WO2020088403A1 - 针对Her2和CD3的同源二聚体型双特异性抗体及其用途 - Google Patents

针对Her2和CD3的同源二聚体型双特异性抗体及其用途 Download PDF

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WO2020088403A1
WO2020088403A1 PCT/CN2019/113671 CN2019113671W WO2020088403A1 WO 2020088403 A1 WO2020088403 A1 WO 2020088403A1 CN 2019113671 W CN2019113671 W CN 2019113671W WO 2020088403 A1 WO2020088403 A1 WO 2020088403A1
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amino acid
bispecific antibody
antibody
cells
cancer
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PCT/CN2019/113671
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French (fr)
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李强
贾世香
马心鲁
严源
张玉华
李媛丽
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安源医药科技（上海）有限公司
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Priority to CN201980071433.7A priority Critical patent/CN112955461B/zh
Priority to US17/290,728 priority patent/US20230073411A1/en
Priority to EP19879228.5A priority patent/EP3889174A1/en
Publication of WO2020088403A1 publication Critical patent/WO2020088403A1/zh

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Definitions

  • the present invention relates to the field of immunology, and more specifically, to bispecific antibodies against human epidermal growth factor receptor 2 (Her2) and cluster determinant 3 (CD3), and the use of such antibodies, especially in the treatment of Uses in tumors.
  • Her2 human epidermal growth factor receptor 2
  • CD3 cluster determinant 3
  • T cells T cells to kill tumor cells.
  • the first signal comes from the binding of the MHC-antigen complex on the antigen-presenting cells to the T cell receptor TCR-CD3, and the second signal is the mutual stimulation of the co-stimulatory molecules expressed by the T cells and the antigen-presenting cells.
  • Non-antigen-specific costimulatory signal generated after the action. Because the expression of MHC on the surface of most tumor cells is down-regulated or even missing, tumor cells escape immune killing.
  • Bispecific antibodies can be divided into double-signal blocking type and cell-mediated function type from the mechanism of action.
  • cell-functional bispecific antibodies refer to anti-CD3 bispecific antibodies that mediate T cell killing.
  • the CD3 molecule is expressed on the surface of all mature T cells and non-covalently binds to the TCR to form a complete TCR-CD3 complex. Together, it participates in the immune response to antigen stimulation. It is currently the most widely used in bispecific antibodies. Trigger molecules on the surface of successful immune effector cells.
  • Bispecific antibodies targeting CD3 can respectively bind CD3 on the surface of T cells and antigens on the surface of tumor cells, thereby shortening the distance between cytotoxic T cells (Tc or CTL) and tumor cells, and directly activating T cells.
  • Tc or CTL cytotoxic T cells
  • T cells Induce T cells to directly kill cancer cells without relying on the dual activation signals of traditional T cells.
  • the agonistic antibody targeting T cell antigen CD3 for example, the first generation of clinical monoclonal antibody OKT3 targeting human CD3 (Kung P et al., Science, 206: 347-349,1979)
  • T cells are excessively activated to release a large number of inflammatory factors, such as interleukin-2 (IL-2), TNF- ⁇ , IFN- ⁇ and interleukin-6 (IL-6), which can cause serious "cytokine storm synthesis Syndrome ”(Hirsch R et al., J.
  • bispecific antibodies In recent years, in order to solve the problem of correct assembly of two different half antibodies, scientists have designed and developed bispecific antibodies with various structures. In summary, there are two major categories.
  • One type of bispecific antibody does not contain an Fc region.
  • the advantage of this type of structured double antibody is that it has a small molecular weight and can be expressed in prokaryotic cells, without the need to consider the problem of correct assembly; the disadvantage is that because there is no antibody Fc segment, the molecular weight is low, resulting in a short half-life, and this form of double antibody It is extremely easy to polymerize, has poor stability and low expression, so its clinical application is limited.
  • Such bispecific antibodies that have been reported so far include BiTE, DART, TrandAbs, bi-Nanobody and so on.
  • bispecific antibodies retains the Fc domain.
  • Such double antibodies form an IgG-like structure with a larger molecular structure, and the endocytosis and recycling processes mediated by FcRn have a longer half-life; while retaining some or all of the effector functions mediated by Fc, Such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cell phagocytosis (ADCP).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cell phagocytosis
  • Such bispecific antibodies that have been reported so far include Triomabs, kih IgG, Cross-mab, orthoFab IgG, DVD IgG, IgG scFv, scFv 2 -Fc and so on.
  • anti-CD3 bispecific antibodies except for TandAb and scFv-Fv-scFv configurations, the design of other anti-CD3 bispecific antibodies widely uses monovalent anti-CD3 forms, mainly because bivalent anti-CD3 bispecific antibodies are easy It leads to excessive activation and induces T cell apoptosis and the instantaneous release of a large number of cytokines (Kuhn C et al., Immunotherapy, 8: 889-906, 2016), and more seriously, it may also trigger the activation of T cells in an antigen-independent manner. Break the immune balance. Therefore, the anti-CD3 bispecific antibodies in the prior art mostly avoid the introduction of bivalent anti-CD3 antibodies.
  • the bispecific antibodies of triFab-Fc, DART-Fc and BiTE-Fc configurations all adopt an asymmetric design (i.e. different Source dimer type double antibody) (Z Wu et al., Pharmacology and Therapeutics, 182: 161-175, 2018), but this brings many challenges to its downstream production, such as the production of undesirable homodimers or faults
  • the matching impurity molecules increase the difficulty of double antibody expression and purification.
  • knock-into-holes technology solves the problem of heavy chain mismatch between heterodimeric diabody molecules to a certain extent, "light chain / heavy chain mismatch” brings another challenge. .
  • One strategy to prevent heavy-light chain mismatch is to exchange the light chain of one of the Fab of the bispecific antibody and the partial domain of the heavy chain to form a Crossmab (hybrid antibody).
  • This method can allow light chain / heavy chain Selective pairing between chains.
  • the disadvantage of these methods is that the generation of mismatch products cannot be completely eliminated, and the residual fractions of any mismatch molecules are difficult to separate from the products, and this method requires a large number of genetic engineering modifications such as mutations for the two antibody sequences. , Can not achieve simple and universal purpose.
  • T cells due to their Fc ⁇ R binding ability, it may lead to unlimited and continuous activation of T cells, and this activation is non-target cell-restricted, whether or not Antigen binding, in tissues expressing Fc ⁇ R (for example, in the hematopoietic, lymphatic, and reticuloendothelial systems), activated T cells are found.
  • This systemic activation of T cells will be accompanied by a large amount of cytokine release, which is a serious adverse reaction during the therapeutic application of T cell activation cytokines or antibodies.
  • the epidermal growth factor receptor family plays an important role in the process of cell growth, development and differentiation, and its overexpression will lead to normal cell dysfunction, often closely related to the occurrence and development of tumors.
  • human epidermal growth factor receptor 2 human epidermal growth factor receptor 2, Her2 / ErbB2
  • Her2 / ErbB2 human epidermal growth factor receptor 2
  • Her2 / ErbB2 is closely related to the degree of malignancy of many epithelial cell carcinomas, and Her2-overexpressing tumors show extremely strong migration and infiltration , Poor sensitivity of chemotherapy drugs, poor recovery, easy to relapse and so on.
  • the expression level of Her2 is extremely low in normal cells, but the expression level is very high during embryonic development.
  • trastuzumab has become the standard treatment for Her-2 positive operable breast cancer (Romond EH et al., N Engl J Med, 2005, 353 (16): 1673-1684; Perez EA et al., J Clin Oncol, 2011,29 (34): 4491-4497).
  • trastuzumab has synergistic effects with platinum, docetaxel, and vinorelbine, with doxorubicin, paclitaxel, and cyclic Phosphoramide has an additive effect. Because Her2 is expressed in a variety of tissues and organs, the side effects of trastuzumab have also attracted attention.
  • CN104558192A discloses an anti-Her2 ⁇ CD3 bispecific antibody with the MSBODY (monomer and ScFv-Fc bispecific antibody) configuration, whose monovalent unit variable region sequence is derived from Containing Fab and Fc domains, the single chain unit is in the form of anti-CD3 ScFv-Fc, although the heavy chain Fc of the monovalent unit and the Fc region of the single chain unit have been modified to make it difficult for them to form homodimers, It is easy to form heterodimers. Inevitably, a small amount of by-products of homodimeric antibodies formed due to mismatch between heavy chains will still be produced, which increases the difficulty of purification and separation. In addition, its anti-CD3 single chain antibody fuses the heavy chain Fc of human IgG1 and retains FcR binding capacity, which will inevitably increase its risk of inducing cytokine storms.
  • bispecific antibodies against Her2 ⁇ CD3 dual targets have been published, their safety and effectiveness still need to be improved, such as the use of humanized antibodies to avoid HAMA reactions, while improving CD3 antibody molecules to weaken their induced cells The risk of factor storms.
  • bifunctional antibody formed by combining the antibody against Her2 with the anti-CD3 antibody has the expected biological function and can effectively kill breast cancer and other related tumor cells.
  • two functional domains are required to properly bind to their binding sites. Even if each antibody can correctly bind to its antigen alone, it cannot be guaranteed that it will retain the original binding capacity after forming the bispecific antibody, and due to the steric hindrance, it cannot be guaranteed that the bispecific antibody can be successfully formed. Synergistically.
  • the purpose of the present invention is to provide a tetravalent, homodimeric bispecific antibody molecule targeting immune effector cell antigens CD3 and Her2.
  • This bispecific antibody can significantly inhibit or kill tumor cells in vivo, but it The non-specific killing effect of normal cells expressing Her2 is significantly reduced, and at the same time it has controlled 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 is provided.
  • the bispecific antibody molecule is composed of two identical polypeptide chains covalently bonded to form a tetravalent homodimer, each polypeptide chain extending from the N-terminus to The C-terminal includes a first single-chain Fv (anti-Her2scFv) that specifically binds Her2, a second single-chain Fv (anti-CD3 scFv) that specifically binds to the effector cell antigen CD3, and an Fc fragment;
  • the single-chain Fv is connected by a connecting peptide, while the second single-chain Fv is directly connected to the 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 Her2, and the VH domain and the VL domain contained in it are connected by a linking peptide (L1), and the VH, L1 and VL are VH-L1-VL or VL-L1-
  • the sequence of 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 from 1-5, and n is preferably 3.
  • Table 5-9 of the present invention exemplifies the amino acid sequences of some preferred anti-Her2scFv VH domains and their complementarity determining regions (HCDR1, HCDR2 and HCDR3), and VL domains and their complementarity determining regions (LCDR1, LCDR2 and LCDR3) amino acid sequence.
  • the complementarity determining regions (HCDR1, HCDR2, and HCDR3) of the VH domain and the complementarity determining regions of the VL domain (LCDR1, LCDR2, and LCDR3) included in the first single-chain Fv of Her2 are selected from the group consisting of:
  • the 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, 21 and 22, 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: 25, 26, and 27, 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: 28, 29 and 30, 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).
  • VH domain and VL domain included in the first single-chain Fv for Her2 are selected from the group consisting of:
  • 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: 23, 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: 24, 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: 31, 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: 32, 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: 49) or a truncated fragment thereof (hereinafter collectively referred to as For CTP).
  • the CTP rigid peptide comprises 10 amino acids at the N-terminal of SEQ ID NO: 49, namely SSSSKAPPPS (CTP 1 ); or the CTP rigid peptide comprises 14 amino acids at the C-terminal of SEQ ID NO: 49, namely SRLPGPSDTPILPQ (CTP 2 );
  • the CTP rigid peptide includes 16 amino acids at the N-terminus of SEQ ID NO: 49, 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 5-11 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: 50, 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 50 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 nM in an in vitro FACS binding analysis assay; more preferably
  • 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.
  • the bispecific antibody specifically binds to effector cells with an EC 50 value of 132.3 nM.
  • Table 5-10 of the present invention exemplifies the amino acid sequences of some preferred anti-CD3 scFv VH domains and their complementarity determining regions (HCDR1, HCDR2 and HCDR3), and VL domains and their complementarity determining The amino acid sequence of the regions (LCDR1, LCDR2 and LCDR3).
  • 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: 33, 34 and 35, 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: 36, 37, and 38, 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 contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 41, 42 and 43, respectively, 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 (eg conservative substitutions)) sequences;
  • the LCDR1, LCDR2, and LCDR3 contained in the VL domain are shown in SEQ ID NO: 44, 45, and 46, 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 its VH domain contains the amino acid sequence shown in SEQ ID NO: 39, 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 40, 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 second single-chain Fv specifically binds to CD3, and the VH domain thereof includes the amino acid sequence shown in SEQ ID NO: 47, 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 48, 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 simultaneously modified, for example, L234A and L235A (L234A / L235A) (according to EU numbering, the amino acid sequence is shown in SEQ ID NO: 54).
  • 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 CH2 of the IgG molecule, thereby losing the binding ability to 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. A certain percentage of amino acids may be lost during antibody production, resulting in charge heterogeneity, which may affect 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 5-12 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: 55, 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 Her2 and CD3, and its amino acid sequence is as follows:
  • sequence shown in SEQ ID NO: 8 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%, A sequence of 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-mentioned bispecific antibody is provided.
  • the DNA molecule encoding the bispecific antibody is represented by the nucleotide sequence shown in SEQ ID NO: 56.
  • 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, NS0 cells or other mammalian cells, preferably CHO cells;
  • a pharmaceutical composition comprising the above bispecific antibody and a pharmaceutically acceptable excipient, carrier or diluent.
  • a preferred embodiment of the present invention discloses a solution preparation containing the above-mentioned bispecific antibody stabilization, the solution preparation further includes a pH adjuster, a stabilizer and a surfactant; preferably, the pH adjustment
  • the agent is citrate buffer or histidine buffer, and the stabilizer is sucrose, and the surfactant is Tween-80; more preferably, the formulation contains 0.5 mg / mL of the above double Specific antibodies, and 20 mM citrate or histidine and 8% sucrose (w / v) and 0.02% PS80 (w / v); the pH of the formulation is 5.5.
  • the sixth aspect of the present invention also provides a method for preparing the bispecific antibody of the present invention, which includes: (a) obtaining a fusion gene of the bispecific antibody and constructing an expression vector of the bispecific antibody; (b) by Genetic engineering methods transfect the above expression vector into host cells; (c) cultivate the above host cells under conditions allowing the production of the bispecific antibody; (d) isolate and purify the produced antibodies.
  • the expression vector in step (a) is selected from one or more of plasmids, bacteria and viruses, preferably, 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 seventh aspect of the present invention provides the use of the bispecific antibody in the preparation of drugs for treating, preventing or alleviating tumors;
  • the tumors include but are not limited to breast cancer, prostate cancer, non-small cell lung cancer, bladder Cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, and soft tissue cancer; preferably, the tumor is selected from breast Cancer, gastric cancer, rectal cancer; more preferably, the tumor is selected from breast cancer.
  • a method for treating a tumor, delaying its progress, and reducing / suppressing its recurrence which comprises administering or administering an effective amount of the bispecific antibody or the pharmaceutical composition to the tumor
  • the tumors include but are not limited to breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, head and neck scales Squamous cell carcinoma, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, and soft tissue cancer; preferably, the tumor is selected from breast cancer, gastric cancer, and rectal cancer; more preferably, the tumor is selected from breast cancer.
  • a method for enhancing or stimulating an immune response or function which comprises administering to a patient / subject individual a therapeutically effective amount of the bispecific antibody or the pharmaceutical composition.
  • a method for treating, preventing or ameliorating an immune disorder or disease in a patient / subject individual which comprises administering to the individual a therapeutically effective amount of the bispecific antibody or The pharmaceutical composition.
  • the anti-Her2scFv contained in the bispecific antibody provided by the present invention is located at the N-terminus of the double antibody, the spatial conformation changes, and the binding ability to Her2 may be weakened under certain conditions, and it is particularly difficult to bind weak or low expression Her2 Of normal cells, the non-specific killing is reduced, but the binding specificity of the cells overexpressing or overexpressing Her2 is not significantly reduced, showing a good in vivo killing effect.
  • 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 maximum safe starting dose of the preclinical toxicology evaluation test has been much higher than other doses at the same target, and no systemic immunotoxicity has occurred, suggesting that the bispecific antibody provided by the present invention has a wider safety window for medication.
  • 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 about 50 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 nM).
  • the CTP rigid peptide contained in the N-terminal linker peptide L3 and the Fc fragment located at the C-terminal of the anti-TAA scFv and Fc are partially "covered” or “shielded” by the anti-CD3 scFv antigen
  • the binding domain this steric hindrance effect makes it bind to CD3 with weaker binding affinity (eg, greater than 1 ⁇ M), which weakens the activation stimulation ability of T cells, thus limiting the excessive release of cytokines, and thus has more High safety;
  • the anti-CD3 scFv used in the present invention can simultaneously bind to the CD3 natural antigens of human and cynomolgus monkeys and / or rhesus monkeys, so that the preclinical toxicology evaluation does not require the construction of replacement molecules, and the obtained
  • the effective dose, toxic dose and toxic side effects are more objective and accurate, and the clinical dose can be directly converted to reduce the risk of clinical research.
  • 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 uncontrollably overactivated, so anti-CD3 bispecific antibodies are designed Generally avoid introducing divalent anti-CD3 structure.
  • 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 circulation half-life in vivo due to the inclusion of Fc fragments. Pharmacokinetic tests have shown that the circulation half-life in mice and cynomolgus monkeys is about 40h and 8h respectively, which will greatly Reduce its frequency of clinical administration.
  • 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.
  • Her2 human epidermal growth factor receptor 2
  • Her2 human epidermal growth factor receptor 2
  • the upregulation of her2 can also activate two downstream signal transduction pathways, triggering a cascading chain reaction, promoting unlimited cell proliferation, and ultimately leading to cancer.
  • her2 can initiate a variety of metastasis-related mechanisms to increase the metastatic capacity of tumor cells.
  • the amplification or overexpression of the Her2 gene occurs in various tumors such as breast cancer, ovarian cancer, gastric cancer, lung adenocarcinoma, prostate cancer, aggressive uterine cancer, and the like.
  • the indications for the Her2 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 homologues of Her2, which are expressed naturally by cells-including tumor cells-or by cells transfected with Her2 gene or cDNA.
  • Species homologues include rhesus monkey Her2.
  • CD3 refers to being a part of the T cell receptor complex, consisting of three different chains CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • CD3 produces clustering on T cells by, for example, the immobilization of anti-CD3 antibodies on it, resulting in T cell activation, which is 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 CD3, a receptor on the surface of T cells, is not particularly limited as long as it can specifically recognize CD3.
  • the anti-human CD3 antibody used in the present invention is cross-reactive with cynomolgus monkey and / or rhesus monkey.
  • 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 of the 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, it is well within the ordinary skill of those in the art to determine the numbering scheme for any variable region sequence in the sequence list.
  • 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.
  • dsFv disulfide bond stable protein
  • antibody fragment refers to an antigen-binding fragment and antibody analog of an antibody that retains the specific binding ability to an antigen (eg, Her2), 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.
  • light chain constant region includes the amino acid sequence from the light chain of an antibody.
  • the light chain constant region includes at least one of a constant kappa domain and a constant lambda domain.
  • 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.
  • Binding defines the affinity interaction between a specific epitope on an antigen and its corresponding antibody, and is generally understood as “specific recognition”.
  • Specific recognition means that the bispecific antibody of the invention does not cross-react with any polypeptide other than the target antigen.
  • the degree of specificity can be judged by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry, etc.
  • the specific recognition is preferably determined by flow cytometry, and the standard of specific recognition may be judged by a person of ordinary skill in the art based on the common knowledge in the art.
  • 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.
  • isolated antibody molecule refers to an antibody molecule that has been identified and separated and / or recovered from a component of its natural environment.
  • the polluting components of its natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • nucleic acids such as DNA or RNA refers to molecules that are separated from other DNA or RNA that exists as macromolecules of natural origin, respectively.
  • isolated as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or are substantially free of chemical precursors or other chemicals when prepared by chemical synthesis.
  • isolated nucleic acid refers to nucleic acid fragments that are not naturally produced as fragments, and these fragments do not exist in a natural state.
  • isolated is also used herein to refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptide refers to both purified and recombinant polypeptides.
  • 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).
  • the cytoplasmic domain of FcyRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see M. Annu. Rev. Immunol., 15: 203-234 (1997)).
  • 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.
  • Fc receptor or "FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer RL et al., J. Immunol., 117: 587, 1976) and (Kim YJ et al., J. Immunol., 24: 249, 1994)).
  • the term "functional domain” refers to a three-dimensional structure capable of specifically recognizing and / or binding to an epitope, such as an antibody or antibody fragment, including natural intact antibody, single chain antibody (scFv), Fd fragment, Fab fragment, F ( ab ') 2 fragments, single domain antibody fragments, isolated CDR fragments and derivatives thereof.
  • Single-stranded here means that the first and second functional domains are covalently linked, preferably formed by a line of co-linear amino acid sequences that can be encoded by one nucleic acid molecule.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain.
  • two or more VH regions are covalently joined with a peptide linker to produce a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • mAb monoclonal antibody
  • monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, except for a few occurrences of possible naturally occurring mutations, the individual antibodies contained in the population are the same, showing a single binding specificity and affinity for a specific epitope .
  • the modifier "monoclonal” indicates the nature of the antibody obtained from a substantially identical group of antibodies, and no specific method is required to produce the antibody.
  • Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing myeloma cells and immune spleen cells to produce hybrid antibody-producing cells. Synthesized by culturing hybridomas, it will not contaminate any other antibodies.
  • Monoclonal antibodies can also be obtained by recombination, such as recombinant technology, phage display technology, synthetic technology, or other existing technologies.
  • chimeric antibody means that a portion of the heavy chain and / or light chain is the same as or homologous to the corresponding sequence in an antibody derived from a specific species or belongs to a specific antibody class or subclass, while the remaining portion of the chain is derived from another
  • the corresponding sequences in antibodies of one species or of another antibody class or subclass are the same or homologous, and fragments of such antibodies, as long as they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison SL et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855, 1984).
  • chimeric antibody may include antibodies (eg, human and murine chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from the first antibody (eg, murine antibody), and the heavy chain and The light chain constant region is from a second antibody (eg, human antibody).
  • first antibody eg, murine antibody
  • second antibody eg, human antibody
  • intact antibody refers to an antibody composed of two antibody heavy chains and two antibody light chains.
  • “Intact antibody heavy chain” is composed of antibody heavy chain variable domain (VH), antibody constant heavy chain domain 1 (CH1), antibody hinge region (HR), antibody heavy chain in the N-terminal to C-terminal direction Consisting of constant domain 2 (CH2) and antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and in the case of antibodies of the IgE subclass, optionally also including antibody heavy Chain constant domain 4 (CH4).
  • VH antibody heavy chain variable domain
  • CH1 constant heavy chain domain 1
  • HR antibody heavy chain constant domain 3
  • VH-CH1-HR-CH2-CH3 antibody heavy chain constant domain 4
  • the "intact antibody heavy chain” is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
  • “Intact antibody heavy chain” is a polypeptide composed of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.
  • the antibody light chain constant domain (CL) may be ⁇ (kappa) or ⁇ (lambda).
  • the intact antibody chain is connected by an inter-polypeptide disulfide bond between the CL domain and the CH1 domain (ie, between the light chain and the heavy chain) and an inter-polypeptide disulfide bond between the hinge regions of the intact antibody heavy chain.
  • Examples of typical intact antibodies are natural antibodies such as IgG (eg, IgG1 and IgG2), IgM, IgA, IgD, and IgE.
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase the sequence homology with the human antibody. Most or all amino acids outside the CDR domain of non-human antibodies, such as mouse antibodies, are replaced with corresponding amino acids from human immunoglobulins, while most or all amino acids in one or more CDR regions are unchanged. The addition, deletion, insertion, substitution or modification of small molecule amino acids is allowed, as long as they do not eliminate the antibody's ability to bind a specific antigen. "Humanized” antibodies retain antigen specificity similar to the original antibody.
  • the source of the CDR is not particularly limited, and can be derived from any animal.
  • antibodies derived from mouse antibodies, rat antibodies, rabbit antibodies, or non-human primates can be used.
  • human frameworks that can be used in this disclosure are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., Supra).
  • KOL and NEWM can be used for the heavy chain
  • REI can be used for the light chain and EU
  • LAY and POM can be used for both the heavy chain and the light chain.
  • human germline sequences can be used; these are available at the following URL: http://www2.mrc-lmb.cam.ac.uk/vbase/list2.php.
  • the receptor heavy and light chains do not necessarily need to be derived from the same antibody, and if necessary, may comprise a composite chain having framework regions derived from different chains.
  • human antibody refers to an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • epitope determining region is usually composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and usually has specific three-dimensional structural properties and specific charge properties.
  • polypeptides or polynucleotides refers to forms of polypeptides or polynucleotides that do not exist in nature.
  • One non-limiting example can be obtained by combining polynucleotides or polypeptides that do not normally occur together. Combined to achieve.
  • conjugated and linked refer to the association of two or more molecules.
  • the connection can also be genetic (ie recombinant fusion).
  • the term includes a reference to link ligands such as antibody moieties to effector molecules.
  • the connection can be achieved using a variety of art-recognized techniques, for example, by chemical or recombinant means.
  • “Chemical way” refers to the reaction between the antibody portion and the effector molecule, so that a covalent bond is formed between the two molecules to form a molecule.
  • 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.
  • cytotoxic agent 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).
  • EC 50 refers to the maximum response of the concentration of an antibody or antigen-binding fragment that induces a 50% response in an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof, that is, half between the maximum response and the baseline .
  • 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
  • immune cell includes cells having a hematopoietic origin and a role in the immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils Red blood cells, mast cells, basophils and granulocytes.
  • lymphocytes such as B cells and T cells
  • natural killer cells such as myeloid cells, such as monocytes, macrophages, eosinophils Red blood cells, mast cells, basophils and granulocytes.
  • “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 functions.
  • 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.
  • immunogenicity refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps clear tumor cells through an immune response. Examples of enhancing tumor immunogenicity include, but are not limited to, treatment with certain inhibitors (eg, bispecific antibodies).
  • immunotherapy refers to the treatment of subjects with or at risk of disease or relapse of the disease by methods that include inducing, enhancing, inhibiting or otherwise modifying the immune response.
  • 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).
  • tumor immunity refers to the process by which tumors escape immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated” when such escapes are weakened, and the tumor is recognized and attacked by the immune system. Examples of tumor identification include tumor binding, tumor contraction, and tumor clearance.
  • host cell is a cell in which a vector can proliferate and its DNA can be expressed, and the cell may be a prokaryotic cell or a eukaryotic cell.
  • the term also includes any progeny of the test host cell. It should be understood that not all progeny are the same as the parent cell because mutations may occur during the replication process and such progeny are included.
  • Host cells include prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • viral vector Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • vectors can direct the expression of genes to which they are effectively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply “expression vectors”).
  • expression vectors useful in recombinant DNA technology usually exist in the form of plasmids.
  • viral vectors eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses
  • 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, citrate buffer and histidine 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.
  • 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.
  • prevention refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (eg, tumor or infection) in a subject or if its effect is minimized.
  • treatment refers to a method performed in order to obtain beneficial or desired clinical results.
  • Beneficial or desired clinical outcomes include, but are not limited to, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis, whether detectable or undetectable.
  • the amount of therapeutic agent effective to relieve the symptoms of any particular disease may vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to elicit the desired response in the subject. Whether a symptom of the disease is alleviated can be assessed by any clinical measurement, which is usually used by a doctor or other skilled healthcare provider to assess the severity or progress of the symptom.
  • cytotoxic molecules such as immunotoxins are toxic to cells intended to be targeted, rather than toxic to other cells of the organism.
  • 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 vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, and the like.
  • an effective amount to prevent disease means that when used alone or in combination with another therapeutic agent or agents, it is sufficient to prevent, prevent, or delay the occurrence of disease (eg, tumor or infection)
  • the amount effective to treat a disease refers to an amount, when used alone or in combination with another therapeutic agent or agents, sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. It is well within the ability of those skilled in the art to determine such an effective amount.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously and many more.
  • the terms "effective” and “effective” with regard to treatment include both pharmacological effectiveness and physiological safety.
  • Pharmacological effectiveness refers to the ability of a drug to promote the resolution of a patient's condition or symptoms.
  • Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the level of cells, organs and / or organisms due to drug administration.
  • 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.
  • site is an amino acid site within the amino acid sequence described herein. This site may be indicated relative to the sequence of a similar natural sequence, such as a naturally occurring IgG domain or chain.
  • corresponding as used herein also includes sites that do not necessarily or depend solely on the number of preceding nucleotides or amino acids. Therefore, the position of a given amino acid according to the present invention that may be substituted may be changed due to the deletion or addition of amino acids elsewhere in the antibody chain.
  • binding protein refers to a protein that specifically binds a specific part or target with high affinity.
  • binding proteins include, but are not limited to, antibodies, antigen-binding fragments of antibodies, adnectin, minibodies, affibodies, affilin, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cells Factors, and chemokines.
  • the binding protein comprises the Fc region of the antibody.
  • complement system refers to a large number of small proteins found in the blood-called complement factors, which generally circulate as inactive precursors (pre-proteins). This term refers to the ability of this invariant and incompatible system to "supplement" antibodies and phagocytic cells to remove pathogens such as bacteria and antigen-antibody complexes from the organism.
  • An example of a complement factor is the complex C1, which contains C1q and two serine proteases C1r and C1s.
  • Complex C1 is a component of the CDC pathway.
  • C1q is a hexavalent molecule with a molecular weight of approximately 460,000, and its structure resembles a bouquet of tulips, in which six collagen "stems" are connected to six spherical head regions.
  • Clq In order to activate the complement cascade, Clq must bind to at least two molecules of IgG1, IgG2 or IgG3.
  • 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-Her2 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 of 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 Her2 ⁇ 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 enhanced 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 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. KR 2010-0099179 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. For example, those skilled in the art can perform appropriate ELISA assays. As explained in Example 5.6, an appropriate ELISA assay enables comparison of the binding strength of variants and parents to FcRn. At a pH of 7.0, comparing the specific signal detected for the variant and the parent polypeptide, if the specific signal of the variant is at least 1.9 times weaker than the specific signal of the parent polypeptide, it is the preferred variant of the present invention, It is more suitable for clinical application.
  • 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 conjugates 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.
  • 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) .
  • Various cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as E. coli or Bacilisstearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), insects 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, humans) Hepatocellular carcinoma cells or 293 cells, etc.).
  • prokaryotic cells such as E. coli or Bacilisstearothermophilus
  • fungal cells such as Saccharomyces cerevisiae or Pichia pastoris
  • insects Cells such as Lepidoptera insect cells including Spodoptera frugiperda cells
  • mammalian cells such as Chinese hamster ovary (CHO) cells, NS0 cells, baby
  • 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, etc. , Immunology Today, 4:72, 1983) and EBV-hybridoma technology (Cole SPC, 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 a bispecific antibody of the present invention that specifically recognizes the antigen, or an antibody partial antigen, and detecting the binding of the antigen An antibody (or antibody portion), or an 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.
  • 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 or immunoconjugate or pharmaceutical composition or combination therapy encoding the antibody of the present application 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.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or immunoconjugate or pharmaceutical composition or combination therapy can be used to treat malignant or premalignant conditions and to prevent the development of neoplastic or malignant states, These include, but are not limited to those conditions described above. Indicate such uses in conditions that are known or suspected to have previously progressed to neoplasia or cancer, specifically, where non-neoplastic cell growth consists of hyperplasia, metaplasia, or most specifically, developmental abnormalities (for For a review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology., 2nd Edition, WB Saunders Co., Philadelphia, pages 68-79, 1976).
  • 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 (e.g., two or more different) bispecific antibodies of the present invention.
  • the pharmaceutical composition of the present invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) 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: cancer, lymphatic system diseases, autoimmune diseases, inflammatory diseases, infectious diseases, immunodeficiency syndrome, and Other related diseases or symptoms.
  • TAAs that can be targeted include but are not limited to those 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 such as toxins, cytotoxic agents, radioisotopes, Immunotherapy agents or vaccines.
  • Therapeutic agents include but are not limited to anti-tumor agents such as doxorubicin (doxorubicin), cisplatin bleomycin sulfate, nitrosourea mustard, chlorambucil and cyclophosphamide hydroxyurea, these
  • the therapeutic agent itself is only effective when it has a toxic or sub-toxic level to the patient.
  • the above therapeutic agents are well known in the art, and also include therapeutic agents that may be developed in the future.
  • the bispecific antibody of the present invention can be combined with, for example, corresponding effector cell costimulatory molecules, effector cells, DC cell surface molecules, DC cells, and T cell-activating molecules (Hutloff A et al., Nature, 397: 262-266, 1999) Apply.
  • the targeted 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.
  • the bispecific antibodies of the invention can be transfected with, for example, immunomodulators, immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) or with genes encoding immunostimulatory cytokines Cell combination administration (He YF et al., J. Immunol., 173: 4919-28, 2004).
  • immunomodulators such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) or with genes encoding immunostimulatory cytokines Cell combination administration (He YF et al., J. Immunol., 173: 4919-28, 2004).
  • 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.
  • 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 present invention encompasses that bispecific antibodies or nucleic acids or polynucleotides or immunoconjugates or pharmaceutical compositions encoding antibodies of the present application may be combined with one or more active therapeutic agents (e.g. chemotherapeutic agents) or other modes of prevention or treatment (e.g. , Radiation) combined use.
  • active therapeutic agents e.g. chemotherapeutic agents
  • other modes of prevention or treatment e.g. , Radiation
  • 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 encoding the antibodies of the present application or immunoconjugates or pharmaceutical compositions can be administered sequentially.
  • the bispecific antibody or nucleic acid or polynucleotide encoding the antibody of the present application or immunoconjugate or pharmaceutical composition can be administered simultaneously.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide or immunoconjugate or pharmaceutical composition encoding the antibody of the present application may be used in any combination with at least one other (active) agent.
  • Treatment with the bispecific antibody of the invention can be combined with other treatments that can be effective against the condition to be treated.
  • Non-limiting examples of antibody combination therapy of the present invention include surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, and adjuvant therapy.
  • the combination therapy also includes all other known technologies and possible future development.
  • the bispecific antibodies of the present application or nucleic acids or polynucleotides encoding the antibodies of the present application or immunoconjugates or pharmaceutical compositions or combination therapies can be used in conjunction with the cancer, autoimmune diseases, inflammation described herein
  • such treatment includes the administration of the bispecific antibody of the present application to patients, such as animals, breastfeeding Animals 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 encoding the antibody of the present application or the immunoconjugate or pharmaceutical composition can be used for diagnostic purposes.
  • the antibodies of the present invention are used in in vitro diagnostic tests, such as laboratory tests that detect useful antigens or in care tests that 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 may be used to prepare an immunoconjugate.
  • immunoconjugates include but are not limited to those described above for immunoconjugates.
  • the connection method includes but is not limited to the immunoconjugate described above.
  • the bispecific antibody of the present invention or the nucleic acid or polynucleotide or immunoconjugate encoding the antibody of the present application can be used for the preparation of a medicament.
  • 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 or immunoconjugate may be used alone, or may be used together with one or more other therapeutic agents, including but not limited As mentioned above.
  • the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application or the immunoconjugate and the additional pharmaceutically active agent as separate components or as components of the same composition Provided, or may be co-administered with other known therapies.
  • Other known therapies include but are not limited to those described above.
  • the combination method, treatment method and administration method of the pharmaceutical composition include but are not limited to the above.
  • containers and instructions containing the bispecific antibodies of the present invention or nucleic acids or polynucleotides encoding the antibodies of the present application or immunoconjugates or pharmaceutical compositions 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 an antibody of the present application or an immunoconjugate or pharmaceutical composition.
  • Combination therapy includes but is not limited to the above.
  • the present invention also provides related detection methods and kits comprising the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application or immunoconjugate or pharmaceutical composition.
  • Related content includes but is not limited to the above.
  • the bispecific antibody of the present application or the nucleic acid or polynucleotide or immunoconjugate or pharmaceutical composition encoding the antibody of the present application can be used for diagnosis, treatment, suppression or prevention of diseases or symptoms, including malignant diseases, disorders, or Conditions related to such diseases or conditions, such as those related to increased cell survival or inhibition of apoptosis, such as, but not limited to, cancer, autoimmune diseases, inflammatory diseases, lymphatic system diseases, infections or infectivity as described above Diseases, immunodeficiency syndrome and other related diseases or symptoms.
  • 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 associated with increased cell survival can be treated, prevented, diagnosed and / or predicted with the bispecific antibody of the present application or the nucleic acid or polynucleotide or immunoconjugate or pharmaceutical composition encoding the antibody of the present application, These include, but are 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, lymphatic system diseases, or infections or infectious diseases) described above.
  • diseases or symptoms associated with increased cell survival can be treated, prevented, diagnosed and / or predicted with the bispecific antibody of the present application or the nucleic acid or polynucleotide or immunoconjugate or pharmaceutical composition encoding the antibody of the present application, These include, but are 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, lymphatic system diseases, or infections or infectious diseases) described
  • the form of the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application or the immunoconjugate or 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 immunoconjugate or 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 immunoconjugates 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 intravascularly to mammals.
  • the infusion of the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application or the immunoconjugate or pharmaceutical composition lasts less than about 4 hours, and more preferably lasts less than about 3 hours time.
  • 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 antibodies of the present application or nucleic acids or polynucleotides encoding the antibodies of the present application or immunoconjugates or pharmaceutical compositions, for example, encapsulating them in liposomes, microparticles In 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 a reversal Record viruses or other vectors.
  • 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 a bispecific antibody or a nucleic acid or polynucleotide encoding an antibody of the present application or an immunoconjugate or pharmaceutical composition can be administered.
  • the amount of bispecific antibody or polynucleotide or immunoconjugate or pharmaceutical composition encoding the antibody of the present application that constitutes a therapeutically effective dose can vary depending on the indication being treated, the weight of the patient, and the calculated skin area of the patient .
  • the administration can be adjusted to achieve the desired effect. In many cases, repeated administration may be required.
  • 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 antibody of the present invention or an antigen-binding fragment thereof and / or instructions for use.
  • the immunoconjugate of the present invention can be used to prepare a diagnostic or therapeutic kit, which includes the immunoconjugate of the present invention and / or instructions for use.
  • 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 The configurations of the bispecific antibodies AB7K, AB7K4, AB7K5, AB7K6, AB7K7, and AB7K8 are shown as a, b, c, d, e, and f, respectively.
  • Figure 1-2 shows the map of the bispecific antibody AB7K7 expression plasmid.
  • the expression plasmid is 9293bp in length and contains 9 major gene fragments, including 1.hCMV promoter; 2. target gene; 3. EMCVIRES; 4. mDHFR screening gene; 5. Syn termination sequence; 6. SV40 promoter; 7 . Karamycin resistance gene; 8. SV40 termination sequence; 9. PUC replicon.
  • Figure 1-3 SEC-HPLC detection results of AB7K7 purified samples.
  • Figure 1-4 SDS-PAGE electrophoresis results of AB7K7 purified samples.
  • Figure 1-6 SDS-PAGE results of AB7K7 in freeze-thaw experiments.
  • FIG. 2-1 FACS tests the ability of bispecific antibodies AB7K and AB7K4 to bind to tumor cells BT474.
  • FIG. 2-2 FACS detects the ability of bispecific antibodies AB7K and AB7K5 to bind to tumor cells BT474.
  • FIG. 1 FACS detects the ability of bispecific antibodies AB7K and AB7K6 to bind to tumor cells BT474.
  • FIG. 2-4 FACS detects the ability of bispecific antibodies AB7K and AB7K7 to bind to tumor cells BT474.
  • FIG. 1 FACS detects the ability of bispecific antibody AB7K8 to bind to tumor cells BT474.
  • FIG. 1 FACS detects the ability of bispecific antibodies AB7K and AB7K4 to bind to effector cell CIK.
  • FIG. 1-7 FACS detects the ability of bispecific antibodies AB7K and AB7K5 to bind to effector cell CIK.
  • FIG. 1 FACS detects the ability of bispecific antibody AB7K6 to bind to effector cell CIK.
  • FIG. 1 FACS detects the ability of bispecific antibodies AB7K and AB7K7 to bind to effector cell CIK.
  • FIG. 1 FACS detects the ability of bispecific antibody AB7K8 to bind to effector cell CIK.
  • FIG. 1 FACS test the ability of bispecific antibody AB7K to bind to cynomolgus monkey T cells.
  • ELISA detects the ability of five Anti-Her2 ⁇ CD3 bispecific antibodies to bind to CD3 and Her2 molecules.
  • FIG. 1 A microplate reader detects the ability of five Anti-Her2 ⁇ CD3 bispecific antibodies to activate the reporter cell line Jurkat T cells.
  • Figure 2-15 Modeling of GS linker peptide and anti-CD3 scFv VH structure.
  • Figure 2-16 Molecular docking model of anti-CD3 Fv and CD3 epsilon chain.
  • Figure 3-1 In vivo antitumor effect of double antibodies AB7K4 and AB7K7 in a transplanted tumor model in which human CIK cells and HCC1954 cells were co-inoculated subcutaneously in NCG mice.
  • Figure 3-2 In vivo anti-tumor effect of double antibody AB7K7 in a transplanted tumor model in which NPG mice were co-inoculated with human CIK cells and human breast cancer cells HCC1954.
  • Figure 3-3 In vivo anti-tumor effect of double antibodies AB7K7 and AB7K8 in a transplanted tumor model in which NCI mice were co-inoculated with human CIK cells and human breast cancer cells HCC1954 under subcutaneous administration.
  • Figure 3-4 In vivo antitumor effect of double antibody AB7K7 in a transplanted tumor model in which NCI mice were co-inoculated with human CIK cells and SK-OV-3 cells.
  • Figure 3-5 In vivo antitumor effect of the dual antibody AB7K7 in a transplanted tumor model in which NPG mice were co-inoculated with human CIK cells and HT-29 cells.
  • Figure 3-6 In vivo antitumor effect of double antibody AB7K7 in a transplanted tumor model in which CD34 immune reconstructed NPG mice were subcutaneously inoculated with human breast cancer HCC1954 cells.
  • Figure 3-7 The anti-tumor effect of the double antibody AB7K7 in a transplanted tumor model inoculated with human breast cancer HCC1954 cells in NPG mice immunized with PBMC.
  • Figure 4-1 Anti-tumor effect of dual antibodies AB7K4 and AB7K7 in a transplanted tumor model in which NCG mice were co-inoculated with human CIK cells and human Burkkit ’s lymphoma Raji cells
  • Figure 4-3 The weight change curve of normal cynomolgus monkeys with multiple antibodies AB7K7 and AB7K8.
  • Figure 5-1 The drug-time curve of the dual antibody AB7K7 in SD rats when detected by two ELISA methods.
  • Figure 5-2 The drug-time curve of the dual antibody AB7K8 in SD rats detected by two ELISA methods.
  • Figure 5-3 Drug-time curves of the dual antibodies AB7K7 and AB7K8 in cynomolgus monkeys.
  • Figure 5-4 Determination of the binding ability of the dual antibodies AB7K, AB7K5 and AB7K7 to FcRn at pH 6.0.
  • Figure 5-5 Determination of the binding ability of the dual antibodies AB7K, AB7K5 and AB7K7 to FcRn at pH 7.0.
  • Example 1 Design and preparation of Anti-Her2 ⁇ CD3 bispecific antibodies with different structures
  • bispecific antibodies of suitable configuration In order to screen out bispecific antibodies of suitable configuration, we designed six bispecific antibodies of different configurations for Her2 and CD3, among which AB7K5, AB7K6 and AB7K8 are single-chain bivalent bispecific antibodies, AB7K, AB7K4 and AB7K7 is a double-chain tetravalent bispecific antibody (see Figure 1-1), of which only AB7K8 does not contain an Fc fragment.
  • Table 1-1 The specific structural characteristics of the six bispecific antibodies are described as follows:
  • the bispecific antibody AB7K is composed of the C-terminus of the two heavy chains of the full-length anti-Her2 antibody connected to an anti-CD3 scFv domain through a connecting peptide (L1).
  • L1 connecting peptide
  • the connecting peptide L1 is composed of a flexible peptide and a rigid peptide, and the flexible peptide is composed of GS (GGGGS) 3 and the rigid peptide is SSSSKAPPPSLPSPSRLPGPSDTPILPQ; wherein the connecting peptide L2 between the VH and VL of the anti-CD3 scFv is (GGGGS) 3 .
  • the bispecific antibody AB7K4 is composed of the C-terminus of the two light chains of the full-length antibody against Her2 connected to an anti-CD3 scFv domain by a connecting peptide (L1).
  • L1 connecting peptide
  • the monoclonal antibody for the amino acid sequence of the heavy chain variable region of the complete antibody against Her2 included in AB7K4 Variable region sequence the light chain amino acid sequence of monoclonal antibody Light chain amino acid sequence (IMGT database INN 7637).
  • the Fc fragment contained in the AB7K4 heavy chain is derived from human IgG1, and has multiple amino acid substitutions / substitutions, namely L234A, L235A, T250Q, N297A, P331S, and M428L (EU numbering), and also deletes / deletes the C-terminal of the Fc fragment K447 (EU number).
  • the connecting peptide L1 is composed of a flexible peptide and a rigid peptide, and the flexible peptide is composed of G 2 (GGGGS) 3 and the rigid peptide is SSSSKAPPPS; wherein the connecting peptide L2 between the VH and VL of the anti-CD3 scFv is (GGGGS) 3 .
  • the bispecific antibody AB7K5 is composed of anti-Her2scFv, Fc fragment, connecting peptide L2 and anti-CD3 scFv in series, and the internal VH and VL of anti-Her2scFv and anti-CD3 scFv are connected by connecting peptides L1 and L3, respectively .
  • the Fc fragment contained in AB7K5 is derived from human IgG1 and has multiple amino acid substitutions / replacements, namely C226S, C229S, L234A, L235A, T250Q, N297A, P331S, T366R, L368H, K409T, and M428L (EU numbering).
  • mutations at the five positions of C226S, C229S, T366R, L368H, and K409T can prevent the polymerization of Fc fragments, thereby promoting the formation of single-chain bivalent bispecific antibodies;
  • Fc fragments carrying L234A / L235A / P331S mutations Removed ADCC and CDC activities;
  • carrying the T250Q / M428L mutation can enhance the binding affinity of the Fc fragment to the receptor FcRn, thereby extending its half-life;
  • the N297A mutation avoids antibody glycosylation and loses its ability to bind Fc ⁇ Rs.
  • 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 .
  • the bispecific antibody AB7K6 is composed of anti-Her2scFv, connecting peptide L2, anti-CD3 scFv and Fc fragments in series, and the anti-Her2scFv and anti-CD3 scFv are connected by connecting peptides L1 and L3 respectively .
  • the Fc fragment contained in AB7K6 is derived from human IgG1 and has multiple amino acid substitutions / substitutions, namely C226S, C229S, L234A, L235A, T250Q, N297A, P331S, T366R, L368H, K409T, and M428L (EU numbering).
  • mutations at the five positions of C226S, C229S, T366R, L368H, and K409T can prevent the polymerization of Fc fragments, thereby promoting the formation of single-chain bivalent bispecific antibodies;
  • Fc fragments carrying L234A / L235A / P331S mutations Removed ADCC and CDC activities;
  • carrying the T250Q / M428L mutation can enhance the binding affinity of the Fc fragment to the receptor FcRn, thereby extending its half-life;
  • the N297A mutation avoids antibody glycosylation and loses its ability to bind Fc ⁇ Rs.
  • 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 .
  • the bispecific antibody AB7K7 is composed of anti-Her2scFv, connecting peptide L2, anti-CD3 scFv and Fc fragments in series, and the anti-Her2scFv and anti-CD3 scFv are internally connected by connecting peptides L1 and L3, respectively .
  • the Fc fragment contained in AB7K7 is derived from human IgG1 and has multiple amino acid substitutions / substitutions, namely L234A, L235A, T250Q, N297A, P331S, and M428L (EU numbering), and also deletes / deletes K447 at the C-terminus of the Fc fragment (EU number).
  • 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 .
  • the bispecific antibody AB7K8 is composed of anti-Her2scFv, linking peptide L2, anti-CD3 scFv and His tags in series, and the anti-Her2scFv and anti-CD3 scFv are connected by connecting peptides L1 and L3 respectively .
  • AB7K8 adds a His tag to the C-terminus of anti-CD3 scFv to form HHHHHHHH, which facilitates antibody purification.
  • 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 .
  • the anti-CD3-scFv VH and VL amino acid sequences contained in the above six bispecific antibodies are shown in SEQ ID NO: 247 and SEQ ID NO: 248, respectively, and the VH and VL are connected by (GGGGS) 3 , the single The cloned antibody (named CD3-3) specifically binds to human and cynomolgus monkey CD3 antigens, and has weak binding affinity to CD3.
  • Table 1-1 Four different structures of bispecific antibodies against Her2 and CD3
  • Ln represents connecting peptides between different structural units
  • n is numbered in the order of arrangement of connecting peptides contained between different structural units from N-terminal to C-terminal of bispecific antibody.
  • the coding genes of the above five 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, Among them, the heavy and light chains of AB7K and AB7K4 can be constructed in one vector or on two different vectors. For example, insert the cDNA sequence encoding AB7K7 (as shown in SEQ ID NO: 56) into the expression plasmid shown in Figure 1-2. This plasmid contains the cytomegalovirus early promoter, which is a high-level expression of mammalian cells.
  • the 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 exceeding 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.
  • Antibody purification generally adopts a three-step purification strategy: crude purification (sample capture), intermediate purification, and fine purification.
  • crude purification stage affinity chromatography is usually used to capture the antibody of interest, which can effectively remove a large amount of impurities in the sample, such as miscellaneous proteins and nucleic acids, endotoxins and viruses.
  • intermediate purification step hydrophobic chromatography or CHT hydroxyapatite chromatography is often used to remove most of the remaining impurity proteins and polymers.
  • ion exchange chromatography or gel filtration chromatography molecular sieve
  • the culture supernatant of the bispecific antibody AB7K8 fused with His-tag can be roughly purified by using a metal chelating affinity chromatography column (for example, HisTrap FF of GE Company).
  • Protein A / G affinity chromatography columns (such as Mabselect SURE from GE, etc.) can be used for crude purification of Fc-containing bispecific antibodies AB7K4, AB7K5, AB7K6, AB7K, and AB7K7.
  • the above crude product is then subjected to intermediate purification and fine purification steps to finally obtain a high-purity, high-quality purified target antibody, and then use a desalting column (such as HiTrap desaulting of GE Corporation) to replace the bispecific antibody storage buffer with PBS Or other suitable buffer.
  • a desalting column such as HiTrap desaulting of GE Corporation
  • the first step, affinity chromatography use MabSelect Sure affinity chromatography medium of GE company or other commercially available affinity medium (such as Diamond protein of Borgron company, etc.) for sample capture, concentration and partial contaminants Remove.
  • the second step hydrophobic chromatography: using Butyl HP of Burglong Company or other commercially available hydrophobic chromatography media (such as Butyl HP of GE, etc.) for intermediate purification to reduce the polymer content.
  • Butyl HP of Burglong Company or other commercially available hydrophobic chromatography media (such as Butyl HP of GE, etc.) for intermediate purification to reduce the polymer content.
  • the second step is anion
  • the target protein separated by the exchange chromatography is adjusted to conductance of 40-50ms / cm with 2M (NH 4 ) 2 SO 4 solution, and then the sample is loaded, and the load is controlled to ⁇ 20mg / ml; after the sample is completed, use a balanced buffer (20mM PB , 0.3M (NH 4 ) 2 SO 4 , pH 7.0), flush the chromatography column with 3-5 column volumes (CV) at a linear flow rate of 100-200 cm / h; Finally, elute the target protein using the elution buffer ( 20mM PB, pH 7.0), eluting buffer at 40%, 80% and 100% respectively, eluting 3-5 column volumes (CV) at a linear flow rate not higher
  • 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 , Second's Generik MC-Q, Merck's Fractogel EMD TMAE, Pall's Q Ceramic (HyperD) F) purification, separation of structural variants, further removal of HCP, DNA and other contaminants.
  • anion exchange chromatography media such as GE ’s QHP, TOSOH ’s Toyopearl GigaCap Q-650, Tiandirenhe ’s DEAE Beads 6FF , Second's Generik MC-Q, Merck's Fractogel EMD TMAE, Pall's Q Ceramic (HyperD) F
  • the SEC-HPLC purity results and SDS-PAGE electrophoresis results of the samples are shown in Figures 1-3 and 1-4.
  • 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 (180KDa), after the reduction can get a clear (90KDa) single-stranded band.
  • AB7K6 Another single-chain bivalent bispecific antibody AB7K6 also has the problem of difficulty in process development.
  • AB7K6 was purified by Protein A affinity chromatography and molecular sieve chromatography Superdex 200 in two steps. After SEC-HPLC detection, it was found to be more pure Difficult to quantify, there is a clear "shoulder peak" in the main peak; in addition, its expression yield is extremely low and very unstable. After placing it in a refrigerator at 4 °C for 24h, SEC-HPLC analysis showed that its peak shape changed from two peaks. It becomes a main peak, and it may be caused by the conversion from single-stranded to double-stranded structure according to the peak-out time. In summary, the current process development of AB7K6 is more difficult, and it is difficult to achieve process amplification and industrialization.
  • AB7K7 has significant advantages in process development, and has the advantages of high yield, simple and efficient purification methods, and stable downstream processes.
  • AB7K7 protein The stability of AB7K7 protein in citrate (20 mM citrate, pH 5.5) and histidine buffer system (20 mM histidine, pH 5.5) were investigated respectively.
  • the AB7K7 protein was stored under accelerated conditions at 25 ° C for 4 weeks to evaluate the stability of the protein.
  • the AB7K7 protein was changed into the above citrate (F2) and histidine (F3) buffers, and the concentration was adjusted to 0.5mg / mL. 8% sucrose (w / v) was added to the above two buffer systems ) And 0.02% PS80 (w / v) as auxiliary materials. Filter with a 0.22 ⁇ m PES membrane needle filter, and aliquot each into 2mL vials, 0.8mL per vial, stoppering and capping immediately after dispensing. Put the samples into different stability measuring boxes according to the scheme in Table 1-2, and take samples at each sampling point for testing and analysis.
  • the testing items include sample appearance, concentration, SEC-HPLC test sample purity, HMW% and LMW%, and Turbidity measurement (A340).
  • the appearance, concentration, turbidity and SEC-HPLC test results of the two formulations stored at 25 °C for 0 to 4 weeks are shown in Tables 1-3 and 1-4, and the SDS-PAGE (reduced / non-reduced) results are shown in Figure 1. -5.
  • the appearance and concentration results of the two prescriptions did not change significantly; in the SEC-HPLC results, the SEC results of the F2 and F3 formulas did not change significantly, and the purity was 97.9% and 98.2% after 4 weeks, respectively.
  • the SDS-PAGE (reduced / non-reduced) results were basically consistent with the LMW% results, with slight changes in F2 and F3.
  • T0 turbidity test sample is the sample after 1 round of freeze-thaw
  • Tm unfolding temperature
  • Tmonset temperature at which the protein begins to unfold
  • Table 1-6 freeze-thaw experiment appearance, concentration, turbidity and SEC-HPLC results
  • Tumor cells BT-474 with positive Her2 expression (Shanghai Chinese Academy of Sciences Cell Bank) were cultured, digested with 0.25% trypsin, and 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 h.
  • the supernatant was removed by centrifugation and washed 3 times with PBS solution (PBSB) containing 1% BSA.
  • PBSB PBS solution
  • AF488-labeled goat anti-human IgG antibody or mouse anti-6 ⁇ His IgG antibody incubate at 4 ° C in the dark for 1 h; centrifuge to remove supernatant, wash twice with 1% PBSB, and then use 100 ⁇ l of 1% paraformaldehyde (PF) per well Resuspend and measure the signal strength by flow cytometry.
  • PF paraformaldehyde
  • FIGS 2-1 to 2-5 show the binding curves of bispecific antibodies with different structures and tumor cells BT474. According about 5nM, AB7K7 tumor cell binding shown in Table, AB7K AB7K4 tumor cells and binding to EC 2-1 50 EC nearly 50 50nM, AB7K5 AB7K8 tumor cells and binding to EC 50 is 100nM, and the tumor AB7K6 Cells BT474 have an EC 50 of more than 200 nM.
  • PBMC peripheral blood mononuclear cells
  • CIK cells Cytokine-Induced Killer cells
  • 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, OriginPro 8 analyzed by software, calculate the bispecific antibodies to human EC cells CIK 50 binding value.
  • PBMC peripheral blood mononuclear cells
  • the bispecific antibody AB7K can also bind well to cynomolgus monkey T cells, and its ability to bind to cynomolgus monkey T cells is roughly equivalent to that of human T cells. Flow cytometry cytometry which bound EC 50 about 26nM.
  • the bispecific antibodies AB7K4, AB7K5, AB7K6, AB7K7 and AB7K8, like AB7K, can specifically bind to cynomolgus monkey T cells.
  • bispecific antibodies to soluble CD3 and Her2 was identified by double antigen sandwich ELISA.
  • the Her2 protein (Beijing Yiqiao Shenzhou, Catalog No. 10004-H08H4) was diluted with PBS to a concentration of 0.1 ⁇ 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, a 4-fold gradient dilution, a total of 11 concentration gradients. Then wash the 96-well plate with PBST, add the diluted bispecific antibody, set the control well without antibody, and incubate for 1 hour at room temperature.
  • 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. 8 were analyzed by the software OriginPro, EC 50 values were calculated with the two bispecific antibody antigen binding.
  • each bispecific antibody can specifically bind CD3 and Her2 molecules simultaneously, and exhibits a good dose dependence with changes in antibody concentration (Figure 2-12).
  • the binding capacity of several bispecific antibodies to soluble CD3 and Her2 is shown in Table 2-3, and their EC 50 values range from 0.03 nM to 3.8 nM, which differs by two orders of magnitude.
  • AB7K has the best binding activity
  • AB7K4 and AB7K7 differ by an order of magnitude
  • AB7K5 and AB7K8 have the weakest binding activity.
  • Jurkat T cells BPS Bioscience, Catalog No. 60621
  • NFAT RE gene can overexpress luciferase in the presence of bispecific antibodies and target cells, and quantify Jurkat T by detecting luciferase activity The degree of cell activation.
  • 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.
  • the Her2 monoclonal antibody targeting Her2 cannot activate Jurkat T cells. Only when both antibodies are present will T cells be activated.
  • the ability of each antibody to activated Jurkat T cells are shown in Table 2-4, wherein the T cell activation ability AB7K4 strongest ability AB7K8 weakest T cell activation, which EC 50 values differ by an order of magnitude.
  • SK-BR-3, MCF-7, HCC1937, NCI-N87, and HCC1954 cells were digested with 0.25% trypsin to prepare single cells
  • the suspension was adjusted to a cell density of 2 ⁇ 10 5 cells / ml, added to a 96-well cell culture plate, 100 ⁇ l / well, and cultured 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.
  • effector cells human PBMC or expanded cultured CIK cells
  • control well set the control well
  • the supernatant was discarded in a 96-well plate, washed 3 times with PBS, added with 10% CCK-8 complete medium, 100 ⁇ l / well, incubated at 37 ° C for 4h, and the absorbance value of A450-620nm was detected with a microplate reader.
  • the software OriginPro 8 was used to analyze, calculate and compare the ability of each bispecific antibody and the same target monoclonal antibody Herceptin to kill tumor cells.
  • EC 50 values of killing tumor cells bispecific antibody-mediated effector cells are summarized in Table 2-5, results are shown for each antibody specific for bis-Her2 tumor cells highly expressed (e.g. SK-BR-3, NCI- N87 and HCC1954) showed a very significant killing effect and were dose-dependent.
  • Each bispecific antibody (especially AB7K7) also showed a good killing effect on MCF-7 breast cancer cells with low Her2 expression.
  • Herceptin-resistant cell line HCC1954 each bispecific antibody also has a good killing effect, while for the cell line HCC1937 that is negative for Her2 expression (very small amount of expression), each bispecific antibody is only at the highest two concentrations Only showed a lethal effect.
  • ⁇ means approximately equal to;-means no detection.
  • the computer software was used to model the structure of anti-CD3 scFv VH containing GS-CTP connecting peptide, and the spatial conformation simulation and prediction of the molecular docking of anti-CD3 scFv and its antigen CD3epsilon chain.
  • the sequence of the GS-CTP connecting peptide between anti-Her2scFv and anti-CD3 scFv in the double antibody AB7K7 is (GGGGGGSGGGGSGGGGSSSSSKAPPPS), the first half is the GS flexible peptide (GGGGGGSGGGGSGGGGGGS), and the second half is the CTP rigid peptide (SSSSKAPPPS).
  • the rigid CTP part (SSSSKAPPPS) is connected to the N terminal of the anti-CD3 scFv VH.
  • the structural CTP peptide is overlaid on the CDR1 region of anti-CD3 scFv VH ( Figure 2-14), which may hinder or hinder the binding of the CD3 antibody to its antigen.
  • the VH of anti-CD3scFv connected to the GS linker peptide was modeled in three dimensions using phyre2 software. It will affect the binding of antigen and antibody. Even if the GS linking peptide is close to the CDR region, due to its own flexibility, it can freely move away from the antigen-antibody binding region, so it will not affect the antigen-antibody binding.
  • Discovery Studio software was used to simulate the molecular docking of anti-CD3 scFv and its antigen CD3 epsilon chain. Because the structure of double-chain anti-CD3 FV and anti-CD3 scFv is highly similar, the use of double-chain anti-CD3 Fv instead of anti-CD3 scFv for structural simulation.
  • the simulation results show that the antigen CD3 epsilon chain binds to the CDR2 and CDR3 of the anti-CD3 Fv VH and does not bind to the CDR1 region ( Figure 2-16), which seems to indicate that the CTP covering the VH CDR1 region does not interfere with the anti-CD3 scFv and Antigen binding.
  • the CD3 molecule is a complex, including a CD3gamma chain, a CD3delta chain and two CD3 epsilon chains
  • the CD3 molecule together with the TCR and Zeta chains, constitutes a T cell receptor complex.
  • the CTP peptide covering the anti-CD3 scFv VH CDR1 does not directly interfere with the binding of the anti-CD3 scFv to its antigen CD3 epsilon chain
  • the CTP peptide may be spaced by a component protein of the T cell receptor complex Structural contact indirectly affects the binding of anti-CD3 scFv to its antigen CD3 epsilon chain.
  • NCG mice were co-inoculated with human CIK cells and human breast cancer HCC1954 cell transplantation tumor model
  • Human breast cancer HCC1954 cells with positive Her2 expression were selected to observe the in vivo tumor suppressive effect of double antibody in the transplanted tumor model in which human CIK cells and HCC1954 cells were co-inoculated subcutaneously in NCG mice.
  • mice purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • HCC1954 cells ATCC
  • 5 ⁇ 10 6 HCC1954 cells and 5 ⁇ 10 5 CIK cells were mixed and inoculated subcutaneously on the right back of NCG mice.
  • mice were randomly divided into 7 groups according to their body weight, and 5 mice in each group were given the corresponding drugs intraperitoneally.
  • the positive control group and the PBS control group were given twice a week for a total of 3 times.
  • Herceptin (Herceptin, Roche) was administered at doses of 1 mg / kg and 3 mg / kg, and the PBS control group was given the same volume of PBS solution.
  • the administration group was given double antibodies AB7K4 and AB7K7 every day at a dose of 0.1 mg / kg and 1 mg / kg, respectively, for a total of 10 times.
  • the average tumor volume of the PBS control group was 1494.61 ⁇ 500.28mm 3 ; the average tumor volume of the 1mg / kg Herceptin administration group was 1327.29 ⁇ 376.65mm 3 ; 3mg / kg
  • the average tumor volume of the Herceptin-administered group was 510.49 ⁇ 106.07 mm 3 and the TGI was 65.84%, which was not significantly different from that of the PBS control group.
  • the average tumor volume of the 0.1mg / kg and 1mg / kg AB7K4 administration groups was 304.10 ⁇ 108.50mm 3 and 79.70 ⁇ 58.14mm 3 respectively, and the TGI was 79.65% and 94.67%, respectively, which were significantly different from the PBS control group ( P ⁇ 0.05).
  • the average tumor volume of the 0.1mg / kg and 1mg / kg AB7K7 administration groups was 385.82 ⁇ 95.41mm 3 and 209.98 ⁇ 51.74mm 3 respectively, and the TGI was 74.19% and 85.95%, respectively, which were significantly different from the PBS control group ( P ⁇ 0.05).
  • NPG mice were co-inoculated with human CIK cells and human breast cancer HCC1954 cell transplantation tumor model
  • Human breast cancer HCC1954 cells with positive Her2 expression were selected to observe the in vivo tumor suppressive effect of double antibodies on transplanted tumor models of NPG mice subcutaneously co-inoculated with human CIK cells and human breast cancer cells HCC1954.
  • CIK cells were obtained according to the method in Example 3.1. Seven to eight-week-old female NPG mice (purchased from Beijing Viton Biotech Co., Ltd.) were selected to collect HCC1954 cells (ATCC) in the logarithmic growth phase, and 5 ⁇ 10 6 HCC1954 cells and 5 ⁇ 10 5 cells were collected. CIK cells were mixed and inoculated subcutaneously on the right back of NPG mice. After 6 days of tumor growth, the mice were randomly divided into 3 groups according to tumor volume and body weight. Each group had 6 mice, and the corresponding drugs were given intraperitoneally. Specifically, the dosage of the AB7K7 administration group was 0.1 mg / kg and 1 mg / kg, respectively.
  • the average tumor volume of the PBS control group was 821.73 ⁇ 201.82mm 3 ; the average tumor volume of the 0.1mg / kg AB7K7 administration group was 435.60 ⁇ 51.04mm 3 , TGI 50.83%, no significant difference compared with the control group; the average tumor volume of the AB7K7 administration group at 1 mg / kg was 40.98 ⁇ 12.64mm 3 , TGI was 95.37%, which was extremely significant compared to the control group (P ⁇ 0.01).
  • the double antibody AB7K7 still has a good therapeutic effect, and a low dose of 0.1 mg / kg has a 50% tumor suppressive effect, while 6 mice in the 1 mg / kg administration group Tumors of 4 mice completely regressed, and the tumor volume of the other 2 was also less than 100 mm 3 , which was smaller than the volume of the group (the average tumor volume of this group at the time of grouping was 161.37 ⁇ 18.98 mm 3 ).
  • the double antibody AB7K7 had a good treatment The role of tumors.
  • CIK cells were obtained according to the method described above. Seven to eight-week-old female NPG mice were selected, and 5 ⁇ 10 6 HCC1954 cells and 5 ⁇ 10 5 CIK cells were mixed and inoculated subcutaneously on the right back of NPG mice. After 1h, the mice were randomly divided into 6 groups according to body weight, and 6 mice in each group were given the corresponding drugs intraperitoneally.
  • the control group and the Herceptin administration group are administered twice a week, the dosage of Herceptin is 3mg / kg, the control group is administered the same volume of PBS solution; the administration dose of bispecific antibody AB7K7 is 1mg / kg The dosage of AB7K8 is 0.7mg / kg, and two dosage frequencies are set.
  • the QD group is given once a day for 10 consecutive days, and the BIW group is given twice a week.
  • the day of administration is recorded as day 0.
  • the maximum diameter (D) and minimum diameter (d) of the tumor are measured twice a week, and the tumor volume (mm 3 ) of each group and the tumor growth of each administration group are calculated according to the above formula Inhibition rate TGI (%).
  • the average tumor volume of the PBS control group was 1588.12 ⁇ 120.46 mm 3 ; the average tumor volume of the 3 mg / kg Herceptin administration group was 361.72 ⁇ 134.70 mm 3 ; AB7K7
  • the average tumor volume of the QD administration group and the BIW administration group were 260.18 ⁇ 45.96mm 3 and 239.39 ⁇ 40.62mm 3 , respectively, and the TGI was 83.62% and 84.93%, respectively, which were significantly different from the PBS control group (P ⁇ 0.01); the average tumor volume of the QD administration group and the BIW administration group of AB7K8 were 284.98 ⁇ 26.62mm 3 and 647.14 ⁇ 118.49mm 3 respectively, and the TGI was 82.06% and 59.25%, respectively, which were extremely significant compared to the PBS control group.
  • NPG mice were co-inoculated with human CIK cells and human ovarian cancer SK-OV-3 cell transplantation tumor model
  • Human ovarian cancer SK-OV-3 cells with positive Her2 expression were selected to observe the in vivo tumor suppressive effect of double antibodies in a transplanted tumor model in which NPG mice were co-inoculated with human CIK cells and SK-OV-3 cells.
  • mice Seven to eight-week-old female NPG mice were selected to collect SK-OV-3 cells in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences), and 3 ⁇ 10 6 SK-OV-3 cells and 3 ⁇ 10 Five CIK cells were mixed and inoculated subcutaneously on the right back of NPG mice. After 1h of inoculation, the mice were randomly divided into 7 groups according to body weight, each group of 6 were given the corresponding drugs intraperitoneally. Herceptin and AB7K7 administration groups were given 1mg / kg, 0.2mg / kg and 0.04mg / kg, respectively. The drug frequency was given twice a week, and the control group was given the same volume of PBS. The day of administration is recorded as day 0. The maximum diameter (D) and minimum diameter (d) of the tumor are measured twice a week, and the tumor volume (mm 3 ) of each group and each administration are calculated according to the formula in Example 3.1 The tumor growth inhibition rate TGI (%) of the group.
  • the average tumor volume of the PBS control group was 834.09 ⁇ 45.64 mm 3 ; the average tumor volume of Herceptin at the doses of 1 mg / kg, 0.2 mg / kg, and 0.04 mg / kg They were 644.84 ⁇ 58.22mm 3 , 884.95 ⁇ 38.63mm 3 and 815.79 ⁇ 78.39mm 3 respectively ; the tumors of all AB7K7 administration groups completely regressed.
  • the above results show that in the ovarian cancer SK-OV-3 model, AB7K7 can still completely regress the tumor at a very low dose of 0.04 mg / kg, showing an excellent anti-tumor effect.
  • mice were subcutaneously co-inoculated with human CIK cells and human colon cancer cell HT-29 cell transplantation tumor model
  • Human colon cancer HT-29 cells with positive Her2 expression were selected to observe the antitumor effect of double antibody in the transplanted tumor model in which NPG mice were co-inoculated with human CIK cells and HT-29 cells.
  • CIK cells were obtained according to the method in Example 3.1. Seven- to eight-week-old female NPG mice were selected to collect HT-29 cells in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences). 3 ⁇ 10 6 HT-29 cells and 3 ⁇ 10 6 CIK cells were collected. Mix and inoculate subcutaneously on the right back of NPG mice. After 1h of inoculation, the mice were randomly divided into 5 groups according to body weight, each group of 6 were given the corresponding drugs intraperitoneally. Specifically, the dose of Herceptin was 3mg / kg, and the dose of AB7K7 administration group was 3mg / kg, respectively.
  • the average tumor volume of the PBS control group was 1880.52 ⁇ 338.26mm 3 ; the average tumor volume of 3mg / kg Herceptin was 1461.36 ⁇ 177.94mm 3 ; AB7K7 at 3mg / kg,
  • the average tumor volume in the 1 mg / kg and 0.3 mg / kg dose groups was 13.94 ⁇ 7.06 mm 3 , 26.31 ⁇ 10.75 mm 3 and 10.47 ⁇ 6.71 mm 3 , of which 4 mice in the 0.3 mg / kg group had tumors Complete regression, tumors in 3 mice in the 1 mg / kg administration group completely resolved, and tumors in 4 mice in the 3 mg / kg administration group completely resolved.
  • the above results show that in the colon cancer HT-29 model, Herceptin has basically no effect on this tumor model, while AB7K7 has tumor regression in mice at all three doses, and very low doses also show excellent resistance The effect of tumor.
  • Human breast cancer HCC1954 cells with positive Her2 expression were selected to observe the antitumor effect of the double antibody in the transplanted tumor model in which CD34 immune reconstructed NPG mice were inoculated with human breast cancer HCC1954 cells subcutaneously.
  • CD34-positive selection magnetic beads purchased from Germany Mitani Biotechnology Co., Ltd.
  • CD34-positive hematopoietic stem cells obtained from fresh umbilical cord blood, and female NPG mice of three to four years of age (purchased from Beijing Viton Biotech) Co., Ltd.), injected CD34-positive hematopoietic stem cells in the tail vein to reconstruct the human immune system in mice.
  • blood was collected from the posterior orbital venous plexus of mice for flow cytometry. Mice with a proportion of human CD45 greater than 15% were regarded as successful in immune reconstruction.
  • HCC1954 cells in the logarithmic growth phase were collected, and 5 ⁇ 10 6 HCC1954 cells were inoculated subcutaneously on the right back of the immunized mice. After 1h, the mice were randomly divided into 3 groups according to body weight, 6 mice in each group. AB7K7 and Herceptin were administered intraperitoneally at a dose of 1 mg / kg, respectively.
  • the control group was given the same volume of PBS, twice a week for a total of 6 Times. The day of administration is recorded as day 0.
  • the maximum diameter (D) and minimum diameter (d) of the tumor are measured twice a week, and the tumor volume (mm 3 ) of each group and each administration are calculated according to the formula in Example 3.1
  • the average tumor volume of the PBS control group was 475.23 ⁇ 58.82mm 3 ; the average tumor volume of the Herceptin administration group was 293.27 ⁇ 66.35mm 3 , and the TGI was 38.29%, relative There was no significant difference between the control group; the average tumor volume of the AB7K7 administration group was 0.67 ⁇ 0.67mm 3 , TGI was 99.86%, all tumors had basically subsided, and there was a very significant difference compared with the control group (P ⁇ 0.01) .
  • the double antibody AB7K7 has an excellent anti-tumor effect in the CD34 immune reconstruction model.
  • the HCC1954 cells with positive Her2 expression were selected to observe the antitumor effect of the double antibody in a transplanted tumor model inoculated with human breast cancer HCC1954 cells in NPG mice immunized with PBMC.
  • mice Peripheral blood of normal people was taken and human PBMC cells were isolated by density gradient centrifugation. Five to six-week-old female NPG mice were selected and injected intraperitoneally with human PBMC cells to reconstruct the human immune system in the mice. Seven days after PBMC injection, HCC1954 cells in the logarithmic growth phase were collected, and 5 ⁇ 10 6 HCC1954 cells were inoculated subcutaneously on the right back of mice. Thirteen days after PBMC injection, blood was collected from the retro-orbital venous plexus for flow cytometry. Mice with a human CD45 ratio greater than 15% were considered successful in immune reconstruction. After 14 days of PBMC injection, the successfully immunized mice were randomly divided into 2 groups according to tumor volume and body weight.
  • Each group had 6 mice. AB7K7 was administered intraperitoneally at a dose of 1 mg / kg. The control group was given PBS three times a week. The day of administration is recorded as day 0. The maximum diameter (D) and minimum diameter (d) of the tumor are measured twice a week, and the tumor volume (mm 3 ) of each group and each administration are calculated according to the formula in Example 3.1 The tumor growth inhibition rate TGI (%) of the group.
  • the average tumor volume of the PBS control group was 1224.05 ⁇ 224.39mm 3 ; the average tumor volume of the AB7K7 administration group was 32.00 ⁇ 0.00mm 3 , and the TGI was 97.41%, all The tumors have subsided, and there is a very significant difference compared with the control group (P ⁇ 0.001).
  • the bifunctional specific antibody AB7K7 has an excellent anti-tumor effect in the PBMC immune reconstruction model.
  • Human Burkkit ’s lymphoma Raji cells with negative Her2 expression were selected to observe whether the double antibody would inhibit tumor growth in a transplanted tumor model in which NCG mice were co-inoculated with human CIK cells and human Burkkit ’s lymphoma Raji cells.
  • CIK cells were obtained according to the method in Example 3.1. Seven to eight-week-old female NCG mice were selected, Raji cells in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were collected, 5 ⁇ 10 6 Raji cells and 2 ⁇ 10 6 CIK cells were mixed, and inoculated in NCG mice were subcutaneously on the right back. After 1h, the mice were randomly divided into 3 groups according to body weight, 5 in each group. The administration group was given AB7K4 and AB7K7 at a dose of 1 mg / kg intraperitoneally. The control group was given the same volume of PBS solution once a day, continuously Medicine for 10 days. The day of administration is recorded as day 0. The maximum diameter (D) and minimum diameter (d) of the tumor are measured twice a week, and the tumor volume (mm 3 ) of each group and each administration are calculated according to the formula in Example 3.1 The tumor growth inhibition rate TGI (%) of the group.
  • TGI tumor growth inhibition rate
  • the average tumor volume of the PBS control group was 2439.88 ⁇ 193.66mm 3 ; the average tumor volume of the AB7K4 administration group was 2408.81 ⁇ 212.44mm 3 , the average of the AB7K7 administration group The tumor volume was 2598.11 ⁇ 289.35 mm 3 , and the average tumor volume of the two administration groups was not different from that of the control group.
  • the double antibodies AB7K4 and AB7K7 did not observe non-specific killing on Her2 negative cell lines, indicating that AB7K4 and AB7K7 will not mediate the killing of T cells against non-target tissues in vivo (ie specificity) Depending on the binding of the bispecific antibody to the corresponding target antigen), without off-target toxicity and high safety.
  • Human breast cancer HCC1954 cells with positive Her2 expression were selected to observe whether the double antibody inhibited tumor growth in a transplanted tumor model in which NPG mice were inoculated with human breast cancer HCC1954 cells alone.
  • mice Seven- to eight-week-old female NPG mice were selected, HCC1954 cells in the logarithmic growth phase were collected, and 5 ⁇ 10 6 HCC1954 cells and Matrigel matrigel (Corning, article number: 354234) were mixed in a volume ratio of 1: 1.
  • the drug group was given Herceptin at a dose of 3 mg / kg and AB7K7 at 1 mg / kg, and the control group was given the same volume PBS is given twice a week. The day of administration is recorded as day 0.
  • the maximum diameter (D) and minimum diameter (d) of the tumor are measured twice a week, and the tumor volume (mm 3 ) of each group and each administration are calculated according to the formula in Example 3.1
  • the average tumor volume of the PBS control group was 1311.35 ⁇ 215.70mm 3 ; the average tumor volume of the Herceptin administration group was 273.98 ⁇ 60.10mm 3 ; the average of the AB7K7 administration group The tumor volume was 1243.20 ⁇ 340.31mm 3 , which was no difference from the control group.
  • Table 4-1 Acute toxicity assessment of cynomolgus monkeys
  • the cynomolgus monkeys in the AB7K8 administration group experienced drowsiness and pupil diminishment, and returned to normal the next day, and there were no abnormalities in the other groups; Symptoms of vomiting appeared and returned to normal on the second day of administration. No abnormalities occurred in other groups; after D21 administration, cynomolgus monkeys in the AB7K7 administration group developed vomiting food symptoms 3 hours after administration and excreted jelly-like stool. Symptoms of vomiting food appeared 1 hour after administration.
  • the different degrees of diarrhea observed during this experiment may be related to the expression of related receptors in the intestine. It is presumed that the double antibody inhibits the heterodimer of Her1 / Her2 or Her2 / Her3 and causes the imbalance of chloride ions in the intestine. , which is an extension of the pharmacological effect, can return to normal after 24 hours of administration.
  • AB7K7 reaches a high dose of 3mg / kg, cynomolgus monkeys are still well tolerated, and the results of pharmacodynamic experiments in mice show that low doses of AB7K7 show good anti-tumor effects, indicating that AB7K7 The treatment window is wider and the safety is higher.
  • AB7K7 was injected into 4 healthy SD rats (purchased from Shanghai Slake Experimental Animal Co., Ltd.) at a dose of 1 mg / kg via tail vein administration.
  • Blood sampling time points are: 1h, 3h, 6h, 24h, 72h, 96h, 120h, 168h, 216h and 264h. Take a certain amount of whole blood at each time point, separate the serum, and then use two ELISA methods to determine the drug concentration in the serum.
  • Method 1 Use anti-AB7K7 antibody A (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) to coat the plate, the concentration of the plate is 0.5 ⁇ g / mL.
  • AB7K7 was 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 and established a standard curve.
  • Anti-AB7K7 antibody B (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., anti-herceptin-HRP) was labeled with HRP, the concentration was 1: 5000, and finally developed with TMB.
  • Method 2 Detect the drug concentration in the serum of SD rats. Plates were coated with anti-AB7K7 antibody A (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) at a concentration of 0.5 ⁇ g / mL.
  • AB7K7 was configured at 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.3125 ng / mL, 0.156 ng / mL, and 0.078 ng / mL and established a standard curve.
  • mice anti-human IgG Fc-HRP Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd.
  • PKSolver software was used to calculate the pharmacokinetic parameters. The specific parameters are shown in Table 5-2.
  • Figure 5-1 shows the detection of AB7K7 blood concentration in rats by two different detection methods, and the detection of AB7K7 concentration in blood by two different detection methods.
  • the obtained blood concentration is basically the same, and the calculated drug generation The parameters are roughly the same, indicating that AB7K7 can be metabolized in the form of a complete molecule in the body to ensure its biological function.
  • NPG mice purchased from Beijing Weitongda Biotechnology Co., Ltd.
  • HCC1954 cells purchased from the Institute of Chinese Academy of Sciences
  • CIK cells were resuscitated two days before the administration, and the cells were collected and injected into mice intravenously after 24 hours of culture.
  • the mice were randomly divided into three groups, with four mice in each group.
  • the dosages of the three administration groups are: 0.3 mg / kg, 1 mg / kg and 3 mg / kg.
  • Blood sampling time points were 1h, 3h, 6h, 24h, 48h, 72h, 96h, 120h, 168h, 216h and 264h. A certain amount of whole blood was collected at each time point, the serum was separated, and then the drug concentration in the serum was determined by ELISA.
  • AB7K7 Anti-AB7K7 antibody A (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) at a concentration of 0.5 ⁇ g / mL.
  • AB7K7 was configured at 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 and a standard curve was established.
  • Anti-AB7K7 antibody B (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) was labeled with HRP at a concentration of 1: 5000, and finally developed with TMB for color development.
  • PKSolver software is used to calculate the pharmacokinetic parameters. The specific parameters are shown in Table 5-3.
  • AB7K8 was injected into 3 healthy SD rats via tail vein administration at doses of 1 mg / kg and 3 mg / kg, respectively.
  • Blood sampling time points are: 0.25h, 0.5h, 1h, 2h, 3h, 4h, 5h and 7h. Take a certain amount of whole blood at each time point, separate the serum, and then use the ELISA method to detect the drug concentration in the serum.
  • AB7K8 obtained pharmacokinetic parameters T 1/2 that were basically consistent, indicating that it exhibited linear metabolic kinetics in SD rats. Because AB7K8 does not contain Fc, its T 1/2 is very short, which is about 20 times shorter than AB7K7.
  • AB7K was injected into 4 healthy SD rats via tail vein administration at a dose of 0.8 mg / kg.
  • Blood sampling time points are: 2h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 216h and 264h. Take a certain amount of whole blood at each time point, separate the serum, and then use two ELISA methods to determine the drug concentration in the serum.
  • Method one Encapsulate the plate with anti-AB7K antibody A (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) at a concentration of 1 ⁇ g / mL.
  • AB7K was configured at 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and 0.3125 ng / mL and established a standard curve.
  • Add 25ng / mL biotin-labeled human CD3E & CD3D (Acro, Catalog No.
  • CDD-H82W0 CDD-H82W0
  • Method 2 Use anti-AB7K antibody A (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) to coat the plate, the concentration of the plate is 1 ⁇ g / mL.
  • AB7K was configured at 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and 0.3125 ng / mL and established a standard curve.
  • Add mouse anti-human IgG Fc-HRP (1: 10000 dilution) Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd.
  • incubate for 1h and finally develop color with TMB.
  • Figure 5-2 shows the detection of AB7K plasma concentration in rats by two different detection methods.
  • the results show that the results of the two detection methods are quite different.
  • the concentration of the first two points (2h, 1D) of the curve is close, but the difference in concentration measured by the two methods becomes larger after the next day, presumably because of the connecting peptide between the anti-CD3 scFv and the anti-Her2 antibody heavy chain Caused by fracture.
  • the structure of AB7K is unstable in the body, so that it cannot play its biological function, and the improved AB7K7 can be metabolized in a complete form in the body, so that it can normally play its biological function.
  • the cynomolgus monkey (purchased from Guangzhou Xiangguan Biotechnology Co., Ltd.) is divided into three groups, one in each group, female and female, weighing 3-4 kg.
  • the first group (G1-1) is the blank control group;
  • the second group (G2-1) AB7K7 is the administration group, the dosage is 0.3mg / kg;
  • the third group (G3-1) is the AB7K8 administration group,
  • the administered dose is 0.2 mg / kg.
  • Blood sampling time points were 15min, 1h, 3h, 6h, 24h, 48h, 72h, 96h, 144h, 192h, 240h and 288h, a total of 13 time points. Blood was collected to collect serum, frozen at -80 ° C, and then the drug concentration in the serum was determined by ELISA.
  • AB7K7 Anti-AB7K7 antibody A (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) at a concentration of 0.5 ⁇ g / mL.
  • AB7K7 was 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 and established a standard curve.
  • Anti-AB7K7 antibody B (Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd., mouse-anti-herceptin) was labeled with HRP at a concentration of 1: 5000, and finally developed with TMB for color development.
  • HRP a concentration of 1: 5000
  • TMB color development.
  • Figure 5-3 shows the blood concentration of AB7K7 in rats.
  • the T 1/2 of AB7K7 in normal cynomolgus monkeys is only about 8 hours.
  • AB7K8 cannot calculate the pharmacokinetic parameters due to too few points on the drug-time curve. However, it can be seen from the drug-time curve that AB7K7 has a much longer half-life than AB7K8 in normal cynomolgus monkeys.
  • Each antibody was diluted with PBS solution to a concentration of 10 ⁇ 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.
  • the biotin biotin-labeled FcRn protein (ACRO Biosystem, Catalog No. FCM-H8286) was diluted with dilutions of pH 6.0 and 7.0, respectively, and was diluted 4 times in a total of 11 concentration gradients.
  • Table 5-7 and Table 5-8 show the measurement results of the binding ability of each antibody to FcRn at pH 6.0 and 7.0, respectively.
  • Table 5-7 Determination of the binding ability of the dual antibodies AB7K, AB7K5 and AB7K7 to FcRn at pH 6.0
  • Table 5-8 Determination of the binding ability of the dual antibodies AB7K, AB7K5 and AB7K7 to FcRn at pH 7.0
  • bispecific antibodies of scFv1-scFv2-Fc configuration such as AB7K7 are easy to prepare, the purification method is simple and efficient, and their preparation And good stability during storage. More beneficially, it has a weak non-specific killing effect on normal cells, and has significant advantages such as the control of toxic and side effects that may be caused by excessive activation of effector cells, and has good drug-forming properties.
  • Table 5-9 lists some preferred amino acid sequences for the VH domain and its complementarity determining regions (HCDR1, HCDR2, and HCDR3) of the first single-chain Fv of Her2, and the VL domain and its complementarity determining regions (LCDR1, The amino acid sequence of the LCDR2 and LCDR3), the amino acid residues contained in the CDR regions are defined according to the Kabat rules.
  • the anti-CD3 scFv binds to the effector cells with an EC 50 value of greater than about 50 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 nM in the in vitro FACS binding analysis assay; more preferably, the bispecific antibody Two single-chain Fv can not only bind to human CD3, but also specifically bind to CD3 of cynomolgus monkey or rhesus monkey.
  • Table 5-10 lists the amino acid sequences of some preferred anti-CD3 scFv VH domains and their complementarity determining regions (HCDR1, HCDR2 and HCDR3), and VL domains and their complementarity determining regions (LCDR1, LCDR2 and LCDR3 ), The amino acid residues contained in the CDR regions are defined according to Kabat rules.
  • the connecting peptide connecting the anti-Her2scFv and the anti-CD3 scFv is composed of a flexible peptide and a rigid peptide; preferably, the amino acid composition of the flexible peptide has the general structure G x S y (GGGGS) z , where x, y and z are 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: 49) or a truncated fragment thereof; preferably, the CTP
  • the rigid peptide composition is SSSSKAPPPS (CTP 1 ).
  • Table 5-11 illustrates the amino acid sequences of some preferred connecting peptides connecting anti-Her2scFv 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.).
  • Tables 5-12 illustrate the amino acid sequences of some Fc fragments with one or more amino acid mutations.

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Abstract

一种同时靶向免疫效应细胞抗原CD3和人表皮生长因子受体2(Her2)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端依次包含能够特异性结合Her2的第一单链Fv、能够特异性结合CD3的第二单链Fv和Fc片段，第一和第二单链Fv通过连接肽相连，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。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。临床前毒理学评价试验的最大安全起始剂量已大大高于其它同靶点剂量，且没有发生系统性免疫毒性，提示双特异性抗体的用药安全窗较宽；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Her2和CD3的同源二聚体型双特异性抗体及其用途
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，涉及针对人表皮生长因子受体2(Her2)和簇集决定子3(CD3)的双特异性抗体，以及这类抗体的用途，特别是其在治疗肿瘤中的用途。
背景技术
一、双特异性抗体
1985年，利用T细胞杀死肿瘤细胞的概念就已被提出(Stearz UD等,Nature,314:628-631,1985)。通常认为有效激活T细胞需要双重信号，第一信号来自抗原呈递细胞上MHC-抗原复合物与T细胞受体TCR-CD3的结合，第二信号为T细胞与抗原呈递细胞表达的共刺激分子相互作用后产生的非抗原特异性共刺激信号。由于多数肿瘤细胞表面MHC的表达下调甚至缺失，从而使肿瘤细胞逃逸免疫杀伤。
双特异性抗体从作用机制上可分为双重信号阻断型和介导细胞功能型。通常，介导细胞功能型双特异性抗体指介导T细胞杀伤的抗CD3双特异性抗体。CD3分子表达于所有成熟T细胞表面，并与TCR呈非共价结合，形成完整的TCR-CD3复合物，共同参于对抗原刺激的免疫应答，是目前在双特异性抗体中应用最多且最成功的免疫效应细胞表面的触发分子。靶向CD3的双特异性抗体则能够分别结合T细胞表面CD3和肿瘤细胞表面抗原，从而拉近细胞毒性T细胞(cytotoxic T cell,Tc或CTL)与肿瘤细胞的距离，并直接激活T细胞，诱导T细胞直接杀伤癌细胞，而不再依赖于传统的T细胞的双重激活信号。但是，靶向T细胞抗原CD3的激动型抗体，例如，第一代应用于临床的靶向人CD3的小鼠单克隆抗体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，诸如白介素-2(IL-2)、TNF-α、IFN-γ和白介素-6(IL-6)，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)，导致以发烧、寒战、头疼、恶心、呕吐、腹泻、呼吸窘迫、无菌性脑膜炎和血压过低为特征的“流感样”综合征。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。
近年来，为了解决将两个不同的半抗体进行正确装配问题，科学家们设计开发了多种结构的双特异性抗体。总体归结起来有两大类，一类双特异性抗体不含Fc区。这类结构双抗优点是分子量小，可以在原核细胞中表达，不需要考虑正确装配的问题；缺点是由于没有抗体Fc段，分子量较低，导致其半衰期较短，且 这种形式的双抗极易聚合、稳定性差且表达量低，因而临床应用受到一定限制。目前已有报道的此类双特异性抗体包括BiTE、DART、TrandAbs、bi-Nanobody等。
另一类双特异性抗体保留Fc结构域。此类双抗形成IgG样结构，分子结构较大，并且由FcRn介导的细胞内吞和再循环过程，使其具有更长的半衰期；同时保留了Fc介导的部分或全部效应子功能，如抗体依赖细胞介导细胞毒性(ADCC)、补体依赖细胞毒性(CDC)和抗体依赖细胞吞噬(ADCP)。目前已有报道的此类双特异性抗体包括Triomabs、kih IgG、Cross-mab、orthoFab IgG、DVD IgG、IgG scFv、scFv 2-Fc等。而对于抗CD3双特异性抗体，目前除了TandAb和scFv-Fv-scFv构型外，其它抗CD3双特异性抗体的设计广泛采用单价抗CD3形式，主要因为二价抗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)，并且更为严重的是它还可能引发非抗原依赖性地激活T细胞而打破免疫平衡。因此，现有技术中的抗CD3双特异性抗体多避免引入二价抗CD3抗体，例如triFab-Fc、DART-Fc和BiTE-Fc构型的双特异性抗体都采用非对称性设计(即异源二聚体型双抗)(Z Wu等,Pharmacology and Therapeutics,182:161-175,2018)，但这对其下游的生产带来很多挑战，如产生不希望出现的同源二聚体或错配的杂质分子，增加了双抗表达和纯化的难度。尽管“knobs-into-holes”技术的运用一定程度上解决了异源二聚体型双抗分子的重链间错配的问题，然而“轻链/重链错配”又带来了另一个挑战。防止重链-轻链错配的一种策略是将双特异性抗体的其中一条Fab的轻链和重链的部分结构域互换，形成Crossmab(杂交抗体)，该方法可以允许轻链/重链间选择性地配对。但这些方法的缺点是不能完全杜绝错配产物的生成，而任何错配分子的残留级分都很难从产物中分离，并且这种方法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，对于含CD3特异性的IgG样结构的双特异性抗体，因具有FcγR结合能力，可能导致无限制的、持续性激活T细胞，且这种激活是非靶细胞限制性的，无论是否与靶抗原结合，在表达FcγR的组织内(例如在造血、淋巴和网状内皮系统内)，都发现了活化的T细胞。这种T细胞的全身性激活，将伴随着细胞因子的大量释放，这是一种在T细胞激活细胞因子或抗体的治疗应用过程中的严重不良反应。因此，对于这类介导T细胞杀伤的抗CD3双特异性抗体需要避免Fc介导的T细胞全身性激活，从而允许免疫效应细胞在靶细胞组织内的限制性激活，即专一性地依赖双特异性抗体与相应靶抗原的结合。
二、人表皮生长因子受体2(Her2/ErbB2)
表皮生长因子受体家族(epidermal growth factor receptor family,ErbB family)在细胞生长、发育及分化过程中起着重要作用，其过量表达会导致细胞正常功能紊乱，常与肿瘤的发生发展密切相关。其中，人表皮生长因子受体2(human epidermal growth factor receptor 2,Her2/ErbB2)的过表达与许多上皮细胞癌的恶性化程度关系密切，Her2高表达肿瘤表现出极强的迁移性和浸润性、化疗药物敏感性差、愈后差、易复发等特点。在正常细胞中Her2表达水平极低，但在胚胎发育期表达量很高，可以自发形成同二聚受体或由配体 诱导形成异二聚受体，触发信号转导网络(Arteaga CL等,Cancer Cell,2014,25:282-303；Roskoski R Jr,Pharmacol Res,2014,79:34-74)，对细胞的增殖、分化、发育、黏附及迁移起重要的调节作用。在原发乳腺癌患者中，近20％～25％的患者存在Her2过表达。除乳腺癌外，在卵巢癌、胃癌、结直肠癌、前列腺癌和肺癌等多种癌症中也都存在Her2过表达的现象。
1998年美国FDA批准了第一个用于Her2阳性转移性乳腺癌(metastatic breast cancer,MBC)临床治疗的单克隆抗体药物曲妥珠单抗
Figure PCTCN2019113671-appb-000001
上市，该抗体识别Her2胞外IV区近膜端表位，一方面通过阻止Her-2受体二聚化来抑制Her-2介导的信号转导系统，另一方面可以激活ADCC效应来杀伤肿瘤细胞。目前，曲妥珠单抗已经成为Her-2阳性可手术乳腺癌的标准治疗方案(Romond EH等,N Engl J Med,2005,353(16):1673-1684；Perez EA等,J Clin Oncol,2011,29(34):4491-4497)。但对于Her2低表达乳腺癌患者的疗效不佳，并且有相当一部分最初抗体治疗有效的患者会在使用1年内产生耐药性(Thery JC等,Eur J Cancer,2014,50:892-901)。因而为了提高有效率，临床上抗Her2治疗通常与化疗药物联合使用，临床数据证实曲妥珠单抗与铂类、多西他赛和长春瑞滨有协同作用，与阿霉素、紫杉醇和环磷酰胺有加和作用。由于Her2表达于多种组织和器官，因此曲妥珠单抗的副作用也引起了人们的关注，常见的副反应如发热、恶心、腹泻、皮疹和头痛等，最严重的是其心脏毒性，单药治疗心功能及病理异常发生率为3％左右，而与蒽环类药物联用时发生率上升为26％～28％(显著高于单用蒽环类药物者的6.0％～9.6％)。因此临床需要研发出疗效更佳，不良反应更低的新型抗Her2药物。
针对
Figure PCTCN2019113671-appb-000002
的应答率较低、临床给药剂量较高、心脏毒性等副作用较大的问题，已利用其可变区序列开发了几种新型的Her2×CD3双特异性抗体，以期提高其治疗有效率、降低用药剂量，减少临床副反应的发生。例如，CN104558192A中公开了一种MSBODY(monomer and ScFv-Fc bispecific antibody)构型的抗Her2×CD3双特异性抗体，其单价单元可变区序列来自
Figure PCTCN2019113671-appb-000003
含有Fab和Fc结构域，单链单元为抗CD3的ScFv-Fc形式，虽然其中单价单元的重链Fc和单链单元的Fc区都经过改造，使各自不易形成同二聚体(homodimer)，而易于形成异二聚体(heterodimer)。但不可避免地，仍然会产生少量的因重链间错配形成的同二聚体抗体的副产物，这增加了纯化分离的难度。另外，它的抗CD3单链抗体融合了人IgG1的重链Fc，且保留了FcR结合能力，这也必然会增加其诱发细胞因子风暴的风险。
尽管已有针对Her2×CD3双靶点的双特异性抗体被公开，但其安全性和有效性仍需要改善，如采用人源化抗体以避免HAMA反应，同时改良CD3抗体分子以削弱其诱发细胞因子风暴的风险。然而，针对Her2的抗体与抗CD3抗体组合形成的双功能抗体是否都具有期待的生物学功能，并能有效地杀伤乳腺癌等相关肿瘤细胞是无法预料的。双特异性抗体中，需要两个功能域分别与其结合位点正确结合。而即使每个抗体单独能与其抗原正确结合，也不能保证其在形成双特异性抗体后仍能保留原来的结合能力，并且由于空间位阻的关系，不能保证形成双特异性抗体后，能顺利地形成协同作用。
综上，本领域仍需要开发一种更适宜临床应用的抗Her2×CD3的多特异性分子，能够高特异地靶向并杀死表达Her2的肿瘤细胞，尤其是低表达Her2的肿瘤细胞，同时在半衰期、稳定性、安全性和可生产性方面具有改善性能的新型双特异性分子。
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Her2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Her2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。
本发明第一方面，提供一种双特异性抗体，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Her2的第一单链Fv(抗-Her2scFv)、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Her2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。
例如，本发明表5-9中示例性的例举了一些优选的抗-Her2scFv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，针对Her2的第一单链Fv包含的VH结构域的互补决定区(HCDR1、HCDR2和HCDR3)和VL结构域的互补决定区(LCDR1、LCDR2和LCDR3)，其选自下组：
(i)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；
(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、21和22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：25、26和27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：28、29和30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，所述针对Her2的第一单链Fv包含的VH结构域和VL结构域，其选自下组：
(i)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；
(ii)VH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；和其VL结构域包含如SEQ ID NO：24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：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；和其VL结构域包含如SEQ ID NO：32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：49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：49N端的10个氨基酸，即SSSSKAPPPS(CTP 1)；或所述CTP刚性肽包含SEQ ID NO：49C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：49N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5-11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：50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50nM，或大于100nM，或大于300nM，或大于500nM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132.3nM的EC 50值与效应细胞特异性结合。
例如，本发明表5-10中示例性的例举了一些优选的抗-CD3 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。
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：33、34和35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：36、37和38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：41、42和43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：44、45和46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：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；和其VL结构域包含如SEQ ID NO：40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。
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：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；和其VL结构域包含如SEQ ID NO：48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)(根据EU编号，氨基酸序列如SEQ ID NO：54所示)。
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末端的赖氨酸可能在抗体生产过程中出现一定比例的缺失，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5-12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：55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Her2和CD3，其氨基酸序列如下：
(i)SEQ ID NO：8所示的序列；
(ii)与SEQ ID NO：8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：8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。
本发明的第二方面，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。
本发明的第三方面，提供了包含上述DNA分子的载体。
本发明的第四方面，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；
本发明的第五方面，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以及可药用赋形剂、载体或稀释剂。
示例性地，本发明一优选实施例中公开了一种包含上述双特异性抗体稳定的溶液制剂，所述溶液制剂还包含pH调节剂、稳定剂及表面活性剂；优选地，所述pH调节剂为柠檬酸盐缓冲液或组氨酸盐缓冲液，和所述稳定剂为蔗糖，和所述表面活性剂为吐温-80；更优选地，所述制剂包含0.5mg/mL的上述双特异性抗体，和20mM柠檬酸盐或组氨酸盐以及8％蔗糖(w/v)和0.02％的PS80(w/v)；所述制剂的pH为5.5。
本发明的第六方面，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。
本发明的第七方面，提供了所述双特异性抗体在制备治疗、预防或缓解肿瘤的药物中的用途；所述肿瘤的实例包括但不限于乳腺癌、前列腺癌、非小细胞肺癌、膀胱癌、卵巢癌、胃癌、结肠直肠癌、食管癌、头和颈的鳞状细胞癌瘤、子宫颈癌、胰腺癌、睾丸癌、恶性黑色素瘤和软组织癌；优选地，所述肿瘤选自乳腺癌、胃癌、直肠癌；更优选地，所述肿瘤选自乳腺癌。
本发明第八方面，提供了一种用于治疗肿瘤、延迟其进展、降低/抑制其复发的方法，其包括将有效量的所述双特异性抗体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；所述肿瘤的实例包括但不限于乳腺癌、前列腺癌、非小细胞肺癌、膀胱癌、卵巢癌、胃癌、结肠直肠癌、食管癌、头和颈的鳞状细胞癌瘤、子宫颈癌、胰腺癌、睾丸癌、恶性黑色素瘤和软组织癌；优选地，所述肿瘤选自乳腺癌、胃癌、直肠癌；更优选地，所述肿瘤选自乳腺癌。
本发明的第九方面，提供了一种用于增强或刺激免疫应答或功能的方法，其包含对患者/受试者个体施用治疗有效量的所述双特异性抗体或所述药物组合物。
本发明的第十方面，提供了一种用于治疗、预防或改善患者/受试者个体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药物组合物。
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-Her2scFv位于双抗的N端，空间构像发生变化，与Her2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Her2的正常细胞，减少了非特异性杀伤，但对过表达或高表达Her2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。临床前毒理学评价试验的最大安全起始剂量已大大高于其它同靶点剂量，且没有发生系统性免疫毒性，提示本发明提供的双特异性抗体的用药安全窗较宽。
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50nM，或大于100nM，或大于300nM，或大于500nM)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，药代动力学试验表明其在小鼠和食蟹猴体内的循环半衰期分别约40h和8h，这将大大降低其临床给药频率。
发明详述
缩写和定义
Her2    人表皮生长因子受体2
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。
“Her2(人类表皮生长因子受体2)”是人类表皮生长因子受体家族成员，许多肿瘤的发生、发展和病情轻重与其活性大小密切相关。除了可发生基因突变或扩增，her2的上调还可激活下游两个信号转导通路，引发瀑布式连锁反应，促进细胞无限增殖，最终导致癌症。此外，her2可启动多种转移相关机制增加肿瘤细胞转移能力。Her2基因的扩增或过表达发生在例如乳腺癌、卵巢癌、胃癌、肺腺癌、前列腺癌、侵袭性子宫癌等多种肿瘤中。针对Her2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Her2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Her2基因或cDNA转染的细胞表达。物种同源物包括恒河猴Her2。
“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集聚(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。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。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“二硫键稳定性蛋白(dsFv)”在VH和VL区分别引入一个半胱氨酸突变点，从而在VH和VL之间形成二硫键而实现结构稳定性。
术语“抗体片段”或“抗原结合片段”是指保留与抗原(如，Her2)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“轻链恒定区”包括来自抗体轻链的氨基酸序列。优选地，所述轻链恒定区包括恒定kappa结构域和恒定lambda结构域中的至少一个。
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。
“结合”定义抗原上的特定表位与其对应抗体之间的亲和性相互作用，一般也理解为“特异性识别”。 “特异性识别”的意思是本发明的双特异性抗体不与或基本上不与目标抗原以外的任意多肽交叉反应。和特异性的程度可以通过免疫学技术来判断，包括但不限于免疫印迹，免疫亲和层析，流式细胞分析等。在本发明中，特异性识别优选通过流式细胞技术来确定，而具体情况下特异性识别的标准可由本领域一般技术人员根据其掌握的本领域常识来判断。
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“分离的抗体分子”指的是已经从其自然环境的组分中识别和分离和/或回收的抗体分子。其自然环境的污染组分是会干扰抗体的诊断或治疗用途的物质，并可能包括酶、激素和其他蛋白质性的或非蛋白质性的溶质。
本文使用的关于细胞、核酸(如DNA或RNA)的术语“分离的”，是指分别从其他的以天然来源的大分子存在的DNA或RNA分离的分子。本文使用的术语“分离的”也指通过重组DNA技术生产时基本不含细胞材料，病毒材料或培养基的核酸或肽，或经化学合成制备时基本不含化学前体或其他化学品。此外，“分离的核酸”是指包括非自然产生为片段的核酸片段，且这些片段自然状态下不存在。本文中术语“分离的”也用于指从其他细胞蛋白或组织分离的细胞或多肽。分离的多肽是指包括纯化的和重组的多肽。
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 Biotechnology,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)和(Kim YJ等,J.Immunol.,24:249,1994))。
术语“功能域”指的是能够特异性识别和/或结合到表位上的三维结构，例如抗体或抗体片段，包括天然完整抗体、单链抗体(scFv)、Fd片段、Fab片段、F(ab’) 2片段、单结构域抗体片段、分离的CDR片段及其衍生物。此处“单链”意思是第一和第二功能域共价连接，优选地以一个核酸分子可编码的共线性氨基酸序列行形成。
术语“结构域抗体”是仅含有重链的可变区或轻链的可变区的免疫功能免疫球蛋白片段。在一些情况 下，两个或更多个VH区用肽接头共价接合，以产生二价结构域抗体。二价结构域抗体的两个VH区可靶向相同或不同的抗原。
术语“单克隆抗体(mAb)”指获自基本均一抗体群体的抗体，即除了少数出现可能的天然产生突变外，群体包含的单独抗体是相同的，对特定表位显示单一结合特异性和亲和力。修饰语“单克隆”表示从实质上一致的抗体组中获得的抗体的性质，不需要通过特定方法生产抗体。单克隆抗体由本领域技术人员所知晓的方法产生，例如通过将骨髓瘤细胞和免疫脾细胞融合制备杂合的抗体产生细胞。通过培养杂交瘤合成，不会污染任何其他抗体。单克隆抗体也可以用如重组技术、噬菌体展示技术、合成技术，或其他现有技术进行重组得到。
术语“嵌合抗体”是指重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性(美国专利No 4,816,567；Morrison SL等,Proc.Natl.Acad.Sci.USA,81:6851-6855,1984)。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
术语“完整抗体”是指由两条抗体重链和两条抗体轻链组成的抗体。“完整抗体重链”是在N-端到C-端方向上由抗体重链可变结构域(VH)、抗体恒定重链结构域1(CH1)、抗体铰链区(HR)、抗体重链恒定结构域2(CH2)和抗体重链恒定结构域3(CH3)组成，缩写为VH-CH1-HR-CH2-CH3；并且在IgE亚类的抗体的情形中，任选地还包括抗体重链恒定结构域4(CH4)。优选地“完整抗体重链”是在N-端到C-端方向上由VH、CH1、HR、CH2和CH3组成的多肽。“完整抗体重链”是在N-端到C-端方向上由抗体轻链可变结构域(VL)和抗体轻链恒定结构域(CL)组成的多肽，缩写为VL-CL。所述抗体轻链恒定结构域(CL)可以是κ(kappa)或λ(lambda)。完整抗体链通过在CL结构域和CH1结构域之间(即轻链和重链之间)的多肽间二硫键和完整抗体重链的铰链区之间的多肽间二硫键连接在一起。典型的完整抗体的实例是天然抗体如IgG(例如，IgG1和IgG2)、IgM、IgA、IgD和IgE。
术语“人源化抗体”是指经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。非人抗体的CDR域外的大部分或全部氨基酸，例如小鼠抗体被来自人免疫球蛋白的相应氨基酸置换，而一个或多个CDR区内的大部分或全部氨基酸未改变。小分子氨基酸的添加，删除，插入，替换或修饰是允许的，只要它们不会消除抗体结合特定抗原的能力。“人源化”抗体保留与原始抗体类似的抗原特异性。CDR的来源没有特别限制，可来源于任何动物。例如，可以利用源于小鼠抗体、大鼠抗体、兔抗体或非人灵长类动物(例如，食蟹猴)抗体。可用于本公开的人类框架的实例是KOL、NEWM、REI、EU、TUR、TEI、LAY和POM(Kabat等人，同上)。例如，KOL和NEWM可以用于重链，REI可以用于轻 链和EU、LAY和POM可以用于重链和轻链二者。或者，可以使用人种系序列；这些可在以下网址获得：http://www2.mrc-lmb.cam.ac.uk/vbase/list2.php。在本公开的人源化抗体分子中，受体重链和轻链不一定需要衍生自相同的抗体，并且如果需要，可以包含具有衍生自不同链的框架区的复合链。
术语“人”抗体(HuMab)是指具有可变区的抗体，其中构架区和CDR区均源自人种系免疫球蛋白序列。此外，如果抗体含有恒定区，恒定区也来源于人种系免疫球蛋白序列。本发明的人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，如本文所用，术语“人抗体”不打算包括其中来源于另一种哺乳动物物种如小鼠的种系的CDR序列已经嫁接到人框架序列上的抗体。
术语“表位”或“抗原决定簇”指具有抗原性(即可诱发特异性免疫应答)的分子上的特定化学基团或肽序列，是免疫球蛋白或抗体特异性结合的抗原(例如Her2)上的位点。免疫球蛋白或抗体特异性结合多肽(例如Her2)上的特定抗原表位。OK表位决定区通常由分子的化学活性表面基团(如氨基酸或糖基侧链)组成且通常有特定的三维结构性质以及特定的电荷性质。
术语“重组”，在涉及多肽或多核苷酸时指自然状态下不存在的多肽或多核苷酸的形式，其中一个非限制性的例子，可以通过将通常不会一起出现的多核苷酸或多肽组合在一起来实现。
术语“缀合”、“连接”是指两个或更多个分子的缔合。连接也可以是遗传的(即重组融合)。在具体上下文中，所述术语包括提及连接配体例如抗体部分与效应分子。所述连接这种连接可以使用多种本领域公认的技术来实现，例如可通过化学或重组的方式。“化学方式”是指所述抗体部分与效应分子之间的反应，使所述两个分子之间形成共价键以形成一个分子。
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“细胞毒剂”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“EC 50”是指在使用抗体或其抗原结合片段进行的体外或体内测定中，诱导50％应答的抗体或其抗原结合片段的浓度的最大响应，即在最大响应和基线之间的一半。
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。
术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞，例如淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
“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“免疫原性”指特定物质引发免疫应答的能力。肿瘤是免疫原性的，并且增强肿瘤免疫原性有助于通过免疫应答清除肿瘤细胞。增强肿瘤免疫原性的例子包括但不限于用某些抑制剂(例如双特异性抗体)处理。
术语“免疫疗法”是指通过包括诱导，增强，抑制或以其他方式修饰免疫应答的方法来治疗患有或有患病风险或疾病复发的受试者。
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“肿瘤免疫”指肿瘤逃避免疫识别和清除的过程。如此，作为治疗概念，肿瘤免疫在此类逃避减弱时得到“治疗”，并且肿瘤被免疫系统识别并攻击。肿瘤识别的例子包括肿瘤结合，肿瘤收缩和肿瘤清除。
术语“宿主细胞”在其中载体可以增殖并且其DNA可以表达的细胞，所述细胞可以是原核细胞或者真核细胞。该术语还包括受试宿主细胞的任何后代。应理解，并不是所有的后代都与亲本细胞相同，因为在复制过程中可能会发生突变，这类后代被包括在内。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。
术语“载体”是指能够运输与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指其中可以连接另外的DNA区段的环状双链DNA环。另一种类型的载体是病毒载体，其中额外的DNA区段可以连接到病毒基因组中。某些载体能够在它们被导入的宿主细胞中自主复制(例如，具有细菌复制起点和游离型哺乳动物载体的细菌载体)。其他载体(例如非附加型哺乳动物载体)可以在导入宿主细胞后整合到宿主细胞的基因组中，并由此与宿主基因组一起复制。此外，某些载体能够指导它们有效连接的基因的表达。这种载体在本文中被称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常以质粒的形式存在。然而，也包括其他形式的表达载体，如病毒载体(例如，复制缺陷型逆转录病毒，腺病毒和腺伴随病毒)，其起到等同的功能。
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、柠檬酸盐缓冲液和组氨酸盐缓冲液。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。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“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤或感染)在受试者体内的发生或如果它发生作用减到最小而实施的方法。术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。有益或所需的临床结果包括但不限于，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后，无论是可检测或是不可检测的。有效缓解任何特定疾病症状的治疗剂的量可以根据诸如患者的疾病状态，年龄和体重以及药物在受试者中引起期望的反应的能力等因素而变化。疾病症状是否得到缓解可以通过任何临床测量来评估，这些测量通常由医生或其他熟练的医疗保健提供者用于评估该症状的严重程度或进展状态。
术语“细胞毒性”分子例如免疫毒素对意欲靶向的细胞的毒性，而不是对生物其他细胞的毒性。
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。
术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤或感染)有效量是指，当单独使用或与另一种或多种治疗剂组合使用时，足以预防，阻止，或延迟疾病(例如，肿瘤或感染)的发生的量；治疗疾病有效量是指，当单独使用或与另一种或多种治疗剂组合使用时，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。术语关于治疗的“有效”和“有效性”包括药理学有效性和生理学安全性二者。药理学有效性是指药物促进患者病症或症状消退的能力。生理学安全性是指由于药物施用导致的细胞、器官和/或生物体水平上的毒性或者其它不良生理效果(不良作用)的水平。
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“位点”是本文描述的氨基酸序列内的氨基酸位点。这个位点可以相对于类似天然序列例如天然存在的IgG结构域或链的序列指示。本文使用的术语“相应的”还包括不一定或不仅取决于前面的核苷酸或氨基酸的数量的位点。因此，可能被置换的根据本发明的给定氨基酸的位点可由于在抗体链中别处缺失或添加氨基酸而变。
术语“结合蛋白”是指以高亲和力特异性结合特定的部分或靶标的蛋白质。结合蛋白的实例包括，但不仅限于，抗体、抗体的抗原结合片段、adnectin、微型抗体、亲和体(affibodies)、affilin、受体的靶结合区、细胞粘附分子、配体、酶、细胞因子、和趋化因子。在本发明的优选实施方案中，结合蛋白包含抗体的Fc区。
术语“补体系统”指在血液中发现的大量的小蛋白质——称为补体因子，其通常以不活跃的前体(前-蛋白)循环。这一术语指的是这不变和不适应性系统的下述能力：“补充”抗体和吞噬细胞从生物体中清除病原体如细菌以及抗原-抗体复合物的能力。补体因子的一个实例是复合物C1，其包含C1q和两个丝氨酸蛋白酶C1r和C1s。复合物C1是CDC途径的组分。C1q是分子量约为460,000的六价分子，且其结构类似 郁金香的花束，其中六个胶原“茎”连接至六个球状的头部区域。为了激活补体级联，C1q必须结合至IgG1、IgG2或IgG3的至少两个分子。
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Her2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Her2×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)结合亲和力增强的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。
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。例如，本领域技术人员可以进行适当的ELISA测定。如实施例5.6中所阐述，适当的ELISA测定使得能够比较变体和亲本与FcRn的结合强度。在PH为7.0时，比较针对变体和亲本多肽检测到的特异性信号，如果变体的特异性信号比亲本多肽的特异性信号弱至少1.9倍，则它是本发明的优选的变体，更适于临床应用。
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、抗体片段和缀合物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。
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(Bacilisstearothermophilus))、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。
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免疫缀合物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。
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免疫缀合物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)。
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Her2xCD3)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体片段(或免疫缀合物)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：癌症、淋巴系统疾病、自身免疫性疾病、炎症性疾病、感染性疾病、免疫缺陷综合症以及其他相关疾病或症状。
可被靶向的TAA包括但不限于上文所述。
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，所述治疗剂例如毒素、细胞毒剂、放射性同位素、免疫治疗剂或疫苗。治疗剂包括但不限于抗肿瘤剂，诸如多柔比星(阿霉素)、顺铂博来霉素硫酸盐、亚硝基脲氮芥、苯丁酸氮芥和环磷酰胺羟脲，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。
本发明的双特异性抗体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。靶向的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。
本发明的双特异性抗体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)。
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。
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。
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免疫缀合物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免疫缀合物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免疫缀合物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免疫缀合物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治疗可以与可有效针对待治疗病症的其他治疗组合。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。
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。
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免疫缀合物或药物组合物或组合疗法可用于与本文所述的癌症、自身免疫性疾病、炎症性疾病、淋巴系统疾病、感染或感染性疾病、免疫缺陷综合症及其他相关疾病或症状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或免疫缀合物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或可用于制备免疫缀合物。所述免疫缀合物包括但不限于上文免疫缀合物所述。所述连接方式包括但不限于上文免疫缀合物所述。
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或免疫缀合物可用于制备药物。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或免疫缀合物可单独使用，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或免疫缀合物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免疫缀合物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免疫缀合物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免疫缀合物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免疫缀合物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癌症、自身免疫性疾病、炎症性疾病、淋巴系统疾病、感染或感染性疾病、免疫缺陷综合症及其他相关疾病或症状。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免疫缀合物或药物组合物治疗，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、淋巴系统疾病或感染或感染性疾病)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免疫缀合物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或免疫缀合物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，双特异性抗体或免疫缀合物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免疫缀合物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免疫缀合物或药物组合物，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免疫缀合物或药物组合物。构成治疗有效剂量的双特异性抗体或编码本申请抗体的多核苷酸或免疫缀合物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，其包括本发明所述抗体或其抗原结合片段和/或使用说明书。本发明所述免疫缀合物可用于制备诊断性或治疗性试剂盒，其包括本发明所述免疫缀合物和/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.双特异性抗体AB7K、AB7K4、AB7K5、AB7K6、AB7K7和AB7K8的构型分别如a、b、c、d、e和f所示。
图1-2、显示了双特异性抗体AB7K7表达质粒图谱。该表达质粒全长9293bp，含有9个主要基因片断，包括1.hCMV启动子；2.目标基因；3.EMCV IRES；4.mDHFR筛选基因；5.Syn中止序列；6.SV40启动子；7.卡拉霉素抗性基因；8.SV40中止序列；9.PUC复制子。
图1-3、AB7K7纯化样品的SEC-HPLC检测结果。
图1-4、AB7K7纯化样品的SDS-PAGE电泳结果。
图1-5、AB7K7在25℃加速实验的SDS-PAGE结果。
图1-6、AB7K7在冻融实验的SDS-PAGE结果。
图2-1、FACS检测双特异性抗体AB7K和AB7K4与肿瘤细胞BT474结合的能力。
图2-2、FACS检测双特异性抗体AB7K和AB7K5与肿瘤细胞BT474结合的能力。
图2-3、FACS检测双特异性抗体AB7K和AB7K6与肿瘤细胞BT474结合的能力。
图2-4、FACS检测双特异性抗体AB7K和AB7K7与肿瘤细胞BT474结合的能力。
图2-5、FACS检测双特异性抗体AB7K8与肿瘤细胞BT474结合的能力。
图2-6、FACS检测双特异性抗体AB7K和AB7K4与效应细胞CIK结合的能力。
图2-7、FACS检测双特异性抗体AB7K和AB7K5与效应细胞CIK结合的能力。
图2-8、FACS检测双特异性抗体AB7K6与效应细胞CIK结合的能力。
图2-9、FACS检测双特异性抗体AB7K和AB7K7与效应细胞CIK结合的能力。
图2-10、FACS检测双特异性抗体AB7K8与效应细胞CIK结合的能力。
图2-11、FACS检测双特异性抗体AB7K与食蟹猴T细胞结合的能力。
图2-12、ELISA检测5种Anti-Her2×CD3双特异性抗体与CD3和Her2分子结合的能力。
图2-13、酶标仪检测5种Anti-Her2×CD3双特异性抗体活化报告基因细胞株Jurkat T细胞的能力。
图2-14、CTP连接肽与抗-CD3 scFv VH结构建模。
图2-15、GS连接肽与抗-CD3 scFv VH结构建模。
图2-16、抗-CD3 Fv与CD3 epsilon链的分子对接模型。
图3-1、双抗AB7K4和AB7K7在NCG小鼠皮下共接种人CIK细胞和HCC1954细胞的移植瘤模型中的体内抑瘤效果。
图3-2、双抗AB7K7在NPG小鼠皮下共接种人CIK细胞和人乳腺癌细胞HCC1954的移植瘤模型中的体内抑瘤效果。
图3-3、在不同给药频次下双抗AB7K7和AB7K8在NPG小鼠皮下共接种人CIK细胞和人乳腺癌细胞HCC1954的移植瘤模型中的体内抑瘤效果。
图3-4、双抗AB7K7在NPG小鼠皮下共接种人CIK细胞和SK-OV-3细胞的移植瘤模型中的体内抑瘤效果。
图3-5、双抗AB7K7在NPG小鼠皮下共接种人CIK细胞和HT-29细胞的移植瘤模型中的体内抑瘤效果。
图3-6、双抗AB7K7在CD34免疫重建的NPG小鼠皮下接种人乳腺癌HCC1954细胞的移植瘤模型中的体内抑瘤效果。
图3-7、双抗AB7K7在PBMC免疫重建的NPG小鼠接种人乳腺癌HCC1954细胞的小鼠移植瘤模型中的体内抑瘤效果。
图4-1、双抗AB7K4和AB7K7在NCG小鼠皮下共接种人CIK细胞和人Burkkit’s淋巴瘤Raji细胞的移植瘤模型中的抑瘤效果。
图4-2、双抗AB7K7在NPG小鼠皮下单独接种人乳腺癌HCC1954细胞的移植瘤模型中的抑瘤效果。
图4-3、正常食蟹猴多次给予双抗AB7K7和AB7K8的体重变化曲线。
图5-1、双抗AB7K7在SD大鼠中用两种ELISA方法检测时的药时曲线。
图5-2、双抗AB7K8在SD大鼠中用两种ELISA方法检测时的药时曲线。
图5-3、双抗AB7K7和AB7K8在食蟹猴体内的药时曲线。
图5-4、pH 6.0时双抗AB7K、AB7K5和AB7K7与FcRn结合能力测定。
图5-5、pH 7.0时双抗AB7K、AB7K5和AB7K7与FcRn结合能力测定。
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、不同结构的Anti-Her2×CD3双特异性抗体的设计和制备
1.1、不同结构双特异性抗体的设计
为了筛选到适宜构型的双特异性抗体，我们针对Her2和CD3设计了六种不同构型的双特异性抗体，其中AB7K5、AB7K6和AB7K8为单链二价双特异性抗体，AB7K、AB7K4和AB7K7为双链四价双特异性 抗体(参见图1-1)，其中仅AB7K8不含Fc片段。具体地，上述四种构型的双特异性抗体的构型及其从N端至C端方向的组成及其氨基酸序列编号如表1-1所示。六种双特异性抗体的具体结构组成特性描述如下：
其中，双特异性抗体AB7K是由抗Her2的全长抗体的两条重链的C端通过连接肽(L1)各自连接一个抗CD3的scFv结构域所组成。AB7K包含的针对Her2的完整抗体的氨基酸序列参照单克隆抗体
Figure PCTCN2019113671-appb-000004
的序列(IMGT数据库INN 7637)，其所包含的Fc片段来自人IgG1，且具有D356E/L358M突变(EU编号)。其连接肽L1由柔性肽和刚性肽组成，且柔性肽组成为GS(GGGGS) 3，刚性肽为SSSSKAPPPSLPSPSRLPGPSDTPILPQ；其中抗CD3 scFv的VH和VL之间的连接肽L2的组成为(GGGGS) 3。
其中，双特异性抗体AB7K4是由抗Her2的全长抗体的两条轻链的C端通过连接肽(L1)各自连接一个抗CD3的scFv结构域所组成。AB7K4包含的针对Her2的完整抗体的重链可变区氨基酸序列参照单克隆抗体
Figure PCTCN2019113671-appb-000005
的可变区序列，其轻链氨基酸序列参照单克隆抗体
Figure PCTCN2019113671-appb-000006
的轻链氨基酸序列(IMGT数据库INN 7637)。AB7K4重链包含的Fc片段来自人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L1由柔性肽和刚性肽组成，且柔性肽组成为G 2(GGGGS) 3，刚性肽为SSSSKAPPPS；其中抗CD3 scFv的VH和VL之间的连接肽L2的组成为(GGGGS) 3。
其中，双特异性抗体AB7K5是由抗-Her2scFv、Fc片段、连接肽L2和抗-CD3 scFv依次串联组成，抗-Her2scFv和抗-CD3 scFv内部VH和VL之间分别由连接肽L1和L3连接。AB7K5所包含的针对Her2的scFv的氨基酸序列参照单克隆抗体
Figure PCTCN2019113671-appb-000007
的可变区序列。AB7K5所包含的Fc片段来自人IgG1，且具有多个氨基酸的替换/取代，分别为C226S、C229S、L234A、L235A、T250Q、N297A、P331S、T366R、L368H、K409T和M428L(EU编号)。其中，C226S、C229S、T366R、L368H和K409T这5个位点的突变，可以防止Fc片段间发生聚合，从而促使其形成单链二价双特异性抗体；携带L234A/L235A/P331S突变的Fc片段去除了ADCC和CDC活性；携带T250Q/M428L突变可以增强Fc片段与受体FcRn的结合亲和力，从而延长其半衰期；N297A突变避免了抗体糖基化，且丧失对FcγRs的结合能力。另外，还删除/缺失了Fc片段C末端的K447(EU编号)，消除了抗体的电荷异质性。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。
其中，双特异性抗体AB7K6是由抗-Her2scFv、连接肽L2、抗-CD3 scFv和Fc片段依次串联组成，抗-Her2scFv和抗-CD3 scFv内部VH和VL之间分别由连接肽L1和L3连接。AB7K6所包含的Fc片段来自人IgG1，且具有多个氨基酸的替换/取代，分别为C226S、C229S、L234A、L235A、T250Q、N297A、P331S、T366R、L368H、K409T和M428L(EU编号)。其中，C226S、C229S、T366R、L368H和K409T这5个位点的突变，可以防止Fc片段间发生聚合，从而促使其形成单链二价双特异性抗体；携带L234A/L235A/P331S突变的Fc片段去除了ADCC和CDC活性；携带T250Q/M428L突变可以增强Fc片段与受体FcRn的结合 亲和力，从而延长其半衰期；N297A突变避免了抗体糖基化，且丧失对FcγRs的结合能力。另外，还删除/缺失了Fc片段C末端的K447(EU编号)，消除了抗体的电荷异质性。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。
其中，双特异性抗体AB7K7是由抗-Her2scFv、连接肽L2、抗-CD3 scFv和Fc片段依次串联组成，抗-Her2scFv和抗-CD3 scFv内部VH和VL之间分别由连接肽L1和L3连接。AB7K7包含的针对Her2的scFv的氨基酸序列参照单克隆抗体
Figure PCTCN2019113671-appb-000008
的可变区序列。AB7K7所包含的Fc片段来自人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。
其中，双特异性抗体AB7K8是由抗-Her2scFv、连接肽L2、抗-CD3 scFv和His标签依次串联组成，抗-Her2scFv和抗-CD3 scFv内部VH和VL之间分别由连接肽L1和L3连接。AB7K8包含的针对Her2的scFv的氨基酸序列参照单克隆抗体
Figure PCTCN2019113671-appb-000009
的可变区序列。AB7K8在抗-CD3 scFv的C末端添加His标签，组成为HHHHHHHH，以便于抗体纯化。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。
上述六种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：247和SEQ ID NO：248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。
表1-1：四种不同结构的针对Her2和CD3的双特异性抗体
Figure PCTCN2019113671-appb-000010
备注：表中Ln代表不同结构单元之间的连接肽，n是以从双特异性抗体从N端至C端不同结构单元间所包含的连接肽的排列顺序依次编号。
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五种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，其中AB7K和AB7K4的重链和轻链可以构建到一个载体中，或者分别构建在两个不同的载体上。例如，将编码AB7K7的cDNA序列(如SEQ ID NO：56所示)插入图1-2所示的表达质粒中，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。
下文分别对几种构型的双特异性抗体的纯化工艺、稳定性、体外和体内生物学功能、安全性及药代动力学等进行成药性评价，以筛选出适宜构型的双特异性抗体。
1.4、双特异性抗体的纯化工艺及稳定性试验
抗体纯化一般采用三步纯化策略：粗纯(样品捕获)、中间纯化和精细纯化。在粗纯阶段，通常利用亲和层析对目的抗体进行捕获，可有效去除样品中的大量杂质，如杂蛋白和核酸、内毒素和病毒。中间纯化步骤较多地采用疏水层析或CHT羟基磷灰石层析以去除大部分的残留的杂质蛋白以及聚合体。精细纯化多采用离子交换层析或凝胶过滤层析(分子筛)以去除与目的抗体性质相近的残留的少量或微量的杂质蛋白，并进一步去除HCP、DNA等污染物。
本发明可以利用金属鳌合亲和层析柱(例如GE公司的HisTrap FF等)对融合His-tag的双特异性抗体AB7K8的培养上清进行粗纯。可以利用Protein A/G亲和层析柱(例如GE公司的Mabselect SURE等)对包含Fc的双特异性抗体AB7K4、AB7K5、AB7K6、AB7K和AB7K7进行粗纯。上述粗纯产物再经过中间纯化及精细纯化步骤，最终获得高纯度、高质量的纯化目的抗体，然后利用脱盐柱(例如GE公司的HiTrap desaulting等)将上述双特异性抗体保存缓冲液置换为PBS或其它合适的缓冲液。
(a)双链四价双特异性抗体AB7K7的纯化
以AB7K7为例，阐明该类四价同源二聚体构型双特异性抗体的具体纯化步骤和方案。
我们采用三步层析法对双特异性抗体AB7K7进行纯化。分别为亲和层析、疏水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GE公司的MabSelect Sure亲和层析介质或其它市售的亲和介质(例如博格隆公司的Diamond protein A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，140mM 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/m；上样完毕后，使用平衡buffer(20mM PB，140mM 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(40mM 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，疏水层析：使用博格隆公司的Butyl HP或其它市售的疏水层析介质(例如GE的Butyl HP等)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，包括电荷特性以及疏水性，我们使用疏水性的差异对二者进行分离。首先，使用平衡buffer(20mM PB，0.3M(NH 4) 2SO 4，pH 7.0)，以100-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；第二步阴离子交换层析分离得到的目标蛋白用2M(NH 4) 2SO 4溶液调电导40-50ms/cm，然后上样，载量控制在<20mg/ml；上样完毕后，使用平衡buffer(20mM PB，0.3M(NH 4) 2SO 4，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，pH 7.0)，分别以40％、80％和100％洗脱buffer，以不高于100cm/h的线性流速洗脱3-5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90％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，分离结构变异体、进一步去除HCP、DNA等污染物。首先使用平衡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)；经第二步疏水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(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)；对流穿组分进行分段收集，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电泳结果分见图1-3和图1-4，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(180KDa)，还原后可得清晰的(90KDa)单链条带。
(b)单链二价双特异性抗体AB7K5和AB7K6的纯化
以Protein A亲和层析和羟基磷灰石(CHT)层析对AB7K5进行纯化，经SEC-HPLC检测发现其纯度较低，且收率不高，还存在表达产量极低的问题。
另一单链二价双特异性抗体AB7K6同样存在工艺开发难度大的问题，AB7K6以Protein A亲和层析和分子筛层析Superdex 200进行两步纯化后，经SEC-HPLC检测后发现其纯度较难定量，主峰中有明显的“肩膀峰”；此外，其表达产量极低、且非常不稳定，在4℃冰箱中放置24h后，SEC-HPLC分析显示其峰形变化，由两个峰变成一个主峰，根据出峰时间推测可能是它由单链转换成双链结构所致。综上可知，AB7K6目前的工艺开发难度较大，难以实现工艺放大和产业化。
综上，AB7K7相对于AB7K5和AB7K6，在工艺开发方面具有显著优势，具有产量高、纯化方法简单高效、下游工艺稳定等优点。我们还进一步考察了AB7K7在不同缓冲液体系、和不同储存条件下的理化稳定性。
c)双特异性抗体AB7K7稳定性试验
分别对AB7K7蛋白在柠檬酸盐(20mM柠檬酸盐、pH 5.5)和组氨酸盐缓冲体系(20mM组氨酸盐、pH 5.5)中的稳定性进行考察。将AB7K7蛋白在25℃的加速条件下贮存4周，对蛋白的稳定性进行评估。
将AB7K7蛋白分别换液至上述柠檬酸盐(F2)和组氨酸盐(F3)缓冲液中，调节浓度至0.5mg/mL，上述两种缓冲体系中均加入了8％蔗糖(w/v)和0.02％PS80(w/v)作为辅料。以0.22μm PES膜针式滤 器过滤，各自分装至2mL的西林瓶中，每瓶0.8mL，分装完成后立即压塞轧盖。根据表1-2方案将样品放入不同的稳定性测定箱中，于每个取样点取出样品进行检测分析，检测项目包括样品外观、浓度、SEC-HPLC检测样品纯度、HMW％和LMW％以及浊度测定(A340)。
表1-2：稳定性测试方案
Figure PCTCN2019113671-appb-000011
备注：X＝外观、浓度、SEC-HPLC、SDS-PAGE(还原&非还原)；Y＝浊度(A340)
2种制剂配方在25℃贮存0～4周后的外观、浓度、浊度及SEC-HPLC检测结果见表1-3和表1-4，SDS-PAGE(还原/非还原)结果见图1-5。2个处方的外观、浓度结果均无明显变化；在SEC-HPLC结果中，F2和F3两个配方的SEC结果未发生明显变化，4周后纯度分别为97.9％和98.2％。SDS-PAGE(还原/非还原)结果与LMW％结果趋势基本一致，F2和F3变化较轻微。
表1-3：25℃加速实验外观、浓度、浊度结果
Figure PCTCN2019113671-appb-000012
*T0浊度检测样品为经历1轮冻融后样品
表1-4：25℃加速实验SEC-HPLC结果
Figure PCTCN2019113671-appb-000013
为了解AB7K7蛋白在2种缓冲体系中的解折叠温度，通过DSF的方式检测了2种处方中的Tm(解折叠温度)与Tmonset(蛋白开始解折叠的温度)，结果见表1-5。2个处方的Tmonset值均较低，F2和F3的Tmonset值均小于45℃。
表1-5：DSF结果
  Tmonset(℃) Tm1(℃) Tm2(℃)
F2 42.0 46.0 60.5
F3 41.0 45.0 58.0
同时还进行了3轮冻融实验，考察冻融(-70℃/室温，反复冻融3次)过程中AB7K7蛋白在上述2种缓冲体系中的稳定性情况，样品准备及测试方案同上。
样品外观、浓度、浊度和SEC-HPLC检测结果见表1-6，SDS-PAGE(还原/非还原)结果见图1-6。在SDS-PAGE(非还原)结果中，F2和F3两个处方经历3轮冻融后，各检项结果无明显变化。
表1-6：冻融实验外观、浓度、浊度与SEC-HPLC结果
Figure PCTCN2019113671-appb-000014
实施例二、Anti-Her2×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Her2阳性肿瘤细胞BT-474的结合活性
培养Her2表达阳性的肿瘤细胞BT-474细胞(上海中国科学院细胞库)，用0.25％胰酶消化，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，用含1％BSA的PBS溶液(PBSB)洗3遍，加入稀释好的AF488标记的山羊抗人IgG抗体或鼠抗6×His 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BT-474结合的EC 50值。
结果显示，不同结构的双特异性抗体和Her2过表达肿瘤细胞均具有良好的结合活性。图2-1～图2-5展示了不同结构的双特异性抗体和肿瘤细胞BT474的结合曲线。根据表2-1所示，AB7K和AB7K4与肿瘤细 胞结合的EC 50在5nM左右，AB7K7与肿瘤细胞结合的EC 50接近50nM，AB7K5和AB7K8与肿瘤细胞结合的EC 50为100nM，而AB7K6与肿瘤细胞BT474结合的EC 50高达200nM以上。
表2-1：Anti-Her2×CD3双特异性抗体与肿瘤细胞BT474结合能力的测定
  AB7K AB7K4 AB7K5 AB7K6 AB7K7 AB7K8
EC 50(nM) 5.009 4.388 125.0 239.9 51.98 125.3
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 OKT3活化24h后，加入250IU/ml IL-2扩增培养7天，制备得到CIK细胞(Cytokine-Induced Killer cells)，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OriginPro 8进行分析，计算各双特异性抗体与人CIK细胞结合的EC 50值。
结果显示各双特异性抗体与CIK细胞的结合存在较大差异(图2-6～图2-10)。如表2-2所示，AB7K的EC 50约20nM，AB7K4的结果与其相当，AB7K7与其相差6倍以上，AB7K5、AB7K6、AB7K8与其均相差10倍以上。
表2-2：Anti-Her2×CD3双特异性抗体与效应细胞CIK结合能力的测定
  AB7K AB7K4 AB7K5 AB7K6 AB7K7 AB7K8
EC 50(nM) 20.51 19.44 375.2 241.7 132.3 504.1
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，接下来的实验过程与上述实施例相同。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OriginPro 8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2-11所示，双特异性抗体AB7K与食蟹猴T细胞也能很好的结合，并且其与食蟹猴T细胞结合的能力和人T细胞结合的能力大致相当，流式细胞仪检测其结合的EC 50大约在26nM。双特异性抗体AB7K4、AB7K5、AB7K6、AB7K7和AB7K8同AB7K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Her2的结合。
将Her2蛋白(北京义翘神州，货号10004-H08H4)以PBS稀释成0.1μg/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，4倍梯度稀释，共11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1h。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OriginPro 8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Her2分子，并且随抗体浓度的变化呈现良好的剂量依赖性(图2-12)。几种双特异性抗体与可溶CD3和Her2的结合能力如表2-3所示，其EC 50值从0.03nM至3.8nM，相差了两个数量级。其中，AB7K的结合活性最好，AB7K4和AB7K7与其相差一个数量级，AB7K5和AB7K8的结合活性最弱。
表2-3：Anti-Her2×CD3双特异性抗体与CD3和Her2分子结合能力的测定
  AB7K AB7K4 AB7K5 AB7K7 AB7K8
EC 50(nM) 0.03128 0.1518 1.004 0.1398 3.815
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2-13的实验结果表明，靶向Her2的单抗Herceptin并不能被活化Jurkat T细胞。只有当两个抗体都存在的情况下，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，其中AB7K4活化T细胞的能力最强，AB7K8活化T细胞的能力最弱，其EC 50值相差一个数量级。
表2-4：Anti-Her2×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
  AB7K AB7K4 AB7K5 AB7K7 AB7K8 Herceptin
EC 50(nM) 0.02263 0.01338 0.05357 0.08952 0.1575 0.009907
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SK-BR-3、MCF-7、HCC1937、NCI-N87、HCC1954细胞(均购自上海中科院细胞库)作为靶细胞，用0.25％的胰酶消化，制备单细胞悬液，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，96孔板弃上清，用PBS洗3遍，加入含10％CCK-8完全培养基，100μl/孔，37℃孵育4h，用酶标仪检测A450-620nm的吸光值。通过软件OriginPro 8进行分析，计算并比较各双特异性抗体和同靶点单抗Herceptin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中，结果显示各双特异性抗体对Her2高表达的肿瘤细胞(例如SK-BR-3、NCI-N87和HCC1954)均呈现非常显著的杀伤作用，并且呈剂量依赖性。各双特异性抗体(尤其是AB7K7)对于低表达Her2的乳腺癌细胞MCF-7也表现出较好的杀伤效果。对于Herceptin耐药的细胞株HCC1954，各双特异性抗体也具有很好的杀伤作用，而对于Her2表达阴性(极少量表达)的细胞株HCC1937，各双特异性抗体只有在最高的两个浓度下才表现出杀伤作用。
表2-5：双特异性抗体介导PBMC杀伤不同肿瘤细胞的EC 50值
Figure PCTCN2019113671-appb-000015
备注：～表示约等于；-表示未进行检测。
2.5、采用计算机技术评估GS-CTP连接肽对抗-CD3 scFv与CD3分子结合能力的影响
采用计算机软件对含GS-CTP连接肽的抗-CD3 scFv VH进行结构建模，并对抗-CD3 scFv与其抗原CD3epsilon链的分子对接进行空间构象模拟和预测。
位于双抗AB7K7中抗-Her2scFv和抗-CD3 scFv之间的GS-CTP连接肽序列为 (GGGGGGSGGGGSGGGGSSSSSKAPPPS)，前半部分为GS柔性肽(GGGGGGSGGGGSGGGGS)，后半部分为CTP刚性肽(SSSSKAPPPS)。刚性CTP部分(SSSSKAPPPS)与抗-CD3 scFv VH的N端相连。通过phyre2软件三维结构建模，结构上CTP肽段覆盖在抗-CD3 scFv VH的CDR1区上(图2-14)，可能会阻碍或不利于CD3抗体与其抗原的结合。对与GS连接肽(去除了CTP，仅包含GS柔性肽)相连接的抗-CD3scFv的VH用phyre2软件进行三维结构建模，发现GS连接肽远离CDR区(图2-15)，并不会对抗原抗体结合造成影响。即使GS连接肽靠近CDR区，由于其自身的灵活性，可以自由移离抗原抗体结合区，因而也不会对抗原抗体结合产生影响。
进一步地，用Discovery Studio软件模拟抗-CD3 scFv及其抗原CD3 epsilon链的分子对接情况。由于双链抗-CD3 Fv与抗-CD3 scFv的结构高度相似，采用双链抗-CD3 Fv代替抗-CD3 scFv进行结构模拟。模拟结果显示抗原CD3 epsilon链与抗-CD3 Fv VH的CDR2和CDR3有结合，与CDR1区不结合(图2-16)，似乎表明覆盖在其VH CDR1区的CTP不会干扰抗-CD3 scFv与抗原的结合。但考虑到CD3分子为一个复合物，包括一个CD3gamma链、一个CD3delta链和2个CD3 epsilon链，该CD3分子与TCR及Zeta链一起构成了T细胞受体复合物。尽管覆盖在抗-CD3 scFv VH CDR1上的CTP肽段不会直接干扰抗-CD3 scFv与其抗原CD3 epsilon链的结合，但CTP肽段可能通过与T细胞受体复合物的某个组成蛋白进行空间结构上的接触，从而间接地影响到抗-CD3 scFv与其抗原CD3 epsilon链的结合。
由于覆盖在抗-CD3 scFv VH的CDR1区上CTP的存在，抗-CD3 scFv与其抗原结合亲和力被大大削弱，从而不会导致因T细胞被过度激活所致的细胞因子大量释放，避免了一些不必要的由T细胞介导的非特异性杀伤。
实施例三、Anti-Her2×CD3双特异性抗体在小鼠移植瘤模型中的药效学研究
3.1、NCG小鼠皮下共接种人CIK细胞和人乳腺癌HCC1954细胞移植瘤模型
选取Her2表达阳性的人乳腺癌HCC1954细胞，观察双抗在NCG小鼠皮下共接种人CIK细胞和HCC1954细胞的移植瘤模型中的体内抑瘤效果。
取正常人外周血，用密度梯度离心法(LymphoprepTM，人淋巴细胞分离液，STEMCELL)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的人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培养细胞，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7～8周龄的雌性NCG小鼠(购自江苏集萃药康生物科技有限公司)，收集处于对数生长期的HCC1954细胞(ATCC)，将5×10 6个HCC1954细胞和5×10 5个CIK细胞混合，接种于NCG小鼠右侧背部皮下。1h后，小鼠按体重随机分为7组，每组5只，分别腹腔给予相应药物，阳性对照组和PBS对照组均为每周给药两次，共给药 3次，阳性对照组分别给予剂量为1mg/kg和3mg/kg的Herceptin(赫赛汀，罗氏)，PBS对照组给予相同体积的PBS溶液。给药组每天给予双抗AB7K4和AB7K7，给药剂量分别为0.1mg/kg和1mg/kg，共给药10次。给药当天记为第0天，每周用电子游标卡尺测量两次肿瘤的最大直径(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33天，PBS对照组平均肿瘤体积为1494.61±500.28mm 3；1mg/kg Herceptin给药组的平均肿瘤体积为1327.29±376.65mm 3；3mg/kg Herceptin给药组的平均肿瘤体积为510.49±106.07mm 3，TGI为65.84％，相对于PBS对照组无显著性差异。0.1mg/kg和1mg/kg AB7K4给药组的平均肿瘤体积分别为304.10±108.50mm 3和79.70±58.14mm 3，TGI分别为79.65％和94.67％，相对于PBS对照组均有显著性差异(P<0.05)。0.1mg/kg和1mg/kg AB7K7给药组的平均肿瘤体积分别为385.82±95.41mm 3和209.98±51.74mm 3，TGI分别为74.19％和85.95％，相对于PBS对照组均有显著性差异(P<0.05)。综上结果表明，不同剂量的双抗AB7K4和AB7K7均能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，显示出良好的抗肿瘤效果；在同样1mg/kg的剂量下，双抗的抑瘤效果要明显优于单抗Herceptin。
3.2、NPG小鼠皮下共接种人CIK细胞和人乳腺癌HCC1954细胞移植瘤模型
选取Her2表达阳性的人乳腺癌HCC1954细胞，观察双抗对NPG小鼠皮下共接种人CIK细胞和人乳腺癌细胞HCC1954的移植瘤模型中的体内抑瘤效果。
根据实施例3.1中的方法获得CIK细胞。选取七至八周龄的雌性NPG小鼠(购自北京维通达生物技术有限公司)，收集处于对数生长期的HCC1954细胞(ATCC)，将5×10 6个HCC1954细胞和5×10 5个CIK细胞混合，接种于NPG小鼠右侧背部皮下。待肿瘤生长6天后，小鼠按肿瘤体积和体重随机分为3组，每组6只，分别腹腔给予相应药物，具体的，AB7K7给药组的剂量分别为0.1mg/kg和1mg/kg，每周给药2次；对照组给予相同体积的PBS溶液。给药当天记为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据实施例3.1中的公式计算各组的肿瘤体积(mm 3)和各给药组的肿瘤生长抑制率TGI(％)。
如图3-2所示，在给药后第21天，PBS对照组平均肿瘤体积为821.73±201.82mm 3；0.1mg/kg的AB7K7给药组的平均肿瘤体积为435.60±51.04mm 3，TGI为50.83％，相对于对照组无显著性差异；1mg/kg的AB7K7给药组的平均肿瘤体积分别为40.98±12.64mm 3，TGI为95.37％，相对于对照组均有极显著性差异(P<0.01)。上述结果表明，在肿瘤生长到一定大小后再给予双抗AB7K7仍有良好的治疗效果，0.1mg/kg低剂量下有50％的抑瘤效果，而1mg/kg给药组6只小鼠中有4只小鼠肿瘤完全消退，另外2只的肿瘤体积也小于100mm 3，小于分组时的体积(分组时该组的平均肿瘤体积为161.37±18.98mm 3)，双抗AB7K7具有良好的的治疗肿瘤的作用。
此外，还考察了双抗AB7K7和AB7K8在上述移植瘤模型中，于两种给药频次下对肿瘤生长的抑制作用。根据上文所述方法获得CIK细胞，选取七至八周龄的雌性NPG小鼠，将5×10 6个HCC1954细胞和5×10 5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6组，每组6只，分别腹腔给予相应药物。具体的，对照组和Herceptin给药组均为每周给药2次，Herceptin的给药剂量为3mg/kg，对照组给予相同体积的PBS溶液；双特异性抗体AB7K7给药剂量为1mg/kg，AB7K8的给药剂量为0.7mg/kg，各设置了两种给药频率，QD组每天给药1次，连续给药10天，BIW组每周给药2次。给药当天记为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据上述公式计算各组的肿瘤体积(mm 3)和各给药组的肿瘤生长抑制率TGI(％)。
如图3-3所示，在给药后第25天，PBS对照组平均肿瘤体积为1588.12±120.46mm 3；3mg/kg的Herceptin给药组的平均肿瘤体积为361.72±134.70mm 3；AB7K7的QD给药组和BIW给药组的平均肿瘤体积分别为260.18±45.96mm 3和239.39±40.62mm 3，TGI分别为83.62％和84.93％，相对于PBS对照组均有极显著性差异(P<0.01)；AB7K8的QD给药组和BIW给药组的平均肿瘤体积分别为284.98±26.62mm 3和647.14±118.49mm 3，TGI分别为82.06％和59.25％，相对于PBS对照组均有极显著性差异(P<0.01)。从上述结果中可以看出，双特异性抗体AB7K7无论是QD组还是BIW组，抗肿瘤效果均优于Herceptin；在等摩尔剂量下，QD组AB7K8与AB7K7的抑瘤效果相当，而BIW组AB7K7显示出显著优于AB7K8的抗肿瘤效果，推测是由于AB7K8为BiTE构型的双特异性抗体，无Fc结构域，AB7K7较AB7K8而言半衰期更长，可以预见临床给药频次降低，获得更好的治疗效果。
3.3、NPG小鼠皮下共接种人CIK细胞和人卵巢癌SK-OV-3细胞移植瘤模型
选取Her2表达阳性的人卵巢癌SK-OV-3细胞，观察双抗在NPG小鼠皮下共接种人CIK细胞和SK-OV-3细胞的移植瘤模型中的体内抑瘤效果。
取正常人外周血，用密度梯度离心法分离人PBMC细胞，然后用McCoy’s 5A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，以及250IU/ml人IL-2；第三天后，300g离心5min，去上清，用RPMI-1640加入10％已灭活FBS重悬细胞，同时加入250IU/ml人IL-2进行培养；每2天添加新鲜的培养液，培养至第10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SK-OV-3细胞(购自中科院上海细胞库)，将3×10 6个SK-OV-3细胞和3×10 5个CIK细胞混合，接种于NPG小鼠右侧背部皮下。接种1h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，Herceptin和AB7K7给药组都分别给药1mg/kg、0.2mg/kg和0.04mg/kg，给药频次均为每周给药2次，对照组给予相同体积PBS。给药当天记为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据实施例3.1中的公式计算各组的肿瘤体积(mm 3)和各给药组的肿瘤生长抑制率TGI(％)。
如图3-4所示，在给药后第21天，PBS对照组平均肿瘤体积为834.09±45.64mm 3；Herceptin在1mg/kg、0.2mg/kg和0.04mg/kg剂量下的平均肿瘤体积分别为644.84±58.22mm 3、884.95±38.63mm 3和815.79±78.39mm 3；AB7K7所有给药组的肿瘤均完全消退。上述结果表明，在卵巢癌SK-OV-3模型中，AB7K7在极低剂量0.04mg/kg下仍然能使肿瘤完全消退，显示出了极好的抗肿瘤效果。
3.4、NPG小鼠皮下共接种人CIK细胞和人结肠癌细胞HT-29细胞移植瘤模型
选取Her2表达阳性的人结肠癌HT-29细胞，观察双抗在NPG小鼠皮下共接种人CIK细胞和HT-29细胞的移植瘤模型中的体内抑瘤效果。
根据实施例3.1中的方法获得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HT-29细胞(购自中科院上海细胞库)，将3×10 6个HT-29细胞和3×10 6个CIK细胞混合，接种于NPG小鼠右侧背部皮下。接种1h后，小鼠按体重随机分为5组，每组6只，分别腹腔给予相应药物，具体的，Herceptin的给药剂量为3mg/kg，AB7K7给药组的剂量分别为3mg/kg、1mg/kg和0.3mg/kg，所有的给药组均为每周给药2次，对照组给予相同体积PBS。给药当天记为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据实施例3.1中的公式计算各组的肿瘤体积(mm 3)和各给药组的肿瘤生长抑制率TGI(％)。
如图3-5所示，在给药后第21天，PBS对照组平均肿瘤体积为1880.52±338.26mm 3；3mg/kg Herceptin的平均肿瘤体积为1461.36±177.94mm 3；AB7K7在3mg/kg、1mg/kg和0.3mg/kg剂量给药组的平均肿瘤体积分别为13.94±7.06mm 3、26.31±10.75mm 3和10.47±6.71mm 3，其中0.3mg/kg给药组有4只小鼠肿瘤完全消退，1mg/kg给药组有3只小鼠肿瘤完全消退，3mg/kg给药组有4只小鼠肿瘤完全消退。上述结果显示，在结肠癌HT-29模型中，Herceptin对此肿瘤模型基本无药效，而AB7K7在三个剂量下均有小鼠的肿瘤完全消退，极低剂量也显示出了极好的抗肿瘤效的果。
3.5、CD34免疫重建的NPG小鼠接种人乳腺癌HCC1954移植瘤模型
选取Her2表达阳性的人乳腺癌HCC1954细胞，观察双抗在CD34免疫重建的NPG小鼠皮下接种人乳腺癌HCC1954细胞的移植瘤模型中的体内抑瘤效果。
使用CD34阳性选择磁珠(购自德国美天旎生物技术有限公司)从新鲜的脐带血中富集CD34阳性的造血干细胞，选取三至四周龄的雌性NPG小鼠(购自北京维通达生物技术有限公司)，尾静脉注射CD34阳性的造血干细胞，在小鼠体内重建人的免疫系统。16周后，小鼠眼眶后静脉丛采血进行流式检测，人CD45比例大于15％的小鼠视为免疫重建成功。收集处于对数生长期的HCC1954细胞，将5×10 6个HCC1954细胞接种于免疫重建的小鼠右侧背部皮下。1h后，小鼠按体重随机分为3组，每组6只，分别腹腔给予剂量为1mg/kg的AB7K7和Herceptin，对照组给予相同体积的PBS，每周给药2次，共给药6次。给药当天记 为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据实施例3.1中的公式计算各组的肿瘤体积(mm 3)和各给药组的肿瘤生长抑制率TGI(％)。
如图3-6所示，在给药后第21天，PBS对照组平均肿瘤体积为475.23±58.82mm 3；Herceptin给药组的平均肿瘤体积为293.27±66.35mm 3，TGI为38.29％，相对于对照组无显著性差异；AB7K7给药组的平均肿瘤体积为0.67±0.67mm 3，TGI为99.86％，所有肿瘤都已基本消退，相对于对照组均有极显著性差异(P<0.01)。综上结果表明，双抗AB7K7在CD34免疫重建模型中具有极好的抗肿瘤效果。
3.6、PBMC免疫重建的NPG小鼠接种人乳腺癌HCC1954细胞移植瘤模型
选取Her2表达阳性的HCC1954细胞，观察双抗在PBMC免疫重建的NPG小鼠接种人乳腺癌HCC1954细胞的小鼠移植瘤模型中的体内抑瘤效果。
取正常人外周血，用密度梯度离心法分离人PBMC细胞，选取五至六周龄的雌性NPG小鼠，腹腔注射人PBMC细胞，在小鼠体内重建人免疫系统。PBMC注射7天后，收集处于对数生长期的HCC1954细胞，将5×10 6个HCC1954细胞接种于小鼠右侧背部皮下。PBMC注射13天后，眼眶后静脉丛采血进行流式检测，human CD45比例大于15％的小鼠视为免疫重建成功。PBMC注射14天后，免疫成功的小鼠按肿瘤体积和体重随机分为2组，每组6只，腹腔给予剂量为1mg/kg的AB7K7，对照组给予PBS，每周给药三次。给药当天记为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据实施例3.1中的公式计算各组的肿瘤体积(mm 3)和各给药组的肿瘤生长抑制率TGI(％)。
如图3-7所示，在给药后第23天，PBS对照组平均肿瘤体积为1224.05±224.39mm 3；AB7K7给药组的平均肿瘤体积为32.00±0.00mm 3，TGI为97.41％，所有肿瘤都已消退，相对于对照组均有极显著性差异(P<0.001)。综上结果表明，双功能特异性抗体AB7K7在PBMC免疫重建模型中有极好的抗肿瘤效果。
实施例四、Anti-Her2×CD3双特异性抗体的安全性评价
4.1、双特异性抗体不能介导对Her2表达阴性肿瘤细胞的非特异性杀伤
选取Her2表达阴性的人Burkkit’s淋巴瘤Raji细胞，观察双抗在NCG小鼠皮下共接种人CIK细胞和人Burkkit’s淋巴瘤Raji细胞的移植瘤模型中是否会抑制肿瘤生长。
根据实施例3.1中的方法获得CIK细胞。选取七至八周龄的雌性NCG小鼠，收集处于对数生长期的Raji细胞(购自中科院上海细胞库)，将5×10 6个Raji细胞和2×10 6个CIK细胞混合，接种于NCG小鼠右侧背部皮下。1h后，小鼠按体重随机分为3组，每组5只，给药组分别腹腔给予剂量为1mg/kg的AB7K4和AB7K7，对照组给予相同体积PBS溶液，每天给药1次，连续给药10天。给药当天记为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据实施例3.1中的公式计算各组的肿瘤体积(mm 3) 和各给药组的肿瘤生长抑制率TGI(％)。
如图4-1所示，在给药后第25天，PBS对照组平均肿瘤体积为2439.88±193.66mm 3；AB7K4给药组的平均肿瘤体积为2408.81±212.44mm 3，AB7K7给药组的平均肿瘤体积为2598.11±289.35mm 3，两个给药组平均肿瘤体积相对于对照组均无差异。综上结果表明，双抗AB7K4和AB7K7在Her2表达阴性的细胞株上均未观察到非特性杀伤，说明AB7K4和AB7K7在体内不会介导T细胞对于非靶点组织的杀伤(即专一性地依赖双特异性抗体与相应靶抗原的结合)，没有脱靶毒性，安全性高。
4.2、双特异性抗体杀伤肿瘤细胞依赖于T细胞的激活
选取Her2表达阳性的人乳腺癌HCC1954细胞，观察双抗在NPG小鼠皮下单独接种人乳腺癌HCC1954细胞的移植瘤模型中是否抑制肿瘤生长。
选取七至八周龄的雌性NPG小鼠，收集处于对数生长期的HCC1954细胞，将5×10 6个HCC1954细胞和Matrigel基质胶(Corning，货号：354234)以体积比1：1混匀，接种于NPG小鼠右侧背部皮下。待肿瘤生长6天后，小鼠按肿瘤体积和体重随机分为3组，每组6只，给药组分别腹腔给予剂量为3mg/kg的Herceptin和1mg/kg的AB7K7，对照组给予相同体积的PBS，每周给药2次。给药当天记为第0天，每周测量两次肿瘤的最大直径(D)和最小直径(d)，并根据实施例3.1中的公式计算各组的肿瘤体积(mm 3)和各给药组的肿瘤生长抑制率TGI(％)。
如图4-2所示，在给药后第21天，PBS对照组平均肿瘤体积为1311.35±215.70mm 3；Herceptin给药组的平均肿瘤体积为273.98±60.10mm 3；AB7K7给药组的平均肿瘤体积为1243.20±340.31mm 3，相对于对照组无差异。综上结果显示，AB7K7在没有人免疫细胞存在的情况下并未抑制HCC1954皮下瘤的生长，表明双抗AB7K7需要通过免疫效应细胞介导才能杀伤肿瘤细胞，而不像Herceptin主要是依赖于FcγR介导的ADCC或CDC效应来杀伤肿瘤细胞，同时证明AB7K7所包含的Fc变体不能结合FcγR，则可以避免介导因其受体FcγR广泛表达所致的T细胞全身性激活，因而药物安全性更高。
4.3、双特异性抗体对正常食蟹猴的毒性评价
选取3-4岁的雌性成年食蟹猴(购于广州相观生物科技有限公司)，体重3-4kg，分成三组，每组一只，分别为溶媒对照组、AB7K7给药组和AB7K8给药组。给药方式为蠕动泵静脉滴注1h，分别于第0天(D0)、第7天(D7)、第21天(D21)和第28天(D28)给药，共给药4次，药物剂量逐次递增。同时每周对动物体重进行称量。给药剂量和体积如下表4-1所示。
表4-1：食蟹猴急性毒性评价给药安排
Figure PCTCN2019113671-appb-000016
于D0给药后，AB7K8给药组食蟹猴出现嗜睡、瞳孔缩小现象，第二天恢复正常，其他组无异常；D7给药后，AB7K7给药组食蟹猴在给药后2-3h出现呕吐症状，给药第二天恢复正常，其他组无异常；D21给药后，AB7K7给药组食蟹猴在给药后3h出现呕吐食物症状，并排泄果冻样粪便，AB7K8给药组在给药后1h出现呕吐食物症状，两组食蟹猴在给药后第二天均恢复正常；于D28给药后40到50min，AB7K7和AB7K8给药组均出现呕吐症状，3h后动物均排出的粪便均有胶冻样粘液，6h后，AB7K7给药组动物排腥臭味水样粪；24h后，动物均恢复正常，采食正常。食蟹猴的体重变化如图4-3所示，箭头表示给药时间，可以看到各组体重均无太大变化，且在正常生理值范围内波动。
本实验过程中观察到的不同程度的腹泻，可能和肠道中相关受体的表达有关，推测是双抗抑制Her1/Her2或Her2/Her3的异二聚体后导致肠道氯离子失衡后造成的，属于药理作用的延伸，给药24h后即可恢复正常。而AB7K7达到3mg/kg的高给药剂量时，食蟹猴仍有良好的耐受性，而通过小鼠的药效实验结果显示，低剂量的AB7K7表现出很好的抗肿瘤效果，表明AB7K7的治疗窗口较宽，安全性较高。
实施例五、Anti-Her2×CD3抗体的药代动力学研究
5.1、双抗AB7K7在SD大鼠体内的药代动力学实验
AB7K7以1mg/kg的剂量，通过尾静脉给药方式注射入4只健康SD大鼠(购自上海斯莱克实验动物有限责任公司)。取血时间点分别为：1h、3h、6h、24h、72h、96h、120h、168h、216h和264h。每个时间点取一定量的全血，分离血清，然后采用两种ELISA方法测定血清中的药品浓度。
方法一、用抗AB7K7抗体A(安源医药科技(上海)有限公司，mouse-anti-herceptin)包板，包板浓度为0.5μg/mL。将AB7K7按照100ng/mL、50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.125ng/mL和1.56ng/mL配置并建立标准曲线。用HRP标记抗AB7K7抗体B(安源医药科技(上海)有限公司，anti-herceptin-HRP)，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5-1。
方法二、检测SD大鼠血清中的药品浓度。用抗AB7K7抗体A(安源医药科技(上海)有限公司，mouse-anti-herceptin)包板，包板浓度为0.5μg/mL。将AB7K7按照5ng/mL、2.5ng/mL、1.25ng/mL、0.625ng/mL、0.3125ng/mL、0.156ng/mL和0.078ng/mL配置并建立标准曲线。将鼠抗人IgG Fc-HRP(安源医药科技(上海)有限公司)按照1：5000加入，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5-2。
图5-1显示了用两种不同检测方法检测AB7K7在大鼠体内的血药浓度，用两种不同的检测方法检测血药中的AB7K7浓度，得到的血药浓度基本一致，计算的药代参数大致相同，说明AB7K7在体内能够以完整分子形式进行代谢，从而保证其生物学功能。
表5-1：双抗AB7K7在SD大鼠中的药代动力学参数(方法一)
AB7K7 t 1/2(h) AUC 0-inf_ob(ng/mL*h) Vz_obs(μg)/(ng/mL)) Cl_obs(μg)/(ng/mL)/h
药代参数 42.10 550236.77 0.02351 3.811E-4
表5-2：双抗AB7K7在SD大鼠中的药代动力学参数(方法二)
AB7K7 t 1/2(h) AUC 0-inf_ob(ng/mL*h) Vz_obs(μg)/(ng/mL)) Cl_obs(μg)/(ng/mL)/h
药代参数 41.02 706126.89 0.01720 2.899E-4
5.2、双抗AB7K7在NPG模型鼠体内的药代动力学实验
NPG小鼠(购自北京维通达生物技术有限公司)给药前一周接种HCC1954细胞(购自中科院细胞所)，接种密度为3.5×10 6/只。给药前两天复苏CIK细胞，培养24h后收集细胞静脉注射入小鼠体内。小鼠随机分为3组，每组四只。三个给药组给药剂量分别是：0.3mg/kg、1mg/kg和3mg/kg。采血时间点分别为1h、3h、6h、24h、48h、72h、96h、120h、168h、216h和264h。每个时间点采一定量的全血，分离血清，然后采用ELISA方法测定血清中的药品浓度。
用抗AB7K7抗体A(安源医药科技(上海)有限公司，mouse-anti-herceptin)包板，包板浓度为0.5μg/mL。将AB7K7按照100ng/mL、50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.125ng/mL和1.56ng/mL配 置并建立标准曲线。用HRP标记抗AB7K7抗体B(安源医药科技(上海)有限公司，mouse-anti-herceptin)，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5-3。
从表5-3中可以看出，AB7K7在NPG模型鼠体内的药代参数与在SD大鼠体内的在数值上没有大的差异。
表5-3：双抗AB7K7在NPG模型鼠中的药代动力学参数
Figure PCTCN2019113671-appb-000017
5.3、双抗AB7K8在SD大鼠体内的药代动力学实验
AB7K8分别以1mg/kg和3mg/kg的剂量，通过尾静脉给药方式注射入3只健康SD大鼠。取血时间点分别为：0.25h、0.5h、1h、2h、3h、4h、5h和7h。每个时间点取一定量的全血，分离血清，然后采用ELISA方法检测血清中的药品浓度。
用抗AB7K8抗体C(安源医药科技(上海)有限公司，mouse-anti-herceptin)包板，包板浓度为2.5μg/mL。将AB7K8按照25ng/mL、12.5ng/mL、6.25ng/mL、3.125ng/mL、1.56ng/mL和0.78ng/mL配置并建立标准曲线。用HRP标记抗-his的抗体(安源医药科技(上海)有限公司)，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5-4。
AB7K8在两种剂量下，得到药代参数T 1/2基本一致，说明其在SD大鼠体内呈现线性代谢动力学。由于AB7K8不含Fc，所以其T 1/2非常短，与AB7K7相比短了约20倍。
表5-4：双抗AB7K8在SD大鼠中的药代动力学参数
AB7K8 t 1/2(h) AUC 0-inf_ob(ng/mL*h) Vz_obs(μg)/(ng/mL)) Cl_obs(μg)/(ng/mL)/h
1mg/kg IV 2.27 4623.14 0.17082 0.05191
3mg/kg IV 1.98 20608.77 0.10220 0.03579
5.4、双抗AB7K在SD大鼠体内的药代动力学实验
AB7K以0.8mg/kg的剂量，通过尾静脉给药方式注射入4只健康SD大鼠。取血时间点分别为：2h、24h、48h、72h、96h、120h、144h、168h、216h和264h。每个时间点取一定量的全血，分离血清，然后采用两种ELISA方法测定血清中的药品浓度。
方法一、用抗AB7K抗体A(安源医药科技(上海)有限公司，mouse-anti-herceptin)包板，包板浓度为1μg/mL。将AB7K按照20ng/mL、10ng/mL、5ng/mL、2.5ng/mL、1.25ng/mL、0.625ng/mL和0.3125ng/mL配置并建立标准曲线。加入25ng/mL的生物素标记的人CD3E & CD3D(Acro，货号CDD-H82W0)，孵育1h后加入1：500稀释的HRP标记的链霉亲和素(BD Pharmingen，货号554066)，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5-5。
方法二、用抗AB7K的抗体A(安源医药科技(上海)有限公司，mouse-anti-herceptin)包板，包板浓度为1μg/mL。将AB7K按照20ng/mL、10ng/mL、5ng/mL、2.5ng/mL、1.25ng/mL、0.625ng/mL和0.3125ng/mL配置并建立标准曲线。加入鼠抗人IgG Fc-HRP(1：10000稀释)(安源医药科技(上海)有限公司)，孵箱1h，最后用TMB显色。
图5-2显示了用两种不同检测方法检测AB7K在大鼠体内的血药浓度，结果表明两种检测方法测得结果差异较大。曲线的前两个点(2h，1D)的浓度较接近，但第二天后，两种方法测得浓度差异变大，推测可能是因抗-CD3 scFv与抗-Her2抗体重链间的连接肽断裂所致。AB7K在体内结构不稳定，从而无法发挥其生物学功能，而改进的AB7K7能够在体内以完整形式代谢，从而能正常发挥其生物学功能。
表5-5：双抗AB7K的药代动力学参数
AB7K t 1/2(h) AUC 0-inf_ob(ng/mL*h) Vz_obs(μg)/(ng/mL)) Cl_obs(μg)/(ng/mL)/h
药代参数 60.47 1022788.69 0.01726 1.985E-4
5.5、双抗AB7K7和AB7K8在食蟹猴体内的药代动力学实验
食蟹猴(购自广州相观生物科技有限公司)分成三个组，每组一只，性别雌性，体重在3-4kg。第一组(G1-1)为空白对照组；第二组(G2-1)AB7K7为给药组，给药剂量为0.3mg/kg；第三组(G3-1)为AB7K8给药组，给药剂量为0.2mg/kg。采血时间点分别为15min、1h、3h、6h、24h、48h、72h、96h、144h、192h、240h和288h，共13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。
用抗AB7K7抗体A(安源医药科技(上海)有限公司，mouse-anti-herceptin)包板，包板浓度为0.5μg/mL。将AB7K7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标记抗AB7K7抗体B(安源医药科技(上海)有限公司，mouse-anti-herceptin)，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5-6。
图5-3显示了AB7K7在大鼠体内的血药浓度，AB7K7在正常食蟹猴体内的T 1/2仅为8个小时左右。AB7K8由于药时曲线上的点过少，无法计算药代参数。但是从药时曲线上可以看出，在正常食蟹猴体内AB7K7要比AB7K8半衰期长很多。
表5-6：双抗AB7K7在食蟹猴体内的药代动力学参数
AB7K7 t 1/2(h) AUC 0-inf_obs ng/mL*h) Vz_obs(μg)/(ng/mL)) Cl_obs(μg)/(ng/mL)/h
药代参数 7.95 87995.48 0.1563 0.01364
5.6、通过ELISA技术评价双特异性抗体和FcRn的结合能力
将各个抗体用PBS溶液稀释成10μg/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。同时分别用pH 6.0和7.0的稀释液稀释生物素biotin标记的FcRn蛋白(ACRO Biosystem，货号FCM-H8286)，4倍梯度稀释，共11个浓度梯度。分别用同pH的PBST清洗96孔板，加入使用相同pH稀释液稀释好的双特异性抗体，设不加抗体的对照孔，室温孵育1h。使用同pH的PBST溶液洗板，将链霉亲和素-HRP(BD，货号554066)加入96孔板中，100μl/孔，室温孵育0.5h。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A450nm-620nm的吸光值。通过软件OriginPro 8进行分析，计算双特异性抗体与FcRn结合的EC 50值。
结果显示了不同的pH条件下每个抗体和FcRn的结合能力不同，结合体内PK的数据，可以分析出双特异性抗体AB7K7的半衰期长于AB7K，但短于Herceptin，这可能更有利于临床应用(图5-4和图5-5)。
表5-7和表5-8分别显示了pH 6.0和7.0时各抗体与FcRn结合能力的测定结果。
表5-7：pH 6.0时双抗AB7K、AB7K5和AB7K7与FcRn结合能力的测定
  Herceptin AB7K AB7K5 AB7K7
EC 50(μg/ml) 2.591 0.8027 1.706 0.4630
表5-8：pH 7.0时双抗AB7K、AB7K5和AB7K7与FcRn结合能力的测定
  Herceptin AB7K AB7K5 AB7K7
EC 50(μg/ml) ～287.1 1.651 13.43 4.838
根据上述对六种抗-Her2×CD3双特异性抗体的多方面研究结果，可以确定AB7K7这类scFv1-scFv2-Fc构型的双特异性抗体具有易于制备、纯化方法简单高效、且其在制备及存储过程中稳定性较好。更有利的是，它对正常细胞的非特异性杀伤作用微弱，且具有控制的可能由效应细胞过度活化所致的毒副作用等显著优势，成药性良好。
表5-9例举了一些优选的针对Her2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，其CDR区所含氨基酸残基根据Kabat规则定义。其中，抗-Her2scFv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5-9：双特异性抗体包含的抗-Her2scFv的氨基酸序列及其CDR区氨基酸序列
Figure PCTCN2019113671-appb-000018
其中，抗-CD3 scFv在体外FACS结合分析测定中以大于约50nM，或大于100nM，或大于300nM，或大于500nM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。
表5-10中例举了一些优选的抗-CD3 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-CD3 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5-10：双特异性抗体包含的抗-CD3 scFv的氨基酸序列及其CDR区氨基酸序列
Figure PCTCN2019113671-appb-000019
其中，连接抗-Her2scFv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：49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5-11中例举了一些优选的连接抗-Her2scFv和抗-CD3 scFv的连接肽的氨基酸序列。
表5-11：连接抗-Her2scFv和抗-CD3 scFv的连接肽的氨基酸序列
SEQ ID NO：50 G 2(GGGGS) 3CTP 1 GGGGGGSGGGGSGGGGSSSSSKAPPPS
SEQ ID NO：51 (GGGGS) 3CTP 1 GGGGSGGGGSGGGGSSSSSKAPPPS
SEQ ID NO：52 GS(GGGGS) 2CTP 1 GSGGGGSGGGGSSSSSKAPPPS
SEQ ID NO：53 (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5-12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5-12：人IgG Fc氨基酸序列
Figure PCTCN2019113671-appb-000020
Figure PCTCN2019113671-appb-000021
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 (31)

  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人表皮生长因子受体2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，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或2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和VL结构域选自下组：
    (i)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；
    (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、21和22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：25、26和27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：28、29和30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。
  4. 如权利要求1、2或3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和VL结构域选自下组：
    (i)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；
    (ii)VH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；和其VL结构域包含如SEQ ID NO：24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：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；和其VL结构域包含如SEQ ID NO：32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。
  5. 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。
  6. 如权利要求1或5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50nM，或大于100nM，或大于300nM，或大于500nM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。
  7. 如权利要求6所述的双特异性抗体，其特征在于，所述第二单链Fv的VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：33、34和35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：36、37和38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。
  8. 如权利要求6所述的双特异性抗体，其特征在于，所述第二单链Fv的VH结构域包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：41、42和43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：44、45和46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。
  9. 如权利要求7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：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；和其VL结构域包含如SEQ ID NO：40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。
  10. 如权利要求8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：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；和其VL结构域包含如SEQ ID NO：48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。
  11. 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；所述刚性肽来自如SEQ ID NO：49所示的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。
  12. 如权利要求11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：50、51、52或53所示的氨基酸序列。
  13. 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。
  14. 如权利要求1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。
  15. 如权利要求14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。
  16. 如权利要求15所述的双特异性抗体，其特征在于，所述Fc片段包含根据EU编号系统确定的L234A/L235A/P331S的氨基酸置换。
  17. 如权利要求15或16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。
  18. 如权利要求17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。
  19. 如权利要求18所述的双特异性抗体，其特征在于，所述Fc片段的氨基酸序列如SEQ ID NO：55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。
  20. 如权利要求1任一项所述的双特异性抗体，其特征在于，所述双特异性抗体包含的氨基酸序列如下：
    (i)SEQ ID NO：8所示的序列；
    (ii)与SEQ ID NO：8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：8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。
  21. 编码如权利要求1-20任一项所述双特异性抗体的DNA分子。
  22. 如权利要求21所述的DNA分子具有如SEQ ID NO：56所示的核苷酸序列。
  23. 包含如权利要求21或22所述DNA分子的载体。
  24. 包含如权利要求23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。
  25. 一种药物组合物，所述组合物包含如权利要求1-20任一项所述的双特异性抗体以及可药用赋形剂、载体或稀释剂。
  26. 如权利要求25所述的药物组合物，其特征在于，所述组合物是包含了如权利要求1-20任一项所述双 特异性抗体的溶液制剂，所述制剂还包含pH调节剂、稳定剂及表面活性剂；优选地，所述pH调节剂为柠檬酸盐缓冲液或组氨酸盐缓冲液，和所述稳定剂为蔗糖，和所述表面活性剂为吐温-80；更优选地，所述制剂包含0.5mg/mL的上述双特异性抗体，和20mM柠檬酸盐或组氨酸盐以及8％蔗糖(w/v)和0.02％的PS80(w/v)；所述制剂的pH为5.5。
  27. 制备如权利要求1-20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。
  28. 如权利要求1-20任一项所述双特异性抗体或如权利要求25或26所述药物组合物在制备治疗、预防或缓解肿瘤的药物中的用途，所述肿瘤的实例包括但不限于乳腺癌、前列腺癌、非小细胞肺癌、膀胱癌、卵巢癌、胃癌、结肠直肠癌、食管癌、头和颈的鳞状细胞癌瘤、子宫颈癌、胰腺癌、睾丸癌、恶性黑色素瘤和软组织癌；优选地，所述肿瘤选自乳腺癌、胃癌、直肠癌；更优选地，所述肿瘤选自乳腺癌。
  29. 如权利要求1-20任一项所述双特异性抗体或如权利要求25或26所述药物组合物用于治疗肿瘤、延迟其进展、降低/抑制其复发的方法，其包括将有效量的所述双特异性抗体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；所述肿瘤的实例包括但不限于乳腺癌、前列腺癌、非小细胞肺癌、膀胱癌、卵巢癌、胃癌、结肠直肠癌、食管癌、头和颈的鳞状细胞癌瘤、子宫颈癌、胰腺癌、睾丸癌、恶性黑色素瘤和软组织癌；优选地，所述肿瘤选自乳腺癌、胃癌、直肠癌；更优选地，所述肿瘤选自乳腺癌。
  30. 如权利要求1-20任一项所述双特异性抗体或如权利要求25或26所述药物组合物用于增强或刺激免疫应答或功能的方法，其包含对患者/受试者个体施用治疗有效量的所述双特异性抗体或所述药物组合物。
  31. 如权利要求1-20任一项所述双特异性抗体或如权利要求25或26所述药物组合物用于治疗、预防或改善患者/受试者个体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药物组合物。
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