematopoietic cell lines, RPMI-8226 and U937, which do not express CD117, as negative controls. The results showed that the anti-CD117×CD3 bispecific T cell engager PR004384 could bind to TF-1 cells, but not to RPMI-8226 and U937 cells (Figure 2A). No binding of the PR001310 anti-HEL×CD3 control bispecific T cell engager (HEL: chicken egg lysozyme) to TF1 cells was observed (Figure 2A). In addition, the results showed that the anti-CD117×CD3 bispecific T cell engager PR004384 bound to TF-1 cells in a dose-dependent manner, with a maximum half-maximal effective concentration (EC50) of 0.5266 nM (Figure 2B). These results confirmed the specificity of PR004384 for the CD117 receptor.

[0317] To determine the binding ability of the anti-CD117×CD3 bispecific T cell engager PR004384 to CD3 expressed on T cells, in vitro binding assays were performed using purified human CD3-positive T cells or peripheral blood mononuclear cells (PBMCs). The results showed that both anti-CD117×CD3 PR004384 and anti-HEL×CD3 PR001310 could specifically bind to purified CD3-positive T cells as well as CD3-positive T cells in PBMCs (Figure 2C). Notably, when the CD3 antigen on T cells was blocked with an anti-CD3 monoclonal antibody (purified NA / LE mouse anti-human CD3 clone UCHT1 [Catalog No: 555329, Invitrogen]) targeting the same epitope, the binding of these two bispecific T cell engagers could not be detected (Figure 2C). In addition, no binding of anti-CD117×CD3 PR004384 or anti-HEL×CD3 PR001310 was detected on cells that do not express CD3, such as U937 and RPMI-8226 cells (Figure 2A), which again confirmed the specificity of the bispecific T cell engager for CD3.

[0318] Next, it was tested whether the anti-CD117×CD3 bispecific T cell engager PR004384 could bind to TF-1 cells and T cells simultaneously and activate T cells. Under in vitro cell culture conditions, T cells did not proliferate in the absence of TF-1 cells and the bispecific T cell engager (Figure 3A). In contrast, it was found that the anti-CD117×CD3 bispecific T cell engager PR004384, but not the anti-HEL×CD3 bispecific T cell engager PR001310, could induce T cell proliferation in the presence of TF-1 cells (Figure 3A). These results indicate that T cells co-cultured with TF-1 cells and the anti-CD117×CD3 bispecific T cell engager PR004384 were activated by the anti-CD117×CD3 bispecific T cell engager PR004384 binding to the CD117 and CD3 receptors simultaneously.

[0319] Similarly, it is noteworthy that it is the anti-CD117×CD3 bispecific T cell engager PR004384, rather than the anti-HEL×CD3 bispecific T cell engager PR001310, that induced the expression of activation markers CD69 and CD25 on T cells (Figure 3B), as well as the expression of a series of cytokines related to T cell activation (Figure 3C). In addition, in the presence of both TF-1 cells and T cells, the anti-CD117×CD3 bispecific T cell engager PR004384 upregulated the expression of Th1 and Th2 cytokines in a dose-dependent manner (Figure 3C).

[0320] Collectively, these results indicate that the anti-CD117×CD3 bispecific T cell engager PR004384 can specifically bind to both target cells expressing CD117 and CD3-positive T cells simultaneously and activate T cells.

[0321] Example 2. Cytotoxic activity of PR004384

[0322] Next, it was investigated whether the anti-CD117×CD3 bispecific T cell engager PR004384 has hematopoietic stem cell-specific killing activity in vitro. In this regard, the killing effect on these cells was tested by incubating TF-1 cells expressing CD117 and primary human bone marrow CD34+ hematopoietic stem cells with the anti-CD117×CD3 bispecific T cell engager PR004384 or anti-HEL×CD3 as a control in the presence of T cells. In vitro, the anti-CD117×CD3 bispecific T cell engager PR004384 induced depletion of more than 80% of TF-1 cells after 6 hours and more than 50% of primary human bone marrow CD34+ cells after 24 hours (Figure 4A). When cells were treated with the anti-HEL×CD3 control bispecific T cell engager PR001310, depletion of CD117-positive cells was not observed (Figure 4A). In addition, it was determined by in vitro killing assays that the anti-CD117×CD3 bispecific T cell engager had no cross-reactivity with mouse T cells, as evidenced by its inability to kill TF-1 cells (Figure 4B).

[0323] Example 3. In vivo pharmacokinetics (PK) of PR004384

[0324] Subsequently, the in vivo pharmacokinetics of the anti-CD117×CD3 bispecific T cell engager PR004384 were studied. Briefly, the anti-CD117×CD3 bispecific T cell engager PR004384 (1.0 mg / kg) was intraperitoneally injected into female C57BL6 / J mice, and blood samples were collected from the tail vein at different time points for analysis. The concentration of the anti-CD117×CD3 bispecific T cell engager PR004384 in serum was determined by enzyme-linked immunosorbent assay (ELISA). The results showed that the serum half-life (T½ value) of the anti-CD117×CD3 bispecific T cell engager PR004384 was 2 hours and 19 minutes (Figure 5).

[0325] Example 4. Depletion effect of PR004384 on hematopoietic stem cells in vivo

[0326] In this example, the depletion activity of the anti-CD117×CD3 bispecific T cell engager PR004384 on hematopoietic stem cells in vivo was studied. Humanized mice with more than 70% human CD45-positive cells in peripheral blood (PB) were used in the experiment. These mice were injected with the anti-CD117×CD3 bispecific T cell engager PR004384 every 12 hours for a total of six times (Figure 6A). No bispecific T cell engager was detected in the blood 24 hours after the last injection. Notably, 16 hours after the last treatment, the depletion rate of human CD34+ CD117+ hematopoietic stem cells in the bone marrow of humanized mice treated with the anti-CD117×CD3 bispecific T cell engager PR004384 was as high as 95% (Figure 6B), and CD34+ CD117+ cells were almost completely depleted 4.5 days after the last treatment (Figure 6C). When the mice were treated with the anti-HEL×CD3 bispecific T cell engager, no depletion of CD34+ CD117+ cells was detected (Figure 6C). Six weeks after treatment, very few human CD45+ cells (less than 1.5×10 6 cells, approximately 3%) were observed in the bone marrow of mice treated with CD117×CD3, while approximately 50×10 6 human CD54+ cells were observed on average in the bone marrow of mice treated with the control HEL×CD3 (Figure 6D).

[0327] Example 5. Monoclonal antibody PR004382 binds to CD117 and CD16a.

[0328] Subsequently, the binding characteristics of monoclonal antibody PR004832 were tested. The VH / VL sequences of this antibody are identical to the CD117 binding region of the anti-CD117×CD3 bispecific T cell engager PR004384. Briefly, using monoclonal antibody PR004382 and isotype control human IgG1 monoclonal antibody (CBMAB-0402ZL, purchased from CreativeBiolabs), an ELISA assay was performed with CD117 protein as the target. The results showed that PR004382 bound to CD117 with an EC50 of 0.01548 nM (Figure 7A). In addition, a FACS-based binding assay was performed using TF-1 cells. In this assay, the EC50 of PR004382 was observed to be 0.03313 nM, while human IgG1 did not bind to TF-1 cells (Figure 7B).

[0329] To determine the affinity of PR004382 for the CD16a receptor expressed on NK cells, a biosensor binding assay was performed, and the results showed that PR004382 bound to CD16a with a KD of 27.2 nM (association constant K-on = 3.43E+5 M, dissociation constant K-off = 9.33E-03 M) (Figure 7C).

[0330] Example 6. Cytotoxic potential of PR004382

[0331] To investigate the cytotoxic ability of PR004382, an antibody-dependent cell cytotoxicity (ADCC) reporter gene bioassay was performed. We determined that the ADCC EC50 of PR004382 was 0.009 nM, while no signal was observed for the human IgG1 control antibody CBMAB-0402ZL (Figure 8A). Next, we performed an in vitro cytotoxicity assay to determine the killing ability of PR004382. Using TF-1 cells as the target, we determined that the EC50 of PR004382 was 5.375 nM, and the IgGκ1 control antibody did not show cytotoxicity (Figure 8B). Using human CD34+ bone marrow cells as the target, we determined that the in vitro cytotoxicity mediated by PR004382 was 15%, while no cytotoxicity was observed for the IgGκ1 control antibody ( Figure 8C). In addition, we determined the pharmacokinetics of PR004382 in vivo. For this analysis, we injected female B6.Cg-Fcgrttm1Dcr Tg (CAG-FCGRT) 276Dcr / DcrJ mice with PR004382 and collected blood samples from the tail vein at different time points for analysis. We determined that the serum T½ value of PR004382 was 3.8 days (Figure 8D).

[0332] Example 7. PR004384 clears primary human leukemia cells in vitro

[0333] In this example, the depletion of anti-CD117×CD3 bispecific T cell engager PR004384 on primary c-KIT positive acute leukemia cells (T / myeloid mixed phenotype acute leukemia) was investigated. Specifically, purified leukemia cells (1×10 5 cells) obtained from patients were mixed with normal human T cells (effector cell to target cell ratio of 10:1) in an expansion medium (IMDM medium, FBS (20%), SCF, TPO, FLT3 (all at 100 ng / mL), P / S) and treated with anti-CD117×CD3 bispecific T cell engager PR004384 or anti-HEL×CD3 control and incubated for 24 hours. When using PR004384 at a concentration of 1 μg / mL, approximately 50% cell death was observed, and when using PR004384 at a concentration of 100 μg / mL, approximately 80% cell death was observed, while no depletion of AML cells was observed when cells were treated with anti-HEL×CD3 bispecific T cell engager (Figure 9).

[0334] Example 8. PR004384 clears human AML cells in vivo

[0335] In this example, the ability of anti-CD117xCD3 bispecific T cell engager PR004384 to clear acute myeloid leukemia cells in humanized mice transplanted with patient-derived xenograft (PDX) AML cells (normal karyotype, FLT3 internal tandem duplication [ITD]) was investigated. These mice were bred using NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV IL3, CSF2, KITLG) 1Eav / MloySzJ (NSG-SGM3) mice (The Jackson Laboratory, BarHarbour, ME, USA). Briefly, AML-derived PDX cells (1×106 cells) were intravenously injected (i.v.) into 7- to 9-week-old female NSG-SGM3 mice irradiated with a sublethal dose and allowed to expand for 3 weeks. Human purified T cells were activated for 48 hours using a T cell activation kit (Miltenyi Biotec) according to the manufacturer's instructions. Three weeks after transplantation, 1×10 7 pre-activated human T cells were intraperitoneally injected (i.p.) (day 0), followed by intraperitoneal injection of 1.0 mg / kg of anti-CD117×CD3 bispecific T cell engager or anti-HEL×CD3 control reagent for 3 consecutive days (twice a day). The presence of PDX AML cells and human T cells was evaluated by flow cytometry on day 5. Flow cytometry analysis of processed cells from bone marrow was performed using anti-anti-mCD45 FITC, anti-hCD45 APC-Cy7, anti-hCD3 PE-CF594, anti-hCD33 APC, anti-hCD4 BV650, anti-hCD8 BV785, anti-CD117 PE-CY7 (all from Biolegend) and Hoechst dye for detecting cell viability.

[0336] At week 3 of the protocol, humanized mice were transplanted with PDX AML cells expressing human CD117 and human CD33 ( Figure 10 A, B). After expansion of PDX AML cells, mice were injected with activated human T cells and treated with PR004384 or the control antibody HEL×CD3 (Figure 10A). Significantly reduced human PDX AML cells were observed in the bone marrow of mice treated with PR004384 compared to control-treated mice (Figure 10C).

[0337] Next, the depletion of leukemia-initiating cells in mice treated with PR004384 was investigated. Specifically, human cells were enriched from the bone marrow of PR004384-treated and control-treated mice using anti-human CD45 biotin and ultrapure streptavidin microbeads from Miltenyi. In addition, residual human T cells were removed using the EasySep™ Human CD3 Positive Selection Kit II (STEMCELL Technology). Approximately 0.2×10 6Individual isolated bone marrow cells were transplanted into sub-lethally irradiated female NSG-SGM3 mice aged 4 to 6 weeks. Six months after secondary transplantation, the presence of PDX AML cells in the blood, bone marrow, and spleen of the secondary transplanted mice was evaluated by flow cytometry.

[0338] PDX AML cells were detected in the bone marrow (BM), spleen, and peripheral blood of most mice receiving control-treated bone marrow cells (Figure 10D). However, PDX AML cells were not detected in the mice receiving bone marrow cells treated with PR004384 after transplantation (Figure 10D), indicating that leukemia-initiating cells had been completely eliminated after treatment with PR004384.

[0339] Example 9. Anti-CD117xCD3 bispecific T cell engager PR004384 binds to rhesus CD117-positive hematopoietic stem cells

[0340] In this example, it was investigated whether the bispecific T cell engager PR004384 could bind to rhesus CD117-positive hematopoietic stem cells. Specifically, according to the manufacturer's protocol, CD34⁺ cells were enriched from rhesus whole bone marrow samples using the EasySep APC Positive Selection Kit (STEMCELL Technology) and APC-labeled mouse anti-human antibody against CD34 (clone 563, BD Pharmingen™). Then, the enriched CD34 cells (1×10 6 cells, purity 55%) were washed with FACS wash buffer, resuspended in 50 μL of bispecific T cell engager solution (10 µg / mL), or only resuspended in FACS wash buffer, and incubated on ice for 2 hours. The cells were washed twice and then incubated with R-phycoerythrin-labeled affinity-purified F(ab')2 fragment goat anti-human IgG, F(ab')2 fragment (Jackson ImmunoResearch Laboratories, PA, USA) on ice for 1 hour, followed by two washes with FACS wash buffer and fixation with a solution containing paraformaldehyde (1%) and calcium chloride (1 mM).

[0341] The expression of CD34 and CD117 was determined using a BD LSRII-Fortessa (BD Biosciences), and the data were analyzed using FlowJo (LLC, CA, USA) software ( Figure 11 A). The results showed that anti-CD117xCD3 PR004384 binds to rhesus CD117+ hematopoietic stem cells (Figure 11 B)。

Claims

1. A bispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to CD117 and comprises a first heavy chain variable region (VH_A) and a first light chain variable region (VL_A), and the second binding region binds to CD3 and comprises a second heavy chain variable region (VH_B) and a second light chain variable region (VL_B), wherein: The VH_A comprises HCDR1, HCDR2, and HCDR3 of VH having an amino acid sequence containing SEQ ID NO: 44; The VL_A comprises LCDR1, LCDR2, and LCDR3 of VL having an amino acid sequence containing SEQ ID NO: 47; The VH_B comprises HCDR1, HCDR2, and HCDR3 of VH having an amino acid sequence containing SEQ ID NO: 43; and The VL_B comprises LCDR1, LCDR2, and LCDR3 of VL having an amino acid sequence containing SEQ ID NO:

46.

2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH_A comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively; The VL_A comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs: 26, 32, and 38, respectively; The VH_B comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs: 5, 11, and 17, respectively; and The VL_B comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs: 25, 31, and 37, respectively.

3. The bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The VH_A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 44; The VL_A comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 47; The VH_B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 43; and The VL_B comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

46.

4. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH_A comprises the amino acid sequence as shown in SEQ ID NO: 44; The VL_A comprises the amino acid sequence shown in SEQ ID NO: 47; The VH_B comprises the amino acid sequence shown in SEQ ID NO: 43; and The VL_B comprises the amino acid sequence shown in SEQ ID NO:

46.

5. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody comprises three polypeptide chains, wherein: The first polypeptide chain comprises, from the N-terminus to the C-terminus: VH_A-CH1; the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL_A-CL-L-VH_B-CH1; and the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL_B-CL; or The first polypeptide chain comprises, from the N-terminus to the C-terminus: VH_B-CH1; the second polypeptide chain comprises, from the N-terminus to the C-terminus: VL_B-CL-L-VH_A-CH1; and the third polypeptide chain comprises, from the N-terminus to the C-terminus: VL_A-CL, wherein CH1 represents the first domain of the constant region of the immunoglobulin heavy chain; CL each represents the constant region of the immunoglobulin light chain; and L is absent or represents an optional linker.

6. The bispecific antibody or antigen-binding fragment thereof according to claim 5, wherein CL in the second polypeptide chain and CL in the third polypeptide chain each independently comprise a constant region derived from a λ light chain or a κ light chain.

7. The bispecific antibody or antigen-binding fragment thereof according to claim 5 or 6, wherein CL in the second polypeptide chain and CL in the third polypeptide chain are derived from different types of immunoglobulin light chains.

8. The bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein: CL in the second polypeptide chain comprises a light chain constant region derived from a λ light chain (such as a human λ light chain), and CL in the third polypeptide chain comprises a light chain constant region derived from a κ light chain (such as a human κ light chain); or CL in the second polypeptide chain comprises a light chain constant region derived from a κ light chain (such as a human κ light chain), and CL in the third polypeptide chain comprises a light chain constant region derived from a λ light chain (such as a human λ light chain).

9. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 5-8, wherein the VL_A comprises a light chain variable region derived from the same type of immunoglobulin light chain as CL in the same polypeptide chain, and / or the VL_B comprises a light chain variable region derived from the same type of immunoglobulin light chain as CL in the same polypeptide chain.

10. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 5-9, wherein each CH1 independently comprises a CH1 derived from an immunoglobulin isotype IgG (such as human IgG), and optionally each CH1 independently comprises a CH1 derived from an IgG subtype selected from the group consisting of IgG1, IgG2, and IgG4 (such as human IgG1, IgG2, and IgG4).

11. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 5-10, wherein the linker comprises the amino acid sequence of (G4S)n, where n is an integer selected from 1-5.

12. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 5-11, wherein: the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 57; the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 56; and the third polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:

52.

13. The bispecific antibody or antigen-binding fragment thereof according to claim 5, wherein: the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 57; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 56; and the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

52.

14. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-13, wherein the antibody does not comprise an immunoglobulin Fc region.

15. A nucleic acid encoding the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-14.

16. A vector comprising the nucleic acid according to claim 15, preferably wherein the vector is an expression vector.

17. A host cell comprising the nucleic acid according to claim 15 or the vector according to claim 16.

18. A method for preparing the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-14, the method comprising: a) culturing the host cell according to claim 17 under conditions suitable for producing the bispecific antibody or antigen-binding fragment thereof; and b) obtaining the bispecific antibody or antigen-binding fragment thereof from the culture.

19. An antibody-drug conjugate (ADC) comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-14, and a cytotoxic moiety conjugated to the bispecific antibody or antigen-binding fragment thereof.

20. A composition comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-14 or the ADC according to claim 19.

21. The composition according to claim 20, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier and / or excipient.

22. A method for depleting hematopoietic stem cells in a subject in need thereof, the method comprising administering to the subject a bispecific antibody or antigen-binding fragment thereof as recited in any one of claims 1-14, a nucleic acid as recited in claim 15, a vector as recited in claim 16, an ADC as recited in claim 19, or a composition as recited in claim 20 or 21.

23. A method for treating a hematological disorder in a subject in need thereof, the method comprising: a) administering to the subject a bispecific antibody or antigen-binding fragment thereof as recited in any one of claims 1-14, an ADC as recited in claim 19, or a composition as recited in claim 20 or 21; and b) transplanting hematopoietic stem cells into the subject.

24. The method according to claim 23, wherein step b) is carried out within 24 hours, 48 hours, 72 hours, 5 days, 7 days, 2 weeks, 3 weeks, or 1 month after step a).

25. The method according to claim 23 or 24, wherein the hematological disorder is selected from the group consisting of hemoglobinopathies, immunodeficiencies, metabolic disorders, hematological diseases (such as bone marrow failure syndromes, myelodysplastic syndromes, acute lymphocytic and myelocytic leukemias, and chronic lymphocytic and myelocytic leukemias), hematopoietic cell proliferative diseases, transplant rejection, autoimmune diseases, and autoinflammatory diseases.

Images