WO2021063349A1 - 靶向bcma的抗体、双特异性抗体及其用途 - Google Patents

靶向bcma的抗体、双特异性抗体及其用途 Download PDF

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WO2021063349A1
WO2021063349A1 PCT/CN2020/118780 CN2020118780W WO2021063349A1 WO 2021063349 A1 WO2021063349 A1 WO 2021063349A1 CN 2020118780 W CN2020118780 W CN 2020118780W WO 2021063349 A1 WO2021063349 A1 WO 2021063349A1
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bcma
antibody
seq
heavy chain
human
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PCT/CN2020/118780
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English (en)
French (fr)
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石磊
张雪琨
何云
赵建勋
周伟
陈飞
谭向阳
戎一平
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和铂医药(苏州)有限公司
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Priority to KR1020227014704A priority Critical patent/KR20220075393A/ko
Priority to US17/764,308 priority patent/US20220340673A1/en
Priority to CN202080068742.1A priority patent/CN114502591B/zh
Priority to JP2022520307A priority patent/JP2022551836A/ja
Priority to EP20872247.0A priority patent/EP4039705A4/en
Publication of WO2021063349A1 publication Critical patent/WO2021063349A1/zh

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Definitions

  • the present invention relates to the field of biopharmaceuticals, in particular to an antibody, a bispecific antibody targeting BCMA and its use.
  • BCMA B-cell maturation antigen, TNFRSF17, CD269 is a transmembrane protein belonging to the TNF receptor superfamily. BCMA is a non-glycosylated transmembrane protein that is involved in B cell maturation, growth and survival. BCMA is a receptor for two ligands of the TNF superfamily: the high-affinity ligand APRIL (proliferation-inducing ligand) and the low-affinity ligand BAFF (B cell activating factor). BCMA is a highly differentiated plasma cell selective protein whose expression is limited to the B-cell lineage and mainly exists on plasma cells and plasmablasts, and to a certain extent on memory B-cells, but not on the periphery B-cells. BCMA is expressed in the malignant plasma cells of multiple myeloma (MM) patients and supports the growth and survival of multiple myeloma cells.
  • MM myeloma
  • Multiple myeloma is the second largest hematological malignancy after non-Hodgkin’s lymphoma, accounting for about 1% of all malignancies, 13% of hematological malignancies, and 2% of deaths due to malignant tumors.
  • myeloma cells clonally proliferate in the bone marrow and in the skeletal sponge soft tissue, causing osteolytic bone destruction. The poor recovery is usually accompanied by various damages caused by anemia, renal failure and extramedullary infiltration of myeloma cells.
  • the 5-year relative productivity of MM was about 34%, and all therapies currently used for MM are non-curative.
  • BCMA antibody can be used in MM cells through a variety of mechanisms. The research and development direction of BCMA mainly focuses on monoclonal antibodies, CAR-T therapy, ADC and bispecific antibody therapy.
  • Prior art antibody 1 (also referred to as positive control 1 hereinafter, numbered as PR000274 in the examples) is the anti-BCMA antibody CA8-J6M0 hIgG1 of GlaxoSmithKline (GSK) Company, currently GSK is prepared based on the anti-BCMA antibody CA8-J6M0
  • the antibody-conjugated drug belantamab mafodotin (CA8-J6M0-mcMMAF, GSK2857916) has been used in multiple clinical trials to treat different types of MM patients.
  • the sequence of this control antibody is derived from (Tabs-Therapeutic Antibody Database).
  • bispecific antibodies in the clinical development stage include Amgen's AMG-420, Regeneron's REGN-5458, Xinji's CC-93269, Johnson & Johnson JNJ-64007957, and AbbVie's TNB383B.
  • these bispecific antibodies also have problems such as short half-life and cytokine release syndrome (CRS).
  • Prior art antibody 2 (hereinafter referred to as positive control 2, numbered PR002199 in the examples) is derived from the anti-BCMA (TNB308902) ⁇ CD3 (TNB_F2B) double antibody of Teneobio's patent WO2018052503.
  • Teneobio's TNB383B bispecific antibody entered clinical phase I for MM in 2019.
  • TNB383B uses a weakened CD3 antibody, which has the characteristic of attenuating the release of cytokines.
  • TNB383B does not combine CD3 from cynomolgus monkeys and BCMA from cynomolgus monkeys, and cannot be used for toxicological evaluation in cynomolgus monkeys.
  • the present invention provides an antibody targeting BCMA, a bispecific antibody targeting BCMA and CD3, and applications thereof.
  • an antibody targeting BCMA which includes two heavy chain variable regions, and the heavy chain variable regions include SEQ ID NO: 22, respectively. , HCDR1, HCDR2, and HCDR3 shown in the amino acid sequence of SEQ ID NO: 33 and SEQ ID NO: 42; or, as shown in the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 37 and SEQ ID NO: 41 HCDR1, HCDR2 and HCDR3.
  • the BCMA antibody of the present invention has the activity of binding to human BMCA and cynomolgus BCMA. The size is only half of the traditional IgG antibody, which can be used for the development of bispecific antibodies and solve the problems of light chain mismatch and heterodimerization.
  • the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 59, SEQ ID NO: 60 or a mutation; the mutation is one or more mutations in the original amino acid sequence.
  • the addition, substitution or deletion of three amino acids maintains or improves the binding of the first protein functional region to the BCMA antigen.
  • the mutation is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence shown in SEQ ID NO: 59 and SEQ ID NO: 60 The sequence identity.
  • the BCMA-targeting antibody further includes an Fc fragment.
  • the BCMA-targeting antibody includes two heavy chains.
  • the Fc fragment is an Fc fragment of hlgG1, hlgG2, hlgG3, hlgG4 or a mutation thereof.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 1 or SEQ ID NO: 7 or SEQ ID NO: 8.
  • the technical solution of the second aspect of the present invention is to provide a bispecific antibody comprising a first protein functional region targeting BCMA and a second protein functional region targeting CD3, wherein
  • the first protein functional region includes the two heavy chain variable regions of the BCMA-targeting antibody as described in the first aspect of the present invention; preferably, the two heavy chain variable regions are separated by (G 4 S ) n is connected, and the n is a non-zero natural number, preferably 1-20, more preferably 3 or 4.
  • the BCMA ⁇ CD3 bispecific antibody of the present invention is a unique BCMA ⁇ CD3 bispecific antibody, which has two binding sites with BCMA, thereby improving the affinity and specificity of binding to tumor cell BCMA; bispecific Both protein functional regions of the antibody have good binding activity to cynomolgus monkeys.
  • the second protein functional region includes a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 and a light chain variable region containing LCDR1, LCDR2, and LCDR3.
  • the HCDR1, HCDR2, and The amino acid sequences of HCDR3 are shown in SEQ ID NO: 25, SEQ ID NO: 38, and SEQ ID NO: 44, and/or the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 50, SEQ ID, respectively. Shown in NO:53 and SEQ ID NO:56.
  • the amino acid sequence of the heavy chain variable region of the second protein functional region is shown in SEQ ID NO: 63, SEQ ID NO: 62 or a mutation thereof, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 66 indicates or is a mutation that maintains or improves the binding of the second protein functional region to CD3.
  • the bispecific antibody of the present invention optimizes the activity of the CD3 end, reduces the release of cytokines, and reduces the toxicity of the BCMA ⁇ CD3 bispecific antibody.
  • the bispecific antibody further includes a heavy chain constant region and a light chain constant region, preferably a human heavy chain constant region and a human light chain constant region, and the human light chain constant region is preferably a human ⁇ , ⁇ light chain constant regions, the human heavy chain constant regions are preferably hlgG1, hlgG2, hlgG3, hlgG4 heavy chain constant regions or mutations thereof;
  • the mutation of the human heavy chain constant region is selected from the following group:
  • the first protein functional region includes a heavy chain with an amino acid sequence such as SEQ ID NO: 8 or SEQ ID NO: 7, and the second protein functional region includes an amino acid sequence such as SEQ ID NO: 5 or SEQ.
  • the heavy chain and amino acid sequence shown in ID NO: 4 are the light chain shown in SEQ ID NO: 47.
  • the bispecific antibody of the present invention carries a human Fc fragment, retains the binding effect with FcRn, and thus has a longer half-life.
  • the technical solution of the third aspect of the present invention is to provide a BCMA-targeting antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention.
  • a BCMA-targeting antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention are isolated nucleic acids.
  • the technical solution of the fourth aspect of the present invention is to provide an expression vector containing the isolated nucleic acid according to the third aspect of the present invention.
  • the technical solution of the fifth aspect of the present invention is to provide a host cell comprising the expression vector according to the fourth aspect of the present invention; preferably, the host cell is a prokaryotic cell or a eukaryotic cell. cell.
  • the technical solution of the sixth aspect of the present invention is to provide an antibody drug conjugate comprising the BCMA-targeting antibody as described in the first aspect of the present invention or as the second aspect of the present invention
  • the bispecific antibodies, and cytotoxic agents are provided.
  • the technical solution of the seventh aspect of the present invention is to provide a pharmaceutical composition comprising the BCMA-targeting antibody as described in the first aspect of the present invention, as described in the second aspect of the present invention A bispecific antibody or a drug conjugate of the antibody according to the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.
  • the technical solution of the eighth aspect of the present invention is to provide the BCMA-targeting antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, and the sixth aspect of the present invention.
  • the technical solution of the eighth aspect of the present invention is to provide a kit combination comprising a kit A and a kit B;
  • the kit A contains the target described in the first aspect of the invention.
  • the drug Box B contains other antibodies, bispecific antibodies, host cells, or pharmaceutical compositions that target CD3, BCMA, or other targets.
  • the use of the medicine box A and the medicine box B is in no particular order, or the medicine box A is used first and then the medicine box B, or the medicine box B is used first and then the medicine box A is used.
  • the BCMA-targeting antibody according to the first aspect of the present invention can be administered to patients for the treatment of related tumors.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the BCMA antibody of the present invention is a brand new fully human antibody containing only a "heavy chain", which has the activity of binding to human BMCA and cynomolgus BCMA.
  • the size of the BCMA heavy chain antibody is only half of the traditional IgG antibody. Due to the lack of light chain, the antibody can be used for bispecific antibodies and solves the problem of light chain mismatch and heterodimerization. .
  • the BCMA ⁇ CD3 bispecific antibody of the present invention is a unique BCMA ⁇ CD3 bispecific antibody with two binding sites for BCMA, thereby increasing the affinity and specificity of binding to tumor cell BCMA Sex. b. It is a bispecific antibody structure with a human Fc fragment, which retains the binding effect of Fc and FcRn, thus having a longer half-life. c. Optimize the activity of the CD3 end, reduce the release of cytokines, and reduce the toxicity of the BCMA ⁇ CD3 bispecific antibody. d. BCMA end and CD3 end antibodies have good binding activity to cynomolgus monkeys.
  • Figure 1 shows the binding ability of FACS to detect the cell level of HCAb monoclonal antibody against human BCMA
  • Figure 2 shows the blocking of the binding of human BCMA to the ligand BAFF protein by HCAb monoclonal antibody against human BCMA detected by ELISA;
  • FIG. 3 shows the in vitro binding of the BCMA ⁇ CD3 bispecific antibody detected by FACS on the NCI-H929 cell line with high BCMA expression
  • Figure 4 shows the in vitro binding of BCMA ⁇ CD3 bispecific antibody on HEK293T cell lines overexpressing human and rhesus BCMA by FACS;
  • Figure 5 shows the FACS detection of BCMA ⁇ CD3 double antibody binding to human and rhesus monkey T cells in vitro
  • Figure 6 shows the in vitro killing experiment of the BCMA ⁇ CD3 bispecific antibody on the BCMA highly expressing cell line NCI-H929 and the release of cytokines
  • Figure 7 shows the in vitro killing experiment of the BCMA ⁇ CD3 bispecific antibody on the BCMA low-expressing cell line RPIM8226 and the release of cytokines
  • Figure 8 shows the FACS detection of the binding of BCMA ⁇ CD3 heterogeneous antibody on the BCMA-negative cell line HL-60;
  • Figure 9 shows the non-specific killing and cytokine release of BCMA ⁇ CD3 bispecific antibody on BCMA-negative cell line HL-60 detected by FACS method
  • Figure 10 is the anti-tumor efficacy evaluation of the BCMA ⁇ CD3 bispecific antibody in the NCI-H929/human PBMC mouse model
  • Figure 11 shows BIACORE detecting the affinity of PR003178 with human CD3e/g recombinant protein, cynomolgus CD3e/g recombinant protein, human BCMA recombinant protein, and cynomolgus BCMA recombinant protein.
  • the term “antibody” generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antibody to the antigen. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the "heavy chain antibody” in this application does not contain the VL region, but only contains the VH region.
  • VH or VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH or VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antibodies include, but are not limited to, full-length antibodies, heavy chain antibodies (HCAb), antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and/ Or dAb), immunoconjugates, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they show the desired antigen-binding activity.
  • HCAb heavy chain antibodies
  • Fab antigen-binding fragments
  • Fab' antigen-binding fragments
  • F(ab)2 fragment fragments
  • F(ab')2 fragments
  • scFv fragments
  • di-scFv di-scFv
  • dAb di-scFv
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable regions of the light and heavy chains, called CDRs or hypervariable regions (HVR), and FR is the more highly conserved part of the variable domains.
  • CDRs or hypervariable regions
  • FR is the more highly conserved part of the variable domains.
  • the variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a ⁇ -sheet configuration, connected by three CDRs to form a loop connection, and in some cases form part of a ⁇ -sheet structure.
  • the CDRs in each chain are close together through the FR region, and together with the CDR from the other chain form the antigen binding site of the antibody.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions. , Such as participating in the antibody-dependent cytotoxicity of the antibody.
  • the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997).
  • the combined definition rule including Kabat definition and Chothia definition is also used to determine the amino acid residues in the variable domain sequence and the full-length antibody sequence (Table 1).
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rules) to position bb (Chothia coding rules);
  • Haa-Hbb can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain , The amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule).
  • L24-L34 can refer to the amino acid sequence from the 24th to the 34th starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and following the Chothia coding rules The amino acid sequence from position 26 to position 32.
  • the effector functions mediated by the Fc domain of antibodies such as ADCC and CDC also have very important biological functions.
  • Different IgG subtypes have different ADCC or CDC functions.
  • IgG1 and IgG3 have strong ADCC and CDC functions, while IgG2
  • the effect of IgG4 and IgG4 is relatively weak.
  • changing the binding ability of Fc to Fc receptors through amino acid mutations or modifications can also modulate the original effector functions of Fc.
  • the "LALA" double mutant (L234A/L235A) in IgG1 can significantly reduce the affinity with Fc ⁇ RIIIA (CD16A), thereby reducing the ADCC effect.
  • the P329G mutation can significantly reduce the binding to multiple Fc ⁇ receptors (see, Schlothauer T, Herter S, Koller CF, Grau-Richards S, Steinhart V, Spick C, Kubbies M, Klein C, P, E. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions.Protein EngDes Sel.2016Oct;29(10):457-466.doi:10.1093/protein/gzw040.Epub 2016 Aug 29.PubMed PMID:27578889 ).
  • the Fc of these antibodies introduced "LALA” double mutants (L234A/L235A) or "LALAPG” triple mutants (L234A/L235A/P329G). ).
  • the BCMA antigen can be used to immunize experimental animals to obtain antibody molecules that specifically bind to BCMA.
  • the experimental animals can be mice, rats, rabbits, sheep, camels, and the like.
  • the antibody molecules obtained are of non-human origin. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability.
  • the process of humanization of antibodies has its technical complexity, and molecules that have been humanized will often reduce their affinity for antigens.
  • transgenic technology have made it possible to breed genetically engineered mice that carry human immunoglobulin immune libraries and have their endogenous mouse immune libraries missing.
  • the antibodies produced by the transgenic mice have fully human sequences, so there is no need for further humanization modification, which greatly improves the efficiency of therapeutic antibody development.
  • the Harbour HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) is a transgenic mouse carrying a human immunoglobulin immune library, which can produce a new "heavy chain” antibody only, the size of which is only the size of a traditional IgG antibody half.
  • the antibodies produced only have human antibody "heavy chain” variable domains and mouse Fc constant domains. Due to the lack of light chain, the antibody almost solves the problems of light chain mismatch and heterodimerization, enabling this technology platform to develop products that are difficult to achieve with traditional antibody platforms.
  • the soluble recombinant human BCMA-ECD-Fc fusion protein was used to immunize Harbour HCAb mice for multiple rounds.
  • the antigen protein is mixed with the immune adjuvant to form the immunogenic reagent, and then injected subcutaneously through the inguinal or injected through the abdominal cavity.
  • the total injection dose received by each mouse is 100 microliters.
  • each mouse received 50 micrograms of antigen protein (recombinant human BCMA-ECD-Fc, ACRO, Cat.BC7-H82F0) and complete Freund's adjuvant (Sigma, #F5881) in a volume ratio of 1: 1 Immunization with the prepared immunogen reagent.
  • each mouse was immunized with immunogenic reagents mixed with 25 micrograms of antigen protein and Sigma Adjuvant System adjuvant (Sigma, #S6322).
  • the interval between each round of booster immunity is at least two weeks, usually no more than five rounds of booster immunity.
  • the immunization time was 0, 14, 28, 42, 56, and 70 days; and on the 49th and 77th days, the mouse serum antibody titers were detected.
  • the BCMA-specific antibody titer in the serum of the detected mouse reaches a certain level, the spleen cells of the mouse are taken out to separate the B cells, and the CD138-positive plasma cells and the BCMA antigen-positive B cell populations are sorted with BD FACS AriaII Cell Sorter .
  • RNA was extracted, cDNA was reverse transcribed, and human VH gene was amplified by PCR.
  • the amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc domain sequence.
  • the plasmid was transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and the expressed HCAb
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the antibody supernatant was screened by Mirrorball with recombinant human BCMA-Fc and Avitag recombinant protein (ACRO, Cat.BC7-H82F0), and the obtained Mirrorball-positive monoclonal antibody was further identified by FACS, and compared with the HEK293T cell line overexpressing human BCMA (HEK293T).
  • the clone number of the positive clone obtained by the screening is HBM1005P63B7, and the following number is PR001046.
  • the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence are obtained by conventional sequencing methods.
  • the sequence of the variable domain of the anti-BCMA monoclonal antibody molecule obtained from the immunized Harbour HCAb mouse is a human antibody sequence.
  • Table 2 lists the amino acid sequence of the heavy chain variable domain of the BCMA antibody in this example, the full-length amino acid sequence of the heavy chain, and the amino acid sequence of the CDR defined according to the Combined definition rules. Please refer to the Sequence Listing for the amino acid sequence of the CDR defined by the antibody of the present invention according to other rules.
  • the plasmid encoding the heavy chain of the antibody PR001046 is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and the purified anti-BCMA recombinant heavy chain antibody can be obtained by using conventional recombinant protein expression and purification techniques.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the purified anti-BCMA recombinant heavy chain antibody can be obtained by using conventional recombinant protein expression and purification techniques.
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, A1383504). Before the start of transient transfection, adjust the cell concentration to 6 ⁇ 10 5 cells/ml and incubate for 24 hours at 37°C in an 8% CO 2 shaker. The cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • the target protein was eluted with 0.1M glycine at pH 3.5, then adjusted to neutral with Tris-HCl at pH 8.0, and finally concentrated with an ultrafiltration tube (Millipore, UFC901024) to change the solution to PBS buffer to obtain purified anti-BCMA Heavy chain antibody solution.
  • the following table shows the light and heavy chain sequences of the full-length antibody PR000274, the light and heavy chain variable region sequences, and the amino acid sequence of the CDR defined according to the Combined definition rules.
  • Example 2 FACS detects the binding ability of anti-human BCMA HCAb monoclonal antibody cell level
  • This example is to study the activity of anti-human BCMA HCAb monoclonal antibody binding to human and cyno BCMA.
  • the HEK293T cell line overexpressing human BCMA HEK293T/huBCMA, Beijing Kangyuan
  • the HEK293T cell line overexpressing cynomolgus monkey BCMA HEK293T/cynoBCMA, Beijing Kangyuan
  • CRL-9068 TM NCI-H929
  • HEK293T/huBCMA and HEK293T/cynoBCMA cells were digested and resuspended in DMEM complete medium to collect the NCI-H929 cell suspension.
  • the three cell densities were adjusted to 1 ⁇ 10 6 cells/mL. 100 ⁇ L cells/well were seeded on 96-well V bottom plate (Corning, Cat#:3894), and then 100 ⁇ L/well, 3 times concentration gradient dilution of 2 times the final concentration of the test antibody was added. Place the cells at 4°C and incubate for 1 hour in the dark.
  • the anti-human BCMA HCAb monoclonal antibody PR001046 was used in the HEK293T cell line overexpressing human BCMA (A), the HEK293T cell line overexpressing cynomolgus BCMA (B), and the cell line NCI- which overexpressed human BCMA.
  • the binding ability on H929(C) is better than the positive control 1 (PR000274).
  • the EC50 of the PR001046 binding curve is less than that of the positive control 1, and the specific maximum fluorescence value and EC50 are shown in (D) in Figure 1.
  • Example 3 ELISA detection of anti-human BCMA HCAb monoclonal antibody blocking the binding of human BCMA to the ligand BAFF protein
  • This example is to evaluate the blocking ability of the anti-human BCMA HCAb monoclonal antibody against the binding of human BCMA (ACRO, BCA-H522y-100 ⁇ g) and the ligand BAFF protein (ACRO, BAF-H5248-50ug).
  • human BCMA ACRO, BCA-H522y-100 ⁇ g
  • ligand BAFF protein ACRO, BAF-H5248-50ug
  • the BAFF protein was biotinylated according to the instructions.
  • 1 ⁇ g/mL human BCMA protein, Fc Tag ACRO, Cat#: BC7-H5254 was used to wrap the plate (Corning, Cat#: 9018), overnight.
  • the anti-human BCMA HCAb monoclonal antibody PR001046 has the ability to block the binding of human BCMA to its ligand BAFF protein comparable to the positive control 1 antibody.
  • IC50 values see (B) in Figure 2.
  • Anti-CD3 antibody is an important part of the construction of T-Cell Engager Bispecific Antibody.
  • the mouse antibody SP34 which is known to bind to human CD3e (CD3 epsilon single subunit or CD3e-containing complex), is humanized.
  • CD3e CD3 epsilon single subunit or CD3e-containing complex
  • This example uses the "CDR grafting" method to humanize the sequence, that is: transplanting the CDR of the mouse anti-VH to the framework region of the human antibody VH, and transplanting the CDR of the mouse anti-VL to the framework region of the human antibody VL .
  • the sequence of the framework region of the human antibody VH or VL can be derived from the human germline gene sequence or the antibody sequence after V(D)J rearrangement or the consensus sequence of the specific VH or VL gene family of the human antibody.
  • the framework region sequence provided by the human germline gene sequence is used as the humanized template sequence, that is, the human germline V gene fragment provides the framework region FR1, FR2, FR3 sequence, and the human germline J gene fragment provides Sequence of FR4 framework region.
  • the humanized variable region (VH or VL) was constructed in an arrangement of (human) FR1- (mouse) CDR1- (human) FR2- (mouse) CDR2- (human) FR3- (mouse) CDR3- (human) FR4 )sequence.
  • human germline V gene fragment IGHV3-73*01 or human germline V gene fragment IGHV3-23*01 combined with the sequence of human germline J gene fragment IGHJ1*01 were used as humanized templates to provide framework region sequences. And introduce one or more amino acid mutations at position 30, 73, 76, 78, 93 or 94 (according to Chothia coding rules) to obtain multiple different VH variants sequence.
  • human germline V gene fragment IGLV7-46*02 combined with the sequence of human germline J gene fragment IGLJ2*01 or human germline V gene fragment IGKV1-39*01 combined with human germline J gene fragment IGKJ4*01
  • the sequence serves as a template for humanization to provide the framework region sequence.
  • introduce zero or more amino acid mutations at position 2, 36, 46, 49, 66, 69, 71 or 87 (according to Chothia coding rules) Get a number of different VL variant sequences.
  • Table 4 below shows the amino acid sequence of one of the CD3 antibodies.
  • the light chain (LC) and heavy chain of the antibody (hereinafter, the heavy chain and heavy chain variable regions of the CD3 antibody of the present invention are referred to as HC1 in the bispecific antibody, respectively).
  • VH1 and the heavy chain of the BCMA antibody of the present invention (hereinafter, the heavy chain and the variable region of the heavy chain of the BCMA antibody of the present invention are referred to as HC2 and VH2 respectively in the bispecific antibody) sequence will constitute the bispecific antibodies PR003178, PR002299 ( See Example 5) for the specific construction method.
  • the anti-BCMA antibodies and anti-CD3 cells selected since Example 4 were used to prepare bispecific antibodies.
  • This BCMA ⁇ CD3 bispecific antibody preparation double antibody can simultaneously bind to two targets, one end of which can recognize BCMA specifically expressed on the surface of tumor cells, and the other end can bind to CD3 molecules on T cells.
  • the BCMA ⁇ CD3 double antibody molecule binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing the tumor cells.
  • each bispecific antibody three protein chains are involved, which respectively comprise the heavy chain of the corresponding anti-BCMA antibody, and the heavy and light chains of the aforementioned anti-CD3 antibody.
  • a mutated heterodimer Fc region was used, which carries "knob-hole” mutations and modifications The disulfide bond, as described in WO2009080251 and WO2009080252.
  • the BCMA ⁇ CD3 bispecific antibody is IgG1 with Fc mutations L234A and L235A (numbered according to the EU index).
  • Each bispecific antibody is produced by co-transfection of three different mammalian expression vectors at the same time, respectively encoding: 1) The heavy chain of the corresponding BCMA antibody, which carries the "Hole” mutation in the Fc region to produce heterodimers For antibodies, CH3 of Fc carries L234A and L235A mutations. 2) The heavy chain of the corresponding CD3 antibody, which carries a "knob” mutation in the Fc region to produce a heterodimeric antibody, and the CH3 of Fc carries L234A and L235A mutations. 3) The light chain of the corresponding CD3 antibody.
  • the "knob” mutation in the Fc region of human IgG1 is composed of the following: T366W, and the "Hole” mutation is composed of the following: T366S, L368A, Y407V.
  • S354C and "Hole” Y349C which can include the "knob” Fc region, form a pair of disulfide bonds to increase stability and yield of heterodimeric antibodies.
  • the following tables 5-7 are the sequence information of the bispecific antibody of the present invention and the positive control 2 respectively.
  • the NCI-H929 cell line with high expression of BCMA was used for the cell binding experiment with human BCMA. Briefly, the NCI-H929 cell suspension was collected. The cell density was adjusted to 1 ⁇ 10 6 cells/mL. 100 ⁇ L cells/well were seeded on 96-well V bottom plate (Corning, Cat#:3894), and then 100 ⁇ L/well, 3 times concentration gradient dilution of 2 times the final concentration of the test antibody was added. Place the cells at 4°C and incubate in the dark for 2 hours.
  • the binding of the BCMA ⁇ CD3 bispecific antibody PR003178 to the human BCMA molecule on the NCI-H929 cell line is better than that of the positive control 2, specifically, the EC50 of the binding curve of the antibody PR003178 It is about 8 times smaller than 2 of the positive control.
  • the binding of antibody PR002299 to human BCMA molecules on the NCI-H929 cell line is weaker than antibody PR003178 in terms of the maximum binding fluorescence value and the EC50 of the binding curve.
  • Example 7 FACS detection of BCMA ⁇ CD3 bispecific antibody binding in vitro on HEK293T cell lines overexpressing human and cynomolgus BCMA
  • HEK293T/huBCMA human BCMA
  • HEK293T/cynoBCMA HEK293T/cynoBCMA
  • HEK293T/cynoBCMA HEK293T/cynoBCMA
  • 100 ⁇ L cells/well were seeded on 96-well V bottom plate (Corning, Cat#:3894), and then 100 ⁇ L/well, 3 times concentration gradient dilution of 2 times the final concentration of the test antibody was added. Place the cells at 4°C and incubate for 1 hour in the dark. After that, 100 ⁇ L/well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • the BCMA ⁇ CD3 bispecific antibody PR003178 binds to human BCMA on HEK293T/huBCMA cells stronger than the positive control 2.
  • the EC50 of the binding curve of the antibody PR003178 is about 5 times smaller than that of the positive control, as shown in Figure 4 (C).
  • PR003178 can cross-bind with cyno BCMA on HEK293T/cynoBCMA cells, while the positive control 2 does not have the ability to cross-bind cyno BCMA.
  • Example 8 FACS detection of BCMA ⁇ CD3 double heteroantibodies binding to human and cynomolgus monkey T cells in vitro
  • the BCMA ⁇ CD3 bispecific antibody PR003178 binds to human CD3 on human T cells stronger than the positive control 2. Specifically, under the same antibody concentration, the fluorescence value bound by PR003178 is higher than the fluorescence value bound by the positive control 2. At the same time, as shown in Figure 5 (B), the antibody PR003178 can cross-bind with cynomolgus monkey CD3 on cynomolgus monkey T cells, while the positive control 2 does not have the ability to cross-bind cynomolgus monkey CD3.
  • Example 9 In vitro killing experiment of BCMA ⁇ CD3 bispecific antibody on BCMA highly expressing cell line NCI-H929 and cytokine release
  • PBMC Human PBMC was used as the effector cell, and the cell line NCI-H929 with high BCMA expression was used as the target cell for in vitro killing experiments and cytokine release detection. Specifically, the density of PBMC was adjusted to 1.1 ⁇ 10 6 cells/mL with RPMI1640/5% FBS medium, the density of NCI-H929 was adjusted to 0.11 ⁇ 10 6 cells/mL, and the two cell suspensions were each 90 ⁇ L cells/mL.
  • the wells were seeded in a 96-well U bottom plate (Corning, Cat#: 3799), and then 20 ⁇ L/well, 10 times the final concentration of the 4-fold concentration gradient dilution of the test antibody, the highest final concentration of the antibody is 30nM, each antibody total 10 concentrations, the final effect-to-target ratio is 10:1, and two replicates are set.
  • ER(0) NCI-H929+PBMC+RPMI1640/5% FBS medium
  • ESR PBMC+RPMI1640/5% FBS medium
  • TSR NCI-H929+RPMI1640/ 5% FBS medium
  • CMB RPMI1640/5% FBS medium only
  • TMR NCI-H929+RPMI1640/5% FBS medium + lysis buffer (added after 24 hours of incubation)
  • VCC RPMI1640/5% FBS culture Base + Lysis buffer (added after 24 hours of incubation).
  • the 96-well plate was placed in a carbon dioxide incubator at 37°C and incubated for 24 hours.
  • the cell culture supernatant was collected and used to detect the release of cytokines IL-6 and TNF- ⁇ .
  • the ELISA detection method refers to IL-6 (IL-6 Human Uncoated ELISA Kit, Thermo, Cat#: 88-7066-88) and TNF- ⁇ (TNF alpha Human Uncoated ELISA Kit, Thermo, Cat#: 88-7346-88) Instructions for the kit operation.
  • the PBMC mediated by the BCMA ⁇ CD3 bispecific antibody PR003178 is better than the positive control 2 (A) on killing the cell line NCI-H929 with high BCMA expression.
  • the killing percentage of PR003178 at high concentrations was higher than that of the positive control 2 and the EC50 of the PR003178 killing curve was about 10 times lower than that of the positive control 2 (D).
  • the release of cytokines IL-6 and TNF- ⁇ produced in the killing system of PR003178 was higher than that of the positive control 2 (B, C, E).
  • Example 10 In vitro killing experiment and cytokine release of BCMA ⁇ CD3 bispecific antibody on the low-expressing cell line RPMI8226 of BCMA
  • PBMC Human PBMC was used as the effector cell, and the cell line RPMI8226 (CAS Cell Bank of Chinese Academy of Sciences) with low expression of BCMA was used as the target cell for in vitro killing experiments and cytokine release detection.
  • the density of PBMC was adjusted to 1.1 ⁇ 10 6 cells/mL with RPMI1640/5% FBS medium, and the density of RPMI8226 was adjusted to 0.11 ⁇ 10 6 cells/mL, and the two cell suspensions were each inoculated with 90 ⁇ L cells/well.
  • ER(0) RPMI8226+PBMC+RPMI1640/5%FBS medium
  • ESR PBMC+RPMI1640/5%FBS medium
  • TSR RPMI8226+RPMI1640/5%FBS culture Base
  • CMB RPMI1640/5% FBS medium only
  • TMR RPMI8226+RPMI1640/5% FBS medium + lysis buffer (added after 24 hours incubation)
  • VCC RPMI1640/5% FBS medium + lysis buffer ( Add after 24 hours of incubation).
  • the 96-well plate was placed in a carbon dioxide incubator at 37°C and incubated for 24 hours.
  • the cell culture supernatant was collected and used to detect the release of cytokines IL-6 and TNF- ⁇ .
  • the ELISA detection method refers to IL-6 (IL-6 Human Uncoated ELISA Kit, Thermo, Cat#: 88-7066-88) and TNF- ⁇ (TNF alpha Human Uncoated ELISA Kit, Thermo, Cat#: 88-7346-88) Instructions for the kit operation.
  • the PBMC mediated by the BCMA ⁇ CD3 bispecific antibody PR003178 is better than the positive control 2 (A) in killing the cell line with low BCMA expression.
  • the target cell killing percentage achieved by PR003178 at high concentrations was higher than that of the positive control 2 (D).
  • the release of cytokines IL-6 and TNF- ⁇ produced in the killing system of PR003178 was higher than that of the positive control 2 (B, C, E).
  • Each 90 ⁇ L cells/well of the suspension was seeded on a 96-well U bottom plate (Corning, Cat#: 3799), and then 20 ⁇ L/well, 10 times the final concentration of the test antibody was added, where the final concentration of the antibody was 100 nM, 30 nM, 10 nM and 3 nM A total of 4 concentrations, the final effect-to-target ratio is 10:1, and two replicates are set.
  • the 96-well plate was incubated in a 37°C carbon dioxide incubator for 24 hours. After the incubation is completed, the cell culture supernatant is collected and used to detect the release of cytokines IL-6 and TNF- ⁇ .
  • the BCMA ⁇ CD3 bispecific antibody PR003178 and the positive control 2 can not mediate the two donor PBMCs that are non-specific for the BCMA-negative cell line HL-60. Killing ( Figure 9 A, D), the system can not detect the release of non-specific cytokines IL-6 ( Figure 9 B, E) and TNF- ⁇ ( Figure 9 C, F).
  • mice were injected intraperitoneally (ip) with 3 ⁇ 10 6 human PBMC, and then on the day of tumor cell inoculation, each experimental mouse was inoculated subcutaneously with 5 ⁇ 10 6 NCI -H929 cells, the cells are resuspended in a mixture of PBS and Matrigel (1:1) (0.1 mL/head). When the tumor volume reached 90mm 3 , the mice were randomly divided into groups of 6 mice.
  • tumor volume (mm 3 ) 0.5 ⁇ (tumor long diameter ⁇ tumor short diameter 2 ).
  • the BCMA ⁇ CD3 bispecific antibody PR003178 has the efficacy of inhibiting tumor growth at 10 micrograms/mouse and 3 micrograms/mouse, and the efficacy of the 10 micrograms/mouse group is better than 3 micrograms/mouse.
  • the efficacy of the rat group At the same time, the efficacy of PR003178 at a dose of 10 ⁇ g/mouse is better than that of the positive control 2 of the same dose.
  • the weight changes of the mice in each group were within the normal range.
  • Example 14 BIACORE detects the affinity of PR003178 with human CD3e/g recombinant protein, cynomolgus CD3e/g recombinant protein, human BCMA recombinant protein, and cynomolgus BCMA recombinant protein
  • HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% P20, pH7.4, GE Healthcare, Cat#BR-1006-69) as the running buffer, series S CM5(GE Healthcare, Cat#BR -1005-30) is the experimental chip.
  • Human BCMA-Fc, Avitag recombinant protein (ACRO, Cat.BC7-H82F0), cynomolgus BCMA-Fc, Avitag (ACRO, Cat.BCA-C82F4) were purchased from ACRO, human CD3e/g ECD-hFc recombinant protein (Lot .20190508002), Cynomolgus CD3e/g ECD-hFc recombinant protein and human (Lot.2018040001) were purchased from Ruizhi Chemical.
  • each cycle includes the combination of PR003178 and the regeneration of the chip.
  • Two-fold dilution of PR003178 was injected into four channels at a flow rate of 30 ⁇ l/min, and the binding time was set to 180s, and the dissociation time was 400s or 600s.
  • 10mM glycine-hydrochloric acid pH1.5 (GE Life Sciences, Cat#BR-1003-54) was injected at the same flow rate for 60 seconds to regenerate the chip.
  • the BCMA ⁇ CD3 bispecific antibody PR003178 binds to human BCMA with high affinity, and the affinity to human BCMA-Fc is about 9.493E-11M, and has a good crossover Combines the activity of human and cynomolgus BCMA.
  • the affinity of the BCMA ⁇ CD3 bispecific antibody PR003178 to human CD3e/g ECD-hFc is about 2.541E-08M, and it has a good cross-binding between human and cynomolgus monkeys CD3e/g ECD-hFc activity.
  • (E) in Figure 11 is the specific data of the affinity determination of the BCMA ⁇ CD3 bispecific antibody PR003178 with BCMA and CD3e/g ECD-hFc, including Ka(1/Ms), Kd(1/s), KD.

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Abstract

提供了一种靶向BCMA的抗体、靶向BCMA和CD3的双特异性抗体及其应用。所述靶向BCMA的抗体包括两个重链可变区，所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:33、SEQ ID NO:42的氨基酸序列所示的HCDR1、HCDR2、HCDR3；或如SEQ ID NO:26、SEQ ID NO:37、SEQ ID NO:41的氨基酸序列所示的HCDR1、HCDR2、HCDR3。该抗体有效地靶向人BCMA并结合食蟹猴BCMA；双特异性抗体有效地同时靶向人BCMA和人CD3、并能结合食蟹猴BCMA和食蟹猴CD3。

Description

靶向BCMA的抗体、双特异性抗体及其用途
本申请要求申请日为2019/9/30的中国专利申请2019109413110的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物制药领域，尤其涉及一种靶向BCMA的抗体、双特异性抗体及其用途。
背景技术
BCMA(B-细胞成熟抗原，TNFRSF17，CD269)是一种属于TNF受体超家族的跨膜蛋白。BCMA是一种非糖基化跨膜蛋白，其参与B细胞成熟、生长和存活。BCMA是TNF超家族的两种配体的受体：高亲和力配体APRIL(增殖诱导配体)以及低亲和力配体BAFF(B细胞活化因子)。BCMA是一种高度分化的浆细胞选择性蛋白，其表达限于B-细胞谱系且主要存在于浆细胞和浆母细胞上，并在一定程度上存在于记忆B-细胞上，但是不存在于外周B-细胞上。BCMA在多发性骨髓瘤(MM)患者的恶性浆细胞中表达，支持多发性骨髓瘤细胞的生长和存活。
多发性骨髓瘤是继非霍奇金淋巴瘤的血液***第二大恶性肿瘤，约占所有恶性肿瘤的1％，占血液***恶性肿瘤的13％，占因恶性肿瘤死亡的2％。通常骨髓瘤细胞在骨髓内及骨骼海绵软组织内克隆性增生，引起溶骨性骨骼破坏，愈后不良多伴有贫血、肾衰竭和骨髓瘤细胞髓外浸润所导致的多种损害。截止2006年，MM的5年相对生产率约为34％，目前用于MM的所有疗法是非治愈性的。作为多发性骨髓瘤的一个新兴靶点，BCMA抗体可以通过多种机制作用于MM细胞，BCMA的研发方向主要集中在单克隆抗体、CAR-T疗法、ADC以及双特异性抗体疗法上。
现有技术抗体1(下文又称阳性对照1，在实施例中编号为PR000274)为葛兰素史克(GSK)公司的抗BCMA抗体CA8-J6M0 hIgG1，目前GSK基于抗BCMA抗体CA8-J6M0制备的抗体偶联药物belantamab mafodotin(CA8-J6M0-mcMMAF,GSK2857916)在多项临床试验中治疗不同类型的MM患者。此对照抗体的序列来源于(Tabs-Therapeutic Antibody Database)。来自一项小型临床研究的数据显示，在35例过度预治疗(大多数患者至少接受了5种疗法治疗失败)复发性或难治性(R/R)多发性骨髓瘤(MM)患者中，客观缓解率(ORR)达到了60％，中位无进展生存期(PFS)为12个月。安全性方面，最常见的副作用包括角膜事件、血小板减少和贫血，这些都与ADC中偶联的细胞毒性制剂有关。
目前，处于临床开发阶段的双特异性抗体有安进的AMG-420、再生元的REGN-5458、新基的CC-93269、强生JNJ-64007957、艾伯维的TNB383B。然而这些双特异性抗体还存在半衰期短、细胞因子释放综合征(CRS)等问题。现有技术抗体2(下文称阳性对照2，在实施例中编号为PR002199)来源于Teneobio公司的专利WO2018052503的抗BCMA(TNB308902)×CD3(TNB_F2B)双抗。Teneobio的TNB383B双特异性抗体于2019年进入临床一期用于MM。Teneobio的TNB383B采用了弱化的CD3抗体，具有减弱细胞因子释放的特点。但是TNB383B不结合食蟹猴的CD3和食蟹猴的BCMA，不能在食蟹猴进行毒理学评估。
因此，急需研发更加安全有效的靶向BCMA并能结合食蟹猴的BCMA的单克隆抗体，以及更加安全有效的同时靶向人的BCMA和CD3、并能结合食蟹猴的BCMA和CD3的双特异性抗体。
发明内容
为解决现有技术中缺乏安全有效的靶向人的BCMA并能结合食蟹猴的BCMA的单克隆抗体，以及同时靶向人的BCMA和CD3、并能结合食蟹猴的BCMA和CD3的双特异性抗体的缺陷的技术问题，本发明提供一种靶向BCMA的抗体、靶向BCMA和CD3的双特异性抗体及其应用。
为解决上述技术问题，本发明的第一方面的技术方案为：一种靶向BCMA的抗体，其包括两个重链可变区，所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:33和SEQ ID NO:42的氨基酸序列所示的HCDR1、HCDR2和HCDR3；或，如SEQ ID NO:26、SEQ ID NO:37和SEQ ID NO:41的氨基酸序列所示的HCDR1、HCDR2和HCDR3。本发明的BCMA抗体具有与人BMCA和食蟹猴BCMA结合的活性。大小只有传统IgG抗体的一半，可以用于双特异性抗体的开发，并解决轻链错配和异源二聚化的问题。
在一较佳的具体实施例中，所述重链可变区的氨基酸序列如SEQ ID NO:59、SEQ ID NO:60所示或其突变；所述突变为在原氨基酸序列上的一个或多个氨基酸的添加、取代或删除，并保持或改善了所述第一蛋白功能区与所述BCMA抗原的结合。优选地，所述突变与如SEQ ID NO:59、SEQ ID NO:60所示的氨基酸序列有80％、85％、90％、95％、96％、97％、98％、99％或以上的序列同一性。
在一较佳的具体实施例中，所述靶向BCMA的抗体还包括Fc片段。由此，所述靶向BCMA的抗体包括两条重链。较佳地，所述Fc片段为hIgG1、hIgG2、hIgG3、hIgG4的Fc片段或其突变。
在一较佳的具体实施例中，所述重链的氨基酸序列如SEQ ID NO:1或SEQ ID NO: 7或SEQ ID NO:8所示。
为解决上述技术问题，本发明的第二方面的技术方案为：提供一种双特异性抗体，其包括靶向BCMA的第一蛋白功能区和靶向CD3的第二蛋白功能区，其中，所述第一蛋白功能区包括如本发明第一方面所述的靶向BCMA的抗体的两个重链可变区；较佳地，所述两个重链可变区之间以(G 4S) n连接，所述n为非0自然数，优选1～20更优选3或4。本发明的BCMA×CD3双特异性抗体是一种特有的BCMA×CD3双特异性抗体，具有两个与BCMA结合的位点，从而提高了与肿瘤细胞BCMA结合的亲和力和特异性；双特异性抗体的两个蛋白功能区均具有良好的结合食蟹猴的活性。
在一较佳的具体实施例中，所述第二蛋白功能区包括含HCDR1、HCDR2和HCDR3的重链可变区和含LCDR1、LCDR2和LCDR3的轻链可变区，所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:38和SEQ ID NO:44所示，和/或，所述LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:50、SEQ ID NO:53和SEQ ID NO:56所示。
较佳地，所述第二蛋白功能区的重链可变区的氨基酸序列如SEQ ID NO:63、SEQ ID NO:62所示或为其突变，轻链可变区的氨基酸序列如SEQ ID NO:66所示或为其突变，所述突变保持或改善了所述第二蛋白功能区与CD3的结合。本发明的双特异性抗体优化了CD3端的活性，减少细胞因子的释放，降低BCMA×CD3双特异性抗体的毒性。
在一较佳的具体实施例中，所述双特异性抗体还包括重链恒定区和轻链恒定区，优选为人重链恒定区和人轻链恒定区，所述人轻链恒定区优选人λ、κ轻链恒定区，所述人重链恒定区优选hIgG1、hIgG2、hIgG3、hIgG4的重链恒定区或其突变；
较佳地，所述人重链恒定区的突变选自以下组：
(1)L234A/L235A突变；
(2)knob突变T366W和hole突变T366S、L368A和Y407V；
(3)knob突变S354C和Hole突变Y349C；
更佳地，所述第一蛋白功能区包括氨基酸序列如SEQ ID NO:8或SEQ ID NO:7所示的重链，所述第二蛋白功能区包括氨基酸序列如SEQ ID NO:5或SEQ ID NO:4所示的重链和氨基酸序列如SEQ ID NO:47所示的轻链。本发明的双特异性抗体带有人Fc片段，保留了与FcRn的结合作用，从而具有较长的半衰期。
为解决上述技术问题，本发明的第三方面的技术方案为：提供一种编码根据如本发明第一方面所述的靶向BCMA的抗体或如本发明第二方面所述的双特异性抗体的分离的核酸。
为解决上述技术问题，本发明的第四方面的技术方案为：提供一种包含根据如本发 明第三方面所述的分离的核酸的表达载体。
为解决上述技术问题，本发明的第五方面的技术方案为：提供一种宿主细胞，其包含如本发明第四方面所述的表达载体；优选地，所述宿主细胞是原核细胞或真核细胞。
为解决上述技术问题，本发明的第六方面的技术方案为：提供一种抗体的药物偶联物，其包含如本发明第一方面所述的靶向BCMA的抗体或如本发明第二方面所述的双特异性抗体，以及细胞毒性剂。
为解决上述技术问题，本发明的第七方面的技术方案为：提供一种药物组合物，其包含如本发明第一方面所述的靶向BCMA的抗体、如本发明第二方面所述的双特异性抗体或如本发明第六方面所述的抗体的药物偶联物，以及药学上可接受的载体。
为解决上述技术问题，本发明的第八方面的技术方案为：提供如本发明第一方面所述的靶向BCMA的抗体、本发明第二方面所述的双特异性抗体、本发明第六方面所述的抗体的药物偶联物或本发明第七方面所述的药物组合物在制备治疗和/或预防癌症的药物中的应用。
此外，为解决上述技术问题，本发明第八方面的技术方案为：提供一种药盒组合，其包括药盒A和药盒B；所述药盒A包含本发明第一方面所述的靶向BCMA的抗体、第二方面所述的双特异性抗体、第五方面所述的宿主细胞、第六方面所述的抗体药物偶联物或第七方面所述的药物组合物；所述药盒B包含其它抗体、双特异性抗体、宿主细胞或药物组合物，所述其它抗体、双特异性抗体、宿主细胞或药物组合物靶向CD3、BCMA或其它靶点。所述药盒A和药盒B的使用不分先后顺序，或先使用药盒A再使用药盒B，或先使用药盒B再使用药盒A。
本发明的第一方面所述的靶向BCMA的抗体、第二方面所述的双特异性抗体、第五方面所述的宿主细胞、第六方面所述的抗体药物偶联物或第七方面所述的药物组合物可施用于病人，用于治疗相关肿瘤。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
1、本发明的BCMA抗体是一种全新的仅含“重链”的全人抗体，具有与人BMCA和食蟹猴BCMA结合的活性。该BCMA重链抗体的大小只有传统IgG抗体的一半，由于不含轻链的这一特点，使得该抗体可以用于双特异性抗体，并解决了轻链错配和异源二聚化的问题。
2、本发明的BCMA×CD3双特异性抗体：a.是一种特有的BCMA×CD3双特异性抗 体，具有两个与BCMA结合的位点，从而提高了与肿瘤细胞BCMA结合的亲和力和特异性。b.是一种带有人Fc片段的双特异性抗体结构，保留了Fc与FcRn的结合作用，从而具有较长的半衰期。c.优化了CD3端的活性，减少细胞因子的释放，降低BCMA×CD3双特异性抗体的毒性。d.BCMA端和CD3端抗体具有良好的结合食蟹猴的活性。
附图说明
图1为FACS检测抗人BCMA的HCAb单抗细胞水平的结合能力；
图2为ELISA检测抗人BCMA的HCAb单抗对人BCMA与配体BAFF蛋白结合的阻断；
图3为FACS检测BCMA×CD3双特异性抗体在高表达BCMA的NCI-H929细胞系上的体外结合；
图4为FACS检测BCMA×CD3双特异性抗体在过表达人和猕猴BCMA的HEK293T细胞株上的体外结合；
图5为FACS检测BCMA×CD3双体异性抗体与人和猕猴T细胞体外结合；
图6为BCMA×CD3双特异性抗体对BCMA高表达细胞系NCI-H929的体外杀伤实验以及细胞因子的释放；
图7为BCMA×CD3双特异性抗体对BCMA低表达细胞系RPIM8226的体外杀伤实验以及细胞因子的释放；
图8为FACS检测BCMA×CD3双体异性抗体在BCMA阴性细胞系HL-60上的结合；
图9为FACS方法检测BCMA×CD3双特异性抗体对BCMA阴性细胞系HL-60的非特异杀伤及细胞因子的释放；
图10为NCI-H929/人PBMC小鼠模型中BCMA×CD3双特异性抗体的抗肿瘤药效评估；
图11为BIACORE检测PR003178与人CD3e/g重组蛋白、食蟹猴CD3e/g重组蛋白及人BCMA重组蛋白、食蟹猴BCMA重组蛋白的亲和力。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
在本申请中，术语“抗体”通常是指包含结合抗原的部分的蛋白质，以及任选地允许 结合抗原的部分采用促进抗体与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。例如本申请中的“重链抗体”不含VL区，仅含VH区。VH或VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH或VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的实例包括但不限于全长抗体、重链抗体(HCAb)、抗原结合片段(Fab，Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，称为CDR或高变区(HVR)，FR为可变域中更高度保守的部分。天然重链和轻链的可变结构域各自包含四个FR区，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点，恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)。在本申请中，还使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基(表1)。
表1本申请抗体CDR定义方法(可参见http://bioinf.org.uk/abs/)
CDR区 Kabat定义 Chothia定义 Combined定义
LCDR1 L24--L34 L24--L34 L24--L34
LCDR2 L50--L56 L50--L56 L50--L56
LCDR3 L89--L97 L89--L97 L89--L97
HCDR1 H31--H35 H26--H32 H26--H35
HCDR2 H50--H65 H52--H56 H50--H65
HCDR3 H95--H102 H95--H102 H95--H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。
抗体Fc结构域介导的效应子功能如ADCC和CDC也有非常重要的生物学功能，不同的IgG亚型有着不同的ADCC或CDC功能，例如IgG1和IgG3有较强的ADCC和CDC作用，而IgG2和IgG4的作用相对较弱。另外，通过氨基酸突变或者修饰来改变Fc与Fc受体的结合能力也可以调节Fc原有的效应子功能。例如，IgG1中的“LALA”双突变体(L234A/L235A)能够显著降低与FcγRIIIA(CD16A)的亲和力，进而降低ADCC作用。另外，P329G突变能够显著降低与多种Fcγ受体的结合(参见，Schlothauer T,Herter S,Koller CF,Grau-Richards S,Steinhart V,Spick C,Kubbies M,Klein C,
Figure PCTCN2020118780-appb-000001
P,
Figure PCTCN2020118780-appb-000002
E.Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions.Protein EngDes Sel.2016Oct；29(10):457-466.doi:10.1093/protein/gzw040.Epub 2016 Aug 29.PubMed PMID:27578889)。在申请中，为了减少BCMA×CD3双特异性抗体与Fcγ受体的结合，这些抗体的Fc引入了“LALA”双突变体(L234A/L235A)或者“LALAPG”三突变体(L234A/L235A/P329G)。
实施例1.小鼠免疫和抗BCMA抗体分子的获得
可以利用BCMA抗原对实验动物进行免疫以获得针对BCMA特异性结合的抗体分子，该实验动物可以是小鼠、大鼠、兔、羊、骆驼等。通常，其得到的抗体分子是非人源的。在获得非人源抗体后，需要对这些分子利用抗体工程技术进行人源化改造，以降低免疫原性并提高成药性。然而，抗体的人源化过程有其技术复杂性，经过人源化改造的分子往往会降低对抗原的亲和力。另一方面，转基因技术的进步使得可以培育出基因工程化小鼠，其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。这种转基因小鼠产生的抗体具有全人源的序列，因而无需再进一步做人源化改造，大大提高了治疗性抗体开发的效率。Harbour HCAb小鼠(Harbour Antibodies BV，WO 2002/085945 A3)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生全新的仅“重链”抗体，该抗体的大小只有传统IgG抗体的一半。其产生的抗体仅具有人的抗体“重链”可变结构域和小鼠Fc恒定结构域。由于不含轻链的这一特点，该抗体几乎解决了轻链错配和异源二聚化的问题，使得这一技术平台能够开发出传统抗体平台难以实现的产品。
1.1用BCMA抗原免疫小鼠
用可溶的重组人BCMA-ECD-Fc融合蛋白对Harbour HCAb小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100微升。在首轮免疫中，每只小鼠接受用50微克抗原蛋白(重组人BCMA-ECD-Fc，ACRO，Cat.BC7-H82F0)与完全弗氏佐剂(Sigma,#F5881)以体积比1:1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中，每只小鼠接受用25微克抗原蛋白与Sigma Adjuvant System佐剂(Sigma,#S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周，通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天；并且在第49、77天，检测小鼠血清抗体滴度。在进行HCAb小鼠脾B细胞分离前5天，以每只小鼠25微克抗原蛋白的剂量进行最后一次增强免疫。
1.2获得HCAb单克隆和抗体序列
当检测小鼠血清中BCMA特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用BD FACS AriaII Cell Sorter分选CD138阳性的浆细胞和BCMA抗原阳性的B细胞群。提取RNA，反转录cDNA后PCR扩增人VH基因。扩增的VH基因片段构建到编码人IgG1抗体重链Fc结构域序列的哺乳动物细胞表达质粒pCAG载体中，质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，表达的HCAb的抗体上清与重组人BCMA-Fc,Avitag重组蛋白(ACRO，Cat.BC7-H82F0)进行Mirrorball筛选，获得的Mirrorball阳性单克隆抗体用FACS进一步的鉴定，与过表达人BCMA的HEK293T细胞株(HEK293T/huBCMA,北京康源)、过表达食蟹猴BCMA的HEK293T细胞株(HEK293T/cynoBCMA,北京康源)和高表达人BCMA的细胞系NCI-H929(
Figure PCTCN2020118780-appb-000003
CRL-9068 TM)细胞的结合能力。筛选获得的阳性克隆的克隆号HBM1005P63B7，下文编号为PR001046。利用常规的测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。在本实施例中，从免疫的Harbour HCAb小鼠得到的抗BCMA单克隆抗体分子可变结构域的序列是人源抗体序列。
表2列出了本实施例中BCMA抗体的重链可变结构域氨基酸序列、重链全长氨基酸序列，和根据Combined定义规则定义的CDR的氨基酸序列。本发明抗体根据其他规则定义的CDR的氨基酸序列请参见序列表。
表2抗BCMA的HCAb抗体相关氨基酸序列
Figure PCTCN2020118780-appb-000004
Figure PCTCN2020118780-appb-000005
1.3制备抗BCMA全人重组抗体
将编码抗体PR001046重链的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到纯化的抗BCMA重组重链抗体。具体说来，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo,A1383504)扩培。瞬时转染开始之前，调节细胞浓度至6×10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞，将上述编码PR001046重链的质粒30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo,31985088)，再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,Inc,Cat#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300G转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)平衡含有MabSelect TM(GE Healthcare Life Science,Cat#71-5020-91AE)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品过柱。用5-10倍柱体积的PBS冲洗柱子。再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,UFC901024)浓缩换液至PBS缓冲液，得到纯化的抗BCMA重链抗体溶液。
下表为阳性对照1即全长抗体PR000274的轻、重链序列，轻、重链可变区序列以及根据Combined定义规则定义的CDR的氨基酸序列。
表3全人重组抗体相关氨基酸序列
Figure PCTCN2020118780-appb-000006
Figure PCTCN2020118780-appb-000007
实施例2.FACS检测抗人BCMA的HCAb单抗细胞水平的结合能力
本实施例是为了研究抗人BCMA的HCAb单抗体外结合人和食蟹猴BCMA的活性。采用过表达人BCMA的HEK293T细胞株(HEK293T/huBCMA,北京康源)、过表达食蟹猴BCMA的HEK293T细胞株(HEK293T/cynoBCMA,北京康源)和高表达人BCMA的细胞系NCI-H929(
Figure PCTCN2020118780-appb-000008
CRL-9068 TM)进行细胞水平上的抗体结合实验。简言之，消化细胞HEK293T/huBCMA和HEK293T/cynoBCMA细胞，并用DMEM完全培养基重悬，收集NCI-H929细胞悬液。将3种细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific,Jackson,Cat#:109-545-06,1：500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值。
如图1所示，抗人BCMA的HCAb单抗PR001046在过表达人BCMA的HEK293T 细胞株(A)、过表达食蟹猴BCMA的HEK293T细胞株(B)和高表达人BCMA的细胞系NCI-H929(C)上的结合能力均优于阳性对照1(PR000274)。具体表现为，PR001046结合曲线的EC50均小于阳性对照1，具体最大荧光值和EC50参见图1中的(D)。
实施例3.ELISA检测抗人BCMA的HCAb单抗对人BCMA与配体BAFF蛋白结合的阻断
本实施例是为了评价抗人BCMA的HCAb单抗对人BCMA(ACRO,BCA-H522y-100μg)与配体BAFF蛋白(ACRO,BAF-H5248-50ug)结合的阻断能力。利用生物素化试剂盒(ThermoFisher，A39257，EZ-Link Sulfo-NHS-LC-Biotin)，按照说明书要求对BAFF蛋白进行生物素化。采用1μg/mL人BCMA蛋白，Fc Tag(ACRO,Cat#:BC7-H5254)包板(Corning,Cat#:9018)，过夜。PBST洗涤3次后加2％BSA室温封闭1小时。PBST洗涤3次后加入100μL/孔3倍浓度梯度稀释的待测抗体，起始浓度50nM,室温孵育1小时后，弃上清，加入100μL/孔0.5μg/mL生物素化的人BAFF蛋白(根据本领域常规方法制备)，室温孵育1小时。弃上清，加入100μL/孔1:4000稀释的链霉亲和素耦连的HRP(Bio-RAD,Cat#:1610380)。室温孵育1小时后，PBST洗涤3次。加入100μL/孔TBM显色，15分钟后加入终止液终止反应。使用Enspire(Perkin Elmer)取OD450值。
如图2中(A)所示，抗人BCMA的HCAb单抗PR001046具有与阳性对照1抗体相当的阻断人BCMA与其配体BAFF蛋白结合的能力。具体IC50值参见图2中的(B)。
实施例4.CD3抗体的人源化改造
抗CD3抗体是构建T细胞桥接器双特异性抗体(T-Cell Engager Bispecific Antibody)的重要组成部分。针对已知的结合人CD3e(CD3 epsilon单亚基或者含有CD3e的复合物)的小鼠抗体SP34进行人源化改造。如WO2017084495A1(江苏恒瑞)或CN104136610B(中外制薬株式会社)中所述，并简述如下：
本实施例使用“CDR移植”的方法进行序列的人源化，即：将鼠抗的VH的CDR移植到人抗体VH的框架区，将鼠抗的VL的CDR移植到人抗体VL的框架区。人抗体VH或VL的框架区的序列可以来源于人的胚系基因序列或者经过V(D)J重排后的抗体序列或者人抗体特定VH或VL基因家族的一致性(consensus)序列。本实施例使用人的胚系基因序列提供的框架区序列作为人源化模板序列，即：人的胚系V基因片段提供框架区FR1，FR2，FR3的序列，人的胚系J基因片段提供框架区FR4的序列。最后以(人)FR1-(鼠)CDR1-(人)FR2-(鼠)CDR2-(人)FR3-(鼠)CDR3-(人)FR4的排列方式构建人源化可变区(VH或VL)序列。
本实施例使用人胚系V基因片段IGHV3-73*01或人胚系V基因片段IGHV3-23*01结合人胚系J基因片段IGHJ1*01的序列作为人源化模板提供框架区序列。并且在第30 位、第73位、第76位、第78位、第93位或第94位(按照Chothia编码规则)引入一个或者多个位点的氨基酸突变，得到多个不同的VH变体序列。
本实施例使用人胚系V基因片段IGLV7-46*02结合人胚系J基因片段IGLJ2*01的序列或者人胚系V基因片段IGKV1-39*01结合人胚系J基因片段IGKJ4*01的序列作为人源化模板提供框架区序列。并且在第2位、第36位、第46位、第49位、第66位、第69位、第71位或第87位(按照Chothia编码规则)引入零个或者多个位点的氨基酸突变，得到多个不同的VL变体序列。下表4为其中一个CD3抗体的氨基酸序列，该抗体的轻链(LC)、重链(下文中，本发明CD3抗体的重链、重链可变区分别在双特异性抗体中简称HC1、VH1)与本发明BCMA抗体的重链(下文中，本发明BCMA抗体的重链、重链可变区分别在双特异性抗体中简称HC2、VH2)序列将构成双特异性抗体PR003178、PR002299(具体构建方法见实施例5)。
表4获得的CD3抗体氨基酸序列
Figure PCTCN2020118780-appb-000009
Figure PCTCN2020118780-appb-000010
实施例5.BCMA×CD3双特异性抗体的制备
自实施例4之后所选的抗BCMA抗体和抗CD3细胞用于制备双特异性抗体。这种BCMA×CD3双特异性抗体的制备双抗可以同时结合两个靶点，其中一端可以识别肿瘤细胞表面特异表达的BCMA，而另一端可以结合T细胞上的CD3分子。当BCMA×CD3双抗分子结合到肿瘤细胞表面后，可以招募并激活肿瘤细胞附近的T细胞，从而杀死肿瘤细胞。
对于每一个双特异性抗体，涉及三条蛋白链，其分别包含相应抗BCMA抗体的重链，以及上述抗CD3抗体的重链和轻链。为了将具有错配的重链(例如抗CD3抗体的两条重链错配)的副产物形成最小化，使用了突变的异源二聚体Fc区，其携带“knob-hole”突变和改造的二硫键，如WO2009080251和WO2009080252中所述。BCMA×CD3双特异性抗体为IgG1，具有Fc突变L234A和L235A(根据EU索引编号)。
通过同时共转染三个不同的哺乳动物表达载体产生每一个双特异性抗体，分别编码：1)相应的BCMA抗体的重链，其在Fc区携带“Hole”突变以产生异源二聚体抗体，Fc的CH3携带L234A、L235A突变。2)相应的CD3抗体的重链，其在Fc区携带“knob”突变以产生异源二聚体抗体,Fc的CH3携带L234A、L235A突变。3)相应的CD3抗体的轻链。人IgG1Fc区的“knob”突变由以下组成：T366W，“Hole”突变由以下组成：T366S、L368A、Y407V。此外，可以包括“knob”Fc区的S354C和“Hole”Y349C形成一对二硫键以增加稳定性和异源二聚体抗体产量。下表5～7分别是本发明的双特异性抗体以及 阳性对照2的序列信息。
表5 PR003178双特异性抗体BCMA链的序列信息
Figure PCTCN2020118780-appb-000011
表6 PR002299双特异性抗体BCMA链的氨基酸序列信息
Figure PCTCN2020118780-appb-000012
表7阳性对照2(PR002199)的氨基酸序列信息
Figure PCTCN2020118780-appb-000013
Figure PCTCN2020118780-appb-000014
实施例6.FACS检测BCMA×CD3双特异性抗体在高表达BCMA的NCI-H929 细胞系上的体外结合
为了研究BCMA×CD3双特异性抗体BCMA臂的结合人BCMA的活性。采用高表达BCMA的NCI-H929细胞系进行细胞上与人BCMA结合实验。简言之，收集NCI-H929细胞悬液。将细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育2小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific,Jackson,Cat#:109-545-06,1：500稀释)，4℃，避光孵育1小时。用100μL/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值。
如图3中(A)和(B)所示，BCMA×CD3双特异性抗体PR003178与NCI-H929细胞系上人BCMA分子的结合优于阳性对照2，具体表现为，抗体PR003178结合曲线的EC50小于阳性对照2约8倍。同时，抗体PR002299与NCI-H929细胞系上人BCMA分子的结合从最大结合荧光值及结合曲线的EC50两方面均弱于抗体PR003178。
实施例7.FACS检测BCMA×CD3双特异性抗体在过表达人和食蟹猴BCMA的HEK293T细胞株上的体外结合
为了研究BCMA×CD3双特异性抗体BCMA臂的体外结合活性。采用过表达人BCMA的HEK293T细胞株(HEK293T/huBCMA)和过表达食蟹猴BCMA的HEK293T细胞株(HEK293T/cynoBCMA)进行细胞水平上的抗体结合实验。简言之，消化细胞HEK293T/huBCMA和HEK293T/cynoBCMA细胞，并用DMEM完全培养基重悬。将2种细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific,Jackson,Cat#:109-545-06,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值.
如图4中(A)所示，BCMA×CD3双特异性抗体PR003178与HEK293T/huBCMA细胞上人BCMA的结合强于阳性对照2。具体表现为，抗体PR003178结合曲线的EC50小于阳性对照2约5倍，如图4中(C)所示。同时，如图4中(B)所示，PR003178能 够与HEK293T/cynoBCMA细胞上食蟹猴BCMA交叉结合，而阳性对照2不具备交叉结合食蟹猴BCMA的能力。
实施例8.FACS检测BCMA×CD3双体异性抗体与人和食蟹猴T细胞体外结合
为了研究BCMA×CD3双特异性抗体CD3臂的体外结合活性。采用人和食蟹猴T细胞进行细胞水平上的抗体结合实验。简言之，将人和食蟹猴T细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g，4℃下离心5分钟，弃上清。再加入100μL/荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific,Jackson,Cat#:109-545-06,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值.
如图5中(A)所示，BCMA×CD3双特异性抗体PR003178与人T细胞上人CD3的结合强于阳性对照2。具体表现为，在相同抗体浓度情况下，PR003178结合的荧光值高于阳性对照2结合的荧光值。同时如图5中(B)所示，抗体PR003178能够与食蟹猴T细胞上食蟹猴CD3交叉结合，而阳性对照2不具备交叉结合食蟹猴CD3的能力。
实施例9.BCMA×CD3双特异性抗体对BCMA高表达细胞系NCI-H929的体外杀伤实验以及细胞因子的释放
为了研究BCMA×CD3双特异性抗体体外介导的靶细胞杀伤能力。采用人PBMC作为效应细胞，高表达BCMA的细胞系NCI-H929作为靶细胞进行体外杀伤实验以及细胞因子释放的检测。具体的，用RPMI1640/5％FBS培养基将PBMC的密度调整为1.1×10 6细胞/mL，NCI-H929的密度调整为0.11×10 6细胞/mL，以两种细胞悬液各90μL细胞/孔接种于96孔U底板(Corning,Cat#:3799)，随后加入20μL/孔，10倍于终浓度的4倍浓度梯度稀释的待测抗体，其中抗体最高终浓度为30nM，每个抗体共10个浓度，最终效靶比为10：1，设置两个重复。同时，在板内设置不同的对照组：ER(0)：NCI-H929+PBMC+RPMI1640/5％FBS培养基；ESR：PBMC+RPMI1640/5％FBS培养基；TSR：NCI-H929+RPMI1640/5％FBS培养基；CMB：仅RPMI1640/5％FBS培养基；TMR:NCI-H929+RPMI1640/5％FBS培养基+裂解缓冲液(孵育24小时后加入)；VCC：RPMI1640/5％FBS培养基+裂解缓冲液(孵育24小时后加入)。96孔板置于37℃二氧化碳培养箱孵育24小时。孵育完成后，取50μl上清液，加入96孔板(Corning,Cat#:3599)，并加入50μl细胞毒性检测试剂(CytoTox
Figure PCTCN2020118780-appb-000015
非放射性细胞毒性检测试剂盒，Promega,USA，cat#G1780)，室温孵育30分钟，加入终止溶液停止反应并用Enspire(Perkin  Elmer)读取OD490值。OD490读值按照以下公式来计算杀伤效果，特异性杀伤％＝{(ER-CMB)-[(ESR-CMB)+(TSR-CMB)]}/(TMR-VCC)*100％。收集细胞培养上清，用于检测细胞因子IL-6和TNF-α的释放。ELISA检测方法参照IL-6(IL-6 Human Uncoated ELISA Kit,Thermo,Cat#:88-7066-88)和TNF-α(TNF alpha Human Uncoated ELISA Kit,Thermo,Cat#:88-7346-88)试剂盒操作说明。
如图6所示，BCMA×CD3双特异性抗体PR003178介导的PBMC对高表达BCMA的细胞系NCI-H929的杀伤优于阳性对照2(A)。具体表现为，PR003178在高浓度所达到杀伤百分比高于阳性对照2以及PR003178杀伤曲线的EC50小于阳性对照2约10倍(D)。同时，PR003178的杀伤体系中产生的细胞因子IL-6和TNF-α的释放均高于阳性对照2(B、C、E)。
实施例10.BCMA×CD3双特异性抗体对BCMA的低表达细胞系RPMI8226的体外杀伤实验以及细胞因子的释放
为了研究BCMA×CD3双特异性抗体体外介导的靶细胞杀伤能力。采用人PBMC作为效应细胞，低表达BCMA的细胞系RPMI8226(中科院CAS细胞库)作为靶细胞进行体外杀伤实验以及细胞因子释放的检测。具体的，用RPMI1640/5％FBS培养基将PBMC的密度调整为1.1×10 6细胞/mL，RPMI8226的密度调整为0.11×10 6细胞/mL，以两种细胞悬液各90μL细胞/孔接种于96孔U底板(Corning,Cat#:3799)，随后加入20μL/孔，10倍于终浓度的4倍浓度梯度稀释的待测抗体，其中抗体最高终浓度为30nM，每个抗体共10个浓度，最终效靶比为10:1，设置两个重复。同时，在板内设置不同的对照组：ER(0)：RPMI8226+PBMC+RPMI1640/5％FBS培养基；ESR：PBMC+RPMI1640/5％FBS培养基；TSR：RPMI8226+RPMI1640/5％FBS培养基；CMB:仅RPMI1640/5％FBS培养基；TMR:RPMI8226+RPMI1640/5％FBS培养基+裂解缓冲液(孵育24小时后加入)；VCC：RPMI1640/5％FBS培养基+裂解缓冲液(孵育24小时后加入)。96孔板置于37℃二氧化碳培养箱孵育24小时。孵育完成后，取50μl上清液，加入96孔板(Corning,Cat#:3599)，并加入50μl细胞毒性检测试剂(CytoTox
Figure PCTCN2020118780-appb-000016
非放射性细胞毒性检测试剂盒，Promega,USA，Cat#:G1780)，室温孵育30分钟，加入终止溶液停止反应并用Enspire(Perkin Elmer)读取OD490值。OD490读值按照以下公式来计算杀伤效果，特异性杀伤％＝{(ER-CMB)-[(ESR-CMB)+(TSR-CMB)]}/(TMR-VCC)*100％。收集细胞培养上清，用于检测细胞因子IL-6和TNF-α的释放。ELISA检测方法参照IL-6(IL-6 Human Uncoated ELISA Kit,Thermo,Cat#:88-7066-88)和TNF-α(TNF alpha Human Uncoated ELISA Kit,Thermo,Cat#:88-7346-88)试剂盒操作说明。
如图7所示，BCMA×CD3双特异性抗体PR003178介导的PBMC对低表达BCMA 的细胞系的杀伤优于阳性对照2(A)。具体表现为，PR003178在高浓度所达到的靶细胞杀伤百分比高于阳性对照2(D)。同时，PR003178的杀伤体系中产生的细胞因子IL-6和TNF-α的释放均高于阳性对照2(B、C、E)。
实施例11.BCMA×CD3双特异性抗体对不表达BCMA细胞系HL-60的结合
为了研究BCMA×CD3双特异性抗体体外结合的特异性。采用不表达人BCMA的细胞系HL-60(
Figure PCTCN2020118780-appb-000017
CCL-240 TM)进行细胞水平上的抗体结合实验。简言之，收集HL-60细胞悬液。将细胞密度分别调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体，抗体最高终浓度为300nM，每个抗体4个浓度。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific,Jackson,Cat#:109-545-06,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值。
如图8所示，在300nM、100nM、33.3nM和11.1nM下，BCMA×CD3双特异性抗体PR003178与阳性对照2均不能与BCMA阴性细胞系HL-60非特异结合。
实施例12.FACS方法检测BCMA×CD3双特异性抗体对BCMA阴性细胞系HL60的非特异杀伤及细胞因子的释放
为了研究BCMA×CD3双特异性抗体体外介导的靶细胞杀伤能力的特异性。采用人PBMC作为效应细胞，不表达BCMA的细胞系HL-60作为靶细胞进行体外杀伤实验以及细胞因子释放的检测。具体的，将靶细胞HL-60用0.5μM的CFSE染料标记。用RPMI1640/5％FBS培养基将3个供体的PBMC的密度调整为1.1×10 6细胞/mL，CFSE标记的HL-60细胞的密度调整为0.11×10 6细胞/mL，以两种细胞悬液各90μL细胞/孔接种于96孔U底板(Corning,Cat#:3799)，随后加入20μL/孔，10倍于终浓度的待测抗体，其中抗体终浓度为100nM、30nM、10nM和3nM共4个浓度，最终效靶比为10:1，设置两个重复。96孔板置于37℃二氧化碳培养箱孵育24小时。孵育完成后，收集细胞培养上清，用于检测细胞因子IL-6和TNF-α的释放。ELISA检测方法参照IL-6(IL-6 Human Uncoated ELISA Kit,Thermo,Cat#:88-7066-88)和TNF-α(TNF alpha Human Uncoated ELISA Kit,Thermo,Cat#:88-7346-88)试剂盒操作说明。将培养的细胞转至96孔V底板(Corning,Cat#:3894)，以100μL/孔的7-AAD(BD,Cat#:559925,1:100)染色体系进行染色，室温孵育30分钟，加入100μL/孔PBS终止反应。使用BD FACS CANTOII进行检测。按照以下公式来计算杀伤效果，特异性杀伤％＝7-AAD+CFSE+百分比/(7- AAD+CFSE+百分比+7-AAD-CFSE+百分比)*100％。
如图9的A-E所示，在100nM、30nM、10nM和3nM下，BCMA×CD3双特异性抗体PR003178和阳性对照2不能介导的2个供体PBMC对BCMA阴性细胞系HL-60产生非特异杀伤(图9的A、D)，该体系中也不能检测到非特异的细胞因子IL-6(图9的B、E)和TNF-α(图9的C、F)的释放。
实施例13.NCI-H929/人PBMC小鼠模型中BCMA×CD3双特异性抗体的抗肿瘤药效评估
为了评估BCMA×CD3双特异性抗体的体内抗肿瘤药效。采用6-8周雌性NCG小鼠，瘤细胞接种前3天，所有小鼠腹腔注射(i.p.)3×10 6人PBMC，然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×10 6NCI-H929细胞，细胞重悬在PBS与Matrigel(1:1)混合液中(0.1mL/只)。当肿瘤体积达到90mm 3时对小鼠进行随机分组，每组6只小鼠。分组后将特定浓度经PBS稀释的药物以静脉注射(i.v.)、每周给药1次总共给药2次(QW*2)的方式进行给药，以PBS为空白对照组。在初次给药后第3天，7天，10天和14天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
如图10中(A)所示，BCMA×CD3双特异性抗体PR003178在10微克/鼠和3微克/鼠均有抑制肿瘤生长的药效，10微克/鼠组的药效优于3微克/鼠组的药效。同时地，在10微克/鼠的给药剂量下PR003178的药效优于同等剂量的阳性对照2。如图10中(B)所示，受试各组小鼠体重变化均在正常范围内。
实施例14.BIACORE检测PR003178与人CD3e/g重组蛋白、食蟹猴CD3e/g重组蛋白及人BCMA重组蛋白、食蟹猴BCMA重组蛋白的亲和力
整个测试使用HBS-EP+(10mM HEPES，150mM NaCl，3mM EDTA和0.05％P20，pH7.4，GE Healthcare，Cat#BR-1006-69)作为运行缓冲液，系列S CM5(GE Healthcare，Cat#BR-1005-30)为实验芯片。人BCMA-Fc,Avitag重组蛋白(ACRO，Cat.BC7-H82F0)、食蟹猴BCMA-Fc,Avitag(ACRO，Cat.BCA-C82F4)购买自ACRO，人CD3e/g ECD-hFc重组蛋白(Lot.20190508002)、食蟹猴CD3e/g ECD-hFc重组蛋白及人(Lot.2018040001)购买自睿智化学。
14.1抗原的偶联
设置流速10μl/min,在两张CM5的4个通道上偶联4种抗原：1)设置注入时间300s，将50mM NHS和200mM EDC以1:1体积比新鲜混合后注入4个通道；2)用pH4.5的醋酸钠(Cat#BR-1003-50)将人BCMA-Fc,Avitag重组蛋白、食蟹猴BCMA-Fc,Avitag、人CD3e/g ECD-hFc重组蛋白、食蟹猴CD3e/g ECD-hFc重组蛋白均稀释至1ug/ml，分别注 入芯片一的2、3、4和芯片二的2通道；3)注入1M pH8.5乙醇胺300s，以封闭芯片表面剩余的活性羧基。封闭后继续用1×HBS-EP+缓冲液平衡仪器两小时，人BCMA-Fc,Avitag重组蛋白、食蟹猴BCMA-Fc,Avitag、人CD3e/g ECD-hFc重组蛋白和食蟹猴CD3e/g ECD-hFc重组蛋白最终偶联量分别为120RU、70RU、200RU和290RU。
14.2亲和力测定
设置多循环动力学模式，每个循环包括PR003178的结合以及芯片的再生。将PR003178两倍梯度稀释注入四个通道，流速为30μl/min，设置结合时间180s，解离时间400s或600s。最后以同样流速注入10mM甘氨酸-盐酸pH1.5(GE Life Sciences,Cat#BR-1003-54)60s，以再生芯片。
用Biacore T200分析软件对实验结果进行分析，1通道作为参比通道扣除，分析模型选用1:1动力学拟合模型。
如图11中的(B)、(D)所示，BCMA×CD3双特异性抗体PR003178与人BCMA具有高亲和力结合，与人BCMA-Fc的亲和力约9.493E-11M，且具有很好的交叉结合人和食蟹猴BCMA的活性。如图11中的(A)、(C)所示，BCMA×CD3双特异性抗体PR003178与人CD3e/g ECD-hFc的亲和力约2.541E-08M，并且具有很好的交叉结合人和食蟹猴CD3e/g ECD-hFc的活性。如图11中的(E)为BCMA×CD3双特异性抗体PR003178与BCMA和CD3e/g ECD-hFc亲和力测定的具体数据，包括Ka(1/Ms),Kd(1/s),KD。

Claims (15)

  1. 一种靶向BCMA的抗体，其特征在于，其包括两个重链可变区，所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:33和SEQ ID NO:42的氨基酸序列所示的HCDR1、HCDR2和HCDR3；或，如SEQ ID NO:26、SEQ ID NO:37和SEQ ID NO:41的氨基酸序列所示的HCDR1、HCDR2和HCDR3。
  2. 如权利要求1所述的靶向BCMA的抗体，其特征在于，所述重链可变区的氨基酸序列如SEQ ID NO:59所示；或所述重链可变区的氨基酸序列如SEQ ID NO:60所示。
  3. 如权利要求1或2所述的靶向BCMA的抗体，其特征在于，所述靶向BCMA的抗体还包括Fc片段；较佳地，所述Fc片段为hIgG1、hIgG2、hIgG3、hIgG4的Fc片段或其突变。
  4. 如权利要求1-3中任一项所述的靶向BCMA的抗体，其特征在于，所述靶向BCMA的抗体的重链的氨基酸序列如SEQ ID NO:1或SEQ ID NO:7或SEQ ID NO:8所示。
  5. 一种双特异性抗体，其包括靶向BCMA的第一蛋白功能区和靶向CD3的第二蛋白功能区，其特征在于，所述第一蛋白功能区包括如权利要求1-4中任一项所述的靶向BCMA的抗体的两个重链可变区；较佳地，两个所述重链可变区之间以(G 4S) n连接，所述n为非0自然数，优选1～20更优选3或4。
  6. 如权利要求5所述的双特异性抗体，其特征在于，所述第二蛋白功能区包括重链可变区和轻链可变区，所述重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:38和SEQ ID NO:44所示，和/或，所述轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:50、SEQ ID NO:53和SEQ ID NO:56所示。
  7. 如权利要求5所述的双特异性抗体，其特征在于，所述第二蛋白功能区的重链可变区的氨基酸序列如SEQ ID NO:63、SEQ ID NO:62所示或为其突变，轻链可变区的氨基酸序列如SEQ ID NO:66所示或为其突变，所述突变保持或改善了所述第二蛋白功能区与CD3的结合。
  8. 如权利要求5-7任一项所述的双特异性抗体，其特征在于，所述双特异性抗体还包括重链恒定区和轻链恒定区，优选为人重链恒定区和人轻链恒定区，所述人轻链恒定区优选人λ、κ轻链恒定区，所述人重链恒定区优选hIgG1、hIgG2、hIgG3、hIgG4的重链恒定区或其突变。
  9. 如权利要求8所述的双特异性抗体，其特征在于，所述人重链恒定区的突变选自 以下组：
    (1)L234A/L235A突变；
    (2)knob突变T366W和hole突变T366S、L368A和Y407V；
    (3)knob突变S354C和Hole突变Y349C；
    较佳地，所述第一蛋白功能区包括氨基酸序列如SEQ ID NO:8或SEQ ID NO:7所示的重链，所述第二蛋白功能区包括氨基酸序列如SEQ ID NO:5或SEQ ID NO:4所示的重链和氨基酸序列如SEQ ID NO:47所示的轻链。
  10. 一种编码根据权利要求1-4中任一项所述的靶向BCMA的抗体或权利要求5-9中任一项所述的双特异性抗体的分离的核酸。
  11. 一种包含根据权利要求10所述的分离的核酸的表达载体。
  12. 一种宿主细胞，其包含根据权利要求11所述的表达载体；优选地，所述宿主细胞是原核细胞或真核细胞。
  13. 一种抗体的药物偶联物，其包含如权利要求1-4中任一项所述的靶向BCMA的抗体或权利要求5-9中任一项所述的双特异性抗体，以及细胞毒性剂。
  14. 一种药物组合物，其包含如权利要求1-4中任一项所述的靶向BCMA的抗体、权利要求5-9中任一项所述的双特异性抗体或权利要求13所述的双特异性抗体的药物偶联物，以及药学上可接受的载体。
  15. 如权利要求1-4中任一项所述的靶向BCMA的抗体、权利要求5-9中任一项所述的双特异性抗体、权利要求13所述的抗体的药物偶联物或权利要求14中所述的药物组合物在制备治疗和/或预防癌症的药物中的应用。
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