Multispecific antibodies targeting dimerizable tumor antigens and immunostimulatory antigens

Abstract

The present invention provides multispecific antibodies that target dimerizable tumor antigens and immunostimulatory antigens.

Description

[Technical Field]

[0001]

[0001] The present invention relates to novel covalent multispecific antibodies that target tumor antigens and immunostimulatory antigens, and uses thereof. [Background technology]

[0002]

[0002] Multifunctional antibodies are constructed based on traditional antibodies through sophisticated design and molecular engineering, enabling them to bind to multiple antigens. Numerous bispecific antibody technology platforms have been developed, including BiTE Bi-specific T-cell Engaging (Micromet, acquired by Amgen in 2012), CrossMab (Roche), DVD-Ig (Abbvie), TandAb (Affimed), and DART (Dual Antigen Re-Targeting, Macrogenics).

[0003]

[0003] In fact, bispecific or multispecific antibodies can exert the same therapeutic effect as the combination of several conventional antibodies. By simultaneously targeting multiple desired targets, bispecific or multispecific antibodies can provide advantages over conventional antibodies through novel and unique mechanisms. For example, blinatumomab (CD3×CD19, Amgen), which targets CD3 and CD19, can efficiently involve T cells in killing CD19-expressing tumor cells through its CD3-recognizing Fv, and has shown superior efficacy in treating ALL (acute lymphoblastic leukemia) and other diseases compared to conventional antibodies. Blinatumomab was approved for sale by the FDA as a treatment for ALL in 2014.

[0004]

[0004] However, despite the advantages offered by bispecific or multispecific antibodies, they also pose challenges, such as side effects, low response rates, and limited efficacy. In particular, certain targets tend to dimerize or aggregate when bound to bispecific or multispecific antibodies, which may cause unwanted side effects. Therefore, there is still a strong need to improve the design of bispecific or multispecific antibodies to achieve both desirable efficacy and reduced side effects. Summary of the Invention

[0005] In one aspect, the present invention provides an engineered antibody comprising: (i) a first polypeptide comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2); (ii) a second polypeptide comprising a second heavy chain variable domain (VH2) linked to the first light chain variable domain (VL1); (iii) a third polypeptide comprising a third heavy chain variable domain (VH3); and (iv) a fourth polypeptide comprising a fourth light chain variable domain (VL3). In some embodiments, the VL1 and VH1 associate to form a first domain capable of binding to a first target, the VL2 and VH2 associate to form a second domain capable of binding to a second target, and the VL3 and VH3 associate to form a third domain capable of binding to a third target. In such embodiments, the second and third polypeptide chains each further comprise a first dimerization domain and a second dimerization domain that associate to form a dimer. In some embodiments, one of the first target and the second target is an immunostimulatory target, and the remaining two targets are different epitopes on the same tumor antigen that are capable of dimerization upon binding to a natural ligand.

[0006] In some embodiments, the N-terminus of the VL1 is linked to the C-terminus of the VH2 and the N-terminus of the VL2 is covalently linked to the C-terminus of the VH1. In some embodiments, the N-terminus of the VH1 is linked to the C-terminus of the VL2 and the N-terminus of the VH2 is linked to the C-terminus of the VL1.

[0007] In some embodiments, the immunostimulatory target is selected from NKG2D, CD3, and CD16.

[0008]

[0008] In some of these embodiments, the immunostimulatory target is NKG2D. In certain embodiments, the domain capable of binding to NKG2D comprises: (a) a VL comprising an LCDR1 having the sequence set forth in SEQ ID NO:29, an LCDR2 having the sequence set forth in SEQ ID NO:30, and an LCDR3 having the sequence set forth in SEQ ID NO:31; and a VH comprising an HCDR1 having the sequence set forth in SEQ ID NO:25, an HCDR2 having the sequence set forth in SEQ ID NO:26, and an HCDR3 having the sequence set forth in SEQ ID NO:27, or (b) a VL comprising an LCDR1 having the sequence set forth in SEQ ID NO:118, an LCDR2 having the sequence set forth in SEQ ID NO:119, and an LCDR3 having the sequence set forth in SEQ ID NO:120; and a VH comprising an HCDR1 having the sequence set forth in SEQ ID NO:114, an HCDR2 having the sequence set forth in SEQ ID NO:115, and an HCDR3 having the sequence set forth in SEQ ID NO:116. In certain embodiments, the VL1 comprises the sequence of SEQ ID NO: 32 and the VH1 comprises the sequence of SEQ ID NO: 28, or the VL comprises the sequence of SEQ ID NO: 121 and the VH comprises the sequence of SEQ ID NO: 117.

[0009] In some other of these embodiments, the immunostimulatory target is CD3. In certain embodiments, the domain capable of binding to CD3 comprises a VL comprising an LCDR1 comprising the sequence set forth in SEQ ID NO:5, an LCDR2 comprising the sequence set forth in SEQ ID NO:6, and an LCDR3 comprising the sequence set forth in SEQ ID NO:7, and a VH comprising an HCDR1 comprising the sequence set forth in SEQ ID NO:1, an HCDR2 comprising the sequence set forth in SEQ ID NO:2, and an HCDR3 comprising the sequence set forth in SEQ ID NO:3. In certain embodiments, the VL1 comprises the sequence set forth in SEQ ID NO:8, and the VH1 comprises the sequence set forth in SEQ ID NO:4.

[0010] In some other of these embodiments, the immunostimulatory target is CD16. In certain embodiments, the domain capable of binding to CD16 comprises a VL comprising an LCDR1 comprising the sequence set forth in SEQ ID NO: 37, an LCDR2 comprising the sequence set forth in SEQ ID NO: 38, and an LCDR3 comprising the sequence set forth in SEQ ID NO: 39, and a VH comprising an HCDR1 comprising the sequence set forth in SEQ ID NO: 33, an HCDR2 comprising the sequence set forth in SEQ ID NO: 34, and an HCDR3 comprising the sequence set forth in SEQ ID NO: 35. In certain embodiments, the VL1 comprises the sequence of SEQ ID NO: 40, and the VH1 comprises the sequence of SEQ ID NO: 36.

[0011]

[0011] In some embodiments, the tumor antigen is HER2.

[0012]

[0012] In some embodiments, one of the domains capable of binding to HER2 is a first HER2-binding domain derived from the antigen-binding domain of trastuzumab, and the other domain capable of binding to HER2 is a second HER2-binding domain derived from the antigen-binding domain of pertuzumab. In some embodiments, the VL2 and the VH2 associate to form the first HER2-binding domain, and the VL3 and the VH3 associate to form the second HER2-binding domain. In some embodiments, the VL2 and the VH2 associate to form the second HER2-binding domain, and the VL3 and the VH3 associate to form the first HER2-binding domain. In some embodiments, the VL1 and the VH1 associate to form the first HER2-binding domain, and the VL3 and the VH3 associate to form the second HER2-binding domain. In some embodiments, the VL1 and VH1 associate to form the second HER2 binding domain, and the VL3 and VH3 associate to form the first HER2 binding domain.

[0013]

[0013] In some embodiments, the first HER2 binding domain comprises an LCDR1 having the sequence shown in SEQ ID NO: 13, an LCDR2 having the sequence shown in SEQ ID NO: 14, and an LCDR3 having the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 97, and the VH2 comprises an HCDR1 having the sequence shown in SEQ ID NO: 9, an HCDR2 having the sequence shown in SEQ ID NO: 10, and an HCDR3 having the sequence shown in SEQ ID NO: 11.

[0014]

[0014] In some embodiments, the second HER2 binding domain comprises an LCDR1 having the sequence shown in SEQ ID NO: 21, an LCDR2 having the sequence shown in SEQ ID NO: 22, and an LCDR3 having the sequence shown in SEQ ID NO: 23, and the VH3 comprises an HCDR1 having the sequence shown in SEQ ID NO: 17, an HCDR2 having the sequence shown in SEQ ID NO: 18, and an HCDR3 having the sequence shown in SEQ ID NO: 19.

[0015] In some embodiments, the VL1 and the VH1 are associated by a first non-native disulfide bond.

[0016] In some embodiments, the first non-native disulfide bond is formed between two non-native cysteine ​​residues in the VL1 and VH1, respectively, and optionally the two non-native cysteine ​​residues are Q100C in the VL1 and G44C in the VH1, where numbering is according to the Kabat index. In some of these embodiments, the VL2 and the VH2 are associated with a native disulfide bond or another non-native disulfide bond formed at a position different from the first non-native disulfide bond.

[0017] In some embodiments, the VL2 and the VH2 are associated by a second non-native disulfide bond.

[0018] In some embodiments, the second non-native disulfide bond is formed between two non-native cysteine ​​residues in the VL2 and VH2, respectively, and optionally, the two non-native cysteine ​​residues are Q100C in the VL2 and G44C in the VH2, where numbering is according to the Kabat index. In some of these embodiments, the VL1 and the VH1 are associated with another non-native disulfide bond formed at a position different from the native disulfide bond or the second non-native disulfide bond.

[0019]

[0019] In some embodiments, the VL1 and the VH1 are further associated with electrostatic interactions between two oppositely charged residues.

[0020]

[0020] In some embodiments, the VL2 and the VH2 are further associated with electrostatic interactions between two oppositely charged residues.

[0021]

[0021] In some embodiments, the two oppositely charged residues are introduced to replace Q38 in the VL1 and Q39 in the VH1, respectively, or to replace Q38 in the VL2 and Q39 in the VH2, respectively, where numbering is according to the Kabat index.

[0022]

[0022] In some embodiments, the two oppositely charged residues are introduced to replace Q40 in the VL1 and Q39 in the VH1, respectively, or to replace Q40 in the VL2 and Q39 in VH2, respectively, where numbering is according to the Kabat index.

[0023]

[0023] In some embodiments, the two oppositely charged residues are introduced to replace Q37 in the VL1 and Q39 in the VH1, respectively, or to replace Q37 in the VL2 and Q39 in VH2, respectively, where numbering is according to the Kabat index.

[0024]

[0024] In some embodiments, the two oppositely charged residues comprise a negatively charged amino acid residue selected from aspartic acid (D) or glutamic acid (E), and a positively charged amino acid residue selected from lysine (K), histidine (H), or arginine (R).

[0025]

[0025] In some embodiments, the two oppositely charged residues comprise Q38D in said VL1 and Q39K in said VH1, respectively, or Q38D in said VL2 and Q39K in said VH2, respectively, where numbering is according to the Kabat index.

[0026] In some embodiments, the VH2 comprises the sequence set forth in SEQ ID NO: 90, and the VL2 comprises the sequence set forth in SEQ ID NO: 91. In such embodiments, the VH3 comprises the sequence set forth in SEQ ID NO: 20, and the VL3 comprises the sequence set forth in SEQ ID NO: 24, or the VH3 comprises the sequence set forth in SEQ ID NO: 106, and the VL3 comprises the sequence set forth in SEQ ID NO: 107.

[0027] In some embodiments, the VH2 comprises the sequence set forth in SEQ ID NO: 92, and the VL2 comprises the sequence set forth in SEQ ID NO: 93. In such embodiments, the VH3 comprises the sequence set forth in SEQ ID NO: 12, and the VL3 comprises the sequence set forth in SEQ ID NO: 16.

[0028] In some embodiments, the VH2 comprises the sequence set forth in SEQ ID NO: 90, and the VL2 comprises the sequence set forth in SEQ ID NO: 98. In such embodiments, the VH3 comprises the sequence set forth in SEQ ID NO: 12, and the VL3 comprises the sequence set forth in SEQ ID NO: 95.

[0029] In some embodiments, the VH2 comprises the sequence set forth in SEQ ID NO: 90, and the VL2 comprises the sequence set forth in SEQ ID NO: 91. In such embodiments, the VH3 comprises the sequence set forth in SEQ ID NO: 12, and the VL3 comprises the sequence set forth in SEQ ID NO: 16.

[0030]

[0030] In some embodiments, the VL1 and VH1 associate to form the first domain capable of binding to NKG2D, the VL2 and VH2 associate to form the first HER2 binding domain, and the VL3 and VH3 associate to form the second HER2 binding domain.

[0031]

[0031] In some of these embodiments, (i) the VL1 comprises the sequence of SEQ ID NO: 32 and the VH1 comprises the sequence of SEQ ID NO: 28, (ii) the VL2 comprises the sequence of SEQ ID NO: 91 and the VH2 comprises the sequence of SEQ ID NO: 90, and (iii) the VL3 comprises the sequence of SEQ ID NO: 24 and the VH3 comprises the sequence of SEQ ID NO: 20.

[0032]

[0032] In some embodiments, the VL1 and VH1 associate to form the first domain capable of binding to CD3, the VL2 and VH2 associate to form the second HER2 binding domain, and the VL3 and VH3 associate to form the first HER2 binding domain.

[0033]

[0033] In some of these embodiments, (i) the VL1 comprises the sequence of SEQ ID NO: 8 and the VH1 comprises the sequence of SEQ ID NO: 4, (ii) the VL2 comprises the sequence of SEQ ID NO: 93 and the VH2 comprises the sequence of SEQ ID NO: 92, and (iii) the VL3 comprises the sequence of SEQ ID NO: 16 and the VH3 comprises the sequence of SEQ ID NO: 12.

[0034]

[0034] In some embodiments, the VL1 and VH1 associate to form the first domain capable of binding to CD16, the VL2 and VH2 associate to form the second HER2 binding domain, and the VL3 and VH3 associate to form the first HER2 binding domain.

[0035]

[0035] In some specific embodiments, (i) the VL1 comprises the sequence of SEQ ID NO: 40, and the VH1 comprises the sequence of SEQ ID NO: 36, (ii) the VL2 comprises the sequence of SEQ ID NO: 93, and the VH2 comprises the sequence of SEQ ID NO: 92, and (iii) the VL3 comprises the sequence of SEQ ID NO: 16, and the VH3 comprises the sequence of SEQ ID NO: 12.

[0036] In some embodiments, the VL1 and VH1 associate to form the first domain capable of binding to CD3, the VL2 and VH2 associate to form a third HER2-binding domain, and the VL3 and VH3 associate to form a fourth HER2-binding domain. In certain embodiments, the third HER2-binding domain is derived from the antigen-binding domain of Trastuzumab and is further modified to reduce affinity for HER2.

[0037]

[0037] In some of these embodiments, (i) the VL1 comprises the sequence of SEQ ID NO: 100 and the VH1 comprises the sequence of SEQ ID NO: 99, (ii) the VL2 comprises the sequence of SEQ ID NO: 98 and the VH2 comprises the sequence of SEQ ID NO: 90, and (iii) the VL3 comprises the sequence of SEQ ID NO: 95 and the VH3 comprises the sequence of SEQ ID NO: 12.

[0038]

[0038] In some embodiments, the VL1 is linked to the VH2 via a first peptide linker, and the VL2 is linked to the VH1 via a second peptide linker.

[0039]

[0039] In certain embodiments, the first peptide linker and the second peptide linker each independently comprise 5 to 9 amino acids.

[0040]

[0040] In certain embodiments, the first peptide linker and the second peptide linker each comprise the sequence set forth in SEQ ID NO:94.

[0041]

[0041] In some embodiments, the first dimerization domain and the second dimerization domain comprise a CH3 domain of an IgG, optionally further comprising one or more mutations that promote heterodimerization.

[0042] In some embodiments, the first dimerization domain comprises a first mutation and the second dimerization domain comprises a second mutation.

[0043] In certain embodiments, a) the first mutation comprises T389W and / or S375C and the second mutation comprises Y438V, T389S, L391A, and / or Y370C; b) the first mutation comprises D427K and / or D377K and the second mutation comprises K420D and / or K440D; c) the first mutation comprises D377K, E378K, and / or D427K and the second mutation comprises K393E, K440D, and / or K470E; d) the first mutation comprises S387H and / or comprises F436A and the second mutation comprises Y370T and / or T422F; e) the first mutation comprises S387H and / or T422F and the second mutation comprises Y422T and / or F436A; f) the first mutation comprises K393D and / or K440D and the second mutation comprises E378K and / or D427K; or g) the first mutation comprises L372D and / or L391E and the second mutation comprises L372K or T389K, wherein numbering is according to the Kabat index.

[0044] In certain embodiments, the first mutation comprises T389W and the second domain comprises T389S, L391A and Y438V, where numbering is according to the Kabat index.

[0045]

[0045] In some embodiments, the first dimerization domain and / or the second dimerization domain further comprise a CH2 domain and / or a hinge region of an IgG.

[0046]

[0046] In some embodiments, the hinge region comprises the sequence set forth in SEQ ID NO: 43, 101 or 102.

[0047] In some embodiments, the third polypeptide further comprises a CH1 region. In some of these embodiments, the CH1 region comprises the sequence set forth in SEQ ID NO:41.

[0048]

[0048] In some embodiments, the fourth polypeptide further comprises a CL region. In some of these embodiments, the CL region comprises the sequence set forth in SEQ ID NO:42.

[0049]

[0049] In some embodiments, one of the first dimerization domain and the second dimerization domain comprises the sequence shown in SEQ ID NO: 44 or 103, and the other comprises the sequence shown in SEQ ID NO: 45 or 104.

[0050]

[0050] In some embodiments, the first dimerization domain and the second dimerization domain retain ADCC activity, wherein one of the first dimerization domain and the second dimerization domain comprises the sequence set forth in SEQ ID NO: 103, and the other comprises the sequence set forth in SEQ ID NO: 104.

[0051]

[0051] In some embodiments, the first dimerization domain and the second dimerization domain do not possess ADCC activity, wherein one of the first dimerization domain and the second dimerization domain comprises the sequence set forth in SEQ ID NO: 44, and the other comprises the sequence set forth in SEQ ID NO: 45.

[0052]

[0052] In some embodiments, the first polypeptide comprises the sequence set forth in SEQ ID NO:58, the second polypeptide comprises the sequence set forth in SEQ ID NO:59, the third polypeptide comprises the sequence set forth in SEQ ID NO:20, and the fourth polypeptide comprises the sequence set forth in SEQ ID NO:24.

[0053]

[0053] In some embodiments, the first polypeptide comprises the sequence set forth in SEQ ID NO: 62, the second polypeptide comprises the sequence set forth in SEQ ID NO: 63, the third polypeptide comprises the sequence set forth in SEQ ID NO: 12, and the fourth polypeptide comprises the sequence set forth in SEQ ID NO: 16.

[0054]

[0054] In some embodiments, the first polypeptide comprises the sequence set forth in SEQ ID NO: 74, the second polypeptide comprises the sequence set forth in SEQ ID NO: 75, the third polypeptide comprises the sequence set forth in SEQ ID NO: 12, and the fourth polypeptide comprises the sequence set forth in SEQ ID NO: 16.

[0055]

[0055] In some embodiments, the engineered antibodies described herein are linked to one or more conjugate moieties.

[0056]

[0056] In some embodiments, the conjugate moiety comprises an agent for detection or isolation, such as a clearance regulator, chemotherapeutic agent, toxin, radioisotope, lanthanide, luminescent label, fluorescent label, enzyme substrate label, DNA alkylating agent, topoisomerase inhibitor, tubulin binding agent, or other anti-cancer agent.

[0057]

[0057] In one aspect, the disclosure provides an isolated polynucleotide encoding an engineered antibody described herein.

[0058]

[0058] In another aspect, the present disclosure provides a vector comprising an isolated polynucleotide described herein.

[0059] In yet another aspect, the present disclosure provides a host cell comprising a vector described herein.

[0060]

[0060] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (i) an engineered antibody described herein, or a polynucleotide encoding the engineered antibody described herein, and (ii) one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0061]

[0061] In some embodiments, the pharmaceutical compositions described herein further comprise an additional therapeutic agent.

[0062]

[0062] In some embodiments, the additional therapeutic agent is an agent for treating a HER2-associated disease or disorder.

[0063]

[0063] In some embodiments, the HER2-associated disease or disorder is a cancer selected from the group consisting of breast cancer, prostate cancer, lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, pancreatic cancer, testicular cancer, urothelial cancer, endometrial cancer, malignant melanoma, and soft tissue cancer (e.g., synovial sarcoma).

[0064]

[0064] In yet another aspect, the present disclosure provides a method for expressing an engineered antibody described herein, comprising culturing a host cell described herein under conditions in which a vector described herein is expressed.

[0065]

[0065] In yet another aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of an engineered antibody described herein.

[0066]

[0066] In yet another aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of an engineered antibody described herein, or a polynucleotide encoding the engineered antibody described herein, and / or a pharmaceutical composition described herein.

[0067] In some embodiments, the disease or disorder is a HER2-associated disease or disorder.

[0068]

[0068] In some embodiments, the subject is a human.

[0069] In some embodiments, the administration is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

[0070]

[0070] In yet another aspect, the present disclosure provides the use of an engineered antibody described herein, a pharmaceutical composition described herein, and / or a polynucleotide encoding said engineered antibody described herein in the manufacture of a medicament for treating, preventing, or alleviating a disease or disorder.

[0071] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized. A description of the drawings is provided below. [Brief explanation of the drawings]

[0072] [Figure 1] FIG. 1 shows a schematic diagram of the DICAD structure. [Figure 2]

[0073] Figure 2 shows a schematic diagram of the TRIAD structure. [Figure 3]

[0074] FIG. 3 shows the effect of Herceptin, Perjeta, 6A1 and 6A2 on BT-474 cell proliferation (dose-effect curves). [Figure 4A]

[0075] FIG. 4A shows the results of killing of SK-BR-3 cells by 6A8, 6A12, 6A14, 6A15 and Herceptin. [Figure 4B]

[0076] FIG. 4B shows the results of killing of SK-BR-3 cells by 6A15, Herceptin, 6A17, 6A14, 6A8, 6A21, 6A19 and 6B1. [Figure 4C]

[0077] FIG. 4C shows the results of SK-BR-3 cell killing by 6A14, 6A17, 6A18, and Trastuzumab. [Figure 4D]

[0078] FIG. 4D shows the results of SK-BR-3 cell killing by 6B1, 6A23, 6A25, and 6A26. [Figure 4E]

[0079] FIG. 4E shows the results of SK-BR-3 cell killing by 6A17, 6A18, and 6A19. [Figure 4F]

[0080] FIG. 4F shows the results of SK-BR-3 cell killing by FT-1, 6B1, and 6A23. [Figure 5A]

[0081] FIG. 5A shows the results of MCF-7 cell killing by 6B1, 6A23, 6A25 and 6A26. [Figure 5B]

[0082] FIG. 5B shows the results of MCF-7 cell killing by 6A17, 6A18, 6A19, FT-1, 6B1, and 6A23. [Figure 6]

[0083] FIG. 6 shows the effects of 6B1, 6A25, 6A19 and 6A26 on the growth of subcutaneously transplanted tumors of human breast cancer KPL-4 cells. [Figure 7]

[0084] FIG. 7 shows the effects of 6B1, 6A19, 6A25 and 6A26 on the growth of subcutaneously transplanted tumors of human breast cancer BT474 cells. [Figure 8]

[0085] FIG. 8 shows the effects of 6B1, 6A19, 6A25 and 6A26 on the growth of subcutaneously transplanted tumors of human colon cancer HT55 cells. [Figure 9]

[0086] FIG. 9 shows the effects of control, 6B1, 6A19, 6A25 and 6A26 on the growth of the subcutaneously implanted prostate cancer cell line PC-3. [Figure 10]

[0087] FIG. 10 shows exemplary constant region sequences of engineered antibodies. [Figure 11]

[0088] FIG. 11 shows the sequences of control antibodies 6A1 and 6A2. [Figure 12]

[0089] FIG. 12 shows the sequence of the control antibody 6B1. [Figure 13-1]

[0090] FIG. 13 shows VH and VL sequences targeting CD3, HER2 (ie, Tratuzumab and Pertuzumab), CD16, and NKG2D, respectively, which have mutations that introduce non-native disulfide bonds and / or charged amino acids. [Figure 13-2]

[0090] Figure 13 shows VH and VL sequences targeting CD3, HER2 (ie, Tratuzumab and Pertuzumab), CD16, and NKG2D, respectively, which have mutations that introduce non-native disulfide bonds and / or charged amino acids. DETAILED DESCRIPTION OF THE INVENTION

[0073]

[0091] Before describing the present invention in detail, the following will be pointed out and defined.

[0074]

[0092] All descriptions provided herein are merely for illustrating various embodiments of the present invention provided in this disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments are included in this specification.

[0075]

[0093] All references cited in this disclosure, including patent applications, issued patents, published articles or other publications, are incorporated by reference in their entirety and are intended to provide methodologies that may be used in connection with the description provided herein. With respect to terms presented in one or more publications that are similar or identical to terms expressly defined in this disclosure, the definition of such terms expressly provided in this disclosure shall control in all respects.

[0076]

[0094] Unless expressly defined otherwise, all technical and scientific terms used in this disclosure are generally assumed to have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0077]

[0095] As used herein, and throughout this disclosure, the terms "a," "an," and "the" are understood to mean "one or more" or "at least one," unless otherwise indicated. By way of example, "a polypeptide complex" means one polypeptide complex or one or more polypeptide complexes.

[0078]

[0096] As used herein, the terms "about," "approximately," or "approximately" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In specific embodiments, the term "about" or "approximately" preceding a numerical value indicates a range of ±15%, 10%, 5%, or 1% of the value.

[0079]

[0097] As used herein, the terms "comprise," "comprises," "comprising," "include," "includes," "including," "contain," "contains," "containing," and "have," "has," and the like, are synonymous and used in an inclusive and open-ended manner and do not exclude additional elements, features, steps, acts, operations, and the like.

[0080]

[0098] As used herein, the term "or" is used in an inclusive (rather than exclusive) sense, so that, for example, when used to connect elements of a list, the term "or" means one, some, or all of the elements in the list.

[0081]

[0099] As used herein, the phrase "at least one" means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The phrase "at least one of" a list of items is understood to refer to any combination of those items, including single members. As an example, "at least one of A, B, or C" is intended to encompass A, B, C, A and B, A and C, B and C, and A, B, and C. Conjunctions such as "at least one of X, Y, and Z" are generally understood to be used to convey that an item, term, etc., can be at least one of X, Y, or Z, unless otherwise specified, from the context. Thus, such conjunctions are generally not intended to imply that at least one of X, at least one of Y, and at least one of Z, respectively, must be present in a particular embodiment.

[0082]

[0100] As used herein, reference to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "additional embodiments," "some embodiments," "particular embodiments," or "a further embodiment," or combinations thereof, is understood to mean that the particular feature, structure, or characteristic described in connection with that particular embodiment is included in at least one embodiment of the present disclosure. Thus, the presence or appearance of such a phrase in various places throughout this disclosure do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0083]

[0101] Conditional language used herein, such as "can," "could," "may," "potential," and "for example," is intended to convey that certain embodiments include certain features, elements, and / or steps, and other embodiments do not, unless expressly stated or understood otherwise within the context in which it is used. Thus, such conditional language is generally not intended to imply that features, elements, and / or steps are in any way required for one or more embodiments.

[0084] I. Definitions and Abbreviations

[0102] In this section, definitions of some general terms are provided. Definitions of other terms may be found in other sections of this disclosure below.

[0085]

[0103] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a linear series of amino acid residues connected to each other by peptide bonds, and include proteins, polypeptides, oligopeptides, peptides, and fragments thereof. Proteins can be composed of naturally occurring amino acids and / or synthetic (e.g., modified or non-naturally occurring) amino acids. Thus, "amino acid," or "peptide residue," as used herein, refers to both naturally occurring and synthetic amino acids. The terms "polypeptide," "peptide," and "protein" include fusion proteins, including, but not limited to, fusion proteins having heterologous amino acid sequences with or without an N-terminal methionine residue, fusions with heterologous and homologous leader sequences, immunologically tagged proteins, and fusion proteins having a detectable fusion partner, such as fusion proteins that include a fluorescent protein, β-galactosidase, luciferase, etc., as a fusion partner.

[0086]

[0104] As used herein, the term "amino acid" refers to a building block of a protein, peptide, polypeptide, or amino acid polymer, and further refers to naturally occurring or synthetic amino acids, as well as any amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. As used herein, naturally occurring amino acids encompass the group of naturally occurring carboxy alpha amino acids, including alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0087]

[0105] As used herein, the term "domain" refers to a globular structure formed by one or more regions of one or more polypeptide chains, including, for example, beta-pleated sheets and / or peptide loops (e.g., containing 3-4 peptide loops) stabilized by intrachain disulfide bonds. Examples may include Fab domains (see below for more details). Note that in this disclosure, the two terms "domain" and "region" may be used interchangeably.

[0088]

[0106] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," or "polynucleotide," etc., are understood to refer to nucleotide polymers of any length, and may include both DNA and RNA, and may be single- or double-stranded.

[0089]

[0107] The term "antibody," as used herein, includes any immunoglobulin, monoclonal, polyclonal, chimeric, humanized, multispecific, bispecific, bivalent, or polyvalent antibody that binds to a specific antigen. Antibodies and related terms are described in more detail below.

[0090]

[0108] In mammals, such as humans, there are five different classes / isotypes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM, corresponding to the five Ig heavy chain types α, δ, ε, γ, and μ, respectively) depending on the different types of heavy chains present in the immunoglobulin. These antibodies typically have different molecular and biological properties, functional distribution, physiological functions, and pathological effects in diseases. A particular antibody class may further include subclasses. For example, in humans, IgA may include the IgA1 and IgA2 subclasses, and IgG may include four subclasses, IgG1, IgG2, IgG3, and IgG4, respectively. With the immunoglobulin monomer as the basic functional unit, mammalian antibodies may exist as monomers (e.g., IgD, IgE, and IgG), dimers (IgA), tetramers (IgM), or pentamers (IgM). In mammals, there are two types of light chains, including kappa (κ) chains and lambda (λ) chains.

[0091]

[0109] Within the basic immunoglobulin unit, a natural or naturally occurring antibody, such as an IgG, typically contains two identical heavy (H) chains and two identical light (L) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond or linkage formed between a pair of cysteine ​​residues present in each of the light and heavy chains, and the two heavy chains are further linked to each other by several disulfide bonds formed between cysteine ​​residues in each heavy chain. The tetramer thus formed is essentially Y-shaped as an antibody, with the end of each fork arm containing an identical antigen-binding site (i.e., paratope) that specifically interacts with a corresponding epitope on the antigen.

[0092]

[0110] More specifically, in natural antibodies, from N- to C-terminus, each heavy chain comprises a variable region (VH, or HCVR) followed by three or four constant regions ("CH", where IgA, IgD, and IgG comprise three CH regions, CH1, CH2, and CH3, and IgE and IgM comprise four CH regions, CH1, CH2, CH3, and CH4), and each light chain comprises a variable region (VL, or LCVR) and a constant region (CL). In Y-shaped antibodies, the variable region of each light chain (i.e., VL region) aligns or associates with the variable region (i.e., VH region) of its paired heavy chain to together form the antigen-binding site of the antibody.

[0093]

[0111] The term "variable region" or "VR," as used herein, refers to the region in an antibody heavy or light chain responsible for antigen binding. In a naturally occurring antibody, the heavy chain variable region (VH or HCVR) contains three highly variable loops called "complementarity-determining regions" (CDRs), i.e., the heavy (H) chain CDRs, including HCDR1, HCDR2, and HCDR3, and the light chain variable region (VL or LCVR) contains three light (L) chain CDRs, including LCDR1, LCDR2, and LCDR3. The CDR boundaries of an antibody can be defined or identified according to the Kabat, Chothia, or Al-Lazikani definitions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28, 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between adjacent sections known as "framework regions" (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. In native antibodies, each VH and VL comprises four FRs, with the CDRs and FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. However, it should be understood that the term "variable region," as used herein, does not necessarily have to include all three CDRs or all four FRs, and should be understood to encompass any variant or derivative of a native variable region derived from a native antibody, so long as such variant or derivative retains antigen-binding activity.

[0094]

[0112] The term "constant region" or "constant portion," as used herein, refers to a region in an antibody heavy or light chain that is not directly involved in antigen binding. It should be understood that the term "constant region" or "constant portion," as used herein, does not necessarily include the entire natural constant region of a natural antibody, but should be understood to encompass any variant or derivative of such a natural constant region or constant portion, so long as such variant or derivative retains the ability to support the stability of the antigen-binding domain or retains its intended biological function, such as secretion, placental transport, Fc receptor binding, complement binding, and other effector functions.

[0095]

[0113] The term "CL region" refers to the constant region of an immunoglobulin light chain adjacent to the VL region. The CL region can span from about Kabat position 108 to about Kabat position 216 in an immunoglobulin light chain. In native antibodies, the constant region of each light chain (i.e., the CL region) is associated with the first constant region (i.e., the CH1 region) of the paired heavy chain.

[0096]

[0114] The term "CH1 region," as used herein, encompasses the first (most amino-terminal) constant region of an immunoglobulin heavy chain extending from about Kabat position 114 to at least about Kabat position 233 (and may extend, for example, to Kabat position 234). The CH1 region is adjacent to the VH region and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0097]

[0115] The term "hinge region," with respect to antibodies, includes the portion of a heavy chain molecule that connects the CH1 region to the CH2 region. The length of the hinge region varies depending on the defined boundaries of the CH1 region and the defined boundaries of the CH2 region. The hinge region is usually flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0098]

[0116] The term "CH2 region," as used herein, refers to the portion of a heavy chain immunoglobulin molecule extending, for example, from about Kabat position 244 to Kabat position 360.

[0099]

[0117] The term "CH3 region," as used herein, refers to the portion of a heavy chain immunoglobulin molecule extending approximately 110 residues from the N-terminus of the CH2 domain, e.g., from about Kabat position 361 to Kabat position 476, 477, or 478. The CH3 domain typically forms the C-terminal portion of antibodies such as IgG, IgA, and IgD. However, in some immunoglobulins, such as IgE and IgM, additional domains may extend from the CH3 domain and form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the ε chain of IgE).

[0100]

[0118] "Fc," as used herein, refers to the portion derived from an antibody, e.g., an IgG, and is composed primarily of the second (CH2) and third (CH3) constant regions of a first heavy chain linked to the CH2 and CH3 of a second heavy chain via one or more covalent bonds that are non-peptide bonds, e.g., disulfide bonds. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0101]

[0119] "Antigen" or "Ag," as used herein, refers to a compound, composition, peptide, polypeptide, protein, or substance (e.g., a polypeptide, carbohydrate, nucleic acid, lipid, or other naturally occurring or synthetic compound) that can be specifically recognized and bound by a component of the immune system, e.g., an antibody. As used herein, the term "antigen" encompasses antigenic epitopes, e.g., fragments of an antigen that are antigenic epitopes. The terms "antigen" and "target" are used interchangeably in this disclosure.

[0102]

[0120] "Epitope" refers to a region of an antigen to which a binding agent (such as an antibody) binds. Epitopes can be formed from contiguous amino acids (also called linear or continuous epitopes) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitopes). Epitopes typically include at least 3, and more usually at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation.

[0103]

[0121] As used herein, the term "antibody fragment" refers to a portion of, or derived from, a full-length antibody. The antibody fragment may be an antigen-binding fragment or a variable region thereof.

[0104]

[0122] As used herein, the term "antigen-binding domain" refers to an antibody fragment or a domain derived from a portion of an antibody, which contains one or more CDRs or binds to an antigen but does not include the intact native antibody structure. Examples of antigen-binding fragments include, without limitation, variable domains, variable regions, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen as the parent full-length antibody. Antigen-binding fragments can contain one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies. Further details and specific formats of antigen-binding moieties are described in Spiess et al., 2015 (Supra) and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated by reference in their entirety.

[0105]

[0123] As used herein, the term "Her2 binding domain" means an antigen-binding domain in which the antigen is HER2.

[0106]

[0124] "Fv," with respect to antibodies, refers to the smallest antibody fragment containing a complete antigen-binding site. Fv fragments consist of a single light-chain variable domain linked to a single heavy-chain variable domain. Numerous Fv designs are available, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond, and scFv, which may be formed by linking the two domains together as a single polypeptide via a peptide linker. Fv constructs containing immunoglobulin heavy or light chain variable domains linked to the corresponding immunoglobulin heavy or light chain variable and constant domains have also been produced. Fvs have also been multimerized to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0107]

[0125] "Fab," as used herein, refers to a single antigen-binding domain derived from an antibody, which has a single heavy chain fragment associated with a single light chain fragment via one or more covalent bonds that are non-peptide bonds. In some embodiments, the single heavy chain fragment in a Fab domain comprises an HCVR and a CH1 region. In some embodiments, the single light chain fragment in a Fab domain comprises an LCVR and a CL domain. In some embodiments, the CH1 region is associated with the HCVR by a covalent bond, such as a disulfide bond. In a native antibody, the Fab domain substantially corresponds to one arm of the antibody and typically retains the ability to recognize and bind to its corresponding antigen.

[0108]

[0126] "Fab'" refers to a Fab fragment that contains part of the hinge region.

[0109]

[0127] "F(ab')2" refers to a Fab' dimer.

[0110]

[0128] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other either directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)).

[0111]

[0129] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv connected to the Fc region of an antibody.

[0112]

[0130] "Camelized single domain antibody", "heavy chain antibody" or "HCAb" refers to a camelized single domain antibody consisting of two V H It refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38 (1999), Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001), WO94 / 04678, WO94 / 25591, U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies possess a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428):446-8 (1993), Nguyen VK. et al. Immunogenetics. Apr; 54(1):39-47 (2002), Nguyen VK. et al. Immunology. May; 109(1):93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0113]

[0131] "Nanobody" refers to an antibody fragment consisting of a VHH domain and two constant domains CH2 and CH3 from a heavy chain antibody.

[0114]

[0132] A "diabody" or "dAb" comprises a small antibody fragment with two antigen-binding sites, wherein these fragments are V or V2 on the same polypeptide chain. L V connected to the domain H Domaine (V H -V L or V L -V H ) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8 (1993), EP404097, WO93 / 11161). By using a linker that is too short to allow pairing of the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, thereby generating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0115]

[0133] A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. H The domains are covalently linked with a peptide linker to create a bivalent or multivalent domain antibody. H The domains may target the same or different antigens.

[0116]

[0134] In certain embodiments, an "scFv dimer" is a dimer of two scFvs, each of which is composed of two scFvs. H -V L V dimerized with the moiety H -V L (linked by a peptide linker), whereby the V of one part is a bivalent diabody or bispecific scFv (BsFv). H 's another part of V L's to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In another embodiment, an "scFv dimer" is a dimer of V L1 -V H2 (also linked by a peptide linker) and associated V H1 -V L2 (linked by a peptide linker), whereby V H1 and V L1 , and V H2 and V L2 are coordinated, and each coordinated pair has a different antigen specificity.

[0117]

[0135] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. H The moieties are linked by a peptide linker (e.g., a long flexible linker) and each of the two V L In some embodiments, the dsFv-dsFv' is bispecific, with each disulfide paired heavy and light chain having a different antigen specificity.

[0118]

[0136] As used herein, the term "multispecific antibody" refers to an artificial or engineered antibody that can simultaneously bind to at least two different epitopes. Bispecific antibodies are essentially one type of multispecific antibody. In addition, other multispecific antibodies may include trispecific antibodies, which have three different antigen-binding specificities, and tetraspecific antibodies, which have four different antigen-binding specificities, etc.

[0119]

[0137] As used herein, the term "bispecific antibody" refers to an antibody containing two physically separable antigen-binding domains with different antigen specificities. Typically, bispecific antibodies are artificial antibodies that have fragments derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes may be on the same antigen or on two different antigens. This is in contrast to naturally occurring antibodies, which have two structurally identical, physically separable antigen-binding portions and therefore have the same antigen specificity.

[0120]

[0138] The term "affinity," as used herein, refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen. The affinity of an antibody for an antigen can be determined by calculating the equilibrium dissociation constant, K, using methods known in the art. D (see generally Davies et al. Ann. Rev. Biochem. 1990, 59: 439-15 473).

[0121]

[0139] The term "variant" refers, without limitation, to any antibody having a structure or sequence derived from an antibody of the present disclosure whose structure / sequence is sufficiently similar to that disclosed herein and, based on that similarity, would be expected by one of skill in the art to have the same or similar activity and utility as the claimed and / or referenced antibody, and therefore is interchangeably referred to as a "functional equivalent." Modifications to obtain a "variant" include, for example, the addition, deletion, and / or substitution of one or more amino acid residues. A functional equivalent or a fragment of a functional equivalent may contain one or more conservative amino acid substitutions. The term "conservative amino acid substitution" refers to the substitution of an amino acid with another amino acid having similar properties to the original amino acid. Conservative amino acid groups are known in the art.

[0122]

[0140] Conservative substitutions may be introduced at any position in a given peptide or fragment thereof. However, it may also be desirable to introduce non-conservative substitutions, particularly, but not limited to, non-conservative substitutions at any one or more positions. Non-conservative substitutions that result in the formation of functionally equivalent fragments of the peptide differ substantially, for example, in polarity, charge, and / or steric bulk, while maintaining the functionality of the derivative or variant fragment.

[0123]

[0141] Throughout this disclosure, numbers indicating amino acid residue positions in antibody constant regions, e.g., the heavy chain constant region 1 (CH1) and light chain constant region (CL) of the constant portion, are based on the Kabat index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). As mentioned above, some positions use the IMGT numbering or EU numbering as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015). These numberings are also available from the IMGT scientific atlas, accessible from the website of the international ImMunoGeneTics information system.

[0124]

[0142] The term "chimeric," as used herein, refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chain is derived from one species and the remaining portion of the heavy and / or light chain is derived from another species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0125]

[0143] As used herein, the term "vector" refers to a vehicle into which a genetic element is operably inserted to express the genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce, or transfect a host cell to express the genetic element carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector can contain an origin of replication. A vector can also contain materials that aid in cell entry, including, but not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0126]

[0144] The term "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0127]

[0145] "HER2" (from human epidermal growth factor receptor 2), as used herein, refers to a protein encoded by the ErbB2 gene in humans. ErbB is an abbreviation for erythroblastic oncogene B, and belongs to the epidermal growth factor receptor family, consisting of an extracellular domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. In humans, the ErbB family includes four members: ErbB1 (Her1), ErbB2 (Her2), ErbB3 (Her3), and ErbB4 (Her4). Of these, HER2 is a receptor tyrosine-protein kinase erbB-2, and is also known as HER2 / neu, CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human).

[0128] II Engineered Antibodies

[0146] The present invention provides novel engineered antibodies. The term "engineered" with respect to antibodies refers to artificially constructed macromolecules that incorporate antibodies or antigen-binding fragments thereof. Engineered antibodies may be of a different format or structure than conventional antibodies and / or may contain amino acid mutations in the original antibody sequence.

[0129]

[0147] The present invention provides novel engineered antibodies having three antigen-binding domains capable of binding to two epitopes on a first target and a second target, respectively. In some embodiments, the second target is a dimerizable antigen. The second target may be an antigen capable of dimerizing or oligomerizing upon binding to a ligand. The engineered antibodies of the present disclosure are designed to simultaneously bind to two epitopes on the second target, thereby increasing their affinity for the second target and not inducing dimerization or oligomerization of the second target. This design is particularly useful because it increases binding affinity to the second target without causing unwanted activation due to dimerization or oligomerization of the second target.

[0130]

[0148] In certain embodiments, the engineered antibodies provided herein are based on the TRIAD format disclosed, for example, in PCT application WO2019 / 120245, which is incorporated herein in its entirety.

[0131]

[0149] TRIAD is a platform for constructing antibodies with a molecular weight of approximately 153 kDa, which can simultaneously recognize three antigens. In general, antibodies provided by the TRIAD platform are similar to conventional antibodies. They have the following advantages: (1) They retain the properties of bivalent, bispecific antibodies, such as avidity, affinity, and potency; (2) They are highly stable and less prone to aggregation; (3) They are easier to express and purify than other bispecific or multispecific antibodies; (4) Their structure is similar to that of natural IgG, resulting in reduced immunogenicity; and (5) They have been proven to have high potency both in vitro and in vivo, and have a longer half-life than most other bispecific or multispecific antibodies.

[0132]

[0150] The TRIAD-based engineered antibodies provided herein have a structure similar to that of natural IgG. Natural IgG is composed of four polypeptide chains, forming a Y-shaped structure with two arms and a tail. However, the engineered antibodies provided herein are asymmetric, with one arm carrying two antigen-binding sites and another arm carrying a third antigen-binding site.

[0133]

[0151] To increase the binding affinity to a given antigen, two antigen-binding sites on the same arm may be designed to target the same antigen, and a third antigen-binding site on another arm may be designed to target a different antigen. However, the inventors surprisingly discovered that such a design may be disadvantageous when the antigens targeted by the two antigen-binding sites on the same arm are capable of dimerization (or oligomerization). This is because simultaneous binding of the two antigen-binding sites on the same arm to an antigen (such as HER2) may cause unwanted dimerization and activation of the antigen, which is contrary to the intent of antigen inhibition.

[0134]

[0152] Even more surprisingly, the inventors discovered that when two antigen-binding sites targeting the same antigen are designed to be located on different arms, unwanted dimerization and activation of the antigen can be significantly reduced, and at the same time, the binding affinity to the antigen can be increased.

[0135]

[0153] Thus, in some embodiments, an engineered antibody comprises (i) a first polypeptide comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2), (ii) a second polypeptide comprising the second heavy chain variable domain (VH2) linked to the first light chain variable domain (VL1), (iii) a third polypeptide comprising a third heavy chain variable domain (VH3), and (iv) a fourth polypeptide comprising a fourth light chain variable domain (VL3), wherein VL1 and VH1 associate to form a first domain capable of binding to a first target, VL2 and VH2 associate to form a second target, and VL3 and VH3 associate to form a third target, wherein one of the first and second targets is an immunostimulatory target, and the remaining two targets are different epitopes on the same tumor antigen that are capable of dimerization upon binding to natural ligands.

[0136]

[0154] In some embodiments, the first target is an immune target and the second and third targets are different epitopes on the same tumor antigen that are capable of dimerization upon binding to a natural ligand.

[0137]

[0155] In some other embodiments, the second target is an immune target and the first and third targets are different epitopes on the same tumor antigen that are capable of dimerization upon binding to a natural ligand.

[0138]

[0156] In certain embodiments, the engineered antibodies provided herein comprise a second domain and a third domain, both of which target a second target. The second domain comprises a VL2 in the first polypeptide and a VH2 in the second polypeptide, and the third domain comprises a VH3 in the third polypeptide and a VL3 in the fourth polypeptide. In other words, the second domain and the third domain, both of which target a second target, are located in different arms of the engineered antibody.

[0139]

[0157] In some embodiments, the first epitope and the second epitope of the second target in the engineered antibody are different, hi some embodiments, the first epitope and the second epitope of the second target in the engineered antibody are substantially the same or identical.

[0140]

[0158] In some embodiments, the C-terminus of VL1 is linked to the N-terminus of VH2, and the C-terminus of VL2 is covalently linked to the N-terminus of VH1. Figure 2 provides a schematic depicting an engineered antibody. In some embodiments, the C-terminus of VH1 is linked to the N-terminus of VL2, and the C-terminus of VH2 is linked to the N-terminus of VL1.

[0141]

[0159] In some embodiments, VH2 and VL1 and / or VH1 and VL2 are directly covalently linked or indirectly covalently linked, for example, via a linker, e.g., a peptide linker. The term "peptide linker," as used herein, can refer to any suitable polypeptide capable of linking two entities to form a single molecule or maintaining a sufficiently close association of the two entities without substantially interfering with the biological activity of the two entities. The linker can be composed of amino acid residues linked together by peptide bonds and can optionally further include one or more unnatural amino acids. Any suitable polypeptide can be used as a linker. In some embodiments, the polypeptide linker can be composed primarily of sterically unhindered amino acids, such as glycine and alanine. In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (e.g., poly(Gly-Ala)), or a combination of glycine and serine (e.g., poly(Gly-Ser)). In some embodiments, the peptide linker comprises the amino acid sequence of RTVAA (SEQ ID NO: 94).

[0142] A. Target of engineered antibodies

[0160] In certain embodiments, the second target is capable of dimerization, aggregation, or cross-linking upon binding to a ligand. Exemplary antigens include, without limitation, receptor protein tyrosine kinases (RPTKs) and class II cytokine receptors. In some embodiments, RPTKs are receptors of the EGF receptor family (also known as the ErbB receptor family, including EGFR, ERBB2, ERBB3, and ERBB4), the insulin receptor family (including INSR and IGFR), the platelet-derived growth factor receptor (the PDGF receptor family, including PDGFRα, PDGFRβ, M-CSFR, KIT, and FLT3L), the VEGF receptor family (including VEGFR1, VEGFR2, and VEGFR3), the fibroblast growth factor receptor family (the FGF receptor family, including FGFR1, FGFR2, FGFR3, and FGFR4), the CCK receptor family (including CCK4), the nerve growth factor receptor family (the NGF receptor family, including TRKA, TRKB, and TRKC), and the like. Class II cytokine receptors include those of the IL-1 receptor family (including IL-1, IL-2, IL-3, IL-4, IL-5), the HGF receptor family (including MET and RON), the Eph receptor family (including EPHA1-6, EPHB1-6), the AXL receptor family (including AXL, MER, TYRO3), the TIE receptor family (including TIE and TEK), the RYK receptor family (including RYK), the DDR receptor family (including DDR1 and DDR2), the RET receptor family (including RET), the ROS receptor family (including ROS), the LTK receptor family (including LTK and ALK), the ROR receptor family (including ROR1 and ROR2), the MuSK receptor family (including MUSK), the LMR receptors (including AATYK1, AATYK2, AATYK3), and others such as RTK106. In some embodiments, class II cytokine receptors include receptors for IFNα, IFNβ, IFNγ, IL-10, etc.

[0143]

[0161] In most cases, the dimerization of this type of antigen on the cell surface can activate a series of biochemical reactions within the cell, leading to a comprehensive cellular response. For example, HER2 dimerization can lead to cell proliferation, especially cancer cell proliferation.

[0144]

[0162] In some embodiments, the second target recognized by the engineered antibody is a tumor antigen that is capable of dimerizing upon binding to a ligand.

[0145]

[0163] In some embodiments, the tumor antigen is HER2. HER2 belongs to the ErbB receptor family and consists of four membrane-bound receptor tyrosine kinases. HER2 is known to be capable of homodimerization and heterodimerization with HER1, HER3, or HER4. HER2 dimerization causes autophosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, initiating various signal transduction pathways, including the PI3K / Akt and MAPK pathways, thereby regulating tumor cell proliferation, differentiation, migration, and apoptosis. HER2 protein has been shown to form clusters in the cell membrane, which may be involved in tumorigenesis.

[0146]

[0164] Antibodies that target HER2 are considered an effective strategy for treating HER2-related diseases. Trastuzumab, sold under brand names such as Herceptin, is a monoclonal antibody for treating breast and gastric cancer, particularly HER2-positive cancers. Pertuzumab, sold under the brand name Perjeta, is another monoclonal antibody that can be used in combination with trastuzumab and docetaxel to treat metastatic HER2-positive breast cancer.

[0147]

[0165] However, the inventors surprisingly discovered that when a TRIAD-format antibody has two HER2-binding domains located on the same arm of the antibody, the TRIAD-format antibody does not exhibit HER2-blocking effects, but unexpectedly exhibits significant HER2 agonistic effects. For example, 6A1 and 6A2, recorded in Example 2, were constructed using the TRIAD model, where each arm of the antibody contains two HER2-binding sites (Trastuzumab and Pertuzumab, which bind to HER2 at ECD2 and ECD4, respectively), and were ineffective. This surprising finding indicates that when two antigen-binding domains are too close to each other in the same arm of an antibody, such as one constructed through the TRIAD platform, HER2 monomers are brought closer to each other, enhancing HER2 dimer formation and thereby promoting HER2-dependent tumor growth.

[0148]

[0166] In contrast, in the engineered antibodies provided herein, in which the second and third domains, both targeting HER2, are located on different arms of the antibody, HER2 dimerization is significantly reduced compared to when they are located on the same arm, but binding affinity to HER2 is at least maintained or improved compared to HER2 monovalent Ig-like bispecific antibodies.

[0149]

[0167] In some embodiments, in the engineered antibody, one of the second and third domains is a first HER2-binding domain derived from the antigen-binding domain of Trastuzumab, and the other is a second HER2-binding domain derived from the antigen-binding domain of Pertuzumab. In other words, when the second domain is the first HER2-binding domain, the third domain is the second HER2-binding domain, or when the third domain is the first HER2-binding domain, the second domain is the second HER2-binding domain.

[0150]

[0168] In some embodiments, in the engineered antibody, VL2 and VH2 associate to form a first HER2-binding domain, and VL3 and VH3 associate to form a second HER2-binding domain, i.e., the second domain is the first HER2-binding domain, and the third domain is the second HER2-binding domain.

[0151]

[0169] In some embodiments, in the engineered antibody, VL2 and VH2 associate to form the second HER2-binding domain, and VL3 and VH3 associate to form the first HER2-binding domain, i.e., the second domain is the second HER2-binding domain, and the third domain is the first HER2-binding domain.

[0152]

[0170] In some embodiments, the second or third domain comprises the VL and VH of Trastuzumab, a known anti-HER2 antibody that recognizes and binds to HER2 through its ECD2. The amino acid sequences of the CDRs, VH, and VL of Trastuzumab are shown in Table 1.

[0153]

[0171] In some embodiments, the second or third domain comprises the VL and VH of Pertuzumab, a known anti-HER2 antibody that recognizes and binds to HER2 through its ECD4. The amino acid sequences of the CDRs, VH, and VL of Pertuzumab are shown in Table 1.

[0154]

[0172] In some embodiments, the first HER2 binding domain comprises an LCDR1 having the sequence set forth in SEQ ID NO: 13, an LCDR2 having the sequence set forth in SEQ ID NO: 14, and an LCDR3 having the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 97, and the VH2 comprises an HCDR1 having the sequence set forth in SEQ ID NO: 9, an HCDR2 having the sequence set forth in SEQ ID NO: 10, and an HCDR3 having the sequence set forth in SEQ ID NO: 11.

[0155]

[0173] In some embodiments, the second HER2 binding domain comprises an LCDR1 having the sequence set forth in SEQ ID NO: 21, an LCDR2 having the sequence set forth in SEQ ID NO: 22, and an LCDR3 having the sequence set forth in SEQ ID NO: 23, and the VH3 comprises an HCDR1 having the sequence set forth in SEQ ID NO: 17, an HCDR2 having the sequence set forth in SEQ ID NO: 18, and an HCDR3 having the sequence set forth in SEQ ID NO: 19.

[0156]

[0174] In some embodiments, the HER2-binding domain is modified to reduce affinity for HER2 compared to the unmodified domain. In some embodiments, the HER2-binding domain is modified to include H107A on the VL according to the IMGT numbering system. For further details on H107A, see, e.g., Dionysos Slaga, et al., 2018. Science Translational Medicine, 10 (463), incorporated herein in its entirety. In some embodiments, the low-affinity HER2-binding domain comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 13, an LCDR2 comprising the sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the sequence set forth in SEQ ID NO: 97, an HCDR1 comprising the sequence set forth in SEQ ID NO: 9, an HCDR2 comprising the sequence set forth in SEQ ID NO: 10, and an HCDR3 comprising the sequence set forth in SEQ ID NO: 11. In some embodiments, the reduced-affinity HER2-binding domain comprises a VL comprising the sequence set forth in SEQ ID NO: 95 and a VH comprising the sequence set forth in SEQ ID NO: 12.

[0157]

[0175] In some embodiments, the HER2-binding domain is modified to facilitate cognate pairing compared to the unmodified domain. In some embodiments, the HER2-binding domain is modified to include A45L in the VH and W87Y in the VL, each according to the Kabat numbering system. In some embodiments, the HER2-binding domain comprises an LCDR1 comprising the sequence set forth in SEQ ID NO:21, an LCDR2 comprising the sequence set forth in SEQ ID NO:22, an LCDR3 comprising the sequence set forth in SEQ ID NO:23, an HCDR1 comprising the sequence set forth in SEQ ID NO:17, an HCDR2 comprising the sequence set forth in SEQ ID NO:18, and an HCDR3 comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the HER2-binding domain comprises a VL comprising the sequence set forth in SEQ ID NO:107 and a VH comprising the sequence set forth in SEQ ID NO:106.

[0158]

[0176] [Table 1-1] [Table 1-2]

[0159]

[0177] In some embodiments, the first target is an immunostimulatory target that, when activated, exerts a stimulatory effect on the subject's immune system, such as to positively modulate cytokine secretion, NK cell activation, T cell proliferation, or antibody production.

[0160]

[0178] An important mechanism of bispecific antibodies is their ability to mediate killing by immune cells such as natural killer (NK) cells, T cells, and macrophages. In recent years, as our understanding of the mechanisms of cancer cell immune evasion has deepened, research into antibody drugs that activate immune cells, particularly T cells, macrophages, and NK cells, has gained attention. For example, anti-CD3 bispecific antibodies can bind to CD3 molecules on the surface of T cells while simultaneously binding to cancer cell surface antigens, thereby shortening the distance between cytotoxic T cells and cancer cells and directing T cells to kill cancer cells directly, eliminating the need for dual T cell activation signals. This type of bispecific antibody, which has an immune-stimulating target, is believed to have significant advantages in its mechanism of action.

[0161]

[0179] In some embodiments, the immunostimulatory target is associated with activation of a T cell, macrophage, or NK cell signaling pathway. Examples of immunostimulatory targets include, without limitation, CD3, CD16, NKG2D, CD28, CD137 (4-1BB), OX40, CD27, GITR, ICOS, and NKp46.

[0162]

[0180] In some embodiments, the immunostimulatory target is selected from NKG2D, CD3, and CD16.

[0163]

[0181] In some embodiments, the immunostimulatory target is NKG2D.

[0164]

[0182] "NKG2D" refers to an activating receptor (transmembrane protein) belonging to the NKG2 family of C-type lectin-like receptors. NKG2D recognizes induced self-proteins from the MIC and RAET1 / ULBP families that appear on the surface of stressed, malignant, and infected cells. NKG2D is the primary recognition receptor for detecting and eliminating malignantly transformed and infected cells because its ligands are induced during cellular stress, such as by genomic stress in infection or cancer. In NK cells, NKG2D functions as an activating receptor that induces cytotoxicity.

[0165]

[0183] In some embodiments, the antigen-binding domain of an anti-NKG2D antibody comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the sequence set forth in SEQ ID NO: 30, an LCDR3 comprising the sequence set forth in SEQ ID NO: 31, an HCDR1 comprising the sequence set forth in SEQ ID NO: 25, an HCDR2 comprising the sequence set forth in SEQ ID NO: 26, and an HCDR3 comprising the sequence set forth in SEQ ID NO: 27. In some embodiments, the antigen-binding domain of an anti-NKG2D antibody comprises a VL comprising the sequence set forth in SEQ ID NO: 32, and a VH comprising the sequence set forth in SEQ ID NO: 28. Exemplary anti-NKG2D antibodies have the sequences shown in Table 2.

[0166]

[0184] In some embodiments, the antigen-binding domain of an anti-NKG2D antibody comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 118, an LCDR2 comprising the sequence set forth in SEQ ID NO: 119, an LCDR3 comprising the sequence set forth in SEQ ID NO: 120, an HCDR1 comprising the sequence set forth in SEQ ID NO: 114, an HCDR2 comprising the sequence set forth in SEQ ID NO: 115, and an HCDR3 comprising the sequence set forth in SEQ ID NO: 116. In some embodiments, the antigen-binding domain of an anti-NKG2D antibody comprises a VL comprising the sequence set forth in SEQ ID NO: 121, and a VH comprising the sequence set forth in SEQ ID NO: 117. Exemplary anti-NKG2D antibodies have the sequences shown in Table 2.

[0167]

[0185] [Table 2]

[0168]

[0186] In some embodiments, the immunostimulatory target is CD3.

[0169]

[0187] "CD3," as used herein, refers to cytotoxic T cells (CD8 + Naive T cells) and T helper cells (CD4 +CD3 refers to the cluster of differentiation 3 (C3C), a protein complex and T cell coreceptor involved in the activation of both naive T cells and T cells. It is composed of four distinct chains. In mammals, the complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the T-cell receptor (TCR) and CD3-ζ (ζ-chain) to generate activation signals in T lymphocytes. The TCR, CD3-ζ, and other CD3 molecules collectively comprise the TCR complex. Because CD3 is required for T cell activation, drugs targeting it (often monoclonal antibodies) are being investigated as immunosuppressive therapies for cancer and other autoimmune diseases. Novel anticancer therapeutics are being developed based on the CD3 T cell coreceptor, with molecules designed to alter costimulatory signals to enable T cells to recognize cancer cells and fully activate them.

[0170]

[0188] In some embodiments, the antigen-binding domain of the anti-CD3 antibody comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 5, an LCDR2 comprising the sequence set forth in SEQ ID NO: 6, an LCDR3 comprising the sequence set forth in SEQ ID NO: 7, an HCDR1 comprising the sequence set forth in SEQ ID NO: 1, an HCDR2 comprising the sequence set forth in SEQ ID NO: 2, and an HCDR3 comprising the sequence set forth in SEQ ID NO: 3. In some embodiments, the antigen-binding domain of the anti-CD3 antibody comprises a VL comprising the sequence set forth in SEQ ID NO: 8 and a VH comprising the sequence set forth in SEQ ID NO: 4.

[0171]

[0189] In some embodiments, the antigen-binding domain of an anti-CD3 antibody is modified to reduce its affinity for CD3 compared to the unmodified domain. In some embodiments, the antigen-binding domain of an anti-CD3 antibody is modified to include an S113T residue in the VH according to the IMGT numbering system. For further details about S113T, see, for example, Dionysos Slaga, et al., 2018. Science Translational Medicine, 10 (463), incorporated herein in its entirety. In some embodiments, the low-affinity CD3-binding domain comprises an LCDR1 comprising the sequence set forth in SEQ ID NO:5, an LCDR2 comprising the sequence set forth in SEQ ID NO:6, an LCDR3 comprising the sequence set forth in SEQ ID NO:7, an HCDR1 comprising the sequence set forth in SEQ ID NO:1, an HCDR2 comprising the sequence set forth in SEQ ID NO:2, and an HCDR3 comprising the sequence set forth in SEQ ID NO:96. In some embodiments, the low-affinity CD3-binding domain comprises a VL comprising the sequence set forth in SEQ ID NO:8 and a VH comprising the sequence set forth in SEQ ID NO:124.

[0172]

[0190] Exemplary anti-CD3 antibodies have the sequences shown in Table 3.

[0173]

[0191] [Table 3]

[0174]

[0192] In some embodiments, the immunostimulatory target is CD16.

[0175]

[0193] CD16, also known as FcγRIII, is a class of differentiation molecule present on the surface of natural killer cells, neutrophils, monocytes, macrophages, and certain T cells. CD16, the best-studied membrane receptor involved in triggering lysis by NK cells, is a member of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC).

[0176]

[0194] CD16 is crucial for the early activation of natural killer (NK) cells after vaccination. In addition, CD16 mediates NK cell responses, potentially maintaining immune homeostasis through both T cell and antibody-dependent signaling pathways. In normal, healthy individuals, cross-linking of CD16 (FcγRIII) by immune complexes induces antibody-dependent cellular cytotoxicity (ADCC) in NK cells. This pathway can also be targeted in cancer or diseased cells by immunotherapy.

[0177]

[0195] In some embodiments, the antigen-binding domain of an anti-CD16 antibody comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 37, an LCDR2 comprising the sequence set forth in SEQ ID NO: 38, an LCDR3 comprising the sequence set forth in SEQ ID NO: 39, an HCDR1 comprising the sequence set forth in SEQ ID NO: 33, an HCDR2 comprising the sequence set forth in SEQ ID NO: 34, and an HCDR3 comprising the sequence set forth in SEQ ID NO: 35. In some embodiments, the antigen-binding domain of an anti-CD16 antibody comprises a VL comprising the sequence set forth in SEQ ID NO: 40, and a VH comprising the sequence set forth in SEQ ID NO: 36. Exemplary anti-CD16 antibodies have the sequences shown in Table 4.

[0178]

[0196] [Table 4]

[0179] B. Modification of Engineered Antibodies

[0197] The engineered antibodies provided herein may further comprise one or more modifications useful for enhancing binding between the corresponding heavy and light chains, e.g., between VH1 and VL1, or VH2 and VL2. In some embodiments, non-natural covalent bonds may be introduced and / or electrostatic interactions may be introduced at the VH1-VL1 interface or the VH2-VL2 interface.

[0180]

[0198] Disulfide bonds introduced into the VH-VL domain

[0199] In some embodiments, one of the first domain (formed by the association of VH1 and VL1) and the second domain (formed by the association of VH2 and VL2) comprises a first non-naturally occurring covalent bond.

[0181]

[0200] In some of these embodiments, the other of the first domain and the second domain does not comprise a non-naturally occurring covalent bond. Alternatively, the other of the first domain and the second domain comprises a second non-naturally occurring covalent bond that is different from the first non-naturally occurring covalent bond. For example, the second non-naturally occurring covalent bond is formed between a pair of two amino acid residues that are different from the two amino acid residues that form the first non-naturally occurring covalent bond, or is formed between amino acid residues at a pair of positions that are different from the first non-naturally occurring covalent bond.

[0182]

[0201] In some embodiments, the first non-natural covalent bond can be a non-natural disulfide bond. In some embodiments, the first non-natural disulfide bond is formed between two introduced cysteine ​​residues. In such embodiments, at least one of the first and second domains is a disulfide-stabilized Fv. Analysis of antibody crystal structures revealed that cysteine ​​mutations can be introduced into a portion of a relatively conserved sequence at the VL-VH interface to form a disulfide bond between the VL and VH, thereby covalently connecting them. The covalent bond between the VL and VH significantly improved the stability of the antibody. The first disulfide Fvs (dsFvs) were constructed by introducing disulfide bonds at the VH-VL interface through covalent interactions between cysteine ​​residues in the CDRs of each fragment (see Glockshuber, R., Malia, M., Pfitzinger, I., and Pluckthun, A. A comparison of strategies to stabilize immunoglobulin Fv-fragments. (1990) Biochemistry, 29, 1362-1367). While this approach did not affect antibody activity, detailed structural information about the CDRs of the original antibody is required for "customized" design to avoid interference with the antigen recognition / binding ability of the CDRs, making this approach difficult to use as a universal solution for constructing various antibodies. To ensure the widespread application of this approach, it is important that only amino acids at selected sites in the conserved FRs are involved in the construction of dsFvs.

[0183]

[0202] Since 1993, several pairing sites for VH-VL covalent bond formation have been discovered, such as VH44-VL100, VH105-VL43, VH100b-VL49, VH100-VL150, and vH101-vL46 (Reiter, Y., Brinkmann, RJ, Kreitman, RJ, et al. Stabilization of the Fv Fragments in Recombinant Immunotoxins by Disulfide Bonds Engineered Into Conserved Framework Regions. (1994) Biochemistry, 33, 5451-5459; Jung, SH, Pastan, I. and Lee, B. Design of interchain disulfide bonds in the framework region of the Fv fragment of the monoclonal antibody B3. (1994) Proteins, Struc. Func. Genet., 19, 35-47; Glockshuber, (See R., Malia, M., Pfitzinger, I. and Plückthun, A. A comparison of strategies to stabilize immunoglobulin Fv-fragments. (1990) Biochemistry, 29, 1362-1367, and Zhu, Z., Presta, LG, Zapata, G. and Carter, P. Remodeling domain interfaces to enhance heterodimer formation. (1997) Prot. Sci., 6, 781-788.) Among them, VH44-VL100 and VH105-VL43 are more widely used because they are superior to the others in many aspects, such as protein expression level, monolayer rate, Tm, and affinity, although to different degrees.

[0184]

[0203] In some embodiments, the VL2 and VH2 of the engineered antibody are associated with a first non-native disulfide bond. In some embodiments, the first non-native disulfide bond is formed between two non-native cysteine ​​residues in VL2 and VH2, respectively. In some embodiments, the two non-native cysteine ​​residues are located in the framework regions (FR) of VL2 and the FR of VH2, respectively. In some embodiments, the two non-native cysteine ​​residues are located at amino acid residues in FR2 of VL2 and FR4 of VH2, respectively.

[0185]

[0204] In some embodiments, the two non-native cysteine ​​residues are at position 44 in VH2 and position 100 in VL2, or at position 105 in VH2 and position 43 in VL2, or at position 100 in VH2 and position 49 in VL2, or at position 100 in VH2 and position 150 in VL2. Unless otherwise specified, all position numbering in this disclosure is according to the Kabat index. In some embodiments, the non-native covalent bond is formed between the amino acid residues introduced at position 44 in VH2 and position 100 in VL2, or the cysteine ​​residues at position 105 in VH2 and position 43 in VL2. In some embodiments, the non-native covalent bond is formed between the cysteine ​​residues at position 44 in VH2 and position 100 in VL2. In some embodiments, the two non-native cysteine ​​residues are 100C in VL2 and 44C in VH2.

[0186]

[0205] In some embodiments, the VL1 and VH1 of the engineered antibody do not contain any introduced non-native disulfide bonds.

[0187]

[0206] In some other embodiments, the VL1 and VH1 of the engineered antibody are associated with a first non-native disulfide bond. In some embodiments, the first non-native disulfide bond is formed between two non-native cysteine ​​residues in VL1 and VH1, respectively. In some embodiments, the two non-native cysteine ​​residues are in the framework regions (FR) of VL1 and the FR of VH1, respectively. In some embodiments, the two non-native cysteine ​​residues are at amino acid residues in FR2 of VL1 and FR4 of VH1, respectively. In some embodiments, the two non-native cysteine ​​residues are at positions 44 in VH1 and 100 in VL1, or at positions 105 in VH1 and 43 in VL1, or at positions 100 in VH1 and 49 in VL1, or at positions 100 in VH1 and 150 in VL1. Unless otherwise specified, all position numbering in this disclosure is according to the Kabat index. In some embodiments, the non-naturally occurring covalent bond is formed between the introduced amino acid residues at position 44 in VH1 and position 100 in VL1, or between the cysteine ​​residues at position 105 in VH1 and position 43 in VL1. In some embodiments, the non-naturally occurring covalent bond is formed between the cysteine ​​residues at position 44 in VH1 and position 100 in VL1. In some embodiments, the two non-naturally occurring cysteine ​​residues are 100C in VL1 and 44C in VH1. In some of these embodiments, the VL2 and VH2 of the engineered antibody do not comprise any introduced non-naturally occurring disulfide bond.

[0188]

[0207] Substitution with charged amino acids

[0208] The engineered antibodies provided herein are modified to promote or support pairing between VH1 and VL1 or VH2 and VL2 by introducing electrostatic interactions into the first domain (formed by association of VH1 and VL1) or the second domain (formed by association of VH2 and VL2).

[0189]

[0209] In some embodiments, VL2 and VH2 are modified to introduce two oppositely charged residues that enhance the electrostatic interaction between VL2 and VH2, and in some of these embodiments, VL1 and VH1 are free of such oppositely charged residues.

[0190]

[0210] In some embodiments, VL1 and VH1 are modified to introduce two oppositely charged residues that enhance the electrostatic interaction between VL1 and VH1, and in some of these embodiments, VL2 and VH2 are free of such oppositely charged residues.

[0191]

[0211] In some embodiments, the engineered antibodies provided herein are modified to introduce a first pair of two oppositely charged residues that enhance the electrostatic interaction between VL2 and VH2, and a second pair of two oppositely charged residues that enhance the electrostatic interaction between VL1 and VH1, provided that pairing between VH1 and VL2 and VH2 and VL1 is prevented, e.g., by electrostatic repulsion. For example, the introduced charged residues in VL2 and VH1 are similarly charged residues and / or the introduced charged residues in VH2 and VL1 are similarly charged residues, thereby preventing mispairing between VL2 and VH1 or VL1 and VH2.

[0192]

[0212] In some embodiments, the two oppositely charged residues consist of a positively charged residue and a negatively charged residue.

[0193]

[0213] In certain embodiments, the first pair of two oppositely charged residues comprises a negatively charged residue in VL2 and a positively charged residue in VH2, hi certain embodiments, the second pair of two oppositely charged residues comprises a positively charged residue in VL1 and a negatively charged residue in VH1.

[0194]

[0214] In certain embodiments, the first pair of two oppositely charged residues comprises a positively charged residue in VL2 and a negatively charged residue in VH2, hi certain embodiments, the second pair of two oppositely charged residues comprises a negatively charged residue in VL1 and a positively charged residue in VH1.

[0195]

[0215] In some embodiments, the negatively charged amino acid is aspartic acid (D) or glutamic acid (E). In some embodiments, the positively charged amino acid is lysine (K), histidine (H), or arginine (R).

[0196]

[0216] Strategies for enhancing the stable binding of corresponding VH and VL in bispecific antibodies by introducing charged amino acids are known in the art. Tan et al. successfully influenced the stability of scFv (single-chain FV variant) by adjusting amino acids at the VH-VL interface based on electrostatic properties (see Philip H. Tan, Brenda M. Sandmaier, Patrick S. Stayton. Contributions of a Highly Conserved VH VL Hydrogen Bonding Interaction to scFv Folding Stability and Refolding Efficiency. Biophys J. 1998 Sep; 75(3): 1473-1482). Subsequently, Igawa et al. adapted this method to modify scDb. To improve the homogeneity of the product, two pairs of Q39-Q38 in the 4V fragment were replaced with amino acids bearing appropriate electrostatic charges to promote or inhibit specific isoforms (see Igawa T, Tsunoda H, Kikuchi Y, et al. VH / VL interface engineering to promote selective expression and inhibit conformational isomerization of thrombopoietin receptor agonist single-chain diabody. Protein Eng Des Sel. 2010 Aug;23(8):667-77 and WO2006106905A1). Gunasekaran et al. at Amgen further investigated this method and incorporated it into the modification of antibody Fab arms.Modification of the electrostatic steering at the CH1-CL interface, combined with modification of the VH-VL at positions 38-39, promoted specific interactions between CH1-VH and CL-VL (see Gunasekaran K, Pentony M, Shen M, et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J Biol Chem. 2010 Jun 18;285(25):19637-46 and Liu Z, Leng EC2, Gunasekaran K3, et al. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism. J Biol Chem. 2015 Mar 20;290(12):7535-62). These strategies enable each HC of the bispecific antibody to interact with the corresponding LC, thereby enabling the bispecific antibody to simultaneously bind to two antigens.

[0197]

[0217] In some embodiments, the engineered antibodies provided herein are modified with electrostatic steering in selected regions in addition to the introduction of non-native disulfide bonds, thereby successfully minimizing unwanted non-specific interactions. Modifications to introduce electrostatic interactions can improve the pharmacokinetic properties of the engineered antibodies, helping to remove bottlenecks in the downstream development process and increasing the likelihood of successful development of bispecific antibodies.

[0198]

[0218] W103 of VH and P44 of VL are both side chains of the hydrophobic core and are positioned in close proximity. The electrostatic interaction between W103 and P44 was also examined during the development of DICAD and was found to be excellent.

[0199]

[0219] In some embodiments, the introduced charged residues in VL1 or VL2 are in a FR (e.g., FR2). In some embodiments, the introduced charged residues in VH1 or VH2 are in a FR (e.g., FR2).

[0200]

[0220] In some embodiments, a first pair of two oppositely charged residues is introduced to replace Q38 in VL2 and Q39 in VH2, respectively, where numbering is according to the Kabat index. In some embodiments, a second pair of two oppositely charged residues is introduced to replace Q38 in VL1 and Q39 in VH1, respectively, where numbering is according to the Kabat index. In such embodiments, the introduced charged residues in VL2 and VH1 are similarly charged residues and / or the introduced charged residues in VH2 and VL1 are similarly charged residues, thereby preventing mispairing between VL2 and VH1 or VL1 and VH2.

[0201]

[0221] In some embodiments, the first pair of two oppositely charged residues comprises Q38D in VL2 and Q39K in VH2, respectively, where numbering is according to the Kabat index, hi some embodiments, the second pair of two oppositely charged residues comprises Q39D in VH1 and Q40K in VL1, respectively, where numbering is according to the Kabat index.

[0202]

[0222] The introduction of positively or negatively charged amino acids into antibodies is known in the art.

[0203]

[0223] In some embodiments, in addition to the disulfide bonds introduced into VH2 and VL2, charged amino acids are introduced into VH2 and VL2.

[0204]

[0224] In some embodiments, VH2 and VL2 are derived from Trastuzumab. In some embodiments, VH2 and VL2 are derived from low-affinity Tratuzumab. In some embodiments, cysteine ​​residues are introduced at position 100 in VL2 and position 44 in VH2 to form a non-native disulfide bond, and two oppositely charged residues are simultaneously introduced to replace Q38 in VL2 and Q39 in VH2, respectively, where numbering is according to the Kabat index. In some embodiments, VH2 comprises the mutations Q38D and G44C, and VL2 comprises the mutations Q39D and Q100C. In some embodiments, VH2 comprises the sequence set forth in SEQ ID NO:90, and VL2 comprises the sequence set forth in SEQ ID NO:91. In some embodiments, VH2 and VL2 are derived from low-affinity Tratuzumab. In such embodiments, VH2 comprises the sequence set forth in SEQ ID NO:90, and VL2 comprises the sequence set forth in SEQ ID NO:98.

[0205]

[0225] In some embodiments, VH3 and VL3 do not contain any introduced non-native disulfide bonds or two pairs of oppositely charged amino acid residues.

[0206]

[0226] In some embodiments, VH3 and VL3 are derived from Pertuzumab. In some embodiments, VH3 comprises the sequence set forth in SEQ ID NO:20 and VL3 comprises the sequence set forth in SEQ ID NO:24. In some embodiments, VH3 comprises the sequence set forth in SEQ ID NO:106 and VL3 comprises the sequence set forth in SEQ ID NO:107.

[0207]

[0227] In some embodiments, VH3 and VL3 are derived from Trastuzumab. In some embodiments, VH3 comprises the sequence set forth in SEQ ID NO: 12, and VL3 comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, VH3 and VL3 are derived from low-affinity Trastuzumab. In some embodiments, VH3 comprises the sequence set forth in SEQ ID NO: 12, and VL3 comprises the sequence set forth in SEQ ID NO: 95.

[0208]

[0228] In some embodiments, VH2 and VL2 are derived from Pertuzumab. In some embodiments, cysteine ​​residues are introduced at position 100 in VL2 and position 44 in VH2 to form a non-native disulfide bond, and simultaneously, two oppositely charged residues are introduced to replace Q38 in VL2 and Q39 in VH2, respectively, where numbering is according to the Kabat index. In some embodiments, VH2 comprises the sequence set forth in SEQ ID NO:92, and VL2 comprises the sequence set forth in SEQ ID NO:93.

[0209]

[0229] In some embodiments, VH3 and VL3 do not contain any introduced non-native disulfide bonds or two pairs of oppositely charged amino acid residues. In some embodiments, VH3 and VL3 are derived from Tratuzumab. In some embodiments, VH3 comprises the sequence set forth in SEQ ID NO: 12, and VL3 comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, VH3 and VL3 are derived from low-affinity Trastuzumab. In some embodiments, VH3 comprises the sequence set forth in SEQ ID NO: 12, and VL3 comprises the sequence set forth in SEQ ID NO: 95.

[0210]

[0230] In some embodiments, the VH1 and VL1 of the engineered antibody do not comprise any introduced non-natural disulfide bond or do not comprise two pairs of oppositely charged amino acid residues. In some embodiments, the VH1 and VL1 of the engineered antibody comprise two introduced pairs of oppositely charged amino acid residues but do not comprise any introduced non-natural disulfide bond. In some embodiments, the VH1 and VL1 also comprise an introduced non-natural disulfide bond and an introduced pair of two oppositely charged amino acid residues.

[0211]

[0231] In some embodiments, VH1 and VL1 are derived from an antigen-binding fragment that targets NKG2D, CD3, or CD16.

[0212]

[0232] In some embodiments, VH1 and VL1 are derived from the variable regions in exemplary anti-CD3 antibodies shown in Table 3. In some other embodiments, VH1 and VL1 comprise an introduced pair of two oppositely charged amino acid residues. In some embodiments, the two oppositely charged residues are introduced to replace Q40 in VL1 and Q39 in VH1, respectively. In some embodiments, VL1 comprises the mutation Q40K and VH1 comprises the mutation Q39D. In some embodiments, VH1 comprises the sequence set forth in SEQ ID NO:99 and VL1 comprises the sequence set forth in SEQ ID NO:100. In some embodiments, the anti-CD3 antibody has low affinity. In some embodiments, VH1 comprises the sequence set forth in SEQ ID NO:105 and VL1 comprises the sequence set forth in SEQ ID NO:100.

[0213]

[0233] In some embodiments, VH1 and VL1 are derived from the variable regions in exemplary anti-CD16 antibodies shown in Table 4. In some other embodiments, VH1 and VL1 comprise an introduced pair of two oppositely charged amino acid residues. In some embodiments, the two oppositely charged residues are introduced to replace Q37 in VL1 and Q39 in VH1, respectively. In some embodiments, VL1 comprises the mutation Q37K and VH1 comprises the mutation Q39D. In some embodiments, VH1 comprises the sequence set forth in SEQ ID NO:108 and VL1 comprises the sequence set forth in SEQ ID NO:109.

[0214]

[0234] In some embodiments, VH1 and VL1 are derived from the variable regions in exemplary anti-NKG2D antibodies shown in Table 2. In some other embodiments, VH1 and VL1 comprise an introduced pair of two oppositely charged amino acid residues. In some embodiments, the two oppositely charged residues are introduced to replace Q37 in VL1 and Q39 in VH1, respectively. In some embodiments, VL1 comprises the mutation Q37K and VH1 comprises the mutation Q39D. In some embodiments, VH1 comprises the sequence set forth in SEQ ID NO: 122 and VL1 comprises the sequence set forth in SEQ ID NO: 123.

[0215]

[0235] Constant region modifications

[0236] In some embodiments, in the engineered antibodies provided herein, each of the second and third polypeptide chains further comprises a first dimerization domain and a second dimerization domain, respectively, that associate to form a dimer.

[0216]

[0237] In some embodiments, the first dimerization domain and the second dimerization domain comprise an IgG CH3 domain, hi some embodiments, the first dimerization domain and the second dimerization domain further comprise a hinge region.

[0217]

[0238] In some embodiments, the second and third polypeptides of the engineered antibody are covalently linked via a hinge region to form a knobs-in-holes structure.

[0218]

[0239] The knobs-in-holes architecture, also known as the "protuberance-into-cavity" strategy, serves to engineer the interface between a first and second polypeptide for hetero-oligomerization.

[0219]

[0240] Generally, the preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. "Protuberances" are constructed by replacing small amino acid side chains from the interface of a first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory "voids" of identical or similar size to the protuberances are optionally created in the interface of a second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Once a protuberance or void with a suitable location and dimensions exists in the interface of a first or second polypeptide, it is sufficient to engineer a corresponding void or protuberance, respectively, in the adjacent interface. See U.S. Pat. No. 8,216,805, the entire disclosure of which is incorporated herein by reference.

[0220]

[0241] In some embodiments, the first dimerization domain and the second dimerization domain of the engineered antibody further comprise one or more mutations that promote heterodimerization, hi some embodiments, the first dimerization domain comprises a first mutation and the second dimerization domain comprises a second mutation.

[0221]

[0242] In some embodiments, (a) the first mutation comprises T389W and / or S375C and the second mutation comprises Y438V, T389S, L391A, and / or Y370C; (b) the first mutation comprises D427K and / or D377K and the second mutation comprises K420D and / or K440D; (c) the first mutation comprises D377K, E378K, and / or D427K and the second mutation comprises K393E, K440D, and / or K470E; (d) the first mutation comprises S387H and / or (e) the first mutation comprises S387H and / or T422F and the second mutation comprises Y422T and / or F436A; (f) the first mutation comprises K393D and / or K440D and the second mutation comprises E378K and / or D427K; or (g) the first mutation comprises L372D and / or L391E and the second mutation comprises L372K or T389K, wherein numbering is according to the Kabat index.

[0222]

[0243] In some embodiments, the first mutation comprises T389W and the second domain comprises T389S, L391A and Y438V, where numbering is according to the Kabat index. In some embodiments, one of the first dimerization domain and the second dimerization domain that comprises the first mutation comprises the sequence set forth in SEQ ID NO:44, and the other of the first dimerization domain and the second dimerization domain that comprises the second mutation comprises the sequence set forth in SEQ ID NO:45.

[0223]

[0244] In some embodiments, one of the first dimerization domain and the second dimerization domain comprises the sequence set forth in SEQ ID NO: 103, and the first dimerization domain and the second dimerization domain comprise the sequence set forth in SEQ ID NO: 104.

[0224]

[0245] In some embodiments, the first dimerization domain and / or the second dimerization domain further comprise an IgG CH2 domain and / or hinge region. In some embodiments, the hinge region comprises the sequence set forth in SEQ ID NO: 43, 101, or 102.

[0225]

[0246] In some embodiments, the third polypeptide of an engineered antibody disclosed herein further comprises a CH1 region whose N-terminus is linked to the C-terminus of the VH3 of the engineered antibody. In some embodiments, the CH1 region comprises the sequence set forth in SEQ ID NO:41.

[0226]

[0247] In some embodiments, the fourth polypeptide of an engineered antibody disclosed herein further comprises a CL region whose N-terminus is linked to the C-terminus of the VL3 of the engineered antibody. In some embodiments, the CL region comprises the sequence set forth in SEQ ID NO:42.

[0227]

[0248] In some embodiments, the engineered antibodies provided herein are humanized. In some embodiments, the engineered antibodies provided herein are monoclonal antibodies, chimeric antibodies, or labeled antibodies.

[0228] C. Exemplary Engineered Antibodies

[0249] In some embodiments, the engineered antibodies disclosed herein comprise four polypeptide chains having the following structure from N-terminus to C-terminus (see also Figure 2):

[0250] Chain 1: VL2 (HER2)-linker-VH1 (immunostimulatory target)

[0251] Chain 2: VL1 (immunostimulatory target)-linker-VH2 (HER2)-hinge-CH2-CH3

[0252] Strand 3: VH3(HER2)-CH1-hinge-CH2-CH3

[0253] Chain 4: VL3(HER2)-CL

[0229]

[0254] NKG2D×HER2 multispecific antibody

[0255] In certain embodiments, the present disclosure provides particular engineered antibodies that target NKG2D (a first target) and HER2 (a second target). In certain embodiments, such engineered antibodies are characterized in that VL1 and VH1 associate to form a first domain capable of binding to NKG2D, VL2 and VH2 associate to form a first HER2-binding domain, and VL3 and VH3 associate to form a second HER2-binding domain.

[0230]

[0256] In a specific embodiment, the first domain is derived from the binding domain of an anti-NKG2D antibody, the second domain is derived from Trastuzumab, and the third domain is derived from Pertuzumab.

[0231]

[0257] In certain embodiments, the engineered antibody comprises mutations in the variable region or Fc region listed in Table 5.

[0232]

[0258] [Table 5-1] [Table 5-2] [Table 5-3]

[0233]

[0259] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 50, 51, 52, and 53, respectively (also referred to herein as antibody 6A12).

[0234]

[0260] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 54, 55, 56, and 57, respectively (also referred to herein as antibody 6A15).

[0235]

[0261] In certain embodiments, the engineered antibody comprises four polypeptide chains, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 58, 59, 60, and 61, respectively (also referred to herein as antibody 6A17).

[0236]

[0262] CD3×HER2 multispecific antibody

[0263] In certain embodiments, the present disclosure provides specific engineered antibodies that target CD3 (a first target) and HER2 (a second target). In certain embodiments, such engineered antibodies are characterized in that VL1 and VH1 associate to form a first domain capable of binding to CD3, VL2 and VH2 associate to form a second HER2-binding domain, and VL3 and VH3 associate to form a first HER2-binding domain.

[0237]

[0264] In a specific embodiment, the first domain is derived from the binding domain of an anti-CD3 antibody, the second domain is derived from Pertuzumab, and the third domain is derived from Trastuzumab.

[0238]

[0265] In a specific embodiment, the first domain is derived from the binding domain of an anti-CD3 antibody, the second domain is derived from Trastuzumab, and the third domain is derived from Pertuzumab.

[0239] In a particular embodiment, the first domain is derived from the binding domain of an anti-CD3 antibody, and the second and third domains are both derived from Trastuzumab.

[0240]

[0266] In a specific embodiment, the first domain is derived from the binding domain of an anti-CD3 antibody, and the second and third domains are both derived from the low affinity Trastuzumab.

[0241]

[0267] In certain embodiments, the engineered antibody comprises mutations in the variable region or Fc region listed in Table 6.

[0242]

[0268] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0243]

[0269] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 46, 47, 48, and 49, respectively (also referred to herein as antibody 6A8).

[0244]

[0270] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 82, 47, 48, and 49, respectively (also referred to herein as antibody 6A14).

[0245]

[0271] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 62, 63, 64, and 65, respectively (also referred to herein as antibody 6A19).

[0246]

[0272] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 78, 79, 80, and 81, respectively (also referred to herein as antibody 6A25).

[0247]

[0273] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 82, 83, 84, and 85, respectively (also referred to herein as antibody 6A26).

[0248]

[0274] CD16×HER2 multispecific antibody

[0275] In certain embodiments, the present disclosure provides specific engineered antibodies that target CD16 (a first target) and HER2 (a second target). In certain embodiments, such engineered antibodies are characterized in that VL1 and VH1 associate to form a first domain capable of binding to CD16, VL2 and VH2 associate to form a second HER2-binding domain, and VL3 and VH3 associate to form a first HER2-binding domain.

[0249]

[0276] In a specific embodiment, the first domain is derived from the binding domain of an anti-CD16 antibody, the second domain is derived from Pertuzumab, and the third domain is derived from Trastuzumab.

[0250]

[0277] In a specific embodiment, the first domain is derived from the binding domain of an anti-CD16 antibody, the second domain is derived from Trastuzumab, and the third domain is derived from Pertuzumab.

[0251]

[0278] In certain embodiments, the engineered antibody comprises mutations in the variable region or Fc region listed in Table 7. In certain embodiments, the engineered antibody comprises a sequence listed in Table 7.

[0252]

[0279] [Table 7-1] [Table 7-2]

[0253]

[0280] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 66, 67, 68, and 69, respectively (also referred to herein as antibody 6A18).

[0254]

[0281] In certain embodiments, the engineered antibody comprises four polypeptide chains, namely, chain 1, chain 2, chain 3, and chain 4, which comprise the amino acid sequences of SEQ ID NOs: 74, 75, 76, and 77, respectively (also referred to herein as antibody 6A23).

[0255]

[0282] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12] [Table 8-13] [Table 8-14] [Table 8-15]

[0256] D antibody variants

[0283] The engineered antibodies provided herein also encompass various variants of the antibody sequences provided herein.

[0257]

[0284] In certain embodiments, antibody variants comprise one or more modifications or substitutions in one or more of the CDR regions, non-CDR regions, and / or constant regions (e.g., Fc regions) provided in Figure 10 above. Such variants retain the binding specificity of the parent antibody to its corresponding target, but also have one or more desirable properties conferred by the modification or substitution. For example, antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or eliminated effector function, improved FcRn receptor binding, and increased pharmacokinetic half-life, pH sensitivity, and / or conjugation suitability (e.g., one or more introduced cysteine ​​residues).

[0258]

[0285] The parent antibody sequence may be screened by methods known in the art, such as "alanine systematic mutagenesis," to identify suitable or preferred residues to modify or replace (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) may be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibodies are produced and screened for properties of interest. If substitution at a particular amino acid position exhibits a functional change of interest, this position can be identified as a potential residue for modification or replacement. Potential residues may be further evaluated by substituting them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0259] Affinity variants

[0286] Antibody affinity variants may contain modifications or substitutions in one or more CDR or FR regions in the heavy or light chain variable region sequences provided in Tables 1 to 4 above. Because it is known in the art that a CDR region is flanked by two FR regions in the variable region, those skilled in the art can easily identify FR sequences based on the CDR and variable region sequences in Tables 1 to 4 above. Affinity variants retain the specific binding affinity of the parent antibody to a target, and thus have target-specific binding affinity that exceeds that of the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR, FR, or variable region sequences comprises a conservative substitution.

[0260]

[0287] Those skilled in the art will understand that one or more amino acid residues may be substituted in the CDR and variable region sequences provided in Tables 1-4 above, while the resulting antibody or antigen-binding fragment still retains its binding affinity or capacity to the target, and thus may have improved binding affinity or capacity. Various methods known in the art can be used to achieve this goal. For example, phage display technology can be used to generate and express a library of antibody variants (e.g., Fab or scFv variants), which can then be screened for binding affinity to the target. As another example, computer software can be used to virtually simulate the binding of an antibody to a target and identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided for substitution to prevent a decrease in binding affinity, or may be targeted for substitution to provide stronger binding.

[0261]

[0288] In certain embodiments, the engineered antibody comprises one or more amino acid residue substitutions in one or more of the CDR sequences and / or one or more of the FR sequences, hi certain embodiments, the affinity variant comprises a total of no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR and / or FR sequences.

[0262]

[0289] In certain embodiments, the engineered antibodies comprise one, two, or three CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to those listed in Tables 1-4 above, but retain a similar level of specific binding affinity to the corresponding target as the parent antibody, and even a higher level.

[0263]

[0290] In certain embodiments, the engineered antibody comprises one or more variable region sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to those listed in Tables 1-4 above, but retain a similar, and even higher, level of specific binding affinity for the corresponding target as the parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Tables 1-4 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (e.g., FRs).

[0264] Glycosylation variants

[0291] The engineered antibodies provided herein also encompass glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibody.

[0265]

[0292] Engineered antibodies may contain one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, such as an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine. Removal of native glycosylation sites can be conveniently achieved, for example, by altering the amino acid sequence so that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence is substituted. Similarly, new glycosylation sites can be created by introducing such tripeptide sequences or serine or threonine residues.

[0266]

[0293] In certain embodiments, the engineered antibodies provided herein comprise a mutation at N297 (eg, N297A, N297Q, or N297G) to remove a glycosylation site.

[0267] E conjugate

[0294] The engineered antibodies provided herein can be used in unconjugated or conjugated form.

[0268]

[0295] In the conjugated form, the engineered antibody is conjugated to one or more desired conjugating moieties, i.e., heterologous moieties, to perform a specific function, for example, to facilitate target detection, or for imaging or therapy.

[0269]

[0296] Here, the present disclosure provides a conjugate comprising an engineered antibody provided herein and a conjugate moiety (e.g., a payload) conjugated thereto, wherein the payload can be any of the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anti-cancer drug, and a cytotoxic molecule.

[0270]

[0297] A variety of conjugates can be linked to the engineered antibodies provided herein by covalent bonding, affinity binding, intercalation, coordinate binding, complex formation, association, blending, or addition (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)).

[0271]

[0298] In certain embodiments, the engineered antibodies provided herein may be engineered to contain specific sites outside the epitope-binding moiety that can be specifically utilized for binding to one or more conjugates. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent linkage to a conjugate.

[0272]

[0299] In certain embodiments, the N-terminus and / or C-terminus of the engineered antibodies provided herein can also function to provide reactive groups for conjugation, for example, the N-terminus is conjugated to one moiety (such as polyethylene glycol (PEG)) and the C-terminus is conjugated to another moiety (such as biotin).

[0273]

[0300] In certain embodiments, the engineered antibodies provided herein may be directly linked to a conjugate, or may be indirectly linked, for example, via another conjugate or linker.

[0274]

[0301] For example, the engineered antibodies provided herein can be linked to thiol-reactive agents in which the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation partners (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0275]

[0302] As another example, the engineered antibodies provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin. As yet another example, the engineered antibodies may be linked to a linker that is further linked to a conjugate. Examples of linkers include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), and bifunctional coupling agents such as bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide disulfide linkages.

[0276]

[0303] In certain embodiments, the conjugate moiety comprises an agent for detection or isolation, such as a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer agent.

[0277]

[0304] The conjugate moiety can be a detectable label, a pharmacokinetic-modifying moiety, a purification moiety, a cytotoxic moiety, or a therapeutic agent. Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides, luminescent labels), chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0278]

[0305] In certain embodiments, the conjugate moiety may be a pharmacokinetic-modifying moiety such as PEG, which serves to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In certain embodiments, the conjugate may be a purification moiety such as a magnetic bead.

[0279]

[0306] In certain embodiments, the conjugate moiety can be a cytotoxic moiety. A "cytotoxic moiety" can be any agent that is harmful to cells or can damage or kill cells. Examples of cytotoxic moieties include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkyl benzoates, and the like. These include, without limitation, nitrating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). In some embodiments, the conjugate moiety comprises an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Jatropha forsi protein, dianthin protein, Phytolacca americana protein, bitter melon inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0280]

[0307] Therapeutic agents or drugs useful as conjugate moieties can be those useful for treating a disease or disorder associated with HER2.

[0281]

[0308] Methods for conjugating conjugate moieties to proteins such as antibodies, immunoglobulins or fragments thereof are described, for example, in U.S. Pat. No. 5,208,020, U.S. Pat. No. 6,4411,163, WO2005037992, WO2005081711, and WO2006 / 034488, which are incorporated by reference herein in their entireties.

[0282]

[0309] In certain embodiments, the engineered antibodies provided herein are used as the basis for a conjugate.

[0283] F. Polynucleotides and Recombinant Methods

[0310] The present disclosure provides isolated polynucleotides encoding the engineered antibodies provided herein. Unless otherwise indicated, a particular polynucleotide sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly set forth sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0284]

[0311] Polynucleotides encoding the engineered antibodies disclosed herein can be generated by methods known in the art. In certain embodiments, the sequence of the polynucleotide can be obtained based on the amino acid sequence of the engineered antibody, or the nucleic acid can be generated by synthetic methods. Alternatively, the polynucleotides provided herein can be obtained from another available nucleic acid encoding a polypeptide having a sequence homologous to a polypeptide in the engineered antibodies disclosed herein. DNA manipulation processes can then be applied to manipulate the sequence of the nucleic acid encoding the parent antibody to introduce, for example, mutations, insertions, deletions, etc., to obtain nucleic acids encoding the engineered antibodies disclosed herein.

[0285]

[0312] The isolated polynucleotides encoding the engineered antibodies may be inserted into one or more vectors for further cloning (amplification of the DNA) or for expression by recombinant techniques known in the art. Multiple vectors are available. The vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), a transcription termination sequence, and one or more other regulatory elements.

[0286]

[0313] The present disclosure provides vectors comprising the isolated polynucleotides provided herein. In certain embodiments, the polynucleotides provided herein encode engineered antibodies having at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence and at least one selectable marker. Exemplary vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, as well as the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, and pBAD. , pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0287]

[0314] Vectors containing polynucleotide sequences encoding engineered antibodies may be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, e.g., Bacillus subtilis and B. licheniformis, Pseudomonas, e.g., Pseudomonas aeruginosa, and Streptomyces.

[0288]

[0315] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for engineered antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are generally available and useful herein, including Kluyveromyces hosts such as Schizosaccharomyces pombe, e.g., Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces wartii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus, Yarrowia spp. (EP 402,226), Pichia pastoris (EP 183,070), Candida spp., Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces species such as Schwanniomyces occidentalis, and filamentous fungi such as Neurospora crassa, Penicillium, and Tolypocladium, as well as Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger.

[0289]

[0316] Suitable host cells for expressing the glycosylated antibodies provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as the armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of P. truncatula NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used in accordance with the present invention, particularly for transfection of armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0290]

[0317] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, and a human hepatocellular carcinoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line such as CHO, BHK, NS0, 293, and their derivatives.

[0291]

[0318] Host cells are transformed with the above-described antibody-producing expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies may be produced by homologous recombination, as known in the art. In certain embodiments, host cells are capable of producing the antibodies provided herein.

[0292]

[0319] The present disclosure also provides a method of expressing an antibody provided herein, the method comprising culturing a host cell provided herein under conditions in which a vector of the present disclosure is expressed. The host cells used to produce the antibodies provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO90 / 03430, WO87 / 00195, or U.S. Pat. No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0293]

[0320] When using recombinant techniques, antibodies may be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate cell debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed for approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0294]

[0321] The engineered antibody prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being preferred.

[0295]

[0322] In certain embodiments, solid-phase-immobilized protein A is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, Bakerbond ABX® resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the antibody recovered, other protein purification techniques can be used, such as fractional ethanol precipitation on an ion exchange column, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0296]

[0323] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer of about pH 2.5 to 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0297] III. Pharmaceutical Preparation and Administration

[0324] The present disclosure also provides pharmaceutical compositions. In addition to the polypeptide conjugates described above, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier.

[0298]

[0325] As used herein, the term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, salt, and / or vehicle is generally chemically and / or physiologically compatible with other ingredients, e.g., the active ingredient (i.e., the polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof), comprising the formulation, and physiologically compatible with the subject to which the pharmaceutical composition is administered.

[0299]

[0326] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is biologically acceptable and non-toxic to a subject. In the context of the present disclosure, pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or various combinations thereof, known in the art.

[0300]

[0327] As used herein, suitable "ingredients" may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable "antioxidants" may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in the pharmaceutical compositions provided herein reduces oxidation of the polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving protein stability and maximizing shelf life. Thus, in certain embodiments, pharmaceutical compositions are provided that comprise, in addition to the active ingredient (i.e., a polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof disclosed herein), one or more antioxidants, such as methionine.

[0301]

[0328] Pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN®-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents utilized as carriers may be added to pharmaceutical compositions in multidose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0302]

[0329] Pharmaceutically acceptable "diluents" may include saline and aqueous buffer solutions.

[0303]

[0330] Pharmaceutically acceptable "adjuvants" may include preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and the incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin, can prolong the absorption of the injectable dosage form.

[0304]

[0331] The pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0305]

[0332] In embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for preparing a liquid solution, suspension, or emulsion. Preparations for injection can include sterile and / or non-pyrogenic solutions prepared for injection, sterile dry soluble preparations such as lyophilized powders prepared to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions prepared for injection, sterile dry insoluble preparations prepared to be combined with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0306]

[0333] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration shall be sterile and non-pyrogenic, as known and practiced in the art.

[0307]

[0334] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving a polypeptide conjugate disclosed herein in a suitable solvent. The solvent may contain an excipient that improves the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of the polypeptide conjugate. Overfilling the vial by a small amount (e.g., about 10%) beyond that required for a dose or set of doses may be acceptable to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0308]

[0335] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the given selected therapy and can be determined empirically.

[0309]

[0336] In certain embodiments, the composition further provides a composition comprising a pharmaceutically acceptable carrier, diluent, or adjuvant and an active ingredient, which may be an engineered antibody or antigen-binding fragment thereof disclosed herein, or an engineered antibody conjugate disclosed herein.

[0310] IV kit

[0337] In another aspect, the present invention provides a kit comprising an engineered antibody provided herein and instructions for use of the engineered antibody. The kit may also include a container and, optionally, one or more vials, test tubes, flasks, bottles, or syringes. Other formats of kits will be apparent to those of skill in the art and are within the scope of the present invention.

[0311] V Medical Use

[0338] In another aspect, the present invention provides a method for treating, preventing, or alleviating a medical condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered antibody disclosed herein, or a polynucleotide encoding an engineered antibody provided herein, or a pharmaceutical composition provided herein.

[0312]

[0339] As used herein, the terms "subject" or "individual" or "animal" or "patient" refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or disorder. Mammalian subjects include humans, livestock, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, and bears. In certain embodiments, the subject is a human.

[0313]

[0340] As used herein, "treating" a condition may include alleviating the condition, slowing the onset or rate of progression of the condition, slowing the progression of symptoms associated with the condition, reducing or halting symptoms associated with the condition, achieving complete or partial regression of the condition, curing the condition, or any combination thereof.

[0314]

[0341] As used herein, the terms "disorder," "disease," or "condition," or the like, refer to a condition affecting a subject that is amenable to treatment with an engineered antibody.

[0315]

[0342] In certain embodiments, the disease or disorder is a HER2-associated disease or disorder. In some embodiments, the HER2-associated condition or disorder is cancer.

[0316]

[0343] As used herein, "cancer" refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal proliferation, or invasion or metastasis. Cancer includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. Examples of cancers or tumors include hematologic malignancies, cancers of the oral cavity (e.g., lip, tongue, or pharynx), digestive tract (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver and biliary tract, pancreas, respiratory system such as larynx or lung (small cell and non-small cell), bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tubes, uterus, cervix, testes, ovaries, or prostate), urinary tract (e.g., bladder or kidney), brain, and endocrine glands such as the thyroid gland.

[0317]

[0344] In some embodiments, the HER2-associated condition or disorder is a HER2-expressing cancer, a HER2-overexpressing cancer, or a HER-ligand-overexpressing cancer.

[0318]

[0345] A "HER2-expressing cancer" is a cancer involving cancer cells or tumor cells that have the HER2 protein present on their cell surface. In HER2-expressing cancer cells, homodimers formed by HER2 or heterodimers formed by HER2 with HER1, HER3, or HER4 can activate the PI3K / Akt and MAPK pathways in the cells, thereby regulating tumor cell proliferation, differentiation, migration, and apoptosis.

[0319]

[0346] A "HER2-overexpressing cancer" is a cancer that has significantly higher levels of a HER receptor, such as HER2, on the cell surface of cancer or tumor cells compared to non-cancerous cells of the same tissue type. Such overexpression can be induced by gene amplification or increased transcription or translation.

[0320]

[0347] HER2 expression or overexpression may be determined in a diagnostic or prognostic assay that evaluates increased levels of HER2 protein present on the surface of a cell (e.g., by immunohistochemistry, IHC). Alternatively, or additionally, levels of nucleic acid encoding HER2 in cells may be measured by, for example, fluorescent in situ hybridization (FISH, see WO 98 / 45479, published October 1998), Southern blotting, or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR). HER2 receptor overexpression may be studied by measuring shed antigens (e.g., HER2 extracellular domains) in biological fluids such as serum (see, e.g., U.S. Pat. No. 4,933,294, issued June 12, 1990; WO 91 / 05264, published April 18, 1991; U.S. Pat. No. 5,401,638, issued March 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). In addition to the above assays, various in vivo assays are available to the skilled practitioner. For example, cells within a patient may be exposed to an antibody, optionally labeled with a detectable label, e.g., a radioisotope, and binding of the antibody to cells within the patient may be assessed, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from the patient previously exposed to the antibody. In some embodiments, the test sample is derived from cancer cells or tissue.

[0321]

[0348] As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount of the therapeutic agent that, when administered to a subject in an appropriate manner, produces a sufficient therapeutic effect in the subject. As with other therapeutic agents, it is understood that the therapeutically effective amount of the polypeptide conjugates provided above will be affected by various factors known in the art, such as the subject's weight, age, past medical history, current drug treatments, health status and cross-reactions, allergies, hypersensitivity, and potential side effects, as well as the route of administration and the extent of disease onset. Doses can be proportionally increased or decreased by a skilled artisan (e.g., a physician or veterinarian) depending on these and other circumstances or requirements.

[0322]

[0349] In certain embodiments, the engineered antibodies or antigen-binding fragments provided herein may be administered in a therapeutically effective amount of about 0.01 mg / kg to about 100 mg / kg. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, or multiple doses may be administered over time.

[0323]

[0350] The engineered antibodies and antigen-binding fragments disclosed herein can be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous infusion, intramuscular injection, or intravenous, including intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0324]

[0351] In some embodiments, the engineered antibodies and antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents. For example, the engineered antibodies and antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, such as a chemotherapeutic or anti-cancer agent.

[0325]

[0352] In some of these embodiments, the engineered antibodies and antigen-binding fragments disclosed herein that are administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in some of these embodiments, the engineered antibodies and antigen-binding fragments and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, engineered antibodies and antigen-binding fragments that are administered "in combination with" another therapeutic agent are not necessarily administered simultaneously with or in the same composition as that agent. Engineered antibodies and antigen-binding fragments that are administered before or after another agent are considered to be administered "in combination with" that agent, as that term "combination" is used herein, even if the engineered antibodies and antigen-binding fragments and the second agent are administered by different routes. When possible, additional therapeutic agents administered in combination with the antibodies and antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to protocols known in the art, such as the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)).

[0326]

[0353] The following examples are provided to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All specific compositions, materials, and methods described below are within the scope of the present invention, in whole or in part. These specific compositions, materials, and methods are not intended to limit the present invention, but are merely intended to illustrate specific embodiments within the scope of the present invention. Those skilled in the art will be able to develop equivalent compositions, materials, and methods without creative effort and without departing from the scope of the present invention. It will be understood that many variations can be made to the procedures described herein within the scope of the present invention. It is the intention of the inventors that such variations are included within the scope of the present invention. [Example]

[0327] Example 1 Antibody Construction, Expression, and Purification

[0354] 1.1 Structure of each antibody

[0355] According to the structures of DICAD (see patent application CN201711415979.9) and TRIAD (see patent application CN201880081812.X), a bispecific antibody having the structure shown in Figure 1 and a trispecific antibody having the structure shown in Figure 2 were constructed.

[0328]

[0356] The bispecific antibody DICAD contains two antigen-targeting domains: Domain 1 (associated by VH1 and VL1) and Domain 2 (associated by VH2 and VL2). The trispecific antibody TRIAD contains three antigen-targeting domains: Domain 1 (associated by VH1 and VL1), Domain 2 (associated by VH2 and VL2), and Domain 3 (associated by VH3 and VL3).

[0329]

[0357] In the DICAD structure, the structure of each peptide chain from N-terminus to C-terminus is as follows:

[0358] Chain 1: VL1-linker-VH2-hinge region-CH2-CH3

[0359] Chain 2: VL2-linker-VH1

[0330]

[0360] In the TRIAD structure, the structure of each peptide chain from N-terminus to C-terminus is as follows:

[0361] Chain 1: VL2-linker-VH1

[0362] Chain 2: VL1-linker-VH2-hinge region-CH2-CH3

[0363] Chain 3: VH3-CH1-hinge region-CH2-CH3

[0364] Chain 4: VL3-CL

[0331]

[0365] Based on the structures of each of the aforementioned polypeptide chains, DICAD antibodies (6A1 and 6A2) and TRIAD antibodies (6A8, 6A12, 6A14, 6A15, 6A17, 6A19, 6A17, 6A18, 6A21, 6A23, 6A25, and 6A26) were constructed. The antigens bound by each domain of each antibody targeting the antigens are shown in Table 9 below, where Tra stands for Trastuzumab and Per stands for Pertuzumab.

[0332] [Table 9]

[0333] [Table 10]

[0334]

[0366] The Fv sequences used in the construction of the DICAD and TRIAD antibodies and binding to each antigen target (Her2, CD3, NKG2D, and CD16) are shown in Tables 1 to 4, respectively.

[0335]

[0367] In the constructs of the DICAD and TRIAD antibodies, point mutations are introduced at designated points (Kabat) in the VH and VL structural domains listed in Tables 5-7, and 10. In particular, each antibody has the structure and mutations shown in Tables 5-7, where mutations in the CH3 region correspond to mutations in human IgG. The full-length and VH / VL sequences used for each of the TRIAD antibodies are also shown in Tables 5-7. The full-length sequences used for each of the DICAD antibodies are shown in Table 10.

[0336]

[0368] 6A1 comprises the chain 1 sequence set forth in SEQ ID NO: 110 (i.e., VL2(HER2-Tra)-linker-VH1(HER2-Per)) and the chain 2 sequence set forth in SEQ ID NO: 111 (i.e., VL1(HER2-Per)-linker-VH2(HER2-Tra)-hinge-CH2-CH3). 6A2 comprises the chain 1 sequence set forth in SEQ ID NO: 112 and the chain 2 sequence set forth in SEQ ID NO: 113. 6A1 and 6A2 are identical except that 6A2 contains point mutations Q39D in VH1(HER2-Per) and Q38K in VL1(HER2-Per) to introduce electrostatic interactions.

[0337]

[0369] 6B1 contains the strand 1 sequence shown in SEQ ID NO: 86, the strand 2 sequence shown in SEQ ID NO: 87, the strand 3 sequence shown in SEQ ID NO: 88, and the strand 4 sequence shown in SEQ ID NO: 89. 6B1 was used as a positive control.

[0338]

[0370] 1.2 Antibody gene synthesis, expression, and purification

[0371] The amino acid sequences of all of the bispecific antibodies are shown in Table 8 of the present disclosure. Polynucleotides encoding these bispecific antibodies were designed and codon-optimized by OptimumGene prior to synthesis.

[0339]

[0372] The target gene was first constructed in the pUC57 vector and then subcloned into the pTGE5 vector. DNA was prepared for transfection by Maxiprep.

[0340]

[0373] 0.3 × 10 CHO3E7 cells 6 The cells were cultured and subcultured at a cell density of 1.8–2.5 × 10 6Transfection was performed when the concentration reached 100 cells / ml. First, 300 μl of DNA heavy and light chains were added to 50 ml of Freestyle CHO medium and gently mixed by shaking. Next, 3 mg of PEI transfection reagent was added and gently mixed by shaking for at least 3 minutes. The mixture was left to stand at 37°C for 7 minutes and then added to 450 ml of cell suspension to obtain a total volume of 500 ml. After 24 hours, 25 ml of TN1 (master mix concentration 200 g / L) was added to the mixture.

[0341]

[0374] On days 1, 3, and 5 after transfection, 1 ml of the suspension was collected for testing. A 50 μl sample was collected for cell counting, and the remaining sample was processed by centrifugation at 3000 rpm for 5 min, after which the supernatant was placed at -20°C. On day 6, the culture was harvested and processed by centrifugation at 5500 rpm for 30 min. The supernatant was separated and filtered through a 0.22 μm filter to further purify the protein.

[0342]

[0375] Chromatography column: 5 ml Monofinity A resin (GenScript, batch number L00433) chromatography column, equilibration buffer A: 20 mM PB, 150 mM NaCl, pH 7.2, wash buffer B: 50 mM citric acid, pH 3.5, neutralization buffer C: 1 M Tris-HCl, pH 9.0, flow rate: 2 ml / min, gradient: 100% gradient elution. After separation, 0.155 ml of neutralization buffer C was added to each 1 ml fraction. The collected protein solution was dialyzed against PBS (pH 7.2) at 4 °C for 16 h.

[0343]

[0376] The aforementioned samples were analyzed by SDS-PAGE followed by Western blot analysis. Samples were purified and analyzed for purity by SEC.

[0344] Example 2 Detection of growth inhibition of cells with high Her2 expression levels by DICAD antibodies

[0377] To compare the effects of the constructed bispecific antibodies on cells with high Her2 expression levels, 6A1, 6A2, trastuzumab (Herceptin), and pertuzumab (Perjeta) were added to BT-474 cells to observe the inhibitory effects on the growth of BT-474 cells.

[0345]

[0378] The SRB method was applied to detect the inhibitory effect of drugs on tumor cell proliferation and growth. The main steps are as follows:

[0346]

[0379] Logarithmically growing cells were seeded into 96-well culture plates and treated with drugs at different concentrations (3, 10, 30, 100, 300, 1000, 3000, and 10,000 ng / ml). Each concentration was replicated in two wells, and a control with the corresponding solvent concentration was included. Tumor cells were cultured at 37°C and 5% CO for 120 hours. Cells were stained with SRB at room temperature and finally lysed by adding Tris solution. OD values ​​were measured at 510 nm wavelength using a microplate reader (BioTek). The cell growth inhibition rate was calculated using the following formula:

[0347]

[0380]

number

[0348]

[0381] Based on the inhibition rate of each concentration, the median inhibitory concentration IC50 was calculated by nonlinear regression method.

[0349]

[0382] The results are shown in Figure 3. The results show that trastuzumab monoclonal antibody had a blocking effect both alone and in combination with pertuzumab monoclonal antibody. In contrast, the bispecific antibodies 6A1 and 6A2, constructed with DICAD using the aforementioned monoclonal antibody VH and VL, did not exhibit a blocking effect but unexpectedly exhibited a significant agonistic effect.

[0350]

[0383] The above results demonstrated by 6A1 and 6A2 suggest that the 2:2 antibodies constructed using the DICAD model are ineffective. The inventors hypothesize that the possible cause is as follows: The antigen-binding domains corresponding to trastuzumab and pertuzumab, which bind Her2 to ECD2 and ECD4, respectively, are too close in the antibodies constructed using the DICAD platform, bringing Her2 monomers closer to each other and enhancing the formation of Her2 dimers, thereby promoting Her2-dependent tumor growth. Therefore, this construct reverses the Her2 signaling blocking effect of the parent antibodies (trastuzumab and pertuzumab) and instead exhibits agonistic activity.

[0351] Example 3 Performance test of TRIAD antibodies

[0384] 3.1 Killing assay of cells with high Her2 expression levels

[0385] To test the ability of the antibodies to kill cells with high Her2 expression levels, an SK-BR-3 cell killing assay was performed.

[0352]

[0386] RPMI medium 1640 supplemented with 2% HI-FBS was used as the assay medium. Corning 96-well white flat-bottom microplates (Cat. No. 3903) were used as the assay plates. SK-BR-3 cells provided by ATCC were used as cells with high Her2 expression levels for testing.

[0353]

[0387] method

[0388] 1) SK-BR-3 cells were resuspended in RPMI1640 + 2% FBS at a concentration of 3E5 cells / mL. 100 μL was dispensed into each well of plate (3903). The cells were incubated overnight at 37°C.

[0389] 2) PBMC cells were resuspended in RPMI1640 + 2% FBS at a concentration of 3E5 cells / mL, and 100 μL of the suspension was added to an SK-BR-3 plate.

[0390] 3) Antibodies were prepared at a concentration of 180 μg / mL in PBS (9 doses, 3-fold dilutions, duplicates). 10 μL was added to the plate.

[0391] 4) The plate was incubated in an incubator for 72 hours.

[0392] 5) The supernatant was removed from the plate, and the plate was washed twice with 100 μL of PBS. Then, 100 μL of 1640 + 2% FBS was added. The CTG signal was detected.

[0354]

[0393] The results are shown in Figures 4A-4F, and indicate that 6B1 has the highest killing ability. Compared to trastuzumab, 6A19 and 6A26 were slightly weaker, while 6A17, 6A25, 6A23, and 6A15 were slightly stronger. Compared to trastuzumab, the antibodies constructed using the above method have enhanced PBMC-dependent antibody killing effects.

[0355]

[0394] 3.2 Results of cell lines with low Her2 expression levels

[0395] MCF-7 Killing Assay Test Design

[0396] Assay medium: RPMI medium 1640 supplemented with 2% HI-FBS

[0397] Assay plate: Corning 96-well white flat-bottom microplate (Cat. No. 3903)

[0398] MCF-7 cells: ATCC HTB-22

[0399] Culture medium: EMEM + 0.01 mg / mL insulin + 10% FBS + 1% PS.

[0356]

[0400] [Table 11]

[0357]

[0401] method

[0402] 1) MCF-7 cells were resuspended in RPMI1640 + 2% FBS at a concentration of 1.5E5 cells / ml. 100 μL was dispensed into each well of a plate (3903). The cells were incubated overnight at 37°C.

[0403] 2) PBMC cells were resuspended in RPMI1640 + 2% FBS at a concentration of 7.5E5 cells / ml, and 100 μL of the suspension was added to an MCF-7 plate.

[0404] 3) Antibody was prepared at a concentration of 540 μg / mL in PBS (9 doses, 3-fold dilution). 10 μL was added to the plate.

[0405] 4) The plate was incubated in an incubator for 72 hours.

[0406] 5) The supernatant was removed from the plate, and the plate was washed twice with 100 μL of PBS. Then, 100 μL of 1640 + 2% FBS was added. The CTG signal was detected.

[0358]

[0407] The results in Figures 5A and 5B showed that 6B1 had the highest killing ability, followed by 6A19 and 6A26, and then 6A25. 6A23, 6A17, 6A18, and trastuzumab showed no obvious killing effect.

[0359]

[0408] 3.3 In vivo results of cell lines with high Her2 expression levels

[0409] 3.3.1 Animal Model of KPL-4 with High Her2 Expression Levels

[0410] Experimental Protocols and Methods

[0411] Six-week-old female B-NDG mice were used. Each mouse received 1 × 10 5 KPL-4 cells were subcutaneously inoculated, and tumors grew to approximately 150 mm 3 At tumor size, mice were intravenously injected with 5 × 10 PBMCs per mouse. Mice were then divided into groups based on tumor volume and injected with IV drug (5 mg / kg) twice weekly (BIW) at 0.1 mL / 10 g body weight.

[0360]

[0412] The effect of the drug on tumor growth was specifically monitored as T / C% or tumor growth inhibition rate (TGI%). The tumor diameter was measured twice a week with a caliper, and the tumor volume (V) was calculated using the following formula.

[0361]

[0413]

Number

[0362]

[0414] In the formula, a and b represent the length and width, respectively.

[0363]

[0415]

Number

[0364]

[0416] In the formula, T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0365]

[0417]

Number

[0366]

[0418] If the tumor appeared to regress

[0367] ​​​​​​​​​​​​​​​​​​​The results are shown in Figure 6. 6B1 and 6A19 (5 mg / kg IV, BIW, 5 doses in total) significantly inhibited the growth of subcutaneously implanted human breast cancer KPL-4 tumors in human PBMC-reconstituted mice, with tumor inhibition rates of 71% and 92%, respectively. Two of six mice in the 6A19 group experienced partial tumor regression, while 6A25 and 6A26 (5 mg / kg IV, BIW, 5 doses in total) did not have significant efficacy against the growth of subcutaneously implanted KPL-4 tumors in human PBMC-reconstituted mice. Tumor-bearing humanized immune-reconstituted mice tolerated the aforementioned drugs well, with no significant weight loss or other symptoms occurring during treatment.

[0370]

[0423] 3.3.2 BT474 Animal Model with High Her2 Expression Levels

[0424] Experimental Protocols and Methods

[0425] Use 6- to 8-week-old female NCG mice. Each mouse receives 1 × 10 7 BT474 cells and 1 × 10 7 PBMCs were inoculated. Tumors were approximately 150 mm 3 Once the tumors reached the tumor size, the mice were divided into groups according to tumor volume and injected with the drug (5 mg / kg) twice a week (BIW) (IV) in a volume of 0.1 mL / 10 g body weight.

[0371]

[0426] The body weight and tumor size of the mice were measured twice a week. The tumor volume was calculated using the following formula:

[0372]

[0427]

number

[0373]

[0428] During the experiment, data were collected using StudyDirector™ (version number: 3.1.399.19, provided by Studylog System, Inc.), including measurements of tumor diameters and animal weight. Raw data were measured using a balance and calipers and imported directly into the software. Any changes in data were recorded by the software.

[0374]

[0429] Experimental results

[0430] The results are shown in Figure 7. 6B1, 6A19, 6A25, and 6A26 (5 mg / kg IV, BIW, for a total of 2 weeks) significantly inhibited subcutaneously transplanted human breast cancer BT474 tumors in human PBMC-immune reconstituted mice, with tumor inhibition rates of 99.05%, 100.00%, 79.28%, and 98.83%, respectively. Tumors in each group partially regressed in 4 / 5, 5 / 5, 2 / 5, and 4 / 5 mice, respectively. Tumor-bearing mice with reconstituted human immune systems tolerated the drugs well, with no significant weight loss or other symptoms occurring during treatment.

[0375]

[0431] 3.3.3 HT55 Animal Model with Intermediate Her2 Expression

[0432] Experimental Protocols and Methods

[0433] Five- to seven-week-old female NCG mice were used and transfected with HT55 cells (human colon cancer cell line, 5 × 10 6 / mouse) subcutaneously inoculated with PBMC cells (1 × 10 7 The suspension was injected intraperitoneally into the mouse. 3 When the dose reached 100 mg / kg, the mice were mass-administered.

[0376]

[0434] Tumor volume was measured three times a week with a vernier caliper to measure the long and short diameters of the tumor. A reduction in tumor volume of 50% or less was considered a partial response (PR), and complete disappearance of the tumor was considered a complete response (CR).

[0377]

[0435] Experimental results

[0436] One animal in each of the 6B1 and 6A19 groups died of severe graft-versus-host disease (GVHD) during treatment. Other results are shown in Figure 8 , which demonstrate that 6B1, 6A19, 6A25, and 6A26 (5 mg / kg IV, BIW, for a total of 2 weeks) significantly inhibited subcutaneously transplanted human breast cancer HT55 cell tumors in human PBMC-immune reconstituted mice, with tumor inhibition rates of 98.54%, 100.00%, 34.49%, and 82.66%, respectively. Tumors partially regressed in 3 / 5, 5 / 5, 0 / 6, and 2 / 6 mice in each group, respectively. Mice bearing tumors from the reconstituted human immune system tolerated the drugs well, with no significant weight loss or other symptoms occurring during treatment. The negative control in Figure 8 is PBS.

[0378]

[0437] 3.3.4 Animal models with low PC3 expression levels

[0438] Experimental Protocols and Methods

[0439] Five- to seven-week-old female NCG mice were transfected with PC-3 cell line (3 × 10 6 / mouse) were subcutaneously inoculated with PBMC cells (1.5 × 10 6 The suspension was injected intraperitoneally into mice. 3 Drugs were administered to each group at the time of maturation.

[0379]

[0440] Testing indicators:

[0441] Tumor volume was measured three times a week, and the long and short diameters of the tumor were measured with a vernier caliper. A reduction of 50% or more in tumor volume was considered a partial response (PR), and complete disappearance of the tumor was considered a complete response (CR).

[0380]

[0442] Experimental results

[0443] The results are shown in Figure 9, which demonstrate that 6B1 can significantly inhibit PC-3 tumor growth on PC3. 6A19 has a similar effect to 6A26, stronger than 6A25, but slightly weaker than 6B1. The control in Figure 9 is PBS.

Claims

1. (i) A first polypeptide comprising a first heavy chain variable domain (VH1) linked to a second light chain variable domain (VL2), (ii) A second polypeptide comprising a second heavy chain variable domain (VH2) linked to a first light chain variable domain (VL1), (iii) A third polypeptide containing a third heavy chain variable domain (VH3), (iv) A fourth polypeptide containing a fourth light chain variable domain (VL3), Includes, The VL1 and VH1 associate to form a first domain capable of binding to the first target. The VL2 and VH2 associate to form a second domain capable of binding to a second target. The VL3 and VH3 associate to form a third domain capable of binding to a third target, and the second and third polypeptide chains each further comprise a first dimerization domain and a second dimerization domain, which are associated to form a dimer. The N-terminus of VL1 is connected to the C-terminus of VH2, and the N-terminus of VL2 is covalently connected to the C-terminus of VH1, or The N-terminus of VH1 is connected to the C-terminus of VL2, and the N-terminus of VH2 is connected to the C-terminus of VL1. One of the first and second targets is an immunostimulatory target, The remaining two targets are engineered antibodies that are different epitopes on the same tumor antigen that can dimerize when bound to a natural ligand.

2. The aforementioned immunostimulatory targets are selected from CD3, NKG2D, and CD16. Depending on the circumstances, (i) The immunostimulatory target is CD3, Here, the domains that can be bound to CD3 are: (a) VL containing LCDR1 containing the sequence shown in SEQ ID NO: 5, LCDR2 containing the sequence shown in SEQ ID NO: 6, and LCDR3 containing the sequence shown in SEQ ID NO: 7, and VH containing HCDR1 containing the sequence shown in SEQ ID NO: 1, HCDR2 containing the sequence shown in SEQ ID NO: 2, and HCDR3 containing the sequence shown in SEQ ID NO: 3, or (b) VL containing the sequence of sequence number 8 and VH containing the sequence of sequence number 4, Or, (ii) The immunostimulatory target is NKG2D, Here, the domains that can be bound to NKG2D are: (a) VL including LCDR1 containing the sequence shown in SEQ ID NO: 29, LCDR2 containing the sequence shown in SEQ ID NO: 30, and LCDR3 containing the sequence shown in SEQ ID NO: 31, and VH including HCDR1 containing the sequence shown in SEQ ID NO: 25, HCDR2 containing the sequence shown in SEQ ID NO: 26, and HCDR3 containing the sequence shown in SEQ ID NO: 27, (b) VL comprising LCDR1 containing the sequence shown in SEQ ID NO: 118, LCDR2 containing the sequence shown in SEQ ID NO: 119, and LCDR3 containing the sequence shown in SEQ ID NO: 120, and VH comprising HCDR1 containing the sequence shown in SEQ ID NO: 114, HCDR2 containing the sequence shown in SEQ ID NO: 115, and HCDR3 containing the sequence shown in SEQ ID NO: 116, (c) VL containing the sequence of sequence number 32 and VH containing the sequence of sequence number 28, or (d) VL containing the sequence of sequence number 121 and VH containing the sequence of sequence number 117, Or, (iii) The immunostimulatory target is CD16, Here, the domains that can be bound to CD16 are: (a) VL containing LCDR1 containing the sequence shown in SEQ ID NO: 37, LCDR2 containing the sequence shown in SEQ ID NO: 38, and LCDR3 containing the sequence shown in SEQ ID NO: 39, and VH containing HCDR1 containing the sequence shown in SEQ ID NO: 33, HCDR2 containing the sequence shown in SEQ ID NO: 34, and HCDR3 containing the sequence shown in SEQ ID NO: 35, or (b) VL containing the sequence of sequence number 40 and VH containing the sequence of sequence number 36, The manipulated antibody according to claim 1.

3. The tumor antigen is HER2, Here, (a) One of the domains capable of binding to HER2 is the first HER2 binding domain, and the other of the domains capable of binding to HER2 is the second HER2 binding domain, (b) The VL2 and VH2 combine to form the first HER2 binding domain, and the VL3 and VH3 combine to form the second HER2 binding domain, (c) The VL2 and VH2 combine to form the second HER2 binding domain, and the VL3 and VH3 combine to form the first HER2 binding domain, (d) The VL1 and VH1 associate to form the first HER2 binding domain, the VL3 and VH3 associate to form the second HER2 binding domain, or (e) The VL1 and VH1 combine to form the first HER2 binding domain, and the VL3 and VH3 combine to form the second HER2 binding domain, Here, (i) The first HER2 binding domain includes LCDR1 containing the sequence shown in SEQ ID NO: 13, LCDR2 containing the sequence shown in SEQ ID NO: 14, and LCDR3 containing the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 97, and the VH2 includes HCDR1 containing the sequence shown in SEQ ID NO: 9, HCDR2 containing the sequence shown in SEQ ID NO: 10, and HCDR3 containing the sequence shown in SEQ ID NO: 11, or (ii) The second HER2 binding domain includes LCDR1 containing the sequence shown in SEQ ID NO: 21, LCDR2 containing the sequence shown in SEQ ID NO: 22, and LCDR3 containing the sequence shown in SEQ ID NO: 23, and the VH3 includes HCDR1 containing the sequence shown in SEQ ID NO: 17, HCDR2 containing the sequence shown in SEQ ID NO: 18, and HCDR3 containing the sequence shown in SEQ ID NO:

19. The manipulated antibody according to claim 1.

4. (a) The first HER2 binding domain is derived from trastuzumab, and the second HER2 binding domain is derived from the antigen binding domain of pertuzumab (b) Both the first HER2-binding domain and the second HER2-binding domain are derived from the antigen-binding domain of trastuzumab, or (c) Both the first HER2-binding domain and the second HER2-binding domain are derived from the antigen-binding domain of pertuzumab. The manipulated antibody according to claim 3.

5. (a) The VL1 and VH1 are associated with a first unnatural disulfide bond, where, The first unnatural disulfide bond is formed between two unnatural cysteine ​​residues in VL1 and VH1, respectively, and optionally the two unnatural cysteine ​​residues are Q100C in VL1 and G44C in VH1, numbered according to the Kabat index. Here, depending on the circumstances, the VL2 and VH2 are associated with a natural disulfide bond, or another unnatural disulfide bond formed at a different location from the first unnatural disulfide bond, and / or (b) The VL2 and VH2 are associated with a second unnatural disulfide bond, where, The second unnatural disulfide bond is formed between two unnatural cysteine ​​residues in VL2 and VH2, respectively, and optionally the two unnatural cysteine ​​residues are Q100C in VL2 and G44C in VH2, numbered according to the Kabat index. Here, depending on the circumstances, VL1 and VH1 are associated with a natural disulfide bond, or another unnatural disulfide bond formed at a different location from the second unnatural disulfide bond. The manipulated antibody according to claim 1.

6. The VL1 and VH1 are further associated with an electrostatic interaction between two oppositely charged residues, and / or the VL2 and VH2 are further associated with an electrostatic interaction between two oppositely charged residues. In this case, depending on the circumstances, (a) The two oppositely charged residues are introduced such that they replace Q38 in VL1 and Q39 in VH1, respectively, or replace Q38 in VL2 and Q39 in VH2, (b) The two oppositely charged residues are introduced to replace Q40 in VL1 and Q39 in VH1, respectively. (c) The two oppositely charged residues are introduced to replace Q37 in VL1 and Q39 in VH1, respectively, or (d) The two oppositely charged residues are introduced to replace Q37 in VL1 and Q39 in VH1, respectively. Here, the numbering follows the Kabat index. In this case, depending on the circumstances, The two oppositely charged residues include a negatively charged amino acid residue selected from aspartic acid (D) or glutamic acid (E), and a positively charged amino acid residue selected from lysine (K), histidine (H), or arginine (R). In this case, depending on the circumstances, The two oppositely charged residues each include Q38D in VL1 and Q39K in VH1, or Q38D in VL2 and Q39K in VH2, respectively, and the numbering follows the Kabat index. The manipulated antibody according to claim 5.

7. a) VH2 includes the sequence shown in Sequence ID 92, VL2 includes the sequence shown in Sequence ID 93, VH3 includes the sequence shown in Sequence ID 12, and VL3 includes the sequence shown in Sequence ID 16. b) The VH2 includes the sequence shown in sequence number 90, the VL2 includes the sequence shown in sequence number 91, the VH3 includes the sequence shown in sequence number 20, and the VL3 includes the sequence shown in sequence number 24. c) The VH2 includes the sequence shown in sequence number 90, the VL2 includes the sequence shown in sequence number 91, the VH3 includes the sequence shown in sequence number 106, and the VL3 includes the sequence shown in sequence number 107, or d) VH2 includes the sequence shown in sequence number 90, VL2 includes the sequence shown in sequence number 91 or 98, VH3 includes the sequence shown in sequence number 12, and VL3 includes the sequence shown in sequence number 16 or 95. The manipulated antibody according to claim 5.

8. (a) The VL1 and VH1 combine to form the first domain capable of binding to CD3, the VL2 and VH2 combine to form the second HER2 binding domain, and the VL3 and VH3 combine to form the first HER2 binding domain, Here, (i) The VL1 includes the sequence of sequence number 8, and the VH1 includes the sequence of sequence number 4, (ii) The VL2 includes the sequence of sequence number 93, the VH2 includes the sequence of sequence number 92, and (iii) The VL3 includes the sequence of sequence number 16, and the VH3 includes the sequence of sequence number 12. (b) The VL1 and VH1 combine to form the first domain capable of binding to CD16, the VL2 and VH2 combine to form the second HER2 binding domain, and the VL3 and VH3 combine to form the first HER2 binding domain, Here, (i) VL1 includes the sequence of sequence number 40, and VH1 includes the sequence of sequence number 36, (ii) The VL2 includes the sequence of sequence number 93, the VH2 includes the sequence of sequence number 92, and (iii) The VL3 includes the sequence of sequence number 16, and the VH3 includes the sequence of sequence number 12. (c) The VL1 and VH1 combine to form the first domain capable of binding to NKG2D, the VL2 and VH2 combine to form the first HER2 binding domain, and the VL3 and VH3 combine to form the second HER2 binding domain. Here, (i) VL1 includes the sequence of sequence number 32, and VH1 includes the sequence of sequence number 28, (ii) The VL2 includes the sequence of sequence number 91, the VH2 includes the sequence of sequence number 90, and (iii) The VL3 includes the sequence of sequence number 24, and the VH3 includes the sequence of sequence number 20. (d) The VL1 and VH1 combine to form the first domain capable of binding to CD3, the VL2 and VH2 combine to form the third HER2 binding domain, and the VL3 and VH3 combine to form the fourth HER2 binding domain. Here, (i) The VL1 includes the sequence of sequence number 100, and the VH1 includes the sequence of sequence number 99, (ii) The VL2 includes the sequence of sequence number 98, the VH2 includes the sequence of sequence number 90, and (iii) The VL3 includes the sequence of sequence number 95, and the VH3 includes the sequence of sequence number 12. Or, (e) The VL1 and VH1 combine to form the first domain capable of binding to CD3, the VL2 and VH2 combine to form the third HER2 binding domain, and the VL3 and VH3 combine to form the fourth HER2 binding domain. Here, (i) The VL1 includes the sequence of sequence number 100, and the VH1 includes the sequence of sequence number 99, (ii) The VL2 includes the sequence of sequence number 91, the VH2 includes the sequence of sequence number 90, and (iii) The VL3 includes the sequence of sequence number 16, and the VH3 includes the sequence of sequence number 12. The manipulated antibody according to claim 1.

9. The VL1 is linked to the VH2 via a first peptide linker, and the VL2 is linked to the VH1 via a second peptide linker. In this case, depending on the circumstances, The first peptide linker and the second peptide linker each independently contain 5 to 9 amino acids, and optionally the first peptide linker and the second peptide linker each contain the sequence shown in Sequence ID No.

94. The manipulated antibody according to claim 1.

10. (i) The first dimerization domain and the second dimerization domain each comprise the CH3 domain of IgG and further optionally comprise one or more mutations that promote heterodimerization, In this case, depending on the circumstances, The first dimerization domain contains the first mutation, and the second dimerization domain contains the second mutation. Depending on the circumstances, a) The first mutation includes T389W and / or S375C, and the second mutation includes Y438V, T389S, L391A, and / or Y370C, b) The first mutation comprises D427K and / or D377K, and the second mutation comprises K420D and / or K440D, c) The first mutation comprises D377K, E378K, and / or D427K, and the second mutation comprises K393E, K440D, and / or K470E, d) The first mutation comprises S387H and / or F436A, and the second mutation comprises Y370T and / or T422F, e) The first mutation comprises S387H and / or T422F, and the second mutation comprises Y422T and / or F436A, f) The first mutation comprises K393D and / or K440D, and the second mutation comprises E378K and / or D427K, or g) The first mutation comprises L372D and / or L391E, and the second mutation comprises L372K or T389K, especially, The first mutation includes T389W, and the second domain includes T389S, L391A, and Y438V. Here, the numbering follows the Kabat index. (ii) The first dimerization domain and / or the second dimerization domain further comprises the CH2 domain and / or hinge region of IgG, wherein the hinge region optionally comprises the sequence shown in Sequence ID No. 43, 101, or 102. And / or, (iii) One of the first dimerization domain and the second dimerization domain includes the sequence shown in SEQ ID NO: 44, and the other includes the sequence shown in SEQ ID NO: 45, or one of the first dimerization domain and the second dimerization domain includes the sequence shown in SEQ ID NO: 103, and the other includes the sequence shown in SEQ ID NO:

104. The manipulated antibody according to claim 1.

11. (i) The third polypeptide further comprises a CH1 region, which optionally comprises the sequence shown in Sequence ID No. 41, and / or (ii) The fourth polypeptide further comprises a CL region, which optionally comprises the sequence shown in Sequence ID No.

42. The manipulated antibody according to claim 8.

12. (a) The first polypeptide comprises the sequence shown in SEQ ID NO: 62, the second polypeptide comprises the sequence shown in SEQ ID NO: 63, the third polypeptide comprises the sequence shown in SEQ ID NO: 12, and the fourth polypeptide comprises the sequence shown in SEQ ID NO: 16 (b) The first polypeptide comprises the sequence shown in SEQ ID NO: 74, the second polypeptide comprises the sequence shown in SEQ ID NO: 75, the third polypeptide comprises the sequence shown in SEQ ID NO: 12, and the fourth polypeptide comprises the sequence shown in SEQ ID NO:

16. (c) The first polypeptide comprises the sequence shown in SEQ ID NO: 58, the second polypeptide comprises the sequence shown in SEQ ID NO: 59, the third polypeptide comprises the sequence shown in SEQ ID NO: 20, and the fourth polypeptide comprises the sequence shown in SEQ ID NO:

24. (d) The first polypeptide comprises the sequence shown in SEQ ID NO: 78, the second polypeptide comprises the sequence shown in SEQ ID NO: 79, the third polypeptide comprises the sequence shown in SEQ ID NO: 80, the fourth polypeptide comprises the sequence shown in SEQ ID NO: 81, or (e) The first polypeptide comprises the sequence shown in SEQ ID NO: 82, the second polypeptide comprises the sequence shown in SEQ ID NO: 83, the third polypeptide comprises the sequence shown in SEQ ID NO: 84, and the fourth polypeptide comprises the sequence shown in SEQ ID NO:

85. The manipulated antibody according to claim 8.

13. The engineered antibody according to claim 1, wherein one or more conjugate portions are linked, the conjugate portions comprising agents for detection or isolation, such as clearance modifiers, chemotherapeutic agents, toxins, radioisotopes, lanthanides, luminescence labels, fluorescent labels, enzyme substrate labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binders, or other anticancer agents.

14. An isolated polynucleotide encoding the manipulated antibody according to claim 1.

15. A vector comprising the isolated polynucleotide described in claim 14.

16. A host cell comprising the vector according to claim 15.

17. (i) the manipulated antibody according to claim 1, or the polynucleotide encoding the manipulated antibody according to claim 1, (ii) one or more pharmaceutically acceptable carriers, diluents, buffers or excipients, Depending on the circumstances, (iii) Additional therapeutic agents, A pharmaceutical composition further comprising, In some cases, the additional therapeutic agent is a drug for treating HER2-related disease or disorder, wherein the HER2-related disease or disorder is a cancer selected from the group consisting of breast cancer, prostate cancer, lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, pancreatic cancer, testicular cancer, urothelial carcinoma, endometrial cancer, malignant melanoma, and soft tissue cancer (e.g., synovial sarcoma). The aforementioned pharmaceutical composition.

18. A method for expressing an engineered antibody according to any one of claims 1 to 13, comprising culturing host cells under conditions in which a vector is expressed.

19. A pharmaceutical composition for use in a method of treating, preventing or alleviating a target disease or disorder, comprising an engineered antibody according to any one of claims 1 to 13 and / or an isolated polynucleotide according to claim 14, wherein the method comprises administering to the target a therapeutically effective amount of the engineered antibody and / or the polynucleotide encoding the engineered antibody, In some cases, the aforementioned disease or disorder may be a HER2-related disease or disorder. Depending on the circumstances, the subject may be a human being, and / or The aforementioned administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration. The aforementioned pharmaceutical composition.

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