WO2020088605A1 - 针对cd19和cd3的同源二聚体型双特异性抗体及其制备方法和用途 - Google Patents

针对cd19和cd3的同源二聚体型双特异性抗体及其制备方法和用途 Download PDF

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WO2020088605A1
WO2020088605A1 PCT/CN2019/114808 CN2019114808W WO2020088605A1 WO 2020088605 A1 WO2020088605 A1 WO 2020088605A1 CN 2019114808 W CN2019114808 W CN 2019114808W WO 2020088605 A1 WO2020088605 A1 WO 2020088605A1
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amino acid
seq
bispecific antibody
cells
sequences
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PCT/CN2019/114808
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French (fr)
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李强
贾世香
马心鲁
赵丽丽
崔雪原
张玉华
刘雪梅
张贵民
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安源医药科技（上海）有限公司
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Priority to US17/290,753 priority Critical patent/US20220002407A1/en
Priority to CN201980071466.1A priority patent/CN112996807B/zh
Priority to EP19879790.4A priority patent/EP3875479A4/en
Publication of WO2020088605A1 publication Critical patent/WO2020088605A1/zh

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Definitions

  • the present invention relates to the field of immunology, and more specifically, to an anti-CD3 bispecific antibody that mediates T cell killing, and the use of such antibodies, especially in the treatment of cancer, autoimmunity, inflammatory diseases, and immune exhaustion. Different uses.
  • the body eliminates aliens through innate immunity and adaptive immunity to maintain the stability of the internal environment, while adaptive immunity performs its immune function through lymphocyte B cells and T cells.
  • B lymphocytes One of the roles of B lymphocytes is to directly kill target cells through antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Many membrane molecules on the surface of B cells such as mIg, CD79a / CD79b, BCR (B cell receptor), CD19, CD40, etc., play an important role in the process of B cell recognition of antigens, activation, proliferation, and secretion of antibodies.
  • BCR is especially necessary for the specific activation of B cell antigens.
  • CTL is the most potent effector cell of the immune system, which is produced by the proliferation and differentiation of the initial T cells that are effectively activated after being stimulated by the antigen.
  • the initial T cell recognition antigen needs to be presented by APC processing.
  • the combination of the T cell receptor TCR-CD3 complex and the MHC-antigen complex distributed on the surface of the two constitute the first signal to effectively activate T cells.
  • the CD3 molecule is a protein expressed on the surface of all mature T lymphocytes. By combining with the TCR to form a TCR-CD3 complex, it initiates a humoral immune response to antigen stimulation, thus becoming a mediator of immune cell (such as T cells, NK cells, etc.) The most commonly used effector cell trigger molecule in bispecific antibodies.
  • CD3 can recruit CTL cells with killing effect.
  • the bifunctional antibody targeting CD3 binds CD3 molecules on the surface of T cells and surface antigens of target cells to make CTL directly contact with target cells, thereby activating T cells and inducing them to kill target cells effectively .
  • the first-generation clinical anti-CD3 monoclonal antibody OKT3 Kerat P et al., Science, 206: 347-349, 1979
  • due to the excessive activation of T cells and the massive release of inflammatory factors will cause serious clinical "Cytokine Storm Syndrome” (Hirsch R et al., J. Immunol., 142: 737-743, 1989). Therefore, to develop bifunctional antibodies targeting CD3, how to weaken or avoid excessive cytokine storms is the primary consideration.
  • bispecific antibodies that contain an Fc domain has a longer half-life due to FcRn-mediated endocytosis and recycling processes; while retaining some or all of Fc-mediated effector functions, such as ADCC, complement Cytotoxicity-dependent (CDC) and antibody-dependent cell phagocytosis (ADCP); and has better solubility and stability, so it has been widely used in the development of double-antibody drugs, including Triomabs, kih IgG, Crossmab, ortho-Fab IgG , DVD, IgG, IgG scFv, scFv2-Fc, etc.
  • IgG-like anti-CD3 bispecific antibodies is widely used in monovalent form, mainly because bivalent anti-CD3 bispecific antibodies can easily lead to excessive activation and induce T cell apoptosis and transient release of a large number of cytokines (Kuhn C, etc., Immunotherapy , 8: 889-906, 2016), may even trigger a non-antigen-dependent activation of T cell responses and break the immune balance.
  • cytokines Kuhn C, etc., Immunotherapy , 8: 889-906, 2016
  • hybridoma cells are used to express such heterodimers with an asymmetric structure, many closely related but difficult-to-remove non-functional mismatch by-products are simultaneously produced.
  • IgG-like anti-CD3 bispecific antibodies may have unrestricted T cell activation due to their ability to bind Fc ⁇ R, and such activated T cells are found in any tissue expressing Fc ⁇ R (eg, in hematopoietic, lymphatic, and net) Within the endothelial system), even if it does not bind to the target antigen.
  • This systemic activation of T cells will be accompanied by a large amount of cytokine release, leading to serious adverse reactions during therapeutic application. Therefore, the development of such anti-CD3 bispecific antibodies that mediate T cell killing requires only limited activation of immune effector cells within target cells.
  • CD19 (Differentiation Cluster 19), also known as B4 or Leu-12, is a specific marker protein on the surface of B lymphocytes and is expressed in all stages of human B lymphocyte development except for stem cells and plasma cells.
  • CD19 forms a B-cell co-receptor complex with CD21 (CD2) and CD81 (TAPA-1), establishes B-cell signal thresholds by regulating B-cell receptor (BCR) dependent and non-dependent signals, and promotes B-cell proliferation Differentiation and signal transduction play an important regulatory role.
  • the regulation of CD19 complex on the activation and proliferation of B cells is accomplished by cross-linking two other B cell signal transduction complexes, and the corresponding enhancement or inhibition is exerted according to the amount of cross-linking of the two.
  • CD19 antigen is widely expressed in B lymphocyte malignancies, including, for example, acute / chronic lymphocytic leukemia (ALL / CLL), non-Hodgkin's lymphoma (NHL), melanoma, myeloma (Nicholson IC, etc., Mol. Immun. , 34: 1157-1165, 1997; Nigel PM, etc., eCancer Med. Sci., 11: 720, 2017; Grossbard ML, etc., Br.J.
  • ALL / CLL acute / chronic lymphocytic leukemia
  • NHL non-Hodgkin's lymphoma
  • melanoma myeloma
  • the purpose of the present invention is to provide a tetravalent, homodimeric bispecific antibody molecule targeting immune effector cell antigen CD3 and tumor-associated antigen (Tumor-Associated Antigen, TAA) CD19.
  • This bispecific antibody is in vivo It can significantly inhibit or kill tumor cells, but the non-specific killing effect on normal cells with low expression of TAA is significantly reduced, and it has the control of toxic and side effects that may be caused by excessive activation of effector cells, and its physical and chemical and in vivo stability are significantly improved .
  • a bispecific antibody against CD19 is provided.
  • the bispecific antibody molecule is composed of two identical polypeptide chains covalently bonded to form a tetravalent homodimer, each polypeptide chain starting from N
  • the end-to-C-end sequentially contains a first single-chain Fv (anti-TAA scFv) that specifically binds to the tumor-associated antigen CD19, a second single-chain Fv (anti-CD3 scFv) that specifically binds to the effector cell antigen CD3, and an Fc fragment; wherein The first and second single-chain Fv are connected by a connecting peptide, while the second single-chain Fv is directly connected to an Fc fragment or connected by a connecting peptide, and the Fc fragment does not have an effector function.
  • the first single-chain Fv is specific for tumor-associated antigens, and the VH and VL domains contained therein are connected by a linking peptide (L1), and the VH, L1, and VL are VH-L1-VL or VL-
  • L1-VH is arranged, and the amino acid sequence of the connecting peptide L1 is (GGGGX) n , X includes Ser or Ala, X is preferably Ser; n is a natural number of 1-5, n is preferably 3;
  • the tumor-associated antigen is CD19, including but not limited to: any variant, isoform, derivative and species homolog of CD19; preferably, CD19 is derived from human, cynomolgus monkey or rhesus monkey .
  • Table 1-1 of the present invention lists some preferred amino acid sequences for the VH domain and complementarity determining regions (HCDR1, HCDR2, and HCDR3) of the first single-chain Fv of CD19, and the VL domain and its complementarity Determine the amino acid sequence of the regions (LCDR1, LCDR2 and LCDR3).
  • the first single-chain Fv specifically binds to CD19, and the CDR1, CDR2 and CDR3 contained therein are selected from the group consisting of:
  • the HCDR1, HCDR2, and HCDR3 contained in the VH domain are shown in SEQ ID NO: 1, 2, and 3, respectively, or are substantially the same as any of the above sequences (eg, at least 80%, 85%, 90%, 92 %, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g.
  • VL domain contains LCDR1, LCDR2 and LCDR3 respectively As shown in SEQ ID NO: 4, 5 and 6, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or Sequences that are more highly similar or have one or more amino acid substitutions (eg, conservative substitutions));
  • HCDR1, HCDR2 and HCDR3 contained in the VH domain are shown in SEQ ID NO: 9, 10 and 11, respectively, or are substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92 %, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g.
  • VL domain contains LCDR1, LCDR2 and LCDR3 respectively As shown in SEQ ID NO: 12, 13 and 14, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or Sequences that are more highly similar or have one or more amino acid substitutions (eg, conservative substitutions));
  • HCDR1, HCDR2, and HCDR3 contained in the VH domain are shown in SEQ ID NOs: 17, 18, and 19, respectively, or are substantially the same as any of the above sequences (eg, at least 80%, 85%, 90%, 92 %, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g.
  • VL domain contains LCDR1, LCDR2 and LCDR3 respectively As shown in SEQ ID NO: 20, 13 and 21, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or Sequences that are more highly similar or have one or more amino acid substitutions (eg, conservative substitutions));
  • HCDR1, HCDR2, and HCDR3 contained in the VH domain are shown in SEQ ID NOs: 24, 25, and 26, respectively, or are substantially the same as any of the above sequences (eg, at least 80%, 85%, 90%, 92 %, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g.
  • VL domain contains LCDR1, LCDR2 and LCDR3 respectively As shown in SEQ ID NO: 27, 28 and 29, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or Sequences that are more highly similar or have one or more amino acid substitutions (eg, conservative substitutions));
  • the HCDR1, HCDR2, and HCDR3 contained in the VH domain are shown in SEQ ID NOs: 17, 18, and 19, respectively, or are substantially the same as any of the above sequences (eg, at least 80%, 85%, 90%, 92 %, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g.
  • VL domain contains LCDR1, LCDR2 and LCDR3 respectively As shown in SEQ ID NO: 32, 33 and 21, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or Sequences that are more highly similar or have one or more amino acid substitutions (eg, conservative substitutions).
  • the first single-chain Fv specifically binds to CD19, which is selected from the group consisting of:
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 7, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (eg, conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 8, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence;
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 15, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (eg, conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 16, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence;
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 22, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (for example, conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 23, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence;
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 30, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (for example, conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 31, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence;
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 34, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (for example, conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 35, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence;
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 36, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g. conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 37, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence;
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 38, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g. conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 39, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence.
  • the connecting peptide (L2) connecting the first single-chain Fv and the second single-chain Fv of the present invention is composed of a flexible peptide and a rigid peptide.
  • the flexible peptide contains 2 or more amino acids, and is preferably selected from the following amino acids: Gly (G), Ser (S), Ala (A), and Thr (T). More preferably, the flexible peptide contains G and S residues.
  • the structural formula of the amino acid composition of the flexible peptide is G x S y (GGGGS) z , where x, y and z are integers greater than or equal to 0, and x + y + z ⁇ 1.
  • the amino acid sequence of the flexible peptide is G 2 (GGGGS) 3 .
  • the rigid peptide is derived from the full-length sequence consisting of amino acids 118 to 145 of the carboxy terminus of the natural human chorionic gonadotropin ⁇ subunit (as shown in SEQ ID NO: 48) or a truncated fragment thereof (hereinafter collectively referred to as For CTP).
  • the CTP rigid peptide comprises 10 amino acids at the N-terminus of SEQ ID NO: 48, namely SSSSKAPPPS (CTP 1 ); or the CTP rigid peptide comprises 14 amino acids at the C-terminus of SEQ ID NO: 48, namely SRLPGPSDTPILPQ (CTP 2 );
  • the CTP rigid peptide includes 16 amino acids at the N-terminus of SEQ ID NO: 48, that is, SSSSKAPPPSLPSPSR (CTP 3 ); in another embodiment, the CTP rigid peptide includes 28 amino acids It starts at the 118th position of human chorionic gonadotropin ⁇ subunit and ends at the 145th position, namely SSSSKAPPPSLPSPSRLPGPSDTPILPQ (CTP 4 ).
  • Table 1-2 of the present invention exemplifies some preferred amino acid sequences of the connecting peptide L2 connecting the first and second single chain Fv.
  • the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 49, the amino acid composition of its flexible peptide is G 2 (GGGGS) 3 , and the amino acid composition of its rigid peptide is SSSSKAPPPS ( That is CTP 1 ).
  • the second single-chain Fv is specific for the immune effector cell antigen CD3, and the VH domain and the VL domain contained therein are connected by a connecting peptide (L3), and the VH, L3, and VL are VH-L3-VL or The sequence of VL-L3-VH is arranged, and the amino acid sequence of the connecting peptide L3 is (GGGGX) n , X includes Ser or Ala, X is preferably Ser; n is a natural number of 1-5, n is preferably 3;
  • the second single-chain Fv of the bispecific antibody binds to effector cells with an EC 50 value of greater than about 10 nM, or greater than 20 nM, or greater than 40 nM, or greater than about 50 nM in an in vitro FACS binding assay assay; more preferably Specifically, the second single-chain Fv of the bispecific antibody can not only bind to human CD3, but also specifically bind to CD3 of cynomolgus monkey or rhesus monkey. In a preferred embodiment of the present invention, the bispecific antibody specifically binds to effector cells with an EC 50 value of 15.69 nM.
  • the second single-chain Fv specifically binds to CD3, and the HCDR1, HCDR2, and HCDR3 contained in the VH domain thereof are shown in SEQ ID NOs: 40, 41, and 42, respectively, or are substantially the same as any of the above sequences (For example, a sequence of at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (eg, conservative substitutions)); LCDR1, LCDR2 and LCDR3 contained in the VL domain are shown in SEQ ID NO: 43, 44 and 45, respectively, or are substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92% , 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (eg, conservative substitutions)).
  • the second single-chain Fv specifically binds to CD3, and the VH domain thereof contains the amino acid sequence shown in SEQ ID NO: 46, or is substantially the same as any of the above sequences (eg, at least 80%, 85 %, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g., conservative substitutions)); and its VL domain contains such as SEQ ID NO: the amino acid sequence shown in 47, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more) Sequences that are similar or have one or more amino acid substitutions (eg, conservative substitutions).
  • the Fc fragment is directly connected to the second single-chain Fv.
  • the Fc fragment of the present invention comprises a hinge region derived from the constant region of a human immunoglobulin heavy chain, CH2 and CH3 domains.
  • the Fc fragment of the present invention is derived from, for example, selected Heavy chain constant regions from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; specifically selected from, for example, human IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions, and more particularly from human The heavy chain constant region of IgG1 or IgG4; and, the Fc fragment has one or more amino acid substitutions, deletions, or additions compared to the natural sequence from which it is derived (eg, up to 20, up to 15, up to 10 , Or up to 5 substitutions, deletions or additions).
  • the Fc fragment is altered, for example, mutated, to modify the properties of the bispecific antibody molecule of the invention (eg, alter one or more of the following characteristics: Fc receptor binding, antibody Glycosylation, effector cell function or complement function).
  • the bispecific antibodies provided by the present invention comprise Fc variants having amino acid substitutions, deletions, or additions with altered effector functions (eg, reduction or elimination).
  • the Fc region of an antibody mediates several important effector functions, such as ADCC, ADCP, and CDC.
  • Methods for changing the affinity of an antibody to effector ligands (such as Fc ⁇ R or complement C1q) by replacing amino acid residues in the Fc region of an antibody, thereby changing the effector function are known in the art (see, for example, EP 388,151A1 ; US564,8260; US562,4821; Natsume A, etc., Cancer Res., 68: 3863-3872, 2008; Idusogie EE, etc., J.
  • amino acid L235 (EU numbering) on the constant region of the antibody is modified to alter the Fc receptor interaction, such as L235E or L235A.
  • amino acids 234 and 235 on the constant region of the antibody are modified simultaneously, such as L234A and L235A (L234A / L235A) (EU numbering).
  • the bispecific antibodies provided by the present invention may include Fc variants with amino acid substitutions, deletions, or additions that have an extended circulating half-life.
  • Fc variants with amino acid substitutions, deletions, or additions that have an extended circulating half-life.
  • M252Y / S254T / T256E, M428L / N434S or T250Q / M428L can extend the half-life of antibodies in primates.
  • More mutation sites included in Fc variants with enhanced binding affinity to neonatal receptors (FcRn) can be found in Chinese invention patents CN 201280066663.2, US 2005 / 0014934A1, WO 97/43316, US 5,869,046, US 5,747,03, WO 96/32478.
  • amino acid M428 (EU numbering) on the antibody constant region is modified to enhance the binding affinity of the FcRn receptor, such as M428L.
  • amino acids 250 and 428 (EU numbering) on the constant region of the antibody are modified simultaneously, such as T250Q and M428L (T250Q / M428L).
  • the bispecific antibodies provided by the present invention may also include Fc variants having amino acid substitutions, deletions, or additions that can reduce or eliminate Fc glycosylation.
  • Fc variants contain reduced glycosylation of N-linked glycans normally present at amino acid position 297 (EU numbering).
  • EU numbering amino acid position 297
  • the glycosylation at position N297 has a great influence on the activity of IgG. If the glycosylation at this site is removed, it will affect the conformation of the upper half of the CH2 of the IgG molecule, thereby losing the ability to bind Fc ⁇ Rs and affecting antibody-related organisms. active.
  • the amino acid N297 (EU numbering) on the human IgG constant region is modified to avoid glycosylation of the antibody, such as N297A.
  • the bispecific antibodies provided by the present invention may also include Fc variants with amino acid substitutions, deletions, or additions that eliminate charge heterogeneity.
  • Fc variants with amino acid substitutions, deletions, or additions that eliminate charge heterogeneity.
  • Various post-translational modifications that occur during the expression of engineered cells can cause charge heterogeneity in monoclonal antibodies, and the heterogeneity of lysine at the C-terminus of IgG antibodies is one of the main reasons. Lysine at the C-terminus of heavy chains Acid K may be lost in a certain proportion during antibody production, resulting in charge heterogeneity, which affects the stability, effectiveness, immunogenicity or pharmacokinetics of the antibody.
  • K447 (EU numbering) at the C-terminus of the IgG antibody is removed or deleted to eliminate the charge heterogeneity of the antibody and improve the uniformity of the expressed product.
  • the amino acid sequences of some preferred Fc fragments are exemplified in Tables 1-3 of the present invention.
  • the Fc fragment contained in the bispecific antibody provided by the present invention shows reduced affinity for at least one of human Fc ⁇ Rs (Fc ⁇ RI, Fc ⁇ RIIa, or Fc ⁇ RIIIa) and C1q , With reduced effector cell function or complement function.
  • the Fc fragment contained in the bispecific antibody is derived from human IgG1 and has L234A and L235A substitutions (L234A / L235A), showing reduced binding ability to Fc ⁇ RI; in addition, the present invention provides
  • the Fc fragment contained in the bispecific antibody may also contain amino acid substitutions that have altered one or several other characteristics (eg, the ability to bind to the FcRn receptor, antibody glycosylation or antibody charge heterogeneity, etc.).
  • the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 54, which has L234A / L235A / T250Q / N297A / P331S / M428L compared to the natural sequence from which it was derived Amino acid substitutions or substitutions, and K447 was deleted or deleted.
  • the bispecific antibody binds to human CD19 and CD3, and its amino acid sequence is as follows:
  • sequence shown in SEQ ID NO: 55 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences with at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) are conservative substitutions.
  • amino acid sequence is as follows:
  • sequence shown in SEQ ID NO: 57 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences with at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) are conservative substitutions.
  • amino acid sequence is as follows:
  • substitutions described in (ii) are conservative substitutions.
  • amino acid sequence is as follows:
  • sequence shown in SEQ ID NO: 61 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences with at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) are conservative substitutions.
  • a DNA molecule encoding the above bispecific antibody is provided.
  • the DNA molecule encoding the bispecific antibody is represented by the nucleotide sequence shown in SEQ ID NO: 56. In a preferred embodiment of the present invention, the DNA molecule encoding the bispecific antibody is represented by the nucleotide sequence shown in SEQ ID NO: 58.
  • the DNA molecule encoding the bispecific antibody is represented by the nucleotide sequence shown in SEQ ID NO: 60.
  • the DNA molecule encoding the bispecific antibody is represented by the nucleotide sequence shown in SEQ ID NO: 62.
  • a vector comprising the above DNA molecule is provided.
  • a host cell comprising the above vector
  • the host cell comprises prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells.
  • a pharmaceutical composition comprising the aforementioned bispecific antibody, or the aforementioned DNA, or the aforementioned nucleotide sequence, and a pharmaceutically acceptable excipient, carrier, or diluent.
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug used to treat immune-related diseases.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity.
  • the additional pharmaceutically active agent is a drug used to treat autoimmune diseases or inflammatory diseases.
  • the additional pharmaceutically active agent is a drug used to treat a disease or condition associated with transplant rejection.
  • the aforementioned bispecific antibody, or the aforementioned DNA, or the aforementioned nucleotide sequence and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.
  • the pharmaceutical active agent is administered before, after or simultaneously with the above pharmaceutical composition to the subject, wherein the pharmaceutical active agent is selected from antibodies, antibody fragments, drugs, enzymes, cytotoxic agents, toxins, antibiotics, hormones, immunity Regulators, cytokines, chemokines, and radioisotopes.
  • the pharmaceutical active agent is selected from antibodies, antibody fragments, drugs, enzymes, cytotoxic agents, toxins, antibiotics, hormones, immunity Regulators, cytokines, chemokines, and radioisotopes.
  • the drug in the above pharmaceutical composition is selected from the group consisting of cyclophosphamide, nimustine, methotrexate, fluorouracil, capecitabine, gemcitabine, tegio, pemetrexed, fluoride Dalabine, doxorubicin, bleomycin, vinorelbine, paclitaxel, docetaxel, irinotecan, tamoxifen, letrozole, exemestane, fulvestrant, gosere Lin, medroxyprogesterone, cisplatin, carboplatin, platinum oxalate, nedaplatin, oxaliplatin, asparaginase, loproplatin, etoposide, vincristine, irinotecan, tegafur, da Carbazine, mitomycin, teniposide, pirarubicin, mitoxantrone, vindesine, raltitrexed,
  • the toxin in the above-mentioned pharmaceutical composition is selected from the group consisting of gaba toxin, acacia toxin, alpha toxin, saponin, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, Pokeweed antiviral protein, white tree toxin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas endotoxin.
  • the immunomodulator in the above pharmaceutical composition is selected from the group consisting of: cytokines, chemokines, stem cell growth factors, lymphotoxins, colony stimulating factors (CSF), interleukins (IL), erythropoietin , Platelet growth factor, tumor necrosis factor (TNF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon- ⁇ , interferon- ⁇ , interference Interferon- ⁇ or interferon- ⁇ , TGF- ⁇ , TGF- ⁇ , interleukin-1 (IL-1), IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25, LIF, FLT-3, vascular endot
  • the above pharmaceutical composition can be administered in combination with one or more additional other therapies, wherein the other therapies are selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, Viral therapy, adjuvant therapy and their combination.
  • the other therapies are selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, Viral therapy, adjuvant therapy and their combination.
  • a method for enhancing or stimulating an immune response or function comprising administering to the individual a therapeutically effective amount of the bispecific antibody, or the DNA, or the nucleoside Acid sequence, or the pharmaceutical composition.
  • a method for treating, preventing or ameliorating an immune disorder or disease in a cell, tissue, organ or animal which comprises administering to the individual a therapeutically effective amount of the bispecific antibody , Or the DNA, or the nucleotide sequence, or the pharmaceutical composition.
  • a method for preventing / treating a disease, delaying its progress, and reducing / suppressing its recurrence comprising an immune-related disease, a tumor, an autoimmune disease, an inflammatory disease, or a transplant Diseases or disorders such as allergy-related diseases or disorders, which include administering or administering an effective amount of the bispecific antibody, or the DNA, or the nucleotide sequence, or the pharmaceutical composition to the patient Individuals with the above diseases or conditions.
  • the above method can be administered in combination with one or more additional other therapies, wherein the other therapies are selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy , Adjuvant therapy and its combination.
  • the other therapies are selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy , Adjuvant therapy and its combination.
  • a method for preparing the bispecific antibody of the present invention includes: (a) obtaining a fusion gene of the bispecific antibody and constructing an expression vector of the bispecific antibody; (b) by the gene An engineering method is used to transfect the above-mentioned expression vector into a host cell; (c) to cultivate the above-mentioned host cell under conditions allowing the production of the bispecific antibody; (d) to isolate and purify the produced antibody.
  • the expression vector in step (a) is selected from one or more of plasmids, bacteria and viruses.
  • the expression vector is PCDNA3.1.
  • the constructed vector is transfected into a host cell by a genetic engineering method, and the host cell includes prokaryotic cells, yeast, or mammalian cells, such as CHO cells, NS0 cells, or other mammalian cells, preferably CHO cells.
  • the host cell includes prokaryotic cells, yeast, or mammalian cells, such as CHO cells, NS0 cells, or other mammalian cells, preferably CHO cells.
  • step (d) uses conventional immunoglobulin purification methods, including protein A affinity chromatography and ion exchange, hydrophobic chromatography or molecular sieve methods to separate and purify the bispecific antibody.
  • the use of the bispecific antibody, or the DNA, or the nucleotide sequence, in the preparation of a medicament for treating, preventing, or alleviating tumors examples of the cancer include but not Limited to: acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute B-lymphocytic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), B-cell lymphoma (BCL), T Cellular lymphoma (TCL) (e.g.
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • B-ALL acute B-lymphocytic leukemia
  • B-CLL chronic B-lymphocytic leukemia
  • BCL B-cell lymphoma
  • TCL T Cellular lymphoma
  • FL Follicular lymphoma
  • B-PLL juvenile B-lymphocytic leukemia
  • MCL mantle cell lymphoma
  • LPL lymphoplasmacytic lymphoma
  • WM Waldens Tren macroglobulinemia
  • ALL lymphoblastic leukemia
  • ALL lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • B cells plasmablastic lymphoma (PBL) (e.g. B Cells), Hodgkin ’s lymphoma, non-Hodgkin ’s lymphoma, diffuse large B-cell lymphoma (DLBCL) (eg, primary site or inflammation-related), Kitt's lymphoma (BL), multiple myeloma, anaplastic large cell lymphoma and HIV-related lymphoma.
  • PBL plasmablastic lymphoma
  • Hodgkin ’s lymphoma e.g. B Cells
  • non-Hodgkin ’s lymphoma non-Hodgkin ’s lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • Kitt's lymphoma BL
  • multiple myeloma anaplastic large cell lymphoma and HIV-related lymphoma.
  • autoimmune or inflammatory diseases Autoimmune disease or inflammatory disease is selected from: rheumatoid arthritis (RA), osteoarthritis, reactive arthritis, systemic lupus erythematosus (SLE), Crohn's disease, multiple sclerosis, scleroderma Disease, psoriasis, psoriatic arthropathy, ulcerative colitis (eg, chronic), insulin-dependent diabetes (eg, adolescents), thyroiditis (eg, chronic), hyperthyroidism, asthma, allergic disease, sarcoidosis , Autoimmune hemolytic anemia, pernicious anemia, graft-versus-host disease, dermatomyositis, chronic hepatitis, microscopic renal vasculitis, chronic active hepatitis, uveitis, intestinal syn
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • Crohn's disease multiple sclerosis
  • transplant rejection includes acute, superacute, or chronic transplant rejection.
  • the transplant rejection includes organ, tissue, or cell transplant rejection, including but not limited to blood transfusion, vascular transplant, epithelial transplant, endothelial transplant, muscle transplant, Connective tissue transplantation, joint transplantation, heart transplantation, lung transplantation, liver transplantation, kidney transplantation, pancreas transplantation, skin transplantation, intestinal transplantation, corneal transplantation, bone marrow transplantation, graft versus host disease and host versus graft disease.
  • a method for detecting the presence of cancer in a mammal which includes: (a) combining a sample containing one or more cells derived from a mammal with the bispecific antibody, or the DNA, or the nucleotide sequence contacts, thereby forming a complex, and;
  • kits containing the bispecific antibody of the present invention, or the DNA, or the nucleotide sequence.
  • a monoclonal antibody that binds to CD19 and an antigen-binding fragment thereof.
  • the variable region of the monoclonal antibody is derived from the first single-chain Fv of the bispecific antibody described above, and includes CDR1.
  • CDR2 and CDR3 are selected from the group:
  • the HCDR1, HCDR2, and HCDR3 contained in the VH domain are shown in SEQ ID NOs: 17, 18, and 19, respectively, or are substantially the same as any of the above sequences (eg, at least 80%, 85%, 90%, 92 %, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g.
  • VL domain contains LCDR1, LCDR2 and LCDR3 respectively As shown in SEQ ID NO: 32, 33 and 21, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or Sequences that are more highly similar or have one or more amino acid substitutions (eg, conservative substitutions));
  • HCDR1, HCDR2, and HCDR3 contained in the VH domain are shown in SEQ ID NOs: 17, 18, and 19, respectively, or are substantially the same as any of the above sequences (eg, at least 80%, 85%, 90%, 92 %, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g.
  • VL domain contains LCDR1, LCDR2 and LCDR3 respectively As shown in SEQ ID NO: 20, 13 and 21, or substantially the same as any of the above sequences (eg at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or Sequences that are more highly similar or have one or more amino acid substitutions (eg, conservative substitutions).
  • the monoclonal antibody is a humanized antibody or a fully human antibody.
  • the antigen-binding fragment is selected from scFv, Fab, Fab ', (Fab') 2, Fv fragment, disulfide-linked Fv (dsFv).
  • VH domain and VL domain included in the CD19-binding monoclonal antibody or antigen-binding fragment thereof are selected from the group consisting of:
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 36, or is substantially the same as any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g. conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 37, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence;
  • the VH domain contains the amino acid sequence shown in SEQ ID NO: 38, or is substantially identical to any of the above sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or having one or more amino acid substitutions (e.g. conservative substitutions)); and its VL domain contains the amino acid sequence shown in SEQ ID NO: 39, or the above Any of the sequences are substantially identical (e.g. at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more highly similar or have one or more amino acid substitutions (e.g. conservative sexual substitution)) sequence.
  • the anti-TAA scFv contained in the bispecific antibody provided by the present invention is located at the N-terminus of the dual antibody, the spatial conformation changes, and the binding ability with TAA may be weakened under certain conditions, especially difficult to bind weak or low expression Normal cells of TAA have reduced non-specific killing, but the binding specificity of cells overexpressing or highly expressing TAA has not decreased significantly, showing good killing effect in vivo.
  • the target antigen when the target antigen is only expressed on tumor cells or the bispecific antibody of the present invention only specifically binds to tumor cells overexpressing the target antigen, the immune effector cells are restricted to only within the target cell tissue Activation, which allows the non-specific killing of normal cells by the bispecific antibody and the accompanying release of cytokines can be minimized, reducing their toxic and side effects in clinical treatment.
  • the anti-CD3 scFv selected by the bispecific antibody provided by the present invention specifically binds to effector cells with weak binding affinity (EC 50 value greater than approximately greater than approximately 10 nM, or greater than 20 nM, or greater than 40 nM, or greater than approximately 50 nM)
  • weak binding affinity EC 50 value greater than approximately greater than approximately 10 nM, or greater than 20 nM, or greater than 40 nM, or greater than approximately 50 nM
  • the antigen-binding domain of scFv this steric effect makes it bind to CD3 with a weaker binding affinity (eg, greater than 1 ⁇ M), which weakens its ability to activate and stimulate T cells, thus limiting excessive release of cytokines, Therefore, it has higher safety;
  • the anti-CD3 scFv used in the present invention can simultaneously bind to the CD3 natural antigens of human and cyn
  • the bispecific antibody provided by the present invention creatively uses a bivalent anti-CD3 scFv, which makes the bispecific antibody circumvent the heterodimer type commonly used in the prior art in terms of configuration design ( The included anti-CD3 scFv is monovalent), so there is no problem of mismatch between heavy chains, which simplifies the downstream purification steps; and unexpectedly, no anti-CD3 scFv was observed in the in vitro cell binding assay Non-specific binding with T cells, and the degree of cell activation (release of IL-2 and other cytokines) is controlled within a safe and effective range, that is, the bivalent anti-CD3 scFv structure used in the present invention does not cause non-antigen-dependent Induces excessive activation of T cells, and for other bispecific antibodies containing bivalent anti-CD3 domains, it is common for T cells to be un
  • the modified Fc fragment contained in the bispecific antibody provided by the present invention does not have Fc ⁇ R binding ability, avoiding systemic activation of T cells mediated by Fc ⁇ R, thus allowing immune effector cells to be restricted only to target cells Is activated within the organization.
  • the bispecific antibody provided by the present invention is a homodimer type, there is no problem of heavy chain and light chain mismatch, the downstream production process is stable, the purification step is simple and efficient, the expression product is uniform, and its physical and chemical and in vivo stability Have improved significantly.
  • the bispecific antibody provided by the present invention has a longer half-life in vivo circulation due to the inclusion of Fc fragments.
  • FR antibody framework region the immunoglobulin variable region excluding the CDR region
  • V region IgG chain segment with variable sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 residue in the heavy chain.
  • the scientific and technical terms used herein have the meaning commonly understood by those skilled in the art.
  • the antibodies or fragments thereof used in the present invention can be further modified by using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination, and / or other modification methods, alone or in combination. Methods for introducing such modifications in the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: An Experimental Manual, Cold Spring Harbor Laboratory (1989) N.Y. The modifications referred to are preferably performed at the nucleic acid level. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
  • CD19 is a differentiation cluster 19 polypeptide, a single channel type I transmembrane glycoprotein, which contains two Ig-like C2 type (immunoglobulin-like) domains and a relatively large cytoplasmic tail, in mammalian species Highly conservative. CD19 is expressed in almost all B lineage cells and follicular cells, is necessary for B lymphocyte differentiation, and functions as a key B cell co-receptor with CD21, CD81, and CD225. Therefore, CD19 is often used as a biodiagnostic marker for B-lymphocyte development, B-cell lymphoma, and B-lymphocytic leukemia. In addition, mutations in CD19 are associated with severe immunodeficiency syndrome.
  • the indications for the CD19 target also include other related diseases or conditions discovered in the prior art and discovered in the future.
  • the term also includes any variants, isoforms, derivatives, and species homologs of CD19 that are expressed naturally by cells, including tumor cells, or by cells transfected with CD19 gene or cDNA.
  • the CD3 molecule is an important differentiation antigen on the T cell membrane and a characteristic marker of mature T cells. It is composed of 6 peptide chains.
  • the TCR-CD3 complex is composed of a non-covalent bond and a T cell antigen receptor (TCR).
  • TCR-CD3 complex is assembled intracytoplasmically and transmits antigen stimulation signals through the immunoreceptor tyrosine activation motif (Immunoreceptor Tyrosine-based Activation Motif, ITAM) in the cytoplasmic region of each polypeptide chain.
  • ITAM immunoreceptor tyrosine activation motif
  • the main function of the CD3 molecule is to stabilize the TCR structure and transmit T cell activation signals.
  • TCR specifically recognizes and binds to the antigen
  • CD3 participates in transducing the signal into the T cell cytoplasm as the first signal to induce T cell activation. It plays an extremely important role in T cell antigen recognition and immune response generation.
  • CD3 refers to being a part of the T cell receptor complex, consisting of three different chains CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ . Clustering of CD3 on T cells by, for example, the immobilization of anti-CD3 antibodies on it, results in T cell activation, similar to T cell receptor-mediated activation, but does not depend on the specificity of TCR clones. Most anti-CD3 antibodies recognize the CD3 epsilon chain.
  • the second functional domain of the present invention that specifically recognizes the CD3 receptor on the T cell surface is not specifically limited, as long as it can specifically recognize CD3, such as but not limited to the CD3 antibody mentioned in the following patents: US 7,994,289; US 6,750,325; US 6,706,265; US 5,968,509; US 8,076,459; US 7,728,114; US 20100183615.
  • the anti-human CD3 antibody used in the present invention is cross-reactive with cynomolgus monkey and / or rhesus monkey, such as but not limited to the anti-human CD3 antibody mentioned in the following patents: WO2016130726, US20050176028, WO2007042261 Or WO2008119565.
  • the term also includes any CD3 variants, isoforms, derivatives and species homologues, which are naturally expressed by the cell or expressed on cells transfected with the gene or cDNA encoding the aforementioned chain.
  • antibody generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. Typical examples of antibodies are immunoglobulins, and derivatives or functional fragments thereof, as long as they show the desired binding specificity. Techniques for preparing antibodies are well known in the art. "Antibodies” include different classes of natural immunoglobulins (eg, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgG1, lgG2, IgA1, IgA2, etc.).
  • Antibodies also include non-natural immunoglobulins, including, for example, single-chain antibodies, chimeric antibodies (eg, humanized murine antibodies) and heteroconjugate antibodies (eg, bispecific antibodies), and antigen-binding fragments thereof ( For example, Fab ', F (ab'), Fab, Fv and rIgG). See also, for example, Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL; Kuby, J., Immunology, 3rd Ed., 1998, NY; W.H. Freeman & Co ..
  • Antibodies can bind to one antigen, called “monospecific”; or to two different antigens, called “bispecific”; or to more than one different antigen, called “multispecific” ".
  • Antibodies can be monovalent, bivalent or multivalent, ie antibodies can bind to one, two or more antigen molecules at a time.
  • Antibodies "monovalently” bind to a specific protein, that is, one molecule of antibody only binds to one molecule of protein, but the antibody can also bind to different proteins. When an antibody binds only to one molecule of two different proteins, the antibody binds "monovalently” to each protein, and the antibody is “bispecific” and “monovalently” binds to two different proteins every kind.
  • An antibody can be “monomeric”, ie it contains a single polypeptide chain.
  • Antibodies may contain multiple polypeptide chains (“multimeric”) or may contain two (“dimeric”), three (“trimeric”), or four ("tetrameric") Polypeptide chain.
  • the antibody may be a homomulitmer, ie the antibody contains more than one molecule of only one polypeptide chain, including homodimers, homotrimers or homologs Source tetramer.
  • the multimeric antibody may be a heteromultimer, that is, the antibody contains more than one different polypeptide chain, including heterodimer, heterotrimer, or heterotetramer.
  • variable region or “CDR region” or “complementarity determining region” refers to the antibody amino acid residues responsible for antigen binding, which are non-contiguous amino acid sequences.
  • the CDR region sequence can be defined by the IMGT, Kabat, Chothia, and AbM methods or the amino acid residues in the variable region identified by any CDR region sequence determination method well known in the art.
  • a hypervariable region contains the following amino acid residues: amino acid residues from the "complementarity determining region" or "CDR" defined by sequence alignment, for example, 24-34 (L1), 50- of the light chain variable domain 56 (L2) and 89-97 (L3) residues and heavy chain variable domains 31-35 (H1), 50-65 (H2) and 95-102 (H3) residues, see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, Public Health, Service, National Institutes of Health, Bethesda, Md .; and / or from “hypervariable loops” defined by structure ( HVL) residues, for example, residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and 26-32 (heavy chain variable domain) H1), 53-55 (H2) and 96-101 (H3) residues, see Chothia and Leskl, J.
  • CDR complementarity
  • “Framework” residues or "FR” residues are variable domain residues other than hypervariable region residues as defined herein.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined by the Kabat, Chothia, or IMGT numbering system. Those skilled in the art can explicitly assign each system to any variable domain sequence without relying on any experimental data beyond the sequence itself. For example, the numbering of Kabat residues for a given antibody can be determined by comparing the antibody sequence with each "standard” numbering sequence to the homology region. Based on the numbering of the sequences provided herein, determining the numbering scheme for any variable region sequence in the sequence listing is well within the ordinary skill of those in the art.
  • single-chain Fv antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, and is a heavy chain variable region (VH) and a light chain variable region connected by a linker
  • the recombinant protein of (VL), a linker that cross-links these two domains to form an antigen binding site, and the linker sequence generally consists of a flexible peptide, such as but not limited to G 2 (GGGGS) 3 .
  • the size of scFv is generally 1/6 of a complete antibody.
  • the single chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain.
  • Fab fragment consists of a light chain and a heavy chain CH1 and variable regions.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the size of the "Fab antibody” is 1/3 of the intact antibody, and it contains only one antigen binding site.
  • Fab 'fragment contains a light chain and a heavy chain between the VH domain and the CH1 domain and the constant region between the CH1 and CH2 domains.
  • F (ab ') 2 fragment contains two light chains and two heavy chains, the VH domain and the CH1 domain, and the constant region portion between the CH1 and CH2 domains, thereby forming between the two heavy chains Interchain disulfide bond. Therefore, the F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • Fc region refers to an antibody heavy chain constant region fragment, which comprises at least the hinge region, CH2 and CH3 domains.
  • Fv region includes variable regions from both heavy and light chains, but lacks constant regions, and is the smallest fragment that contains a complete antigen recognition and binding site.
  • Fd fragment consists of the CH1 of a heavy chain and the variable region, and is the portion of the heavy chain remaining after the light chain is removed from the Fab fragment.
  • antibody fragment refers to an antigen-binding fragment and antibody analog of an antibody that retains the specific binding ability to an antigen (eg, CD19), which usually includes at least part of the antigen binding of the parent antibody (Parental Antibody) Zone or variable zone.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed in moles, antibody fragments retain at least 10% of the maternal binding activity. Preferably, the antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the binding affinity of the parent antibody to the target.
  • Antibody fragments include, but are not limited to: Fab fragments, Fab 'fragments, F (ab') 2 fragments, Fv fragments, Fd fragments, complementarity determining region (CDR) fragments, disulfide bond stable proteins (dsFv), etc .; linear antibodies ( Linear Antibody), single chain antibodies (such as scFv monoclonal antibodies) (technology from Genmab), bivalent single chain antibodies, single chain phage antibodies, single domain antibodies (Single Domain Antibody) (such as VH domain antibodies), domain antibodies ( Technology from AbIynx); multispecific antibodies formed by antibody fragments (eg, three-chain antibodies, tetra-chain antibodies, etc.); and engineered antibodies such as chimeric antibodies (Chimeric Antibody) (eg, humanized murine antibodies), heteroconjugation Antibodies (Heteroconjugate Antibody), etc. These antibody fragments are obtained by conventional techniques known to those skilled in the art, and these fragments are screened for utility by the same
  • connecting peptide refers to a peptide connecting two polypeptides, wherein the connecting peptide may be two immunoglobulin variable regions or one variable region.
  • the linker peptide can be 0-30 amino acids or 0-40 amino acids in length. In some embodiments, the linker peptide may be 0-25, 0-20, or 0-18 amino acids in length. In some embodiments, the linker peptide may be a peptide no longer than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids long. In other embodiments, the linker peptide may be 0-25, 5-15, 10-20, 15-20, 20-30, or 30-40 amino acids long.
  • the linker peptide may be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long.
  • Linking peptides are known to those skilled in the art.
  • the linker peptide can be prepared by any method in the art.
  • the linker peptide may be of synthetic origin.
  • heavy chain constant region includes amino acid sequences from immunoglobulin heavy chains.
  • the heavy chain constant region-containing polypeptide includes at least one of the following: CH1 domain, hinge (eg, upper hinge region, middle hinge region, and / or lower hinge region) domain, CH2 domain, CH3 domain, or a variant thereof Body or fragment.
  • the antigen-binding polypeptide used in the present application may include a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a part of a hinge domain and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; A polypeptide chain having a CH1 domain, at least a part of a hinge domain, and a CH3 domain, or a polypeptide chain having a CH1 domain, at least a part of a hinge structure, a CH2 domain, and a CH3 domain.
  • the polypeptide of the present application includes a polypeptide chain having a CH3 domain.
  • the antibodies used in this application may lack at least a portion of the CH2 domain (eg, all or a portion of the CH2 domain).
  • the heavy chain constant regions may be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.
  • VH domain includes the amino-terminal variable domain of an immunoglobulin heavy chain
  • CH1 domain includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain.
  • the CH1 domain is adjacent to the VH domain and is the amino terminus of the hinge region of immunoglobulin heavy chain molecules.
  • hinge region includes the part of the heavy chain molecule that connects the CH1 domain to the CH2 domain.
  • the hinge region contains about 25 residues and is flexible, allowing the two N-terminal antigen binding regions to move independently.
  • the hinge area can be divided into three different structural domains: upper, middle, and lower hinge structural domains (Roux KH et al., J. Immunol., 161: 4083, 1998).
  • disulfide bond includes a covalent bond formed between two sulfur atoms.
  • the amino acid cysteine contains a sulfhydryl group, which can form a disulfide bond or bridge with the second sulfhydryl group.
  • the CH1 and CK regions are connected by disulfide bonds and the two heavy chains are connected by two disulfide bonds, at positions 239 and 242 corresponding to the Kabat numbering system (positions 226 or 229, EU numbering system) connection.
  • in vivo half-life refers to the biological half-life of the polypeptide of interest in the circulation of a given animal and represents the time required for the animal to clear half of the amount present in the circulation of the animal from the circulation and / or other tissues.
  • identity is used to refer to the sequence matching between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • each molecule is the same at this position.
  • the "percent identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions for comparison x 100. For example, if 6 of the 10 positions of the two sequences match, the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • comparisons are made when two sequences are aligned to produce maximum identity.
  • Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol., 48: 443-453, which is conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl.
  • Fc region or “Fc fragment” refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the hinge region, the CH2 domain and the CH3 domain, which mediate the interaction between the immunoglobulin and the host tissue or factor Binding, including binding to Fc receptors located on various cells of the immune system (eg, effector cells) or to the first component (C1q) of the classical complement system, includes the native sequence Fc region and the variant Fc region.
  • the Fc region of the human IgG heavy chain is the segment from the amino acid residue at the position of Cys226 or Pro230 to the carboxyl terminal, but the boundary may vary.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may or may not be present.
  • Fc can also refer to this isolated region, or in the case of Fc-containing protein polypeptides, such as "binding proteins containing Fc regions", also known as "Fc fusion proteins” (eg, antibodies or immunoadhesins).
  • the native sequence Fc region of the antibody of the present invention includes mammalian (eg, human) IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.
  • mammalian eg, human
  • the substitution, insertion, and / or deletion of a single amino acid having about 10 amino acids per 100 amino acids in the amino acid sequences of the two Fc polypeptide chains relative to the sequence of the mammalian Fc polypeptide amino acid sequence.
  • the above differences may be changes in Fc that extend half-life, changes that increase FcRn binding, changes that inhibit Fc ⁇ receptor (Fc ⁇ R) binding, and / or changes that reduce or remove ADCC and CDC.
  • the Fc region contains the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the IgM and IgE Fc regions contain the three heavy chain constants in each polypeptide chain Domain (CH domain 2-4).
  • Fc receptor refers to a receptor that binds to the Fc region of an immunoglobulin.
  • the FcR may be a natural sequence human FcR, or it may be an FcR (gamma receptor) that binds an IgG antibody, as well as allelic variants and alternative splicing forms of these receptors.
  • the FcyR family consists of three activated receptors (FcyRI, FcyRIII and FcyRIV in mice; FcyRIA, FcyRIIA and FcyRIIIA in humans) and an inhibitory receptor (FcyRIIb or equivalent FcyRIIB).
  • FcyRII receptors include FcyRIIA ("activated receptor") and FcyRIIB ("inhibited receptor”), which have similar amino acid sequences.
  • the cytoplasmic domain of FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • NK cells selectively express an activated Fc receptor (Fc ⁇ RIII in mice and Fc ⁇ RIIIA in humans), but mice and The inhibitory Fc ⁇ RIIb is not expressed in humans.
  • Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a in terms of the type of activated Fc receptors it binds.
  • the term "FcR” encompasses other FcRs herein, including those that will be identified in the future. Methods for measuring binding to FcRn are known (see, for example, Ghetie V et al., Immunol.
  • Fc receptor or "FcR” also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer RL et al., J. Immunol., 117: 587, 1976; Kim YJ et al., J. Immunol. , 24: 249, 1994).
  • target cell is a cell that binds to an antibody and participates in mediating disease.
  • the target cell may be a cell that is generally involved in mediating an immune response and also mediating disease.
  • the B cells that are usually involved in mediating the immune response may be target cells.
  • the target cells are cancer cells, pathogen-infected cells, or cells involved in mediating autoimmune diseases or inflammatory diseases (eg, fibrotic diseases).
  • the antibody may bind to the target cell by binding to the antigen on the "target cell protein", which is a protein displayed on the surface of the target cell and may be a highly expressed protein.
  • toxin refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells.
  • the term is intended to include radioisotopes (eg, I 131 , I 125 , Y 90 , Re 186 ), chemotherapeutic agents, and toxins (such as enzymatically active toxins of bacterial, fungal, plant, or animal origin), or fragments thereof.
  • cytokine generally refers to a protein released by a cell population that acts as an intercellular medium on another cell or has autocrine effects on cells that produce the protein.
  • cytokines include lymphokines, mononuclear factors; interleukins (“IL”), such as IL-2, IL-6, IL-17A-F; tumor necrosis factors, such as TNF- ⁇ or TNF- ⁇ ; and Other polypeptide factors, such as leukemia inhibitory factor ("LIF").
  • chemotherapy refers to the administration of any chemical agent for therapeutic use in the treatment of diseases characterized by abnormal cell growth.
  • diseases include, for example, tumors, new organisms and cancer, as well as diseases characterized by proliferative growth.
  • “Chemotherapeutic agents” specifically target cells involved in cell division, but not cells that are not involved in cell division. Chemotherapeutic agents directly interfere with processes closely related to cell division, such as DNA replication, RNA synthesis, protein synthesis, or the assembly, breakdown, or function of mitotic spindles, and / or molecules (such as nucleotides or Amino acid) synthesis or stability. Therefore, chemotherapeutic agents have cytotoxic or cytostatic effects on cancer cells and other cells involved in cell division. Combination chemotherapy gives more than one agent to treat cancer.
  • immunobinding and “immunobinding properties” refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen (for which the immunoglobulin is specific).
  • the strength or affinity of the immune binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction, where the smaller the K D value, the higher the affinity.
  • K D equilibrium dissociation constant
  • the immunological binding properties of the selected polypeptide can be quantified using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. Both "association rate constant" (K a or K on ) and “dissociation rate constant” (K d or K off ) can be calculated from the concentration and the actual rate of association and dissociation.
  • cross-reactivity refers to the ability of the antibodies described herein to bind tumor-associated antigens from different species.
  • the antibodies described herein that bind to human TAA can also bind TAA from other species (eg, cynomolgus monkey TAA).
  • Cross-reactivity can be measured by detecting specific reactivity with purified antigens in binding assays (eg, SPR, ELISA), or binding to or physiologically interacting with TAA-expressing cells or otherwise functionally interacting with TAA-expressing cell functions .
  • binding assays eg, SPR, ELISA
  • binding to or physiologically interacting with TAA-expressing cells or otherwise functionally interacting with TAA-expressing cell functions include surface plasmon resonance (eg, Biacore) or similar techniques (eg, Kinexa or Octet).
  • immune response is composed of cells of the immune system (eg T lymphocytes, B lymphocytes, NK cells, antigen presenting cells, macrophages, eosinophils, mast cells, DC cells or neutrophils) and The role of soluble macromolecules produced by immune cells or the liver is produced by any of these cells or liver (including antibodies, cytokines and complements).
  • T lymphocytes eg, T lymphocytes, B lymphocytes, NK cells, antigen presenting cells, macrophages, eosinophils, mast cells, DC cells or neutrophils
  • the role of soluble macromolecules produced by immune cells or the liver is produced by any of these cells or liver (including antibodies, cytokines and complements).
  • This effect leads to the invasion of invasive pathogens, cells or tissues infected by pathogens, cancer Cells, or normal human cells or tissues in the case of pathological inflammation, selectively damage, destroy, or remove them from the body.
  • the immune response includes, for example, activation or suppression
  • “Effector cell” refers to a cell of the immune system that expresses one or more FcRs and mediates one or more effector functions.
  • the cell expresses at least one type of activating Fc receptor, such as human Fc ⁇ RIII, and performs ADCC effector function.
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), NK cells, monocytes, macrophages, neutrophils, and eosinophils.
  • Effector cells also include, for example, T cells. They can be derived from any organism including but not limited to humans, mice, rats, rabbits and monkeys.
  • effector function refers to those biological activities that can be attributed to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region), and it varies with the antibody isotype.
  • antibody effector functions include, but are not limited to: Fc receptor binding affinity, ADCC, ADCP, CDC, down regulation of cell surface receptors (eg, B cell receptors), B cell activation, cytokine secretion, antibodies, and antigens -Half-life / clearance rate of antibody complexes, etc.
  • Methods for changing the effector function of antibodies are known in the art, for example, by introducing mutations in the Fc region.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Ig binds to FcR present on cytotoxic cells (such as NK cells, neutrophils, or macrophages) to make These cytotoxic effector cells specifically bind to the target cells to which the antigen is attached, and then kill the target cells by secreting cytotoxin.
  • cytotoxic cells such as NK cells, neutrophils, or macrophages
  • Methods for detecting ADCC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the antibody to be tested and FcR (eg, CD16a).
  • ADCP antibody-dependent cell-mediated phagocytosis
  • complement dependent cytotoxicity refers to a form of cytotoxicity that activates the complement cascade by binding the complement component C1q to the antibody Fc.
  • Methods for detecting the CDC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the antibody to be tested and the Fc receptor (eg, Clq).
  • pharmaceutically acceptable carrier and / or excipient and / or stabilizer refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient and / or Stabilizers, which are non-toxic to the cells or mammals exposed to them at the dosages and concentrations employed.
  • a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient and / or Stabilizers, which are non-toxic to the cells or mammals exposed to them at the dosages and concentrations employed.
  • pH adjusting agents include but are not limited to phosphate buffer.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include but are not limited to sodium chloride.
  • Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like.
  • Agents for maintaining osmotic pressure include but are not limited to sugar, NaCl and the like.
  • Agents that delay absorption include but are not limited to monostearate and gelatin.
  • Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), and the like.
  • Preservatives include but are not limited to various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredients in the medicine, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , Lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate) and so on.
  • patient and “subject” “individual” “subject” refer to any human or non-human animal, especially human, receiving prophylactic or therapeutic treatment.
  • methods and compositions described herein can be used to treat subjects with cancer.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles and the like.
  • Treatment or “therapy” for a subject means to reverse, alleviate, ameliorate, inhibit, slow down, or prevent the appearance, progression, development, severity, or recurrence of disease-related symptoms, complications, disorders, or biochemical indicators Subjects are subjected to any type of intervention or treatment for the purpose, or to administer active agents to them.
  • autoimmune in the state of immune tolerance, a certain amount of self-reactive T cells and autoantibodies are ubiquitous in the peripheral immune system of all individuals, which is helpful to help clear the aging and degeneration of its own components, to maintain the immune system's own Immune stability has important physiological significance.
  • mutated refers to the replacement, deletion or insertion of one or more of the natural nucleic acid or polypeptide (ie, a reference sequence that can be used to define the wild type) Nucleotides or amino acids.
  • T cell receptor is a special receptor that exists on the surface of T cells, that is, T lymphocytes.
  • T cell receptors in the body exist as a complex of several proteins.
  • T cell receptors usually have two separate peptide chains, usually T cell receptor ⁇ and ⁇ (TCR ⁇ and TCR ⁇ ) chains, and on some T cells T cell receptor ⁇ and ⁇ (TCR ⁇ and TCR ⁇ ).
  • the other proteins in the complex are CD3 proteins: CD3 ⁇ and CD3 ⁇ heterodimers, and most importantly, CD3 ⁇ homodimers with six ITAM motifs.
  • the ITAM motif on CD3 ⁇ can be phosphorylated by Lck, which in turn recruits ZAP-70.
  • Lck and / or ZAP-70 can also phosphorylate tyrosine on many other molecules, especially CD28, LAT and SLP-76, which allows the aggregation of signaling complexes surrounding these proteins.
  • bispecific antibody refers to a bispecific antibody of the present invention, for example, an anti-CD19 antibody or antigen-binding fragment thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (such as TAA, Cytokines and cell surface receptors) to generate bispecific molecules that bind to at least two different binding sites or target molecules.
  • another functional molecule such as another peptide or protein (such as TAA, Cytokines and cell surface receptors) to generate bispecific molecules that bind to at least two different binding sites or target molecules.
  • the antibody of the invention can be functionally linked (eg, by chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other binding molecules, such as another An antibody, antibody fragment, peptide, or binding mimetics to produce a bispecific molecule.
  • bispecific antibody refers to the inclusion of two variable domains or scFv units so that the resulting antibody recognizes two different antigens.
  • Many different forms and uses of bispecific antibodies are known in the art (Chames P, et al., Curr. Opin. Drug, Disc. Dev., 12: 276, 2009; Spiess C, et al., Mol. Immunol., 67: 95-106 , 2015).
  • hCG- ⁇ carboxy terminal peptide is a short peptide derived from the carboxy terminal of the ⁇ -subunit of human chorionic gonadotropin (hCG).
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • TSH thyrotropin
  • hCG chorionic gonadotropin
  • CTP contains 37 amino acid residues, it has 4 O-glycosylation sites, and the sugar side chain terminal is a sialic acid residue. Negatively charged, highly sialylated CTP can resist renal clearance, thereby prolonging the half-life of the protein in the body (Fares FA et al., Proc. Natl. Acad. Sci. USA, 89: 4304-4308, 1992).
  • glycosylation means that oligosaccharides (carbohydrates containing two or more monosaccharides linked together, for example 2 to about 12 monosaccharides linked together) are attached to form glycoproteins. Oligosaccharide side chains are usually connected to the backbone of glycoproteins via N- or O-linkages. The oligosaccharides of the antibodies disclosed herein are usually CH2 domains attached to the Fc region as N-linked oligosaccharides. "N-linked glycosylation” refers to the attachment of carbohydrate moieties to asparagine residues of glycoprotein chains.
  • the technician can recognize that each of the CH2 domains of murine IgG1, IgG2a, IgG2b, and IgG3 and human IgG1, IgG2, IgG3, IgG4, IgA, and IgD has a residue for N-linked glycosylation at residue 297 Single place.
  • the heavy chain and light chain variable regions contained in the antibody of the present invention contain amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibody retains the present invention, such as CD19 ⁇ Desired functional properties of the CD3 bispecific antibody.
  • conservative modification is intended to mean that the amino acid modification does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated advantages. Conservative amino acid substitution refers to the replacement of amino acid residues with amino acid residues having similar side chains.
  • the family of amino acid residues with similar side chains has been described in detail in the art. These families include those with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), and uncharged polar side chains (e.g.
  • glycine Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains e.g. alanine, valine, leucine, isoleucine , Proline, phenylalanine, methionine
  • ⁇ -branched side chains such as threonine, valine, isoleucine
  • aromatic side chains such as tyrosine, phenylalanine , Tryptophan, histidine. Therefore, one or more amino acid residues in the CDR regions of the antibody of the invention can be replaced with other amino acid residues from the same side chain family.
  • Fc variants with altered binding affinity to neonatal receptors (FcRn)
  • FcRn refers to a protein that binds to at least part of the Fc region of an IgG antibody encoded by the FcRn gene.
  • FcRn can be derived from any organism including but not limited to humans, mice, rats, rabbits and monkeys.
  • the functional FcRn protein contains two polypeptides often referred to as heavy and light chains.
  • the light chain is ⁇ -2-microglobulin, and the heavy chain is encoded by the FcRn gene.
  • the present invention relates to antibodies whose binding to FcRn is regulated (modulation includes increasing and decreasing binding). For example, in some cases, increased binding can cause cells to recycle antibodies and thereby prolong, for example, the half-life of therapeutic antibodies. Sometimes, it is desirable to reduce FcRn binding, for example, as a diagnostic or therapeutic antibody containing a radiolabel. In addition, antibodies that show increased binding to FcRn, while binding to other Fc receptors, such as Fc ⁇ Rs, can be used in the present invention.
  • the present application relates to antibodies containing amino acid modifications that modulate the binding force to FcRn. It is of special significance that at lower pH, the binding affinity for FcRn shows an increase, while at higher pH, the binding basically does not show a change.
  • the antibody or functional change that contains the Fc region minimally body.
  • Fc variants with enhanced binding affinity to neonatal receptors (FcRn)
  • the plasma half-life of IgG depends on its binding to FcRn. It generally binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). By studying the binding sites of the two, the binding site of IgG on FcRn was modified to increase its binding capacity at pH 6.0. It has been shown that mutation of some residues of the human Fc ⁇ domain important for binding FcRn can increase serum half-life. It has been reported that mutations in T250, M252, S254, T256, V308, E380, M428, and N434 (EU numbering) can increase or decrease FcRn binding affinity (Roopenian DC et al., Nat. Rev. Immunol., 7: 715-725, 2007 ). Korean Patent No.
  • KR 10-1027427 discloses trastuzumab (Herceptin, Genentech) variants with increased binding affinity for FcRn, and these variants comprise selected from 257C, 257M, 257L, 257N, 257Y, 279Q, One or more amino acid modifications of 279Y, 308F and 308Y.
  • Korean Patent Publication No. KR2010-0099179 provides bevacizumab (Avastin, Genentech) variants and these variants show increased in vivo by amino acid modifications contained in N434S, M252Y / M428L, M252Y / N434S and M428L / N434S half life.
  • Hinton et al also found that T250Q and M428L 2 mutants increased the binding to FcRn by 3 and 7 times, respectively. Mutation of 2 sites at the same time increases the binding by 28-fold. In rhesus monkeys, M428L or T250QM / 428L mutants showed a 2-fold increase in plasma half-life (Hinton PR et al., J. Immunol., 176: 346-356, 2006). More mutation sites included in Fc variants with enhanced binding affinity to neonatal receptors (FcRn) can be found in the Chinese invention patent CN 201280066663.2.
  • Other antibodies cause enhanced affinity to FcRn in the present invention include, but are not limited to point mutations following amino acid modifications: 226,227,230,233,239,241,243,246,259,264,265,267,269,270,276,284,285,288,289,290,291,292,294,298,299,301,302,303,305,307,309,311,315,317,320,322,325,327,330,332,334,335,338,340,342,343,345,347,350,352,354,355,356,359,360,361,362,369,370,371,375,378,382,383,384,385,386,387,389,390,392,393,394,395,396,397,398,399,400,401,403,404,408,411,412,414,415,416,418,419,420,421,422,424,426,433,438,439,440,443,444,44
  • Fc variants with enhanced binding affinity to FcRn also include all other known amino acid modification sites and amino acid modification sites that have not yet been discovered.
  • IgG variants can be optimized to have increased or decreased FcRn affinity, as well as increased or decreased human Fc ⁇ R, including but not limited to Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIc, Fc ⁇ RIIIa, and alleles including them Gene variant Fc ⁇ RIIIb affinity.
  • the Fc ligand specificity of the IgG variant will determine its therapeutic application.
  • the use of a given IgG variant for therapeutic purposes will depend on the epitope or form of the target antigen, and the disease or indication to be treated.
  • enhanced FcRn binding may be more preferred, because enhanced FcRn binding may result in increased serum half-life. Longer serum half-life allows for lower frequency and dose administration during treatment. This property may be particularly preferred when the therapeutic agent is administered in response to an indication requiring repeated administration.
  • reduced FcRn affinity may be particularly preferred when variant Fc is required to have increased clearance or decreased serum half-life, such as when Fc polypeptides are used as imaging agents or radiotherapy agents.
  • the affinity of the polypeptide for FcRn can be evaluated by methods known in the art.
  • FcRn can come from any organism including but not limited to humans, mice, rats, rabbits and monkeys.
  • inhibiting changes in Fc [gamma] R binding refers to one or more insertions, deletions, or substitutions in the Fc polypeptide chain that inhibit the binding of Fc [gamma] RIIA, Fc [gamma] RIIB, and / or Fc [gamma] RIIIA, such as by, for example, based on a competitive binding experiment , Waltham, MA) determination.
  • Fc ⁇ receptor (Fc ⁇ R) binding include L234A, L235A, or any change that inhibits glycosylation at position N297, including any substitution at N297.
  • the bispecific antibody provided by the present invention inhibits changes in Fc ⁇ R binding.
  • the Fc fragments contained therein exhibit reduced affinity for at least one of human Fc ⁇ Rs (Fc ⁇ RI, Fc ⁇ RIIa, or Fc ⁇ RIIIa) and C1q, with reduced effector cell function or complement Features.
  • Fc [gamma] R can be from any organism including but not limited to humans, mice, rats, rabbits and monkeys.
  • half-life extending Fc change refers to a change in the half-life of an Fc polypeptide chain that prolongs the in vivo half-life of a protein that contains an altered Fc polypeptide chain compared to the half-life of an identical Fc protein that contains the same Fc polypeptide, but it does not include the altered .
  • the alteration can be included in the Fc polypeptide chain as part of the bispecific antibody.
  • M428L and N434S are half-life Fc changes and can be used in combination, alone or in any combination. These changes and others are described in detail in US Patent Application Publication 2010/0234575 and US Patent 7,670,600. Portions describing this change in US Patent Application Publication 2010/0234575 and US Patent 7,670,600 are incorporated herein by reference.
  • any substitution at one of the following sites can be considered as a half-life extending Fc change: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428, 430, 434 , 436.
  • Each of these changes or a combination of these changes can be used to extend the half-life of the bispecific antibodies described herein.
  • Other changes that can be used to extend half-life are described in detail in International Application PCT / US2012 / 070146 (Publication No. WO 2013/096221) filed on December 17, 2012. The part of this application describing the above changes is incorporated herein by reference.
  • Fc changes that extend half-life also include sites and modifications that include well-known techniques and may be discovered in the future.
  • Fc can come from any organism including but not limited to humans, mice, rats, rabbits and monkeys.
  • the present invention provides bispecific antibodies, nucleic acids, nucleic acid variants, derivatives and species homologs encoding antibodies, antibody fragments and fusion proteins thereof.
  • nucleic acid sequences encoding immunoglobulin regions comprising VH, VL, hinge, CH1, CH2, CH3 and CH4 regions are known in the art. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD, 1991. According to the teaching provided herein, a person skilled in the art can combine the nucleic acid sequence and / or other nucleic acid sequences known in the art to construct a nucleic acid sequence encoding the bispecific antibody of the present invention. Exemplary nucleotides encoding the bispecific antibody of the present invention include (1) SEQ ID NO: 56, (2) SEQ ID NO: 58, (3) SEQ ID NO: 60, and (4) SEQ ID NO: 62.
  • nucleic acid sequence encoding the bispecific antibody of the present invention can determine the nucleic acid sequence encoding the bispecific antibody of the present invention.
  • companies such as DNA 2.0 (Menlo Park, CA, USA) and Blue Heron (Bothell, WA, USA) usually use chemical synthesis to produce any desired sequence Sort DNA of gene size, thereby simplifying the process of producing the DNA.
  • the bispecific antibodies of the invention can be prepared by any method known in the art. Early methods for constructing bispecific antibodies include chemical cross-linking methods or hybrid hybridoma or tetravalent tumor methods (eg, Staerz UD et al., Nature, 314: 628-31, 1985; Milstein C et al., Nature, 305: 537 -540, 1983; Karpovsky B et al., J. Exp. Med., 160: 1686-1701, 1984).
  • the chemical coupling method is to connect two different monoclonal antibodies by chemical coupling to prepare a bispecific monoclonal antibody. For example, chemical binding of two different monoclonal antibodies, or, for example, chemical binding of two antibody fragments such as two Fab fragments.
  • the hybrid-hybridoma method is to produce bispecific monoclonal antibodies through the method of cell hybridization or ternary hybridoma. These cell hybridomas or ternary hybridomas are fused through the established hybridoma, or the established hybridoma and childhood The lymphocytes obtained from mice are fused. Although these techniques are used to manufacture BiAbs, various production problems make such complexes difficult to use, such as producing mixed populations containing different combinations of antigen binding sites, difficulties in protein expression, the need to purify the target BiAb, low yields, production The cost is high.
  • Recent methods utilize genetically engineered constructs that can produce a homogeneous product of a single BiAb without thorough purification to remove unwanted by-products.
  • Such constructs include tandem scFv, diabody, tandem diabody, dual variable domain antibodies, and heterodimerization using motifs such as Ch1 / Ck domain or DNLTM (Chames & Baty, Curr. Opin. Drug.Discov. Devel., 12: 276-83, 2009; Chames & Baty, mAbs, 1: 539-47).
  • Related purification techniques are well known.
  • the single lymphocyte antibody method can also be used to generate antibodies by cloning and expressing immunoglobulin variable region cDNA produced by a single lymphocyte selected for the production of specific antibodies, for example, by Babcook J et al., Proc. Natl. Acad. Sci. USA. 93: 7843-7848, 1996; WO 92/02551; WO 2004/051268 and WO 2004/106377.
  • Antigen polypeptides used to generate, for example, immunized hosts or for panning for antibodies such as for phage display (or yeast cell or bacterial cell surface expression) can be genetically engineered from a host containing an expression system by methods well known in the art Cells are prepared, or they can be recovered from natural biological sources.
  • nucleic acids encoding one or two polypeptide chains of a bispecific antibody can be introduced into the cultured host cells by various known methods (such as transformation, transfection, electroporation, bombardment with nucleic acid-coated particles, etc.).
  • the nucleic acid encoding the bispecific antibody can be inserted into a vector suitable for expression in the host cell before being introduced into the host cell.
  • the vector may contain sequence elements that enable expression of the inserted nucleic acid at the RNA and protein levels.
  • the host cell containing the nucleic acid can be cultured under conditions that enable the cell to express the nucleic acid, and the resulting BiAb can be collected from a cell population or culture medium.
  • BiAb can be produced in vivo, for example, in plant leaves (see, for example, Scheller J et al., Nature Biotechnol., 19: 573-577, 2001 and references cited therein), in bird eggs (see, eg Zhu et al., Nature Biotechnol., 23: 1159-1169, 2005 and references cited therein), or mammalian milk (see, eg, Laible G et al., Reprod. Fertil. Dev., 25: 315, 2012) .
  • a variety of cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as E. coli or Bacilis stearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), Insect cells (such as Lepidoptera insect cells including Spodoptera frugiperda cells) or mammalian cells (such as Chinese hamster ovary (CHO) cells, NS0 cells, baby hamster kidney (BHK) cells, monkey kidney cells, Hela cells, Human hepatocellular carcinoma cells or 293 cells, etc.).
  • prokaryotic cells such as E. coli or Bacilis stearothermophilus
  • fungal cells such as Saccharomyces cerevisiae or Pichia pastoris
  • Insect cells such as Lepidoptera insect cells including Spodoptera frugiperda cells
  • mammalian cells such as Chinese hamster ovary (CHO) cells,
  • Bispecific antibodies can be prepared by immunizing suitable subjects (eg, rabbits, goats, mice, or other mammals, including transgenic and deleted mammals) by immunogenic preparations of bispecific antigens.
  • a suitable immunogenic preparation may be, for example, a bispecific antigen chemically synthesized or recombinantly expressed.
  • the formulation may further contain an adjuvant, such as Freund's complete or incomplete adjuvant or similar immunostimulating compound.
  • the bispecific antigens of the present invention can be used alone or preferably as a conjugate with a carrier protein. This method of enhancing the antibody response is well known in the art.
  • different animal hosts can be used for in vivo immunization. It is possible to use a host that expresses useful endogenous antigen by itself, or a host that has caused a defect in the useful endogenous antigen.
  • Bispecific antibodies can be prepared by binding the methods described above.
  • the bispecific antibody molecule of the present invention can be used as a monoclonal antibody (MAb) for each target.
  • the antibody is chimeric, humanized, or fully human.
  • Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, Nature, 256: 495-497, 1975), three-source hybridoma technology, human B-cell hybridoma technology (Kozbor D et al., Immunology Today, 4:72, 1983) and EBV-hybridoma technology (Cole, SPC, etc., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan Riss, Inc., 1985).
  • the bispecific antibodies of the invention can be used to detect any or all of these antigens by conventional immunological analysis methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry (For example in biological samples such as serum or plasma).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • tissue immunohistochemistry Form in biological samples such as serum or plasma.
  • the present invention provides a method for detecting an antigen in a biological sample. The method includes: contacting the biological sample with the bispecific antibody of the present invention that specifically recognizes the antigen, or contacting the antibody portion with the antigen, and detecting binding to the antigen Antibody (or antibody portion), or unbound antibody (or antibody portion), thereby detecting the antigen in the biological sample.
  • the antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody.
  • Suitable detectable substances include various enzymes, repair groups, fluorescent substances, luminescent substances and radioactive substances.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase;
  • suitable repair group complexes include streptavidin / biotin and Avidin / Biotin;
  • suitable fluorescent substances include 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, Alkaline Rusin B, dichlorotriazinylamine fluorescein, dan Sulfonyl chloride or phycoerythrin;
  • luminescent substances include 3-aminophthaloyl hydrazine;
  • suitable radioactive substances include I 125 , I 131 , 35 S or 3 H.
  • Bispecific antibodies can bind to a molecule expressed on the surface of immune effector cells (referred to herein as "effector cell protein”) and another molecule expressed on the surface of target cells (referred to herein as “target cell protein”).
  • the immune effector cells may be T cells, NK cells, macrophages or neutrophils.
  • the effector cell protein is a protein contained in the T cell receptor (TCR) -CD3 complex.
  • TCR-CD3 complex is composed of homodimers including TCR ⁇ and TCR ⁇ or TCR ⁇ and TCR ⁇ and from CD3 ⁇ (CD3 ⁇ ) chain, CD3 ⁇ (CD3 ⁇ ) chain, CD3 ⁇ (CD3 ⁇ ) chain and Various CD3 chains such as CD3 ⁇ (CD3 ⁇ ) chain.
  • the effector cell protein may be a human CD3 ⁇ (CD3 ⁇ ) chain, which may be part of a dimeric protein.
  • the effector cell protein may be human and / or cynomolgus monkey and / or rhesus monkey TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ .
  • the bispecific antibody can also bind to the CD3 epsilon chain of non-human species (such as mouse, rat, rabbit, New World monkey, and / or Old World monkey species).
  • the species include, but are not limited to the following mammal species: Musmusculus; Rattus rattus; Rattus norvegicus; Crab-eating macaque; Hamadryasbaboon, Papiohamadryas; Large Baboon (Guineababoon), Guinea baboon (Papiopapio); olive baboon (olivebaboon), East African baboon (Papioanubis); yellow baboon (yellowbaboon), prairie baboon (Papiocynocephalus); Marmoset (Callithrixjacchus); Tamarind Tamarin (Saguinus Oedipus) and Squirrel Monkey (Saimirisciureus).
  • Known from the field of protein therapeutics include, but
  • a partial fragment of the above-mentioned CD3 chain may be bound to the homodimer of the present invention
  • the CD3 may be a human CD3 ⁇ chain or a CD3 ⁇ chain derived from a different species, preferably derived from the above One of the mammal species listed.
  • the epitope to which the antibody binds can be determined by alanine scanning or other well-known techniques, which are described, for example, in US Patent Application Publication 2010/183615, where relevant portions are incorporated herein by reference.
  • the effector cell proteins to which the bispecific antibody can bind include, but are not limited to, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , and TCR ⁇ .
  • NK cells or cytotoxic T cells are immune effector cells, then for example NKG2D, CD352, NKp46 or CD16a may be effector cell proteins.
  • CD 8+ T cells are immune effector cells, then, for example, 4-1BB or NKG2D may be effector cell proteins.
  • the bispecific antibody can bind to other effector cell proteins expressed on T cells, NK cells, macrophages, or neutrophils.
  • Target cells and target cell proteins expressed on target cells are Target cells and target cell proteins expressed on target cells
  • bispecific antibodies can bind to effector cell proteins and target cell proteins.
  • the target cell protein can be expressed on the surface of cancer cells, pathogen-infected cells, or cells that mediate diseases (eg, inflammatory, autoimmune diseases).
  • the target cell protein is highly expressed on the surface of the target cell, although high levels of expression are not necessary.
  • the target cell protein is not expressed or underexpressed on the surface of the target cell.
  • the bispecific antibody of the homodimer as described herein can bind to the cancer cell antigen as described above.
  • Cancer cell antigens can be human proteins or proteins derived from other species.
  • the target cell protein may be a cell surface protein that mediates diseases related to the lymphatic system.
  • the target cell protein may be a protein that is selectively expressed or overexpressed or not expressed on the surface of tumor cells.
  • the target cell may be a cell that mediates an autoimmune disease or an inflammatory disease.
  • human eosinophils in asthma can be target cells, in which case, for example, EGF-like motif-containing mucin-like hormone receptor (EMR1) can be used as the target cell protein.
  • EGF-like motif-containing mucin-like hormone receptor (EMR1) can be used as the target cell protein.
  • excess human B cells can be used as target cells in patients with systemic lupus erythematosus, in this case, such as CD19 or CD20 can be used as target cell proteins.
  • excessive human Th2T cells can be used as target cells, in this case, such as CCR4 can be used as target cell protein.
  • the target cell may be mediated such as atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, renal transplantation fibrosis, renal allograft nephropathy, or pulmonary fibrosis (including idiopathic) Fibrotic cells of chronic pulmonary fibrosis and / or idiopathic pulmonary hypertension).
  • COPD chronic obstructive pulmonary disease
  • scleroderma scleroderma
  • renal transplantation fibrosis fibrosis
  • renal allograft nephropathy or pulmonary fibrosis (including idiopathic) Fibrotic cells of chronic pulmonary fibrosis and / or idiopathic pulmonary hypertension.
  • pulmonary fibrosis including idiopathic Fibrotic cells of chronic pulmonary fibrosis and / or idiopathic pulmonary hypertension.
  • FAP ⁇ fibroblast activation protein alpha
  • the target cells may be cells that mediate other related diseases, including diseases or disorders related to transplant rejection.
  • Bispecific antibodies can bind to target cell proteins from mice, rats, rabbits, New World monkeys, and / or Old World monkey species.
  • the species include but are not limited to the following species: Musmusculus; Rattus rattus; Rattus norvegicus; Crab-eating macaque, Macacafascicularis; Hamadryasbaboon, Egyptian baboon (Papiohamadryas); big baboon (Guineababoon), Guinea baboon (Papiopapio); olive baboon (olivebaboon), east African baboon (Papioanubis); yellow baboon (yellowbaboon), grassland baboon (Papiocynocephalus); (Papioursinus), common marmoset (Callithrixjacchus), tamarind tamarin (Saguinus Oedipus) and squirrel monkey (Saimirisciureus).
  • cancer refers to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body.
  • Cancer includes benign and malignant cancers and dormant tumors or micrometastasis. Cancer includes primary malignant cells or tumors (e.g., tumors that have not migrated to a site other than the original malignant disease or tumor site in the subject) and secondary malignant cells or tumors (e.g., tumors resulting from metastasis, metastasized to malignant Cells or tumor cells migrate to a secondary site that is different from the original tumor site). Cancer also includes hematological malignancies. "Hematological malignancies” include lymphomas, leukemias, myeloma or lymphoid malignancies, as well as splenic and lymph node tumors.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition or combination therapy is useful for the treatment, prevention or remission of cancer.
  • cancer include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphocytic malignancies.
  • squamous cell carcinoma eg, epithelial squamous cell carcinoma
  • Ewing's sarcoma e.g. Ewing's sarcoma
  • Wilms' tumor astrocytoma
  • lung cancer including small cell lung cancer, Non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma
  • peritoneal cancer hepatocellular carcinoma
  • gastric cancer or gastric cancer including gastrointestinal cancer
  • pancreatic cancer glioblastoma multiforme
  • cervical cancer ovarian cancer Cancer
  • liver cancer bladder cancer
  • hepatocellular carcinoma hepatocellular carcinoma
  • neuroendocrine tumor medullary thyroid carcinoma
  • differentiated thyroid cancer breast cancer, ovarian cancer, colon cancer
  • rectal cancer endometrial cancer or uterine cancer
  • salivary adenocarcinoma Kidney or kidney cancer, prostate cancer, vulvar cancer, anal cancer, penile cancer and head and neck cancer.
  • cancer or malignant diseases include, but are not limited to: acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, adult (primary) hepatocytes Cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's lymphoma, adult lymphocytic lymphoma, adult non-Hodgkin's lymphoma, adult primary Liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant disease, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, renal pelvis And ureteral cancer, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral
  • the bispecific antibody of the present invention or the nucleic acid or pharmaceutical composition or combination therapy encoding the antibody of the present application can be used to treat malignant or premalignant conditions and to prevent the development of neoplastic or malignant states, including but not limited to those described above Those illnesses.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition or combination therapy is useful for the treatment, prevention or alleviation of diseases of the lymphatic system.
  • diseases of the lymphatic system include, but are not limited to:
  • B-cell lymphoma is a solid tumor of B cell development, including Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL).
  • Hodgkin lymphoma mainly includes classic Hodgkin lymphoma and nodular lymphoma.
  • Diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia, and mantle cell lymphoma (MCL) are the five most common B cells Non-Hodgkin's lymphoma accounts for 3/4 of non-Hodgkin's lymphoma.
  • B-cell non-Hodgkin's lymphomas include, but are not limited to: a) Prodromal lymphoma: B-lymphoblastic leukemia / lymphoma and accompanying diseases.
  • Mature B-cell tumors include but are not limited to: chronic lymphocytic leukemia / small lymphocytic lymphoma, B-prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, lymphoplasma Cell lymphoma, heavy chain disease, plasma cell myeloma / plasma cell tumor, extranodal mucosa-associated lymphoid tissue marginal zone B-cell lymphoma (MALT lymphoma), primary cutaneous follicular center lymphoma, follicular lymphoma (follicular lymphoma) Gastrointestinal follicular lymphoma, childhood follicular lymphoma, "in situ
  • Some B-cell lymphomas are caused by infection.
  • infectious viruses include Kaposi's sarcoma-associated herpes virus (primary exudative lymphoma), Epstein-Barr virus (lymphoma-like granuloma, post-transplant lymphoma) Proliferative diseases), HIV (AIDS-related lymphoma) and Helicobacter pylori (mucosa-associated lymphoma).
  • B-cell lymphomas are divided into indolent lymphomas and invasive lymphomas.
  • Indolent lymphoma usually develops slowly and can maintain stable disease and long-term survival for many years, but it cannot be cured.
  • Invasive lymphoma usually requires a stronger treatment, but there is a possibility of cure.
  • the prognosis and treatment of B-cell lymphoma depends on the specific type and stage of lymphoma.
  • T cell lymphoma T cell lymphoma
  • T-cell lymphoma includes four types of lymphoma that affect T cells: Angiocentric lymphoma (Angiocentric lymphoma), skin T cell lymphoma (Cutaneous T cell lymphoma): Sézary's disease And mycosis granuloma (Mycosis fungoides), anaplastic large cell lymphoma (Anaplastic) large-cell lymphoma and angioimmunoblastic T cell lymphoma (Angioimmunoblastic T-cell lymphoma).
  • Angiocentric lymphoma Angiocentric lymphoma
  • Skin T cell lymphoma Cutaneous T cell lymphoma
  • Mycosis fungoides Sézary's disease And mycosis granuloma
  • Anaplastic large cell lymphoma Anaplastic large cell lymphoma (Anaplastic) large-cell lymphoma
  • angioimmunoblastic T cell lymphoma
  • Non-limiting examples include: acute lymphoblastic leukemia (such as acute pre-T-lymphocytic leukemia and lymphoma), immature lymphocytic leukemia (such as immature T-lymphocytic leukemia), CD30 + marker leukemia (such as anaplastic large cell lymphoma) , Lymphoma-like papulosis), skin diseases (such as mycosis fungoides, Sezary's disease), liver and spleen diseases, vascular diseases, intestinal related diseases, and infectious diseases (such as human T lymphotropic virus type (such Adult T-cell leukemia and lymphoma)).
  • acute lymphoblastic leukemia such as acute pre-T-lymphocytic leukemia and lymphoma
  • immature lymphocytic leukemia such as immature T-lymphocytic leukemia
  • CD30 + marker leukemia such as anaplastic large cell lymphoma
  • Lymphoma-like papulosis
  • Illustrative examples include: acute B-lymphocytic leukemia (precursor B-cell lymphocytic leukemia), ordinary B-cell leukemia (B-cell chronic lymphocytic leukemia), young B-lymphocytic leukemia, and CD11c hairy cell leukemia.
  • Illustrative examples include: acute T-lymphocytic leukemia, acute pre-T-lymphocytic leukemia and lymphoma, juvenile T-lymphocytic leukemia, adult T-cell leukemia and lymphoma.
  • Illustrative examples include: lymphatic system and bone marrow system (eg, acute biphenotype leukemia), hyperlymphocytic disease, such as lymphoproliferative diseases (X-linked lymphoproliferative diseases, autoimmune lymphoproliferative syndrome), leukemia-like reactions sexual diseases and pseudolymphoma.
  • lymphoproliferative diseases X-linked lymphoproliferative diseases, autoimmune lymphoproliferative syndrome
  • leukemia-like reactions sexual diseases and pseudolymphoma.
  • Bispecific antibodies or nucleic acids or polynucleotides encoding the antibodies of the present application or pharmaceutical compositions or combination therapies play a key role in the pathology associated with various diseases including immune and inflammatory factors.
  • the above diseases include but are not limited to rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease , Ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes, thyroiditis, asthma, allergic diseases, psoriasis, scleroderma dermatitis, graft-versus-host disease, organ transplant rejection, organ transplant-related Acute or chronic immune diseases, sarcoidosis, atherosclerosis, disseminated vascular coagulation, Kawasaki disease, Grave disease, nephrotic syndrome, chronic fatigue syndrome, orbital necrotizing granulomatosis, Dutch purpur
  • Transplant rejection refers to hyperacute, acute, or chronic rejection of allografts or xenografts of cells, tissues, or solid organs.
  • the graft can include one or more organs (eg, kidney transplantation) Or heart and lung transplantation), parts of organs (eg, skin grafts), cells (eg, bone marrow transplants, islet cells, other endocrine or exocrine cells) or tissues (eg, skin, or connective tissues such as cartilage, ligaments, or tendons).
  • organs eg, kidney transplantation
  • parts of organs eg, skin grafts
  • cells eg, bone marrow transplants, islet cells, other endocrine or exocrine cells
  • tissues eg, skin, or connective tissues such as cartilage, ligaments, or tendons.
  • More specific grafts include, but are not limited to, stem cells, corneal tissue, heart, lung, heart-lung combination, kidney, liver, intestine (or other parts of the digestive tract), pancreas (especially islets), trachea or esophagus, blood vessels, Nerve, bone, bone marrow, cartilage, joint, tendon, ligament, muscle, fat, breast, gastrointestinal lining, skin, epithelium, endothelium, connective tissue, etc.
  • the present invention can be used with body parts including various tissue types, for example, for replacement of eyes, ears, nose, fingers (fingers or toes), joints, blood vessels, nerves, muscles, or other surgical changes or Rebuild limbs or other body parts.
  • Indications include conditions related to transplant rejection, such as treatment (including improvement, reduction, elimination or cure of the cause or symptom) or prevention (including substantial or complete) or avoidance of the following: a) acute transplant rejection, such as treatment Recipients of heart, lung, cardiopulmonary, liver, kidney, pancreas, skin, intestine or corneal transplantation, especially to prevent and / or treat T cell-mediated rejection, and graft-versus-host disease, such as bone marrow transplantation. b) Chronic transplant rejection, for example in particular to prevent graft vascular disease, for example, which is characterized by graft arterial stenosis due to intimal thickening due to smooth muscle cell proliferation and related effects.
  • acute transplant rejection such as treatment Recipients of heart, lung, cardiopulmonary, liver, kidney, pancreas, skin, intestine or corneal transplantation, especially to prevent and / or treat T cell-mediated rejection
  • graft-versus-host disease such as bone marrow transplantation.
  • Chronic transplant rejection for example in
  • Xenotransplant rejection including acute, hyperacute or chronic rejection of organs when the organ donor and recipient are different species, most particularly B-cell-mediated rejection or antibody-mediated rejection .
  • Indications also include, for example, adverse reactions after blood transfusions, including but not limited to fever reactions, allergic reactions, hemolytic reactions, graft-versus-host disease after blood transfusions, complications after massive blood transfusions (overloading of circulation, bleeding tendency), bacterial contamination Transfusion reactions caused by blood transfusion-borne diseases.
  • the drug can be administered to patients who have just received transplants or are about to receive transplants by any conventional route, including but not limited to intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral administration.
  • the CD19 bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition or combination therapy will be administered
  • Receptors include any animal that can exhibit related diseases or conditions such as autoimmune diseases, inflammatory diseases, transplant rejection diseases and symptoms.
  • the recipient is a mammal. More precisely, the mammal includes at least humans, apes, rodents, sheep, cows, ruminants, rabbits, pigs, goats, horses, dogs, rabbits, felines, birds and so on. Among them, the most preferred is human.
  • the host may be a mammal of commercial importance, or a companion animal or other valuable animal, such as a member of an endangered species.
  • the non-human animals are preferably murine, such as guinea pigs, hamsters, gerbils, rats and mice. Therefore, human doctors and veterinarians can use the bispecific antibodies of the present invention.
  • the CD19 bispecific antibody of the present invention or the nucleic acid or polynucleotide or pharmaceutical composition encoding the antibody of the present application can be used with any type of tissue transplantation or transplantation procedure, especially where the donor (transplanted) tissue is immunized by the recipient host The risk of system failure or rejection or risky procedures.
  • the CD19 bispecific antibody of the present invention or the nucleic acid or polynucleotide or pharmaceutical composition encoding the antibody of the present application can be used in any environment where the donor tissue and the recipient host do not have histocompatibility.
  • the transplant donor can be a non-homologous member of the same phylogenetic species as the transplant recipient (ie, the allogeneic donor, which provides the allograft tissue), or a member of a different phylogenetic species (that is, the xenogeneic donor, which provides Xenograft tissue).
  • Donor tissue can come from conventional means, such as volunteers or other living donors.
  • the donor can achieve histocompatibility as well as the recipient host. Therefore, in the case where the recipient host is human, autologous and allogeneic donor tissues are preferred.
  • a xenogeneic donor is used as the source of transplanted tissue, it is preferred that the donor is compatible with the recipient host relative to MHC; for example, baboons or chimpanzees will be preferred as donors for transplanting tissue into the human body.
  • the CD19 bispecific antibody of the present invention or the nucleic acid or polynucleotide or pharmaceutical composition encoding the antibody of the present application can be used to promote the implantation of any body tissue or organ type, regardless of whether the donor (graft) tissue is the entire organ, organ or tissue Part or part of it is still isolated cells.
  • the donor tissue includes an organ or body part.
  • the donor tissue includes a portion, part or biopsy of the donor organ or tissue.
  • the donor tissue contains cells, especially isolated or suspended cells, including cells removed or excised from the donor host, cells maintained in primary culture, or immortalized cell lines.
  • the donor tissue may include cells carrying foreign genetic material, such as transfected or transformed host cells, which have (or derived from ancestral cells that have been engineered) include the genetics necessary to produce a polypeptide of therapeutic value substance.
  • the donor tissue may be derived from a transgenic mammal that has been engineered to include the genetic material necessary to produce a polypeptide of therapeutic value to the recipient host in some or all of its body tissues .
  • Exemplary polypeptides that have therapeutic value for the receptor include: hormones, such as insulin or growth hormone; cytokines; growth and differentiation factors; enzymes; structural proteins; and the like.
  • the effective dose to inhibit rejection of transplantation is approximately 0.001 mg to 10.0 mg per kg of body weight.
  • the selected dose between approximately 0.001 mg-10.0 mg / kg body weight is effective and non-toxic.
  • the dose used will be given to the patient 1 to 6 times a day.
  • the present invention provides a method for inhibiting transplant rejection.
  • the method includes administering an effective amount of a CD19 bispecific antibody as defined herein or a nucleic acid or polynucleotide encoding a antibody of the present application or a pharmaceutical composition for inhibiting transplant rejection.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application can be used to prepare a pharmaceutical composition or a sterile composition, for example, a bispecific antibody and a pharmaceutically acceptable carrier, The excipients or stabilizers are mixed.
  • the pharmaceutical composition may include one or a combination (such as two or more different) bispecific antibodies of the present invention.
  • the pharmaceutical composition of the present invention may comprise a combination of antibodies or antigen-binding fragments with complementary activities that bind to different epitopes on the target antigen.
  • the formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions.
  • pharmaceutically acceptable means that when the molecular body, molecular fragment, or composition is properly administered to an animal or human, they will not produce adverse, allergic, or other adverse reactions.
  • pharmaceutically acceptable carriers or components include sugars (such as lactose), starch, cellulose and derivatives thereof, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, and the like.
  • the pharmaceutical compositions of the present invention are used to treat, prevent, or alleviate diseases including, but not limited to, immune-related diseases, cancer, autoimmune diseases, inflammatory diseases, transplant rejection rejection-related diseases or disorders.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application may be used alone, or may be used together with one or more other pharmaceutically active agents, such as antibodies, antibody fragments, drugs , Enzymes, cytotoxic agents, toxins, antibiotics, hormones, immunomodulators, cytokines, chemokines and radioisotopes.
  • other pharmaceutically active agents such as antibodies, antibody fragments, drugs , Enzymes, cytotoxic agents, toxins, antibiotics, hormones, immunomodulators, cytokines, chemokines and radioisotopes.
  • Drugs include but are not limited to cyclophosphamide, nimustine, methotrexate, fluorouracil, capecitabine, gemcitabine, tegio, pemetrexed, fludarabine, doxorubicin, bleomycin , Vinorelbine, paclitaxel, docetaxel, irinotecan, tamoxifen, letrozole, exemestane, fulvestrant, goserelin, medroxyprogesterone, cisplatin, carboplatin , Platinum oxalate, nedaplatin, oxaliplatin, asparaginase, loplatin, etoposide, vincristine, irinotecan, tegafur, dacarbazine, mitomycin, teniposide , Pirarubicin, mitoxantrone, vindesine, raltitrexed, methotrexate, cis
  • Toxins include, but are not limited to: ginatoxin, acacia toxin, alpha toxin, saponin, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, white tree toxin, Diphtheria toxin, Pseudomonas exotoxin and Pseudomonas endotoxin.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application may, for example, co-stimulate molecules with corresponding effector cells, effector cells, DC cell surface molecules, DC cells, molecules that activate T cells (Hutloff A, etc., Nature, 397: 262-266, 1999) co-administered.
  • the effector cells may be human leukocytes, such as macrophages, neutrophils or monocytes. Other cells include eosinophils, NK cells and other cells with IgG or IgA receptors. If necessary, effector cells can be obtained from the subject to be treated.
  • Target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution.
  • the number of cells administered can be in the order of 10 8 -10 9 but may vary according to the purpose of treatment. In general, this amount is sufficient to achieve localization on target cells (eg, cells expressing CD19 tumors) and achieve cell killing.
  • target cells eg, cells expressing CD19 tumors
  • the composition of the present invention having a complement binding site such as a portion from IgG1, IgG2, or IgG4 or IgM that binds to complement may also be used.
  • the composition of the invention can also be administered together with complement, for example in combination with CIq.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application may be combined with, for example, immunomodulators, immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules) or Cells transfected with genes encoding immunostimulatory cytokines are administered in combination (He YF et al., J. Immunol., 173: 4919-28, 2004).
  • Immunomodulators may include, but are not limited to, cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSF), interleukins (IL), erythropoietin, platelet growth factors, tumor necrosis Factor- ⁇ (TNF), TNF- ⁇ , granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon- ⁇ , interferon- ⁇ , interferon - ⁇ , interferon- ⁇ , interferon- ⁇ , stem cell growth factor called “S1 factor”, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, Relaxin, relaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), liver growth factor, prostaglandin, fibroblast growth factor, prolactin, place
  • bispecific antibodies or antibody fragments can be linked to immunomodulators, such as cytokines.
  • immunomodulators such as cytokines.
  • Cytokine complexes are disclosed in, for example, US Patent Nos. 7,906,118 and 8,034,3522, the example portion of each of which is incorporated herein by reference.
  • Preferred immunomodulators include interferon- ⁇ , interferon- ⁇ and interferon- ⁇ .
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application can also be administered in combination with, for example, standard cancer treatments (eg, surgery, radiation, and chemotherapy).
  • standard cancer treatments eg, surgery, radiation, and chemotherapy
  • anti-tumor therapy using the compositions of the present invention and / or effector cells equipped with these compositions is used in combination with chemotherapy.
  • Non-limiting examples of antibody combination treatments of the present invention include surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and combinations thereof.
  • the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application may be linked to the agent (as an immune complex) or administered separately from the agent. In the latter case, the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application may be administered before, after or co-administered with the agent, or may be combined with other known therapies (such as anti-cancer therapy , Such as radiation) co-administered.
  • the pharmaceutical composition of the present application contains a nucleic acid or polynucleotide encoding a protein, and the nucleic acid can be administered in vivo to promote the expression of the encoded protein by constructing it as part of a suitable nucleic acid expression vector and administering it so that It enters the cell, for example, by using a retroviral vector (see US Patent No. 4,980,286), or by direct injection, or by using particle bombardment (such as gene gun, Biolistic, Dupont), or by using lipids or cell surface.
  • a retroviral vector see US Patent No. 4,980,286
  • particle bombardment such as gene gun, Biolistic, Dupont
  • transfection agent by linking it to a homeobox peptide and applying it, the homeobox peptide is known to enter the nucleus (see, for example, Joliot A et al., Proc. Natl. Acad. Sci .USA, 88: 1864-1868,1991) etc.
  • the nucleic acid can be introduced into the cell and incorporated into the host cell DNA for expression by homologous recombination.
  • composition of the present invention may be in various forms. It includes, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersing or suspending tablets, pills, powders, liposomes, and suppositories.
  • liquid solutions eg, injectable and infusible solutions
  • dispersing or suspending tablets pills, powders, liposomes, and suppositories.
  • Typical preferred compositions are injectable or infusible solutions, such as those similar to those used to passively immunize humans with other antibodies.
  • the route of administration can take many forms, including oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, Intraocular, inhalation, insufflation, topical, skin, percutaneous, or intraarterial.
  • the preferred form of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the antibody is administered by intravenous injection or injection.
  • the antibody is injected intramuscularly or subcutaneously.
  • the determination of the appropriate dose is performed by a clinician, for example using parameters or factors known or suspected in the art to affect the treatment. Generally, the dose starts with an amount slightly less than the optimal dose, and it is then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects.
  • Important diagnostic measures include, for example, symptoms of inflammation or measures of the level of inflammatory cytokines produced.
  • the present invention provides a container (such as a plastic or glass vial, such as a lid or a chromatographic column, a hollow hole needle, or a syringe cylinder) that contains any antibody of the present invention or a nucleic acid or polynucleotide encoding the antibody of the present application and instructions.
  • a container such as a plastic or glass vial, such as a lid or a chromatographic column, a hollow hole needle, or a syringe cylinder
  • the present invention also provides an injection device comprising any antibody of the present invention or a nucleic acid or polynucleotide encoding the antibody of the present application.
  • the present invention encompasses that bispecific antibodies or nucleic acids or polynucleotides or pharmaceutical compositions encoding antibodies of the present application can be combined with one or more active therapeutic agents (eg, chemotherapeutic agents) or other preventive or therapeutic modalities (eg, radiation) use.
  • active therapeutic agents eg, chemotherapeutic agents
  • combination therapies include therapeutic agents that affect the immune response (eg, enhance or activate the response) and therapeutic agents that affect (eg, inhibit or kill) tumors / cancer cells.
  • Combination therapy can reduce the possibility of drug-resistant cancer cells.
  • Combination therapy may allow the dose of one or more of the agents to be reduced to reduce or eliminate the adverse effects associated with one or more of the agents.
  • Such combination therapy may have a synergistic treatment or prevention effect on the underlying disease, disorder or condition.
  • “Combination” includes therapies that can be administered separately, for example, formulated separately for single administration (eg, can be provided in a kit), and therapies that can be administered together in a single formulation (ie, "co-formulation").
  • the bispecific antibodies of the present invention or the nucleic acids or polynucleotides or pharmaceutical compositions encoding the antibodies of the present application can be administered sequentially.
  • the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition can be administered simultaneously.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition can be used in combination with at least one other (active) agent in any manner.
  • the treatment with the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition can be combined with other treatments that can be effective against the condition to be treated.
  • Non-limiting examples of other treatment combinations include surgery, chemotherapy, and radiotherapy , Immunotherapy, gene therapy, DNA therapy, RNA therapy, nano therapy, virus therapy, adjuvant therapy.
  • the combination therapy also includes all other known technologies and possible future development.
  • the invention provides (eg, in vitro or in vivo) detection of TAA in samples (eg, biological samples, eg, blood, serum, semen, or urine or tissue biopsy samples (eg, from hyperproliferative or cancerous lesions) ) Is present or at its level.
  • samples eg, biological samples, eg, blood, serum, semen, or urine or tissue biopsy samples (eg, from hyperproliferative or cancerous lesions)
  • This method can be used to evaluate (e.g., monitor the treatment or progression of the disease (e.g., immune disorder, cancer) of the invention, its diagnosis and / or staging in the subject).
  • the method may include: i. Incubating the sample with an antibody specific to TAA; ii. Detecting the TAA complex using a detectable probe; iii.
  • the method may involve, for example, immunohistochemistry, immunocytochemistry, flow cytometry, magnetic beads complexed with antibody molecules, ELISA assay, PCR technology (eg, RT-PCR).
  • PCR technology eg, RT-PCR.
  • bispecific antibody molecules or nucleic acids or polynucleotides encoding dual antibodies used in in vivo and in vitro diagnostic methods are directly or indirectly labeled with a detectable substance to output a detection signal.
  • Suitable detectable substances include but are not limited to various biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • kits for diagnosing or detecting one or more bispecific antibodies or nucleic acids or polynucleotides encoding biantibodies of the invention wherein, in one embodiment, the bispecific antibodies or bispecific antibodies
  • the resistant nucleic acid or polynucleotide and a pharmaceutically acceptable carrier are optionally combined with one or more therapeutic agents, optionally formulated together in a pharmaceutical composition. It is used in a variety of detection assays, including for example immunoassays, such as ELISA (sandwich or competitive format).
  • the kit may include other additives, such as stabilizers, buffers (eg, blocking buffer or lysis buffer), and the like.
  • the reagent may be provided as a dry powder, which is usually lyophilized, including excipients, which, after dissolution, will provide a reagent solution with an appropriate concentration.
  • the components of the kit may be pre-attached to the solid support, or may be applied to the surface of the solid support when the kit is used.
  • the signal generating device may be pre-associated with the bispecific antibody or nucleic acid or polynucleotide encoding the dual antibody of the present invention, or one or more components may need to be combined before use, For example, buffers, antibody-enzyme conjugates, enzyme substrates, etc.
  • enzymes that catalyze the formation of chemiluminescent or chromogenic products or the reduction of chemiluminescent or chromogenic substrates are components of the signal generating device.
  • Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction products generated by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored labels.
  • a detectable label as described above can be attached to the recombinant protein of the present invention through linkers of different lengths to reduce potential steric hindrance. Such enzymes are well known in the art.
  • the kit may also include additional reagents, such as blocking reagents, washing reagents, enzyme substrates, etc. for reducing non-specific binding to the solid phase surface.
  • additional reagents such as blocking reagents, washing reagents, enzyme substrates, etc. for reducing non-specific binding to the solid phase surface.
  • the solid surface can be in the form of tubes, beads, microtiter plates, microspheres, or other materials suitable for immobilizing proteins, peptides, or polypeptides.
  • the kit components can be packaged together, or packed in two or more containers.
  • the container may be a vial containing a sterile lyophilized formulation suitable for reconstitution.
  • Other containers that can be used include, but are not limited to bags, trays, boxes, tubes, and the like.
  • the kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents.
  • the kit contains a bispecific antibody packaged in a container or a nucleic acid or polynucleotide encoding an antibody of the present application.
  • the kit includes a bispecific antibody or a nucleic acid or polynucleotide encoding an antibody of the present application, and one or more pharmaceutically acceptable carriers.
  • the kit contains the combination of the invention, which includes the bispecific antibody of the invention or the nucleic acid or polynucleotide encoding the antibody of the application together with one or more pharmaceutically acceptable in a single common container
  • the carrier optionally combined with one or more therapeutic agents, is optionally formulated together in a pharmaceutical composition.
  • the kit contains the bispecific antibody of the invention or the nucleic acid or polynucleotide encoding the antibody of the present application in one container or a pharmaceutical composition thereof and the pharmaceutical composition and / or treatment in another container Agent.
  • Simultaneous administration of the two therapeutic agents does not require the agents to be administered at the same time or through the same route, as long as there is overlap in the time period over which the agents exert their therapeutic effects.
  • the above kit contains a label attached to or packaged with the container, the label describing the contents of the container and providing indications and / or methods for using the contents of the container, which are useful for treatment , Prevention and / or diagnosis of one or more disease states as described herein.
  • the kit may optionally further include a syringe for parenteral, such as intravenous administration.
  • the kit also includes a device for tightly containing the vial for commercial sale and / or packaging and delivery. It also includes devices or equipment for performing the detection or monitoring methods described herein.
  • the bispecific antibodies of the present application or the nucleic acids or polynucleotides encoding the antibodies of the present application or pharmaceutical compositions or combination therapies can be used for immune-related diseases, cancers, autoimmune diseases, and inflammatory diseases described herein
  • diseases or diseases related to transplant rejection such treatment includes administration of the bispecific antibody or nucleic acid or polynucleotide encoding the dual antibody or pharmaceutical composition or combination therapy to the patient,
  • animals, mammals, and humans are used to treat or diagnose one or more diseases or symptoms described herein.
  • Therapeutic compounds of the present application include, but are not limited to, antibodies of the present application (including their variants, isoforms, derivatives, and species homologs as described herein) or nucleic acids or polynucleotides encoding the antibodies of the present application (Including their variants, isoforms, derivatives and species homologues as described herein).
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide or pharmaceutical composition encoding the antibody of the present application can be used for diagnostic purposes.
  • the antibodies of the present invention are used in in vitro diagnostic tests, such as laboratory tests to detect useful antigens or in care tests to detect useful antigens. Established in vitro tests using antibodies include ELISAs, RIAs, Western blots, etc.
  • the antibodies of the invention are used in in vivo diagnostic tests, such as in vivo imaging tests.
  • the antibody is labeled with a detectable substance that can be detected in vivo
  • the labeled antibody can be administered to a subject, and the labeled antibody can be detected in vivo, thereby enabling in vivo imaging.
  • Related diagnostic technologies also include other well-known technologies and technologies that may be developed.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application can be used to prepare a medicine.
  • the pharmaceutical composition includes but is not limited to the pharmaceutical composition described above.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application may be used alone, or may be used together with one or more other therapeutic agents, including but not limited to those described above.
  • the bispecific antibody or nucleic acid or polynucleotide encoding the antibody of the present application and the additional pharmaceutically active agent are provided as separate components or as components of the same composition, or may be combined with Other known therapies are co-administered.
  • a container and instructions including the bispecific antibody of the present invention or the nucleic acid or polynucleotide or pharmaceutical composition encoding the antibody of the present application are also provided.
  • the present invention provides a combination therapy comprising a bispecific antibody of the present invention or a nucleic acid or polynucleotide encoding a antibody of the present application or a pharmaceutical composition.
  • Combination therapy includes but is not limited to the above.
  • the present invention also provides related detection methods and kits containing the bispecific antibody of the present invention or the nucleic acid or polynucleotide or pharmaceutical composition encoding the antibody of the present application.
  • Related content includes but is not limited to the above.
  • the bispecific antibodies of the present application or the nucleic acids or polynucleotides or pharmaceutical compositions encoding the antibodies of the present application can be used for diagnosis, treatment, suppression or prevention of diseases or symptoms, including malignant diseases, disorders, or with such diseases or disorders Related conditions, such as diseases related to increasing cell survival or inhibiting apoptosis, such as, but not limited to, immune-related diseases, cancer, autoimmune diseases, inflammatory diseases, transplant rejection-related diseases or disorders described above.
  • diseases related to increasing cell survival or inhibiting apoptosis such as, but not limited to, immune-related diseases, cancer, autoimmune diseases, inflammatory diseases, transplant rejection-related diseases or disorders described above.
  • the disease involves unregulated and / or inappropriate proliferation of cells, sometimes accompanied by the destruction of adjacent tissues and the growth of new blood vessels, which may allow cancer cells to invade, ie metastasize, to new areas.
  • diseases or symptoms related to increased cell survival that can be treated, prevented, diagnosed, and / or predicted by the bispecific antibodies of the present application or nucleic acids or polynucleotides or pharmaceutical compositions encoding the antibodies of the present application, including but not limited to, The development and / or metastasis of malignant tumors, as well as the development and / or metastasis of other related diseases or symptoms (eg, inflammatory diseases or diseases of the lymphatic system) described above.
  • the form of the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition includes but is not limited to the above.
  • the method of administering the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition includes but is not limited to the above.
  • Treatment methods and combination therapies include but are not limited to those described above.
  • bispecific antibodies or nucleic acids or polynucleotides encoding the antibodies of the present application or pharmaceutical compositions can be formulated for intravenous administration by, for example, bolus injection or continuous infusion, or even by other parenteral routes, such as intravenous , Intramuscular, intraperitoneal or intravascular administration to mammals.
  • the infusion of the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application or the pharmaceutical composition lasts less than about 4 hours, and more preferably lasts less than about 3 hours.
  • injectable preparations can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with an added preservative.
  • composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulations such as suspensions, stabilizers and / or dispersants.
  • active ingredient may be in powder form for reconstitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.
  • Various delivery systems are known and can be used to administer the antibodies of the present application or nucleic acids or polynucleotides or pharmaceutical compositions encoding the antibodies of the present application, for example, encapsulate them in liposomes, microparticles, microcapsules, Recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, for example, Wu GY et al., J. Biol. Chem., 262: 4429-4432, 1987), construct nucleic acids as retroviruses or other vectors Wait.
  • the delivery system also includes all other known technologies and possible future developments.
  • Additional pharmaceutical methods can be used to control the duration of action of the therapeutic composition, such as a controlled release system.
  • controlled release systems are pumps (see, for example, Sefton, CRC, Crit. Ref. Biomed. Eng., 14: 201, 1987; Buchwald H et al., Surgery, 88: 507, 1980) or polymeric materials (such as See, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres. Boca Raton, Fla., 1974), etc.
  • This pharmaceutical method also includes all other well-known technologies and possible future developments.
  • a therapeutically effective dose of bispecific antibody or nucleic acid or polynucleotide encoding the antibody of the present application or pharmaceutical composition can be administered.
  • the amount of the bispecific antibody or the polynucleotide or pharmaceutical composition encoding the antibody of the present application which constitutes a therapeutically effective dose may vary according to the indication being treated, the weight of the patient, and the calculated skin surface area of the patient.
  • the administration can be adjusted to achieve the desired effect. In many cases, repeated administration may be required. For example, it can be administered three times or twice a week, once a week, once every two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks or ten weeks, or Dosing every two months, three months, four months, five months or six months.
  • the bispecific antibody of the present application or the nucleic acid or polynucleotide encoding the antibody of the present application is usually administered to a patient at a dosage of about 0.001-100 mg / kg, such as about 0.1-50 mg / kg, for example, about 0.1-20 mg / kg, such as about 0.1 -10 mg / kg, for example about 0.5 mg / kg, such as about 0.3 mg / kg, about 1 mg / kg or about 3 mg / kg.
  • the dosage of the antibody administered can be adjusted according to the patient's estimated skin surface area or according to the patient's body weight.
  • the administration schedule can optionally be repeated at other time intervals, and the dosage can be administered by various parenteral routes with appropriate adjustments to the dosage and schedule.
  • human antibodies have a longer half-life in the human body than antibodies from other species, due to the immune response to foreign polypeptides. Therefore, lower doses of human antibodies are generally allowed to reduce the frequency of administration.
  • the dosage and frequency of administration of the antibody of the present application can be reduced by enhancing antibody absorption and tissue penetration (for example, into the brain), and the enhancement is achieved by modification such as lipidation.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application can be used to prepare a diagnostic or therapeutic kit, which includes the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application and / Or instruction manual.
  • the pharmaceutical composition of the present invention can be used to prepare a diagnostic or therapeutic kit, which includes the pharmaceutical composition and / or instructions for use of the present invention.
  • FIG. 1-1 SEC-HPLC detection results of CD19 double antibody purified samples.
  • FIG. 1-2 SDS-PAGE electrophoresis results of purified samples of CD19 double antibody.
  • FIG. 2-1 FACS detects the ability of bispecific antibody AB23P7 to bind to tumor cells Raji.
  • FIG. 2-2 FACS detects the ability of bispecific antibody AB23P8 to bind to tumor cells Raji.
  • Figure 2-3 FACS tests the ability of bispecific antibodies AB23P9 and AB23P10 to bind to tumor cells Raji.
  • FIG. 2-4 FACS detects the ability of bispecific antibodies AB23P7 and AB23P8 to bind to effector cell CIK.
  • FIG. 1 FACS detects the ability of bispecific antibodies AB23P9 and AB23P10 to bind to effector cells CIK.
  • FIG. 1 FACS detects the ability of bispecific antibodies AB23P7 and AB23P10 to bind to cynomolgus monkey T cells.
  • ELISA detects the ability of four Anti-CD19 ⁇ CD3 bispecific antibodies to bind to CD3 and CD19 molecules.
  • FIG. 2-8 The microplate reader detects the ability of AB23P7 and AB23P8 bispecific antibodies to activate the reporter cell line Jurkat T cells.
  • FIG. 1 A microplate reader detects the ability of four Anti-CD19 ⁇ CD3 bispecific antibodies to activate the reporter cell line Jurkat T cells.
  • Figure 3-1 In vivo anti-tumor effect of the dual antibodies AB23P9 and AB23P10 in a transplanted tumor model in which NPG mice were co-inoculated with human CIK cells and Raji cells.
  • Figure 3-2 In vivo antitumor effects of the dual antibodies Blincyto, AB23P9 and AB23P10 in a transplanted tumor model in which NPG mice were co-inoculated with human CIK cells and Daudi cells.
  • Figure 4-1A Determination of serum cytokine IL-2 levels in normal male cynomolgus monkeys given bispecific antibody AB23P7.
  • FIG. 4-1B Determination of serum cytokine IL-2 levels in normal female cynomolgus monkeys given bispecific antibody AB23P10.
  • Figure 4-1C Determination of serum cytokine IL-6 levels in normal male cynomolgus monkeys given bispecific antibody AB23P7.
  • FIG. 4-1D Determination of serum cytokine IL-6 levels in normal female cynomolgus monkeys given bispecific antibody AB23P10.
  • Figure 4-1E Determination of serum cytokine IFN- ⁇ content in normal male cynomolgus monkeys given bispecific antibody AB23P7.
  • Figure 4-1F Determination of serum cytokine IFN- ⁇ content in normal female cynomolgus monkeys given bispecific antibody AB23P10.
  • Example 1 Design and preparation of Anti-CD19 ⁇ CD3 bispecific antibody
  • the seven antibodies including the bispecific antibodies AB23P7, AB23P8, AB23P9 and AB23P10, are composed of anti-CD19 scFv, linker peptide L2, anti-CD3 scFv and Fc fragments in series, anti-CD19 scFv and anti-CD3 scFv inside VH and VL are connected by connecting peptides L1 and L3, respectively.
  • the VH domain and the VL domain are sequentially referred to: the bispecific antibody Blinatumomab (sequence from the US patent US The antibody of CD19 in 10/554852, whose heavy chain variable region and light chain variable region are shown in SEQ ID NOs: 7 and 8 respectively herein), antibody conjugate Coltuximab (sequence from US patent US 14/117806 Antibody HuB4 that binds to CD19, its heavy chain variable region and light chain variable region are shown in SEQ ID NOs: 15 and 16, respectively), and antibody conjugate Denintuzumab (sequence from Chinese patent CN 201580017717.X that binds CD19
  • the antibody hBU12, the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 22 and 63, respectively, and the monoclonal antibody Inebilizumab (se
  • the chain variable region and the light chain variable region are shown in SEQ ID NOs: 30 and 31, respectively, and the bispecific antibody Duvortuxizumab (sequence comes from the antibody that binds CD19 in Chinese Patent CN 201580051198.9, and its heavy chain variable region and light region
  • the chain variable regions are as SEQ ID NO: 34 herein FIG 35).
  • the Fc fragments contained in the bispecific antibody are all derived from human IgG1, and have multiple amino acid substitutions / substitutions, namely L234A, L235A, T250Q, N297A, P331S, and M428L (EU number), and also delete / delete Fc fragments K447 (EU number) at the end of C.
  • the connecting peptide (L2) is composed of a flexible peptide and a rigid peptide, and the flexible peptides are both G 2 (GGGGS) 3 and the rigid peptide is SSSSKAPPPS.
  • the composition of the connecting peptides L1 and L3 in each scFv is (GGGGS) 3 .
  • Table 1-1 lists the amino acid sequences of the anti-CD19-scFv VH domain and its complementarity determining regions (HCDR1, HCDR2, and HCDR3) of seven bispecific antibodies, and the VL domain and its complementarity determining regions (LCDR1, LCDR2 And LCDR3), the amino acid residues contained in the CDR regions are defined according to the Kabat rule.
  • the anti-CD3 scFv binds to effector cells with an EC 50 value of greater than about 10 nM, or greater than 20 nM, or greater than 40 nM, or greater than about 50 nM in an in vitro FACS binding assay; more preferably, the bispecific antibody ’s
  • the second single-chain Fv can not only bind to human CD3, but also specifically bind to CD3 of cynomolgus monkey or rhesus monkey.
  • the VH and VL amino acid sequences of the anti-CD3-scFv included in the bispecific antibody are shown in SEQ ID NO: 46 and SEQ ID NO: 47, respectively, and between (HGGGGS) 3 connection, the monoclonal antibody (named CD3-3) specifically binds to human and cynomolgus monkey CD3 antigens, and has weak binding affinity to CD3.
  • the connecting peptide connecting the anti-CD19 scFv and the anti-CD3 scFv is composed of a flexible peptide and a rigid peptide; preferably, the amino acid composition structure of the flexible peptide is G x S y (GGGGS) z , where x, y and z Is an integer greater than or equal to 0, and x + y + z ⁇ 1.
  • the rigid peptide is derived from the full-length sequence consisting of amino acids 118 to 145 of the carboxy terminus of the natural human chorionic gonadotropin ⁇ subunit (as shown in SEQ ID NO: 48) or a truncated fragment thereof; preferably, the CTP
  • the rigid peptide composition is SSSSKAPPPS (CTP 1 ).
  • Table 1-2 lists the amino acid sequences of some preferred connecting peptides connecting anti-CD19 scFv and anti-CD3 scFv.
  • Table 1-2 Amino acid sequence of connecting peptides connecting anti-TAA scFv and anti-CD3 scFv
  • the Fc fragment is directly or through a connecting peptide connected to the anti-CD3 scFv
  • the Fc fragment is preferably selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, and IgG4, more specifically selected from the heavy chain constant regions of human IgG1 or IgG4; and Fc is mutated to modify the properties of the bispecific antibody molecule, for example , Showing reduced affinity for at least one of human Fc ⁇ Rs (Fc ⁇ RI, Fc ⁇ RIIa or Fc ⁇ RIIIa) and C1q, with reduced effector cell function or complement function.
  • the Fc fragment may also include amino acid substitutions that alter one or several other characteristics (eg, ability to bind to the FcRn receptor, antibody glycosylation or antibody charge heterogeneity, etc.).
  • Table 1-3 exemplifies the amino acid sequences of some Fc fragments with one or more amino acid mutations.
  • the coding genes of the above bispecific antibodies were synthesized according to conventional molecular biology methods, and the coding cDNAs of the obtained fusion genes were inserted between the corresponding restriction sites of the eukaryotic expression plasmid pCMAB2M modified by PCDNA3.1.
  • pCMAB2M modified by PCDNA3.1.
  • cytomegalovirus early promoter which is the enhancer required for high-level expression of foreign genes in mammalian cells.
  • Plasmid pCMAB2M also contains a selectable marker, so that it can have kanamycin resistance in bacteria and G418 resistance in mammalian cells.
  • the pCMAB2M expression vector contains the mouse dihydrofolate reductase (DHFR) gene, so that the target gene and DHFR gene can be co-amplified in the presence of methotrexate (MTX) (See US Patent 4,399,216).
  • DHFR mouse dihydrofolate reductase
  • MTX methotrexate
  • the expression plasmid constructed above is transfected into a mammalian host cell line to express the bispecific antibody.
  • the preferred host cell line is a DHFR enzyme-deficient CHO-cell (see US Pat. No. 4,818,679).
  • the host cell is CHO-derived cell line DXB11.
  • a preferred method of transfection is electroporation, but other methods can also be used, including calcium phosphate co-sedimentation, lipofection.
  • the DHFR gene inhibited by MTX In order to achieve higher levels of fusion protein expression, it is advisable to use the DHFR gene inhibited by MTX for co-amplification.
  • the transfected fusion protein gene was co-amplified with the DHFR gene.
  • Subclones with positive expression of DHFR by limiting dilution were gradually pressurized and screened for transfectants that can grow in up to 6 ⁇ M MTX medium.
  • the secretion rate was measured and cell lines highly expressing foreign proteins were selected. Cell lines with a secretion rate of more than about 5 (preferably about 15) ⁇ g / 10 6 (ie millions) cells / 24h are subjected to adaptive suspension culture using serum-free medium. Then, the cell supernatant was collected and the bispecific antibody was separated and purified.
  • the first step, affinity chromatography use AT Protein, Diamond, or other commercially available affinity media (such as MabSelect Sure affinity chromatography media from GE, etc.) for sample capture, concentration, and some contaminants Removal.
  • the second step hydroxyapatite chromatography: using BIO-RAD's CHT Type II or other commercially available hydroxyapatite media for intermediate purification, used to reduce the polymer content.
  • BIO-RAD's CHT Type II or other commercially available hydroxyapatite media for intermediate purification, used to reduce the polymer content.
  • the third step, anion exchange chromatography use Q-HP from Bogron or other commercially available anion exchange chromatography media (such as GE ’s QHP, TOSOH ’s Toyopearl GigaCap Q-650, Tiandirenhe ’s DEAE Beads 6FF , Generik MC-Q of Saifen Technology, Fractogel EMD TMAE of Merck, QCeramic HyperD of Pall) are refined to further remove contaminants such as HCP and DNA.
  • anion exchange chromatography media such as GE ’s QHP, TOSOH ’s Toyopearl GigaCap Q-650, Tiandirenhe ’s DEAE Beads 6FF , Generik MC-Q of Saifen Technology, Fractogel EMD TMAE of Merck, QCeramic HyperD of Pall
  • the SEC-HPLC purity results and SDS-PAGE electrophoresis results of the samples are shown in Figure 1-1 and Figure 1-2, respectively.
  • the SEC-HPLC results show that the main peak purity of the bispecific antibody after three-step chromatography is more than 95%, SDS -PAGE electrophoresis band pattern is as expected, non-reduced electrophoresis (160KDa), and a clear (80KDa) single-stranded band can be obtained after reduction.
  • CD19 double antibody protein in citrate (20 mM citrate, 8% sucrose, 0.02% Tween-80) at different pH (5.5 and 6.0) was investigated, and the effect of shock on the stability of the sample was also investigated.
  • the CD19 double antibody protein was stored under accelerated conditions at 25 ° C for 2 weeks to evaluate the stability of the protein.
  • the CD19 double antibody protein was changed into citrate buffers at pH 5.5 and 6.0 respectively. Sampling and testing at different time points, taking samples at each sampling point for testing and analysis, testing items include SEC-HPLC and CE-SDS.
  • the SEC-HPLC and CE-SDS test results of the two formulations stored at 25 ° C for 0 to 2 weeks are shown in Table 1-4 and Table 1-5. After the sample was left for two weeks, there was no significant change in the proportions of aggregates, main peaks, shoulder peaks and fragments in the SEC-HPLC detection results. At different pHs, the proportions of the components of the sample were not much different. There was no significant difference in the SEC-HPLC results of the samples in citrate buffers at pH 5.5 and 6.0 for two weeks.
  • the CD19-expressing tumor cells Raji cells (Shanghai Chinese Academy of Sciences Cell Bank) were cultured, and the cells were collected by centrifugation. The collected cells were resuspended with 1% PBSB, the cell density was adjusted to 2 ⁇ 10 6 cells / ml, placed in a 96-well plate, 100 ⁇ l (2 ⁇ 10 5 cells) per well, and blocked at 4 ° C. for 0.5 h. After blocking, the cells were centrifuged and the supernatant was discarded. A series of diluted bispecific antibodies were added and incubated at 4 ° C for 1 hour.
  • AF647-labeled goat anti-human IgG antibody incubated at 4 ° C in the dark for 1 h; centrifuge to remove supernatant, wash twice with 1% PBSB, resuspend each well with 100 ⁇ l 1% paraformaldehyde (PF), and detect signal by flow cytometry strength.
  • the average fluorescence intensity is used as the Y axis and the antibody concentration is used as the X axis.
  • the analysis is performed by the software GraphPad, and the EC 50 value of the bispecific antibody binding to the tumor cell Raji is calculated.
  • FIG. 2-1 to 2-3 show the binding curves of bispecific antibodies with different structures and tumor cells Raji. As shown in Table 2-1, four pairs of molecules that specifically bind to Raji cells EC 50 nM in both horizontal level.
  • PBMC peripheral blood mononuclear cell mononuclear cell mononuclear cell
  • PBMC peripheral blood mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononuclear cell mononucleic acid.
  • the expanded T cells were prepared, and the expression of CD3 on the cell surface was positive by flow cytometry.
  • the preparation and determination methods of the samples to be tested are the same as those in Example 2.1a).
  • the 1% PF detects the resuspended cells, mean fluorescence intensity analyzed by GraphPad Software, EC 50 values were calculated for each bispecific antibody binds to human T cells.
  • PBMC peripheral blood mononuclear cells
  • the bispecific antibodies AB23P7 and AB23P10 have almost no difference in their binding ability to cynomolgus monkey T cells.
  • the flow cytometer detected that the EC 50 they bound was approximately 5.5 nM, and the two bispecific antibodies
  • the binding ability with cynomolgus monkey T cells is stronger than that with human T cells.
  • AB23P8 and AB23P9 like AB23P7 and AB23P10, can specifically bind to cynomolgus monkey T cells.
  • bispecific antibodies to soluble CD3 and CD19 was identified by double antigen sandwich ELISA.
  • the CD19 protein (ACRO Biosystems, catalog number CD9-H5251) was diluted with PBS to a concentration of 1 ⁇ g / ml, added to a 96-well plate, 100 ⁇ l / well, and coated at 4 ° C overnight. Then it was blocked with 1% skimmed milk powder for 1 hour at room temperature. Simultaneously dilute each bispecific antibody with a 5-fold gradient for a total of 10 concentration gradients. Then wash the 96-well plate with PBST, add the diluted bispecific antibody, set the control well without antibody, and incubate at room temperature for 2h.
  • the unbound bispecific antibody was washed away with PBST, and the biotinylated CD3E & CD3D (ACRO Biosystem, Catalog No. CDD-H82W1) was mixed with 50 ng / ml Streptavdin HRP (BD, Catalog No. 554066) into a 96-well plate, 100 ⁇ l / well , Incubate at room temperature for 1h. Thereafter, the 96-well plate was washed with PBST, TMB was added, 100 ⁇ l / well, and the color was developed at room temperature for 15 min, and then 0.2M H 2 SO 4 was added to stop the color reaction. Detect the absorbance of A450-620nm with a microplate reader. Analyzed by the software GraphPad, EC 50 values were calculated with the two bispecific antibody antigen binding.
  • Jurkat T cells BPS Bioscience, Catalog No. 60621
  • NFAT RE reporter gene can overexpress luciferase in the presence of bispecific antibodies and target cells (Raji cells), by detecting luciferase activity
  • Bispecific antibody blinatumomab Amgen
  • the concentration of the bispecific antibody is used as the X axis, and the fluorescein signal is used as the Y axis to fit the four-parameter curve.
  • Normally cultured tumor cell lines including Raji-Luc, NALM6, and Reh cells (all purchased from the Shanghai Chinese Academy of Sciences Cell Bank) as target cells, collect the cell suspension, centrifuge, adjust the cell density to 2 ⁇ 10 5 cells / ml, add to 96 wells In the cell culture plate, 100 ⁇ l / well, culture overnight. Dilute the corresponding antibody according to the experimental design, 50 ⁇ l / well, and add the same volume of medium to the wells without antibody addition. Then add 5 times the number of effector cells (human PBMC or expanded cultured CIK cells), 100 ⁇ l / well, set the control well, and add the same volume of medium to the wells without adding effector cells.
  • effector cells human PBMC or expanded cultured CIK cells
  • Table 2-6 EC bispecific antibody-mediated killing of tumor cells PBMC value 50
  • Table 2-7 CIK bispecific antibody-mediated killing of tumor cells EC 50 values
  • the CD19-positive human Burkkit ’s lymphoma Raji cell xenograft mouse model was selected for anti-CD19-CD3 bifunctional specific antibody to conduct in vivo pharmacodynamic studies on tumor growth inhibition.
  • Peripheral blood of normal people was taken and human PBMC cells were separated by density gradient centrifugation, and then resuspended with RPMI-1640 culture medium added 10% inactivated FBS, and OKT3 and 250IU / ml human IL were added at a final concentration of 1 ⁇ g / ml -2; after the third day of culture, centrifuge at 300g for 5min, change the medium, add 10% inactivated FBS resuspended cells for culture with RPMI-1640, and add 250IU / ml human IL-2; then add fresh culture every 2 days The liquid was cultured until the 10th day, and CIK cells were collected.
  • NPG mice Seven- to eight-week-old female NPG mice (Beijing Viton Biotech Co., Ltd.) were selected to collect Raji cells in the logarithmic growth phase. 5 ⁇ 10 6 Raji cells and 5 ⁇ 10 6 CIK cells were mixed and inoculated. Subcutaneously on the right back of NPG mice. After 1 hour, the mice were randomly divided into 7 groups according to their body weight, and 6 mice in each group were given the corresponding drugs intraperitoneally. All administration groups were administered twice a week, and the doses of the bifunctional antibody AB23P9 and AB23P10 administration groups were 1 mg / kg, 0.1 mg / kg and 0.01 mg / kg.
  • the average tumor volume of the PBS control group was 1679.90 ⁇ 359.05mm 3 ; the average tumor of the AB23P9 administration group at 1 mg / kg, 0.1 mg / kg, and 0.01 mg / kg
  • the volumes were 7.39 ⁇ 7.39mm 3 , 56.84 ⁇ 36.69mm 3 and 124.78 ⁇ 35.03mm 3
  • the TGI was 99.56%, 96.62% and 92.57%, respectively, which were significantly different from the control group (P ⁇ 0.01); 1mg / kg, 0.1mg / kg and 0.01mg / kg of AB23P10 administration group, the average tumor volume was 0.00 ⁇ 0.00mm 3 , 0.00 ⁇ 0.00mm 3 and 196.79 ⁇ 146.03mm 3
  • the TGI was 100%, 100% and 88.29%, compared with the control group, there was a very significant difference (P ⁇ 0.01).
  • the CD19-positive human Burkkit ’s lymphoma Daudi cell transplanted mouse model was selected to perform anti-CD19-CD3 bifunctional specific antibody for in vivo pharmacodynamic study of tumor growth inhibition.
  • Human PBMC was isolated using the method of Example 3.1, and similarly resuspended twice. In the second resuspension culture, fresh culture medium was added every 2 days, and cultured until the 8th day, and CIK cells were collected. Seven- to eight-week-old female NPG mice were selected to collect Daudi cells (cell bank of the Chinese Academy of Sciences) in the logarithmic growth phase. 5 ⁇ 10 6 Daudi cells and 1 ⁇ 10 6 CIK cells were mixed and inoculated into NPG mice Subcutaneously on the right back. After 1h, the mice were randomly divided into 8 groups according to their body weight, and 5 mice in each group were given the corresponding drugs intraperitoneally. All administration groups were given twice a week.
  • the dosage of the Blincyto administration group was 1 mg / kg, and the dosages of the bifunctional antibody AB23P9 and AB23P10 administration groups were 1 mg / kg, 0.1 mg / kg, and 0.01 mg / kg.
  • the average tumor volume of the PBS control group was 1903.03 ⁇ 727.61mm 3 ; the average tumor volume of the 1mg / kg Blincyto administration group was 0.00 ⁇ 0.00mm 3 , and the TGI was 100%, which is significantly different from the control group (P ⁇ 0.05); the average tumor volume of the AB23P9 administration group at 1 mg / kg, 0.1 mg / kg, and 0.01 mg / kg was 0.00 ⁇ 0.00mm 3 , 261.13 ⁇ 178.17, respectively mm 3 and 520.87 ⁇ 133.26mm 3, TGI were 100%, 86.28% and 72.63%, with a significant difference (P ⁇ 0.05) with respect to 1mg / kg administration group control group P9, the remaining two groups antitumor effect But the difference was not significant; the average tumor volume of the AB23P10 administration group at 1 mg / kg, 0.1 mg / kg, and 0.01 mg / kg was 0.00
  • Table 4-1 Dosing regimen for cynomolgus monkey acute toxicity evaluation
  • Table 4-2 Daily observation of cynomolgus monkeys
  • the temperature changes of cynomolgus monkeys are shown in Table 4-3. Monitoring the animal's body temperature on the day of each administration shows that the body temperature of each group does not change much and fluctuates within the range of normal physiological values.
  • the body weight of the test animals did not change as a whole. Compared with the body weight 4 days before the administration (denoted as D-4), only the body weight of the male cynomolgus monkeys in the AB23P7 administration group decreased significantly, but the body weight The weight loss is still within 10%, and the weight loss stops after the drug is stopped.
  • cynomolgus monkeys After administration, cynomolgus monkeys take blood to collect serum and determine the content of cytokines in the serum. The results are shown in Figures 4-1A to 4-1F. After the first administration, IL-2, IL-6 and IFN- ⁇ all increased, but returned to normal within 24h. Subsequent administration again, although the dose gradually increased, but after the cynomolgus monkey immune system adapted, cytokines remained at normal levels. This shows that the cynomolgus monkey has a low-to-high adaptation process to the test drug tolerance. The different degrees of diarrhea observed during the course of this experiment are common side effects of immunopharmaceutical therapy and return to normal after drug withdrawal.
  • the cynomolgus monkey (Guangzhou Xiangguan Biotechnology Co., Ltd.) is divided into two groups, one male and one male in each group, weighing 3-4 kg.
  • the first group is the AB23P7 administration group; the second group is the AB23P10 administration group, the dosage is 50 ⁇ g / kg; the blood sampling time points are 30min, 1h, 3h, 6h, 8h, 24h, 48h, 72h, a total of 8 Point in time. Blood was collected to collect serum, frozen at -80 ° C, and then the drug concentration in the serum was determined by ELISA.
  • AB23P7 and AB23P10 were configured according to 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, and 1.56 ng / mL, respectively, and established standard curves.
  • the goat anti-human IgG antibody was labeled with HRP, the detection concentration was 1: 5000, and the color was finally developed with TMB.

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Abstract

提供了一种同时靶向免疫效应细胞抗原CD3和肿瘤相关抗原CD19的四价、同源二聚体型双特异性抗体分子，所述双特异性抗体分子从N端至C端依次包含第一和第二单链Fv和Fc片段；其中，第一单链Fv能够特异性结合CD19，第二单链Fv能够特异性结合CD3，且第一和第二单链Fv通过连接肽相连，而第二单链Fv与Fc片段直接相连或通过连接肽相连；所述Fc片段不具有CDC、ADCC和ADCP等效应子功能。所述双特异性抗体能够显著抑制或杀伤肿瘤细胞，又具有控制的可能由效应细胞过度活化所致的毒副作用；此外，这种双特异性抗体为同源二聚体型，不存在重链及轻链错配问题，纯化步骤简单高效，表达量高、且其理化和体内稳定性都显著提高。

Description

针对CD19和CD3的同源二聚体型双特异性抗体及其制备方法和用途
相关申请
本申请要求2018年11月1日提交的中国专利申请CN 201811294887.4的优先权。以上所引用的优先权申请的内容全文以引用的方式并入本文。
技术领域
本发明涉及免疫学领域，更具体地，涉及一种介导T细胞杀伤的抗CD3双特异性抗体，以及这类抗体的用途，特别是其在治疗癌症、自身免疫、炎症性疾病以及免疫排异中的用途。
背景技术
机体通过固有免疫和适应性免疫排除异己以维持内环境稳定，而适应性免疫通过淋巴细胞B细胞和T细胞来执行其免疫功能。B淋巴细胞的作用之一是通过抗体依赖细胞介导的细胞毒作用(ADCC)直接杀伤靶细胞。B细胞表面的许多膜分子，如mIg，CD79a/CD79b，BCR(B细胞受体)，CD19，CD40等在B细胞识别抗原、活化、增殖以及分泌抗体等过程中发挥重要作用。BCR尤其是B细胞抗原特异性激活所必须的，其快速激活一系列激酶并启动一个复杂“信号体”的形成，继而激活下游诸如控制B细胞生长、存活和分化等途径。因此，BCR信号的稳态失衡及B细胞的异常活化可能导致相关疾病的产生。由于许多跨膜蛋白质(例如CD19、CD20和CD22)是调节BCR信号转导的特定元件，因而成为B细胞功能性疾病治疗的理想靶标。
CTL是免疫系统最有力的效应细胞，由经抗原刺激后被有效活化的初始T细胞增殖分化后产生。初始T细胞识别抗原需要通过APC的加工提呈，分别分布于两者表面的T细胞受体TCR-CD3复合物与MHC-抗原复合物的结合构成有效激活T细胞的第一信号。CD3分子是表达于所有成熟T淋巴细胞表面的蛋白，通过与TCR结合形成TCR-CD3复合物而启动对抗原刺激的体液免疫应答，因此成为介导免疫细胞(如T细胞，NK细胞等)杀伤型双特异性抗体中应用最多的效应细胞表面触发分子。CD3可以募集具有杀伤作用的CTL细胞，靶向CD3的双功能抗体通过分别结合T细胞表面CD3分子和靶细胞表面抗原，使CTL与靶细胞直接接触，进而激活T细胞并诱导其有效杀伤靶细胞。但是，第一代应用于临床的抗CD3单克隆抗体OKT3(Kung P等,Science,206:347-349,1979)，由于T细胞被过度激活而大量释放炎症因子，在临床上会引起严重的“细胞因子风暴综合症”(Hirsch R等,J.Immunol.,142:737-743,1989)。因而，开发靶向CD3的双功能抗体，如何削弱或避免过度的细胞因子风暴是首要考虑的问题。
一直以来，构建出具有正确重链和轻链组合的产物是双特异性抗体开发中最大的挑战。其中一类包含Fc结构域的双特异性抗体，因为FcRn介导的细胞内吞和再循环过程而具有更长的半衰期；同时保留了Fc 介导的部分或全部效应子功能，如ADCC、补体依赖细胞毒性(CDC)和抗体依赖细胞吞噬(ADCP)作用；并且具有更好的溶解性和稳定性，因而在双抗药物开发中得到广泛应用，包括Triomabs、kih IgG、Crossmab、ortho-Fab IgG、DVD IgG、IgG scFv、scFv2-Fc等。目前的IgG样抗CD3双特异性抗体设计广泛采用单价形式，主要是由于二价抗CD3双特异性抗体很容易导致过度激活而诱发T细胞凋亡和大量细胞因子瞬时释放(Kuhn C等,Immunotherapy,8:889-906,2016)，甚至可能引发非抗原依赖性激活T细胞反应而打破免疫平衡。但是，通常采用杂交瘤细胞表达此类具有非对称结构的异源二聚体时会同时产生许多密切相关却难以去除的非功能性错配副产物。尽管“knobs-into-holes”及其衍生技术的运用部分解决了异源二聚体型双抗分子的重链间错配的问题，然而“重链/轻链错配”又带来了另一个挑战。一种结合“CrossMab+KiH”的策略可以将其错配减到最低程度，但却需要针对两个抗体序列进行大量的突变等基因工程改造，无法达到简单、通用的目的。
另外，类IgG结构的抗CD3双特异性抗体因具有FcγR结合能力，可能导致无限制的T细胞激活，且这种活化的T细胞发现于任何表达FcγR的组织内(例如在造血、淋巴和网状内皮系统内)，即使其未与靶抗原结合。这种T细胞的全身性激活，将伴随着细胞因子的大量释放，导致治疗应用过程中的严重不良反应。因此，开发这类介导T细胞杀伤的抗CD3双特异性抗体仅需要免疫效应细胞在靶细胞内的限制性激活。
CD19(分化簇19)又称B4或Leu-12，是B淋巴细胞表面特异标记蛋白，表达于除干细胞和浆细胞外人类B淋巴细胞发育的各个阶段。CD19与CD21(CD2)，CD81(TAPA-1)形成B细胞共受体复合物，通过调节B细胞受体(B cell recertor,BCR)依赖和非依赖信号建立B细胞信号阈值，对B细胞增殖分化及信号转导起重要调控作用。CD19复合物对B细胞激活及增殖的调控是通过交联另外两种B细胞信号转导复合物来完成的，根据二者交联的量发挥相应的增强或抑制作用。CD19抗原广泛表达于B淋巴细胞恶性肿瘤，包括例如，急/慢性淋巴细胞白血病(ALL/CLL)、非霍奇金淋巴瘤(NHL)、黑色素瘤、骨髓瘤(Nicholson IC等,Mol.Immun.,34:1157-1165,1997；Nigel PM等,eCancer Med.Sci.,11:720,2017；Grossbard ML等,Br.J.Haematol.,102:509-15,1998)，此外被发现与免疫系统疾病(如类风湿性关节炎、多发性硬化症)(Kunz M等,Mediators Inflamm.,2009；2009:979258.doi:10.1155/2009/979258)和免疫排异(US 926,0530)相关，且仅暴露于异常细胞表面，可作为理想靶标用于相关疾病的诊断、预后判断及治疗。因此，开发在产品半衰期、稳定性、安全性和可生产性方面具有优越性能的抗CD19/CD3双特异性抗体将会在许多依赖于B细胞活化的疾病(例如，自身免疫或恶性肿瘤)以及移植排异中具有治疗益处。
发明内容
本发明目的是提供一种靶向免疫效应细胞抗原CD3和肿瘤相关抗原(Tumor-Associated Antigen,TAA)CD19的四价、同源二聚体型双特异性抗体分子，这种双特异性抗体在体内能够显著抑制或杀伤肿瘤细胞， 但对低表达TAA的正常细胞的非特异性杀伤作用显著降低，同时具有控制的可能由效应细胞过度活化所致的毒副作用，且其理化和体内稳定性都显著提高。
本发明一个方面，提供一种针对CD19的双特异性抗体，所述双特异性抗体分子由两条相同的多肽链以共价键结合形成四价同源二聚体，每条多肽链从N端至C端依次包含特异性结合肿瘤相关抗原CD19的第一单链Fv(抗-TAA scFv)、特异性结合效应细胞抗原CD3的第二单链Fv(抗-CD3 scFv)和Fc片段；其中，第一和第二单链Fv通过连接肽相连，而第二单链Fv与Fc片段直接相连或通过连接肽相连，且所述Fc片段不具有效应子功能。
其中，第一单链Fv针对肿瘤相关抗原具有特异性，其所包含的VH结构域和VL结构域通过连接肽(L1)连接，所述VH、L1和VL以VH-L1-VL或VL-L1-VH的顺序排列，且所述连接肽L1的氨基酸序列为(GGGGX) n，X包含Ser或Ala，X优选Ser；n为1-5的自然数，n优选3；
示例性地，所述肿瘤相关抗原为CD19，包含但不限于：CD19的任何变体、同工型、衍生物和物种同源物；优选地，CD19来源于人、食蟹猴或恒河猴。
例如，本发明表1-1中例举了一些优选的针对CD19的第一单链Fv的VH结构域及其互补决定区(HCDR1、HCDR2和HCDR3)的氨基酸序列，和VL结构域及其互补决定区(LCDR1、LCDR2和LCDR3)的氨基酸序列。
优选地，所述第一单链Fv特异性结合CD19，其包含的CDR1、CDR2和CDR3选自下组：
(i)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：1、2和3所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：4、5和6所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(ii)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：9、10和11所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：12、13和14所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(iii)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或 具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：20、13和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(iv)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：24、25和26所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：27、28和29所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(v)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：32、33和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
更优选地，所述第一单链Fv特异性结合CD19，其选自下组：
(i)VH结构域包含如SEQ ID NO：7所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：8所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(ii)VH结构域包含如SEQ ID NO：15所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：16所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(iii)VH结构域包含如SEQ ID NO：22所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例 如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：23所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(iv)VH结构域包含如SEQ ID NO：30所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：31所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(v)VH结构域包含如SEQ ID NO：34所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：35所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(vi)VH结构域包含如SEQ ID NO：36所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：37所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(vii)VH结构域包含如SEQ ID NO：38所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：39所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
其中，连接本发明所述第一单链Fv和第二单链Fv的连接肽(L2)由柔性肽和刚性肽组成。
进一步地，所述柔性肽包含2个或更多个氨基酸，并优选自下列几种氨基酸：Gly(G)、Ser(S)、Ala(A)和Thr(T)。更优地，所述柔性肽包含G和S残基。最优地，所述柔性肽的氨基酸组成结构通式为G xS y(GGGGS) z，其中x，y和z是大于或等于0的整数，且x+y+z≥1。例如，在一优选实施例中，所述柔性肽的氨基酸序列为G 2(GGGGS) 3。
进一步地，所述刚性肽来自天然人绒毛膜促性腺激素β亚基羧基末端第118至145位氨基酸组成的全 长序列(如SEQ ID NO：48所示)或其截短的片段(以下统称为CTP)。优选地，所述CTP刚性肽包含SEQ ID NO：48N端的10个氨基酸，即SSSSKAPPPS(CTP 1)；或所述CTP刚性肽包含SEQ ID NO：48C端的14个氨基酸，即SRLPGPSDTPILPQ(CTP 2)；又如，另一实施例中，所述CTP刚性肽包含SEQ ID NO：48N端的16个氨基酸，即SSSSKAPPPSLPSPSR(CTP 3)；再如，另一些实施例中，所述CTP刚性肽包含28个氨基酸并开始于人绒毛膜促性腺激素β亚基的第118位，终止于第145位，即SSSSKAPPPSLPSPSRLPGPSDTPILPQ(CTP 4)。
例如，本发明表1-2中示例性的例举了一些优选的连接第一和第二单链Fv的连接肽L2的氨基酸序列。
在本发明的一优选实施例中，所述连接肽的氨基酸序列如SEQ ID NO：49所示，其柔性肽的氨基酸组成为G 2(GGGGS) 3，和其刚性肽的氨基酸组成为SSSSKAPPPS(即CTP 1)。
其中，第二单链Fv针对免疫效应细胞抗原CD3具有特异性，其所包含的VH结构域和VL结构域通过连接肽(L3)连接，所述VH、L3和VL以VH-L3-VL或VL-L3-VH的顺序排列，且所述连接肽L3的氨基酸序列为(GGGGX) n，X包含Ser或Ala，X优选Ser；n为1-5的自然数，n优选3；
优选地，所述双特异性抗体的第二单链Fv在体外FACS结合分析测定中以大于约10nM，或大于20nM，或大于40nM，或大于约50nM的EC 50值结合于效应细胞；更优选地，所述双特异性抗体的第二单链Fv不仅能与人CD3结合，还可与食蟹猴或恒河猴的CD3特异性结合。在本发明的一优选实施例中，所述双特异性抗体以15.69nM的EC 50值与效应细胞特异性结合。
优选地，所述第二单链Fv特异性结合CD3，其VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：40、41和42所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：43、44和45所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
更优选地，所述第二单链Fv特异性结合CD3，其VH结构域包含如SEQ ID NO：46所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：47所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
其中，本发明所述Fc片段直接或通过连接肽L4与第二单链Fv相连，且所述连接肽L4包含1-20个氨 基酸，并优选自下列几种氨基酸：Gly(G)、Ser(S)、Ala(A)和Thr(T)；较优地地，所述连接肽L4选自Gly(G)和Ser(S)；更优选地，所述连接肽L4组成为(GGGGS)n，n＝1，2，3或4。本发明的一优选实施例中，所述Fc片段与第二单链Fv直接相连。
另一方面，本发明所述Fc片段包含来源于人免疫球蛋白重链恒定区的铰链区、CH2和CH3结构域，例如，在某些实施方案中，本发明所述Fc片段来源于例如选自人IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；特别地选自例如人IgG1、IgG2、IgG3和IgG4的重链恒定区，更特别地选自人IgG1或IgG4的重链恒定区；并且，所述Fc片段与其所源自的天然序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个置换、缺失或添加)。
在一些优选实施方案中，所述Fc片段被改变，例如被突变，以修饰本发明所述双特异性抗体分子的性质(例如改变下列中的一个或更多个特性：Fc受体结合、抗体糖基化、效应细胞功能或补体功能)。
例如，本发明提供的双特异性抗体包含具有改变的效应子功能(例如降低或消除)的氨基酸置换、缺失或添加的Fc变体。抗体的Fc区介导几种重要的效应子功能，例如ADCC、ADCP、CDC等。通过替换抗体的Fc区中的氨基酸残基，以改变抗体对效应子配体(如FcγR或补体C1q)的亲和力，从而改变效应子功能的方法是本领域已知的(参见，例如EP 388,151A1；US 564,8260；US 562,4821；Natsume A等,Cancer Res.,68:3863-3872,2008；Idusogie EE等,J.Immunol.,166:2571-2575,2001；Lazar GA等,PNAS,103:4005-4010,2006；Shields RL等,JBC,276:6591-6604,2001；Stavenhagen JB等,Cancer Res.,67:8882-8890,2007；Stavenhagen JB等,Advan.Enzyme.Regul.,48:152-164,2008；Alegre ML等,J.Immunol.,148:3461-3468,1992；和Kaneko E等,Biodrugs,25:1-11,2011)。在本发明一些优选实施例中，对抗体恒定区上的氨基酸L235(EU编号)进行修饰以改变Fc受体相互作用，例如L235E或L235A。在另一些优选实施例中，对抗体恒定区上的氨基酸234和235同时进行修饰，例如L234A和L235A(L234A/L235A)(EU编号)。
例如，本发明提供的双特异性抗体可包含具有延长的循环半衰期的氨基酸置换、缺失或添加的Fc变体。研究发现M252Y/S254T/T256E、M428L/N434S或者T250Q/M428L都能够延长抗体在灵长类动物中的半衰期。更多的与新生儿受体(FcRn)结合亲和力增强的Fc变体所包含突变位点可以参见中国发明专利CN 201280066663.2、US 2005/0014934A1、WO 97/43316、US 5,869,046、US 5,747,03、WO 96/32478。在本发明一些优选实施例中，对抗体恒定区上的氨基酸M428(EU编号)进行修饰以增强FcRn受体的结合亲和力，例如M428L。在另一些优选实施例中，对抗体恒定区上的氨基酸250和428(EU编号)同时进行修饰，例如T250Q和M428L(T250Q/M428L)。
例如，本发明提供的双特异性抗体也可包含具有可以降低或消除Fc糖基化的氨基酸置换、缺失或添加的Fc变体。例如，Fc变体包含正常存在于氨基酸位点297(EU编号)处的N-连接聚糖降低的糖基化。N297位糖基化对IgG的活性有很大影响，如果该位点糖基化被移除，则会影响IgG分子CH2上半部分的构象， 从而丧失对FcγRs的结合能力，影响抗体相关的生物活性。在本发明的一些优选实施例中，对人IgG恒定区上的氨基酸N297(EU编号)进行修饰以避免抗体的糖基化，例如N297A。
例如，本发明提供的双特异性抗体也可包含具有消除电荷异质性的氨基酸置换、缺失或添加的Fc变体。在工程细胞表达过程中发生的多种翻译后修饰会都会引起单克隆抗体的电荷异质性，而IgG抗体C末端赖氨酸的不均一性是其中的一个主要原因，重链C端的赖氨酸K可能在抗体生产过程中出现一定比例的缺失，从而造成电荷异质性，从而影响抗体的稳定性、有效性、免疫原性或药代动力学。在本发明的一些优选实施例中，将IgG抗体C末端的K447(EU编号)去除或缺失，以消除抗体的电荷异质性，提高表达产物的均一性。
本发明表1-3中示例性的例举了一些优选的Fc片段的氨基酸序列。与包含野生型人IgG Fc区的双特异性抗体相比，本发明提供的双特异性抗体所包含的Fc片段对人FcγRs(FcγRI、FcγRIIa或FcγRIIIa)和C1q的至少一种显示出降低的亲和力，具有减少的效应细胞功能或补体功能。例如，在本发明的一优选实施例中，双特异性抗体包含的Fc片段来自人IgG1，且具有L234A和L235A取代(L234A/L235A)，显示出对FcγRI降低的结合能力；此外，本发明提供的双特异性抗体包含的所述Fc片段还可以包含具有使其它一种或几种特性(例如，与FcRn受体结合能力、抗体糖基化或抗体电荷异质性等)改变的氨基酸取代。例如，在本发明的一优选实施例中，所述Fc片段的氨基酸序列如SEQ ID NO：54所示，它与其所源自的天然序列相比具有L234A/L235A/T250Q/N297A/P331S/M428L的氨基酸置换或取代，且K447被缺失或删除。
本发明一优选实施例中，所述双特异性抗体结合人CD19和CD3，其氨基酸序列如下：
(i)SEQ ID NO：55所示的序列；
(ii)与SEQ ID NO：55所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：55所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些优选的实施方案中，(ii)中所述的置换是保守置换。
同样优选地，其氨基酸序列如下：
(i)SEQ ID NO：57所示的序列；
(ii)与SEQ ID NO：57所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：57所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些优选的实施方案中，(ii)中所述的置换是保守置换。
同样优选地，其氨基酸序列如下：
(i)SEQ ID NO：59所示的序列；
(ii)与SEQ ID NO：59所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：59所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些优选的实施方案中，(ii)中所述的置换是保守置换。
同样优选地，其氨基酸序列如下：
(i)SEQ ID NO：61所示的序列；
(ii)与SEQ ID NO：61所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：61所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些优选的实施方案中，(ii)中所述的置换是保守置换。
本发明另一方面，提供了编码上述双特异性抗体的DNA分子。
本发明的优选实施例中，编码上述双特异性抗体的DNA分子如SEQ ID NO：56所示的核苷酸序列。本发明的优选实施例中，编码上述双特异性抗体的DNA分子如SEQ ID NO：58所示的核苷酸序列。
本发明的优选实施例中，编码上述双特异性抗体的DNA分子如SEQ ID NO：60所示的核苷酸序列。
本发明的优选实施例中，编码上述双特异性抗体的DNA分子如SEQ ID NO：62所示的核苷酸序列。
本发明另一方面，提供了包含上述DNA分子的载体。
本发明另一方面，提供了包含上述载体的宿主细胞；所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，优选为CHO细胞。
本发明另一方面，提供了一种药物组合物，所述组合物包含上述双特异性抗体、或上述DNA、或上述核苷酸序列以及可药用赋形剂、载体或稀释剂。
优选地，药物组合物还包含另外的药学活性剂。
优选地，另外的药学活性剂是用于治疗免疫相关疾病的药物。
优选地，另外的药学活性剂是具有抗肿瘤活性的药物。
优选地，另外的药学活性剂是用于治疗自身免疫性疾病或炎症性疾病的药物。
优选地，另外的药学活性剂是用于治疗移植排异反应相关疾病或病症的药物。
优选地，上述双特异性抗体、或上述DNA、或上述核苷酸序列与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
优选地，向受试者施用上述药物组合物之前、之后或同时施用药学活性剂，其中所述药学活性剂选自抗体、抗体片段、药物、酶、细胞毒性剂、毒素、抗生素、激素、免疫调节剂、细胞因子、趋化因子和放射性同位素。
优选地，上述药物组合物中的药物选自由以下组成的组：环磷酰胺、尼莫司汀、氨甲蝶呤、氟尿嘧啶、卡培他滨、吉西他滨、替吉奥、培美曲塞、氟达拉滨、阿霉素、博来霉素、长春瑞滨、紫杉醇、多西他赛、伊利替康、他莫昔芬、来曲唑、依西美坦、氟维司群、戈舍瑞林、甲羟孕酮、顺铂、卡铂、草酸铂、奈达铂、奥沙利铂、门冬酰胺酶、洛铂、依托泊苷、长春新碱、伊立替康、替加氟、达卡巴嗪、丝裂霉素、替尼泊苷、吡柔比星、米托蒽醌、长春地辛、雷替曲塞、甲氨蝶呤、顺铂博来霉素硫酸盐、亚硝基脲氮芥、苯丁酸氮芥、环磷酰胺羟脲和道诺霉素。
优选地，上述药物组合物中的毒素选自由以下组成的组：篦麻毒素、相思子毒素、α毒素、皂素、核糖核酸酶(RNA酶)、DNA酶I、葡萄球菌肠毒素-A、美洲商陆抗病毒蛋白、白树毒素、白喉毒素、假单胞菌外毒素和假单胞菌内毒素。
优选地，上述药物组合物中的免疫调节剂选自由以下组成的组：细胞因子、趋化因子、干细胞生长因子、淋巴毒素、集落刺激因子(CSF)、白细胞介素(IL)、红细胞生成素、血小板生长因子、肿瘤坏死因子(TNF)、粒细胞-集落刺激因子(G-CSF)、粒细胞巨噬细胞-集落刺激因子(GM-CSF)、干扰素-α、干扰素-β、干扰素-γ或干扰素-λ、TGF-α、TGF-β、白细胞介素-1(IL-1)、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12；IL-13、IL-14、IL-15、IL-16、IL-17、IL-18、IL-21、IL-23、IL-25、LIF、FLT-3、血管内皮生长因子、血小板反应蛋白和内皮抑制素。
优选地，上述药物组合物可与一种或多种额外的其他疗法组合施用，其中其他疗法选自下组：手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。
本发明另一方面，提供了一种用于增强或刺激免疫应答或功能的方法，其包含对所述个体施用治疗有效量的所述双特异性抗体、或所述DNA、或所述核苷酸序列、或所述药物组合物。
本发明另一方面，提供了一种用于治疗、预防或改善细胞、组织、器官或动物中的免疫病症或疾病的方法，其包括对所述个体施用治疗有效量的所述双特异性抗体、或所述DNA、或所述核苷酸序列、或所述药物组合物。
本发明另一方面，提供了一种用于预防/治疗疾病、延迟其进展、降低/抑制其复发的方法，所述疾病包含免疫相关疾病、肿瘤、自身免疫性疾病、炎症性疾病或移植排异反应相关疾病或病症等疾病或病症，其包括将有效量的所述双特异性抗体、或所述DNA、或所述核苷酸序列、或所述药物组合物给予或施用至所述患有以上疾病或病症的个体。
优选地，上述方法可与一种或多种额外的其他疗法组合施用，其中其他疗法选自下组：手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。
本发明另一方面，还提供了制备本发明所述双特异性抗体的方法，其包括：(a)获得双特异性抗体的融合基因，构建双特异性抗体的表达载体；(b)通过基因工程方法将上述表达载体转染到宿主细胞中；(c)在允许产生所述双特异性抗体的条件下培养上述宿主细胞；(d)分离、纯化产生的所述抗体。
其中，步骤(a)中所述表达载体选自质粒、细菌和病毒中的一种或多种，优选地，所述表达载体为PCDNA3.1。
其中，步骤(b)通过基因工程方法将所构建的载体转染入宿主细胞中，所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，优选为CHO细胞。
其中，步骤(d)通过常规的免疫球蛋白纯化方法，包含蛋白质A亲和层析和离子交换、疏水层析或分子筛方法分离、纯化所述双特异性抗体。
本发明另一方面，提供了所述双特异性抗体、或所述DNA、或所述核苷酸序列，在制备治疗、预防或缓解肿瘤的药物中的用途；所述癌症的实例包括但不限于：急性骨髓性白血病(AML)、慢性骨髓性白血病(CML)、急性B淋巴细胞性白血病(B-ALL)、慢性B淋巴细胞白血病(B-CLL)、B细胞淋巴瘤(BCL)、T细胞淋巴瘤(TCL)(例如皮肤)、骨髓增生异常综合征(MDS)、小淋巴细胞淋巴瘤(SLL)、毛细胞白血病(HCL)、边缘区淋巴瘤(MZL)(例如结外或脾)、滤泡性淋巴瘤(FL)(例如儿童型或胃肠道)、幼B淋巴细胞白血病(B-PLL)、套细胞淋巴瘤(MCL)、淋巴浆细胞淋巴瘤(LPL)/瓦尔登斯特伦巨球蛋白血症(WM)、淋巴母细胞白血病(ALL)(例如B细胞)、淋巴母细胞淋巴瘤(LBL)(例如B细胞)、浆母细胞性淋巴瘤(PBL)(例如B细胞)、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫大B细胞淋巴瘤(DLBCL)(例如，原发部位或炎症相关)、伯基特淋巴瘤(BL)、多发性骨髓瘤、间变性大细胞淋巴瘤和HIV相关淋巴瘤。
本发明另一方面，提供了所述双特异性抗体、或所述DNA、或所述核苷酸序列，在制备治疗、预防或缓解自身免疫性或炎症性疾病的药物中的用途，所述自身免疫性疾病或炎症性疾病选自：类风湿性关节炎(RA)、骨关节炎、反应性关节炎、系统性红斑狼疮(SLE)、克罗恩氏病、多发性硬化症、硬皮病、牛皮癣、牛皮癣关节病、溃疡性结肠炎(例如，慢性)、胰岛素依赖性糖尿病(例如，青少年)、甲状腺炎(例 如，慢性)、甲状腺机能亢进、哮喘、变态反应性疾病、结节病、自身免疫溶血性贫血、恶性贫血、移植物抗宿主病、皮肌炎、慢性肝炎、微观肾脉管炎、慢性活动性肝炎、葡萄膜炎、肠滑膜炎、自身免疫肠疾病、特发性白细胞减少症、自身免疫性肾小球肾炎、自身免疫溶血性贫血、自身免疫性肝炎、间质肺炎、慢性天疱疮、寻常型天疱疮、动脉炎、结节性多动脉炎和强直性脊柱炎。
本发明另一方面，提供了所述双特异性抗体、或所述DNA、或所述核苷酸序列，在制备治疗、预防或缓解移植排异反应相关疾病或病症的药物中的用途，所述移植排异反应包括急性、超急性或慢性移植排异反应，所述移植排异包括器官、组织或细胞移植排异，包括但不限于输血、血管移植、上皮移植、内皮移植、肌肉移植、结缔组织移植、关节移植、心脏移植、肺移植、肝移植、肾移植、胰腺移植、皮肤移植、肠移植、角膜移植、骨髓移植、移植物抗宿主病和宿主抗移植物病。
本发明另一方面，提供了一种检测哺乳动物中癌症存在的方法，其包括：(a)将包含一个或多个源自哺乳动物的细胞的样品与所述双特异性抗体、或所述DNA、或所述核苷酸序列接触，从而形成复合物，和；
(b)检测所述复合物，其中复合物的检测表明哺乳动物中癌症的存在。
本发明另一方面，提供了一种试剂盒，其含有本发明所述的双特异性抗体、或所述的DNA、或所述的核苷酸序列。
本发明另一方面，还提供了一种结合CD19的单克隆抗体及其抗原结合片段，所述单克隆抗体可变区来自上述双特异性抗体的第一单链Fv，且所包含的CDR1、CDR2和CDR3选自下组：
(i)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：32、33和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(ii)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：20、13和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序 列。
优选地，所述单克隆抗体为人源化抗体或全人抗体。
优选地，所述抗原结合片段选自scFv、Fab、Fab’、(Fab’)2、Fv片段、二硫键连接的Fv(dsFv)。
更优选地，所述结合CD19的单克隆抗体或其抗原结合片段所包含的VH结构域和VL结构域选自下组：
(i)VH结构域包含如SEQ ID NO：36所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：37所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
(ii)VH结构域包含如SEQ ID NO：38所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：39所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
本发明公开的技术方案，取得了有益的技术效果，概况如下：
1、本发明提供的双特异性抗体包含的抗-TAA scFv位于双抗的N端，空间构像发生变化，与TAA的结合能力在某些条件下可能减弱，尤其不易结合弱表达或低表达TAA的正常细胞，减少了非特异性杀伤，但对过表达或高表达TAA的细胞的结合特异性没有显著下降，表现出良好的体内杀伤效果。由此亦知，当靶抗原仅表达于肿瘤细胞上或本发明所述双特异性抗体仅与过表达靶抗原的肿瘤细胞特异性结合时，使得免疫效应细胞限制性仅在靶细胞组织内被激活，这使得所述双特异性抗体对正常细胞的非特异性杀伤以及细胞因子的伴随释放能够被降至最低，减小其在临床治疗中的毒副作用。
2、本发明提供的双特异性抗体选择的抗-CD3 scFv以微弱的结合亲和力(EC 50值大于约大于约10nM，或大于20nM，或大于40nM，或大于约50nM)与效应细胞特异性结合，此外因被包埋在抗-TAA scFv和Fc之间，且位于其N端的连接肽L3包含的CTP刚性肽和位于其C端的Fc片段，都部分“遮盖”或“屏蔽”了抗-CD3 scFv的抗原结合域，这种位阻效应使其以更微弱的结合亲和力(例如以大于1μM)与CD3结合，这使其对T细胞的活化刺激能力减弱，因而限制了细胞因子的过度释放，因而具有更高的安全性；另外，本发明采用的抗-CD3 scFv可同时结合人和食蟹猴和/或恒河猴的CD3天然抗原，使其临床前毒理学评价不需要再构建替代分子，且获得的有效剂量、毒性剂量和毒副反应更客观、准确，可以直接进行临床剂量的转换，降低临床研究的风险。再者，本发明提供的双特异性抗体创造性地采用了二价抗-CD3 scFv， 这使得所述双特异性抗体在构型设计上规避了现有技术普遍所采用的异源二聚体型(所包含的抗-CD3 scFv为单价)的非对称结构，因而也不存在重链间错配的问题，简化了下游纯化步骤；并且出人意料地，在体外细胞结合试验中未观察到抗-CD3 scFv与T细胞的非特异性结合，且细胞激活程度(IL-2等细胞因子的释放)控制在安全、有效的范围内，即本发明采用的二价抗-CD3 scFv结构并未引起非抗原依赖地诱导T细胞的过度活化，而对其他包含二价抗-CD3结构域的双特异性抗体而言，T细胞被不可控地过度激活是普遍存在的，因而抗-CD3双特异性抗体在设计时一般避免引入二价抗-CD3结构。
3、本发明提供的双特异性抗体所包含的经修饰的Fc片段不具有FcγR结合能力，避免了由FcγR所介导的T细胞全身性激活，因而允许免疫效应细胞限制性地仅在靶细胞组织内被激活。
4、本发明提供的双特异性抗体为同源二聚体型，不存在重链及轻链错配的问题，下游生产工艺稳定，纯化步骤简单高效，表达产物均一，且其理化和体内稳定性都显著提高。
5、本发明提供的双特异性抗体因包含Fc片段，而具有较长的体内循环半衰期。
发明详述
缩写和定义
BiAb   双特异性抗体(bispecific antibody)
CDR    用Kabat编号系统界定的免疫球蛋白可变区中的互补决定区
EC 50   产生50％功效或结合的浓度
ELISA  酶联免疫吸附测定
FR     抗体构架区：将CDR区排除在外的免疫球蛋白可变区
HRP    辣根过氧化物酶
IL-2   白细胞介素2
IFN    干扰素
IC 50   产生50％抑制的浓度
IgG    免疫球蛋白G
Kabat  由Elvin A Kabat倡导的免疫球蛋白比对及编号系统
mAb    单克隆抗体
PCR    聚合酶链式反应
V区    在不同抗体之间序列可变的IgG链区段。其延伸到轻链的109位Kabat残基和重链的第113位残基。
VH     免疫球蛋白重链可变区
VK     免疫球蛋白κ轻链可变区
K D    平衡解离常数
k a    结合速率常数
k d    解离速率常数
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。本发明使用的抗体或其片段可单独或联合使用本领域已知的常规技术，例如氨基酸缺失、插入、取代、增加、和/或重组以及/或其他修饰方法作进一步修饰。根据一种抗体的氨基酸序列在其DNA序列中引入这种修饰的方法对本领域技术人员来说是众所周知的；见例如，Sambrook，分子克隆：实验手册，Cold Spring Harbor Laboratory(1989)N.Y.。所指的修饰优选在核酸水平上进行。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
“CD19”是分化簇19多肽，是单通道I型跨膜糖蛋白，其含有两个Ig样C2型(免疫球蛋白样)结构域和一个相对较大的胞质尾，在哺乳动物物种中高度保守。CD19在几乎所有的B谱系细胞中和滤泡细胞中表达，对于B淋巴细胞分化是必需的，并且作为B细胞关键共受体与CD21、CD81和CD225一起发挥作用。因此，CD19常被用作B淋巴细胞发育、B细胞淋巴瘤以及B淋巴细胞白血病的生物诊断标志。此外，CD19中的突变与严重的免疫缺陷综合症相关。针对CD19靶点的适应症还包括其他现有技术中发现的以及未来发现的相关疾病或病症。该术语还包括CD19的任何变体、同工型、衍生物和物种同源物，其由细胞-包括肿瘤细胞-天然地表达，或由以CD19基因或cDNA转染的细胞表达。
CD3分子是T细胞膜上的重要分化抗原，是成熟T细胞的特征性标志，由6条肽链组成，以非共价键与T细胞抗原受体(TCR)组成TCR-CD3复合体，不仅参与TCR-CD3复合体的胞浆内组装，而且通过各多肽链胞浆区的免疫受体酪氨酸活化基序(Immunoreceptor Tyrosine-based Activation Motif,ITAM)传递抗原刺激信号。CD3分子的主要功能为：稳定TCR结构，传递T细胞活化信号，当TCR特异性识别并结合抗原后，CD3参与将信号转导到T细胞胞浆内，作为诱导T细胞活化的第一信号，在T细胞抗原识别和免疫应答产生过程中具有极其重要的作用。
“CD3”指的是作为T细胞受体复合物的一部分，由三个不同的链CD3ε，CD3δ和CD3γ组成。CD3在T细胞上通过例如抗CD3抗体对其的固定作用而产生的集中(clustering)，导致T细胞的活化，与T细胞受体介导的活化类似，但是不依赖于TCR克隆的特异性。绝大多数抗CD3抗体识别CD3ε链。本发明的特异性识别T细胞表面受体CD3的第二功能域不受具体的限制，只要其能够特异性地识别CD3，例如但不限于在下列专利中提到的CD3抗体：US 7,994,289；US 6,750,325；US 6,706,265；US 5,968,509；US 8,076,459；US 7,728,114；US20100183615。优选地，本发明中使用的抗人CD3抗体与食蟹猴和/或恒河猴具有交叉反应 性，例如但不限于下列专利中提到的抗人CD3抗体：WO 2016130726，US 20050176028，WO 2007042261或WO 2008119565。该术语还包括任何CD3变体、同工型、衍生物和物种同源物，其由细胞天然地表达，或在以编码前述的链的基因或cDNA转染的细胞上表达。
术语“抗体”通常指具有免疫球蛋白-类功能的蛋白质性结合分子。抗体的典型实例是免疫球蛋白，以及其衍生物或功能片段，只要其显示所需的结合特异性即可。用于制备抗体的技术是本领域熟知的。“抗体”包括不同类的天然免疫球蛋白(例如IgA、IgG、IgM、IgD和IgE)和亚类(如IgG1、lgG2、IgA1、IgA2等)。“抗体”还包括非天然免疫球蛋白，包括例如单链抗体，嵌合抗体(例如，人源化鼠抗体)和异源偶联抗体(例如，双特异性抗体)，以及其抗原结合片段(例如，Fab’，F(ab’)，Fab，Fv和rIgG)。还参见，例如，Pierce Catalog and Handbook,1994-1995,Pierce Chemical Co.,Rockford,IL；Kuby,J.,Immunology,3rd Ed.,1998,NY；W.H.Freeman&Co.。
抗体可以结合至一种抗原，称为“单特异性”；或结合至两种不同的抗原，称为“双特异性”；或结合至多于一种的不同的抗原，称为“多特异性”。抗体可以是单价、二价或多价的，即抗体可以一次结合至一个、两个或多个抗原分子。
抗体“单价地”结合至某特定蛋白质，即一分子的抗体仅结合至一分子的蛋白质，但是该抗体也可以结合到不同的蛋白质。当抗体仅结合至一分子的两种不同的蛋白质，该抗体为“单价地”结合至每一种蛋白质，并且该抗体是“双特异性的”且“单价地”结合至两种不同蛋白质的每一种。抗体可以是“单体的”，即其包含单个多肽链。抗体可包含多个多肽链(“多聚体的”)或可包含两个(“二聚体的”)、三个(“三聚体的”)或四个(“四聚体的”)多肽链。若抗体为多聚体的，则该抗体可以是同源多聚体(homomulitmer)，即抗体包含多于一分子的仅一种多肽链，包括同源二聚体、同源三聚体或同源四聚体。可选的，多聚体抗体可以是异源多聚体，即抗体包含多于一种不同的多肽链，包括异源二聚体、异源三聚体或异源四聚体。
术语“超变区”或“CDR区”或“互补决定区”是指负责抗原结合的抗体氨基酸残基，是非连续的氨基酸序列。CDR区序列可以由IMGT、Kabat、Chothia和AbM方法来定义或本领域熟知的任何CDR区序列确定方法而鉴定的可变区内的氨基酸残基。例如，超变区包含以下氨基酸残基：来自序列比对所界定的“互补决定区”或“CDR”的氨基酸残基，例如，轻链可变结构域的24-34(L1)、50-56(L2)和89-97(L3)位残基和重链可变结构域的31-35(H1)、50-65(H2)和95-102(H3)位残基，参见Kabat等，1991，Sequences of Proteins of Immunological Interest(免疫目的物的蛋白质序列)，第5版，Public Health Service，National Institutes of Health，Bethesda，Md.；和/或来自根据结构来界定的“超变环”(HVL)的残基，例如，轻链可变结构域的26-32(L1)、50-52(L2)和91-96(L3)位残基和重链可变结构域的26-32(H1)、53-55(H2)和96-101(H3)位残基，参见Chothia和Leskl,J.Mol.Biol.,196:901-917,1987。“构架”残基或“FR”残基为除本文定义的超变区残基之外的可变结构域残基。在某些实施方案中，本发明的抗体或其抗原结合片段含有 的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。本领域技术人员可以明确地将每种系统赋予任何可变结构域序列，而不依赖于超出序列本身之外的任何实验数据。例如，给定抗体的Kabat残基编号方式可通过将抗体序列与每种“标准”编号序列对比同源区来确定。基于本文提供的序列的编号，确定序列表中任何可变区序列的编号方案完全在本领域技术人员的常规技术范围内。
术语“单链Fv抗体”(或“scFv抗体”)是指包含抗体的VH和VL结构域的抗体片段，是通过接头(linker)连接的重链可变区(VH)和轻链可变区(VL)的重组蛋白，接头使得这两个结构域相交联以形成抗原结合位点，接头序列一般由柔性肽组成，例如但不限于G 2(GGGGS) 3。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一个核苷酸链编码的一条氨基酸链序列。对于scFv综述，可参见Pluckthun(1994)The Pharmacology of Monoclonal Antibodies(单克隆抗体药理学),第113卷,Rosenburg和Moore主编,Springer-Verlag,New York,第269-315页。还参见国际专利申请公开号WO 88/01649和美国专利第4,946,778号和第5,260,203号。
术语“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fab抗体”的大小是完整抗体的1/3，其只包含一个抗原结合位点。
术语“Fab’片段”含有一条轻链和一条重链的VH结构域和CH1结构域以及CH1和CH2结构域之间的恒定区部分。
术语“F(ab’)2片段”含有两条轻链和两条重链的VH结构域和CH1结构域以及CH1和CH2结构域之间的恒定区部分，由此在两条重链间形成链间二硫键。因此，F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。
术语“Fc”区指抗体重链恒定区片段，其包含至少铰链区、CH2和CH3结构域。
术语“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区，是包含完整抗原识别和结合位点的最小片段。
术语“Fd片段”由一条重链的CH1及可变区组成，是Fab片段除去轻链后剩下的重链部分。
术语“抗体片段”或“抗原结合片段”是指保留与抗原(如，CD19)特异性结合能力的抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(Parental Antibody)的抗原结合区或可变区。抗体片段保留母体抗体的至少某些结合特异性。通常，当用摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％的母体抗体对靶标的结合亲和力。抗体片段包括但不限于：Fab片段、Fab’片段、F(ab’)2片段、Fv片段、Fd片段、互补决定区(CDR)片段、二硫键稳定性蛋白(dsFv)等；线性抗体(Linear Antibody)、单链抗体(例如scFv单抗体)(技术来自Genmab)、二价单链抗体、单链噬菌体抗体、单域抗体(Single Domain Antibody)(例如VH结构域抗体)、结构域抗体(技术来自AbIynx)；由抗体片段形成的多特异性抗体(例如三链抗体、四链抗体等)；和工程改造 抗体如嵌合抗体(Chimeric Antibody)(例如人源化鼠抗体)、异缀合抗体(Heteroconjugate Antibody)等。这些抗体片段用本领域技术人员已知的常规技术获得，并用与完整抗体相同的方法对这些片段的实用性进行筛选。
术语“连接肽”是指连接两个多肽的肽，其中所述连接肽可以是两个免疫球蛋白可变区或一个可变区。连接肽的长度可以是0-30个氨基酸或0-40个氨基酸。在一些实施方案中，连接肽可以是0-25、0-20或0-18个氨基酸长度。在一些实施方案中，连接肽可以是不多于14、13、12、11、10、9、8、7、6或5个氨基酸长的肽。在其它实施方案中，连接肽可以是0-25、5-15、10-20、15-20、20-30或30-40个氨基酸长。在其它实施方案中，连接肽可以是约0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个氨基酸长。连接肽是本领域技术人员已知的。连接肽的制备可以采用本领域任何方法。例如，连接肽可以是合成来源的。
术语“重链恒定区”包括来自免疫球蛋白重链的氨基酸序列。包含重链恒定区的多肽至少包含以下一种：CH1结构域，铰链(例如，上部铰链区、中间铰链区，和/或下部铰链区)结构域，CH2结构域，CH3结构域，或其变体或片段。例如，本申请中使用的抗原结合多肽可包含具有CH1结构域的多肽链；具有CH1结构域、至少一部分的铰链结构域和CH2结构域的多肽；具有CH1结构域和CH3结构域的多肽链；具有CH1结构域、至少一部分铰链结构域和CH3结构域的多肽链，或者具有CH1结构域，至少一部分铰链结构，CH2结构域，和CH3结构域的多肽链。在另一个实施例中，本申请的多肽包括具有CH3结构域的多肽链。另外，在本申请中使用的抗体可能缺少至少一部分CH2结构域(例如，所有的或一部分的CH2结构域)。如上文所述，但本技术领域的普通技术人员应理解，重链恒定区可能会被修改，使得它们在氨基酸序列上与天然存在的免疫球蛋白分子不同。
术语“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，而术语“CH1结构域”包括免疫球蛋白重链的第一(多数为氨基末端)恒定区。CH1结构域邻近VH结构域并且是免疫球蛋白重链分子的铰链区的氨基末端。
术语“铰链区”包括重链分子的将CH1结构域连接至CH2结构域的那一部分。该铰链区包含约25个残基并且是柔性的，从而使两个N-末端抗原结合区独立地移动。铰链区可分为三个不同的结构域：上部、中部、和下部铰链结构域(Roux KH等,J.Immunol.,161:4083,1998)。
术语“二硫键”包括两个硫原子之间形成的共价键。氨基酸半胱氨酸含有巯基，该巯基可以与第二个巯基形成二硫键或桥连。在大多数天然存在的IgG分子中，CH1和CK区由二硫键连接并且两个重链由两个二硫键连接，在对应于使用Kabat编号系统的239和242处(位置226或229，EU编号系统)连接。
术语“体内半衰期”指目的多肽在给定动物的循环中的生物半衰期，并表示为在动物中从循环和/或其他组织清除存在于该动物循环中的量的一半所需的时间。
术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.,48:443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.,4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J.MoI.Biol.,48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
术语“Fc区”或“Fc片段”是指免疫球蛋白重链的C端区，其含有铰链区的至少一部分、CH2结构域和CH3结构域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体结合或与经典补体系统的第一组分(C1q)结合，包括天然序列Fc区和变异Fc区。
通常，人IgG重链Fc区为自其Cys226或Pro230位置的氨基酸残基至羧基末端的区段，但其边界可能有变化。Fc区的C-末端赖氨酸(残基447，依照EU编号系统)可以存在或可以不存在。Fc还可以指隔离的这一区域，或在包含Fc的蛋白多肽的情况下，例如“包含Fc区的结合蛋白”，还称为“Fc融合蛋白”(例如，抗体或免疫粘合素)。本发明的抗体中天然序列Fc区包括哺乳动物(例如人)IgG1、IgG2(IgG2A，IgG2B)、IgG3和IgG4。在人IgG1Fc区中，至少两个等位基因类型是已知的。在某些实施方案中，相对于哺乳动物Fc多肽氨基酸序列的序列，两条Fc多肽链的氨基酸序列中每100个氨基酸中具有10个左右氨基酸的单一氨基酸的置换、插入和/或缺失。在一些实施方案中，上述不同可以是延长半衰期的Fc改变、增加FcRn结合的改变、抑制Fcγ受体(FcγR)结合的改变和/或降低或去除ADCC和CDC的改变。
在IgG、IgA和IgD抗体同种型中，Fc区包含抗体的两条重链的每一条的CH2和CH3恒定结构域；IgM和IgE Fc区包含在每条多肽链中的三个重链恒定结构域(CH结构域2-4)。
术语“Fc受体”或“FcR”指结合免疫球蛋白Fc区的受体。FcR可以是天然序列人FcR，也可以是结合IgG抗体的FcR(γ受体)，以及这些受体的等位基因变体和可变剪接形式。FcγR家族由三种活化受体(小鼠中的FcγRI，FcγRIII和FcγRIV；人类中的FcγRIA，FcγRIIA和FcγRIIIA)和一种抑制性受体(FcγRIIb 或等同的FcγRIIB)组成。FcγRII受体包括FcγRIIA(“活化受体”)和FcγRIIB(“抑制受体”)，它们具有相似的氨基酸序列。FcγRIIA的胞质结构域中包含免疫受体基于酪氨酸的活化基序(ITAM)。FcγRIIB的胞质结构域中包含免疫受体基于酪氨酸的抑制基序(ITIM)(参见M.Annu.Rev.Immunol.,15:203-234,1997)。大多数天然效应细胞类型共表达一种或多种活化FcγR和抑制性FcγRIIb，而NK细胞选择性表达一种活化性Fc受体(小鼠中的FcγRIII和人中的FcγRIIIA)，但小鼠和人中不表达抑制性FcγRIIb。人类IgG1与大多数人类Fc受体结合，在其结合的活化性Fc受体的类型方面被认为等同于鼠类IgG2a。术语“FcR”在本文中涵盖其它FcR，包括那些未来将会鉴定的。测量对FcRn的结合的方法是已知的(参见例如Ghetie V等,Immunol.Today,18:592-8,1997；Ghetie V等,Nature Biotechnol.,15:637-40,1997)。可测定人FcRn高亲和力结合多肽与FcRn的体内结合和血清半衰期，例如在表达人FcRn的转基因小鼠或经转染的人细胞系中。术语“Fc受体”或“FcR”还包括新生儿受体FcRn，它负责将母体IgG转移给胎儿(Guyer RL等,J.Immunol.,117:587,1976；Kim YJ等,J.Immunol.,24:249,1994)。
术语“靶细胞”是与抗体结合的并参与介导疾病的细胞。在一些情况下，靶细胞可以是通常参与介导免疫反应，还参与疾病的介导的细胞。例如在B细胞淋巴瘤中，通常参与介导免疫反应的B细胞可以是靶细胞。在一些实施方案中，靶细胞为癌细胞、病原体感染的细胞或参与介导自身免疫性疾病或炎性疾病(例如纤维化疾病)的细胞。所述抗体可通过结合至“靶细胞蛋白”上的抗原而结合至靶细胞，所述靶细胞蛋白是在靶细胞表面显示的蛋白质，且可能为高度表达的蛋白质。
术语“毒素”用于本文时指抑制或防止细胞的功能和/或引起细胞破坏的物质。该术语意图包括放射性同位素(例如I 131、I 125、Y 90、Re 186)、化学治疗剂、和毒素(诸如细菌、真菌、植物或动物起源的酶活毒素)，或其片段。
术语“细胞因子”一般指由一种细胞群体释放的，作为细胞间介质对另一细胞起作用或者对生成该蛋白质的细胞具有自分泌影响的蛋白质。此类细胞因子的例子包括淋巴因子，单核因子；白介素(“IL”)，例如IL-2，IL-6，IL-17A-F；肿瘤坏死因子，例如TNF-α或TNF-β；和其它多肽因子，例如白血病抑制因子(“LIF”)。
术语“化学治疗”或“化疗”指在特征为异常细胞生长的疾病的治疗中施以有治疗用途的任何化学剂。这样的疾病包括例如肿瘤、新生物和癌症以及特征为增生性生长的疾病。“化疗剂”特异性地靶向参与细胞分裂的细胞，而不靶向未参与细胞分裂的细胞。化疗剂直接干扰与细胞分裂密切相关的过程，例如DNA复制、RNA合成、蛋白质合成、或有丝分裂纺锤体的组装、分解或功能，和/或在这些过程中发挥作用的分子(如核苷酸或氨基酸)的合成或稳定性。因此，化疗剂对癌细胞和其它参与细胞分裂的细胞都具有细胞毒性或细胞抑制效应。联合化疗给予多于一种的试剂来治疗癌症。
术语“免疫结合”和“免疫结合性质”是指一种非共价相互作用，其发生在免疫球蛋白分子和抗原(对于该抗原而言免疫球蛋白为特异性的)之间。免疫结合相互作用的强度或亲和力可以相互作用的平衡解离 常数(K D)表示，其中K D值越小，表示亲和力越高。所选多肽的免疫结合性质可使用本领域中公知的方法定量。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(K a或K on)和“解离速率常数”(K d或K off)两者都可通过浓度及缔合和解离的实际速率而计算得出。(参见Malmqvist M等,Nature,361:186-187,1993)。k d/k a的比率等于解离常数K D(通常参见Davies C等,Annual.Rev.Biochem.,59:439-473,1990)。可用任何有效的方法测量K D、k a和k d值。
术语“交叉反应”是指本文所述的抗体结合来自不同物种的肿瘤相关抗原的能力。例如，本文所述的结合人TAA的抗体还可结合来自其它物种的TAA(例如，食蟹猴TAA)。交叉反应性可通过检测在结合测定法(例如，SPR、ELISA)中与纯化抗原的特定反应性，或与生理表达TAA的细胞的结合或以其它方式与生理表达TAA的细胞功能相互作用来测量。本领域中已知测定结合亲和力的分析的实例包括表面等离子共振(例如，Biacore)或类似技术(例如，Kinexa或Octet)。
术语“免疫应答”是由免疫系统细胞(例如T淋巴细胞，B淋巴细胞，NK细胞，抗原呈递细胞、巨噬细胞，嗜酸性粒细胞，肥大细胞，DC细胞或嗜中性粒细胞)以及由免疫细胞或肝脏所产生的可溶性大分子的作用，由这些细胞或肝脏中(包括抗体，细胞因子和补体)的任何一个产生，该作用导致对侵入性病原体、被病原体感染的细胞或组织、癌细胞、或病理炎症情况下的正常人类细胞或组织的选择性损害、破坏或将它们从人体中清除。免疫反应包括例如T细胞(例如效应T细胞或Th细胞，如CD 8+或CD 4+T细胞)的活化或抑制，或抑制或耗竭NK细胞或Treg细胞。
“效应细胞”是指免疫系统的一种细胞，其表达一种或多种FcR并介导一种或多种效应器功能。优选地，该细胞表达至少一种类型的激活性Fc受体，例如人FcγRIII，并执行ADCC效应器功能。介导ADCC的人白细胞的实例包括外周血单个核细胞(PBMC)、NK细胞、单核细胞、巨噬细胞、中性粒细胞和嗜酸性粒细胞。效应细胞也包括例如T细胞。他们可以来源于包括但不限于人、小白鼠、大鼠、兔子和猴的任何生物体。
术语“效应子功能”是指，那些可归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性，且其随抗体同种型而变化。抗体效应子功能的例子包括但不限于：Fc受体结合亲和性、ADCC、ADCP、CDC、细胞表面受体(例如B细胞受体)的下调、B细胞活化、细胞因子分泌、抗体和抗原-抗体复合物的半衰期/清除率等。改变抗体的效应子功能的方法是本领域已知的，例如通过在Fc区引入突变来完成。
术语“抗体依赖性细胞介导的细胞毒性(ADCC)”是指一种细胞毒性形式，Ig通过与细胞毒性细胞(例如NK细胞、中性粒细胞或巨噬细胞)上存在的FcR结合，使这些细胞毒性效应细胞特异性结合到抗原附着的靶细胞上，然后通过分泌细胞毒素杀死靶细胞。检测抗体的ADCC活性的方法是本领域已知的，例如可通过测定待测抗体与FcR(例如CD16a)之间的结合活性来评价。
术语“抗体依赖细胞介导的吞噬作用(ADCP)”指一种细胞介导的反应，在该反应中，表达FcγR的非特异性细胞毒活性细胞识别靶细胞上结合的抗体并随后引起该靶细胞的吞噬。
术语“补体依赖的细胞毒性(CDC)”是指通过使补体成分C1q与抗体Fc结合来激活补体级联的细胞毒性形式。检测抗体的CDC活性的方法是本领域已知的，例如可通过测定待测抗体与Fc受体(例如C1q)之间的结合活性来评价。
术语“药学上可接受的载体和/或赋形剂和/或稳定剂”，是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂/或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
术语“患者”和“受试者”“个体”“对象”是指接受预防性或治疗性治疗的任何人类或非人类动物，尤其是人。例如，本文所述的方法和组合物可用于治疗患有癌症的受试者。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物，绵羊，狗，牛，鸡，两栖动物，爬行动物等。
对受试者的“治疗”或“疗法”是指以逆转、减轻、改善、抑制、减缓或防止与疾病有关的症状、并发症、病症或生化指标的出现、进展、发展、严重程度或复发为目的对受试者进行任何类型的干预或处理，或者向其施用活性剂。
术语“自身免疫”在免疫耐受状态下，一定量的自身反应性T细胞和自身抗体普遍存在于所有个体的外周免疫系统中，有利于协助清除衰老变性的自身成分，对维持免疫系统的自身免疫稳定具有重要的生理学意义。
关于核酸和多肽的术语“突变的”、“突变体”和“突变”分别指与天然核酸或多肽相比(即可以用来定义野生型的参照序列)，更换、缺失或插入一个或多个核苷酸或氨基酸。
术语“T细胞受体(TCR)”是存在于T细胞即T淋巴细胞表面上的特殊受体。体内T细胞受体以几个 蛋白质的复合物存在。T细胞受体通常具有两个单独的肽链，通常是T细胞受体α和β(TCRα和TCRβ)链，在一些T细胞上是T细胞受体γ和δ(TCRγ和TCRδ)。复合物中其他蛋白质是CD3蛋白质：CD3εγ和CD3εδ异源二聚体，最重要的是，有六个ITAM基序的CD3ζ同源二聚体。CD3ζ上的ITAM基序可以被Lck磷酸化，反过来募集ZAP-70。Lck和/或ZAP-70也可以在许多其他分子上磷酸化酪氨酸，尤其是CD28、LAT和SLP-76，这允许围绕这些蛋白质的信号传导复合物聚集。
术语“双特异性抗体”指本发明的双特异性抗体，例如抗CD19抗体或其抗原结合片段可以进行衍生化或连接至另一功能性分子上，例如另一种肽或蛋白质(例如TAA、细胞因子和细胞表面受体)以生成与至少两种不同结合位点或靶分子结合的双特异性分子。为创建本发明的双特异性分子，可以将本发明的抗体在功能上连接(例如通过化学偶联、基因融合、非共价结合或其它方式)至一种或多种其它结合分子，诸如另一种抗体、抗体片段、肽或结合模仿物，从而产生双特异性分子。例如，“双特异性抗体”是指包含两个可变结构域或scFv单位使得所得抗体识别两种不同抗原。本领域已知双特异性抗体的许多不同的形式和用途(Chames P等,Curr.Opin.Drug Disc.Dev.,12:276,2009；Spiess C等,Mol.Immunol.,67:95-106,2015)。
术语“hCG-β羧基末端肽(CTP)”是一段来自人绒毛膜促性腺激素(hCG)的β-亚基羧基末端的短肽。四种与生殖相关的多肽类激素促卵泡激素(FSH)、黄体生成素(LH)、促甲状腺素(TSH)和绒毛膜促性腺激素(hCG)含有相同的α-亚基和各自特异的β-亚基。与其它三种激素相比，hCG体内半衰期明显延长，这主要来源于其β-亚基上特有的羧基末端肽(CTP)。CTP含有37个氨基酸残基，它具有4个O-糖基化位点，糖侧链终端是唾液酸残基。带负电、高度唾液酸化的CTP能够抵抗肾脏对其的清除作用，从而延长蛋白在体内的半衰期(Fares FA等,Proc.Natl.Acad.Sci.USA,89:4304-4308,1992)。
术语“糖基化”意思是低聚糖(含有连接在一起的两个或更多个单糖、例如连接在一起的2个到约12个单糖的碳水化合物)附着形成糖蛋白。低聚糖侧链通常通过N-或O-连接连接到糖蛋白的骨架上。本文公开的抗体的低聚糖通常是连接到Fc区的CH2结构域，作为N-连接的低聚糖。“N-连接的糖基化”是指碳水化合物类部分连接到糖蛋白链的天冬酰胺残基上。例如，技术人员可以识别鼠IgG1、IgG2a、IgG2b和IgG3以及人IgG1、IgG2、IgG3、IgG4、IgA和IgD的CH2结构域中的每一个在残基297处有用于N-连接的糖基化的单一位点。
同源抗体
在又一方面，本发明抗体包含的重链和轻链可变区所包含的氨基酸序列与本文所述的优选抗体的氨基酸序列同源，且其中所述抗体保留了本发明所述，例如CD19×CD3双特异性抗体的期望的功能特性。
具有保守修饰的抗体
术语“保守修饰”意图指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。此类保守修饰包括氨基酸的取代、添加和缺失。修饰可以通过本领域已知的标准技术，例如定点诱变和PCR介导的优点引入到本发明的抗体中。保守氨基酸取代指氨基酸残基用具有类似侧链的氨基酸残基替换。本领域中对具有类似侧链的氨基酸残基家族已有详细说明。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区中的一个或多个氨基酸残基。
与新生儿受体(FcRn)结合亲和力改变的Fc变体
这里使用的“FcRn”指结合IgG抗体Fc区的至少部分由FcRn基因编码的蛋白。FcRn可以来源于包括但不限于人、小白鼠、大鼠、兔子和猴的任何生物体。功能性FcRn蛋白包含经常被称为重链和轻链的两条多肽，轻链是β-2-微球蛋白，重链由FcRn基因编码。
本发明涉及对FcRn的结合被调节的抗体(调节包括增加以及降低结合)。例如：在有些情况下，增加的结合会导致细胞再循环抗体，并由此延长，例如治疗抗体的半衰期。有时，降低FcRn结合是合乎需要的，例如用作包含放射标记的诊断抗体或治疗抗体。另外，对FcRn的结合显示出增加，同时对其他Fc受体，例如FcγRs的结合被改变的抗体可以用于本发明。
本申请涉及包含调节对FcRn的结合力的氨基酸修饰的抗体。具有特殊意义的是在较低的pH时，对FcRn的结合亲和力显示出增加，而在更高的pH时，结合基本上不显示出改变的最低限度地包含Fc区的抗体或其功能性变体。
与新生儿受体(FcRn)结合亲和力增强的Fc变体
IgG的血浆半衰期取决于它与FcRn的结合，一般在pH 6.0时结合，在pH 7.4(血浆pH)时解离。通过对两者结合位点的研究，改造IgG上与FcRn结合的位点，使之在pH 6.0时结合能力增加。已经证明对于结合FcRn重要的人Fcγ结构域的一些残基的突变可增加血清半衰期。已报道T250、M252、S254、T256、V308、E380、M428和N434(EU编号)中的突变可增加或降低FcRn结合亲和力(Roopenian DC等,Nat.Rev.Immunol.,7:715-725,2007)。韩国专利号KR 10-1027427公开了具有增加的FcRn结合亲和力的曲妥珠单抗(赫赛汀，Genentech)变体，并且这些变体包含选自257C、257M、257L、257N、257Y、279Q、279Y、308F和308Y的一个或更多个氨基酸修饰。韩国专利公开号KR 2010-0099179提供了贝伐单抗(阿瓦斯汀， Genentech)变体并且这些变体通过包含在N434S、M252Y/M428L、M252Y/N434S和M428L/N434S的氨基酸修饰显示增加的体内半衰期。此外，Hinton等也发现T250Q和M428L2个突变体分别使与FcRn的结合增加3和7倍。同时突变2个位点，则结合增加28倍。在恒河猴体内，M428L或T250QM/428L突变体显示血浆半衰期增加2倍(Hinton PR等,J.Immunol.,176:346-356,2006)。更多的与新生儿受体(FcRn)结合亲和力增强的Fc变体所包含突变位点可以参见中国发明专利CN 201280066663.2。此外，有研究对五种人源化抗体的Fc段进行T250Q/M428L突变不仅改善了Fc与FcRn的相互作用，且在随后的体内药代动力学试验中，发现以皮下注射给药，Fc突变抗体与野生型抗体相比药代动力学参数有所改善，如体内暴露量增加、清除率降低、皮下生物利用度提高(Datta-Mannan A等,MAbs.Taylor&Francis,4:267-273,2012)。
其他可引起本发明抗体与FcRn亲和力增强的突变点包括但不限于以下氨基酸修饰：226,227,230,233,239,241,243,246,259,264,265,267,269,270,276,284,285,288,289,290,291,292,294,298,299,301,302,303,305,307,309,311,315,317,320,322,325,327,330,332,334,335,338,340,342,343,345,347,350,352,354,355,356,359,360,361,362,369,370,371,375,378,382,383,384,385,386,387,389,390,392,393,394,395,396,397,398,399,400,401,403,404,408,411,412,414,415,416,418,419,420,421,422,424,426,433,438,439,440,443,444,445,446，其中Fc区中氨基酸的编号是Kabat中的EU索引的编号。
与FcRn结合亲和力增强的Fc变体还包括其他一切公知的氨基酸修饰位点以及尚未被发现的氨基酸修饰位点。
在可选择的实施方式中，可以优化IgG变体使其具有增加或降低的FcRn亲和力，以及增加或降低的人FcγR，包括但不限于FcγRI、FcγRIIa、FcγRIIb、FcγRIIc、FcγRIIIa和包括他们的等位基因变异的FcγRIIIb亲和力。
优先地，IgG变体的Fc配体特异性将决定它的治疗应用。给定IgG变体用于治疗目的将取决于靶抗原的表位或形式，以及待治疗的疾病或适应症。对大多数靶和适应症来说，增强的FcRn结合可是更优选的，因为增强的FcRn结合可以导致血清半衰期延长。较长的血清半衰期允许治疗时以较低的频率和剂量给药。为了使需要重复给药的适应症作出反应而施用该治疗剂时，这种特性可是特别优选的。对一些靶和适应症来说，当需要变体Fc具有增加的清除或降低的血清半衰期时，例如当Fc多肽用作显象剂或放射治疗剂时，降低的FcRn亲和力可是特别优选的。
可以通过现有技术的公知方法来评价该多肽对FcRn的亲和力。
FcRn可以来自包括但不限于人类、小鼠、大鼠、兔和猴的任意生物。
抑制FcγR结合的改变
本文所述“抑制FcγR结合的改变”是指Fc多肽链中抑制FcγRIIA、FcγRIIB和/或FcγRIIIA的结合的 一个或多个插入、缺失或置换，所述结合如通过例如基于的竞争结合实验(PerkinElmer,Waltham,MA)测定。这些改变可包含在作为双特异性抗体一部分的Fc多肽链中。更具体地，抑制Fcγ受体(FcγR)结合的改变包括L234A、L235A或抑制N297位糖基化的任意改变，包括在N297的任意置换。此外，连同抑制N297位糖基化的改变一起，通过建立另外的二硫桥来稳定二聚体Fc区的另外的改变也被预期。抑制FcγR结合的改变的进一步实例包括在一条Fc多肽链中的D265A改变和在另一条Fc多肽链中的A327Q改变。上述一些突变描述于，如Xu D等,Cellular Immunol.,200:16-26,2000中，其中描述上述突变及其活性评估的部分以引用的方式并入本文。以上是根据EU编号。
例如，本发明提供的双特异性抗体抑制FcγR结合的改变所包含的Fc片段对人FcγRs(FcγRI、FcγRIIa或FcγRIIIa)和C1q的至少一种显示出降低的亲和力，具有减少的效应细胞功能或补体功能。
其他抑制FcγR结合的改变包含公知技术及未来可能被发现的位点及其修饰。
FcγR可以来自包括但不限于人类、小鼠、大鼠、兔和猴的任意生物。
延长半衰期的Fc改变
本文所述“延长半衰期的Fc改变”是指与包含相同Fc多肽、但其不包含改变的相似Fc蛋白质的半衰期相比，Fc多肽链中延长包含改变的Fc多肽链的蛋白质的体内半衰期的改变。所述改变可包含在作为双特异性抗体一部分的Fc多肽链中。改变T250Q、M252Y、S254T和T256E(第250位的苏氨酸变为谷氨酰胺；第252位的甲硫氨酸变为酪氨酸；第254位的丝氨酸变为苏氨酸；和第256位的苏氨酸变为谷氨酸；根据EU编号进行编号)为延长半衰期的Fc改变并能联合、单独或任意组合使用。这些改变及其它一些改变详细描述于美国专利7,083,784中。美国专利7,083,784中描述这种改变的部分以引用的方式并入本文。
同样地，M428L和N434S为延长半衰期的Fc改变并能联合、单独或任意组合使用。这些改变及其它一些改变详细描述于美国专利申请公开文本2010/0234575和美国专利7,670,600中。美国专利申请公开文本2010/0234575和美国专利7,670,600中描述这种改变的部分以引用的方式并入本文。
此外，按照本文含义，在以下位点之一处的任何置换都可被认为是延长半衰期的Fc改变：250、251、252、259、307、308、332、378、380、428、430、434、436。这些改变中的每一个或者这些改变的组合可用于延长本文所述双特异性抗体的半衰期。其它可用于延长半衰期的改变详细描述于2012年12月17日提交的国际申请PCT/US2012/070146(公开号:WO 2013/096221)中。这一申请中描述上述改变的部分以引用的形式并入本文。
延长半衰期的Fc改变还包括包含公知技术及未来可能被发现的位点及其修饰。
Fc可以来自包括但不限于人类、小鼠、大鼠、兔和猴的任意生物。
编码双抗的核酸
使用本文描述的治疗剂和抗体或抗体片段，本领域技术人员可容易地构建含有功能等价核酸(例如序列不同、但编码相同的效应部分或抗体序列的核酸)的多个克隆。因此，本发明提供了双特异性抗体、编码抗体、抗体片段及其融合蛋白的核酸、核酸变体、衍生物和物种同源物。
本领域已知许多编码包含VH、VL、铰链、CH1、CH2、CH3和CH4区的免疫球蛋白区的核酸序列。参见，如，Kabat等.,Sequences of Proteins of Immunological Interest,Public Health Service N.I.H.,Bethesda,MD,1991。根据本文提供的教导，本领域技术人员可将所述核酸序列和/或本领域已知的其它核酸序列结合，以构建编码本发明双特异性抗体的核酸序列。编码本发明双特异性抗体的示例性核苷酸包括(1)SEQ ID NO：56，(2)SEQ ID NO：58，(3)SEQ ID NO：60和(4)SEQ ID NO：62。
此外，基于本文和其它地方提供的氨基酸序列及本领域常识，本领域技术人员可以确定编码本发明双特异性抗体的核酸序列。除较传统的生产编码特定氨基酸序列的克隆DNA片段的方法外，现今如DNA 2.0(Menlo Park,CA,USA)和Blue Heron(Bothell,WA,USA)等公司通常采用化学合成来生产任意期望顺序排序的基因大小的DNA，从而简化生产所述DNA的过程。
制备双特异性抗体的方法
可采用本领域任何已知的方法制备本发明双特异性抗体。早期构建双特异性抗体的方法有化学交联法或杂合杂交瘤或四价体瘤法(例如，Staerz UD等,Nature,314:628-31,1985；Milstein C等,Nature,305:537-540,1983；Karpovsky B等,J.Exp.Med.,160:1686-1701,1984)。化学偶联法是将2个不同的单克隆抗体用化学偶联的方式连接在一起，制备出双特异性单克隆抗体。例如两种不同单克隆抗体的化学结合，或例如两个抗体片段如两个Fab片段的化学结合。杂合—杂交瘤法是通过细胞杂交法或者三元杂交瘤的方式产生双特异性单克隆抗体，这些细胞杂交瘤或者三元杂交瘤是通过建成的杂交瘤融合，或者建立的杂交瘤和从小鼠得到的淋巴细胞融合而得到的。虽然这些技术用于制造BiAb，但各种产生问题使得此类复合物难以使用，诸如产生含有抗原结合位点的不同组合的混合群体、蛋白质表现方面的困难、需要纯化目标BiAb、低产率、生产费用高等。
最近的方法利用经过基因工程改造的构建体，其能够产生单一BiAb的均质产物而无需彻底纯化以去除不需要的副产物。此类构建体包括串联scFv、二抗体、串联二抗体、双可变结构域抗体和使用诸如Ch1/Ck结构域或DNLTM的基元的异源二聚(Chames&Baty,Curr.Opin.Drug.Discov.Devel.,12:276-83,2009；Chames&Baty,mAbs,1:539-47)。相关纯化技术是公知的。
还可以使用单淋巴细胞抗体方法通过克隆和表达由选择用于产生特异性抗体的单个淋巴细胞产生的免疫球蛋白可变区cDNA来产生抗体，例如由Babcook J等人,Proc.Natl.Acad.Sci.USA.93:7843-7848,1996； WO 92/02551；WO 2004/051268和WO 2004/106377所述的方法。
用于产生例如用于免疫宿主或用于淘选诸如用于噬菌体展示(或酵母细胞或细菌细胞表面表达)的抗体的抗原多肽可以通过本领域熟知的方法从包含表达系统的遗传工程改造的宿主细胞制备，或者它们可以是从天然生物来源回收。例如，可将编码双特异性抗体的一条或两条多肽链的核酸通过多种已知的方法(如转化、转染、电穿孔、用核酸包被的微粒轰击等)引入培养的宿主细胞。在一些实施方案中，编码双特异性抗体的核酸在被引入宿主细胞前可先插入至适于在宿主细胞中表达的载体中。典型的所述载体可包含使插入的核酸能够在RNA和蛋白质水平上表达的序列元件。
所述载体是本领域公知的，并且许多是商购可获得的。含有所述核酸的宿主细胞可在能够使细胞表达该核酸的条件下培养，且得到的BiAb可从细胞群或培养基中收集。可选地，BiAb可在体内生产，例如，在植物叶片中(参见，如Scheller J等,Nature Biotechnol.,19:573-577,2001和其中引用的参考文献)，鸟蛋中(参见，如Zhu L等,Nature Biotechnol.,23:1159-1169,2005和其中引用的参考文献)，或哺乳动物的奶中(参见，如Laible G等,Reprod.Fertil.Dev.,25:315,2012)。
可以使用的多种培养的宿主细胞包括，例如，原核细胞、真核细胞、细菌细胞(如大肠杆菌或嗜热脂肪芽胞杆菌(Bacilis stearothermophilus))、真菌细胞(如酿酒酵母或毕赤酵母)、昆虫细胞(如包括草地夜蛾细胞在内的鳞翅目昆虫细胞)或哺乳动物细胞(如中国仓鼠卵巢(CHO)细胞、NS0细胞、小仓鼠肾(BHK)细胞、猴肾细胞、Hela细胞、人肝细胞癌细胞或293细胞等等)。
双特异性抗体可通过双特异性抗原的免疫原性制剂免疫合适的受试者(例如，兔，山羊，小鼠，或其它哺乳动物，包括转基因的和经剔除的上述哺乳动物)制备。合适的免疫原性制剂可以是，例如化学合成的或重组表达的双特异性抗原。所述的制剂可进一步包含佐剂，例如弗氏完全或不完全佐剂或类似的免疫刺激化合物。而且，当用于制备抗体时，特别是通过体内免疫的方式，本发明的双特异性抗原可以单独使用，或优选地作为与载体蛋白的偶联物。这种加强抗体应答的方法是本领域所公知的。根据所需的抗体不同，可使用不同的动物宿主进行体内免疫。可使用自身表达有用的内源抗原的宿主，或使用已导致有用内源抗原缺陷的宿主。
双特异性抗体可通过结合的以上所述的方法制备。
本发明所述双特异性抗体分子可以作为关于每个靶的单克隆抗体(MAb)。在一些实施方案中，抗体是嵌合的、人源化的或全人的。
单克隆抗体可以通过本领域已知的任何方法制备，诸如杂交瘤技术(Kohler&Milstein,Nature,256:495-497,1975)，三源杂交瘤技术，人B细胞杂交瘤技术(Kozbor D等,Immunology Today,4:72,1983)和EBV-杂交瘤技术(Cole SPC等,Monoclonal Antibodies and Cancer Therapy,pp77-96,Alan R Liss,Inc.,1985)。
本发明的双特异性抗体，或其部分可通过常规的免疫学分析方法，例如酶联免疫吸附试验(ELISA)， 放射免疫分析(RIA)或组织免疫组织化学用于检测任一或所有这些抗原(例如在生物样品，如血清或血浆中)。本发明提供检测生物样品中的抗原的方法，该方法包括：使所述生物样品与本发明的可特异识别所述抗原的双特异性抗体，或抗体部分与抗原相接触，并检测与抗原结合的抗体(或抗体部分)，或非结合抗体(或抗体部分)，由此检测所述生物样品中的所述抗原。所述抗体用可检测的物质进行直接或间接的标记，以便于检测结合或非结合抗体。合适的可检测物质包括多种酶，修复基团，荧光物质，发光物质和放射性物质。合适的酶的例子包括，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，乙酰胆碱酯酶；合适的修复基团复合物的例子包括链霉抗生物素蛋白/生物素和抗生物素蛋白/生物素；合适的荧光物质的例子包括7-羟基香豆素，荧光素，荧光素异硫氰酸盐，硷性蕊香红B，二氯三嗪基胺荧光素，丹磺酰氯或藻红蛋白；发光物质的例子包括3-氨基邻苯二甲酰环肼；合适的放射性物质的例子包括I 125、I 131、 35S或 3H。
免疫效应细胞和效应细胞蛋白
双特异性抗体可结合至在免疫效应细胞表面表达的分子(本文称为“效应细胞蛋白”)和在靶细胞表面表达的另一分子(本文称为“靶细胞蛋白”)。所述免疫效应细胞可以是T细胞、NK细胞、巨噬细胞或嗜中性粒细胞。在一些实施方案中，所述效应细胞蛋白为包含在T细胞受体(TCR)-CD3复合物中的蛋白。所述TCR-CD3复合物是包含同源二聚体，所述同源二聚体包含TCRα和TCRβ或TCRγ和TCRδ及来自CD3ζ(CD3ζ)链、CD3ε(CD3ε)链、CD3γ(CD3γ)链和CD3δ(CD3δ)链等的多种CD3链。在一些实施方案中，所述效应细胞蛋白可以是人CD3ε(CD3ε)链，该链可以是二聚体蛋白的一部分。可选地，所述效应细胞蛋白可以是人和/或食蟹猴和/或恒河猴TCRα、TCRβ、TCRδ、TCRγ、CD3β、CD3γ、CD3δ或CD3ζ。
此外，在一些实施方案中，双特异性抗体还可结合至非人物种(如小鼠、大鼠、兔、新世界猴和/或旧世界猴物种)的CD3ε链。所述物种包括但不限于以下哺乳动物物种：小家鼠(Musmusculus)；黑家鼠(Rattusrattus)；褐家鼠(Rattusnorvegicus)；食蟹猕猴；阿拉伯狒狒(hamadryasbaboon)，埃及狒狒(Papiohamadryas)；大狒狒(Guineababoon)，几内亚狒狒(Papiopapio)；橄榄狒狒(olivebaboon)，东非狒狒(Papioanubis)；黄狒狒(yellowbaboon)，草原狒狒(Papiocynocephalus)；南非大狒狒(Chacmababoon)，豚尾狒狒(Papioursinus)；普通狨(Callithrixjacchus)；绒顶柽柳猴(SaguinusOedipus)和松鼠猴(Saimirisciureus)。由蛋白质治疗剂发展领域已知，在通常用于临床前研究的人和物种(如小鼠和猴)中具有相似活性的治疗剂能简化和加快药物的开发，这对将药物推向市场极其重要。
在一些优选实施方案中，可将上述CD3链的部分片段结合至本发明的同源二聚体，所述CD3可以是人CD3ε链或源自不同物种的CD3ε链，优选为源自上文所列哺乳动物物种中的一种。抗体所结合的表位可以通过丙氨酸扫描或其他公知技术确定，所述丙氨酸扫描描述于，如美国专利申请公开文本2010/183615中，其中相关的部分以引用的方式并入本文。
当T细胞为免疫效应细胞时，双特异性抗体可结合的效应细胞蛋白包括但不限于，CD3ε、CD3γ、CD3δ、CD3ζ、TCRα、TCRβ、TCRγ和TCRδ。当NK细胞或细胞毒性T细胞为免疫效应细胞时，则例如NKG2D、CD352、NKp46或CD16a可以为效应细胞蛋白。当CD 8+T细胞为免疫效应细胞时，则例如4-1BB或NKG2D可以是效应细胞蛋白。可选地，双特异性抗体可结合至在T细胞、NK细胞、巨噬细胞或嗜中性粒细胞上表达的其它效应细胞蛋白。
靶细胞和在靶细胞上表达的靶细胞蛋白
如上文所述，双特异性抗体可结合至效应细胞蛋白和靶细胞蛋白。例如，所述靶细胞蛋白可以在癌细胞、病原体感染的细胞或介导疾病(例如炎性、自身免疫性疾病)的细胞表面表达。在一些实施方案中，该靶细胞蛋白能在靶细胞表面高度表达，尽管高水平的表达不是必需的。在一些实施方案中，该靶细胞蛋白在靶细胞表面不表达或低表达。
当靶细胞为癌细胞时，如本文所述的同源二聚体的双特异性抗体可结合至如上文所述的癌细胞抗原。癌细胞抗原可以是人蛋白或源自其它物种的蛋白。
在一些实施例中，所述靶细胞蛋白可以是介导淋巴系统相关疾病的细胞表面的蛋白。
在一些实施例中，所述靶细胞蛋白可以是在肿瘤细胞表面选择性表达或过表达或不表达的蛋白。
在其他方面，靶细胞可以是介导自身免疫性疾病或炎症性疾病的细胞。例如，哮喘中的人嗜酸性粒细胞可以是靶细胞，在这种情况下，如含EGF样模体粘液样激素受体(EMR1)可作为靶细胞蛋白。可选地，在全身性红斑狼疮患者中过量人B细胞可作为靶细胞，在这种情况下，如CD19或CD20可作为靶细胞蛋白。在其它自身免疫性疾病中，过量人Th2T细胞可作为靶细胞，在这种情况下，如CCR4可作为靶细胞蛋白。同样地，靶细胞可以是介导如动脉粥样硬化、慢性阻塞性肺疾病(COPD)、肝硬化、硬皮病、肾移植纤维化、肾同种异体移植肾病或肺纤维化(包括特发性肺纤维化和/或独特型肺动脉高压)的纤维化细胞。对于所述纤维化病症，如成纤维细胞活化蛋白α(FAPα)可以是靶细胞蛋白。
在一些实施例中，靶细胞可以是介导其他相关疾病的细胞，包括移植排异反应相关的疾病或病症。
双特异性抗体可结合至来自小鼠、大鼠、兔、新世界猴和/或旧世界猴物种等的靶细胞蛋白。所述物种包括但不限于以下物种：小家鼠(Musmusculus)；黑家鼠(Rattusrattus)；褐家鼠(Rattusnorvegicus)；食蟹猕猴，食蟹猴(Macacafascicularis)；阿拉伯狒狒(hamadryasbaboon)，埃及狒狒(Papiohamadryas)；大狒狒(Guineababoon)，几内亚狒狒(Papiopapio)；橄榄狒狒(olivebaboon)，东非狒狒(Papioanubis)；黄狒狒(yellowbaboon)，草原狒狒(Papiocynocephalus)；南非大狒狒(Chacmababoon)，豚尾狒狒(Papioursinus)，普通狨(Callithrixjacchus)，绒顶柽柳猴(SaguinusOedipus)和松鼠猴(Saimirisciureus)。
癌症
术语“癌症”是指以体内异常细胞的不受控生长为特征的一大类疾病。“癌症”包括良性和恶性癌症以及休眠肿瘤或微转移。癌症包括原发性恶性细胞或肿瘤(例如细胞未迁移至受试者体内原始恶性疾病或肿瘤部位以外的部位的肿瘤)和继发性恶性细胞或肿瘤(例如由转移产生的肿瘤，转移为恶性细胞或肿瘤细胞迁移至与原始肿瘤部位不同的次级部位)。癌症也包括血液学恶性肿瘤。“血液学恶性肿瘤”包括淋巴瘤，白血病，骨髓瘤或淋巴恶性肿瘤，以及脾癌和淋巴结肿瘤。
在优选的实施方案中，本发明双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物或组合疗法对癌症的治疗、预防或缓解是有用的。癌症的实例包括但不限于癌瘤、淋巴瘤、成胶质细胞瘤、黑素瘤、肉瘤和白血病、骨髓瘤或淋巴恶性疾病。此类癌症的更特定实例如下所述并且包括：鳞状细胞癌(例如上皮鳞状细胞癌)、尤因氏肉瘤、韦尔姆斯氏肿瘤、星形细胞瘤、肺癌(包括小细胞肺癌、非小细胞肺癌、肺腺癌和肺鳞状癌)、腹膜癌、肝细胞癌、胃部癌或胃癌(包括胃肠癌)、胰腺癌、多形性成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞瘤、肝细胞癌瘤、神经内分泌肿瘤、甲状腺髓样癌、甲状腺分化癌、乳癌、卵巢癌、结肠癌、直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾脏癌或肾癌、前列腺癌、阴门癌、肛门癌、阴茎癌以及头颈癌。
癌症或恶性病的其他实例包括但不限于：急性儿童成淋巴细胞性白血病、急性成淋巴细胞性白血病、急性淋巴细胞性白血病、急性骨髓性白血病、肾上腺皮质癌、成人(原发性)肝细胞癌、成人(原发性)肝癌、成人急性淋巴细胞性白血病、成人急性骨髓性白血病、成人霍奇金氏淋巴瘤、成人淋巴细胞性淋巴瘤、成人非霍奇金氏淋巴瘤、成人原发性肝癌、成人软组织肉瘤、AIDS相关性淋巴瘤、AIDS相关性恶性病、肛门癌、星形细胞瘤、胆管癌、膀胱癌、骨癌、脑干神经胶质瘤、脑瘤、乳腺癌、肾盂和输尿管癌、中枢神经系统(原发性)淋巴瘤、中枢神经系统淋巴瘤、小脑星形细胞瘤、大脑星形细胞瘤、宫颈癌、儿童(原发性)肝细胞癌、儿童(原发性)肝癌、儿童急性成淋巴细胞性白血病、儿童急性骨髓性白血病、儿童脑干神经胶质瘤、儿童小脑星形细胞瘤、儿童大脑星形细胞瘤、儿童颅外胚细胞瘤、儿童霍奇金氏病、儿童霍奇金氏淋巴瘤、儿童下丘脑和视通路神经胶质瘤、儿童成淋巴细胞性白血病、儿童成神经管细胞瘤、儿童非霍奇金氏淋巴瘤、儿童松果体和幕上原始神经外胚层瘤、儿童原发性肝癌、儿童横纹肌肉瘤、儿童软组织肉瘤、儿童视通路和下丘脑神经胶质瘤、慢性淋巴细胞性白血病、慢性髓细胞性白血病、结肠癌、皮肤T细胞淋巴瘤、内分泌胰岛细胞癌、子宫内膜癌、室管膜瘤、上皮癌、食道癌、尤因氏肉瘤和相关肿瘤、外分泌胰腺癌、颅外胚细胞瘤、性腺外胚细胞瘤、肝外胆管癌、眼癌、女性乳腺癌、高歇氏病、胆囊癌、胃部癌、胃肠道良性肿瘤、胃肠道肿瘤、胚细胞瘤、妊娠性滋养层细胞瘤、毛细胞白血病、头颈癌、肝细胞癌、霍奇金氏淋巴瘤、高丙种球蛋白血症、下咽癌、肠癌、眼内黑素瘤、胰岛细胞癌、胰岛细胞胰腺癌、卡波济氏肉瘤、肾癌、喉癌、唇口腔癌、肝癌、肺癌、淋巴增生性病症、巨球蛋白血症、男性乳腺 癌、恶性间皮瘤、恶性胸腺瘤、成神经管细胞瘤、黑素瘤、间皮瘤、转移性原发灶隐匿性鳞状颈癌、转移性原发性鳞状颈癌、转移性鳞状颈癌、多发性骨髓瘤、多发性骨髓瘤/浆细胞赘瘤、骨髓发育不良综合症、髓细胞性白血病、骨髓性白血病、骨髓增生性病症、鼻腔和副鼻窦癌、鼻咽癌、成神经细胞瘤、非霍奇金氏淋巴瘤、非黑素瘤皮肤癌、非小细胞肺癌、转移性原发灶隐匿性鳞状颈癌、口咽癌、骨肉瘤/恶性纤维肉瘤、骨肉瘤/恶性纤维组织细胞瘤、骨肉瘤/骨骼的恶性纤维组织细胞瘤、卵巢上皮癌、卵巢胚细胞瘤、卵巢低恶性潜能肿瘤、胰腺癌、病变蛋白血症、真性红细胞增多症、副甲状腺癌、阴茎癌、嗜铬细胞瘤、垂体肿瘤、原发性中枢神经系统淋巴瘤、原发性肝癌、前列腺癌、直肠癌、肾细胞癌、肾盂和输尿管癌、成视网膜细胞瘤、横纹肌肉瘤、唾液腺癌、肉状瘤病肉瘤、塞扎里综合症、皮肤癌、小细胞肺癌、小肠癌、软组织肉瘤、鳞状颈癌、胃癌、幕上原始神经外胚层和松果体瘤、T细胞淋巴瘤、睾丸癌、胸腺瘤、甲状腺癌、肾盂和输尿管的移行迁移细胞癌、移行迁移肾盂和输尿管癌、滋养层细胞瘤、输尿管和肾盂细胞癌、输尿管癌、子宫癌、子宫肉瘤、阴道癌、视通路和下丘脑神经胶质瘤、阴门癌、瓦尔登斯特伦巨球蛋白血症、韦尔姆斯氏瘤和位于以上所列出的器官系统中的除赘瘤以外任何其他过度增生性疾病。
本发明的双特异性抗体或编码本申请抗体的核酸或药物组合物或组合疗法可用于治疗恶性或恶变前病状以及用于预防发展成赘生性或恶性状态，包括但不限于上文所述的那些病症。指示此类用途在已知的或怀疑先前进展至瘤形成或癌症的病状中，具体来说，其中非瘤性细胞生长由过度增生、化生组成，或最具体来说，出现发育异常(针对此类异常生长病状的综述，参见Robbins和Angell,Basic Pathology.,第2版,W.B.Saunders Co.,Philadelphia,第68-79页,1976)。
淋巴系统疾病
在优选的实施方案中，本发明双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物或组合疗法对淋巴系统疾病的治疗、预防或缓解是有用的。淋巴系统疾病的实例包括但不限于：
B细胞淋巴瘤
B细胞淋巴瘤(B-cell lymphoma)是B细胞发生的实体肿瘤，包括霍奇金淋巴瘤(HL)和非霍奇金淋巴瘤(NHL)。霍奇金淋巴瘤主要包括经典霍奇金淋巴瘤和结节性淋巴细胞瘤。弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、黏膜相关淋巴组织淋巴瘤(MALT)、小淋巴细胞淋巴瘤/慢性淋巴细胞白血病、套细胞淋巴瘤(MCL)是5种最常见的B细胞非霍奇金淋巴瘤，占非霍奇金淋巴瘤的3/4。
B细胞非霍奇金淋巴瘤包括但不限于：a)前驱淋巴性肿瘤：B淋巴母细胞白血病/淋巴瘤及伴随症。b)成熟B细胞肿瘤包括但不限于：慢性淋巴细胞性白血病/小淋巴细胞性淋巴瘤、B-前淋巴细胞性白血病、脾边缘带淋巴瘤、毛细胞白血病、脾淋巴瘤/白血病、淋巴浆细胞淋巴瘤、重链病、浆细胞骨髓瘤/浆细胞瘤、结外粘膜相关淋巴组织边缘带B细胞淋巴瘤(MALT淋巴瘤)、原发皮肤滤泡中心淋巴瘤、滤泡性淋巴瘤(胃 肠道滤泡性淋巴瘤、儿童滤泡性淋巴瘤、“原位”滤泡性淋巴瘤)、结内边缘带B细胞淋巴瘤、套细胞淋巴瘤、弥漫大B细胞淋巴瘤、T细胞/组织细胞丰富的大B细胞淋巴瘤、老年人EBV阳性的弥漫大B细胞淋巴瘤、慢性炎症相关的弥漫大B细胞淋巴瘤、脓胸相关淋巴瘤、慢性骨髓炎相关淋巴瘤、植入物相关淋巴瘤、原发中枢神经弥漫大B细胞淋巴瘤、淋巴瘤样肉芽肿、原发纵膈(胸腺)大B细胞淋巴瘤、血管内大B细胞淋巴瘤、原发皮肤大B细胞淋巴瘤，腿型、浆母细胞性淋巴瘤、原发渗漏性淋巴瘤、ALK阳性弥漫大B细胞淋巴瘤、起源于HHV8阳性的多中心Castleman病的大B细胞淋巴瘤、伯基特淋巴瘤、介于弥漫大B细胞淋巴瘤和伯基特淋巴瘤之间的不能分类的B细胞淋巴瘤、介于弥漫大B细胞淋巴瘤和经典霍奇金淋巴瘤之间的不能分类的B细胞淋巴瘤。
有一部分B细胞淋巴瘤是感染引起的，感染性病毒的非限制实例包括卡波西氏肉瘤相关疱疹病毒(原发性渗出性淋巴瘤)、EB病毒(淋巴瘤样肉芽肿、移植后淋巴增殖性疾病)、HIV(艾滋病相关淋巴瘤)和幽门螺杆菌(粘膜相关淋巴瘤)。
另外，根据临床行为的不同，B细胞淋巴瘤分为惰性淋巴瘤和侵袭性淋巴瘤。惰性淋巴瘤通常发展缓慢，可保持多年疾病稳定及长期生存，但无法治愈。侵袭性淋巴瘤通常需要较强烈的治疗方法，但有治愈的可能。B细胞淋巴瘤的预后和治疗取决于淋巴瘤的具体类型以及分期分级。
T细胞淋巴瘤
T细胞淋巴瘤(T-cell lymphoma)包括四种影响T细胞的淋巴瘤类型：血管中心性淋巴瘤(Angiocentric lymphoma)、皮肤T细胞淋巴瘤(Cutaneous T cell lymphoma)：塞扎病(Sézary's disease)及蕈样肉芽肿(Mycosis fungoides)、间变性大细胞淋巴瘤(Anaplastic large-cell lymphoma)和血管免疫母细胞性T细胞淋巴瘤(Angioimmunoblastic T-cell lymphoma)。
其非限制性实例包括：急性淋巴细胞白血病(例如急性前T淋巴细胞白血病及淋巴瘤)、幼淋巴细胞白血病(例如幼T淋巴细胞白血病)、CD30 +标志性白血病(例如间变性大细胞淋巴瘤、淋巴瘤样丘疹病)、皮肤疾病(例如蕈样肉芽肿、塞扎里氏病)、肝脾疾病、血管疾病、肠道相关疾病和感染性疾病(例如人类嗜T淋巴球病毒型(例如成人T细胞白血病及淋巴瘤))。
B淋巴细胞白血病
示例性实例包括：急性B淋巴细胞白血病(前体B细胞淋巴细胞白血病)、普通B细胞白血病(B细胞慢性淋巴细胞白血病)、幼B淋巴细胞白血病和CD11c毛细胞白血病。
T淋巴细胞白血病
示例性实例包括：急性T淋巴细胞白血病、急性前T淋巴细胞白血病及淋巴瘤、幼T淋巴细胞白血病、 成人T细胞白血病及淋巴瘤。
其他淋巴相关疾病
示例性实例包括：淋巴系统及骨髓系统(例如急性双表型白血病)、淋巴球过多症，如淋巴增生性疾病(X连锁淋巴增生性疾病、自身免疫性淋巴增生综合征)、类白血病反应性疾病和假性淋巴瘤。
自身免疫性疾病和炎症性疾病
双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物或组合疗法在与包括免疫和炎症因素的多种疾病的相关病理学中起关键作用。上述的疾病包括但不限于类风湿病关节炎，骨关节炎、少年慢性关节炎、Lyme关节炎、牛皮癣的关节炎、反应性关节炎、脊椎关节病、系统性红斑狼疮、克罗恩氏病、溃疡性结肠炎、炎症性肠疾病、胰岛素依赖性糖尿病、甲状腺炎、哮喘、变态反应性疾病、牛皮癣、硬皮病皮炎、移植物抗宿主病、器官移植排异反应、与器官移植相关的急性或慢性免疫疾病、结节病、动脉粥样硬化、散播性血管性凝血、Kawasaki病、Grave病、肾变病综合症、慢性疲劳综合症、眶坏死性肉芽肿病、荷-索紫癜症、微观肾脉管炎、慢性活动性肝炎、葡萄膜炎、脓毒性休克、毒性休克综合症、脓毒综合症、恶病质、传染性疾病、寄生虫病、获得性免疫缺陷综合症、急性横贯性脊髓炎、慢性舞蹈病、帕金森氏病、阿尔察默病、中风、夏科氏肝硬变、溶血性贫血、恶性肿瘤、心力衰竭、心肌梗塞、阿狄森病、偶发性I型多腺缺陷和II型多腺缺陷、施密特氏综合症、成人(急性)呼吸困难综合症、脱发、斑秃、血清反应阴性关节病、关节病、莱特尔氏病、牛皮癣关节病、溃疡性鲕状结构关节病、肠滑膜炎(enteropathic synovitis)、衣原体、耶尔森氏菌属和沙门氏菌属相关的关节病、脊椎结核关节病、粥样疾病/动脉硬化、特异反应性过敏、自身免疫肠疾病、慢性天疱疮、落叶状天疱疮、类天疱疮、线性IgA疾病(linear IgA disease)、自身免疫溶血性贫血、库姆斯阳性溶血性贫血、获得性恶性贫血、幼年恶性贫血、肌痛性脑炎/Royal Free Disease、慢性皮肤粘膜假丝酵母病、巨细胞性动脉炎、原发性硬化性肝炎(primary sclerosing hepatitis)、隐原性自身免疫肝炎、获得性免疫缺陷疾病综合症、获得性免疫缺陷相关疾病、丙型肝炎、共有变换免疫缺陷(common varied immunodeficiency)(共有可变化的血内丙种免疫球蛋白过少)、扩张性心肌病、女性不育症、卵巢衰竭、卵巢功能早期衰退、纤维变性的肺部疾病、隐原性纤维肺泡炎、炎症后的间质肺疾病(postinflammatory interstitial lung disease)、间质肺炎、与结缔组织疾病相关的间质肺部疾病、混合结缔组织疾病相关的肺部疾病、全身硬化相关的间质肺病、类风湿病关节炎相关的间质肺病、系统性红斑狼疮相关的肺部疾病、皮肌炎/多肌炎相关的肺部疾病、病相关的肺部疾病、强直性脊柱炎相关的肺部疾病、脉管炎弥漫性肺部疾病、含铁血黄素沉着病相关的肺部疾病、药物诱导的间质肺部疾病、辐射纤维化、闭塞性支气管炎、慢性嗜酸性肺炎、淋巴细胞浸润的肺部疾病、感染后间质肺部疾病、痛风性关节炎、自身免疫性肝炎、I型自身免疫 性肝炎(典型地自身免疫的或狼疮状肝炎)、II型自身免疫的肝炎(抗-LKM抗体肝炎)、自身免疫介导的低血糖、B型抗胰岛素微黑的棘皮症、甲状旁腺机能减退、与器官移植相关的急性免疫疾病、与器官移植相关的慢性免疫疾病、骨关节病、初期的致硬化胆管炎、I型牛皮癣、II型牛皮癣、自发性leucopaenia、自身免疫性中性白细胞减少症、肾脏疾病NOS、肾小球肾炎(glomerulonephritides)、肾微观vasulitis、莱姆病、盘状红斑狼疮、自发的或NOS雄性不育、精子自身免疫、多发性硬化(所有的亚型)、交感性眼炎、肺动脉高血压症继发性结缔组织疾病(pulmonary hypertension secondary to connective tissue disease)、古德帕斯彻氏综合症、结节性动脉周围炎的肺部表现、急性风湿热、类风湿性脊椎炎、斯提耳病、系统性硬化、Sjorgren综合症、高安氏病/动脉炎、自身免疫血小板病、自发性血小板病、自身免疫甲状腺病、甲状腺机能亢进、致甲状腺肿的自身免疫的甲状腺机能减退(Hashimoto病)、萎缩的自身免疫甲状腺功能减退、初期的粘液水肿、晶状体眼色素层炎、初期的结节性脉管炎、白癜风、中枢神经系统疾病(例如，抑郁、精神分裂症、阿尔采默氏病和帕金森氏症等)、急性和慢性疼痛，和脂质失调。
移植排异相关疾病
移植排异指的是细胞、组织或实体器官的同种异体移植物或异种移植物的超急性、急性或慢性排异，根据常规用法，移植物可包括一个或多个器官(例如，肾移植或心肺移植)，器官的部分(例如，皮肤移植物)，细胞(例如，骨髓移植，胰岛细胞，其他内分泌或外分泌细胞)或组织(例如皮肤，或结缔组织，如软骨，韧带或肌腱)。更具体的移植物包括但不限于干细胞、角膜组织、心、肺、心-肺联合、肾、肝、肠(或消化道的其他部分)、胰腺(特别是胰岛)、气管或食管、血管、神经、骨、骨髓、软骨、关节、肌腱、韧带、肌肉、脂肪、乳腺、胃肠道衬里、皮肤、上皮，内皮、结缔组织等。类似地，此外，本发明可以与包括多种组织类型的身体部位一起使用，例如用于眼睛、耳朵、鼻子、手指(手指或脚趾)、关节、血管、神经、肌肉的替换或其他手术改变或重建肢体或其他身体部位。
适应症包括与移植排异相关的病症，例如治疗(包括改善，减少，消除或治愈病因或症状)或预防(包括实质性或完全性)或避免以下情况：a)急性移植排异，例如治疗心脏，肺，心肺，肝，肾，胰腺，皮肤，肠或角膜移植的接受者，尤其是预防和/或治疗T细胞介导的排异反应，以及移植物抗宿主病，如骨髓移植。b)慢性移植排异，例如特别是预防移植血管疾病，例如，其特征在于由于平滑肌细胞增殖和相关作用导致的内膜增厚导致的移植物动脉狭窄。c)异种移植排异，包括当器官供体与受体不同的物种时发生的器官的急性，超急性或慢性排异，最特别是由B细胞介导的排异或抗体介导的排异。适应症还包括例如输血后的不良反应，包括但不限于发热反应，过敏反应，溶血反应，输血后移植物抗宿主病，大量输血后的并发症(循环负荷过重、出血倾向)，细菌污染引起的输血反应，输血传播的疾病。
药物可以用任何常规的途径给予刚接受移植或准备接受移植的患者，其途径包括但不限于静脉内、肌 内、口服、皮下、皮内和非肠道给药。
因为移植排异过程中常伴随产生自身免疫疾病，炎症性疾病等相关疾病或病症，因此本发明CD19双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物或组合疗法将要给药的受体包括可以表现出例如自身免疫疾病，炎症性疾病，移植排异疾病及症状等相关疾病或病症的任何动物。较理想地，受体是哺乳动物。更确切地说，该哺乳动物至少包含人类，猿，啮齿动物，羊，牛，反刍动物，兔类动物，猪，山羊，马，犬，兔，猫科的动物，鸟类等等。其中，最优选是人。在其他实施方案中，宿主可以是具有商业重要性的哺乳动物，或伴侣动物或其他有价值的动物，例如濒危物种的成员。据本发明最佳实施例，非人类的动物最好为鼠科，例如豚鼠，仓鼠，沙鼠，大鼠和小鼠。所以，人类的医生和兽医均可使用本发明双特异性抗体。
本发明CD19双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可以与任何类型的组织移植或移植程序一起使用，特别是其中供体(移植的)组织受到受体宿主免疫系统的失败或排异的风险或有风险的程序。特别地，本发明CD19双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可用于供体组织与受体宿主不具有组织相容性的任何环境中。移植供体可以是与移植受体相同的系统发育物种的非同源成员(即同种异体供体，提供同种异体移植组织)，或不同系统发育物种的成员(即，异种供体，提供异种移植组织)。供体组织可以通过常规手段来自，例如志愿者或其他活体供体。优选地，供体与受体宿主一样可实现组织相容性。因此，在受体宿主是人的情况下，优选自体和同种异体供体组织。如果使用异种供体作为移植组织来源，优选供体与受体宿主相对MHC相容；例如，狒狒或黑猩猩将优选作为将组织移植到人体内的供体。
本发明CD19双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可用于促进任何身体组织或器官类型的植入，无论供体(移植物)组织是整个器官，器官或组织的部分或部分，还是分离的细胞。在一些实施方案中，供体组织包括器官或身体部分。在其他实施方案中，供体组织包括供体器官或组织的部分，部分或活组织检查。在其他实施方案中，供体组织包含细胞，特别是分离的或悬浮的细胞，包括从供体宿主取出或切除的细胞，维持在原代培养物中的细胞，或永生化细胞系。任选地，供体组织可以包括携带外源遗传物质的细胞，例如转染或转化的宿主细胞，其已经(或源自已经被改造的祖先细胞)包括产生具有治疗价值的多肽所必需的遗传物质。在其他实施例中，供体组织可以衍生自转基因哺乳动物，所述转基因哺乳动物已被工程化以包括在其一些或所有身体组织中产生对受体宿主具有治疗价值的多肽所必需的遗传物质。对受体具有治疗价值的示例性多肽包括：激素，例如胰岛素或生长激素；细胞因子；生长和分化因素；酶；结构蛋白；等等。
抑制移植排异反应的有效剂量大约为每kg体重0.001mg-10.0mg。在大约0.001mg-10.0mg/kg体重间所选的剂量是有效而无毒的。所用的剂量将以每天1～6次给予患者。
本发明提供了一种抑制移植排异反应的方法，该方法包括给予抑制移植排异反应有效量的本文定义的CD19双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物。
药物组合物
本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸(例如CD19xCD3)可以应用于制备药物组合物或无菌组合物，例如，将双特异性抗体与药学上可接受的载体、赋形剂或稳定剂混合。药物组合物可包括一种或组合的(如两种或更多不同的)本发明的双特异性抗体。例如，本发明的药物组合物可包含与靶抗原上的不同表位结合的具有互补活性的抗体或抗原结合片段的组合。治疗和诊断剂的制剂可通过以例如冻干粉末、浆液、水性溶液或悬浮液的形式与药学可接受的载体、赋形剂或稳定剂混合来制备。
术语“药学上可接受的”指当分子本体、分子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其他不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括糖类(如乳糖)、淀粉、纤维素及其衍生物、植物油、明胶、多元醇(如丙二醇)、海藻酸等。
在一些优选实施方案中，本发明药物组合物用于治疗、预防或缓解的疾病包括但不限于：免疫相关疾病、癌症、自身免疫性疾病、炎症性疾病、移植排异反应相关疾病或病症。
本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸可单独使用，或可与一种或更多种其他药学活性剂共同使用，所述药学活性剂例如抗体、抗体片段、药物、酶、细胞毒性剂、毒素、抗生素、激素、免疫调节剂、细胞因子、趋化因子和放射性同位素。
药物包括但不限于环磷酰胺、尼莫司汀、氨甲蝶呤、氟尿嘧啶、卡培他滨、吉西他滨、替吉奥、培美曲塞、氟达拉滨、阿霉素、博来霉素、长春瑞滨、紫杉醇、多西他赛、伊利替康、他莫昔芬、来曲唑、依西美坦、氟维司群、戈舍瑞林、甲羟孕酮、顺铂、卡铂、草酸铂、奈达铂、奥沙利铂、门冬酰胺酶、洛铂、依托泊苷、长春新碱、伊立替康、替加氟、达卡巴嗪、丝裂霉素、替尼泊苷、吡柔比星、米托蒽醌、长春地辛、雷替曲塞、甲氨蝶呤、顺铂博来霉素硫酸盐、亚硝基脲氮芥、苯丁酸氮芥、环磷酰胺羟脲和道诺霉素，这些治疗剂本身仅在对患者具有毒性或亚毒性水平时才有效。以上治疗剂是本领域公知的，也包括未来可能发展的治疗剂。
毒素包括但不限于：篦麻毒素、相思子毒素、α毒素、皂素、核糖核酸酶(RNA酶)、DNA酶I、葡萄球菌肠毒素-A、美洲商陆抗病毒蛋白、白树毒素、白喉毒素、假单胞菌外毒素和假单胞菌内毒素。
本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸可与例如相应的效应细胞共刺激分子、效应细胞、DC细胞表面分子、DC细胞、活化T细胞的分子(Hutloff A等,Nature,397:262-266,1999)共同施用。效应细胞可以是人白细胞，诸如巨噬细胞、嗜中性粒细胞或单核细胞。其他细胞包括嗜酸性粒细胞、NK细胞和其他带有IgG或IgA受体的细胞。如果需要，可从要治疗的受试者获取效应细胞。靶特异性效应 细胞可作为生理可接受的溶液中的细胞的悬浮液来施用。施用的细胞数可在10 8-10 9数量级范围内，但是可根据治疗目的而有所不同。一般而言，该量足以实现在靶细胞(如表达CD19的肿瘤的细胞)的定位并实现细胞杀伤。存在补体的情况下，也可使用具有补体结合位点的本发明的组合物，所述补体结合位点诸如来自与补体结合的IgG1、IgG2或IgG4或IgM的部分。本发明的组合物还可与补体一起施用，例如与C1q组合施用。
本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸可与例如免疫调节剂、免疫原性剂，例如癌症细胞、纯化的肿瘤抗原(包括重组蛋白、肽和碳水化合物分子)或用编码免疫刺激性细胞因子的基因转染细胞组合施用(He YF等,J.Immunol.,173:4919-28,2004)。
免疫调节剂可包括但不限于细胞因子、趋化因子、干细胞生长因子、淋巴毒素、成血因子、集落刺激因子(CSF)、白细胞介素(IL)、红细胞生成素、血小板生长因子、肿瘤坏死因子-α(TNF)、TNF-β、粒细胞-集落刺激因子(G-CSF)、粒细胞巨噬细胞-集落刺激因子(GM-CSF)、干扰素-α、干扰素-β、干扰素-γ、干扰素-λ、称为“S1因子”的干细胞生长因子、人生长激素、N-甲硫氨酰人生长激素、牛生长激素、甲状旁腺激素、甲状腺素、胰岛素、胰岛素原、松弛素、松弛素原、卵泡刺激素(FSH)、促甲状腺激素(TSH)、促黄体激素(LH)、肝生长因子、前列腺素、成纤维细胞生长因子、催乳激素、胎盘催乳素、OB蛋白质、苗勒氏管抑制物质、小鼠促性腺素相关联肽、抑制素、活化素、血管内皮生长因子、整联蛋白、NGF-β、血小板生长因子、TGF-α、TGF-β、胰岛素样生长因子-I、胰岛素样生长因子-II、巨噬细胞-CSF(M-CSF)、IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、IL-16、IL-17、IL-18、IL-21、IL-23、IL-25、LIF、FLT-3、血管内皮生长因子、血小板反应蛋白、内皮抑制素。在某些实施方案中，双特异性抗体或抗体片段可连接至免疫调节剂，如细胞因子。细胞因子复合物公开于例如美国专利号7,906,118和8,034,3522中，其中每一个的实施例部分以引用方式并入本文。优选免疫调节剂包括干扰素-α、干扰素-β和干扰素-λ。
本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸还可与例如标准癌症治疗(例如，手术、放射和化学疗法)组合施用。例如，使用本发明的组合物和/或装备了这些组合物的效应细胞的抗肿瘤疗法与化学疗法联合使用。本发明抗体组合治疗的非限制性实例包括手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。
双特异性抗体或编码本申请抗体的核酸或多核苷酸可连接至所述剂(作为免疫复合体)或与所述剂分开施用。在后一种情况下，双特异性抗体或编码本申请抗体的核酸或多核苷酸可在所述剂之前、之后或与所述剂共同施用，或可与其他已知疗法(如抗癌疗法、如辐射)共同施用。
本申请的药物组合物包含编码蛋白的核酸或多核苷酸，该核酸可以体内给予，以促进其编码的蛋白的表达，通过将其构建为合适的核酸表达载体的一部分并将其给予，以使其进入细胞内，例如，通过使用逆 转录病毒载体(见美国专利第4,980,286号)，或通过直接注射，或通过使用微粒轰击(如基因枪，Biolistic，Dupont)，或用脂类或细胞表面受体或转染剂涂覆，通过将其连接至同源异型框类肽并施用，该同源异型框类肽已知能进入核内(见，例如，Joliot A等,Proc.Natl.Acad.Sci.USA,88:1864-1868,1991)等。或者，可将核酸经细胞内引入，并结合至宿主细胞DNA中，通过同源重组进行表达。
本发明的组合物可以是多种形式。其包括例如，液体，半固体和固体的剂量形式，例如液体溶液(例如，可注射的和不熔化的溶液)分散剂或悬浮剂片剂，丸剂，粉剂，脂质体和栓剂。优选的方式依赖于施用方式和治疗用途。典型的优选组合物是可注射的或不熔化的溶液，例如那些类似于用其他抗体对人进行被动免疫的组合物。施用路径可以有多种形式，包括经口、直肠、经粘膜、经肠、肠胃外；肌肉内、皮下、皮内、髓内、鞘内、直接心室内、静脉内、腹膜内、鼻内、眼内、吸入、吹入、局部、皮肤、经皮或动脉内。优选的施用形式是非肠道的(例如静脉内，皮下，腹膜内，肌内)。在优选的实施方案中，所述的抗体通过静脉内注入或注射施用。在另一优选的实施方案中，所述的抗体通过肌内或皮下注射。
以上组合方法、治疗方法及施用方法是公知的，也包括未来可能发展的组合、治疗及施用方法。
适当剂量的测定通过临床医师，例如使用本领域中已知或怀疑影响治疗的参数或因素进行。通常，剂量以略小于最佳剂量的量开始，且其随后以较小增量增加，直至相对于任何负面的副作用实现所要或最佳作用。重要的诊断量度包括例如炎症的症状或所产生炎性细胞因子的水平的量度。
本发明提供容器(例如塑料或玻璃小瓶，例如具有盖或色谱柱、中空孔针或注射器圆筒)，其包含任一本发明的抗体或编码本申请抗体的核酸或多核苷酸及说明书。本发明还提供注射装置，其包含任一本发明的抗体或编码本申请抗体的核酸或多核苷酸。
组合疗法
本发明涵盖双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可与一或多种活性治疗剂(例如化学治疗剂)或其他预防或治疗模式(例如，辐射)组合的用途。在此类组合疗法中，各种活性剂经常具有不同的互补作用机制，组合疗法可能导致协同效应。组合疗法包含影响免疫反应(例如增强或活化反应)之治疗剂及影响(例如抑制或杀死)肿瘤/癌细胞之治疗剂。组合疗法可降低抗药性癌细胞发生的可能性。组合疗法可允许试剂中的一或多种试剂剂量减少，以减少或消除与试剂中之一或多种相关的不良作用。此类组合疗法可对潜在疾病、病症或病状具有协同的治疗或预防作用。
“组合”包括可以分开施用的疗法，例如针对单独投药分开调配(例如，可以在套组中提供)，及可以按单一调配物(亦即，“共调配物”)一起投与的疗法。在某些实施例中，本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可依次序施用。在其他实施例中，双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可同时施用。本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸 或药物组合物可以与至少一种其他(活性)药剂以任何方式组合使用。
用本发明双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物治疗可以与可有效针对待治疗病症的其他治疗组合，其他治疗组合的非限制性实例包括手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法。
组合疗法还包括其他一切公知技术中已有的以及未来可能发展的部分。
检测方法和试剂盒
在一些方面，本发明提供了(例如，体外或体内)检测TAA在样品(例如，生物样品，例如，血液、血清、精液或尿或组织活检样品(例如，来自过度增生性或癌性病灶))中存在或其水平的方法。该方法可以用来评价(例如，监测对象中本发明所述疾病(例如，免疫病症、癌症)的治疗或进展、其诊断和/或分期)。该方法可以包括：i.将样品与特异性针对TAA的抗体温育；ii.使用可检测的探针检测TAA复合物；iii.将(ii)的量与从含有已知量的TAA的参比样品获得的标准曲线进行比较；并且iv.从所述标准曲线计算所述样品中TAA的量。复合物的形成表示存在TAA，并且可以显示本文所述治疗的适宜性或需求。该方法可以涉及，例如免疫组织化学、免疫细胞化学、流式细胞术、抗体分子复合的磁珠、ELISA测定法、PCR技术(例如，RT-PCR)。一般，体内和体外诊断方法中所用的双特异性抗体分子或编码双抗的核酸或多核苷酸直接或间接地用可检测物质标记以输出检测信号。合适的可检测物质包括但不限于各种生物活性酶、辅基、荧光物质、发光物质和放射性物质。
还提供包含一种或多种本发明的双特异性抗体或编码双抗的核酸或多核苷酸的诊断或检测的试剂和试剂盒，其中，在一个实施方案中，双特异性抗体或编码双抗的核酸或多核苷酸和药学上可接受的载体，任选地与一种或多种治疗剂组合，任选地一起配制于药物组合物中。其用于多种检测测定中，包括例如免疫测定，诸如ELISA(夹心型或竞争形式)。试剂盒可包括其它添加剂，诸如稳定剂、缓冲剂(例如阻断缓冲液或裂解缓冲液)等等。具体地，试剂可作为干燥粉末提供，其通常冻干，包括赋形剂，其在溶解后，将提供具有适当浓度的试剂溶液。
试剂盒的组分可预附接至固体支持物，或可以当使用试剂盒时施加至固体支持物的表面。在本发明的一些实施方案中，信号产生装置可与本发明的双特异性抗体或编码双抗的核酸或多核苷酸预缔合，或在使用前可能需要组合一种或多种组分，例如缓冲剂、抗体-酶缀合物、酶底物等。在具体方面，催化化学发光或发色产物形成或化学发光或发色底物的还原的酶是信号产生装置的组分。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，可通过不同长度的接头将如上所述的可检测的标记连接至本发明的重组蛋白，以降低潜在的位阻。此类酶是本领域中众所周知的。
试剂盒也可包括额外试剂，例如用于减少与固相表面的非特异性结合的封闭试剂、洗涤试剂、酶底物等。固相表面可呈管、珠粒、微量滴定板、微球体或适合于固定蛋白、肽或多肽的其它材料的形式。
试剂盒组分可以包装在一起，或分装在两个或多个容器中。在一些实施例中，容器可以是含有适合进行重组的组合物的无菌冻干制剂的小瓶。可使用的其他容器包括但不限于袋、盘、盒、管等。试剂盒还可含有一种或多种适于复原和/或稀释其他试剂的缓冲液。
在一个实施方案中，试剂盒包含包装在容器中的双特异性抗体或编码本申请抗体的核酸或多核苷酸。
在一个实施方案中，试剂盒包含包装在容器中的双特异性抗体或编码本申请抗体的核酸或多核苷酸，和一种或多种药学上可接受的载体。
在一个实施方案中，试剂盒包含本发明的组合，其在单一常用容器中包括本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸连同一种或多种药学上可接受的载体，任选地与一种或多种治疗剂组合，任选地一起配制于药物组合物中。
在一个实施方案中，试剂盒包含一个容器中的本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸或其药物组合物和另一容器中的其药物组合物和/或治疗剂。
两种治疗剂的同时施用不要求药剂同时或通过相同途径施用，只要药剂发挥其治疗作用的时间段存在重叠。
在另一个方面，以上试剂盒包含附接至容器或与容器一起包装的标签，所述标签描述容器的内含物且提供适应症和/或关于使用容器内含物的方法，为可用于治疗、预防和/或诊断如本文所述的一种或多种疾病状态的说明书。
试剂盒可任选地进一步包括用于肠胃外，例如静脉内施用的注射器。试剂盒也包括用于严密地容纳小瓶用于商业销售和/或便于包装和递送的装置。还包括用于进行本文所述的检测或监测方法的装置或设备。
用途
本文所述的，本申请的双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物或组合疗法可用于与本文所述的免疫相关疾病、癌症、自身免疫性疾病、炎症性疾病、移植排异反应相关疾病或病症的治疗和诊断方法中，此种治疗包括将本申请的双特异性抗体或编码双抗的核酸或多核苷酸或药物组合物或组合疗法给予至患者，例如动物、哺乳动物和人，用于治疗或诊断本文描述的一种或多种疾病或症状。本申请的治疗化合物包括但不限于，本申请的抗体(包括如本文所述的它们的变体、同工型、衍生物和物种同源物)或编码本申请的抗体的核酸或多核苷酸(包括如本文所述的它们的变体、同工型、衍生物和物种同源物)。
本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可用于诊断目的。在一个实 施方案中，本发明的抗体用于体外诊断试验，例如检测有用抗原的实验室试验或在检测有用抗原的保健试验(care test)中。已建立的应用抗体的体外试验包括ELISAs，RIAs，Western印迹等。在又一实施方案中，本发明的抗体用于体内诊断试验，例如体内成象试验。例如所述的抗体用能在体内检测的可检测的物质标记，所述的标记抗体可施用于受试者，并且所述的标记抗体可在体内检测，由此可以进行体内成像。相关诊断技术还包括其他公知技术及可能发展的技术。
本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸可用于制备药物。所述药物组合物包括但不限于上文药物组合物所述。本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸可单独使用，或可与一种或更多种其他治疗剂共同使用，所述治疗剂包括但不限于上文所述。在一些优选实施方案中，所述双特异性抗体或编码本申请抗体的核酸或多核苷酸与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供，或可与其他已知疗法共同施用。其他已知疗法包括但不限于上文所述。药物组合物的组合方法、治疗方法及施用方法包括但不限于上文所述。还提供包含本发明双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物的容器及说明书。
本发明提供包含本发明双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物的组合疗法。组合疗法包含但不限于上文所述。
本发明还提供包含本发明双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物的相关检测方法和试剂盒。相关内容包含但不限于上文所述。
本申请的双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可以用于诊断、治疗，抑制或预防疾病或症状，包括恶性的疾病，病症，或与这样的疾病或病症相关的状况，如与增加细胞存活或抑制细胞凋亡相关的疾病，例如但不限于上文所述免疫相关疾病、癌症、自身免疫性疾病、炎症性疾病、移植排异反应相关疾病或病症。例如，所述疾病涉及细胞的无调节和/或不适当增殖，有时伴随有临近组织和新血管生长的破坏，这可能使得癌细胞可向新的区域入侵，即转移。
可以用本申请的双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物治疗，预防，诊断和/或预测的与增加细胞存活相关的其他疾病或症状，包括但不限于，恶性肿瘤的发展和/或转移，以及上文所述其他相关疾病或症状(例如，炎症性疾病或淋巴系统疾病)的发展和/或转移。
双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物的形式包括但不限于上文所述。给予本发明的双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物的方法包括但不限于上文所述。治疗方法及组合疗法包括但不限于上文所述。
例如，双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物可配制成用于通过例如推注或连续输注进行静脉内施用，或甚至通过其他肠胃外途径，如静脉内、肌肉内、腹膜内或血管内来施用至哺乳动物。优选地，双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物的输注持续少于约4个 小时的时间，并且更优选持续少于约3个小时的时间。注射制剂可以呈单位剂型提供，例如，在安瓿或多剂量容器中，其中添加防腐剂。组合物可采用诸如于油性或水性媒剂中的混悬液、溶液或乳液等形式，并且可含有配制剂，如混悬剂、稳定剂和/或分散剂。替代地，活性成分可呈在使用前用适合媒剂(例如无菌无热原质水)复原的粉末形式。
各种递送系统是已知的，并可用于给予本申请的抗体或编码本申请抗体的核酸或多核苷酸或药物组合物，例如，将其包封在脂质体，微粒，微胶囊中，能表达该化合物的重组细胞，受体介导的内吞作用(参见，例如，Wu GY等,J.Biol.Chem.,262:4429-4432,1987)，构建核酸作为逆转录病毒或其他载体等。递送系统还包括一切其他公知技术及未来可能发展的部分。
可以采用另外的药学方法控制治疗组合物的作用持续时间，例如控制释放系统。控制释放系统的非限制性实例为泵(例如参见Sefton,CRC,Crit.Ref.Biomed.Eng.,14:201,1987；Buchwald H等,Surgery,88:507,1980)或聚合物材料(例如参见，Medical Applications of Controlled Release,Langer和Wise(编),CRC Pres.Boca Raton,Fla.,1974)等。此药学方法还包括一切其他公知技术及未来可能发展的部分。
可施用治疗有效剂量的双特异性抗体或编码本申请抗体的核酸或多核苷酸或药物组合物。构成治疗有效剂量的双特异性抗体或编码本申请抗体的或多核苷酸或药物组合物的量可根据所治疗的适应症、患者的体重、计算得到的患者的皮肤表面积而变化。可调节其给药从而达到预期效果。在许多情况下，可能需要重复给药。例如，其可每周三次、两次给药，每周一次给药，每两周、三周、四周、五周、六周、七周、八周、九周或十周一次给药，或每两个月、三个月、四个月、五个月或六个月一次给药。本申请的双特异性抗体或编码本申请抗体的核酸或多核苷酸对于患者的给药剂量通常为约0.001-100mg/kg，如约0.1-50mg/kg，例如约0.1-20mg/kg，如约0.1-10mg/kg，例如约0.5mg/kg，如约0.3mg/kg、约1mg/kg或约3mg/kg。可选的，抗体的给药剂量可以根据患者的估计皮肤表面积进行调整或根据患者的体重进行调整。给药时程可任选地以其他时间间隔重复，并且剂量可在对剂量和时程进行适当调整下通过各种胃肠外途径给予。
一般来说，人抗体比来自其他物种的抗体在人体内有更长的半衰期，由于有对于外源多肽的免疫反应。因此，低剂量的人抗体，减少给药频率通常是允许的。另外，本申请的抗体的给药剂量和频率可通过加强抗体的吸收和组织渗透(例如，进入大脑)来降低，该加强通过修饰例如脂化来实现。
本发明双特异性抗体或编码本申请抗体的核酸或多核苷酸可用于制备诊断性或治疗性试剂盒，其包括本发明所述双特异性抗体或编码本申请抗体的核酸或多核苷酸和/或使用说明书。本发明所述药物组合物可用于制备诊断性或治疗性试剂盒，其包括本发明所述药物组合物和/或使用说明书。
附图说明
图1-1、CD19双抗纯化样品的SEC-HPLC检测结果。
图1-2、CD19双抗纯化样品的SDS-PAGE电泳结果。
图2-1、FACS检测双特异性抗体AB23P7与肿瘤细胞Raji结合的能力。
图2-2、FACS检测双特异性抗体AB23P8与肿瘤细胞Raji结合的能力。
图2-3、FACS检测双特异性抗体AB23P9和AB23P10与肿瘤细胞Raji结合的能力。
图2-4、FACS检测双特异性抗体AB23P7和AB23P8与效应细胞CIK结合的能力。
图2-5、FACS检测双特异性抗体AB23P9和AB23P10与效应细胞CIK结合的能力。
图2-6、FACS检测双特异性抗体AB23P7和AB23P10与食蟹猴T细胞结合的能力。
图2-7、ELISA检测4种Anti-CD19×CD3双特异性抗体与CD3和CD19分子结合的能力。
图2-8、酶标仪检测AB23P7和AB23P8双特异性抗体活化报告基因细胞株Jurkat T细胞的能力。
图2-9、酶标仪检测4种Anti-CD19×CD3双特异性抗体活化报告基因细胞株Jurkat T细胞的能力。
图3-1、双抗AB23P9和AB23P10在NPG小鼠皮下共接种人CIK细胞和Raji细胞的移植瘤模型中的体内抑瘤效果。
图3-2、双抗Blincyto、AB23P9和AB23P10在NPG小鼠皮下共接种人CIK细胞和Daudi细胞的移植瘤模型中的体内抑瘤效果。
图4-1A、正常雄性食蟹猴给与双特异性抗体AB23P7后血清中细胞因子IL-2含量的测定。
图4-1B、正常雌性食蟹猴给与双特异性抗体AB23P10后血清中细胞因子IL-2含量的测定。
图4-1C、正常雄性食蟹猴给与双特异性抗体AB23P7后血清中细胞因子IL-6含量的测定。
图4-1D、正常雌性食蟹猴给与双特异性抗体AB23P10后血清中细胞因子IL-6含量的测定。
图4-1E、正常雄性食蟹猴给与双特异性抗体AB23P7后血清中细胞因子IFN-γ含量的测定。
图4-1F、正常雌性食蟹猴给与双特异性抗体AB23P10后血清中细胞因子IFN-γ含量的测定。
具体实施方式
通过下列实施例进一步说明本发明，所述实施例不应解释为进一步限制。在此将整篇申请中引用的所有附图和所有参考文献、专利和已公开专利申请的内容明确收入本文作为参考。
实施例一、Anti-CD19×CD3双特异性抗体的设计和制备
1.1、双特异性抗体的设计
在本文的优先权文件中国专利申请CN 201811294887.4中，针对示例性双特异性抗体Her2和CD3设计了六种不同的构型，本文中选用其中一种类似AB7K7的构型并进行了优化，由此衍生出具有以下结构的Anti-CD19×CD3双特异性抗体。具体地，上述双特异性抗体的构型及其从N端至C端方向的组成特性描述如下：
七个抗体包含双特异性抗体AB23P7、AB23P8、AB23P9和AB23P10在内均是由抗-CD19 scFv、连接肽L2、抗-CD3 scFv和Fc片段依次串联组成，抗-CD19 scFv和抗-CD3 scFv内部VH和VL之间分别由连接肽L1和L3连接。其中，表1-1中列举的双特异性抗体所包含的前五个抗-CD19 scFv的氨基酸序列中，其VH结构域和VL结构域依次参照：双特异性抗体Blinatumomab(序列来自美国专利US 10/554852中结合CD19的抗体，其重链可变区和轻链可变区分别如本文SEQ ID NO：7和8所示)，抗体偶联物Coltuximab(序列来自美国专利US 14/117806中结合CD19的抗体HuB4，其重链可变区及轻链可变区分别如本文SEQ ID NO：15和16所示)，抗体偶联物Denintuzumab(序列来自中国专利CN 201580017717.X中结合CD19的抗体hBU12，其重链可变区及轻链可变区分别如本文SEQ ID NO：22和63所示)，单克隆抗体Inebilizumab(序列来自美国专利US 15/863144中结合CD19的抗体，其重链可变区及轻链可变区分别如本文SEQ ID NO：30和31所示)和双特异性抗体Duvortuxizumab(序列来自中国专利CN 201580051198.9中结合CD19的抗体，其重链可变区及轻链可变区分别如本文SEQ ID NO：34和35所示)。双特异性抗体所包含的Fc片段均来自人IgG1，且具有多个氨基酸的替换/取代，分别为L234A、L235A、T250Q、N297A、P331S和M428L(EU编号)，同时还删除/缺失了Fc片段C末端的K447(EU编号)。其连接肽(L2)由柔性肽和刚性肽组成，且柔性肽均为G 2(GGGGS) 3，刚性肽为SSSSKAPPPS。而每个scFv内部的连接肽L1和L3的组成均为(GGGGS) 3。
表1-1列举了七个双特异性抗体的抗CD19-scFv的VH结构域及其互补决定区(HCDR1、HCDR2和HCDR3)的氨基酸序列，和VL结构域及其互补决定区(LCDR1、LCDR2和LCDR3)的氨基酸序列，其CDR区所含氨基酸残基根据Kabat规则定义。其中，抗CD19-scFv的VH和VL之间的连接肽氨基酸组成为(GGGGS)n，n＝1，2，3，4或5。
表1-1：双特异性抗体包含的抗CD19-scFv的氨基酸序列及其CDR区氨基酸序列
Figure PCTCN2019114808-appb-000001
Figure PCTCN2019114808-appb-000002
Figure PCTCN2019114808-appb-000003
其中，抗-CD3 scFv在体外FACS结合分析测定中以大于约10nM，或大于20nM，或大于40nM，或大于约50nM的EC 50值结合于效应细胞；更优选地，所述双特异性抗体的第二单链Fv不仅能与人CD3结合，还可与食蟹猴或恒河猴的CD3特异性结合。
在一个优选实施例中，上述双特异性抗体包含的抗CD3-scFv的VH和VL氨基酸序列分别如SEQ ID  NO：46和SEQ ID NO：47所示，且VH和VL之间由(GGGGS) 3连接，该单克隆抗体(命名为CD3-3)特异性结合人类和食蟹猴CD3抗原，且与CD3具有微弱的结合亲和力。
其中，连接抗-CD19 scFv和抗-CD3 scFv的连接肽由柔性肽和刚性肽组成；优选地，柔性肽的氨基酸组成结构通式为G xS y(GGGGS) z，其中x，y和z是大于或等于0的整数，且x+y+z≥1。而刚性肽来自天然人绒毛膜促性腺激素β亚基羧基末端第118至145位氨基酸组成的全长序列(如SEQ ID NO：48所示)或其截短的片段；优选地，所述CTP刚性肽组成为SSSSKAPPPS(CTP 1)。表1-2中例举了一些优选的连接抗-CD19scFv和抗-CD3 scFv的连接肽的氨基酸序列。
表1-2：连接抗-TAA scFv和抗-CD3 scFv的连接肽的氨基酸序列
SEQ ID NO：49 G 2(GGGGS) 3CTP 1 GGGGGGSGGGGSGGGGSSSSSKAPPPS
SEQ ID NO：50 (GGGGS) 3CTP 1 GGGGSGGGGSGGGGSSSSSKAPPPS
SEQ ID NO：51 GS(GGGGS) 2CTP 1 GSGGGGSGGGGSSSSSKAPPPS
SEQ ID NO：52 (GGGGS) 1CTP 4 GGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ
其中，Fc片段直接或通过连接肽与抗-CD3 scFv相连，连接肽包含1-20个氨基酸，并优选自下列几种氨基酸：Gly(G)、Ser(S)、Ala(A)和Thr(T)，更优地自Gly(G)和Ser(S)，最优选地，所述连接肽组成为(GGGGS)n，n＝1，2，3或4。
Fc片段优选自人IgG1、IgG2、IgG3和IgG4的重链恒定区，更特别地选自人IgG1或IgG4的重链恒定区；并且Fc是突变的，以修饰双特异性抗体分子的性质，例如，对人FcγRs(FcγRI、FcγRIIa或FcγRIIIa)和C1q的至少一种显示出降低的亲和力，具有减少的效应细胞功能或补体功能。此外，Fc片段还可以包含具有使其它一种或几种特性(例如，与FcRn受体结合能力、抗体糖基化或抗体电荷异质性等)改变的氨基酸取代。
表1-3中例举了一些具有一个或多个氨基酸突变的Fc片段的氨基酸序列。
表1-3：人IgG Fc氨基酸序列
Figure PCTCN2019114808-appb-000004
Figure PCTCN2019114808-appb-000005
1.2、双特异性抗体分子表达载体的构建
按常规分子生物学方法合成上述双特异性抗体的编码基因，并将获得的融合基因的编码cDNA分别插入到经PCDNA3.1改造后的真核表达质粒pCMAB2M的相应酶切位点间，该质粒含巨细胞病毒早期启动子，它是哺乳动物细胞高水平表达外源基因所需的增强子。质粒pCMAB2M还含有选择性标记物，从而在细菌中可以具有卡那霉素抗性，而在哺乳动物细胞中可以具有G418抗性。另外，当宿主细胞是DHFR基因表达缺陷型时，pCMAB2M表达载体含有小鼠的二氢叶酸还原酶(DHFR)基因，从而在存在氨甲蝶呤(MTX)时能共扩增目的基因和DHFR基因(参见美国专利US 4,399,216)。
1.3、双特异性抗体分子的表达
将上述构建的表达质粒转染哺乳动物宿主细胞系，以表达双特异性抗体。为了稳定高水平的表达，优选的宿主细胞系是DHFR酶缺陷型CHO-细胞(参见美国专利US 4,818,679)，本实施例中宿主细胞选取CHO衍生细胞株DXB11。一种优选的转染方法是电穿孔，也可以使用其它方法，包括磷酸钙共沉降、脂转染。在电穿孔中，用设置为300V电场和1500μFd电容的Gene Pulser电穿孔仪(Bio-Rad Laboratories，Hercules，CA)，在比色杯内的5×10 7个细胞中加入50μg表达载体质粒DNA。在转染两天后，将培养基改成含0.6mg/mL G418的生长培养基。用极限稀释亚克隆转染子，并用ELISA方法测定各细胞系的分泌率。筛选出高水平表达双特异性抗体的细胞株。
为了实现融合蛋白较高水平的表达，宜用受MTX药物抑制的DHFR基因进行共扩增。在含有递增浓度MTX的生长培养基中，用DHFR基因共扩增转染的融合蛋白基因。极限稀释DHFR表达阳性的亚克隆，逐步加压并筛选出能在高达6μM MTX培养基中生长的转染子，测定其分泌率，筛选出高表达外源蛋白的细胞系。将分泌率超过约5(较佳地约15)μg/10 6(即百万)个细胞/24h的细胞系使用无血清培养基的进行 适应性悬浮培养。然后，收集细胞上清并分离纯化双特异性抗体。
下文分别对几种双特异性抗体的纯化工艺、稳定性、体外和体内生物学功能等进行评价。
1.4、双特异性抗体的纯化工艺及稳定性试验
a)双链四价双特异性抗体的纯化
我们采用三步层析法对CD19双特异性抗体进行纯化。分别为亲和层析、羟基磷灰石层析和阴离子交换层析(本实施例采用的蛋白纯化仪为美国GE公司的AKTA pure 25M。本实施例中采用的试剂均购自国药集团化学试剂有限公司，纯度均为分析级)。
第一步，亲和层析：采用博格隆公司的AT Protein A Diamond或其它市售的亲和介质(例如GE公司的MabSelect Sure亲和层析介质等)进行样品捕获、浓缩以及部分污染物的去除。首先使用平衡buffer(20mM PB，150mM NaCl，pH 7.4)，以100-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；将经过澄清后的发酵液以100-200cm/h的线性流速上样，载量不高于20mg/mL填料；上样完毕后，使用平衡buffer(20mM PB，150mM NaCl，pH 7.4)以100-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)，冲洗未结合的组份；使用去污buffer 1(50mM NaAc-HAc，1M NaCl，pH 5.0)，以100-200cm/h的线性流速冲洗层析柱3-5个柱体积，去除部分污染物；使用去污buffer 2(50mM NaAc-HAc，pH 5.0)，以100-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；之后使用洗脱buffer(50mM NaAc-HAc，pH 3.5)，以不高于100cm/h的线性流速洗脱目标产物，收集目标峰。
第二步，羟基磷灰石层析：使用BIO-RAD公司的CHT TypeⅡ或其它市售的羟基磷灰石介质进行中间纯化，用于降低聚合体含量。目标蛋白聚合以后，聚合体和单体之间存在性质上的差异，包括电荷特性以及钙离子螯合等作用，我们使用电荷特性等的差异对二者进行分离。首先，使用平衡buffer(20mM PB，pH 7.0)，以100-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；第一步亲和层析分离得到的目标蛋白调pH 7.0，然后上样，载量控制在<5mg/ml；上样完毕后，使用平衡buffer(20mM PB，pH 7.0)，以100-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；最后进行目标蛋白洗脱，使用洗脱buffer(20mM PB，1M NaCl，pH 7.0)，以0-25％梯度洗脱，以不高于100cm/h的线性流速洗脱10个柱体积(CV)，对洗脱组分进行分段收集，分别送检SEC-HPLC。将单体百分比大于95％的目标组分合并进行下一步层析。
第三步，阴离子交换层析：使用博格隆公司的Q-HP或其它市售的阴离子交换层析介质(例如GE的Q HP、TOSOH的Toyopearl GigaCap Q-650、天地人和的DEAE Beads 6FF，赛分科技的Generik MC-Q、Merck的Fractogel EMD TMAE、Pall的Q Ceramic HyperD F)进行精细纯化，进一步去除HCP、DNA等污染物。首先使用平衡buffer(20mM PB，0.15M NaCl，pH 7.0)，以100-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；经第二步羟基磷灰石层析分离得到的目标蛋白上样，收集流穿，上样完毕，使用平衡buffer(20 mM PB，0.15M NaCl，pH 7.0)，以100-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；对流穿组分进行收集，分别送样进行蛋白含量、SEC-HPLC和电泳检测。
样品的SEC-HPLC纯度结果及SDS-PAGE电泳结果分别见图1-1和图1-2，其中SEC-HPLC结果显示，三步层析后双特异性抗体的主峰纯度达95％以上，SDS-PAGE电泳带型符合预期，非还原电泳(160KDa)，还原后可得清晰的(80KDa)单链条带。
b)CD19双特异性抗体稳定性试验
对CD19双抗蛋白在不同pH(5.5和6.0)的柠檬酸盐(20mM柠檬酸盐、8％蔗糖、0.02％Tween-80)中的稳定性进行考察，同时探究震荡对样品稳定性的影响。将CD19双抗蛋白在25℃的加速条件下贮存2周，对蛋白的稳定性进行评估。
将CD19双抗蛋白分别换液至pH 5.5和6.0的柠檬酸盐缓冲液中。不同时间点取样检测，每个取样点取出样品进行检测分析，检测项目包括SEC-HPLC及CE-SDS。
2种制剂配方在25℃贮存0～2周后的SEC-HPLC和CE-SDS检测结果见表1-4和表1-5。样品在放置两周后，SEC-HPLC检测结果中聚体、主峰、肩峰及片段的比例无明显变化，在不同pH下，样品各组分的比例也相差不大。样品在pH 5.5和6.0的柠檬酸盐缓冲液中两周内SEC-HPLC结果无明显差异。
表1-4：25℃加速实验SEC-HPLC结果
Figure PCTCN2019114808-appb-000006
表1-5：25℃加速实验CE-SDS结果
Figure PCTCN2019114808-appb-000007
Figure PCTCN2019114808-appb-000008
将两个CD19双抗蛋白分别放置于稳定性培养箱中，其中一个设定转速300rpm，一个转速为零，其余条件保持一致。300rpm，3d条件下SEC-HPLC检测结果见表1-6。对照组和样品组聚体、主峰、肩峰及片段的比例基本一致，因此，300rpm转速对CD19双抗样品蛋白稳定性无明显影响。
表1-6：300rpm三天对照试验SEC-HPLC结果
Figure PCTCN2019114808-appb-000009
实施例二、Anti-CD19×CD3双特异性抗体的体外生物学功能评价
2.1、双特异性抗体与效应细胞和靶细胞结合活性的测定(FACS)
a)利用流式分析法检测双特异性抗体与CD19阳性肿瘤细胞Raji的结合活性
培养CD19表达阳性的肿瘤细胞Raji细胞(上海中科院细胞库)，离心收集细胞。将收集的细胞用1％PBSB重悬，调整细胞密度为2×10 6个/ml，置于96孔板中，每孔100μl(2×10 5个细胞)，4℃封闭0.5h。封闭后的细胞离心弃上清，加入稀释好的一系列浓度的双特异性抗体，4℃孵育1h；离心去上清，用1％BSA的PBS溶液(PBSB)洗3遍，加入稀释好的AF647标记的山羊抗人IgG抗体，4℃避光孵育1h；离心去上清，1％PBSB洗两遍，每孔再用100μl 1％多聚甲醛(PF)重悬，流式细胞仪检测信号强度。再以平均荧光强度作为Y轴，抗体浓度作为X轴，通过软件GraphPad进行分析，计算双特异性抗体与肿瘤细胞Raji结合的EC 50值。
结果显示，不同结构的双特异性抗体和CD19过表达肿瘤细胞均具有良好的结合活性。图2-1～图2-3展示了不同结构的双特异性抗体和肿瘤细胞Raji的结合曲线。如表2-1所示，四个双特异性分子与Raji细胞结合的EC 50均在nM级水平。
表2-1：Anti-CD19×CD3双特异性抗体与肿瘤细胞Raji结合能力的测定
AB23P7 AB23P8 AB23P9 AB23P10
EC 50(nM) 1.393 1.924 2.600 2.678
b)利用FACS检测双特异性抗体与人T细胞的结合活性
采用密度梯度离心法从人新鲜血液制备PBMC，用含10％热灭活FBS的1640培养基重悬，加入2μg/ml CD3抗体活化24h后，加入250IU/ml IL-2扩增培养7天，制备得到扩增的T细胞，经流式细胞分析仪检测细胞表面CD3表达呈阳性。待测样品制备及测定方法同实施例2.1a)。将1％PF重悬的细胞上机检测，以平均荧光强度，通过软件GraphPad进行分析，计算各双特异性抗体与人T细胞结合的EC 50值。
结果显示各双特异性抗体和CIK均具有良好的结合活性(图2-4～图2-5)。如表2-2所示，AB23P7的EC 50约为16nM，AB23P8的结果与其相当，AB23P9、AB23P10的EC 50约为50nM和30nM。
表2-2：Anti-CD19×CD3双特异性抗体与效应细胞CIK结合能力的测定
AB23P7 AB23P8 AB23P9 AB23P10
EC 50(nM) 15.69 16.69 49.52 32.41
c)通过FACS检测双特异性抗体与食蟹猴CIK细胞膜表面CD3的交叉反应性
采用密度梯度离心法从食蟹猴新鲜血液制备PBMC，用含10％热灭活FBS的1640培养基重悬，加入2μg/ml OKT3活化24h后，加入250IU/ml IL-2扩增培养7天，得到食蟹猴CIK细胞备用。将人CIK细胞和食蟹猴CIK细胞离心收集，待测样品制备及测定方法同实施例2.1a)。将1％多聚甲醛溶液重悬的细胞上机检测，以平均荧光强度，通过软件GraphPad进行分析，计算双特异性抗体分别与人CIK细胞和食蟹猴CIK细胞结合的EC 50值。
如图2-6所示，双特异性抗体AB23P7和AB23P10与食蟹猴T细胞的结合能力几乎没有差别，流式细胞仪检测其结合的EC 50大约在5.5nM，且两个双特异性抗体与食蟹猴T细胞的结合能力强于与人T细胞的结合能力。AB23P8、AB23P9同AB23P7、AB23P10一样可以与食蟹猴T细胞特异性结合。
2.2、双特异性抗体与抗原的结合能力测定
通过双抗原夹心ELISA法鉴定双特异性抗体与可溶CD3和CD19的结合。
将CD19蛋白(ACRO Biosystems，货号CD9-H5251)以PBS稀释成1μg/ml的浓度，加入96孔板中，100μl/孔，4℃包被过夜。然后用1％脱脂奶粉室温封闭1h。同时稀释各双特异性抗体，用5倍梯度稀释，共10个浓度梯度。然后用PBST清洗96孔板，加入稀释好的双特异性抗体，设不加抗体的对照孔，室温孵育2h。将未结合的双特异性抗体以PBST洗去，将生物素化的CD3E&CD3D(ACRO Biosystem，货号CDD-H82W1)以50ng/ml混合Streptavdin HRP(BD，货号554066)加入96孔板中，100μl/孔，室温孵育1h。其后，将96孔板以PBST清洗，加入TMB，100μl/孔，室温显色15min，然后加入0.2M H 2SO 4终止显色反应。用酶标仪检测A450-620nm的吸光值。通过软件GraphPad进行分析，计算双特异性抗体与两个 抗原结合的EC 50值。
结果显示，各个双特异性抗体都能同时特异性地结合CD3和CD19分子，并且随抗体浓度的变化呈现良好的剂量依赖性(图2-7)。几种双特异性抗体与可溶CD3和CD19的结合能力如表2-3所示，其EC 50值从0.19nM至0.47nM，两端结合能力几乎相差无几。
表2-3：Anti-CD19×CD3双特异性抗体与CD3和CD19分子结合能力的测定
AB23P7 AB23P8 AB23P9 AB23P10
EC 50(nM) 0.2185 0.1925 0.2211 0.4704
2.3、报告基因细胞株评价双特异性抗体活化T细胞的能力
含有NFAT RE报告基因的Jurkat T细胞(BPS Bioscience，货号60621)，在双特异性抗体和靶细胞(Raji细胞)同时存在的情况下可以过表达萤光素酶，通过检测萤光素酶的活性来定量Jurkat T细胞的活化程度。以双特异性抗体blinatumomab(
Figure PCTCN2019114808-appb-000010
Amgen)为阳性对照。以双特异性抗体的浓度做X轴，荧光素信号作为Y轴，拟合四参数曲线。
根据图2-8～图2-9的实验结果显示，在没有CD19过表达靶细胞的存在下，Jurkat T细胞几乎不能被活化，只有当双抗及两端靶细胞都存在的情况下，T细胞才会被活化。各抗体活化Jurkat T细胞的能力显示在表2-4中，各双特异性抗体活化Jurkat T细胞的能力几乎相当。
表2-4：Anti-CD19×CD3双特异性抗体活化报告基因细胞株Jurkat T细胞能力的测定
AB23P7 AB23P8 AB23P9 AB23P10 Blincyto
EC 50(nM) 1.080 1.123 0.8527 0.7093 2.714
2.4、双特异性抗体介导T细胞杀伤肿瘤细胞的能力
正常培养的肿瘤细胞系，包括Raji-Luc、NALM6和Reh细胞(均购自上海中科院细胞库)作为靶细胞，收集细胞悬液，离心，调整细胞密度2×10 5个/ml，加入96孔细胞培养板中，100μl/孔，培养过夜。按实验设计稀释相应抗体，50μl/孔，无需加入抗体的孔则用相同体积的培养基补入。然后加入5倍于靶细胞数的效应细胞(人PBMC或者扩增培养的CIK细胞)，100μl/孔，设置对照孔，无需加入效应细胞的孔则用相同体积的培养基补入。培养48h后，Raji-Luc细胞用Steady-Glo Luciferase Assay System(Promega)检测，其他细胞使用CytoTox96 Non-Radio Cytotoxicity Assay(Promega)检测。以检测结果作为Y轴，双特异性抗体浓度作为X轴，通过软件GraphPad进行分析，计算并比较各双特异性抗体介导杀伤肿瘤细胞的能力。
各双特异性抗体介导效应细胞杀伤肿瘤细胞的EC 50值归纳在表2-5～表2-7中，结果显示各双特异性抗体对CD19高表达的肿瘤细胞均呈现非常显著的杀伤作用，其EC 50达到pM级别，并且呈剂量依赖性。
表2-5：双特异性抗体介导CIK杀伤肿瘤细胞的EC 50值
Figure PCTCN2019114808-appb-000011
备注：-表示未进行检测。
表2-6：双特异性抗体介导PBMC杀伤肿瘤细胞的EC 50值
Figure PCTCN2019114808-appb-000012
备注：-表示未进行检测。
表2-7：双特异性抗体介导CIK杀伤不同肿瘤细胞的EC 50值
EC 50(pM) AB23P7 AB23P10 Blincyto
NALM6 - 4.402 77.29
Reh 1.709 1.640 11.87
备注：-表示未进行检测。
实施例三、Anti-CD19×CD3双特异性抗体的体内生物学功能评价
3.1、AB23P9和AB23P10(Anti-CD19×CD3)在体内皮下移植人Burkkit’s淋巴瘤Raji模型中的药效学研究
选取CD19表达阳性的人Burkkit’s淋巴瘤Raji细胞小鼠移植瘤模型对抗CD19-CD3双功能特异性抗体进行体内抑制肿瘤生长的药效学研究。
取正常人外周血，用密度梯度离心法分离人PBMC细胞，然后用RPMI-1640培养液加入10％已灭活的FBS重悬，并且加入终浓度为1μg/ml的OKT3和250IU/ml人IL-2；培养第三天后，300g离心5min后换液，用RPMI-1640加入10％已灭活FBS重悬细胞进行培养，同时加入250IU/ml人IL-2；之后每2天添加新鲜的培养液，培养到第10天，收集CIK细胞。选取七至八周龄的雌性NPG小鼠(北京维通达生物技术有限公司)，收集处于对数生长期的Raji细胞，将5×10 6个Raji细胞和5×10 6个CIK细胞混合，接种于NPG小鼠右侧背部皮下。1小时后，小鼠按体重随机分为7组，每组6只，分别腹腔给予相应药物。所有给药组均为每周给药两次，双功能抗体AB23P9和AB23P10给药组的剂量均为1mg/kg、0.1mg/kg和0.01mg/kg。给药当天记为第0天，每周用电子游标卡尺测量肿瘤的最大直径(D)和最小直径(d)，使用以下 公式计算肿瘤体积(mm 3)＝[D×d 2]/2，并根据公式计算各给药组的肿瘤生长抑制率TGI(％)＝(1-给药组体积/对照组体积)×100％。
如图3-1所示，在给药后第26天，PBS对照组平均肿瘤体积为1679.90±359.05mm 3；1mg/kg、0.1mg/kg和0.01mg/kg的AB23P9给药组的平均肿瘤体积分别为7.39±7.39mm 3、56.84±36.69mm 3和124.78±35.03mm 3，TGI分别为99.56％、96.62％和92.57％，相对于对照组均有极显著性差异(P<0.01)；1mg/kg、0.1mg/kg和0.01mg/kg的AB23P10给药组的平均肿瘤体积分别为0.00±0.00mm 3、0.00±0.00mm 3和196.79±146.03mm 3，TGI分别为100％、100％和88.29％，相对于对照组均有极显著性差异(P<0.01)。
上述结果表明，双特异性抗体AB23P9和AB23P10在动物体内通过激活人类免疫细胞来抑制肿瘤细胞的生长，在相同的剂量下，AB23P9和AB23P10抗肿瘤效果无差异。
3.2、Blincyto、AB23P9和AB23P10(Anti-CD19×CD3)在体内皮下移植人Burkkit’s淋巴瘤Daudi模型中的药效学研究
选取CD19表达阳性的人Burkkit’s淋巴瘤Daudi细胞小鼠移植瘤模型对抗CD19-CD3双功能特异性抗体进行体内抑制肿瘤生长的药效学研究。
采用实施例3.1的方法分离人PBMC，并同样进行两次重悬培养。第二次重悬培养时每2天添加新鲜的培养液，培养到第8天，收集CIK细胞。选取七至八周龄的雌性NPG小鼠，收集处于对数生长期的Daudi细胞(中科院细胞库)，将5×10 6个Daudi细胞和1×10 6个CIK细胞混合，接种于NPG小鼠右侧背部皮下。1h后，小鼠按体重随机分为8组，每组5只，分别腹腔给予相应药物，所有给药组均为每周给药两次。Blincyto的给药组剂量为1mg/kg，双功能抗体AB23P9和AB23P10给药组的剂量均为1mg/kg、0.1mg/kg和0.01mg/kg。给药当天记为第0天，每周用电子游标卡尺测量肿瘤的最大直径(D)和最小直径(d)，使用以下公式计算肿瘤体积(mm 3)＝[D×d 2]/2，并根据公式计算各给药组的肿瘤生长抑制率TGI(％)＝(1-给药组体积/对照组体积)×100％。
如图3-2所示，在给药后第25天，PBS对照组平均肿瘤体积为1903.03±727.61mm 3；1mg/kg的Blincyto给药组的平均肿瘤体积为0.00±0.00mm 3，TGI为100％，相对于对照组有显著性差异(P<0.05)；1mg/kg、0.1mg/kg和0.01mg/kg的AB23P9给药组的平均肿瘤体积分别为0.00±0.00mm 3、261.13±178.17mm 3和520.87±133.26mm 3，TGI分别为100％、86.28％和72.63％，相对于对照组P9的1mg/kg给药组有显著性差异(P<0.05)，其余两组有抑瘤效果但差异不显著；1mg/kg、0.1mg/kg和0.01mg/kg的AB23P10给药组的平均肿瘤体积分别为0.00±0.00mm 3、58.46±36.67mm 3和599.28±199.80mm 3，TGI分别为100％、96.93％和68.51％，相对于对照组P10的1mg/kg和0.1mg/kg给药组均有显著性差异(P<0.05)，0.01mg/kg给药组有抑瘤效果但差异不显著。
上述结果表明，双特异性抗体Blincyto、AB23P9和AB23P10在动物体内通过激活人类免疫细胞来抑制肿瘤细胞的生长。在1mg/kg的剂量下，Blincyto、AB23P9和AB23P10抗肿瘤效果无差异。P9和P10在同一剂量下组间比较无差异，且对肿瘤的抑制效果呈现出一定的剂量依赖性。
实施例四、Anti-CD19×CD3双特异性抗体的安全性评价
4.1、双特异性抗体对正常食蟹猴的毒性评价
选取3-4岁的成年食蟹猴(广州相观生物科技有限公司)，体重3-4kg，分成两组，每组雌雄各一只，分别为AB23P7给药组和AB23P10给药组。使用0.9％的生理盐水稀释样品，给药方式为蠕动泵静脉滴注20ml/kg/h，分别于第1天(D1)、第4天(D4)、第8天(D8)和第11天(D11)给药，共给药4次，药物剂量按照下表4-1。同时每周对动物体重进行称量。给药浓度和体积如表4-1所示。
表4-1：食蟹猴急性毒性评价给药方案
Figure PCTCN2019114808-appb-000013
如表4-2所示，开始给药后，两只雌性食蟹猴出现粪便软化，其中AB23P7给药组频率较高并出现水样粪便，AB23P10在第一次给药后马上恢复正常。观察到的其他副作用，如厌食、呕吐在发生后第二天恢复正常。
表4-2：食蟹猴的日常观察
Figure PCTCN2019114808-appb-000014
备注：F4：粪便软化；F5：水样粪便；K2：厌食；F11：呕吐；空白：无副作用。
食蟹猴的体温变化如表4-3所示，每次给药的当天监测动物的体温，可以看到各组体温均无太大变化，且在正常生理值范围内波动。
表4-3：食蟹猴的体温监测(℃)
Figure PCTCN2019114808-appb-000015
如表4-4所示，测试动物的体重整体上变化不大，相对于给药之前4天(记作D-4)体重，仅AB23P7给药组雄性食蟹猴体重下降较为明显，但体重减轻仍在10％之内，停药后体重停止下降。
表4-4：食蟹猴的体重监测(Kg)
Figure PCTCN2019114808-appb-000016
食蟹猴在给药后，取血收集血清，测定血清中细胞因子的含量。结果如图4-1A～4-1F所示。在第一次给药后，IL-2、IL-6和IFN-γ都有升高，但是在24h内恢复正常。随后再次给药，虽然剂量逐渐增加，然而在食蟹猴免疫系统适应后，细胞因子仍然维持在正常水平。说明食蟹猴对测试药物的耐受有一个从低到高的适应过程。本实验过程中观察到的不同程度的腹泻，是免疫药物治疗中常见的副作用，在停药后即恢复正常。在高达1mg/kg的给药剂量下，食蟹猴未出现严重的毒副作用，表明两个候选药物都有很好的耐受。体内药效实验结果显示，低剂量的AB23P7和AB23P10表现出很好的抗肿瘤效果，表明候选药物的治疗窗口较宽，安全性较高。
4.2、双特异性抗体在食蟹猴体内的药代动力学实验
食蟹猴(广州相观生物科技有限公司)分成两个组，每组雌雄各一只，体重在3-4kg。第一组为AB23P7给药组；第二组为AB23P10给药组，给药剂量为50μg/kg；采血时间点分别为30min、1h、3h、6h、8h、24h、48h、72h，共8个时间点。取血收集血清，-80℃冻存，然后采用ELISA方法测定血清中的药品浓度。
用人CD19抗原(Acrobiosystems，CD9-H5258)包板，包板浓度为0.5μg/mL。分别将AB23P7、AB23P10按照100ng/mL、50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.125ng/mL、1.56ng/mL配置并建立标准曲线。用HRP标记羊抗人IgG抗体，检测使用浓度为1：5000，最后用TMB显色。利用PKSolver软件计算药代动力学参数，具体参数见表4-5。
结果显示，AB23P7在食蟹猴中的半衰期为15.85±5.3h，AB23P10在食蟹猴中的半衰期为15.9±1.8h，两个双特异性抗体在食蟹猴中表现出一致的半衰期。
表4-5：双抗在食蟹猴体内的药代动力学参数
Figure PCTCN2019114808-appb-000017
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (42)

  1. 一种双特异性抗体，所述双特异性抗体分子由两条相同的多肽链以共价键结合形成四价同源二聚体，每条多肽链从N端至C端依次包含特异性结合肿瘤相关抗原CD19的第一单链Fv、特异性结合效应细胞抗原CD3的第二单链Fv和Fc片段；其中，第一和第二单链Fv通过连接肽相连，而第二单链Fv与Fc片段直接相连或通过连接肽相连，且所述Fc片段不具有CDC、ADCC和ADCP等效应子功能。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述第一单链Fv所包含的VH结构域和VL结构域通过连接肽连接，且所述连接肽的氨基酸序列为(GGGGX)n，X包含Ser或Ala，X优选Ser；n为1-5的自然数，n优选3。
  3. 如权利要求1所述的双特异性抗体，其特征在于，所述肿瘤相关抗原为CD19，包含但不限于：CD19的任何变体、同工型、衍生物和物种同源物；优选地，CD19来源于人、食蟹猴或恒河猴。
  4. 如权利要求1所述的双特异性抗体，其特征在于，所述第一单链Fv特异性结合CD19，其包含的CDR1、CDR2和CDR3选自下组：
    (i)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：1、2和3所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：4、5和6所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (ii)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：9、10和11所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：12、13和14所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (iii)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：20、13和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (iv)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：24、25和26所示，或与上述 序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：27、28和29所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (v)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：32、33和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
  5. 如权利要求1或4所述的双特异性抗体，其特征在于，所述第一单链Fv特异性结合CD19，其选自下组：
    (i)VH结构域包含如SEQ ID NO：7所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：8所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (ii)VH结构域包含如SEQ ID NO：15所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：16所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (iii)VH结构域包含如SEQ ID NO：22所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：23所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (iv)VH结构域包含如SEQ ID NO：30所示的氨基酸序列，或与上述序列中的任何基本上相同(例如 至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：31所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (v)VH结构域包含如SEQ ID NO：34所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：35所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (vi)VH结构域包含如SEQ ID NO：36所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：37所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (vii)VH结构域包含如SEQ ID NO：38所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：39所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
  6. 一种能够特异性结合CD19的单克隆抗体或其抗原结合片段，其包含如权利要求4所述双特异性抗体第一单链Fv所包含的CDR1、CDR2和CDR3，其选自下组：
    (i)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：32、33和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (ii)VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：17、18和19所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的 或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：20、13和21所示，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    优选地，所述单克隆抗体是人源化抗体；
    优选地，所述抗原结合片段选自scFv、Fab、Fab’、(Fab’)2、Fv片段、二硫键连接的Fv(dsFv)。
  7. 如权利要求6所述的单克隆抗体或其抗原结合片段，其所包含的VH结构域和VL结构域选自下组：
    (i)VH结构域包含如SEQ ID NO：36所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：37所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；
    (ii)VH结构域包含如SEQ ID NO：38所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：39所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
  8. 如权利要求1所述的双特异性抗体，其特征在于，所述第二单链Fv所包含的VH结构域和VL结构域通过连接肽连接，且所述连接肽的氨基酸序列为(GGGGX)n，X包含Ser或Ala，X优选Ser；n为1-5的自然数，n优选3。
  9. 如权利要求1或8所述的双特异性抗体，其特征在于，所述第二单链Fv在体外结合亲和力分析中以大于约10nM，或大于20nM，或大于40nM，或大于约50nM的EC 50值结合于效应细胞；更优选地，所述双特异性抗体的第二单链Fv不仅能与人CD3结合，还可与食蟹猴或恒河猴的CD3特异性结合。
  10. 如权利要求9所述的双特异性抗体，其特征在于，所述第二单链Fv特异性结合CD3，其VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：40、41和42所示，或与上述序列至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代的序列；和其VL结构域包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：43、44和45所示，或与上述序列至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代的序列。
  11. 如权利要求10所述的双特异性抗体，其特征在于，所述第二单链Fv特异性结合CD3，其VH结构域 包含如SEQ ID NO：46所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列；和其VL结构域包含如SEQ ID NO：47所示的氨基酸序列，或与上述序列中的任何基本上相同(例如至少80％、85％、90％、92％、95％、97％、98％、99％或更高度相似的或具有一个或更多个氨基酸取代(例如保守性取代))的序列。
  12. 如权利要求1所述的双特异性抗体，其特征在于，所述连接第一单链Fv和第二单链Fv的连接肽由柔性肽和刚性肽组成；且所述柔性肽包含2个或更多个氨基酸，并优选自下列几种氨基酸：Gly(G)、Ser(S)、Ala(A)和Thr(T)；更优地，所述柔性肽包含G和S残基；最优地，所述柔性肽的氨基酸组成结构通式为GxSy(GGGGS)z，其中x，y和z是大于或等于0的整数，且x+y+z≥1；所述刚性肽来自天然人绒毛膜促性腺激素β亚基羧基末端第118至145位氨基酸组成的全长序列或其截短的片段；优选地，所述刚性肽包含SSSSKAPPPS。
  13. 如权利要求12所述的双特异性抗体，其特征在于，所述连接肽包含如SEQ ID NO：49所示的氨基酸序列。
  14. 如权利要求1所述的双特异性抗体，其特征在于，连接所述Fc片段与第二单链Fv的连接肽包含1-20个氨基酸，并优选自下列几种氨基酸：Gly(G)、Ser(S)、Ala(A)和Thr(T)；较优选自Gly(G)和Ser(S)；更优选地，所述连接肽组成为(GGGGS)n，n＝1，2，3或4。
  15. 如权利要求1或14所述的双特异性抗体，其特征在于，所述Fc片段包含来源于人免疫球蛋白重链恒定区的铰链区、CH2和CH3结构域；较优地，Fc片段选自人IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；较优地，Fc片段选自人IgG1、IgG2、IgG3和IgG4的重链恒定区；更优地，Fc片段选自人IgG1或IgG4的重链恒定区；并且，所述Fc片段与其所源自的天然序列相比具有一个或多个氨基酸的置换、缺失或添加。
  16. 如权利要求15所述的双特异性抗体，其特征在于，所述Fc片段包含具有降低或消除的效应子功能(ADCP、ADCC和CDC效应)的氨基酸置换、缺失或添加。
  17. 如权利要求16所述的双特异性抗体，其特征在于，所述Fc片段包含根据EU编号系统确定的L234A/L235A/P331S的氨基酸置换。
  18. 如权利要求16或17所述的双特异性抗体，其特征在于，所述Fc片段还包含具有以下一种或多种性质的氨基酸的置换、缺失或添加：
    (i)与新生儿受体(FcRn)的结合亲和力增强；
    (ii)降低或消除的糖基化；
    (iii)降低或消除的电荷异质性。
  19. 如权利要求18所述的双特异性抗体，其特征在于，所述Fc片段还包含以下一个或多个氨基酸的置换、缺失或添加：
    (i)根据EU编号系统确定的M428L、T250Q/M428L、M428L/N434S或M252Y/S254T/T256E的氨基酸置换；
    (ii)根据EU编号系统确定的N297A的氨基酸置换；
    (iii)根据EU编号系统确定的K447的氨基酸缺失。
  20. 如权利要求18所述的双特异性抗体，其特征在于，所述Fc片段的氨基酸序列如SEQ ID NO：54所示，它与其所源自的天然序列相比具有根据EU编号系统确定的以下6个氨基酸的置换或取代：L234A/L235A/N297A/P331S/T250Q/M428L；且缺失或删除了根据EU编号系统确定的K447。
  21. 如权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体结合人CD19和CD3，其氨基酸序列包含下组：
    (a)组：
    (i)SEQ ID NO：55所示的序列；
    (ii)与SEQ ID NO：55示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：55所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    (b)组：
    (i)SEQ ID NO：57所示的序列；
    (ii)与SEQ ID NO：57所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：57所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    (c)组：
    (i)SEQ ID NO：59所示的序列；
    (ii)与SEQ ID NO：59所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4 个或5个置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：59所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    (d)组：
    (i)SEQ ID NO：61所示的序列；
    (ii)与SEQ ID NO：61所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：61所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
  22. 编码如权利要求1-5、8-21任一项所述双特异性抗体的DNA分子。
  23. 如权利要求22所述的DNA分子具有如SEQ ID NO：56、58、60或62所示的核苷酸序列。
  24. 包含如权利要求22或23所述DNA分子的载体。
  25. 包含如权利要求24所述载体的宿主细胞；所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，优选为CHO细胞。
  26. 一种药物组合物，所述组合物包含如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列以及药学上可接受的赋形剂、载体或稀释剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是用于治疗免疫相关疾病的药物；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物；
    优选地，所述另外的药学活性剂是用于治疗自身免疫性疾病或炎症性疾病的药物；
    优选地，所述另外的药学活性剂是用于治疗移植排异反应相关疾病或病症的药物；
    优选地，所述抗体与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
  27. 如权利要求26所述的药物组合物还包括向受试者施用所述药物组合物之前、之后或同时施用药学活性剂，其中所述药学活性剂选自抗体、抗体片段、药物、酶、细胞毒性剂、毒素、抗生素、激素、免疫调节剂、细胞因子、趋化因子和放射性同位素。
  28. 如权利要求27所述的药物组合物，其中所述药物选自由以下组成的组：环磷酰胺、尼莫司汀、氨甲蝶呤、氟尿嘧啶、卡培他滨、吉西他滨、替吉奥、培美曲塞、氟达拉滨、阿霉素、博来霉素、长春瑞滨、 紫杉醇、多西他赛、伊利替康、他莫昔芬、来曲唑、依西美坦、氟维司群、戈舍瑞林、甲羟孕酮、顺铂、卡铂、草酸铂、奈达铂、奥沙利铂、门冬酰胺酶、洛铂、依托泊苷、长春新碱、伊立替康、替加氟、达卡巴嗪、丝裂霉素、替尼泊苷、吡柔比星、米托蒽醌、长春地辛、雷替曲塞、甲氨蝶呤、顺铂博来霉素硫酸盐、亚硝基脲氮芥、苯丁酸氮芥、环磷酰胺羟脲和道诺霉素。
  29. 如权利要求27所述的药物组合物，其中所述毒素选自由以下组成的组：篦麻毒素、相思子毒素、α毒素、皂素、核糖核酸酶(RNA酶)、DNA酶I、葡萄球菌肠毒素-A、美洲商陆抗病毒蛋白、白树毒素、白喉毒素、假单胞菌外毒素和假单胞菌内毒素。
  30. 如权利要求27所述的药物组合物，其中所述免疫调节剂选自由以下组成的组：细胞因子、趋化因子、干细胞生长因子、淋巴毒素、集落刺激因子(CSF)、白细胞介素(IL)、红细胞生成素、血小板生长因子、肿瘤坏死因子(TNF)、粒细胞-集落刺激因子(G-CSF)、粒细胞巨噬细胞-集落刺激因子(GM-CSF)、干扰素-α、干扰素-β、干扰素-γ或干扰素-λ、TGF-α、TGF-β、白细胞介素-1(IL-1)、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12；IL-13、IL-14、IL-15、IL-16、IL-17、IL-18、IL-21、IL-23、IL-25、LIF、FLT-3、血管内皮生长因子、血小板反应蛋白和内皮抑制素。
  31. 如权利要求26-30任一项所述的药物组合物，其特征在于施用一种或多种额外的其他疗法，其中其他疗法选自下组：手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。
  32. 一种用于增强或刺激免疫应答或功能的方法，其包含对所述个体施用治疗有效量的如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列或如权利要求26-30任一项所述的药物组合物。
  33. 一种用于治疗、预防或改善细胞、组织、器官或动物中的免疫病症或疾病的方法，其包括对所述个体施用治疗有效量的如权利要求1-5、8-21任一项所述的双特异性抗体、或如权利要求22所述的DNA、或如权力要求23所述的核苷酸序列或如权利要求26-30任一项所述的药物组合物。
  34. 一种用于预防/治疗疾病、延迟其进展、降低/抑制其复发的方法，所述疾病包含免疫相关疾病、肿瘤、自身免疫性疾病、炎症性疾病或移植排异反应相关疾病或病症等疾病，其包括将有效量的如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列或如权利要求26-30任一项所述的药物组合物给予或施用至所述患有以上疾病或病症的个体。
  35. 如权利要求32-34任一项所述的方法，其特征在于施用一种或多种额外的其他疗法，其中其他疗法选自下组：手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。
  36. 制备如权利要求1-5、8-21任一项所述双特异性抗体的方法，其包括：(a)获得双特异性抗体的融合基因，构建双特异性抗体的表达载体；(b)通过基因工程方法将上述表达载体转染到宿主细胞中；(c)在允许产生所述双特异性抗体的条件下培养上述宿主细胞；(d)分离、纯化产生的所述抗体；
    其中，步骤(a)中所述表达载体选自质粒、细菌和病毒中的一种或多种，优选地，所述表达载体为pCDNA3.4载体；
    其中，步骤(b)通过基因工程方法将所构建的载体转染入宿主细胞中，所述宿主细胞包含原核细胞、酵母或哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，优选为CHO细胞；
    其中，步骤(d)通过常规的免疫球蛋白纯化方法，包含蛋白质A亲和层析和离子交换、疏水层析或分子筛方法分离、纯化所述双特异性抗体。
  37. 如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列在制备药物中的用途，所述药物用于：
    (1)在受试者(例如人)中治疗免疫相关疾病；
    (2)在受试者(例如人)中治疗肿瘤；
    (3)在受试者(例如人)中治疗自身免疫性疾病或炎症性疾病；
    (4)在受试者(例如人)中治疗移植排异反应相关疾病或病症。
  38. 如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列在制备治疗、预防或缓解肿瘤的药物中的用途，所述癌症包括：急性骨髓性白血病(AML)、慢性骨髓性白血病(CML)、急性B淋巴细胞性白血病(B-ALL)、慢性B淋巴细胞白血病(B-CLL)、B细胞淋巴瘤(BCL)、T细胞淋巴瘤(TCL)(例如皮肤)、骨髓增生异常综合征(MDS)、小淋巴细胞淋巴瘤(SLL)、毛细胞白血病(HCL)、边缘区淋巴瘤(MZL)(例如结外或脾)、滤泡性淋巴瘤(FL)(例如儿童型或胃肠道)、幼B淋巴细胞白血病(B-PLL)、套细胞淋巴瘤(MCL)、淋巴浆细胞淋巴瘤(LPL)/瓦尔登斯特伦巨球蛋白血症(WM)、淋巴母细胞白血病(ALL)(例如B细胞)、淋巴母细胞淋巴瘤(LBL)(例如B细胞)、浆母细胞性淋巴瘤(PBL)(例如B细胞)、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫大B细胞淋巴瘤(DLBCL)(例如，原发部位或炎症相关)、伯基特淋巴瘤(BL)、多发性骨髓瘤、间变性大细胞淋巴瘤和HIV相关淋巴瘤。
  39. 如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列在制备治疗、预防或缓解自身免疫性疾病或炎症性疾病的药物中的用途，所述自身免疫性或炎症性疾病选自：类风湿性关节炎(RA)、骨关节炎、反应性关节炎、系统性红斑狼疮(SLE)、克罗恩氏病、多发性硬化症、硬皮病、牛皮癣、牛皮癣关节病、溃疡性结肠炎(例如，慢性)、胰岛素依赖性 糖尿病(例如，青少年)、甲状腺炎(例如，慢性)、甲状腺机能亢进、哮喘、变态反应性疾病、结节病、自身免疫溶血性贫血、恶性贫血、移植物抗宿主病、皮肌炎、慢性肝炎、微观肾脉管炎、慢性活动性肝炎、葡萄膜炎、肠滑膜炎、自身免疫肠疾病、特发性白细胞减少症、自身免疫性肾小球肾炎、自身免疫溶血性贫血、自身免疫性肝炎、间质肺炎、慢性天疱疮、寻常型天疱疮、动脉炎、结节性多动脉炎和强直性脊柱炎。
  40. 如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列在制备治疗、预防或缓解移植排异反应相关疾病或病症的药物中的用途，所述移植排异反应包括急性、超急性或慢性移植排异反应，所述移植排异包括器官、组织或细胞移植排异，包括但不限于输血、血管移植、上皮移植、内皮移植、肌肉移植、结缔组织移植、关节移植、心脏移植、肺移植、肝移植、肾移植、胰腺移植、皮肤移植、肠移植、角膜移植、骨移植、骨髓移植、移植物抗宿主病或宿主抗移植物病等。
  41. 检测哺乳动物中癌症的存在的方法，其包括：
    (a)将包含一个或多个源自哺乳动物的细胞的样品与如权利要求1-5、8-21任一项所述的双特异性抗体、或如权力要求22所述的DNA、或如权力要求23所述的核苷酸序列接触，从而形成复合物，和；
    (b)检测所述复合物，其中复合物的检测表明哺乳动物中癌症的存在。
  42. 一种试剂盒，其含有如权利要求1-5、8-21任一项所述的双特异性抗体、或如权利要求22所述的DNA、或如权力要求23所述的核苷酸序列。
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