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WO2024254757A1 - Anticorps bispécifiques dirigés contre folr1 et leurs utilisations - Google Patents

Anticorps bispécifiques dirigés contre folr1 et leurs utilisations Download PDF

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Publication number
WO2024254757A1
WO2024254757A1 PCT/CN2023/099866 CN2023099866W WO2024254757A1 WO 2024254757 A1 WO2024254757 A1 WO 2024254757A1 CN 2023099866 W CN2023099866 W CN 2023099866W WO 2024254757 A1 WO2024254757 A1 WO 2024254757A1
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amino acid
seq
inhibitor
acid sequence
antigen binding
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Zuoxiang XIAO
Dongwen ZHOU
Wei Zhou
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Zhejiang Shimai Pharmaceutical Co Ltd
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Zhejiang Shimai Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention is directed to bispecific antibodies targeting FOLR1 and CD3, and uses of such antibodies, in particular their use in the treatment of cancers.
  • CD3 bispecific antibodies hold potential as potent cancer therapeutics as they recruit and activate a broad repertoire of T cells against tumor cells expressing a tumor-associated cell surface antigen. They circumvent the need for T-cell receptor engagement with MHC Class I in complex with antigenic peptide, and instead recruit T cells to target cells expressing cell surface antigen.
  • One arm of the bispecific antibody binds to a tumor-associated cell surface antigen, and the other arm binds to the CD3 protein on T cells, leading to a cytotoxic T lymphocyte (CTL) response against tumor cells.
  • CTL cytotoxic T lymphocyte
  • the folate receptor- ⁇ (FR ⁇ , also known as FOLR1) is a glycosylphosphatidylinositol-linked cell-surface glycoprotein that has high affinity for folates. Most normal tissues do not express FOLR1, and transport of physiologic folates into most cells is thought to be mediated by several other proteins, most notably, reduced folate carrier. High levels of FOLR1 have been found in serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, and non-small cell lung cancer of the adenocarcinoma subtype.
  • FOLR1 expression is maintained in metastatic foci and recurrent carcinomas in ovarian cancer patients, and after chemotherapy in epithelial ovarian and endometrial cancers. These properties show that FOLR1 may offer a promising target antigen for tumor-specific activation of systemically administered T cell redirection therapies.
  • the present disclosure provides FOLR1 ⁇ CD3 bispecific antibodies that is in a form of a bispecific T cell engager (BiTE) , FR1-V4-LLG-P329G-1.1 and FR1-V4-LFLE-P329G.
  • BiTE bispecific T cell engager
  • FOLR1 positive cancers e.g., colon cancer, kidney cancer, colorectal cancer, lung cancer, gastric cancer, ovarian cancer, endometrial cancer, fallopian tube carcinoma, urothelium carcinoma, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer and liver cancer
  • FOLR1 positive cancers e.g., colon cancer, kidney cancer, colorectal cancer, lung cancer, gastric cancer, ovarian cancer, endometrial cancer, fallopian tube carcinoma, urothelium carcinoma, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer and liver cancer
  • the present disclosure provides a bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region that binds to FOLR1 comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1) and a second antigen binding region that binds to CD3 comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) , wherein the VL1 comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 1-3 respectively; the VH1 comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 5-7 respectively; the VL2 comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 9-11 respectively; and the VH2 comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 13-15 respectively.
  • the VL1 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: 4;
  • the VH1 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: 8;
  • the VL2 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: 12;
  • the VH2 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: 16.
  • the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 8; the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 12; and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 16.
  • the first antigen binding region comprises a scFv comprising the VL1 and VH1, and the scFv is linked to the N terminal of the VL2 or the VH2, optionally via a linker.
  • the bispecific antibody comprises: a first polypeptide chain comprising from the N terminal to C terminal: the VH2, a heavy chain constant region 1 (CH1) , a hinge region, a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and a second polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL2, a light chain constant region (CL) , a hinge region, a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .
  • each of the CH1, CH2, CH3 and hinge region is independently derived from immunoglobulin isotype IgG (e.g. human IgG) , preferably derived from IgG subtype selected from the group consisting of IgG1, IgG2 and IgG4 (e.g. human IgG1, IgG2 and IgG4) .
  • immunoglobulin isotype IgG e.g. human IgG
  • IgG subtype selected from the group consisting of IgG1, IgG2 and IgG4 e.g. human IgG1, IgG2 and IgG4 .
  • the CL is derived from ⁇ light chain or ⁇ light chain.
  • the hinge region each independently comprises an amino acid sequence selected from any one of SEQ ID NOs: 23-25.
  • one or both of the CH2 comprise at least one amino acid mutation that is capable of decreasing the effector function of the bispecific antibody, preferably the at least one mutation is selected from L234A, L235A, G237A, P329G or any combination thereof or selected from L234F, L235E, P329G or combination thereof.
  • one or both of the CH3 comprise at least one amino acid mutation that is capable of decreasing homodimerization of the first and second polypeptide chains.
  • the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as shown in SEQ ID NO: 21 or 22.
  • 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: 17, and 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: 18.
  • 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: 19, and 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: 20.
  • the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 17, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 18. In other embodiments, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 19, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 20.
  • the bispecific antibody is a bispecific T-cell engager (BiTE) .
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the bispecific antibody or the antigen binding fragment thereof disclosed herein.
  • the present disclosure provides a vector comprising the nucleic acid disclosed herein.
  • the present disclosure provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition further comprises a second therapeutic agent.
  • the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.
  • BTK Bruton’s tyrosine kinase
  • the present disclosure provides a conjugate comprising the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.
  • the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.
  • the present disclosure provides a method of treating a cancer in a subject, comprising administering to the subject an effective amount of the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.
  • the cancer is an FOLR1 positive cancer.
  • the cancer is selected from the group consisting of ovarian cancer (e.g., ovarian epithelial carcinoma, such as serous and endometrioid epithelial ovarian cancer) , endometrial cancer (e.g., endometrial adenocarcinoma) , fallopian tube carcinoma, urothelium carcinoma, breast cancer (e.g., triple-negative breast cancer) and lung cancer (e.g., non-small cell lung cancer) .
  • ovarian cancer e.g., ovarian epithelial carcinoma, such as serous and endometrioid epithelial ovarian cancer
  • endometrial cancer e.g., endometrial adenocarcinoma
  • fallopian tube carcinoma e.g., urothelium carcinoma
  • breast cancer e.g., triple-negative breast cancer
  • lung cancer e.g., non-small cell lung cancer
  • the method further comprises administering to the subject a second therapeutic agent.
  • the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.
  • BTK Bruton’s tyrosine kinase
  • the present disclosure provides use of the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein in the manufacture of a medicament for treating a cancer in a subject.
  • the present disclosure provides the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein for use in treating a cancer in a subject.
  • the cancer is an FOLR1 positive cancer.
  • the cancer is selected from the group consisting of ovarian cancer (e.g., ovarian epithelial carcinoma, such as serous and endometrioid epithelial ovarian cancer) , endometrial cancer (e.g., endometrial adenocarcinoma) , fallopian tube carcinoma, urothelium carcinoma, breast cancer (e.g., triple-negative breast cancer) and lung cancer (e.g., non-small cell lung cancer) .
  • the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is in combination with a second therapeutic agent.
  • the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.
  • BTK Bruton’s tyrosine kinase
  • Figure 1 shows schematic representation of one example of FOLR1 ⁇ CD3 BiTEs of the present invention.
  • Figure 2 shows binding of FOLR1 ⁇ CD3 BiTEs against recombinant human FOLR1 as measured by ELISA.
  • Figure 3 shows binding of FOLR1 ⁇ CD3 BiTEs against recombinant human CD3 as measured by ELISA.
  • Figure 4A shows binding of FOLR1 ⁇ CD3 BiTEs against FOLR1 expressing cell line SKOV3 as measured by flow cytometry.
  • Figure 4B shows binding of FOLR1 ⁇ CD3 BiTEs against HT1080-FR1 stable cell line as measured by flow cytometry.
  • Figure 5A shows FOLR1 ⁇ CD3 BiTEs induced T cell activation in the presence of FOLR1 expressing cell line HT1080-FR1.
  • Figure 5B shows FOLR1 ⁇ CD3 BiTEs induced T cell activation in the presence of FOLR1 expressing cell line SKOV3.
  • FR1-mAb, CD3-mAb, and the combination of FR1-mAb and CD3-mAb are used as control.
  • Figure 5C shows FOLR1 ⁇ CD3 BiTEs induced T cell activation in the presence of FOLR1-negative cell line HT1080.
  • FR1-mAb, CD3-mAb, and the combination of FR1-mAb and CD3-mAb are used as control.
  • Figure 5D shows FOLR1 ⁇ CD3 BiTEs induced T cell activation in absence of target cells.
  • Figure 6 shows killing of FOLR1-positive HT1080-FR1 cells by FOLR1 ⁇ CD3 BiTEs in the presence of human PBMCs.
  • Figure 7 shows killing of FOLR1-negative HT1080 cells by FOLR1 ⁇ CD3 BiTEs in the presence of human PBMCs.
  • Figure 8A shows tumor volume in the B-NDG mice xenografted with HT1080-FR1/PBMC treated with FR1-V4-LFLE-P329G or FR1-V4-LLG-P329G-1.1.
  • Five mice per group were randomized into groups and intravenously treated with 500 ⁇ g/kg of FR1-V4-LFLE-P329G or FR1-V4-LLG-P329G-1.1.
  • Tumor volume was measured three times weekly for 15 days. Data represent mean tumor volume ⁇ SEM.
  • Figure 8B shows body weight of the B-NDG mice xenografted with HT1080-FR1/PBMC treated with FR1-V4-LFLE-P329G or FR1-V4-LLG-P329G-1.1 over 15 days. Data represent mean body weight ⁇ SEM.
  • an antibody includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.
  • the term “antibody” refers to an immunoglobulin molecule which has the ability to specifically bind to a specific antigen. Such molecule often comprises two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL.
  • variable regions of the heavy and light chains of antibodies contain a binding domain that interacts with an antigen.
  • the constant regions of antibodies may 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.
  • the heavy chain of immunoglobulins can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (fragment crystallizable) .
  • the Fd region comprises the VH and CH1 domains and, in combination with the light chain, forms Fab (antigen-binding fragment) .
  • the Fc fragment is responsible for the immunoglobulin effector functions, which includes, for example, complement fixation and binding to cognate Fc receptors of effector cells.
  • the hinge region found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc region.
  • the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses.
  • the immunoglobulin hinge region can be further subdivided structurally and functionally into three regions: the upper hinge, the core hinge, and the lower hinge (Shin et al., Immunological Reviews 130: 87, 1992) .
  • the upper hinge includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains.
  • the length of the upper hinge region correlates with the segmental flexibility of the antibody.
  • the core hinge region contains the inter-heavy chain disulfide bridges.
  • the lower hinge region joins the amino terminal end of, and includes residues in the CH2 domain.
  • the core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys that, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may affect the effector functions of the Fc portion of the antibody.
  • a “light chain variable region” (VL) or “heavy chain variable region” (VH) consists of a “framework” region interrupted by three “complementarity determining regions” or “CDRs” .
  • the framework regions serve to align the CDRs for specific binding to an epitope of an antigen.
  • the CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDRs 1, 2, and 3 of a VL domain are also referred to herein, respectively, as LCDR1, LCDR2, and LCDR3;
  • CDRs 1, 2, and 3 of a VH domain are also referred to herein, respectively, as HCDR1, HCDR2, and HCDR3.
  • 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.
  • the present disclosure can use CDRs defined according to any of these numbering systems, although preferred embodiments use Kabat defined CDRs.
  • an immunoglobulin molecule can be divided 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.
  • the light chain of the antibody can be classified as a lambda ( ⁇ ) chain or a kappa ( ⁇ ) chain, based on the amino acid sequence of the light chain.
  • antibody as used herein should be understood in its broadest meaning, and includes monoclonal antibodies (including full-length monoclonal antibodies) , polyclonal antibodies, antibody fragments, and multi-specific antibodies containing at least two different antigen binding regions (e.g., bispecific antibodies) .
  • the antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in Fc regions, and mutations in glycosylation sites.
  • Antibodies also include post-translation modified antibodies, fusion proteins containing the antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit desired biological activity.
  • bispecific antibody in the context of the present invention is to be understood as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as different target binding but includes as well binding to different epitopes in one target.
  • the term "bispecific antibody” as used herein should be understood in its broadest meaning, and includes full-length bispecific antibodies and antigen binding fragments thereof.
  • the bispecific antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in Fc regions, and mutations in glycosylation sites.
  • Bispecific antibodies also include post-translation modified antibodies, fusion proteins containing the antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit desired biological activity.
  • the term “antigen binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.
  • antigen binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR) ; and (ix) a nanobody,
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ) .
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment" of an antibody.
  • the term also includes a "linear antibody” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) , which forms an antigen binding region together with a complementary light chain polypeptide, and a modified version of any of the foregoing fragments, which retains antigen binding activity.
  • antigen binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • binding refers to a non-random binding reaction between two molecules, such as between an antibody and its target antigen.
  • the binding specificity of an antibody can be determined based on affinity and/or avidity.
  • the affinity represented by the equilibrium constant for the dissociation of an antigen with an antibody (KD) , is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antibody: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antibody.
  • KD equilibrium constant for the dissociation of an antigen with an antibody
  • the affinity can also be expressed as the affinity constant (KA) , which is 1/KD.
  • Avidity is the measure of the strength of binding between an antibody and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody and the number of pertinent binding sites present on the antibody.
  • an antibody will bind to an antigen with a dissociation constant (KD) of 10 -5 to 10 -12 M or less, and preferably 10 -7 to 10 -12 M or less and more preferably 10 -8 to 10 -12 M, and/or 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 .
  • KD dissociation constant
  • Any K D value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specifically binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA) , enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.
  • Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA) , enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.
  • epitope refers to a site on an antigen to which an antibody binds.
  • An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the smallest binding site of an antibody and therefore is the specific target of the antibody or antigen binding fragment thereof.
  • sequence identity refers to the extent to which two sequences (amino acid) have the same residue at the same positions in an alignment.
  • amino acid sequence is X%identical to SEQ ID NO: Y refers to X%identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X%of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y.
  • computer programs are employed for such calculations.
  • Exemplary programs that compare and align pairs of sequences 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) .
  • conservative amino acid substitutions which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.
  • Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within 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.
  • Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
  • bispecific T-cell engager 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 target cells (See, PCT Publication WO 05/061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321: 974-977, which are incorporated herein by reference in their entireties) .
  • the BiTE of the disclosure has an antigen binding region that binds to FOLR1 and a second antigen binding region that is directed towards a T cell antigen.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the term "host cell” refers to a cell into which an expression vector has been introduced.
  • pharmaceutically acceptable means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof and/or that such carrier or excipient is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.
  • treatment refers to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptom of the disease.
  • Treatment may include treatment of a disease or disorder (e.g.
  • cancer in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease) ; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician.
  • treating includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases (e.g. cancers) .
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • an effective amount means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.
  • subject refers to any mammalian subject for whom diagnosis, treatment or therapy is desired.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc.
  • the present disclosure provides a bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region that binds to FOLR1 comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1) and a second antigen binding region that binds to CD3 comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) , wherein the VL1 comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 1-3 respectively; the VH1 comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 5-7 respectively; the VL2 comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 9-11 respectively; and the VH2 comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 13-15 respectively.
  • CDR sequences are defined according to Kabat numbering system.
  • the VL1 of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 1 (SVSSSISSNNLH) , SEQ ID NO: 2 (GTSNLAS) and SEQ ID NO: 3 (QQWSSYPYMYT) respectively
  • the VH1 of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 5 (GYGLS) , SEQ ID NO: 6 (MISSGGSYTYYADSVKG) and SEQ ID NO: 7 (HGDDPAWFAY) respectively
  • the VL2 of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 9 (RSSTGAVTTSNYAN) , SEQ ID NO: 10 (GANKRAP) and SEQ ID NO: 11 (ALWYSNLWV) respectively
  • SEQ ID NO: 9 RSSTGAVTTS
  • the VL1 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: 4;
  • the VH1 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: 8;
  • the VL2 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: 12;
  • the VH2 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: 16.
  • the VL1 comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 4 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to FOLR1.
  • the VH1 comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 8 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to FOLR1.
  • the VL2 comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 12 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3.
  • the VH2 comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 16 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3.
  • 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 parent polypeptide.
  • the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of the inserted, deleted and/or substituted amino acid can be 1-20, preferably 1-10, more preferably 1-7, still more preferably 1-5, and most preferably 1-2.
  • the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.
  • the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3, and/or FR4.
  • FR framework
  • the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) .
  • conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within 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.
  • Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
  • the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 8; the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 12; and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 16.
  • the first antigen binding region comprises a scFv comprising the VL1 and VH1, and the scFv is linked to the N terminal of the VL2 or the VH2, optionally via a linker.
  • the scFv is linked to the N terminal of the VL2 optionally via a linker.
  • the scFv is linked to the N terminal of the VH2 optionally via a linker.
  • the scFv is formed by linking the VL1 and the VH1 via a linker.
  • the linker may be any flexible linker.
  • the linker comprises an amino acid sequence of (G4S) n, wherein n is an integer selected from 1-5.
  • the linker may comprise an amino acid sequence of GGGGS (SEQ ID NO: 26) .
  • the linker may comprise an amino acid sequence of GGGGSGGGGS (SEQ ID NO: 27) .
  • the linker may comprise an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 28) .
  • the linker may comprise an amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 21) .
  • the linker may comprise an amino acid sequence of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 29) .
  • the linker comprises an amino acid sequence as shown in SEQ ID NO: 21.
  • the linker comprises an amino acid sequence of GS (G4S) n, wherein n is an integer selected from 1-5.
  • the linker may comprise an amino acid sequence of GSGGGGS (SEQ ID NO: 30) .
  • the linker may comprise an amino acid sequence of GSGGGGSGGGGS (SEQ ID NO: 22) .
  • the linker may comprise an amino acid sequence of GSGGGGSGGGGSGGGGS (SEQ ID NO: 31) .
  • the linker may comprise an amino acid sequence of GSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32) .
  • the linker may comprise an amino acid sequence of GSGGGGSGGGGSGGGGSGGGGGGSGGGGS (SEQ ID NO: 33) .
  • the linker comprises an amino acid sequence as shown in SEQ ID NO: 22.
  • the bispecific antibody disclosed herein may comprise a hinge region and an Fc region comprising CH2 and CH3 of an antibody.
  • the hinge region of an IgG class antibody refers to a short amino acid sequence region between the CH1 and CH2 portions of the heavy chain that is relatively flexible in the antibody native state.
  • the hinge region may comprise part or all of a wild type hinge sequence or a variant thereof having one or more substitutions.
  • the hinge region may comprise one of the following amino acid sequences: EPKSCDKTHTCPPCP (SEQ ID NO: 34) ; PKSCDKTHTCPPCP (SEQ ID NO: 35) ; KSCDKTHTCPPCP (SEQ ID NO: 36) ; SCDKTHTCPPCP (SEQ ID NO: 37) ; CDKTHTCPPCP (SEQ ID NO: 24) ; DKTHTCPPCP (SEQ ID NO: 38) ; KTHTCPPCP (SEQ ID NO: 39) ; THTCPPCP (SEQ ID NO: 40) ; HTCPPCP (SEQ ID NO: 41) ; TCPPCP (SEQ ID NO: 42) ; or CPPCP (SEQ ID NO: 23) ; or a variant thereof having one or more substitutions (e.g., 1-6 substitutions, for example, 1-5, 1-4, 1, 2, 3, 4,
  • the Fc region may be of any isotype, including, but not limited to, IgG1, IgG2, IgG3 and IgG4, and may comprise one or more mutations or modifications.
  • the Fc region is of IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications.
  • the Fc region is human IgG1 Fc.
  • the Fc region has a decreased effector function, e.g., decreased ADCC, ADCP, CDC, and/or C1q, Fc ⁇ RI, Fc ⁇ RII, or Fc ⁇ RIIIA binding.
  • the Fc region may be of an IgG1 isotype, or a non-IgG1 type, e.g. IgG2, IgG3 or IgG4, which has been mutated such that the ability to mediate effector function has been reduced or even eliminated.
  • Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol.
  • the Fc region may comprise the amino acid sequence having one or more of the following amino acid substitutions: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G, as compared with the wild type sequence.
  • the Fc region comprises a mutation removing the acceptor site for Asn-linked glycosylation or is otherwise manipulated to change the glycosylation properties.
  • an N297Q mutation can be used to remove an Asn-linked glycosylation site.
  • Fc region comprises an IgG1 sequence with an N297Q mutation.
  • the Fc region is glyco-engineered to reduce fucose and thus enhance ADCC, e.g. by addition of compounds to the culture media during antibody production as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105: 350 or by using FUT8 knockout cells, e.g. as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87: 614.
  • ADCC may alternatively be optimized using the method described by et al. (1999) Nature Biotech 17: 176.
  • the Fc region has been engineered to enhance complement activation, e.g. as described in Natsume et al. (2009) Cancer Sci. 100: 2411.
  • the Fc region comprises modifications or mutations that can inhibit Fc homodimerization.
  • the Fc region comprises a variant of a human IgG1 Fc wildtype sequence.
  • the variant can comprise amino acid substitutions at positions T366 and Y407 of human IgG1 (Kabat numbering) .
  • T366 is substituted with L (Leucine) .
  • Y407 is substituted with I (Isoleucine) , F (Phenylalanine) , L (Leucine) , M (Methionine) , H (Histidine) , K (Lysine) , S (Serine) , Q (Glutamine) , T (Threonine) , W (Tryptophan) , A (Alanine) , G (Glycine) or N (Asparagine) . More preferably, Y407 is substituted with H. In one embodiment, T366 is substituted with L, and Y407 is substituted with H.
  • the Fc region can be a monomeric human IgG1 Fc (e.g., mFc7.2) as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.
  • the bispecific antibody comprises: a first polypeptide chain comprising from the N terminal to C terminal: the VH2, a heavy chain constant region 1 (CH1) , a hinge region, a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) ; and a second polypeptide chain comprising from the N terminal to C terminal: the scFv, an optional linker, the VL2, a light chain constant region (CL) , a hinge region, a heavy chain constant region 2 (CH2) , and a heavy chain constant region 3 (CH3) .
  • each of the CH1, CH2, CH3 and hinge region is independently derived from immunoglobulin isotype IgG (e.g. human IgG) , preferably derived from IgG subtype selected from the group consisting of IgG1, IgG2 and IgG4 (e.g. human IgG1, IgG2 and IgG4) .
  • the CL is derived from ⁇ light chain or ⁇ light chain.
  • the hinge region each independently comprises an amino acid sequence selected from any one of SEQ ID NOs: 23-25 and 34-42. In a preferred embodiment, the hinge region each independently comprises an amino acid sequence of SEQ ID NO: 23. In another preferred embodiment, the hinge region each independently comprises an amino acid sequence of SEQ ID NO: 24. In another preferred embodiment, the hinge region each independently comprises an amino acid sequence of SEQ ID NO: 25.
  • one or both of the CH2 comprise at least one amino acid mutation that is capable of decreasing the effector function of the bispecific antibody.
  • the CH2 may comprise at least one amino acid substitution selected from E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G, or any combination thereof.
  • the at least one mutation is selected from L234A, L235A, G237A, P329G or any combination thereof.
  • the at least one mutation is selected from L234A, L235A, G237A, and P329G.
  • the at least one mutation is selected from L234F, L235E, P329G or combination thereof.
  • the at least one mutation is selected from L234F, L235E, and P329G.
  • one or both of the CH3 comprise at least one amino acid mutation that is capable of decreasing homodimerization of the first and second polypeptide chains.
  • amino acid T366 of the one or both CH3 is substituted with L (Leucine)
  • amino acid Y407 of the one or both CH3 is substituted with I (Isoleucine) , F (Phenylalanine) , L (Leucine) , M (Methionine) , H (Histidine) , K (Lysine) , S (Serine) , Q (Glutamine) , T (Threonine) , W (Tryptophan) , A (Alanine) , G (Glycine) or N (Asparagine) .
  • amino acid T366 of the one or both CH3 is substituted with L
  • amino acid Y407 of the one or both CH3 is substituted with H.
  • T366 is substituted with L and Y407 is substituted with H.
  • the linker can be those as describe above.
  • the linker may be any flexible linker.
  • the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5.
  • the linker comprises an amino acid sequence as shown in SEQ ID NO: 21 or 22.
  • the bispecific antibody disclosed herein may comprise a constant domain comprising CH1, a hinge region, CH2 and CH3 as described above. In some embodiments, the bispecific antibody disclosed herein may comprise a constant domain comprising CL, a hinge region, CH2 and CH3 as described above. In some embodiments, the first polypeptide chain of the bispecific antibody disclosed herein may comprise a constant domain comprising CH1, a hinge region, CH2 and CH3 as described above, and the second polypeptide chain the bispecific antibody disclosed herein may comprise a constant domain comprising CL, a hinge region, CH2 and CH3 as described above. In some embodiments, the constant domain may comprise the amino acid sequence as shown in any one of SEQ ID NOs: 43-46.
  • the first polypeptide chain may comprise a constant domain comprising the amino acid sequence as shown in SEQ ID NO: 43 or 44.
  • the second polypeptide chain may comprise a constant domain comprising the amino acid sequence as shown in SEQ ID NO: 45 or 46.
  • 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: 17, and 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: 18.
  • 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: 19, and 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: 20.
  • the first polypeptide chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 17 or 19 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3.
  • the second polypeptide chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 18 or 20 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to FOLR1 and CD3.
  • 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 parent polypeptide.
  • the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of the inserted, deleted and/or substituted amino acid can be 1-50, preferably 1-20, more preferably 1-10, still more preferably 1-5.
  • the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.
  • the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3 and/or FR4; and/or constant regions, e.g., CL, CH1, CH2 and/or CH3.
  • FR framework
  • constant regions e.g., CL, CH1, CH2 and/or CH3.
  • the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) .
  • conservative substitutions are as described above.
  • the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 17; and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 18.
  • the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 19, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 20.
  • the bispecific antibody is a bispecific T cell engager (BiTE) .
  • the bispecific antibody is in form of an HBiTE as described in PCT application No. PCT/US2018/016524 (which is incorporated herein by reference in its entirety) .
  • the light chain from N-terminus to C-terminus, comprises an anti-target VL domain, an anti-CD3 VL-CL and a monomeric human IgG1 Fc (e.g., mFc7.2) ; and the heavy chain, from N-terminus to C-terminus, comprises an anti-target VH domain, an anti-CD3 VH-CH1 and a monomeric human IgG1 Fc (e.g., mFc7.2) .
  • Monomeric Fc7.2 contains two amino acid mutations (T366L and Y407H) capable of inhibiting Fc homodimerization.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the bispecific antibody or the antigen binding fragment thereof disclosed herein.
  • nucleic acid includes both single-stranded and double-stranded nucleotide polymers.
  • the nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • the invention provides nucleic acid molecules encoding any one of the heavy chain variable region sequences disclosed herein.
  • the invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the heavy chain variable region sequences disclosed herein.
  • the invention provides nucleic acid molecules encoding any one of the light chain variable region sequences disclosed herein.
  • the invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the light chain variable region sequences disclosed herein.
  • the 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 invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to 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.
  • the invention provides nucleic acid molecules encoding a heavy chain variable region sequence that comprises the CDR sequences of any one of the heavy chain variable region sequences disclosed herein.
  • the invention also provides nucleic acid molecules that encode a heavy chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein.
  • the invention provides nucleic acid molecules encoding a light chain variable region sequence that comprises the CDR sequences of any one of the light chain variable region sequences disclosed herein.
  • the invention also provides nucleic acid molecules that encode a light chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the light chain variable region sequences disclosed herein.
  • the invention provides nucleic acid molecules encoding: (i) a heavy chain variable region sequence that comprises the CDR sequences of any one of the heavy chain variable region sequences disclosed herein and (ii) a light chain variable region sequence that comprises the CDR sequences of any one of the light chain variable region sequences disclosed herein.
  • the invention also provides nucleic acid molecules that encode: (i) a heavy chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein and (ii) a light chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the light chain variable region sequences disclosed herein.
  • the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) .
  • the invention provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding the bispecific antibody disclosed herein.
  • the invention provides a deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding the bispecific antibody disclosed herein.
  • the deoxyribonucleic acid (DNA) may be introduced into the cells of a human body in vivo.
  • the deoxyribonucleic acid (DNA) of the invention is comprised in a vector or a delivering agent.
  • the deoxyribonucleic acid (DNA) of the invention is integrated into the genome of a cell.
  • the ribonucleic acid (RNA) may be introduced into the cells of a human body in vivo.
  • the ribonucleic acid (RNA) of the invention is comprised in a vector or a delivering agent.
  • the present disclosure provides a vector comprising the nucleic acid disclosed herein.
  • the vector is an expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of the bispecific antibody.
  • the invention provides expression vectors comprising any of the nucleic acid molecules mentioned above.
  • the vector is a viral vector.
  • the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV) , a lentiviral vector, or any combination thereof.
  • AAV adenovirus associated vector
  • Suitable exemplary vectors include e.g., pGAR, pBABE-puro, pBABE-neo largeTcDNA, 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.
  • An expression vector may be any suitable recombinant expression vector. Suitable vectors comprise those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • a vector may be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md. ) , the pBluescript series (Stratagene, LaJolla, Calif. ) , the pET series (Novagen, Madison, Wis. ) , the pGEX series (Pharmacia Biotech, Uppsala, Sweden) , and the pEX series (Clontech, Palo Alto, Calif. ) .
  • Bacteriophage vectors such as ⁇ GT10, ⁇ GT11, ⁇ ZapII (Stratagene) , ⁇ EMBL4, and ⁇ NM1149, also may be used.
  • plant expression vectors useful in the context of the disclosure comprise pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech) .
  • animal expression vectors useful in the context of the disclosure comprise pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech) .
  • Recombinant expression vectors may be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 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 expression vectors, which are circular or linear may be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems may be derived, e.g., from ColEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • the vector may be an adenoviral vector comprising a nucleotide sequence encoding the bispecific antibody disclosed herein.
  • the vector may be administered into the body of a subject, and then enter into a cell of the subject in vivo, thereby the nucleotide sequence encoding the bispecific antibody disclosed herein is integrated into the genome of the cell, and subsequently the cell expresses the bispecific antibody disclosed herein.
  • the present disclosure provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.
  • any cell may be used as a host cell for the nucleic acids or the vectors of the present disclosure.
  • the cell can be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cells such as a mammalian cell.
  • Suitable prokaryotic cells include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E.
  • the cell is a human cell.
  • the cell is an immune cell.
  • host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T and HEK293FS.
  • the host cell of the invention is prepared by introducing the vector disclosed herein or the nucleic acid disclosed herein in vitro or ex vivo.
  • the host cell of the invention may be administered into the body of a subject, and the host cell expresses the bispecific antibody disclosed herein in vivo.
  • the invention provides host cells into which any of the vectors mentioned above have been introduced.
  • the invention further provides a method of preparing the bispecific antibody of the invention, wherein the method comprises a) culturing the host cell of the fourth aspect of the invention under a condition suitable for the production of the bispecific antibody; and b) obtaining the bispecific antibody from the culture.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the bispecific antibody or antigen binding fragment thereof or agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Preferred examples of such carriers or excipients include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5%human serum albumin.
  • Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the pharmaceutical composition further comprises a second therapeutic agent.
  • the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.
  • BTK Bruton’s tyrosine kinase
  • the therapeutic agent is a chemotherapeutic agent.
  • the chemotherapeutic agents can include, for example, cytotoxic agents, anti-metabolite agents (e.g., folate antagonists, purine analogs, pyrimidine analogs, etc. ) , topoisomerase inhibitors (e.g., camptothecin derivatives, anthracenedione, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.
  • anti-microtubule agents e.g., taxanes, vinca alkaloids
  • protein synthesis inhibitors e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids
  • alkylating agents e.g., alkyl sulfonates, ethylenimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.
  • alkaloids, terpenoids, and kinase inhibitors e.g., kinase inhibitors.
  • the pharmaceutical composition of the invention can be formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation) , transdermal (i.e., topical) , transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA) ; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J. ) or phosphate buffered saline (PBS) .
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture 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, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the active 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.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the invention provides therapeutic compositions comprising the bispecific antibody or antigen binding fragment thereof of the present invention.
  • Therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN TM ) , DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (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.
  • the present disclosure provides a conjugate comprising the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.
  • a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety.
  • the chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, carbohydrate or recombinant virus.
  • An antibody conjugate is often referred to as an “immunoconjugate” .
  • the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent)
  • the conjugate is often referred to as an "antibody-drug conjugate" or "ADC.
  • conjugated may refer to making two polypeptides into one contiguous polypeptide molecule.
  • an antibody is joined to a chemical moiety.
  • an antibody joined to a chemical moiety is further joined to a lipid or other molecule to a protein or peptide to increase its half-life in the body.
  • the linkage can be either by chemical or recombinant means.
  • the linkage is chemical, wherein a reaction between the antibody moiety and the chemical moiety has produced a covalent bond formed between the two molecules to form one molecule.
  • a peptide linker short peptide sequence
  • a chemical moiety can be linked to the antibody of the invention using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used.
  • the procedure for attaching a chemical moiety to the antibody varies according to the chemical structure of the chemical moiety.
  • Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH) , free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the chemical moiety.
  • the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules.
  • the linker can be any molecule used to join the antibody to the chemical moiety.
  • the linker is capable of forming covalent bonds to both the antibody and to the chemical moiety.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site.
  • Cleavage of the linker to release the chemical moiety from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • the antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein) .
  • the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling.
  • the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody) , a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a strep tavidin core region or a polyhistidine tag) .
  • One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types) .
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate) .
  • Such linkers are commercially available.
  • the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.
  • the therapeutic agent includes but is not limited to immunomodulators, radioactive compounds, enzymes (for example perforin) , chemotherapeutic agents (for example cis-platin) , or a toxin.
  • the therapeutic agent can be such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g., auristatins) , or minor groove binding agents such as calicheamicin.
  • cytotoxic agents include such as, small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents.
  • agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e.
  • radio-nuclides i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of a or ⁇ particles, or ⁇ rays, for example, iodine-131 , rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225 and astatine-213; chelating agents may be used to facilitate the association of these radionuclides to the molecules, or multimers thereof.
  • the detectable moiety can be selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule.
  • a detectable moiety for diagnostic purposes includes for instance, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.
  • the bispecific antibody can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT) , computed axial tomography (CAT) scans, magnetic resonance imaging (MRI) , nuclear magnetic resonance imaging NMRI) , magnetic resonance tomography (MTR) , ultrasound, fiberoptic examination, and laparoscopic examination) .
  • detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI) .
  • useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.
  • Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) .
  • the bispecific antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • enzymes that are useful for detection
  • a bispecific antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned.
  • the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable.
  • the bispecific antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.
  • the bispecific antibody may be fused to a self-labelling protein tag (e.g. HaloTag) .
  • a self-labelling protein tag e.g. HaloTag
  • the protein tag could be cloned at the end of a constant region.
  • HaloTag is a self-labelling protein tag derived from a bacterial enzyme (a haloalkane dehalogenase) , designed to covalently bind to a synthetic ligand.
  • the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3(6) : 373-82) .
  • the bispecific antibody may be labeled with a magnetic agent, such as gadolinium.
  • Antibodies can also be labeled with lanthanides (such as europium and dysprosium) , and manganese.
  • Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels.
  • the bispecific antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) .
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • the bispecific antibody can also be labeled with a radiolabeled amino acid.
  • the radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I.
  • the immune stimulatory molecule is an immune effector molecule which stimulates immune response.
  • the immune stimulatory molecule can be cytokines such as IL-2 and IFN- ⁇ , chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, complement activators; viral/bacterial protein domains, or viral/bacterial peptides.
  • the present disclosure provides a method of treating a cancer in a subject, comprising administering to the subject an effective amount of the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.
  • the cancer is a solid tumor. In some embodiments, the cancer is an FOLR1 positive cancer.
  • cancers include: bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma) , fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancers, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, glioblastoma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acous
  • adenocarcinoma subtype squamous cell carcinoma (epidermoid carcinoma) , adenocarcinoma, large-cell carcinoma and small-cell lung cancer (SCLC) ; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical) , spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor) , prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; genital cancers such as penile cancer; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.
  • the cancer is selected from the group consisting of ovarian cancer (e.g., ovarian epithelial carcinoma, such as serous and endometrioid epithelial ovarian cancer) , endometrial cancer (e.g., endometrial adenocarcinoma) , fallopian tube carcinoma, urothelium carcinoma, breast cancer (e.g., triple-negative breast cancer) and lung cancer (e.g., non-small cell lung cancer) .
  • ovarian cancer e.g., ovarian epithelial carcinoma, such as serous and endometrioid epithelial ovarian cancer
  • endometrial cancer e.g., endometrial adenocarcinoma
  • fallopian tube carcinoma e.g., urothelium carcinoma
  • breast cancer e.g., triple-negative breast cancer
  • lung cancer e.g., non-small cell lung cancer
  • dosage administered to a subject may vary with the embodiment, the medicament employed, the method of administration, and the site and subject being treated. However, a dose should be sufficient to provide a therapeutic response.
  • a clinician may determine the effective amount to be administered to a human or other subject in order to treat a medical condition. The precise amount required to be therapeutically effective may depend upon numerous factors, e.g., such as the activity of the antibody, and the route of administration.
  • a dose of the antibodies, compositions or conjugates described herein may be administered to a mammal at one time or in a series of sub-doses administered over a suitable period of time, e.g., on a daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annual, or annual basis, as needed.
  • a dosage unit comprising an effective amount of antibodies, compositions or conjugates may be administered in a single daily dose, or the total daily dosage may be administered in two, three, four, or more divided doses administered daily, as needed.
  • a suitable means of administration may be selected by a medical practitioner.
  • Route of administration may be parenteral, for example, administration by injection, transnasal administration, transpulmonary administration, or transcutaneous administration. Administration may be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.
  • the antibodies, compositions or conjugates are selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as orally. Dose and method of administration may vary depending on the weight, age, condition, and the like of the subject, and may be suitably selected.
  • the method further comprises administering to the subject a second therapeutic agent.
  • the antibody, composition or conjugate disclosed herein is administered prior to, substantially simultaneously with, or after the administration of the second therapeutic agent.
  • the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.
  • BTK Bruton’s tyrosine kinase
  • the second therapeutic agent is a chemotherapeutic agent.
  • the chemotherapeutic agents can include, for example, cytotoxic agents, anti-metabolite agents (e.g., folate antagonists, purine analogs, pyrimidine analogs, etc. ) , topoisomerase inhibitors (e.g., camptothecin derivatives, anthracenedione, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.
  • anti-microtubule agents e.g., taxanes, vinca alkaloids
  • protein synthesis inhibitors e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids
  • alkylating agents e.g., alkyl sulfonates, ethylenimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.
  • alkaloids, terpenoids, and kinase inhibitors e.g., kinase inhibitors.
  • the present disclosure provides use of the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein in the manufacture of a medicament for treating a cancer in a subject.
  • the present disclosure also provides the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein for use in treating a cancer in a subject.
  • the cancer is a solid tumor or a hematologic malignancy.
  • the cancer is an FOLR1 positive cancer.
  • the cancer is selected from the group consisting of ovarian cancer (e.g., ovarian epithelial carcinoma, such as serous and endometrioid epithelial ovarian cancer) , endometrial cancer (e.g., endometrial adenocarcinoma) , fallopian tube carcinoma, urothelium carcinoma, breast cancer (e.g., triple-negative breast cancer) and lung cancer (e.g., non-small cell lung cancer) .
  • ovarian cancer e.g., ovarian epithelial carcinoma, such as serous and endometrioid epithelial ovarian cancer
  • endometrial cancer e.g., endometrial adenocarcinoma
  • fallopian tube carcinoma e.g., urothelium carcinoma
  • breast cancer e.g., triple-negative breast cancer
  • the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is in combination with a second therapeutic agent.
  • the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, a LAG3 inhibitor and glucocorticoid.
  • BTK Bruton’s tyrosine kinase
  • the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as the bispecific antibodies or the antigen binding fragment disclosed herein.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as the bispecific antibodies or the antigen binding fragment disclosed herein.
  • associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the kit comprises a first container containing the bispecific antibodies disclosed herein.
  • the kit comprises a first container that is a vial containing the bispecific antibodies as a lyophilized sterile powder under vacuum, and the kit further comprises a second container comprising a pharmaceutically acceptable fluid.
  • an injection device containing the bispecific antibodies.
  • the injection device comprises the bispecific antibody in sterile solution.
  • the injection device is a syringe.
  • 293 free style (293FS) cells CHO-S cells and protein A agarose were purchased from ThermoFisher Scientific.
  • FOLR1-positive cell line SKOV3 was donated by R&D Center U.S.A.
  • FOLR1-negative cell line HT1080 was purchased from National Collection of Authenticated Cell Cultures.
  • Human FOLR1 protein, His tag (MALS verified) , and human CD3 protein were purchased from ACRO.
  • PE anti-FOLR1 (Folate Binding Protein) antibody was purchased from Bio-Legend.
  • Anti-human IgG ( ⁇ -chain specific) -R-Phycoerythrin antibody produced in goat, anti-human IgG (Fc-specific) -Peroxidase antibody produced in goat were purchased from Sigma.
  • Anti-His tag antibody (HRP) mouse monoclonal was purchased from Sino Biological.
  • a stable cell line HT1080-FR1 was generated to facilitate in vitro and in vivo efficacy study. Briefly, the commercial FOLR1 recombinant plasmid pCMV-FOLR1 (Sino Biological) was transiently transfected into HT1080 cells with the agent Lipofectamine TM LTX Reagent with PLUS TM Reagent (Thermo) and transfection-specific media Opti-MEM TM I (Gibco) . Cell culture was supplemented with hygromycin B afterwards to select positive clones. 2-3 weeks later, single positive clones were gradually separated and verified with flow cytometry. A FOLR1 positive stable cell line HT1080-FR1 was obtained.
  • Bispecific T cell engager is a novel class of bispecific antibodies that can guide cytotoxic T cells to kill cancer cells by simultaneously binding to a tumor antigen and a T cell antigen, such as CD3 molecule on T cell surface.
  • scFv single-chain Fv
  • scFv single-chain Fv
  • the anti-CD3 Fab was further fused to the N terminus of monomeric Fc (e.g., mFc7.2) .
  • monomeric Fc7.2 contains two amino acid mutations (T366L and Y407H) capable of inhibiting Fc homodimerization as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.
  • the light chain of FOLR1 ⁇ CD3 BiTE is composed of anti-FOLR1 scFv, anti-CD3 VL, CL, a hinge region, CH2 and CH3 domains.
  • the heavy chain is composed of anti-CD3 VH, CH1, a hinge region, CH2 and CH3 domains ( Figure 1) .
  • Two FOLR1 ⁇ CD3 BiTEs termed as FR1-V4-LFLE-P329G and FR1-V4-LLG-P329G-1.1 respectively, were construed based on different hinge region, CH2 and CH3 domains.
  • the anti-FOLR1 scFv fragment was cloned into pBY plasmids containing an anti-CD3 hSP34 VL-CL and a complete engineered Fc by in-fusion cloning.
  • the heavy chain was constructed into a single vector pBY for expression in mammalian cells.
  • the two plasmids containing heavy chain and light chain gene were co-transfected to 293FS or CHO-S cells.
  • the plasmids and transfection agent PEI were mixed at ratio 1: 3 and then added into 293FS or CHO-S cell culture dropwise.
  • the cells were continued to grow for 5-7 days after transfection.
  • the cell culture was harvested by centrifugation at 8000 rpm for 20 min.
  • the culture supernatant containing target proteins were loaded onto Protein A Sepharose 4 Fast Flow column (GE Healthcare) . The subsequent purification was carried out according to the manufacturer’s instructions.
  • FOLR1 ⁇ CD3 BiTEs were well expressed in transiently transfected 293 free style (293FS) or CHO-S cells and secreted into the culture supernatants.
  • FR1-V4-LFLE-P329G displays an apparent molecular weight (aMW) of approximately 127 kDa
  • FR1-V4-LLG-P329G-1.1 displays an apparent molecular weight (aMW) of approximately 128 kDa.
  • the CDR sequences of FR1-V4-LFLE-P329G and FR1-V4-LLG-P329G-1.1 according to the Kabat numbering system are shown in Table 1.
  • the amino acid sequences of light chain variable region (VL) and heavy chain variable region (VH) of FR1-V4-LFLE-P329G and FR1-V4-LLG-P329G-1.1 are shown in Table 2.
  • the whole light chain and heavy chain sequences of FR1-V4-LFLE-P329G and FR1-V4-LLG-P329G-1.1 are shown in Table 3.
  • ELISA was performed according to standard protocols to determine the binding affinity of FOLR1 ⁇ CD3 BiTEs to both human FOLR1 and human CD3. Briefly, human antigen CD3, His-tag, or human FOLR1 was coated on Corning EIA/RIA high-binding 96-well plates (Corning Inc. ) at 100 ng per well overnight at 4°C and blocked with 3%nonfat milk in PBS (pH7.4) . Five-fold serially diluted antibodies from 50 ⁇ g/mL were added and incubated at room temperature for 2 h. The plates were washed with PBS containing 1%nonfat milk. Bound antibodies were detected by anti-His tag antibody (HRP) (Sigma) .
  • HRP anti-His tag antibody
  • the assay was developed at room temperature with TMB substrate (Solarbio) and monitored at 450 nm with a microplate reader.
  • the half-maximal binding (EC 50 ) was calculated by fitting the data to the Langmuir adsorption isotherm. The results were shown in Figures 2-3.
  • FOLR1 ⁇ CD3 BiTEs To measure the binding ability of FOLR1 ⁇ CD3 BiTEs to cell surface-associated FOLR1, flow cytometry was carried out using the FOLR1-positive cell lines SKOV3 and HT1080-FR1. About 5 ⁇ 10 5 cells were incubated with different concentrations of antibodies (100 ⁇ g/mL, 20 ⁇ g/mL, 4 ⁇ g/mL, 0.8 ⁇ g/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, 1.28 ng/mL, 0.256 ng/mL, 0 ng/mL) on ice for 1 h.
  • antibodies 100 ⁇ g/mL, 20 ⁇ g/mL, 4 ⁇ g/mL, 0.8 ⁇ g/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, 1.28 ng/mL, 0.256 ng/mL, 0 ng/mL
  • the cells were washed once with PBS containing 0.5%bovine serum albumin (PBSA) and resuspended in 100 ⁇ l PBSA. Then 1 ⁇ l /test anti-human IgG ( ⁇ -chain specific) -R-Phycoerythrin antibody (Sigma) was added and incubated for 30 min. The cells were washed once with PBSA and then used for flow cytometry analysis. The results were shown in Figures 4A-4B.
  • PBSA bovine serum albumin
  • HT1080 The ability and specificity of FOLR1 ⁇ CD3 BiTEs to activate human T cells in the presence of target cells (HT1080-FR1, SKOV3, HT1080) were evaluated by using Bio-Glo TM Luciferase Assay System with TCR/CD3 effector cells (Jurkat-NFAT-CD3) .
  • HT1080 cells do not express human FOLR1, while both SKOV3 cells and FOLR1 transfected stable cells HT1080-FR1 have a high level of FOLR1 expression.
  • the TCR/CD3 effector cells (Jurkat-NFAT-CD3) express endogenous TCR and CD3 receptors.
  • the TCR transduces intracellular signals, resulting in TCR-mediated T cell activation and producing an enhanced fluorescence signal.
  • Target cells were plated on 96-well plates at a density of 1 ⁇ 10 4 (for HT1080-FR1) or 1.5 ⁇ 10 4 (for SKOV3 and HT1080) cells in 100 ⁇ l RMPI 1640 complete medium per well for overnight. After removal of the supernatant, 50 ⁇ l antibodies (FR1-V4-LFLE-P329G, FR1-V4-LLG-P329G-1.1, FR1-mAb, CD3-mAb and FR1-mAb+CD3-mAb) were added into each well in a 5-fold gradient dilution at the maximum concentration of 100 ⁇ g/mL.
  • effector cells Jurkat-NFAT-CD3 were added at a density of 1.2 ⁇ 10 5 cells in 50 ⁇ l RMPI 1640 complete medium per well.
  • the plates were incubated for 6 h at 37 °C in a humidified incubator.
  • Stable-Lite Luciferase Assay System solution Vazyme
  • Luminescence was detected using ELISA reader (Molecular Devices) . The results were shown in Figures 5A-5D.
  • Jurkat-NFAT-CD3 cells were efficiently activated by both FR1-V4-LFLE-P329G and FR1-V4-LLG-P329G-1.1 with EC 50 of approximately 82.98 ng/mL and 97.89 ng/mL, respectively ( Figure 5A) .
  • the FOLR1-positive SKOV3 cells Jurkat-NFAT-CD3 cells were also efficiently activated by both FR1-V4-LFLE-P329G and FR1-V4-LLG-P329G-1.1 with EC 50 of approximately 1012 ng/mL and 684.8 ng/mL, respectively (Figure 5B) .
  • FOLR1 ⁇ CD3 BiTEs The In vitro efficacy of FOLR1 ⁇ CD3 BiTEs was assessed by CCK8 assay using FOLR1 transfected cell line HT1080-FR1, and FOLR1-negative HT1080 cell line was used as negative control.
  • 5 ⁇ 10 3 target cells were seeded in 100 ⁇ l RPMI 1640 complete medium, and incubated for approximately 20 h. Then, effector cells, human PBMCs (hPBMCs, 1 ⁇ 10 5 cells per well in 50 ⁇ l RPMI 1640 complete medium) , were added. Meanwhile, 50 ⁇ l antibodies 5-fold serially diluted from 4 ⁇ g/ml were added into each well.
  • the in vivo efficacy of FOLR1 ⁇ CD3 BiTEs was evaluated by using B-NDG mice. Considering the high FOLR1 expression and excellent tumorigenic characteristics of the stable cell line HT1080-FR1, a tumor model in B-NDG mice was established by mixing HT1080-FR1 and hPBMCs. A total 200 ⁇ L mixture of 1.7 ⁇ 10 6 HT1080-FR1 cells (100 ⁇ l) and 1.7 ⁇ 10 6 freshly isolated human PBMCs (100 ⁇ L) was subcutaneously inoculated into the right-side abdomen of B-NDG mice. These mice were randomized and staged at tumor size of 100 to 150 mm 3 .
  • mice were dosed subcutaneously with physiological saline, while the experiment group mice were intravenously treated with single dose of FR1-V4-LFLE-P329G at 500 ⁇ g/kg or FR1-V4-LLG-P329G-1.1 at 500 ⁇ g/kg.
  • the mice were dosed three times per week, with a total of 15 days of treatment. At the same time, tumor volumes and body weight of mice were measured.
  • TGI Tumor growth inhibition rates
  • both FR1-V4-LFLE-P329G and FR1-V4-LLG-P329G-1.1 have excellent pharmacodynamic functions in vitro and in vivo. Therefore, these FOLR1 ⁇ CD3 BiTEs are expected to conduct clinical research.

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Abstract

Sont divulgués des anticorps bispécifiques dirigés contre FOLR1 et leurs utilisations, en particulier des anticorps bispécifiques dirigés contre FOLR1 et CD3, des acides nucléiques codant pour les anticorps, des vecteurs comprenant les acides nucléiques, et une cellule hôte comprenant les acides nucléiques ou les vecteurs. Sont divulgués également des compositions pharmaceutiques et des conjugués anticorps-médicament comprenant les anticorps, et des méthodes thérapeutiques utilisant les anticorps.
PCT/CN2023/099866 2023-06-13 2023-06-13 Anticorps bispécifiques dirigés contre folr1 et leurs utilisations Pending WO2024254757A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103025758A (zh) * 2010-05-14 2013-04-03 Abbvie公司 Il-1结合蛋白
WO2016128912A1 (fr) * 2015-02-12 2016-08-18 Acerta Pharma B.V. Combinaisons thérapeutiques d'un inhibiteur de btk, d'un inhibiteur de pi3k, d'un inhibiteur de jak-2, d'un inhibiteur de pd-1, et/ou d'un inhibiteur de pd-l1
CN107074955A (zh) * 2014-11-20 2017-08-18 豪夫迈·罗氏有限公司 针对FolR1和CD3的T细胞活化性双特异性抗原结合分子
WO2019244107A1 (fr) * 2018-06-21 2019-12-26 Daiichi Sankyo Company, Limited Compositions comprenant des fragments de liaison à l'antigène cd3 et leurs utilisations
CN112424227A (zh) * 2018-03-14 2021-02-26 阿特根公司 与folr1特异性结合的抗体及其用途
CN113260379A (zh) * 2018-12-21 2021-08-13 浙江时迈药业有限公司 蛋白酶可切割的双特异性抗体及其用途
CN114423789A (zh) * 2021-12-24 2022-04-29 浙江时迈药业有限公司 针对间皮素的抗体及其用途
CN115916826A (zh) * 2020-06-19 2023-04-04 豪夫迈·罗氏有限公司 与CD3和FolR1结合的抗体

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103025758A (zh) * 2010-05-14 2013-04-03 Abbvie公司 Il-1结合蛋白
CN107074955A (zh) * 2014-11-20 2017-08-18 豪夫迈·罗氏有限公司 针对FolR1和CD3的T细胞活化性双特异性抗原结合分子
WO2016128912A1 (fr) * 2015-02-12 2016-08-18 Acerta Pharma B.V. Combinaisons thérapeutiques d'un inhibiteur de btk, d'un inhibiteur de pi3k, d'un inhibiteur de jak-2, d'un inhibiteur de pd-1, et/ou d'un inhibiteur de pd-l1
CN112424227A (zh) * 2018-03-14 2021-02-26 阿特根公司 与folr1特异性结合的抗体及其用途
WO2019244107A1 (fr) * 2018-06-21 2019-12-26 Daiichi Sankyo Company, Limited Compositions comprenant des fragments de liaison à l'antigène cd3 et leurs utilisations
CN113260379A (zh) * 2018-12-21 2021-08-13 浙江时迈药业有限公司 蛋白酶可切割的双特异性抗体及其用途
CN115916826A (zh) * 2020-06-19 2023-04-04 豪夫迈·罗氏有限公司 与CD3和FolR1结合的抗体
CN114423789A (zh) * 2021-12-24 2022-04-29 浙江时迈药业有限公司 针对间皮素的抗体及其用途

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