WO2020088164A1 - 双特异性抗体及其用途 - Google Patents

双特异性抗体及其用途 Download PDF

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WO2020088164A1
WO2020088164A1 PCT/CN2019/108057 CN2019108057W WO2020088164A1 WO 2020088164 A1 WO2020088164 A1 WO 2020088164A1 CN 2019108057 W CN2019108057 W CN 2019108057W WO 2020088164 A1 WO2020088164 A1 WO 2020088164A1
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Prior art keywords
bispecific antibody
amino acid
sequence
cells
cell
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PCT/CN2019/108057
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English (en)
French (fr)
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***
贾世香
赵丽丽
张贵民
马心鲁
严源
李振宇
胡兴霞
张玉华
李斌
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山东新时代药业有限公司
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Priority to JP2021525317A priority Critical patent/JP7410143B2/ja
Priority to KR1020217015342A priority patent/KR20210087472A/ko
Priority to MX2021005155A priority patent/MX2021005155A/es
Priority to AU2019370339A priority patent/AU2019370339A1/en
Priority to PE2021000645A priority patent/PE20211867A1/es
Priority to BR112021008486-0A priority patent/BR112021008486A2/pt
Application filed by 山东新时代药业有限公司 filed Critical 山东新时代药业有限公司
Priority to US17/290,401 priority patent/US20230416385A1/en
Priority to CA3118397A priority patent/CA3118397A1/en
Priority to EP19879912.4A priority patent/EP3889179A4/en
Publication of WO2020088164A1 publication Critical patent/WO2020088164A1/zh
Priority to CONC2021/0006970A priority patent/CO2021006970A2/es
Priority to ZA2021/03717A priority patent/ZA202103717B/en

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Definitions

  • the present invention relates to the field of immunology, and more specifically, to an anti-CD3 bispecific antibody that mediates T cell killing, and the use of such antibodies, particularly in the treatment of cancer.
  • myeloma is the second most common hematological malignancy.
  • the uncontrolled proliferation of monoclonal plasma cells in the bone marrow leads to the overproduction of monoclonal immunoglobulins and immunosuppression, as well as osteolysis and end organ damage.
  • monoclonal antibodies have been approved for clinical use.
  • multiple myeloma treatment programs have significantly improved the survival rate of patients. Nonetheless, the current treatment options still do not meet current treatment needs, especially for relapsed / refractory patients who are resistant to current therapies.
  • BCMA B cell maturation antigen
  • APRIL proliferation-inducing ligand
  • BCMA regulates different aspects of humoral immunity, B cell development and homeostasis.
  • the expression of BCMA occurs at a later stage of B-cell differentiation and is beneficial to the long-term survival of plasmablasts and plasma cells in the bone marrow.
  • the targeted deletion of the BCMA gene in mice resulted in a significant reduction in the number of long-lived plasma cells in the bone marrow, indicating that BCMA is important for its survival.
  • Overexpression of BCMA or stimulation of APRIL by BCMA in multiple myeloma cells can directly up-regulate key immune checkpoint molecules, which may help immunosuppress the bone marrow microenvironment.
  • T lymphocytes play an important role.
  • the cellular immunity mediated by T cells mainly recognizes the antigen peptide presented by the major histocompatibility complex (MHC) on the cell surface through the T cell receptor (TCR) to activate the intracellular T cells Signal to specifically kill the target cell.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • T-cell binding bispecific antibodies represent a very effective way to redirect activated cytotoxic T cells to tumors.
  • CD3 is expressed in mature T cells and can transduce the activation signal generated by TCR recognition antigen.
  • TCBs can simultaneously bind the surface tumor antigen and the CD3 ⁇ subunit of T cell receptors, providing a physical connection between T cells and tumor cells, thereby effectively activating resting T cells to kill tumor cells and achieve the effect of treating tumors (Smits N C, Sentman C, L, Journal of Clinical Oncology, 2016: JCO649970.). Because T cell bispecific bypasses TCR antigen recognition and T cell activation co-stimulation requirements, they eliminate the need for tumor-specific immunity and overcome many of the obstacles faced by T cells in the tumor microenvironment.
  • 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 general, there are two major categories. One type of bispecific antibody does not contain Fc region, including BiTE, DART, TrandAbs, bi-Nanobody and so on.
  • 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.
  • bispecific antibodies retain Fc domains, such as Triomabs, kih IgG, Cross-mab, orthoFab IgG, DVD IgG, IgG scFv, scFv 2 -Fc and other configurations.
  • Fc domains such as Triomabs, kih IgG, Cross-mab, orthoFab IgG, DVD IgG, IgG scFv, scFv 2 -Fc and other configurations.
  • 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
  • the present invention aims to develop a BCMA bispecific molecule with improved performance in terms of product half-life, stability, safety and manufacturability.
  • the purpose of the present invention is to provide a tetravalent homodimeric bispecific antibody molecule targeting immune effector cell antigen CD3 and tumor antigen BCMA.
  • This bispecific antibody can significantly inhibit or kill tumor cells in vivo, but The non-specific killing effect of normal cells with low expression of BCMA is significantly reduced, while the controllability of toxic and side effects caused by excessive activation of effector cells is increased, and their physical and chemical properties and in vivo stability are significantly improved.
  • a bispecific antibody is disclosed.
  • the bispecific antibody molecule is composed of two identical polypeptide chains covalently bonded to form a tetravalent homodimer, each The polypeptide chain includes, from the N-terminus to the C-terminus, a first single-chain Fv that specifically binds to the tumor antigen BCMA, a second single-chain Fv that specifically binds to the effector cell antigen CD3, and an Fc fragment; It is connected by a connecting peptide, and the second single-chain Fv is directly connected to an Fc fragment or connected by a connecting peptide, and the Fc fragment does not have effector functions such as CDC, ADCC, and ADCP.
  • the first single-chain Fv includes a VH domain and a VL domain, and is connected by a linking peptide L1, and the amino acid sequence of the linking peptide L1 is (GGGGX) n , X includes a group selected from Ser or Ala, and n is Natural number of 1-5; X is preferably Ser, n is preferably 3.
  • the amino acid sequence of the linker peptide L1 is (GGGGS) 3.
  • the amino acid sequence of the linker peptide L1 further includes (GGGGS) 1 or (GGGGS) 2 Or (GGGGS) 4 or (GGGGS) 5 or (GGGGA) 1 or (GGGGA) 2 or (GGGGA) 3 or (GGGGA) 4 or (GGGGA) 5.
  • the first single-chain Fv includes:
  • VH domain which contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, 2 and 3, respectively, or 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
  • VL domain which contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, 5 and 6, respectively, or 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 first single-chain Fv includes:
  • amino acid sequence is the VH domain shown in SEQ ID NO: 7, or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to the above sequence Or one or more amino acid substitutions (eg conservative substitutions)); and
  • the amino acid sequence is the VL domain shown in SEQ ID NO: 8, or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to the above sequence Or a sequence with one or more amino acid substitutions (eg conservative substitutions).
  • 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 terminal of the natural human chorionic gonadotropin ⁇ subunit (as shown in SEQ ID NO: 9) or a truncated fragment thereof (hereinafter collectively referred to as For CTP).
  • the CTP1 rigid peptide contains 10 amino acids at the N-terminal of SEQ ID NO: 9 SSSSKAPPPS; or the CTP2 rigid peptide contains 14 amino acids at the C-terminal of SEQ ID NO: 9 SRLPGPSDTPILPQ; the CTP3 rigid peptide contains 16 at the N-terminal of SEQ ID NO: 9 Amino acids, namely SSSSKAPPPSLPSPSR; CTP4 rigid peptide contains 28 amino acids and starts at position 118 of human chorionic gonadotropin ⁇ subunit and ends at position 145, namely SSSSKAPPPSLPSPSRLPGPSDTPILPQ.
  • the rigid peptide is SSSSKAPPPS, namely CTP1 rigid peptide.
  • the rigid peptide sequence also includes CTP2 (SRLPGPSDTPILPQ), CTP3 (SSSSKAPPPSLPSPSR), CTP4 (SSSSKAPPPSLPSPSRLPGPSDTPILPQ).
  • the amino acid sequence of the linker peptide L2 is shown in SEQ ID NO: 10, the amino acid composition of its flexible peptide is G 2 (GGGGS) 3 , and the amino acid composition of its rigid peptide is SSSSKAPPPS , Namely CTP1 rigid peptide.
  • the second single-chain Fv of the bispecific antibody specifically binds CD3, and binds to the effector cells with an EC 50 value of greater than 50 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 nM in the in vitro FACS binding assay; more preferably Specifically, the second single-chain Fv of the bispecific antibody can not only bind to human CD3, but also specifically bind to CD3 of cynomolgus monkey or rhesus monkey. In a preferred embodiment of the present invention, the bispecific antibody specifically binds to effector cells with an EC 50 value of 132.3 nM.
  • the second single-chain Fv includes:
  • VH domain which contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, 12, and 13, respectively, or 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
  • VL domain which contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, 15 and 16, respectively, or 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-stranded Fv comprises an amino acid sequence as shown in SEQ ID NO: 17 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% of the above sequence Or a VH domain that is more highly similar or has one or more amino acid substitutions (eg, conservative substitutions); and
  • amino acid sequence is shown in SEQ ID NO: 18 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to the above sequence or has one or more VL domains of amino acid substitutions (eg conservative substitutions).
  • the VH domain and the VL domain of the second single-chain Fv are connected by a connecting peptide L3, the VH, L3 and VL are arranged in the order of VH-L3-VL or VL-L3-VH, and the
  • the amino acid sequence of the linker peptide L3 is (GGGGX) n , X is selected from Ser or Ala, n is a natural number of 1-5; X is preferably Ser, and n is preferably 3.
  • the amino acid sequence of the linker peptide L3 is (GGGGS) 3.
  • the amino acid sequence of the linker peptide L3 further includes (GGGGS) 1 or (GGGGS) 2 Or (GGGGS) 4 or (GGGGS) 5 or (GGGGA) 1 or (GGGGA) 2 or (GGGGA) 3 or (GGGGA) 4 or (GGGGA) 5.
  • the Fc fragment is directly connected to the second single-chain Fv.
  • the Fc fragment is connected to the second single-chain Fv via a linker peptide L4, and the linker peptide L4 amino acid sequence includes (GGGGS) 1 or (GGGGS) 2 or (GGGGS) 3 or (GGGGS) 4.
  • 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, human IgG1.
  • the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 19, which has the following 6 amino acid substitutions or substitutions determined according to the EU numbering system compared to the natural sequence from which it was derived: L234A / L235A / N297A / P331S / T250Q / M428L; and K447 determined according to the EU numbering system is missing or deleted.
  • the EU numbering system comes from the website: http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.htm.
  • the Fc fragment is altered, for example, mutated, to modify the properties of the bispecific antibody molecule of the invention (eg, alter one or more of the following characteristics: Fc receptor binding, antibody Glycosylation, effector cell function or complement function).
  • the bispecific antibodies provided by the present invention comprise Fc variants having amino acid substitutions, deletions, or additions with altered effector functions (eg, reduction or elimination).
  • the Fc region of an antibody mediates several important effector functions, such as ADCC, ADCP, and CDC.
  • Methods for changing the affinity of an antibody to effector ligands (such as Fc ⁇ R or complement C1q) by replacing amino acid residues in the Fc region of an antibody, thereby changing the effector function are known in the art (see, for example, EP 388,151A1 ; US564,8260; US562,4821; Natsume A, etc., Cancer Res., 68: 3863-3872, 2008; Idusogie EE, etc., J.
  • amino acid L235 (EU numbering) on the constant region of the antibody is modified to alter the Fc receptor interaction, such as L235E or L235A.
  • amino acids 234 and 235 on the constant region of the antibody are modified simultaneously, such as L234A and L235A (L234A / L235A) (EU numbering).
  • the bispecific antibodies provided by the present invention may include Fc variants with amino acid substitutions, deletions, or additions that have an extended circulating half-life.
  • Fc variants with amino acid substitutions, deletions, or additions that have an extended circulating half-life.
  • M252Y / S254T / T256E, M428L / N434S or T250Q / M428L can extend the half-life of antibodies in primates.
  • More mutation sites included in Fc variants with enhanced binding affinity to neonatal receptors (FcRn) can be found in Chinese invention patents CN 201280066663.2, US 2005 / 0014934A1, WO 97/43316, US 5,869,046, US 5,747,03, WO 96/32478.
  • amino acid M428 (EU numbering) on the antibody constant region is modified to enhance the binding affinity of the FcRn receptor, such as M428L.
  • amino acids 250 and 428 (EU numbering) on the constant region of the antibody are modified simultaneously, such as T250Q and M428L (T250Q / M428L).
  • the bispecific antibodies provided by the present invention may also include Fc variants having amino acid substitutions, deletions, or additions that can reduce or eliminate Fc glycosylation.
  • Fc variants contain reduced glycosylation of N-linked glycans normally present at amino acid position 297 (EU numbering).
  • EU numbering amino acid position 297
  • the glycosylation at position N297 has a great influence on the activity of IgG. If the glycosylation at this site is removed, it will affect the conformation of the upper half of 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 will cause charge heterogeneity in monoclonal antibodies, and the heterogeneity of lysine at the C-terminus of IgG antibodies is one of the main reasons.
  • Acid K may be lost in a certain proportion during antibody production, resulting in charge heterogeneity, which affects the stability, effectiveness, immunogenicity or pharmacokinetics of the antibody.
  • K447 (EU numbering) at the C-terminus of the IgG antibody is removed or deleted to eliminate the charge heterogeneity of the antibody and improve the uniformity of the expressed product.
  • the 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: 19, 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 molecule of the present invention is composed of two identical polypeptide chains joined by an interchain disulfide bond in the hinge region of the Fc fragment to form a tetravalent homodimer, and each polypeptide chain is sequentially -Composed of BCMA scFv, linker peptide, anti-CD3 scFv and Fc fragments.
  • the bispecific antibody binds to human BCMA and CD3, and its amino acid sequence is as follows:
  • sequence shown in SEQ ID NO: 20 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences with at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (2) 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: 21.
  • a vector comprising the above DNA molecule is provided.
  • a host cell comprising the above vector; the host cell comprises a prokaryotic cell, yeast or mammalian cell, preferably, the host cell is a mammalian cell, such as CHO cell, NS0 cell or other The mammalian cell is further preferably a CHO cell.
  • a pharmaceutical composition comprising the above bispecific antibody and a pharmaceutically acceptable excipient and / or carrier and / or diluent.
  • a method for preparing the bispecific antibody of the present invention which comprises:
  • the expression vector in step (a) is selected from one or more of plasmids, bacteria, and viruses.
  • the expression vector is a plasmid, and more preferably, the expression vector is PCDNA3.1;
  • the constructed vector is transfected into a host cell by a genetic engineering method.
  • the host cell includes a prokaryotic cell, a yeast, or a mammalian cell.
  • the host cell is a mammalian cell, such as CHO
  • the cells, NS0 cells or other mammalian cells are further 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 treatment or improvement of a drug selected from plasma cell disorders, other B cell disorders related to BCMA expression, and autoimmune diseases
  • the Plasma cell disorders include but are not limited to multiple myeloma, plasma cell tumor, plasma cell leukemia, macroglobulin leukemia, amyloidosis, Fahrenheit macroglobulin leukemia, solitary osteoplasmoma, extramedullary plasmacytoma, Osteosclerotic myeloma, heavy chain disease, monoclonal gamma globulinopathy of undefined significance, and smoldering multiple myeloma.
  • the bispecific antibody provided by the present invention is not easy to bind to normal cells with weak or low expression of BCMA, reducing non-specific killing, but the binding specificity of cells overexpressing or overexpressing BCMA is not significantly reduced, showing good In vivo killing effect. It is also known that when the target antigen is only expressed on tumor cells or the bispecific antibody of the present invention only specifically binds to tumor cells overexpressing the target antigen, the immune effector cells are restricted to only within the target cell tissue Activation, which allows the non-specific killing of normal cells by the bispecific antibody and the accompanying release of cytokines can be minimized, reducing their toxic and side effects in clinical treatment.
  • the anti-CD3 scFv selected by the bispecific antibody provided by the present invention specifically binds to the effector cells with weak binding affinity (EC 50 value greater than 50 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 nM).
  • the bispecific antibody provided by the present invention creatively adopts the bivalent anti-CD3 scFv, which makes the bispecific antibody circumvent the heterodimer type commonly used in the prior art (so The included anti-CD3 scFv is monovalent), so there is no problem of mismatch between heavy chains, which simplifies downstream purification steps; and unexpectedly, no anti-CD3 scFv and Non-specific binding of T cells, and the degree of cell activation (release of cytokines such as IL-2) is controlled within a safe and effective range, that is, the bivalent anti-CD3 scFv structure used in the present invention does not cause antigen-independent induction Over-activation of T cells, and for other bispecific antibodies containing bivalent anti-CD3 domains, it is common for T cells to be uncontrollably over-activated, so anti-CD3 bispecific antibodies are generally designed Avoid introducing divalent anti-CD3 structures.
  • 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 to only 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.
  • FR antibody framework region the immunoglobulin variable region excluding the CDR region
  • Region V IgG chain segments with variable sequence between different antibodies extends to Kabat residue 109 of the light chain and 113th of 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.
  • BCMA is a B cell maturation antigen, a member of the tumor necrosis factor receptor superfamily, preferentially expressed in mature B lymphocytes, and expressed on the surface of plasmablasts (ie, plasma cell precursors) and plasma cells.
  • BCMA RNA can be detected in the spleen, lymph nodes, thymus, adrenal glands, and liver, and the level of BCMA mRNA also increases after multiple B cell lines mature.
  • BCMA is associated with leukemia, lymphoma (such as Hodgkin's lymphoma), multiple myeloma, autoimmune diseases (such as systemic lupus erythematosus) and other diseases, so it can be used as a potential target for related B-cell diseases.
  • BCMA targets also include other related diseases or conditions discovered in the prior art and discovered in the future.
  • the term also includes any variants, isoforms, and species homologs of BCMA, which are expressed naturally by cells-including tumor cells-or by cells transfected with BCMA genes or cDNA.
  • the CD3 molecule is an important differentiation antigen on the T cell membrane and a characteristic marker of mature T cells. It is composed of 6 peptide chains.
  • the TCR-CD3 complex is composed of a non-covalent bond and a T cell antigen receptor (TCR).
  • TCR T cell antigen receptor
  • the TCR-CD3 complex is assembled in the cytoplasm and transmits antigen stimulation signals through the immunoreceptor tyrosine activation motif (Immunoreceptor Tyrosine-based Activation Motif, ITAM) in the cytoplasmic region of each polypeptide chain.
  • ITAM immunoreceptor tyrosine activation motif
  • the main function of the CD3 molecule is to stabilize the TCR structure and transmit T cell activation signals.
  • TCR specifically recognizes and binds to the antigen
  • CD3 participates in transducing the signal into the T cell cytoplasm as the first signal to induce T cell activation. It plays an extremely important role in T cell antigen recognition and immune response generation.
  • CD3 refers to being a part of the T cell receptor complex, consisting of three different chains CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ . Clustering of CD3 on T cells by, for example, the immobilization of anti-CD3 antibodies on it, results in T cell activation, similar to T cell receptor-mediated activation, but does not depend on the specificity of TCR clones. Most anti-CD3 antibodies recognize the CD3 epsilon chain.
  • the second functional domain of the present invention that specifically recognizes the CD3 receptor on the surface of T cells is not particularly limited, as long as it can specifically recognize CD3, such as but not limited to the CD3 antibody mentioned in the following patents: US7,994,289, US6,750,325; US6,706,265; US5,968,509; US8,076,459; US7,728,114; US20100183615.
  • the anti-human CD3 antibody used in the present invention is cross-reactive with cynomolgus monkey and / or rhesus monkey, such as but not limited to the anti-human CD3 antibody mentioned in the following patents: WO2016130726, US20050176028, WO2007042261 Or WO2008119565.
  • the term also includes any CD3 variants, isoforms, derivatives and species homologues, which are naturally expressed by the cell or expressed on cells transfected with the gene or cDNA encoding the aforementioned chain.
  • antibody specifically includes monoclonal antibodies, polyclonal antibodies, and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies.
  • Antigen-binding fragments include fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of intact antibody portions that retain the antigen-binding specificity of the parent antibody molecule.
  • the antigen-binding fragment may comprise at least one variable region (heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a specific antigen.
  • Suitable antigen-binding fragments include, but are not limited to, bispecific antibody bodies and single chain molecules and Fab, F (ab ') 2, Fc, Fabc and Fv molecules, single chain (Sc) antibodies, individual antibody light chains, Separate antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, composed of one heavy chain and one light chain
  • the dimer consisting of VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782, a bivalent fragment comprising two Fab fragments connected by a disulfide bond on the hinge region, consists essentially of Fd fragments composed of VH and CH1 domains; Fv fragments composed essentially of VL and VH domains of one arm of an antibody, dAb fragments (Ward et al., Nature, 1989, 341: 544-54), which are basically composed of VH Domain composition, also called domain
  • antigen-binding fragments can include non-antibody protein frameworks that can successfully incorporate polypeptide fragments into an orientation that imparts affinity for a given antigen of interest (eg, protein scaffold).
  • Antigen-binding fragments can be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies.
  • antibody or antigen-binding fragment thereof may be used to indicate that a given antigen-binding fragment incorporates one or more amino acid fragments of the antibody mentioned in the phrase.
  • 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, determining the numbering scheme for any variable region sequence in the sequence listing is well within the ordinary skill of those in the art.
  • single-chain Fv antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, and is a heavy chain variable region (VH) and a light chain variable region connected by a linker
  • the recombinant protein of (VL), a linker that cross-links these two domains to form an antigen binding site, and the linker sequence generally consists of a flexible peptide, such as but not limited to G 2 (GGGGS) 3 .
  • the size of scFv is generally 1/6 of a complete antibody.
  • the single chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain.
  • Fab fragment consists of a light chain and a heavy chain CH1 and variable regions.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the size of the "Fab antibody” is 1/3 of the intact antibody, and it contains only one antigen binding site.
  • Fab 'fragment contains a light chain and a heavy chain between the VH domain and the CH1 domain and the constant region between the CH1 and CH2 domains.
  • F (ab ') 2 fragment contains two light chains and two heavy chains, the VH domain and the CH1 domain, and the constant region portion between the CH1 and CH2 domains, thereby forming between the two heavy chains Interchain disulfide bond. Therefore, the F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • Fc region refers to an antibody heavy chain constant region fragment, which comprises at least the hinge region, CH2 and CH3 domains.
  • Fv region includes variable regions from both heavy and light chains, but lacks constant regions, and is the smallest fragment that contains a complete antigen recognition and binding site.
  • Fd fragment consists of the CH1 of a heavy chain and the variable region, and is the portion of the heavy chain remaining after the light chain is removed from the Fab fragment.
  • dsFv disulfide bond stable protein
  • 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.
  • 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 amino acid sequences contained in the heavy and light chain variable regions of the antibodies of the present invention are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the present invention Said, for example, the desired functional properties of the Her2 ⁇ CD3 bispecific antibody.
  • conservative modifications is intended to mean that amino acid modifications do not significantly affect or change the binding characteristics of antibodies containing the amino acid sequence.
  • 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.
  • 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.
  • FcRn Fc variants with altered binding affinity to neonatal receptors (FcRn), as used herein, "FcRn” refers to proteins that bind to at least part of the Fc region of IgG antibodies encoded by the FcRn gene.
  • FcRn can be derived from any organism including but not limited to humans, mice, rats, rabbits and monkeys.
  • the functional FcRn protein contains two polypeptides often referred to as heavy and light chains. The light chain is ⁇ -2-microglobulin, and the heavy chain is encoded by the FcRn gene.
  • the present invention relates to antibodies whose binding to FcRn is regulated (modulation includes increasing and decreasing binding). For example, in some cases, increased binding can cause cells to recycle antibodies and thereby prolong, for example, the half-life of therapeutic antibodies. Sometimes, it is desirable to reduce FcRn binding, for example, as a diagnostic or therapeutic antibody containing a radiolabel. In addition, antibodies that show increased binding to FcRn, while binding to other Fc receptors, such as Fc ⁇ Rs, can be used in the present invention.
  • the present application relates to antibodies containing amino acid modifications that modulate the binding force to FcRn. It is of special significance that at lower pH, the binding affinity for FcRn shows an increase, while at higher pH, the binding basically does not show a change.
  • the antibody or functional change that contains the Fc region minimally body.
  • 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, as enhanced FcRn binding can lead to an increase in 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.
  • Fc alteration that extends half-life.
  • prolonged half-life Fc alteration refers to the prolongation of an Fc polypeptide chain containing an altered Fc polypeptide chain in the Fc polypeptide chain compared to the half-life of a similar Fc protein that contains the same Fc polypeptide but does not include the altered. Changes in the half-life of proteins in the body. 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.
  • nucleic acids encoding bispecific antibodies using the therapeutic agents and antibodies or antibody fragments described herein, those skilled in the art can easily construct functionally equivalent nucleic acids (eg, nucleic acids with different sequences but encoding the same effector portion or antibody sequence) Multiple clones. Accordingly, the present invention provides bispecific antibodies, nucleic acids, nucleic acid variants, derivatives and species homologs encoding antibodies, antibody fragments and conjugates and their fusion proteins.
  • 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 SEQ ID NO: 21.
  • 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.
  • any method known in the art may be used to prepare the bispecific antibody of the present invention.
  • 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.
  • 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.
  • 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 host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as E. coli or Bacillus stearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), insect cells (Such as Lepidoptera insect cells including Spodoptera frugiperda cells) or mammalian cells (such as Chinese hamster ovary (CHO) cells, NS0 cells, baby hamster kidney (BHK) cells, monkey kidney cells, Hela cells, human liver Cell cancer cells or 293 cells, etc.).
  • prokaryotic cells such as E. coli or Bacillus stearothermophilus
  • fungal cells such as Saccharomyces cerevisiae or Pichia pastoris
  • insect cells such as Lepidoptera insect cells including Spodoptera frugiperda cells
  • mammalian cells such as Chinese hamster ovary (CHO) cells, NS0 cells, baby hamster kidney
  • 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 include an adjuvant, such as Freund's complete adjuvant or incomplete adjuvant or similar immunostimulating compounds.
  • the bispecific antigens of the present invention can be used alone or preferably as a conjugate with a carrier protein. This method of enhancing the antibody response is well known in the art.
  • different animal hosts can be used for in vivo immunization. It is possible to use a host that expresses useful endogenous antigen by itself, or a host that has caused a defect in the useful endogenous antigen.
  • Bispecific antibodies can be prepared by binding the methods described above.
  • the bispecific antibody molecule of the present invention can be used as a monoclonal antibody (MAb) for each target.
  • the antibody is chimeric, humanized, or fully human.
  • Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, Nature, 256: 495-497, 1975), three-source hybridoma technology, human B cell hybridoma technology (Kozbor D et al., Immunology Today, 4:72, 1983) and EBV-hybridoma technology (Cole SPC, Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan Riss, Inc., 1985).
  • the bispecific antibodies of the invention or parts thereof 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 For example 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 antigen-binding fragment, and detecting the antigen-bound An antibody or part thereof, or an unbound antibody or part thereof, 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 anti-biotin Biotin / biotin;
  • suitable fluorescent substances include 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, alkaline rugosa red B, dichlorotriazinyl fluorescein, dansine Acid chloride or phycoerythrin;
  • luminescent substances include 3-aminophthaloyl hydrazine;
  • suitable radioactive substances include I 125 , I 131 , 35 S or 3 H.
  • the pharmaceutical composition, the bispecific antibody of the present invention or the nucleic acid or polynucleotide encoding the antibody of the present application can be used to prepare a pharmaceutical composition or a sterile composition, for example, a bispecific antibody and a pharmaceutically acceptable carrier, The excipients or stabilizers are mixed.
  • the pharmaceutical composition may include one or a combination (such as two or more different) bispecific antibodies of the present invention.
  • the pharmaceutical composition of the present invention may comprise a combination of antibodies or 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 bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application may be linked to or administered separately from some substances (as immune complexes) of the above-mentioned pharmaceutically acceptable carrier or components thereof.
  • the bispecific antibody or the nucleic acid or polynucleotide encoding the antibody of the present application may be administered before, after or co-administered with some substances of the above pharmaceutically acceptable carrier or its components, or may be administered with other Known therapies (such as anti-cancer therapies, such as radiation) are co-administered.
  • 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 pharmaceutical composition of the present invention may be a combination of two drugs, or may be a combination of products with similar functions that have been marketed or products that increase therapeutic effects.
  • Bispecific antibodies mediate the activation of CD4 + T cells / CD8 + T cells.
  • Fig. 10 In vivo tumor suppressive effect of bispecific antibody co-inoculated with human CIK cells and human myeloma cells RPMI-8226 subcutaneously in NPG mice. Note: The arrow represents the time of each administration; *** indicates that there is a significant difference.
  • FIG. 11 In vivo tumor suppressive effect of a bispecific antibody co-vaccinated with human CIK cells and human Burkkit ’s lymphoma Daudi cell transplanted tumors subcutaneously in NPG mice. Note: The arrow represents the time of each administration; *** indicates that there is a significant difference.
  • the materials used in the experiment can be purchased or can be prepared by referring to the currently disclosed technology; those with unspecified sources and specifications are commercially available; various processes and methods not described in detail are well known in the art normal method.
  • the bispecific antibody AP163 is composed of anti-BCMA scFv, connecting peptide L2, anti-CD3 scFv and Fc fragments in series, and VH and VL inside anti-BCMA scFv and anti-CD3 scFv are connected by connecting peptides L1 and L3, respectively .
  • the VH and VL amino acid sequences of the scFv for BCMA contained in AP163 are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the anti-CD3-scFv-containing VH and VL amino acid sequences of AP163 are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the VH and VL are connected by (GGGGS) 3 .
  • the Fc fragment contained in AP163 is derived from human IgG1, and has multiple amino acid substitutions / substitutions, namely L234A, L235A, T250Q, N297A, P331S, and M428L (EU number), and also deletes / deletes K447 at the C-terminus of 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 coding genes of the above bispecific antibodies were synthesized according to conventional molecular biology methods, and the coding cDNAs of the obtained fusion genes were inserted between the corresponding restriction sites of the eukaryotic expression plasmid pCMAB2M modified by PCDNA3.1. 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 host cell line is a DHFR enzyme-deficient CHO-cell (see US Pat. No. 4,818,679).
  • the host cell is a 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.
  • electroporation using a Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, CA) set to an electric field of 300 V and a capacitance of 1500 ⁇ Fd, 50 ⁇ g of expression vector plasmid DNA was added to 5 ⁇ 10 7 cells in a cuvette. Two days after transfection, the medium was changed to a growth medium containing 0.6 mg / mL G418. Transfectants were subcloned with limiting dilution, and the secretion rate of each cell line was measured by ELISA. Cell lines that express bispecific antibodies at high levels are selected.
  • the operation steps mainly include: co-amplify the transfected fusion protein gene with DHFR gene in growth medium containing increasing concentration of MTX.
  • Subclones with positive expression of DHFR by limiting dilution were gradually pressurized and screened for transfectants that can grow in up to 6 ⁇ M MTX medium.
  • the secretion rate was measured and cell lines highly expressing foreign proteins were selected.
  • Cell lines with a secretion rate of more than 5 (preferably about 15) ⁇ g / 10 6 (ie millions) cells / 24h are used for adaptive suspension culture using serum-free medium. The cell supernatant was collected and the bispecific antibody was isolated and purified.
  • the bispecific antibody AP163 was purified by three-step chromatography. They are affinity chromatography, hydroxyapatite chromatography and anion exchange chromatography (the protein purification instrument used in this example is AKTA pure 25M of GE Corporation. The reagents used in this example are all purchased from Sinopharm Chemical Reagents) Co., Ltd., the purity is analytical grade).
  • affinity chromatography MabSelect Sure affinity chromatography medium (MabSelect Sure, purchased from GE) of GE company or other commercially available affinity medium (such as Diamond protein A of Boglong company, etc.) Sample capture, concentration and removal of some contaminants.
  • the second step hydroxyapatite chromatography: using BIO-RAD's CHT Type II or other commercially available hydroxyapatite media (CHT Type II, purchased from BIO-RAD) for intermediate purification, used to reduce polymer content.
  • CHT Type II commercially available hydroxyapatite media
  • the third step, anion exchange chromatography use Q-HP of Boglong Company or other commercially available anion exchange chromatography media (Q-HP, purchased from Boglong Company) (such as GE's QHP, TOSOH Toyopearl) GigaCap Q-650, the world ’s DEAE Beads 6FF, Racepoint ’s Generik MC-Q, Merck ’s Fractogel EMD TMAE, Pall ’s Q Ceramic Ceramic HyperF) are refined to further remove HCP, DNA and other contaminants.
  • Q-HP commercially available anion exchange chromatography media
  • the second step is hydroxyapatite chromatography separation
  • the obtained target protein was loaded, collected and flowed through.
  • the balance column (20mM, PB, 0.15M, NaCL, pH 7.0) was used to flush the chromatography column with 3-5 column volumes at a linear flow rate of 100-200cm / h ( CV); collect the flow-through components and send samples for protein content, SEC-HPLC and electrophoresis detection.
  • SEC-HPLC purity results and SDS-PAGE electrophoresis results of the samples are shown in Figure 1 and Figure 2, where the SEC-HPLC results show that the main peak purity of the bispecific antibody after three-step chromatography is more than 95%, and the SDS-PAGE electrophoresis band
  • the type is as expected, non-reduced electrophoresis (180KDa), and a clear (90KDa) single-stranded band can be obtained after reduction.
  • AP163 can specifically bind to BCMA-positive cells with a dose-effect relationship; meanwhile, AP163 can be highly specific for human T cells and cynomolgus monkey T cells. Combined, and have a dose-effect relationship.
  • the antigen-coated human and monkey CD3 and BCMA were diluted with PBS to 0.1 ⁇ g / ml, added to 96-well plates, 100 ⁇ l / well, and coated at 2-8 ° C overnight. Discard the liquid in the plate, add PBST containing skimmed milk powder and block at room temperature for 2h, then wash the plate twice with PBST.
  • the bispecific antibody to be tested was diluted 4-fold in total with 12 gradients, 2 replicate wells per concentration, 100 ⁇ l / well was added to 96-well plates, and incubated at room temperature for 2 h.
  • Normally cultured human myeloma NCI-H929 cells were used as target cells, stained with PKH26 staining reagent, and resuspended the cells using 1640 complete medium, adjusting the cell density to 1 ⁇ 10 5 cells / ml, adding 50 ⁇ l / well to 96-well cell culture In the board.
  • the bispecific antibody was gradient diluted with culture medium and added at 50 ⁇ l / well. Add 5 times the number of effector cells (expanded cultured T cells) to the number of target cells, 50 ⁇ l / well. Incubate at 37 ° C in a 5% CO 2 incubator for 1 hour to allow the sample to be tested and the cells to mix well and a bridging reaction to occur.
  • the 96-well plate was washed with DPBS, flow cytometry was used for on-machine detection, and T cells were captured.
  • the T cells with PKH26 signal were bridged cells, and the statistical data was used to calculate the bridge ratio.
  • AP163 can specifically make tumor cells and target cells to have a bridging reaction, and has a dose-effect relationship.
  • Bispecific antibodies mediate the activation of CD4 + T cells / CD8 + T cells
  • PBMC recruit 3 healthy volunteer blood donors, draw peripheral blood and extract PBMC, use CD4 + T cell isolation kit and CD8 + T cell isolation kit to sort and enrich CD4 + T cells and CD8 + T cells in PBMC, use Resuspend the cells in 1640 complete medium containing 10% FBS, adjust the cell density to 1 ⁇ 10 6 cells / ml, and add 50 ⁇ l / well to the 96-well cell culture plate.
  • Culture human myeloma NCI-H929 cells adjust the cell density to 1 ⁇ 10 5 cells / ml, and add 50 ⁇ l per well.
  • the bispecific antibody was diluted in a gradient, 50 ⁇ l / well was added to a 96-well plate, and incubated at 37 ° C.
  • AP163 can mediate the release of granzyme by CD4 + T cells and CD8 + T cells of three healthy volunteers when killing tumor cells, and shows a dose-effect relationship.
  • Jurkat T cells (purchased from BPS Bioscience) containing the NFAT RE reporter gene can overexpress luciferase in the presence of bispecific antibodies and BCMA positive cells, and quantify Jurkat T by detecting luciferase activity The degree of cell activation. Specifically, H929 cells were resuspended by centrifugation, the cell density was adjusted to 2 ⁇ 10 5 cells / ml, and 40 ⁇ l / well was added to a 96-well cell culture plate. NFAT-Jurkat cells adjust the cell density to 2 ⁇ 10 6 cells / ml, and add 40 ⁇ l per well. The bispecific antibody AP163 was diluted with culture medium to 50 ⁇ g / mL.
  • AP163 can specifically activate NFAT-Jurkat cells with an EC 50 value of 3.161 ng / ml, and its concentration is proportional to the signal intensity.
  • Bispecific antibodies mediate the ability of T cells to kill tumor cells
  • AP163 EC mediated T cell killing of tumor cells is 50 8 0.239pM
  • eating mediated EC cynomolgus monkey T cells to kill tumor cells 50 is 0.278pM.
  • AP163 can specifically mediate human and cynomolgus monkey T cells to kill tumor cells
  • H929 cells with high expression of BCMA show significant killing effect, and show a dose-effect relationship.
  • bispecific antibodies to activate T cells alone or dependent on target cells to cause cytokine release is evaluated.
  • the primary T cells were cultured, and the cells were collected by centrifugation and resuspended with 1% PBSB. The cell density was adjusted to 1 ⁇ 10 6 cells / ml and placed in 96-well plates at 90 ⁇ l / well.
  • Maternal monoclonal antibody AB314 (refer to WO2007042261 patent document) and bispecific antibody AP163 that recognize CD3 were diluted with culture medium to 10000ng / mL, after 10-fold dilution, 10 ⁇ l / well was added to 96-well plate and placed at 37 °C, Cultivate in a 5% CO 2 incubator.
  • the culture supernatant was taken at 24h and 48h incubation, and detected and analyzed with LEGENDplex TM human Th1 / Th2 kit, and the signal intensity was detected by flow cytometry. Taking the cytokine concentration as the Y axis and the antibody concentration as the X axis, the analysis was performed by the software GraphPad Prism 6, and the amount of cytokine release induced by AB314 and AP163 activated T cells was calculated. The results showed that in the absence of target cells, AB314 activated primary T cells for 24h, causing the release of cytokines IL-4, IL-5 and TNF- ⁇ ; while AP163 activated primary T cells for 24h and 48h. No significant cytokine release.
  • Human T cells were cultured, and the cells were collected by centrifugation and resuspended in 1640 complete medium of 10% FBS. The cell density was adjusted to 1 ⁇ 10 6 cells / ml and placed in 96-well plates at 50 ⁇ l / well. The density of human myeloma NCI-H929 cells was adjusted to 1 ⁇ 10 5 cells / ml, and 50 ⁇ l / well was added. After AP163 was diluted in a gradient, 50 ⁇ l / well was added to a 96-well plate and incubated at 37 ° C. in a 5% CO 2 incubator for 1, 2, 3, 4, 5, 6, and 24 h, respectively.
  • Bispecific antibodies mediate T cell killing of BCMA positive human tumor cells under different effect target ratios
  • PBMC PBMC
  • PBMC PBMC
  • resuspend the cells with 1640 complete medium containing 10% FBS adjust the cell density to 3 ⁇ 10 6 cells / ml, add 50 ⁇ l per well to 96 well cell culture board.
  • Prepare a dilution of dexamethasone and indomethacin add 50 ⁇ l per well to a 96-well plate, and incubate PBMC for 1 h and 14 h, respectively; add a volume of buffer to the control group.
  • Human myeloma NCI-H929 cells were adjusted to a cell density of 1 ⁇ 10 5 cells / ml, 50 ⁇ l per well.
  • NPG mice were co-inoculated with human CIK cells and human myeloma cells NCI-H929 transplanted tumor model
  • mice Human myeloma NCI-H929 cells and CIK cells (expansion induced by human PBMC activation to day 10) were co-inoculated at different ratios under the skin of the right anterior flank of female NPG mice.
  • the mice were randomly grouped according to the weight of the mice, 4 in each group, for a total of 4 groups. Dosing started on the day of grouping. The route of administration in all groups was intraperitoneal injection. The control group was given the same volume of PBS solution. The dose of AP163 was 0.2 mg / kg. Dosing twice a week for 4 weeks. The tumor volume and body weight were measured every 3 days, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the animal was euthanized, the tumor was stripped and weighed, the photograph was taken, and the relative tumor inhibition rate (TGI%) was calculated.
  • TGI% tumor inhibition rate
  • the average tumor volume of the effective target ratio 1/1 control group was 1501 ⁇ 351mm 3
  • the average tumor volume of the effective target ratio 1/2 control group was 1555 ⁇ 244mm 3
  • the effective target ratio 1 / 1AP163 test group mean tumor volume of 99 ⁇ 38mm 3
  • TGI% 93% efficiency target ratio 1 / 1AP163 test group mean tumor volume was 481 ⁇ 215mm 3
  • TGI% was 70.2%.
  • NPG mice were co-inoculated with human CIK cells and human Burkkit ’s lymphoma Raji cell transplantation tumor model
  • mice Human Burkkit's lymphoma Raji cells and cultured and expanded CIK (Raji cell to CIK cell ratio 1: 1), mixed with Matrigel at a volume ratio of 1: 1, were co-inoculated on the right back of female NPG mice Subcutaneously. One hour after the inoculation, the mice were randomly grouped according to the weight of the mice, 4 in each group, for a total of 4 groups. Dosing started on the day of grouping, and the control group was given the same volume of PBS solution. The doses of the AP163 experimental group were 1 mg / kg, 0.1 mg / kg, and 0.01 mg / kg, respectively. In all groups, the route of administration was intraperitoneal injection, twice a week for a total of 3 weeks.
  • the tumor volume and body weight were measured every 3 days, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the animal was euthanized, the tumor was stripped and weighed, the photograph was taken, and the relative tumor inhibition rate (TGI%) was calculated.
  • the average tumor volume of the control group was 1750 ⁇ 653 mm 3 ; the average tumor volume of the AP163 1 mg / kg, 0.1 mg / kg, and 0.01 mg / kg test groups were all 0.00 ⁇ 0.00 mm 3 , all with The tumor volume of the solvent control group was significantly different (P ⁇ 0.05), and the TGI of all three groups was 100%, indicating that the test bispecific antibody AP163 had extremely significant tumor suppressing effect.
  • NPG mice were co-inoculated with human CIK cells and human myeloma cells RPMI-8226 transplanted tumor model
  • Human myeloma cells RPMI-8226 and CIK were co-inoculated subcutaneously on the right back of female NPG mice. One hour after the inoculation, they were randomly divided into groups according to the body weight of the mice and started to be administered that day. There are 7 in the first group and 8 in the remaining two groups, a total of 3 groups. Dosing started on the day of grouping, and the control group was given the same volume of PBS solution. The dosage of AP163 experimental group was 1 mg / kg and 0.1 mg / kg, respectively. In all groups, the route of administration was intraperitoneal injection, once every two days, 8 consecutive times, and the experiment was terminated 18 days after the last dose.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the animal was euthanized, the tumor was stripped and weighed, the photograph was taken, and the relative tumor inhibition rate (TGI%) was calculated.
  • the body weight of each group of animals showed an increase, and there was no significant difference in the weight of animals in different groups (P> 0.05).
  • the average tumor volume of the control group was 1647.79 ⁇ 247.90mm 3 ; the average tumor volume of the AP163 1mg / kg test group was 0.00 ⁇ 0.00mm 3 and the TGI% was 100%; the average tumor volume of the AP163 0.1mg / kg test group was 8.00 ⁇ 5.24 mm 3 , TGI% is 99.51%.
  • the experimental results show that the antibody AP163 has a very significant tumor suppressing effect. Under the conditions of this experiment, AP163 significantly inhibited tumor growth at all administration concentrations. At the same time, AP163 also showed good safety and did not produce obvious toxic effects on experimental animals.
  • NPG mice were co-inoculated with human CIK cells and human Burkkit ’s lymphoma Daudi cell transplantation tumor model
  • mice Human Burkkit's lymphoma Daudi cells and cultured and expanded CIK (Daudi cell density 5 ⁇ 10 6 cells / ml, CIK cell density 1 ⁇ 10 6 cells / ml) were mixed with Matrigel at a volume ratio of 1: 1 , Co-inoculated subcutaneously on the right back of female NPG mice. One hour after the inoculation, the mice were randomly grouped according to the weight of the mice, with 6 animals in each group, for a total of 4 groups. Dosing started on the day of grouping, and the control group was given the same volume of PBS solution. The doses of the AP163 experimental group were 1 mg / kg, 0.2 mg / kg, and 0.04 mg / kg, respectively.
  • the route of administration in all groups was intraperitoneal injection, once every two days, a total of 8 times, and the experiment was ended 10 days after the last dose.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • the animal was euthanized, the tumor was stripped and weighed, the photograph was taken, and the relative tumor inhibition rate (TGI%) was calculated.
  • a transient increase in Neut, CD3-CD20 +, TNF- ⁇ , IL-2 and IL-6 and a decrease in Lymph, CD3 + , CD3-CD16 + / CD56 + can be seen after the first drug; the maximum tolerated dose ( MTD) ⁇ 2.5mg / kg.
  • Collect venom blood samples (approximately 1 mL) from the non-administration site of the subcutaneous vein of the animal's hind limbs into a tube without anticoagulant.
  • the blood collection points of groups 1 to 3 are: before the first and last administration of each group of animals Immediately after ( ⁇ 1min), 1h, 3h, 6h, 8h, 24h, 48h, 72h after the start of administration.
  • the centrifuge tube (not anticoagulated) is stored in an ice water bath before use; the blood sample is collected and transferred to the above centrifuge tube, and then centrifuged at 3000 ⁇ g for 10 min at 2-8 ° C. After separating the serum sample, divide it into 2 parts and store it under -70 °C. Blood samples must be collected within 2 hours from the completion of centrifugation.
  • the ELISA method was used to detect and analyze the concentration of AP163 in the serum, and the non-compartment model method (NCA) of WinNonlin 8.0 software was used to calculate the kinetic parameters of the drug administration group.
  • NCA non-compartment model method
  • the results showed that the half-life of AP163 in the 0.1, 0.5 and 2.5 mg / kg groups was 7.08, 8.95 and 11.42 hours, respectively.

Abstract

一种双特异性抗体，特异性结合免疫细胞的表面抗原CD3和肿瘤细胞表面的BCMA抗原，其能够以高亲和力与人CD3结合并诱导T细胞增殖，介导肿瘤细胞杀伤作用。所述双特异性抗体在体外测试可用于介导T细胞特异杀伤靶细胞。该双特异性抗体构建方法简单，避免了异源双特异性抗体两组轻链与重链错配的可能性，从而降低了抗体纯化的难度，得到的抗体亲和力高，引起的细胞因子副作用小，安全性高。

Description

双特异性抗体及其用途 技术领域
本发明涉及免疫学领域，更具体地，涉及一种介导T细胞杀伤的抗CD3双特异性抗体，以及这类抗体的用途，特别是其在治疗癌症中的用途。
背景技术
多发性骨髓瘤是第二常见的血液***恶性肿瘤，其骨髓中单克隆浆细胞的不受控制的增殖，导致单克隆免疫球蛋白和免疫抑制的过量产生，以及骨溶解和终末器官损伤。目前有两个单克隆抗体已批注临床使用，在过去十年，多发性骨髓瘤治疗方案已经显著提高了患者的存活率。尽管如此，现有的治疗方案仍未满足目前的治疗需求，特别是对于当前疗法具有抗性的复发/难治性患者。
B细胞成熟抗原(BCMA)是一种高度浆细胞特异性抗原，在调节B细胞成熟和分化为浆细胞方面通过参与增殖诱导配体(APRIL)发挥重要作用。BCMA表达限于B细胞谱系并且主要存在于浆细胞和浆母细胞上，并且在一定程度上存在于记忆B细胞上，但在外周和幼稚B细胞上实质上不存在，也未见在其他正常组织细胞中表达。BCMA也在多发性骨髓瘤细胞上表达并参与白血病和淋巴瘤。连同其家族成员TACI(跨膜活化剂和亲环素受体配体相互作用物)和BAFF-R(B细胞活化因子受体)，BCMA调节体液免疫、B细胞发育和体内稳态的不同方面。BCMA的表达出现在B-细胞分化作用的较后期并有利于浆母细胞和浆细胞在骨髓中的长期存活。小鼠中BCMA基因的靶向缺失导致骨髓中长寿浆细胞的数量显著减少，指示BCMA对其存活具有重要性。BCMA过表达或通过多发性骨髓瘤细胞中的BCMA刺激APRIL可以直接上调关键免疫检查点分子，这可能有助于免疫抑制骨髓微环境。
在细胞免疫过程中，T淋巴细胞扮演着重要的角色。T细胞所介导的细胞免疫主要是通过T细胞受体(T cell receptor，TCR)特异识别细胞表面由主要组织相容性复合物(MHC)所递呈的抗原肽，进而激活T细胞胞内信号，对该靶细胞进行特异杀伤。这对及时清除体内病变的细胞及预防肿瘤的发生起着至关重要的作用。由于多数癌细胞表面的MHC的表达下调甚至缺失，使得肿瘤细胞能够逃逸免疫杀伤，从而发生肿瘤。
T细胞结合双特异性抗体(T cell-engaging bispecific antibodies，TCBs)代表了一种非常有效的将激活的细胞毒性T细胞重定向到肿瘤的方式。CD3作为T细胞受体的一部分，表达于成熟T细胞，能够转导TCR识别抗原所产生的活化信号。TCBs能够同时结合表面肿瘤抗原和T细胞受体的CD3ε亚基，在T细胞和肿瘤细胞之间提供一个物理连接，从而有效的激活静止的T细胞杀伤肿瘤细胞，达到***的效果(Smits N C,Sentman C L,Journal of Clinical Oncology,2016:JCO649970.)。因为T细胞双特异性旁路TCR抗原识别和T细胞活化的共刺激要求，它们消除了对肿瘤特异性免疫的需要，并克服了肿瘤微环境中T细胞面临的 许多障碍。
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，包括BiTE、DART、TrandAbs、bi-Nanobody等。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。另一类双特异性抗体保留Fc结构域，例如Triomabs、kih IgG、Cross-mab、orthoFab IgG、DVD IgG、IgG scFv、scFv 2-Fc等构型。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)。然而这类双抗也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。
因此，本发明旨在开发一种在产品半衰期、稳定性、安全性和可生产性方面具有改善性能的BCMA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和肿瘤抗原BCMA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BCMA的正常细胞的非特异性杀伤作用显著降低，同时增加由效应细胞过度活化所致的毒副作用的可控性，且其理化性质和体内稳定性都显著提高。
具体的，本发明的第一个方面，公开了一种双特异性抗体，所述双特异性抗体分子由两条相同的多肽链以共价键结合形成四价同源二聚体，每条多肽链从N端至C端依次包含特异性结合肿瘤抗原BCMA的第一单链Fv、特异性结合效应细胞抗原CD3的第二单链Fv和Fc片段；其中，第一和第二单链Fv通过连接肽相连，而第二单链Fv与Fc片段直接相连或通过连接肽相连，且所述Fc片段不具有CDC、ADCC和ADCP等效应子功能。
其中，所述第一单链Fv包含VH结构域和VL结构域，并通过连接肽L1连接，且所述连接肽L1的氨基酸序列为(GGGGX) n，X包含选自Ser或Ala，n为1-5的自然数；X优选Ser，n优选3。
本发明的一优选实施例中，所述连接肽L1的氨基酸序列为(GGGGS)3，在其他优选实施例方案中，所述连接肽L1的氨基酸序列还包括(GGGGS)1或(GGGGS)2或(GGGGS)4或(GGGGS)5或(GGGGA)1或(GGGGA)2或(GGGGA)3或(GGGGA)4或(GGGGA)5。
优选的，所述的第一单链Fv包含：
(1)VH结构域，其包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：1、2和3所示，或与上述序列至少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
(2)VL结构域，其包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：4、5和6所示，或与上述序列至少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。
更优选的，所述第一单链Fv，其包含：
(1)氨基酸序列如SEQ ID NO：7所示的VH结构域，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(例如保守性取代))的序列；和
(2)氨基酸序列如SEQ ID NO：8所示的VL结构域，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(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***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：9所示)或其截短的片段(以下统称为CTP)。优选地，CTP1刚性肽包含SEQ ID NO：9N端的10个氨基酸，即SSSSKAPPPS；或CTP2刚性肽包含SEQ ID NO：9C端的14个氨基酸，即SRLPGPSDTPILPQ；CTP3刚性肽包含SEQ ID NO：9N端的16个氨基酸，即SSSSKAPPPSLPSPSR；CTP4刚性肽包含28个氨基酸并开始于人绒毛膜***β亚基的第118位，终止于第145位，即SSSSKAPPPSLPSPSRLPGPSDTPILPQ。
在本发明的一优选实施例中，所述刚性肽为SSSSKAPPPS，即CTP1刚性肽。在其他优选实施方案中，刚性肽序列还包括CTP2(SRLPGPSDTPILPQ)、CTP3(SSSSKAPPPSLPSPSR)、CTP4(SSSSKAPPPSLPSPSRLPGPSDTPILPQ)。
在本发明的一优选实施例中，所述连接肽L2的氨基酸序列如SEQ ID NO：10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CTP1刚性肽。
其中，双特异性抗体的第二单链Fv特异性结合CD3，在体外FACS结合分析测定中以大于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。
优选地，所述第二单链Fv包含：
(1)VH结构域，其包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：11、12和13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(例如保守性取代))的序列；和
(2)VL结构域，其包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：14、15和16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(例如保守性取代))的序列。
更优选地，所述第二单链Fv包含氨基酸序列如SEQ ID NO：17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(例如保守性取代))的VH结构域；和
氨基酸序列如SEQ ID NO：18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(例如保守性取代))的VL结构域。
优选地，所述第二单链Fv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选自Ser或Ala，n为1-5的自然数；X优选Ser，n优选3。
本发明的一优选实施例中，所述连接肽L3的氨基酸序列为(GGGGS)3，在其他优选实施例方案中，所述连接肽L3的氨基酸序列还包括(GGGGS)1或(GGGGS)2或(GGGGS)4或(GGGGS)5或(GGGGA)1或(GGGGA)2或(GGGGA)3或(GGGGA)4或(GGGGA)5。
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。在其他优选实施例中，所述Fc片段通过连接肽L4与第二单链Fv相连，连接肽L4氨基酸序列包括(GGGGS)1或(GGGGS)2或(GGGGS)3或(GGGGS)4。
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)。
所述Fc片段的氨基酸序列如SEQ ID NO：19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***确定的K447。EU编号***来自网址：http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.htm.
在一些优选实施方案中，所述Fc片段被改变，例如被突变，以修饰本发明所述双特异性抗体分子的性质(例如改变下列中的一个或更多个特性：Fc受体结合、抗体糖基化、效应细胞功能或补体功能)。
例如，本发明提供的双特异性抗体包含具有改变的效应子功能(例如降低或消除)的氨基酸置换、缺失或添加的Fc变体。抗体的Fc区介导几种重要的效应子功能，例如ADCC、ADCP、CDC等。通过替换抗体的Fc区中的氨基酸残基，以改变抗体对效应子配体(如FcγR或补体C1q)的亲和力，从而改变效应子功能的方法是本领域已知的(参见，例如EP 388,151A1；US 564,8260；US 562,4821；Natsume A等,Cancer Res.,68:3863-3872,2008；Idusogie EE等,J.Immunol.,166:2571-2575,2001；Lazar GA等,PNAS,103:4005-4010,2006；Shields RL等,JBC,276:6591-6604,2001；Stavenhagen JB等,Cancer Res.,67:8882-8890,2007；Stavenhagen JB等,Advan.Enzyme.Regul.,48:152-164,2008；Alegre ML等,J.Immunol.,148:3461-3468,1992；和Kaneko E等,Biodrugs,25:1-11,2011)。在本发明一些优选实施例中，对抗体恒定区上的氨基酸L235(EU编号)进行修饰以改变Fc受体相互作用，例如L235E或L235A。在另一些优选实施例中，对抗体恒定区上的氨基酸234和235同时进行修饰，例如L234A和L235A(L234A/L235A)(EU编号)。
例如，本发明提供的双特异性抗体可包含具有延长的循环半衰期的氨基酸置换、缺失或添加的Fc变体。研究发现M252Y/S254T/T256E、M428L/N434S或者T250Q/M428L都能够延长抗体在灵长类动物中的半衰期。更多的与新生儿受体(FcRn)结合亲和力增强的Fc变体所包含突变位点可以参见中国发明专利CN 201280066663.2、US 2005/0014934A1、WO 97/43316、US 5,869,046、US 5,747,03、WO 96/32478。在本发明一些优选实施例中，对抗体恒定区上的氨基酸M428(EU编号)进行修饰以增强FcRn受体的结合亲和力，例如M428L。在另一些优选实施例中，对抗体恒定区上的氨基酸250和428(EU编号)同时进行修饰，例如T250Q和M428L(T250Q/M428L)。
例如，本发明提供的双特异性抗体也可包含具有可以降低或消除Fc糖基化的氨基酸置换、缺失或添加的Fc变体。例如，Fc变体包含正常存在于氨基酸位点297(EU编号)处的N-连接聚糖降低的糖基化。N297位糖基化对IgG的活性有很大影响，如果该位点糖基化被移除，则会影响IgG分子CH2上半部分的构象，从而丧失对FcγRs的结合能力，影响抗体相关的生物活性。在本发明的一些优选实施例中，对人IgG恒定区上的氨基酸N297(EU编号)进行修饰以避免抗体的糖基化，例如N297A。
例如，本发明提供的双特异性抗体也可包含具有消除电荷异质性的氨基酸置换、缺失或添加的Fc变体。在工程细胞表达过程中发生的多种翻译后修饰会都会引起单克隆抗体的电荷异质性，而IgG抗体C末端赖氨酸的不均一性是其中的一个主要原因，重链C端的赖氨酸K可能在抗体生产过程中出现一定比例的缺失， 从而造成电荷异质性，从而影响抗体的稳定性、有效性、免疫原性或药代动力学。在本发明的一些优选实施例中，将IgG抗体C末端的K447(EU编号)去除或缺失，以消除抗体的电荷异质性，提高表达产物的均一性。
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：19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。
本发明所述双特异性抗体分子由两条相同的多肽链通过Fc片段铰链区的链间二硫键结合形成四价同源二聚体，每条多肽链自N端至C端依次由抗-BCMA scFv、连接肽、抗-CD3 scFv和Fc片段组成。
本发明一优选实施例中，所述双特异性抗体结合人BCMA和CD3，其氨基酸序列如下：
(1)SEQ ID NO：20所示的序列；
(2)与SEQ ID NO：20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
(3)与SEQ ID NO：20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，(2)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：21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，优选地，所述宿主细胞为哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，进一步优选为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和/或载体和/或稀释剂。
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，所述表达载体为质粒，更优选地，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，优选地，所述宿主细胞为哺乳动物细胞，如CHO细胞、NS0细胞或其它哺乳动物细胞，进一步优选为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用于治疗或改善选自浆细胞障碍、其他与BCMA表达有关的B细胞障碍和自生免疫性疾病的药物中的用途，其中所述浆细胞障碍包括但不限于多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白白血症、淀粉样变性、华氏巨球蛋白白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病及郁积型多发性骨髓瘤。
本发明公开的技术方案，取得了有益的技术效果：
1、本发明提供的双特异性抗体不易结合弱表达或低表达BCMA的正常细胞，减少了非特异性杀伤，但对过表达或高表达BCMA的细胞的结合特异性没有显著下降，表现出良好的体内杀伤效果。由此亦知，当靶抗原仅表达于肿瘤细胞上或本发明所述双特异性抗体仅与过表达靶抗原的肿瘤细胞特异性结合时，使得免疫效应细胞限制性仅在靶细胞组织内被激活，这使得所述双特异性抗体对正常细胞的非特异性杀伤以及细胞因子的伴随释放能够被降至最低，减小其在临床治疗中的毒副作用。
2、本发明提供的双特异性抗体选择的抗-CD3 scFv以微弱的结合亲和力(EC 50值大于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-BCMA 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。
3、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。
4、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。
5、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。
发明详述
缩写和定义
BCMA      B细胞成熟抗原
BiAb      双特异性抗体(bispecific antibody)
CDR       用Kabat编号***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***
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、***、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。
“BCMA”是B细胞成熟抗原，属于肿瘤坏死因子受体超家族成员，优先在成熟B淋巴细胞中表达，并且表达在浆母细胞(即，浆细胞前体)和浆细胞的表面。在脾脏、***、胸腺、肾上腺和肝脏中均能检测到BCMA的RNA，多个B细胞系成熟后其BCMA mRNA的水平也增加。BCMA与白血病、淋巴瘤(如霍奇金淋巴瘤)、多发性骨髓瘤、自身免疫性疾病(如***性红斑狼疮)等多种疾病有关，因此可作为涉及相关B细胞疾病的潜在靶标。针对BCMA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BCMA的任何变体、同工型、和物种同源物，其由细胞-包括肿瘤细胞-天然地表达，或由以BCMA基因或cDNA转染的细胞表达。
CD3分子是T细胞膜上的重要分化抗原，是成熟T细胞的特征性标志，由6条肽链组成，以非共价键与T细胞抗原受体(TCR)组成TCR-CD3复合体，不仅参与TCR-CD3复合体的胞浆内组装，而且通过各多肽链胞浆区的免疫受体酪氨酸活化基序(Immunoreceptor Tyrosine-based Activation Motif，ITAM)传递抗原刺激信号。CD3分子的主要功能为：稳定TCR结构，传递T细胞活化信号，当TCR特异性识别并结合抗原后，CD3参与将信号转导到T细胞胞浆内，作为诱导T细胞活化的第一信号，在T细胞抗原识别和免疫应答产生过程中具有极其重要的作用。
“CD3”指的是作为T细胞受体复合物的一部分，由三个不同的链CD3ε，CD3δ和CD3γ组成。CD3在T细胞上通过例如抗CD3抗体对其的固定作用而产生的集中(clustering)，导致T细胞的活化，与T细胞受体介导的活化类似，但是不依赖于TCR克隆的特异性。绝大多数抗CD3抗体识别CD3ε链。本发明的特异性识别T细胞表面受体CD3的第二功能域不受具体的限制，只要其能够特异性地识别CD3，例如但不限于在下列专利中提到的CD3抗体：US7,994,289，US6,750,325；US6,706,265；US5,968,509；US8,076,459；US7,728,114；US20100183615。优选地，本发明中使用的抗人CD3抗体与食蟹猴和/或恒河猴具有交叉反应性，例如但不限于下列专利中提到的抗人CD3抗体：WO 2016130726，US 20050176028，WO 2007042261或WO 2008119565。该术语还包括任何CD3变体、同工型、衍生物和物种同源物，其由细胞天然地表达，或在以编码前述的链的基因或cDNA转染的细胞上表达。
术语“抗体”具体包括单克隆抗体、多克隆抗体和抗体样多肽，例如嵌合抗体和人源化抗体。“抗原结合 片段”包括通过任何已知技术提供的片段，例如酶裂解，肽合成和重组技术。一些抗原结合片段由保留亲本抗体分子的抗原结合特异性的完整抗体部分组成。例如，抗原结合片段可以包含已知结合特定抗原的抗体的至少一个可变区(重链或轻链可变区)或一个或多个CDR。合适的抗原结合片段的实例包括但不限于双特异性抗体体和单链分子以及Fab，F(ab')2，Fc，Fabc和Fv分子，单链(Sc)抗体，单独的抗体轻链，单独的抗体重链，抗体链或CDR和其他蛋白质之间的嵌合融合物，蛋白质支架，重链单体或二聚体，轻链单体或二聚体，由一个重链和一个轻链组成的二聚体，由VL，VH，CL和CH1结构域，或如WO2007059782中所述的单价抗体，包含通过铰链区上的二硫键连接的两个Fab片段的二价片段，基本上由VH和CH1结构域组成的Fd片段；基本上由抗体单臂的VL和VH结构域组成的Fv片段，dAb片段(Ward等人,Nature,1989,341:544-54)，其基本上由VH结构域组成，也称为结构域抗体(Holt等人,Trends Biotechnol.2003,21(11)：484-90)；或纳米体(Revets等人；Expert Opin Biol Ther.2005Jan；5(1)：111-24)；分离的互补决定区(CDR)等。所有抗体同种型可用于产生抗原结合片段。另外，抗原结合片段可以包括非抗体蛋白质框架，其可以成功地将多肽片段并入赋予给定的感兴趣抗原(例如蛋白质支架)的亲和力的取向。可以通过完整抗体的酶切或化学切割来重组产生或产生抗原结合片段。术语“抗体或其抗原结合片段”可以用于表示给定的抗原结合片段掺入短语中提到的抗体的一个或多个氨基酸片段。
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***确定。本领域技术人员可以明确地将每种***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“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***的239和242处(位置226或229，EU编号***)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“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***(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)、***(LH)、促甲状腺素(TSH)和绒毛膜***(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)结合亲和力改变的Fc变体，这里使用的“FcRn”指结合IgG抗体Fc区的至少部分由FcRn基因编码的蛋白。FcRn可以来源于包括但不限于人、小白鼠、大鼠、兔子和猴的任何生物体。功能性FcRn蛋白包含经常被称为重链和轻链的两条多肽，轻链是β-2-微球蛋白，重链由FcRn基因编码。
本发明涉及对FcRn的结合被调节的抗体(调节包括增加以及降低结合)。例如：在有些情况下，增加的结合会导致细胞再循环抗体，并由此延长，例如治疗抗体的半衰期。有时，降低FcRn结合是合乎需要的，例如用作包含放射标记的诊断抗体或治疗抗体。另外，对FcRn的结合显示出增加，同时对其他Fc受体，例如FcγRs的结合被改变的抗体可以用于本发明。
本申请涉及包含调节对FcRn的结合力的氨基酸修饰的抗体。具有特殊意义的是在较低的pH时，对FcRn的结合亲和力显示出增加，而在更高的pH时，结合基本上不显示出改变的最低限度地包含Fc区的抗体或 其功能性变体。
与新生儿受体(FcRn)结合亲和力增强的Fc变体，IgG的血浆半衰期取决于它与FcRn的结合，一般在pH 6.0时结合，在pH 7.4(血浆pH)时解离。通过对两者结合位点的研究，改造IgG上与FcRn结合的位点，使之在pH 6.0时结合能力增加。已经证明对于结合FcRn重要的人Fcγ结构域的一些残基的突变可增加血清半衰期。已报道T250、M252、S254、T256、V308、E380、M428和N434(EU编号)中的突变可增加或降低FcRn结合亲和力(Roopenian DC等,Nat.Rev.Immunol.,7:715-725,2007)。韩国专利号KR 10-1027427公开了具有增加的FcRn结合亲和力的曲妥珠单抗(赫赛汀，Genentech)变体，并且这些变体包含选自257C、257M、257L、257N、257Y、279Q、279Y、308F和308Y的一个或更多个氨基酸修饰。韩国专利公开号KR 2010-0099179提供了贝伐单抗(阿瓦斯汀，Genentech)变体并且这些变体通过包含在N434S、M252Y/M428L、M252Y/N434S和M428L/N434S的氨基酸修饰显示增加的体内半衰期。此外，Hinton等也发现T250Q和M428L2个突变体分别使与FcRn的结合增加3和7倍。同时突变2个位点，则结合增加28倍。在恒河猴体内，M428L或T250QM/428L突变体显示血浆半衰期增加2倍(Hinton PR等,J.Immunol.,176:346-356,2006)。更多的与新生儿受体(FcRn)结合亲和力增强的Fc变体所包含突变位点可以参见中国发明专利CN 201280066663.2。此外，有研究对五种人源化抗体的Fc段进行T250Q/M428L突变不仅改善了Fc与FcRn的相互作用，且在随后的体内药代动力学试验中，发现以皮下注射给药，Fc突变抗体与野生型抗体相比药代动力学参数有所改善，如体内暴露量增加、清除率降低、皮下生物利用度提高(Datta-Mannan A等,MAbs.Taylor&Francis,4:267-273,2012)。
其他可引起本发明抗体与FcRn亲和力增强的突变点包括但不限于以下氨基酸修饰：226,227,230,233,239,241,243,246,259,264,265,267,269,270,276,284,285,288,289,290,291,292,294,298,299,301,302,303,305,307,309,311,315,317,320,322,325,327,330,332,334,335,338,340,342,343,345,347,350,352,354,355,356,359,360,361,362,369,370,371,375,378,382,383,384,385,386,387,389,390,392,393,394,395,396,397,398,399,400,401,403,404,408,411,412,414,415,416,418,419,420,421,422,424,426,433,438,439,440,443,444,445,446，其中Fc区中氨基酸的编号是Kabat中的EU索引的编号。
与FcRn结合亲和力增强的Fc变体还包括其他一切公知的氨基酸修饰位点以及尚未被发现的氨基酸修饰位点。
在可选择的实施方式中，可以优化IgG变体使其具有增加或降低的FcRn亲和力，以及增加或降低的人FcγR，包括但不限于FcγRI、FcγRIIa、FcγRIIb、FcγRIIc、FcγRIIIa和包括他们的等位基因变异的FcγRIIIb亲和力。
优先地，IgG变体的Fc配体特异性将决定它的治疗应用。给定IgG变体用于治疗目的将取决于靶抗原的表位或形式，以及待治疗的疾病或适应症。对大多数靶和适应症来说，增强的FcRn结合可是更优选的， 因为增强的FcRn结合可以导致血清半衰期延长。较长的血清半衰期允许治疗时以较低的频率和剂量给药。为了使需要重复给药的适应症作出反应而施用该治疗剂时，这种特性可是特别优选的。对一些靶和适应症来说，当需要变体Fc具有增加的清除或降低的血清半衰期时，例如当Fc多肽用作显象剂或放射治疗剂时，降低的FcRn亲和力可是特别优选的。
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。
编码双特异性抗体的核酸，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SEQ ID NO：21。
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***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***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***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(Bacillus stearothermophilus))、真菌细胞(如酿酒酵母或毕赤酵母)、昆虫细胞(如包括草地夜蛾细胞在内的鳞翅目昆虫细胞)或哺乳动物细胞(如中国仓鼠卵巢(CHO)细胞、NS0细胞、小仓鼠肾(BHK)细胞、猴肾细胞、Hela细胞、人肝细胞癌细胞或293细胞等等)。
双特异性抗体可通过双特异性抗原的免疫原性制剂免疫合适的受试者(例如，兔，山羊，小鼠，或其它哺乳动物，包括转基因的和经剔除的上述哺乳动物)制备。合适的免疫原性制剂可以是，例如化学合成的或重组表达的双特异性抗原。所述的制剂可进一步包含佐剂，例如弗氏完全佐剂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点的单克隆抗体(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，或抗原结合片段相接触，并检测与抗原结合的抗体或其部分，或非结合抗体或其部分，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。
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。
双特异性抗体或编码本申请抗体的核酸或多核苷酸可连接至上述药学上可接受的载体或其组分的一些物质(作为免疫复合体)或与其分开施用。在后一种情况下，双特异性抗体或编码本申请抗体的核酸或多核苷酸可在上述药学上可接受的载体或其组分的一些物质之前、之后或与其共同施用，或可与其他已知疗法(如抗癌疗法、如辐射)共同施用。
本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。
本发明的药物组合物可以是两种药物的组合，可以是与已上市的类似功能相同产品或者增加治疗效果的产品的组合。
附图说明
图1、AP163纯化样品的SEC-HPLC检测结果。
图2、AP163纯化样品的SDS-PAGE电泳结果。
图3、双特异性抗体与BCMA阳性细胞结合能力测定。
图4、双特异性抗体与不同T细胞结合能力测定。
图5、双特异性抗体同时结合靶细胞与效应细胞能力测定。
图6、双特异性抗体介导CD4 +T细胞/CD8 +T细胞的激活。
图7、双特异性抗体活化报告基因细胞株Jurkat T细胞的能力测定。
图8、双特异性抗体介导T细胞杀伤肿瘤细胞的能力测定。
图9、不同效靶比下双特异性抗体介导T细胞杀伤BCMA阳性细胞的能力测定。
图10、双特异性抗体在NPG小鼠皮下共接种人CIK细胞和人骨髓瘤细胞RPMI-8226移植瘤模型中的体内 抑瘤效果。注：箭头代表各次给药时间；***表示存在显著性差异。
图11、双特异性抗体在NPG小鼠皮下共接种人CIK细胞和人Burkkit’s淋巴瘤Daudi细胞移植瘤模型中的体内抑瘤效果。注：箭头代表各次给药时间；***表示存在显著性差异。
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。
以下各实施例中，实验所用物料可购买，也可参照现有公开的技术制备；未标明来源和规格的均为市售可得；未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1、双特异性抗体分子表达载体的构建
双特异性抗体AP163由抗-BCMA scFv、连接肽L2、抗-CD3 scFv和Fc片段依次串联组成，抗-BCMA scFv和抗-CD3 scFv内部的VH和VL之间分别由连接肽L1和L3连接。AP163所包含的针对BCMA的scFv的VH和VL氨基酸序列分别如SEQ ID NO：7和SEQ ID NO：8所示。AP163包含的抗CD3-scFv的VH和VL氨基酸序列分别如SEQ ID NO：17和SEQ ID NO：18所示，且VH和VL之间由(GGGGS) 3连接。AP163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。
按常规分子生物学方法合成上述双特异性抗体的编码基因，并将获得的融合基因的编码cDNA分别***到经PCDNA3.1改造后的真核表达质粒pCMAB2M的相应酶切位点间。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)。
实施例2、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。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。
用受MTX药物抑制的DHFR基因进行共扩增，其操作步骤主要包括：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5(较佳地约15)μg/10 6(即百万)个细胞/24h的细胞系使用无血清培养基的进行适应性悬浮培养。收集细胞上清并分离纯化双特异性抗体。
实施例3、双特异性抗体的纯化
采用三步层析法对双特异性抗体AP163进行纯化。分别为亲和层析、羟基磷灰石层析和阴离子交换层析(本实施例采用的蛋白纯化仪为美国GE公司的AKTA pure 25M。本实施例中采用的试剂均购自国药集团化学试剂有限公司，纯度均为分析级)。
第一步，亲和层析：采用GE公司的MabSelect Sure亲和层析介质(MabSelect Sure，购自GE公司)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，pH3.5)，以不高于100cm/h的线性流速洗脱目标产物，收集目标峰。
第二步，羟基磷灰石层析：使用BIO-RAD公司的CHT TypeⅡ或其它市售的羟基磷灰石介质(CHT Type Ⅱ，购自BIO-RAD公司)进行中间纯化，用于降低聚合体含量。目标蛋白聚合以后，聚合体和单体之间存在性质上的差异，包括电荷特性以及钙离子螯合等，我们使用电荷特性等差异对二者进行分离。首先，使用平衡buffer(20mM PB，pH 7.0)，以100-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；第一步亲和层析分离得到的目标蛋白调pH 7.0，然后上样，载量控制在<5mg/ml；上样完毕后，使用平衡buffer(20mM PB，pH 7.0)，以100-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；最后进行目标蛋白洗脱，使用洗脱buffer(20mM PB，1M NaCL，pH 7.0)，以0-50％梯度洗脱，以不高于100cm/h的线性流速洗脱10个柱体积(CV)，对洗脱组分进行分段收集，分别送检SEC-HPLC。将单体百分比大于95％的目标组分合并进行下一步层析。
第三步，阴离子交换层析：使用博格隆公司的Q-HP或其它市售的阴离子交换层析介质(Q-HP，购自博格隆公司)(例如GE的Q HP、TOSOH的Toyopearl GigaCap Q-650、天地人和的DEAE Beads 6FF，赛 分科技的Generik MC-Q、Merck的Fractogel EMD TMAE、Pall的Q Ceramic HyperD F)进行精细纯化，进一步去除HCP、DNA等污染物。首先使用平衡buffer(20mM PB，0.15M NaCL，pH 7.0)，以100-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；经第二步羟基磷灰石层析分离得到的目标蛋白上样，收集流穿，上样完毕，使用平衡buffer(20mM PB，0.15M NaCL，pH 7.0)，以100-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；对流穿组分进行收集，分别送样进行蛋白含量、SEC-HPLC和电泳检测。
样品的SEC-HPLC纯度结果及SDS-PAGE电泳结果分见图1和图2，其中SEC-HPLC结果显示，三步层析后双特异性抗体的主峰纯度达95％以上，SDS-PAGE电泳带型符合预期，非还原电泳(180KDa)，还原后可得清晰的(90KDa)单链条带。
实施例4、Anti-BCMA×CD3双特异性抗体的体外生物学功能评价
(1)双特异性抗体与BCMA阳性细胞和T细胞的结合活性
培养人骨髓瘤NCI-H929细胞、人Jurkat-LUC细胞、人T淋巴细胞白血病HUT-78细胞、人骨髓瘤MM.1S细胞、人早幼粒白血病HL60细胞、人T细胞和食蟹猴T细胞，离心收集细胞用1％DPBS(杜氏磷酸缓冲液)重悬，分别调整细胞密度为2×10 6个/ml，置于96孔板中，每孔100μl。待测的双特异性抗体AP163进行梯度稀释，每孔100μl，37℃、5％CO 2培养箱中孵育1h。离心，每孔加入200μl 1％DPBS清洗2次后离心去上清，每孔加入100μl荧光二抗(Alexa
Figure PCTCN2019108057-appb-000001
647山羊抗人IgG(H+L)抗体)，37℃、5％CO 2培养箱中孵育1h。离心去上清，1％DPBS洗板两遍，每孔加入100μl 1％DPBS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 Prism 6进行分析，计算AP163与BCMA+细胞以及与CD3+细胞的结合活性。
如图3、图4所示，在细胞水平上，AP163可特异性的与BCMA阳性的细胞高度结合且具有量效关系；同时，AP163与人T细胞和食蟹猴T细胞均可特异性的高度结合，且具有量效关系。
(2)双特异性抗体与人猴种属CD3和BCMA蛋白的结合能力及交叉反应性测定
用PBS将包被抗原人和猴的CD3、BCMA分别稀释至0.1μg/ml，加入96孔板中，100μl/孔，2-8℃包被过夜。弃去板中液体，加入含有脱脂奶粉的PBST室温封闭2h后，PBST洗板2次。待测双特异性抗体4倍稀释共12个梯度，每浓度2个复孔，100μl/孔加入96孔板中，室温孵育2h。将未结合的双特异性抗体以PBST洗去，将生物素化的人BCMA蛋白或人CD3蛋白稀释至0.1μg/ml，以1:1000混合HRP标记的链霉亲和素(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，室温避光显色2-3min，然后加入1M HCL终止显色反应。用酶标仪检测OD450nm的吸光值。以样品浓度对数值为横坐标，吸光度值为纵坐标做四参数非线性回归，可变斜率方程。计算双特异性抗体与抗原结合的EC 50值。实验结果如表1所示，双特异性抗体与人源CD3和BCMA蛋白、食蟹猴源CD3和BCMA蛋白结合的EC 50值差距很小，双特异性抗体与不同种属抗原的结合能力基本相同。
表1、双特异性抗体与人猴种属CD3和BCMA蛋白的结合能力及交叉反应性测定结果
Figure PCTCN2019108057-appb-000002
(3)双特异性抗体同时结合靶细胞与效应细胞能力测定
正常培养的人骨髓瘤NCI-H929细胞作为靶细胞，用PKH26染色试剂染色标记，使用1640完全培养基重悬细胞，调整细胞密度为1×10 5个/ml，50μl/孔加入96孔细胞培养板中。将双特异性抗体用培养基梯度稀释，50μl/孔加入。加入5倍于靶细胞数的效应细胞(扩增培养的T细胞)，50μl/孔。37℃、5％CO 2培养箱中孵育1h，使待测样品与细胞充分混匀并发生桥连反应。用DPBS清洗96孔板，流式细胞术上机检测，捕捉T细胞，则带有PKH26信号的T细胞为发生桥连的细胞，统计数据计算桥连比例。从图5中可以看出，AP163可特异性的使肿瘤细胞和靶细胞发生桥连反应，且具有量效关系。
(4)双特异性抗体介导CD4 +T细胞/CD8 +T细胞的激活
招募3名健康的自愿献血者，抽取外周血并提取PBMC，利用CD4 +T细胞分离试剂盒及CD8 +T细胞分离试剂盒分选富集PBMC中的CD4 +T细胞和CD8 +T细胞，用含10％FBS的1640完全培养基重悬细胞，调整细胞密度为1×10 6个/ml，50μl/孔加入96孔细胞培养板中。培养人骨髓瘤NCI-H929细胞，调整细胞密度为1×10 5个/ml，每孔加入50μl。将双特异性抗体进行梯度稀释，50μl/孔加入96孔板，37℃、5％CO 2培养箱中孵育24h。离心取上清液，采用ELISA法颗粒酶检测试剂盒检测上清中颗粒酶释放量。通过软件GraphPad Prism 6进行分析，计算双特异性抗体介导CD4 +T细胞/CD8 +T细胞激活的EC 50。
如图6和表2所示，AP163可介导3名健康志愿者的CD4 +T细胞和CD8 +T细胞杀伤肿瘤细胞时释放颗粒酶，并呈现量效关系。
表2、双特异性抗体介导CD4 +T细胞/CD8 +T细胞激活释放颗粒酶的EC 50
Figure PCTCN2019108057-appb-000003
(5)双特异性抗体活化T细胞能力评价
含有NFAT RE报告基因的Jurkat T细胞(购自BPS Bioscience)，在双特异性抗体和BCMA阳性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。具体的，H929细胞离心重悬，调整细胞密度为2×10 5个/ml，40μl/孔加入96孔细胞培养板中。NFAT-Jurkat细胞调整细胞密度到2×10 6个/ml，每孔加入40μl。双特异性抗体AP163用培养基稀释成50μg/mL，10倍稀释后，每孔加入20μl，37℃、5％CO 2培养箱中孵育48h。洗板后，分别加入100μl/孔
Figure PCTCN2019108057-appb-000004
Luciferase，反应5min后，用酶标仪检测冷发光值。以双特异性抗体的浓度做X轴，荧光素强度作为Y轴，通过软件GraphPad Prism 6进行分析，计算双特异性抗体活化T细胞的EC 50。
如图7所示，AP163可以特异性的活化NFAT-Jurkat细胞，EC 50值为3.161ng/ml，且其浓度和信号强度成正比。
(6)双特异性抗体介导T细胞杀伤肿瘤细胞的能力
培养人、食蟹猴的T细胞，调整细胞密度为1×10 6个/ml，50μl/孔分别加入96孔细胞培养板中。正常培养的人骨髓瘤NCI-H929细胞作为靶细胞，调整细胞密度1×10 5个/ml，50μl/孔加入。之后每孔加入50μl梯度稀释后双特异性抗体AP163，37℃、5％CO 2培养箱中孵育24h。每孔加入40μl Bright-Glo试剂，室温避光静置3min，应用多功能酶标仪检测RLU值，通过软件GraphPad Prism 6进行分析，计算双特异性抗体介导T细胞杀伤H929细胞的EC 50值。
如图8所示，AP163介导人T细胞杀伤肿瘤细胞的EC 50值为0.239pM，介导食蟹猴T细胞杀伤肿瘤细胞的EC 50值为0.278pM。AP163可特异性的介导人和食蟹猴的T细胞杀伤肿瘤细胞，BCMA高表达的H929细胞呈现出显著的杀伤作用，并呈现量效关系。
(7)双特异性抗体引起细胞因子释放的评价
评价双特异性抗体单独或依赖靶细胞活化T细胞引起细胞因子释放的能力。
培养原代T细胞，离心收集细胞用1％PBSB重悬，分别调整细胞密度为1×10 6个/ml置于96孔板中，90μl/孔。识别CD3的母本单抗AB314(具体参考WO2007042261专利文献)与双特异性抗体AP163分别用培养基稀释成10000ng/mL，10倍梯度稀释后，10μl/孔加入96孔板，放入37℃，5％CO 2培养箱中培养。分别在孵育24h和48h时取培养上清液，用LEGENDplex TM人Th1/Th2试剂盒检测分析，流式细胞仪检测信号强度。以细胞因子浓度作为Y轴，抗体浓度作为X轴，通过软件GraphPad Prism 6进行分析，计算AB314与AP163活化T细胞致细胞因子释放的量。结果显示，在无靶细胞存在的情况下，AB314活化原代T细胞24h后，引起细胞因子IL-4、IL-5和TNF-α的释放；而AP163活化原代T细胞，24h，48h均无显著细胞因子的释放。
培养人T细胞，离心收集细胞用10％FBS的1640完全培养基重悬，调整细胞密度为1×10 6个/ml置于96孔板中，50μl/孔。人骨髓瘤NCI-H929细胞密度调整为1×10 5个/ml，50μl/孔加入。AP163梯度稀释后， 50μl/孔加入96孔板，37℃，5％CO 2培养箱中分别培养1、2、3、4、5、6、24h。孵育结束后每孔取50μl上清液，应用8细胞因子检测试剂盒检测上清中8中细胞因子的释放量。实验结果表明，AP163能够活化T细胞释放IL-5、IL-13、IL-2、IL-6、IL-10、IFN-γ、TNF-α、IL-4，并呈现时间依赖性，具体见表3。
表3、双特异性抗体引起细胞因子释放量
Figure PCTCN2019108057-appb-000005
(8)不同效靶比下双特异性抗体介导T细胞杀伤BCMA阳性人肿瘤细胞
培养人T细胞，分别调整细胞密度为2×10 7个/ml、1×10 7个/ml、1×10 6个/ml、1×10 5个/ml、1×10 4个/ml、1×10 3个/ml，每孔50μl分别加入96孔细胞培养板。人骨髓瘤NCI-H929细胞作为靶细胞，调整细胞密度1×10 5个/ml，50μl/孔加入。之后每孔加入50μl梯度稀释后的AP163，37℃、5％CO 2培养箱中孵育24h。每孔加入40μl Bright-Glo试剂，室温避光静置3min，应用多功能酶标仪检测RLU值，通过软件GraphPad Prism 6进行分析，计算双特异性抗体介导T细胞杀伤H929细胞的EC 50值。
如图9所示，效靶比(E/T)高于1/1时，AP163介导T细胞杀伤肿瘤细胞的效率可达到100％杀伤；E/T低于1/1时，EC50值随着E/T的减小而逐渐增大。
(9)评价***及吲哚美辛对AP163介导PBMC杀伤肿瘤细胞的影响作用
招募12名健康的自愿献血者，抽取外周血并提取PBMC，用含10％FBS的1640完全培养基重悬细胞，调整细胞密度为3×10 6个/ml，每孔50μl加入96孔细胞培养板。配制***和吲哚美辛稀释液，每孔50μl加入96孔板，分别孵育PBMC 1h和14h；对照组加入等体积的缓冲液。人骨髓瘤NCI-H929细胞调整细胞密度1×10 5个/ml，每孔50μl。之后每孔加入50μl梯度稀释的AP163，37℃、5％CO 2培养箱中分别孵育4、8、12、24、48h，每孔加入40μl Bright-Glo试剂，室温避光静置3min，应用多功能酶标仪检测RLU值。分析数据结果显示，***或吲哚美辛孵育PBMC对AP163介导PBMC杀伤肿瘤细胞的影响较小。
实施例5、Anti-BCMA×CD3双特异性抗体在小鼠移植瘤模型中的药效学研究
(1)NPG小鼠皮下共接种人CIK细胞和人骨髓瘤细胞NCI-H929移植瘤模型
将人骨髓瘤NCI-H929细胞与CIK细胞(由人PBMC激活诱导扩增至第10天)以不同比例共同接种于雌性NPG小鼠右侧前胁肋部皮下。接种1小时后，根据小鼠体重进行随机分组，每组4只，共4组。分组当天开始给药，所有组给药途径均为腹腔注射，对照组给予相同体积的PBS溶液，AP163给药剂量为0.2mg/kg。每周给药2次，连续给药4周。每3天测量肿瘤体积及体重1次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI％)。
如表4所示，实验结束时，效靶比1/1对照组平均肿瘤体积为1501±351mm 3，效靶比1/2对照组平均肿瘤体积为1555±244mm 3；效靶比1/1AP163试验组平均肿瘤体积为99±38mm 3，TGI％为93％，效靶比1/1AP163试验组平均肿瘤体积为481±215mm 3，TGI％为70.2％。上述结果表明，AP163有明显的抑瘤作用，同时，AP163呈现较好的安全性，未对实验动物产生明显的毒性作用。
表4、AP163对NCI-H929NPG小鼠模型的药效作用
Figure PCTCN2019108057-appb-000006
注：i.p:腹腔注射，biw:每周两次
(2)NPG小鼠皮下共接种人CIK细胞和人Burkkit’s淋巴瘤Raji细胞移植瘤模型
将人Burkkit’s淋巴瘤Raji细胞与培养扩增后的CIK(Raji细胞与CIK细胞比例1:1)，并与Matrigel以体积比为1:1的比例混合，共同接种于雌性NPG小鼠右侧背部皮下。接种1小时后，根据小鼠的体重进行随机分组，每组4只，共4组。分组当天开始给药，对照组给予相同体积的PBS溶液，AP163实验组的给药剂量分别为1mg/kg、0.1mg/kg和0.01mg/kg。所有组给药途径均为腹腔注射，每周给药两次，共给药3周。每3天测量肿瘤体积及体重1次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI％)。
结果见表5，实验结束时，对照组平均肿瘤体积为1750±653mm 3；AP163 1mg/kg、0.1mg/kg和0.01mg/kg试验组的平均肿瘤体积都为0.00±0.00mm 3，均与溶剂对照组的肿瘤体积有显著性差异(P<0.05)，三组的TGI都为100％，表明供试双特异性抗体AP163有极其显著的抑瘤作用。
表5、AP163对Raji NPG小鼠模型的药效作用
Figure PCTCN2019108057-appb-000007
注：i.p:腹腔注射，biw:每周两次
(3)NPG小鼠皮下共接种人CIK细胞和人骨髓瘤细胞RPMI-8226移植瘤模型
将人骨髓瘤细胞RPMI-8226与CIK共同接种于雌性NPG小鼠右侧背部皮下，接种1小时后，根据小鼠的体重进行随机分组，当天开始给药。第一组7只,其余两组各8只，共3组。分组当天开始给药，对照组给予相同体积的PBS溶液，AP163实验组的给药剂量分别为1mg/kg和0.1mg/kg。所有组给药途径均为腹腔注射，每两天给药1次，连续给药8次，末次给药18天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI％)。
如图10所示，实验结束时(首次给药32天后)，各组动物体重均出现增长，不同组别的动物体重比较无显著性差异(P>0.05)。对照组平均肿瘤体积为1647.79±247.90mm 3；AP163 1mg/kg试验组的平均肿瘤体积为0.00±0.00mm 3，TGI％为100％；AP163 0.1mg/kg试验组的平均肿瘤体积为8.00±5.24mm 3，TGI％为99.51％。实验结果表明，抗体AP163有极显著抑瘤作用。在本实验条件下，AP163在各给药浓度下均显著抑制肿瘤生长，同时AP163也呈现较好的安全性性，未对实验动物产生明显的毒性作用。
(4)NPG小鼠皮下共接种人CIK细胞和人Burkkit’s淋巴瘤Daudi细胞移植瘤模型
将人Burkkit’s淋巴瘤Daudi细胞与培养扩增后的CIK(Daudi细胞密度5×10 6个/ml，CIK细胞密度1×10 6个/ml)，并与Matrigel以体积比1:1的比例混合，共同接种于雌性NPG小鼠右侧背部皮下。接种1h后，根据小鼠的体重进行随机分组，每组6只，共4组。分组当天开始给药，对照组给予相同体积的PBS溶液，AP163实验组的给药剂量分别为1mg/kg、0.2mg/kg和0.04mg/kg。所有组给药途径均为腹腔注射，每两天给药1次，共给药8次，末次给药10天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI％)。
结果见图11，实验结束时(首次给药25天后)，各组动物体重均出现增长，与对照组比较无显著性差异(P>0.05)。实验结束时，对照组平均肿瘤体积为970.83±165.40mm 3；AP163 1mg/kg、0.2mg/kg和0.04mg/kg 试验组的平均肿瘤体积分别为171.99±32.60mm 3、190.82±53.60mm 3和228.68±44.96mm 3，对应的TGI％分别为82.28％、80.34％和76.44％，且与对照组相比抑瘤作用均有显著性差异(P<0.05)，表明AP163在各给药浓度下均显著抑制肿瘤生长，且有较好的安全性，未对实验动物产生明显的毒性作用。
实施例6、Anti-BCMA×CD3双特异性抗体安全性评价试验
评价AP163每周给药2次，重复静脉输注给予食蟹猴2周后的毒性反应情况，为后续的毒性试验确定合适的剂量范围及观察指标。6只食蟹猴，3只/性别，分成3组，1只/性别/组，分别给予0.1、0.5和2.5mg/kg的AP163(第1、2或3组)。输注速度为30mL/kg/h，给药容量为10mL/kg。所有动物在14天(D14)给药期结束后D15实施安乐死。
试验期间，周期性的监测动物的临床症状、体重、食量、体温、心电图、血压、临床病理指标(血细胞计数、凝血功能指标和血液生化)、淋巴细胞亚群、细胞因子、药物血浆浓度测定和毒代分析。所有动物均进行大体解剖。大体解剖观察未见明显异常，未进行组织病理学检查。结果表明在本试验条件下，0.1、0.5和2.5mg/kg的AP163每周给药2次，重复静脉输注给予食蟹猴2周，各动物未见死亡或濒死，各给药组动物第1次药后均可见一过性的Neut、CD3-CD20+、TNF-α、IL-2和IL-6升高和Lymph、CD3 +、CD3-CD16 +/CD56 +降低；最大耐受剂量(MTD)≥2.5mg/kg。
实施例7、Anti-BCMA×CD3双特异性抗体药代动力学试验
试验共使用6只食蟹猴(3只/性别)，分成3组，1只/性别/组，分别给予0.1、0.5和2.5mg/kg的AP163。从动物后肢皮下静脉非给药部位采集毒代血样(约1mL)至无抗凝剂的管中，第1～3组的采血时间点为：每组动物首末次给药前、给药结束药后即刻(±1min)、给药开始后1h、3h、6h、8h、24h、48h、72h。
离心管(不抗凝)使用前冰水浴保存；血样采集后转移至上述离心管中，然后于2～8℃，3000×g离心10min。分离血清样本后分装为2份，置于-70℃以下保存。血样采集至离心完成需在2小时内完成。
采用ELISA方法进行检测分析血清中AP163的浓度，采用WinNonlin 8.0软件的非房室模型法(NCA)对给药组的动力学参数进行计算，各组药代动力学参数结果见表6。结果表明，0.1、0.5和2.5mg/kg剂量组AP163的体内半衰期分别为7.08、8.95和11.42小时。
表6、AP163在静脉注射食蟹猴后药代参数计算
Figure PCTCN2019108057-appb-000008
虽然说明并描述了本发明的优选例，应理解本领域的技术人员可根据本文的教导做出各种改变，这些改变不违背本发明的范围。
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，本领域技术人员可对本发明做各种修改或改动，这些等价形式同样落后于本申请所附权利要求书所限定的范围。

Claims (28)

  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，每条多肽链包含特异性结合肿瘤抗原BCMA的第一单链Fv、特异性结合效应细胞抗原CD3的第二单链Fv和Fc片段；其中，第一和第二单链Fv通过连接肽相连，而第二单链Fv与Fc片段直接相连或通过连接肽相连。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述Fc片段不具有CDC、ADCC和ADCP效应子功能。
  3. 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，n为1-5的自然数；X优选Ser，n优选3。
  4. 如权利要求1所述的双特异性抗体，其特征在于，所述第一单链Fv包含：
    (1)VH结构域，其包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：1、2和3所示，或与上述序列至少80相似度或具有一个或更多个氨基酸保守序列取代的序列；和
    (2)VL结构域，其包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：4、5和6所示，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
  5. 如权利要求1或4所述的双特异性抗体，其特征在于，所述第一单链Fv包含：
    (1)氨基酸序列如SEQ ID NO：7所示的VH结构域，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；和
    (2)氨基酸序列如SEQ ID NO：8所示的VL结构域，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
  6. 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，n为1-5的自然数；X优选Ser，n优选3。
  7. 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包含：
    (1)VH结构域，其包含的HCDR1、HCDR2和HCDR3分别如SEQ ID NO：11、12和13所示，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；和
    (2)VL结构域，其包含的LCDR1、LCDR2和LCDR3分别如SEQ ID NO：14、15和16所示，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
  8. 如权利要求1或7所述的双特异性抗体，其特征在于，所述第二单链Fv包含：
    (1)氨基酸序列如SEQ ID NO：17所示的VH结构域，或与上述序列至少80％相似度 或具有一个或更多个氨基酸保守序列取代的序列；和
    (2)氨基酸序列如SEQ ID NO：18所示的VL结构域，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
  9. 如权利要求1或7所述的双特异性抗体，其特征在于，所述第二单链Fv可与人或食蟹猴或恒河猴的CD3特异性结合。
  10. 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)任意两个或两个以上的组合；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；所述刚性肽来自天然人绒毛膜***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，所述刚性肽包含氨基酸序列SSSSKAPPPS。
  11. 如权利要求10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：10所示的氨基酸序列。
  12. 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)中的任意重复组合；较优选自Gly(G)和Ser(S)的任意重复组合；更优选地，所述连接肽组成为(GGGGS)n，n＝1，2，3或4。
  13. 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。
  14. 如权利要求13所述的双特异性抗体，其特征在于，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13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***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：
    (1)与新生儿受体FcRn的结合亲和力增强；
    (2)降低或消除的糖基化；
    (3)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：
    (1)根据EU编号***确定的M428L、T250Q/M428L、M428L/N434S或M252Y/S254T/T256E的氨基酸置换；和/或
    (2)根据EU编号***确定的N297A的氨基酸置换；和/或
    (3)根据EU编号***确定的K447的氨基酸缺失。
  19. 如权利要求17所述的双特异性抗体，其特征在于，所述Fc片段的氨基酸序列如SEQ ID NO：19所示，它与其所源自的天然序列相比具有根据EU编号***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***确定的K447。
  20. 如权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体结合人BCMA和CD3，其氨基酸序列如下：
    (1)SEQ ID NO：20所示的序列；或
    (2)与SEQ ID NO：20所示的序列相比具有一个或几个置换、缺失或添加的序列；或
    (3)与SEQ ID NO：20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. 一种DNA分子，其特征在于，编码如权利要求1-20任一项所述双特异性抗体。
  22. 如权利要求21所述的DNA分子，其特征在于，具有如SEQ ID NO：21所示的核苷酸序列。
  23. 一种表达载体，其特征在于，所述表达载体包含编码权利要求21所述氨基酸的DNA分子或权利要求22所述DNA分子。
  24. 一种宿主细胞，其特征在于，转化权利要求23所述的表达载体，优选地，所述宿主细胞为原核细胞或酵母或哺乳动物细胞，更优选地，所述宿主细胞为哺乳动物细胞，更进一步优选地，所述哺乳动物细胞为CHO细胞或NS0。
  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和/或载体和/或稀释剂。
  26. 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；
  27. 如权利要求1-20任一项所述双特异性抗体在用于治疗或改善选自浆细胞障碍、其他与BCMA表达有关的B细胞障碍和自身免疫性疾病的药物中的用途，其中所述浆细胞障碍包括但不限于多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白白血症、淀粉样变性、华氏巨球蛋白白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病及郁积型多发性骨髓瘤。
  28. 用于治疗权利要求27所述的疾病的组合产品，其中所述的双特异性抗体为AP163。
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Families Citing this family (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7137696B2 (ja) 2019-05-14 2022-09-14 プロヴェンション・バイオ・インコーポレイテッド １型糖尿病を予防するための方法および組成物
WO2021238932A1 (zh) * 2020-05-26 2021-12-02 百奥泰生物制药股份有限公司 多特异性抗体及其应用
TWI806087B (zh) * 2020-06-30 2023-06-21 大陸商和鉑醫藥（上海）有限責任公司 免疫細胞銜接多特異性結合蛋白及其製備和應用
CN111826400A (zh) * 2020-07-21 2020-10-27 中科宝承生物医学科技有限公司 一种双特异性抗体nk细胞制备方法及其细胞和应用
US20230279138A1 (en) * 2020-07-27 2023-09-07 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Novel bispecific anti-cd3/cd20 polypeptide complex formulation
CN112062855A (zh) * 2020-08-26 2020-12-11 康诺亚生物医药科技（成都）有限公司 一种含有衔接器的药物治疗剂的开发和应用
CN112010982B (zh) * 2020-08-31 2023-06-27 重庆金迈博生物科技有限公司 一种抗gpc3/cd3双特异性抗体及其应用
CN116323671A (zh) * 2020-11-06 2023-06-23 安进公司 具有增加的选择性的多靶向性双特异性抗原结合分子
WO2022156739A1 (zh) * 2021-01-20 2022-07-28 江苏恒瑞医药股份有限公司 特异性结合bcma和cd3的抗原结合分子及其医药用途
KR20230157315A (ko) 2021-01-28 2023-11-16 리제너론 파마슈티칼스 인코포레이티드 사이토카인 방출 증후군을 치료하기 위한 조성물 및 방법
CN117062840A (zh) * 2021-04-15 2023-11-14 正大天晴药业集团股份有限公司 靶向bcma的多特异性抗体
CN117242095A (zh) * 2021-04-23 2023-12-15 和铂医药(上海)有限责任公司 一种双特异性抗体的纯化方法
CN115433282A (zh) * 2021-06-02 2022-12-06 启愈生物技术(上海)有限公司 抗cd3抗体变异体、融合蛋白及应用
CN115581765A (zh) * 2021-07-05 2023-01-10 山东新时代药业有限公司 一种重组人源化抗bcma/cd3双特异性抗体注射液
CN115569191A (zh) * 2021-07-05 2023-01-06 山东新时代药业有限公司 一种重组人源化抗bcma/cd3双特异性抗体冻干制剂
CN113527493B (zh) * 2021-07-20 2023-10-27 广州爱思迈生物医药科技有限公司 一种b7-h3抗体及其应用
WO2023122882A1 (zh) * 2021-12-27 2023-07-06 上海鑫湾生物科技有限公司 双特异性t细胞衔接器、其重组溶瘤病毒及其用途
CN116462765A (zh) * 2022-01-20 2023-07-21 上海君实生物医药科技股份有限公司 抗cd3和抗cd20双特异性抗体及其用途
CN116554340A (zh) * 2022-01-28 2023-08-08 江苏众红生物工程创药研究院有限公司 新型长效化和高活性且更安全的抗体构建体
CN114410588B (zh) * 2022-01-29 2022-11-04 西安电子科技大学 一种α1β1整合素依赖增强型CAR巨噬细胞及其制备方法和应用
TW202342549A (zh) * 2022-03-01 2023-11-01 大陸商江蘇恆瑞醫藥股份有限公司 特異性結合ｆｌｔ３和ｃｄ３的抗原結合分子及其醫藥用途
CN115304680B (zh) * 2022-03-11 2024-02-02 四川大学华西医院 基于Pep42构建的双特异性细胞接合器分子的制备及其应用
WO2023201226A1 (en) 2022-04-11 2023-10-19 Regeneron Pharmaceuticals, Inc. Compositions and methods for universal tumor cell killing
CN114716559B (zh) * 2022-04-12 2023-05-26 无锡科金生物科技有限公司 双特异性抗体及其治疗癌症的应用
CN114685675B (zh) * 2022-04-27 2023-02-03 深圳市汉科生物工程有限公司 双特异性抗体及其在治疗癌症中的用途
TW202346368A (zh) * 2022-05-12 2023-12-01 德商安美基研究（慕尼黑）公司 具有增加的選擇性的多鏈多靶向性雙特異性抗原結合分子
CN115286715B (zh) * 2022-05-18 2023-05-23 上海百英生物科技股份有限公司 一种抗cd3的纳米抗体或其抗原结合部分及其制备方法
WO2024027793A1 (en) * 2022-08-05 2024-02-08 I-Mab Biopharma (Hangzhou) Co., Ltd. Bispecific antibodies targeting ifnar1 and blys
CN116023499B (zh) * 2022-10-26 2024-01-23 北京力邦生物医药科技有限公司 一种针对cd3和cd20的双特异性抗体
CN116063526A (zh) * 2022-12-31 2023-05-05 合肥天港免疫药物有限公司 抗pdl1的抗体及其用途
CN116143934B (zh) * 2023-03-21 2023-07-25 诺赛联合(北京)生物医学科技有限公司 一种干细胞外泌体提取试剂盒及其应用
CN117169518B (zh) * 2023-11-03 2024-01-19 赛德特(北京)生物工程有限公司 T淋巴细胞制剂中的cd3抗体残留物的检测方法和试剂盒

Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
WO1988001649A1 (en) 1986-09-02 1988-03-10 Genex Corporation Single polypeptide chain binding molecules
US4818679A (en) 1985-02-19 1989-04-04 The Trustees Of Columbia University In The City Of New York Method for recovering mutant cells
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
EP0388151A1 (en) 1989-03-13 1990-09-19 Celltech Limited Modified antibodies
WO1992002551A1 (en) 1990-08-02 1992-02-20 B.R. Centre Limited Methods for the production of proteins with a desired function
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
WO1996032478A1 (en) 1995-04-14 1996-10-17 Genentech, Inc. Altered polypeptides with increased half-life
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
WO1997043316A1 (en) 1996-05-10 1997-11-20 Beth Israel Deaconess Medical Center, Inc. Physiologically active molecules with extended half-lives and methods of using same
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5968509A (en) 1990-10-05 1999-10-19 Btp International Limited Antibodies with binding affinity for the CD3 antigen
US6706265B1 (en) 1992-03-24 2004-03-16 Btg International Limited Humanized anti-CD3 specific antibodies
US6750325B1 (en) 1989-12-21 2004-06-15 Celltech R&D Limited CD3 specific recombinant antibody
WO2004051268A1 (en) 2002-12-03 2004-06-17 Celltech R & D Limited Assay for identifying antibody producing cells
WO2004106377A1 (en) 2003-05-30 2004-12-09 Celltech R & D Limited Methods for producing antibodies
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
US20050176028A1 (en) 2003-10-16 2005-08-11 Robert Hofmeister Deimmunized binding molecules to CD3
US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
WO2007042261A2 (en) 2005-10-11 2007-04-19 Micromet Ag Compositions comprising cross-species-specific antibodies and uses thereof
WO2007059782A1 (en) 2005-11-28 2007-05-31 Genmab A/S Recombinant monovalent antibodies and methods for production thereof
WO2008119565A2 (en) 2007-04-03 2008-10-09 Micromet Ag Cross-species-specific binding domain
US7728114B2 (en) 2004-06-03 2010-06-01 Novimmune S.A. Anti-CD3 antibodies and methods of use thereof
US20100183615A1 (en) 2007-04-03 2010-07-22 Micromet Ag Cross-species-specific bispecific binders
KR20100099179A (ko) 2007-12-26 2010-09-10 젠코어 인코포레이티드 ＦｃＲｎ에 대한 변경된 결합성을 갖는 Ｆｃ 변이체
US20100234575A1 (en) 2004-11-12 2010-09-16 Xencor, Inc. Fc variants with altered binding to fcrn
KR101027427B1 (ko) 2004-11-12 2011-04-11 젠코어 인코포레이티드 ＦcRn에 대하여 증가된 결합력을 갖는 Ｆc 변이체
US7994289B2 (en) 1998-07-21 2011-08-09 Btg International Limited Humanized anti-CD3 antibodies
US8076459B2 (en) 2003-10-16 2011-12-13 Micromet Ag Multispecfic deimmunized CD3-binders
WO2012066058A1 (en) * 2010-11-16 2012-05-24 Boehringer Ingelheim International Gmbh Agents and methods for treating diseases that correlate with bcma expression
CN102741423A (zh) * 2009-10-23 2012-10-17 雅培制药有限公司 双重可变结构域免疫球蛋白及其用途
WO2013096221A1 (en) 2011-12-21 2013-06-27 Amgen Inc. Variant fc-polypeptides with enhanced binding to the neonatal fc receptor
CN104114578A (zh) * 2011-11-15 2014-10-22 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
CN105658668A (zh) * 2013-10-10 2016-06-08 Ucl商务股份有限公司 分子
WO2016130726A1 (en) 2015-02-10 2016-08-18 Minerva Biotechnologies Corporation Humanized anti-muc1* antibodies
CN105980406A (zh) * 2013-03-15 2016-09-28 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
WO2017134134A1 (en) * 2016-02-03 2017-08-10 Amgen Research (Munich) Gmbh BCMA and CD3 Bispecific T Cell Engaging Antibody Constructs
WO2017223111A1 (en) * 2016-06-21 2017-12-28 Teneobio, Inc. Cd3 binding antibodies
CN107759694A (zh) * 2016-08-19 2018-03-06 安源医药科技（上海）有限公司 双特异性抗体及其制备方法与用途
CN108474793A (zh) * 2015-10-30 2018-08-31 葛兰素史密斯克莱知识产权发展有限公司 预后方法

Family Cites Families (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3522A (en) 1844-04-04 richard son
US530A (en) 1837-12-20 Threshing-machine
US926A (en) 1838-09-17 Machine for threshing and winnowing grain
US8034A (en) 1851-04-08 Cooking-stove
US4980286A (en) 1985-07-05 1990-12-25 Whitehead Institute For Biomedical Research In vivo introduction and expression of foreign genetic material in epithelial cells
DE60124912T2 (de) * 2001-09-14 2007-06-14 Affimed Therapeutics Ag Multimerische, einzelkettige, Tandem-Fv-Antikörper
US7906118B2 (en) 2005-04-06 2011-03-15 Ibc Pharmaceuticals, Inc. Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (DNL) technology
US8444973B2 (en) 2005-02-15 2013-05-21 Duke University Anti-CD19 antibodies and uses in B cell disorders
JP5082104B2 (ja) 2006-03-23 2012-11-28 国立大学法人東北大学 高機能性二重特異性抗体
AU2009228158B2 (en) * 2008-03-27 2014-02-27 Zymogenetics, Inc. Compositions and methods for inhibiting PDGFRbeta and VEGF-A
TWI667257B (zh) * 2010-03-30 2019-08-01 中外製藥股份有限公司 促進抗原消失之具有經修飾的ＦｃＲｎ親和力之抗體
ES2730941T7 (es) * 2010-10-22 2020-05-27 Seattle Genetics Inc Efectos sinérgicos entre los conjugados de conjugados anticuerpo-fármaco a base de auristatina e inhibidores de la ruta PI3K-AKT-mTOR
EP3590965A1 (en) * 2011-03-29 2020-01-08 Roche Glycart AG Antibody fc variants
RS64791B1 (sr) * 2011-05-27 2023-11-30 Glaxo Group Ltd Bcma (cd269/tnfrsf17) - vezujući proteini
HUE038225T2 (hu) 2011-08-23 2018-10-29 Roche Glycart Ag Bispecifikus, T-sejtaktiváló antigénkötõ, molekulák
TW201817745A (zh) * 2011-09-30 2018-05-16 日商中外製藥股份有限公司 具有促進抗原清除之ＦｃＲｎ結合域的治療性抗原結合分子
CN104144949B (zh) * 2011-12-22 2016-08-31 财团法人生物技术开发中心 双特异性t细胞活化剂抗体
EP3586874A1 (en) * 2012-08-14 2020-01-01 IBC Pharmaceuticals, Inc. T-cell redirecting bispecific antibodies for treatment of disease
US9701759B2 (en) * 2013-01-14 2017-07-11 Xencor, Inc. Heterodimeric proteins
US9546203B2 (en) * 2013-03-14 2017-01-17 Amgen Inc. Aglycosylated Fc-containing polypeptides with cysteine substitutions
US9288361B2 (en) 2013-06-06 2016-03-15 Open Text S.A. Systems, methods and computer program products for fax delivery and maintenance
BR112016000666A2 (pt) * 2013-07-12 2017-10-03 Zymeworks Inc Constructos de ligação de antígeno cd3 e cd19 biespecíficos
CN104744592B (zh) * 2013-12-27 2019-08-06 北京韩美药品有限公司 抗HER2-抗CD3 scFv双特异四价抗体
US20170274072A1 (en) * 2014-03-26 2017-09-28 Tohoku University Bispecific antibody targeting human epidermal growth factor receptor
CN104558192B (zh) * 2015-01-21 2018-12-28 武汉友芝友生物制药有限公司 一种双特异性抗体her2xcd3的构建及应用
JP6871155B2 (ja) * 2014-07-25 2021-05-12 メモリアル スローン ケタリング キャンサー センター 二重特異性ｈｅｒ２及びｃｄ３結合分子
AP2017009823A0 (en) * 2014-09-26 2017-03-31 Macrogenics Inc Bi-specific monovalent diabodies that are capable of binding cd19 and cd3, and uses thereof
US10428155B2 (en) 2014-12-22 2019-10-01 Xencor, Inc. Trispecific antibodies
CN104829728B (zh) * 2015-01-21 2019-03-12 武汉友芝友生物制药有限公司 一种双特异性抗体her2xcd3的构建及应用
CN104558191B (zh) * 2015-01-21 2020-08-21 武汉友芝友生物制药有限公司 一种双特异性抗体cd20×cd3的构建及应用
CN104558193B (zh) * 2015-01-21 2019-01-11 武汉友芝友生物制药有限公司 一种靶向鼠t淋巴细胞cd3和人肿瘤抗原her2的双特异性抗体制备方法及应用
CN107223135B (zh) 2015-03-16 2021-11-02 亥姆霍兹慕尼黑中心-德国环境健康研究中心(Gmbh) 用于治疗hbv感染和相关病症的三特异性结合分子
CN104829729B (zh) * 2015-04-03 2018-08-14 复旦大学 一种携带抗Her2/CD3双特异性功能蛋白的人T细胞制备
EP3298042A1 (en) * 2015-05-20 2018-03-28 Amgen Research (Munich) GmbH B-cell depletion as a diagnostic marker
WO2016194992A1 (ja) * 2015-06-05 2016-12-08 中外製薬株式会社 免疫活性化剤の併用
TW202346349A (zh) * 2015-07-31 2023-12-01 德商安美基研究（慕尼黑）公司 Dll3及cd3抗體構築體
DK3333192T3 (da) * 2015-08-03 2021-05-31 Cafa Therapeutics Ltd Antistof imod glypican-3 og anvendelse deraf
AU2016308567B2 (en) * 2015-08-17 2022-10-27 Janssen Biotech, Inc. Anti-BCMA antibodies, bispecific antigen binding molecules that bind BCMA and CD3, and uses thereof
KR101851380B1 (ko) * 2015-10-12 2018-04-23 아주대학교산학협력단 효모접합을 이용한 항체 ch3 도메인 이종이중체 돌연변이쌍 제조 방법 및 이에 의하여 제조된 ch3 돌연변이체 쌍
AU2016371034A1 (en) 2015-12-17 2018-05-31 Janssen Biotech, Inc. Antibodies specifically binding HLA-DR and their uses
KR20230148844A (ko) 2016-03-29 2023-10-25 유니버시티 오브 써던 캘리포니아 암을 표적하는 키메라 항원 수용체
US20170349657A1 (en) * 2016-06-01 2017-12-07 Xencor, Inc. Bispecific antibodies that bind cd20 and cd3
WO2018035084A1 (en) * 2016-08-16 2018-02-22 Epimab Biotherapeutics, Inc. Monovalent asymmetric tandem fab bispecific antibodies
CN106256835A (zh) * 2016-08-19 2016-12-28 安源医药科技（上海）有限公司 高糖基化人生长激素融合蛋白及其制备方法与用途
CN106117370B (zh) * 2016-08-19 2017-05-17 安源医药科技（上海）有限公司 高糖基化Exendin‑4及其类似物的融合蛋白、其制备方法和用途
CN110225924B (zh) 2017-01-25 2024-04-02 免疫医疗有限公司 松弛素融合多肽及其用途
US11046768B2 (en) * 2017-01-27 2021-06-29 Memorial Sloan Kettering Cancer Center Bispecific HER2 and CD3 binding molecules
EP3574012A1 (en) * 2017-01-27 2019-12-04 Memorial Sloan Kettering Cancer Center Bispecific her2 and cd3 binding molecules
AU2018224094A1 (en) * 2017-02-24 2019-09-19 Macrogenics, Inc. Bispecific binding molecules that are capable of binding CD137 and tumor antigens, and uses thereof
CN108623689B (zh) * 2017-03-15 2020-10-16 宜明昂科生物医药技术（上海）有限公司 新型重组双功能融合蛋白及其制备方法和用途
WO2018175279A2 (en) * 2017-03-20 2018-09-27 The General Hospital Corporation MITIGATING Fc-Fc RECEPTOR INTERACTIONS IN CANCER IMMUNOTHERAPY
EP3619235A1 (en) * 2017-04-11 2020-03-11 Inhibrx, Inc. Multispecific polypeptide constructs having constrained cd3 binding and methods of using the same
CN107501412A (zh) * 2017-10-11 2017-12-22 深圳精准医疗科技有限公司 突变型双特异性抗体及其应用
AU2019271219A1 (en) 2018-05-16 2020-11-26 Arbele Limited Composition of bispecific antibodies and method of use thereof

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4818679A (en) 1985-02-19 1989-04-04 The Trustees Of Columbia University In The City Of New York Method for recovering mutant cells
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
WO1988001649A1 (en) 1986-09-02 1988-03-10 Genex Corporation Single polypeptide chain binding molecules
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
EP0388151A1 (en) 1989-03-13 1990-09-19 Celltech Limited Modified antibodies
US6750325B1 (en) 1989-12-21 2004-06-15 Celltech R&D Limited CD3 specific recombinant antibody
WO1992002551A1 (en) 1990-08-02 1992-02-20 B.R. Centre Limited Methods for the production of proteins with a desired function
US5968509A (en) 1990-10-05 1999-10-19 Btp International Limited Antibodies with binding affinity for the CD3 antigen
US6706265B1 (en) 1992-03-24 2004-03-16 Btg International Limited Humanized anti-CD3 specific antibodies
WO1996032478A1 (en) 1995-04-14 1996-10-17 Genentech, Inc. Altered polypeptides with increased half-life
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
WO1997043316A1 (en) 1996-05-10 1997-11-20 Beth Israel Deaconess Medical Center, Inc. Physiologically active molecules with extended half-lives and methods of using same
US7994289B2 (en) 1998-07-21 2011-08-09 Btg International Limited Humanized anti-CD3 antibodies
US7670600B2 (en) 2000-12-12 2010-03-02 MedImmine, LLC Molecules with extended half-lives, compositions and uses thereof
US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
WO2004051268A1 (en) 2002-12-03 2004-06-17 Celltech R & D Limited Assay for identifying antibody producing cells
WO2004106377A1 (en) 2003-05-30 2004-12-09 Celltech R & D Limited Methods for producing antibodies
US8076459B2 (en) 2003-10-16 2011-12-13 Micromet Ag Multispecfic deimmunized CD3-binders
US20050176028A1 (en) 2003-10-16 2005-08-11 Robert Hofmeister Deimmunized binding molecules to CD3
US7728114B2 (en) 2004-06-03 2010-06-01 Novimmune S.A. Anti-CD3 antibodies and methods of use thereof
US20100234575A1 (en) 2004-11-12 2010-09-16 Xencor, Inc. Fc variants with altered binding to fcrn
KR101027427B1 (ko) 2004-11-12 2011-04-11 젠코어 인코포레이티드 ＦcRn에 대하여 증가된 결합력을 갖는 Ｆc 변이체
WO2007042261A2 (en) 2005-10-11 2007-04-19 Micromet Ag Compositions comprising cross-species-specific antibodies and uses thereof
WO2007059782A1 (en) 2005-11-28 2007-05-31 Genmab A/S Recombinant monovalent antibodies and methods for production thereof
WO2008119565A2 (en) 2007-04-03 2008-10-09 Micromet Ag Cross-species-specific binding domain
US20100183615A1 (en) 2007-04-03 2010-07-22 Micromet Ag Cross-species-specific bispecific binders
KR20100099179A (ko) 2007-12-26 2010-09-10 젠코어 인코포레이티드 ＦｃＲｎ에 대한 변경된 결합성을 갖는 Ｆｃ 변이체
CN102741423A (zh) * 2009-10-23 2012-10-17 雅培制药有限公司 双重可变结构域免疫球蛋白及其用途
WO2012066058A1 (en) * 2010-11-16 2012-05-24 Boehringer Ingelheim International Gmbh Agents and methods for treating diseases that correlate with bcma expression
CN104114578A (zh) * 2011-11-15 2014-10-22 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
WO2013096221A1 (en) 2011-12-21 2013-06-27 Amgen Inc. Variant fc-polypeptides with enhanced binding to the neonatal fc receptor
CN105980406A (zh) * 2013-03-15 2016-09-28 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
CN105658668A (zh) * 2013-10-10 2016-06-08 Ucl商务股份有限公司 分子
WO2016130726A1 (en) 2015-02-10 2016-08-18 Minerva Biotechnologies Corporation Humanized anti-muc1* antibodies
CN108474793A (zh) * 2015-10-30 2018-08-31 葛兰素史密斯克莱知识产权发展有限公司 预后方法
WO2017134134A1 (en) * 2016-02-03 2017-08-10 Amgen Research (Munich) Gmbh BCMA and CD3 Bispecific T Cell Engaging Antibody Constructs
WO2017223111A1 (en) * 2016-06-21 2017-12-28 Teneobio, Inc. Cd3 binding antibodies
CN107759694A (zh) * 2016-08-19 2018-03-06 安源医药科技（上海）有限公司 双特异性抗体及其制备方法与用途

Non-Patent Citations (35)

* Cited by examiner, † Cited by third party
Title
ALEGRE ML ET AL., J. IMMUNOL., vol. 148, 1992, pages 3461 - 3468
BABCOOK J ET AL., PROC. NATL. ACAD. SCI. USA., vol. 93, 1996, pages 7843 - 7848
CHAMES P ET AL., CURR. OPIN. DRUG DISC. DEV., vol. 12, 2009, pages 276
CHAMESBATY, CURR. OPIN. DRUG. DISCOV. DEVEL., vol. 12, 2009, pages 276 - 83
CHAMESBATY, MABS, vol. 1, pages 539 - 47
CHOTHIALESKL, J.MOL.BIOL., vol. 196, 1987, pages 901 - 917
COLE SPC ET AL.: "Monoclonal Antibodies and Cancer Therapy", 1985, ALAN RLIS, INC., pages: 77 - 96
DATTA-MANNAN A ET AL., MABS. TAYLOR & FRANCIS, vol. 4, 2012, pages 267 - 273
FARES FA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4304 - 4308
HINTON PR ET AL., J. IMMUNOL., vol. 176, 2006, pages 346 - 356
HOLT ET AL., TRENDS BIOTECHNOLOGY, vol. 21, no. 11, 2003, pages 484 - 90
IDUSOGIE EE ET AL., J. IMMUNOL., vol. 166, 2001, pages 2571 - 2575
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE N.I.H.
KANEKO E ET AL., BIODRUGS, vol. 25, 2011, pages 1 - 11
KARPOVSKY B ET AL., J. EXP. MED., vol. 160, 1984, pages 1686 - 1701
KOHLERMILSTEIN, NATURE, vol. 256, 1975, pages 495 - 497
KOZBOR D ET AL., IMMUNOLOGY TODAY, vol. 4, 1983, pages 72
LAIBLE G ET AL., REPROD. FERTIL. DEV., vol. 25, 2012, pages 315
LAZAR GA ET AL., PNAS, vol. 103, 2006, pages 4005 - 4010
MILSTEIN C ET AL., NATURE, vol. 305, 1983, pages 537 - 540
NATSUME A ET AL., CANCER RES., vol. 68, 2008, pages 3863 - 3872
REVETS ET AL., EXPERT OPIN BIOL THER, vol. 5, no. 1, January 2005 (2005-01-01), pages 111 - 24
ROOPENIAN DC ET AL., NAT. REV. IMMUNOL., vol. 7, 2007, pages 715 - 725
ROSENBURGMOORE: "PluckThun", vol. 113, 1994, SPRINGER-VERLAG, article "The Pharmacology of Monoclonal Antibodies", pages: 269 - 315
ROUX KH ET AL., J.IMMUNOL., vol. 161, 1998, pages 4083
SCHELLER J ET AL., NATURE BIOTECHNOL., vol. 19, 2001, pages 573 - 577
See also references of EP3889179A4
SHIELDS RL ET AL., JBC, vol. 276, 2001, pages 6591 - 6604
SMITS N CSENTMAN C L, JOURNAL OF CLINICAL ONCOLOGY, 2016, pages JC0649970
SPIESS C ET AL., MOL. IMMUNOL., vol. 67, 2015, pages 95 - 106
STAERZ UD ET AL., NATURE, vol. 314, 1985, pages 628 - 31
STAVENHAGEN JB ET AL., ADVAN. ENZYME. REGUL., vol. 48, 2008, pages 152 - 164
STAVENHAGEN JB ET AL., CANCER RES., vol. 67, 2007, pages 8882 - 8890
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 54
ZHU L ET AL., NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1159 - 1169

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