ANTI-HUMAN INTERLEUKIN 5(IL-5) MONOCLONAL ANTIBODY AND USE THEREOF
20220010008 · 2022-01-13
Inventors
- Yizhen YANG (Shanghai, CN)
- Songlin YANG (Shanghai, CN)
- Jian Wu (Shanghai, CN)
- Xinxiu YANG (Shanghai, CN)
- Xiaohui Shao (Shanghai, CN)
- Qin ZHONG (Shanghai, CN)
- Shaoping Hu (Shanghai, CN)
- Qing Duan (Shanghai, CN)
- Lile Liu (Shanghai, CN)
- Dongxu WANG (Shanghai, CN)
- Chaohui DAI (Shanghai, CN)
- Mengying WANG (Shanghai, CN)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
A61K47/6845
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
C07K16/24
CHEMISTRY; METALLURGY
A61K35/17
HUMAN NECESSITIES
Abstract
Disclosed in the present invention are an antibody targeting IL-5, a preparation method therefor and use thereof. In particular, disclosed in the present invention is a novel murine-derived or chimeric monoclonal antibody targeting IL-5. Also disclosed in the present invention is a method for preparing said monoclonal antibody. The monoclonal antibody of the present invention is capable of binding IL-5 antigen with high specificity, has high affinity and can well alleviate a series of asthma symptoms caused by IL-5, thereby achieving the effect of treating asthma.
Claims
1. A heavy chain variable region of an antibody, wherein the heavy chain variable region comprises complementary determining regions or CDRs selected from the group consisting of: VH-CDR1 shown in SEQ ID NO: 10n+3, VH-CDR2 shown in SEQ ID NO: 10n+4, and VH-CDR3 shown in SEQ ID NO: 10n+5; wherein each n is independently 0, 1, 2, 3, or 4; wherein any one of the above amino acid sequences further comprises a derivative sequence which is obtained through optional addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining the binding affinity to IL-5.
2-4. (canceled)
5. An antibody, wherein the antibody comprises: (1) the heavy chain variable region of claim 1; and/or (2) the light chain variable region comprising complementary determining regions or CDRs selected from the group consisting of: VL-CDR1 shown in SEQ ID NO: 10n+8, VL-CDR2 shown in SEQ ID NO: 10n+9, and VL-CDR3 shown in SEQ ID NO: 10n+10; wherein each n is independently 0, 1, 2, 3, or 4; wherein any one of the above amino acid sequences further comprises a derivative sequence which is obtained through optional addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining the binding affinity to IL-5.
6. The antibody of claim 5, wherein the antibody comprises the heavy chain variable region and the light chain variable region; wherein, the heavy chain variable region and the light chain variable region comprise CDRs selected from the group consisting of: TABLE-US-00031 VH-CDR1 VH-CDR2 VH-CDR3 VL-CDR1 VL-CDR2 VL-CDR3 Sequence Sequence Sequence Sequence Sequence Sequence number number number number number number 3 4 5 8 9 10 13 14 15 18 19 20 23 24 25 28 29 30 33 34 35 38 39 40 43 44 45 48 49 50 wherein any one of the above amino acid sequences further comprises a derivative sequence which is obtained through optional addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining the binding affinity to IL-5.
7. The antibody of claim 5, wherein the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 1, 11, 21, 31, or 41; and/or the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 36, or 46.
8. The antibody of claim 6, wherein the antibody is selected from the group consisting of: TABLE-US-00032 Antibody VH sequence VL sequence number Clone number number 1 147D7H2 1 6 2 102A2E11B6 11 16 3 69F2F3 21 26 4 154F10G8 31 36 5 151G3B11 41 46.
9. A recombinant protein, wherein the recombinant protein comprises: (i) the antibody of claim 5; and (ii) an optional tag sequence that assists expression and/or purification.
10. A polynucleotide, wherein the polynucleotide encodes a polypeptide selected from group consisting of: (1) the antibody of claim 5; and (2) the recombinant protein comprising the antibody.
11. The polynucleotide of claim 10, wherein the polynucleotide encoding the heavy chain variable region is shown in SEQ ID NO: 2, 12, 22, 32, or 42; and/or, the polynucleotide encoding the light chain variable region is shown in SEQ ID NOs: 7, 17, 27, 37, or 47.
12. The polynucleotide of claim 11, wherein the polynucleotide encoding the heavy chain variable region and the polynucleotide encoding the light chain variable region are selected from the group consisting of: TABLE-US-00033 Polynucleotide encoding VH Polynucleotide encoding VL Clone Sequence number Sequence number 147D7H2 2 7 102A2E11B6 12 17 69F2F3 22 27 154F10G8 32 37 151G3B11 42 47.
13. A vector, wherein the vector comprises the polynucleotide of claim 10.
14. A genetically engineered host cell, wherein the host cell contains the vector of claim 13.
15. An antibody conjugate, wherein the antibody conjugate comprises: (a) an antibody moiety, which is selected from the group consisting of: the antibody of claim 5, and a combination thereof; and (b) a coupling moiety coupled to the antibody moiety, and the coupling moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, and a combination thereof.
16. An immune cell, which expresses or is exposed outside the cell membrane with the antibody of claim 5.
17. A pharmaceutical composition, wherein the pharmaceutical composition comprises: (i) an active ingredient, wherein the active ingredient is selected from the group consisting of: the antibody of claim 5, the recombinant protein comprising the antibody, the antibody conjugate comprising the antibody, the immune cell expressing the antibody, and a combination thereof; and (ii) a pharmaceutically acceptable carrier.
18. A method for the treatment of diseases associated with abnormal IL-5 expression or function, which comprises administering an effective amount of the antibody of claim 5, the recombinant protein comprising the antibody, the antibody conjugate comprising the antibody, the immune cell expressing the antibody, and a combination thereof, to a subject in need.
19. The method of claim 18, wherein the disease associated with abnormal IL-5 expression or function is asthma, atopic dermatitis, or inflammation diseases.
20. The method of claim 19, wherein the inflammatory disease is chronic obstructive pulmonary disease.
Description
DESCRIPTION OF DRAWINGS
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[0211] Modes for Carrying Out the Present Invention
[0212] Through extensive and intensive research, the inventors prepared a group of specific monoclonal murine antibodies binding to IL-5 by optimized hybridoma technology using human IL-5 as immunogen. The DNA and amino acid sequences of the variable region were determined by molecular biological methods. The variable regions of the murine antibody were combined with the constant region of human antibody to form a mouse-human chimeric antibody molecule, or converted into a humanized antibody molecule through humanization technology, or constructed into other molecular forms such as bispecific antibodies, multispecific antibodies, single chain antibodies, single fragment antibodies, etc. according to specific purposes. These antibodies can bind to human IL-5 protein and inhibit the binding of IL-5 to its receptor IL-5Ra subunit. These antibodies show good biological activity in experiments that neutralize the proliferation of CTLL-2 and TF-1 stimulated by IL-5. The anti-IL-5 antibody exerts the function of inhibiting the proliferation and differentiation of eosinophils by IL-5, which can well relieve a series of asthma symptoms caused by IL-5, thereby playing the role of treating asthma. Especially for severe asthma, it can play an auxiliary role in conventional treatment or even replace conventional therapy. In addition, the present invention also provides the use of these antibodies, including but not limited to the use of neutralizing IL-5 to reduce the differentiation and proliferation of eosinophils, i.e. to reduce the accumulation and infiltration of eosinophils and other asthmatic symptoms, the use alone or in combination with other anti-asthmatic antibodies for the treatment of asthma, and the associated use for the diagnosis of asthmatic patients. The present invention has been completed on the basis of this.
[0213] Terms
[0214] In the present invention, “VH-CDR1” and “CDR-H1” can be used interchangeably and both refer to CDR1 of heavy chain variable region; “VH-CDR2” and “CDR-H2” can be used interchangeably and both refer to CDR2 of heavy chain variable region; “VH-CDR3” and “CDR-H3” can be used interchangeably and both refer to CDR3 of heavy chain variable region. “VL-CDR1” and “CDR-L1” can be used interchangeably and both refer to CDR1 of light chain variable region; “VL-CDR2” and “CDR-L2” can be used interchangeably and both refer to CDR2 of light chain variable region; “VL-CDR3” and “CDR-L3” can be used interchangeably and both refer to CDR3 of light chain variable region.
[0215] Antibody
[0216] As used herein, the term “antibody” or “immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 Da having the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain via a covalent disulfide bond, and different immunoglobulin isotypes have different numbers of disulfide bonds between the heavy chains. There are also regularly spaced intrachain disulfide bonds in each heavy and each light chain. Each heavy chain has a variable region (VH) at one end, followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of a light chain pairs with the first constant region of a heavy chain, and the variable region of a light chain pairs with the variable region of a heavy chain. Special amino acid residues form an interface between the variable regions of a light chain and a heavy chain.
[0217] As used herein, the term “variable” means that antibodies are different from each other in terms of sequence in certain parts of variable regions, which is responsible for the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not distributed evenly throughout the variable regions of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The conserved parts of variable regions are called framework regions (FRs). Each of the variable regions of naturally occurring heavy and light chains comprises four FR regions, which are generally in a β-sheet configuration, joined by the three CDRs forming a linking loop, and in some cases, may form a partial β-sheet structure. The CDRs in each chain are closely linked together via the FR regions, and together with the CDRs of the other chain, form the antigen binding site of an antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of an antibody to an antigen, however, they exhibit different effector functions, for example, involved in the antibody-dependent cytotoxicitiy of an antibody.
[0218] The “light chain” of a vertebrate antibody (immunoglobulin) can be classified into one of the two obviously different classes (referred to as κ and λ) depending on the amino acid sequence of its constant region. Immunoglobulins can be classified into different classes depending on the amino acid sequences of their heavy chain constant regions. There are mainly five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known for those skilled in the art.
[0219] In general, the antigen binding characteristics of an antibody can be described by three specific regions located in the heavy and light chain variable regions, called complementary determining regions (CDRs), which divide the variable region into four framework regions (FRs); the amino acid sequences of the four FRs are relatively conservative and are not directly involved in the binding reaction. These CDRs form a ring structure, and approach to each other in the steric structure by virtue of the β-sheets formed by the FRs between them, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of an antibody. By comparison of the amino acid sequences of antibodies of the same type, it can be determined which amino acids form FRs or CDRs.
[0220] The present invention includes not only an intact antibody, but also the fragments of the antibody having an immunological activity or a fusion protein formed by the antibody and another sequence. Therefore, the present invention also includes fragments, derivatives and analogs of the antibody.
[0221] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies as prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, for example, a chimeric antibody having a variable region from a monoclonal antibody from a mouse and a constant region from a human immunoglobulin (see, for example, U.S. Pat. Nos. 4,816,567 and 4,816,397, which are incorporated herein by reference in its entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, which has one or more complementary determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared by recombinant DNA techniques well known in the art.
[0222] In the present invention, an antibody may be monospecific, bispecific, trispecific, or multispecific.
[0223] In the present invention, the antibody of the present invention further includes a conservative variant thereof, which refers to a polypeptide formed by substitution of at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or analogous property, as compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably formed by carrying out the amino acid substitution according to Table 23.
TABLE-US-00005 TABLE 23 Preferred Initial residue Representative substitution substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu
[0224] Anti-IL-5 Antibody
[0225] In the present invention, the antibody (antibody of the present invention) is an anti-IL-5 antibody. The present invention provides an IL-5 targeting antibody with high specificity and high affinity, which comprises a heavy chain and a light chain. The heavy chain comprises the amino acid sequence of heavy chain variable region (VH), and the light chain comprises the amino acid sequence of light chain variable region (VL).
[0226] Preferably,
[0227] the heavy chain variable region (VH) comprises complementary determining regions or CDRs selected from the group consisting of:
[0228] VH-CDR1 shown in SEQ ID NO: 10n+3,
[0229] VH-CDR2 shown in SEQ ID NO: 10n+4, and
[0230] VH-CDR3 shown in SEQ ID NO: 10n+5;
[0231] wherein each n is independently 0, 1, 2, 3, or 4;
[0232] the light chain variable region (VL) comprises complementary determining regions or CDRs selected from the group consisting of:
[0233] VL-CDR1 shown in SEQ ID NO: 10n+8,
[0234] VL-CDR2 shown in SEQ ID NO: 10n+9, and
[0235] VL-CDR3 shown in SEQ ID NO: 10n+10;
[0236] wherein each n is independently 0, 1, 2, 3, or 4;
[0237] wherein any one of the above amino acid sequences further comprises a derivative sequence which is obtained through optional addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining the binding affinity to IL-5.
[0238] Preferably, the heavy chain variable region (VH) comprises the following three complementary determining regions or CDRs:
[0239] VH-CDR1 shown in SEQ ID NO: 10n+3,
[0240] VH-CDR2 shown in SEQ ID NO: 10n+4, and
[0241] VH-CDR3 shown in SEQ ID NO: 10n+5;
[0242] the light chain variable region (VL) comprises the following three complementary determining regions or CDRs:
[0243] VL-CDR1 shown in SEQ ID NO: 10n+8,
[0244] VL-CDR2 shown in SEQ ID NO: 10n+9, and
[0245] VL-CDR3 shown in SEQ ID NO: 10n+10;
[0246] wherein each n is independently 0, 1, 2, 3, or 4; preferably n is 0 or 1;
[0247] wherein any one of the above amino acid sequences further comprises a derivative sequence which is obtained through optional addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining the binding affinity to IL-5.
[0248] In another preferred embodiment, the sequence with at least one amino acid added, deleted, modified and/or substituted in any of the above amino acid sequences is preferably an amino acid sequence having a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% to the above amino acid sequence.
[0249] Method for determining sequence homology or identity that are well known to the ordinary skilled in the art includes, but are not limited to: Computer Molecular Biology, edited by Lesk, A. M., Oxford University Press, New York, 1988; Biocomputing; Biocomputing: Informatics and Genome Projects, edited by Smith, D. W., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, edited by Griffin, A. M. and Griffin, H. G., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M Stockton Press, New York, 1991 and Carillo, H. & Lipman, D., SIAM J. Applied Math., 48:1073(1988). The preferred method for determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program method for determining identity between two sequences includes, but are not limited to, the GCG software package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is available to the public from NCBI and other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
[0250] The antibody in the present invention can be a full-length protein (such as IgG1, IgG2 (for example IgG2a, IgG2b or IgG2c), IgG3 or IgG4), or a protein fragment containing an antigen-antibody binding domain (such as Fab, F(ab′), sdAb, ScFv fragments). The antibody in the present invention can be a wild-type protein, or a mutant protein that has achieved a certain effect through specific mutations, for example, using mutations to eliminate the effector function of the antibody.
[0251] Preferably, the antibody described herein is one or more of an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody (sdAb) and a Single-domain antibody, as well as a monoclonal antibody or a polyclonal antibody made from the above antibodies. The monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
[0252] The antibody full-length protein is a conventional antibody full-length protein in the art, which comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein and human heavy chain constant region and human light chain constant region constitute a fully human antibody full-length protein. Preferably, the antibody full-length protein is IgG1, IgG2, IgG3 or IgG4.
[0253] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies and humanized antibodies, more preferably be selected from humanized antibodies and human-animal chimeric antibodies, more preferably a fully humanized antibody.
[0254] The antibody derivative of the present invention may be a single-chain antibody, and/or an antibody fragment, for example, Fab, Fab′, (Fab′)2 or other antibody derivatives known in the art, etc., and may be any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtype antibodies.
[0255] The single-chain antibody is a conventional single-chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids. Wherein, the animal is preferably a mammal, such as mouse.
[0256] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR grafted and/or modified antibody that targets IL-5, such as human IL-5.
[0257] In above content of the present invention, the number of the added, deleted, modified and/or substituted amino acids, preferably does not exceed 40%, more preferably does not exceed 35%, is more preferably 1-33%, is more preferably 5-30%, is more preferably 10-25%, and is more preferably 15-20% of the total number of the amino acids of the initial amino acid sequence.
[0258] In the above content of the present invention, more preferably, the number of the added, deleted, modified and/or substituted amino acids, may be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.
[0259] In another preferred embodiment, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, 11, 21, 31, or 41.
[0260] In another preferred embodiment, the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 36, or 46.
[0261] In another preferred embodiment, the amino acid sequences of the heavy chain variable region and/or the light chain variable region of the antibody targeting IL-5 are shown in the following Table 24:
TABLE-US-00006 TABLE 24 Antibody number VH sequence number VL sequence number 1 1 6 2 11 16 3 21 26 4 31 36 5 41 46.
[0262] In another preferred embodiment, the antibody targeting IL-5 is 147D7H2, 102A2E11B6, 69F2F3, 154F10G8, 151G3B11, 100F4E11B3, 144D3C4A5, 90F9G10H2, 136E11F2, or 128G11B3.
[0263] In another preferred embodiment, the antibody targeting IL-5 is 147D7H2.
[0264] In another preferred embodiment, the antibody targeting IL-5 is 102A2E11B6.
[0265] Recombinant Protein
[0266] The invention also provides a recombinant protein comprising one or more of heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of an IL-5 antibody, and/or one or more of light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2) and light chain CDR3 (VL-CDR3) of an IL-5 antibody, The sequences of the heavy chain CDR1-3 are as follows:
[0267] VH-CDR1 shown in SEQ ID NO: 10n+3,
[0268] VH-CDR2 shown in SEQ ID NO: 10n+4,
[0269] VH-CDR3 shown in SEQ ID NO: 10n+5;
[0270] The sequences of the light chain CDR1-3 are as follows:
[0271] VL-CDR1 shown in SEQ ID NO: 10n+8,
[0272] VL-CDR2 shown in SEQ ID NO: 10n+9, and
[0273] VL-CDR3 shown in SEQ ID NO: 10n+10;
[0274] wherein each n is independently 0, 1, 2, 3, or 4; preferably n is 0 or 1;
[0275] wherein any one of the above amino acid sequences further comprises a derivative sequence which is obtained through optional addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining the binding affinity to IL-5.
[0276] In another preferred embodiment, the sequence with at least one amino acid added, deleted, modified and/or substituted in any of the above amino acid sequences is preferably an amino acid sequence having a homology or sequence identity of at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% to the above amino acid sequence.
[0277] In another preferred embodiment, the recombinant protein according to the present invention comprises a heavy chain variable region of an IL-5 antibody and/or a light chain variable region of an IL-5 antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, 11, 21, 31, or 41; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 36, or 46.
[0278] In another preferred embodiment, the recombinant protein according to the present invention comprises a heavy chain variable region of an IL-5 antibody and a light chain variable region of an IL-5 antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, 11, 21, 31, or 41; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 6, 16, 26, 36, or 46.
[0279] In another preferred embodiment, the amino acid sequence numbers of the recombinant protein as well as the heavy chain CDR1-3 and light chain CDR1-3 comprised therein are as shown in Table 25:
TABLE-US-00007 TABLE 25 Amino acid sequence numbers of heavy chain CDR1-3 and light chain CDR1-3 Recombinant Heavy chain protein Light chain protein protein Variable VH- VH- VH- Variable VL- VL- VL- numbers region CDR1 CDR2 CDR3 region CDR1 CDR2 CDR3 1 1 3 4 5 6 8 9 10 2 11 13 14 15 16 18 19 20 3 21 23 24 25 26 28 29 30 4 31 33 34 35 36 38 39 40 5 41 43 44 45 46 48 49 50
[0280] wherein any one of the above amino acid sequences further comprises a derivative sequence which is obtained through optional addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining the binding affinity to IL-5.
[0281] Preferably, the recombinant protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region, wherein the antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat antibody light chain constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.
[0282] The recombinant protein is a conventional protein in the art. Preferably, it is one or more of an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody (sdAb) and a single-domain antibody, as well as a monoclonal antibody or a polyclonal antibody made from the above antibodies. The monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
[0283] The antibody full-length protein is a conventional antibody full-length protein in the art, which comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein and human heavy chain constant region and human light chain constant region constitute a fully human antibody full-length protein. Preferably, the antibody full-length protein is IgG1, IgG2, IgG3 or IgG4.
[0284] The single-chain antibody is a conventional single-chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
[0285] The antigen-antibody binding domain protein fragments are conventional antigen-antibody binding domain protein fragments in the art, which comprise a light chain variable region, a light chain constant region, and an Fd segment of heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F (ab′).
[0286] The single domain antibody is a conventional single domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region.
[0287] The single-domain antibody is a conventional single-domain antibody in the art, which only comprises a heavy chain variable region.
[0288] Wherein, the preparation method of the recombinant protein is a conventional preparation method in the art. Preferably, the preparation method is: isolating and obtaining the protein from an expression transformant that recombinantly expresses the protein or obtaining the protein by artificially synthesizing a protein sequence. The method of isolating and obtaining the protein from an expression transformant that recombinantly expresses the protein is preferably as follows: cloning a nucleic acid molecule encoding the protein carrying a point mutation into a recombinant vector, and transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant, and by culturing the obtained recombinant expression transformant, the recombinant protein can be obtained by separation and purification.
[0289] Nucleic Acid
[0290] The present invention also provides a nucleic acid encoding the above-mentioned antibody (e.g., an anti-IL-5 antibody) or recombinant protein, or the heavy chain variable region or the light chain variable region of the anti-IL-5 antibody.
[0291] Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO: 2, 12, 22, 32, or 42 in the sequence listing; and/or, the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO: 7, 17, 27, 37, or 47 in the sequence listing.
[0292] Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO: 2, 12, 22, 32, or 42 in the sequence listing; and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO: 7, 17, 27, 37, or 47 in the sequence listing.
[0293] The preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it comprises the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-length sequence synthesis.
[0294] Those skilled in the art know that the base sequence encoding the amino acid sequence of the protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog. The homolog of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
[0295] Vector
[0296] The present invention also provides a recombinant expression vector comprising the nucleic acid.
[0297] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors, thus being constructed. The expression vector is one of a variety of conventional vectors in the art, as long as it can carry the above-mentioned nucleic acid molecule. The vector preferably includes: various plasmids, cosmids, phage or virus vectors and the like.
[0298] The present invention also provides a recombinant expression transformant comprising the above-mentioned recombinant expression vector.
[0299] Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably comprising: being obtained by transforming the recombinant expression vector into a host cell. The host cell is one of a variety of conventional host cells in the art, as long as the recombinant expression vector can replicate itself stably and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E.coli TG1 or E.coli BL21 cell (for expressing single-chain antibodies or Fab antibodies), or HEK293 or CHO cell (for expressing full-length IgG antibodies). The above-mentioned recombinant expression plasmid is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
[0300] Antibody Preparation
[0301] The sequence of the DNA molecule for the antibody or a fragment thereof according to the present invention can be obtained by conventional techniques, for example, methods such as PCR amplification or genomic library screening. In addition, the sequences encoding light chain and heavy chain can be fused together, to form a single-chain antibody.
[0302] Once a relevant sequence is obtained, the relevant sequence can be obtained in bulk using a recombination method. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.
[0303] In addition, a relevant sequence can be synthesized artificially, especially when the fragment is short in length. Usually, several small fragments are synthesized first, and then are linked together to obtain a fragment with a long sequence.
[0304] At present, it is possible to obtain a DNA sequence encoding the antibody of the present invention (or fragments thereof, or derivatives thereof) completely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.
[0305] The present invention further relates to a vector comprising said suitable DNA sequence and a suitable promoter or a control sequence. These vectors can be used to transform suitable host cells to enable them to express protein.
[0306] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S, HEK-293 cells.
[0307] In general, under conditions suitable for expression of the antibody according to the present invention, the host cell obtained is cultured. Then, the antibody of the present invention is purified by using conventional immunoglobulin purification steps, for example, the conventional separation and purification means well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
[0308] The monoclonal antibody obtained can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of a monoclonal antibody can be determined by, for example, the Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).
[0309] The antibody according to the present invention can be expressed in a cell or on the cell membrane, or is secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to its physical, chemical, and other properties. These methods are well known to those skilled in the art. The examples of these methods comprise, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, ultrasonic treatment, supercentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and any other liquid chromatography, and the combination thereof.
[0310] Antibody-Drug Conjugate (ADC)
[0311] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody according to the present invention.
[0312] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, and chemical conjugation is preferred. Preferably, the effector molecule is a therapeutically active drug. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small-molecule drug or a radionuclide.
[0313] The antibody according to present invention and the effector molecule may be coupled by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that results in a linkage between an effector molecule and an antibody via a covalent bond, such as glutaraldehyde, etc. The coupling agent utilizing a carboxyl group may be any one or more of cis-aconitic anhydride coupling agents (such as cis-aconitic anhydride) and acyl hydrazone coupling agents (the coupling site is acyl hydrazone).
[0314] Certain residues on an antibody (such as Cys or Lys, etc.) are used to link a variety of functional groups, including imaging agents (such as chromophores and fluorophores), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as poly(ethylene glycol)) and therapeutic agents. An antibody can be conjugated to a functional agent to form a conjugate of the antibody-functional agent. A functional agent (e.g. a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to an antibody. A functional agent can be linked to an antibody either directly or indirectly via a linker.
[0315] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC comprises a linker between a drug and an antibody. The linker can be a degradable or non-degradable linker. Typically, degradable linkers are easily degraded in an intracellular environment, for example, the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by protease (e.g. lysosomal protease or endosomal protease) in a cell, or sugar linkers, for example, glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers may include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g. linkers that are hydrolyzed at a pH of below 5.5, such as hydrazone linkers) and linkers that are degraded under reducing conditions (e.g. disulfide-bond linkers). A non-degradable linker typically releases a drug under conditions that the antibody is hydrolyzed by protease.
[0316] Prior to linkage to an antibody, a linker has a reactive group capable of reacting with certain amino acid residues, and the linkage is achieved by the reactive group. A thiol-specific reactive group is preferred, and includes, for example, a maleimide compound, a halogenated (e.g. iodo-, bromo- or chloro-substituted) amide; a halogenated (e.g. iodo-, bromo- or chloro-substituted) ester; a halogenated (e.g. iodo-, bromo- or chloro-substituted) methyl ketone, a benzyl halide (e.g. iodide, bromide or chloride); vinyl sulfone, pyridyl disulfide; a mercury derivative such as 3,6-di-(mercurymethyl)dioxane, wherein the counter ion is CH.sub.3COO.sup.−, Cl.sup.− or NO.sub.3.sup.−; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide linked to an antibody via thiosuccimide.
[0317] A drug may be any cytotoxic drug which inhibits cell growth or immunosuppression. In an embodiment, an antibody is linked to a drug via a linker, and the drug has a functional group that can form a bond with the linker. For example, a drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group that can form a bond with a linker. When a drug is directly linked to a linker, the drug has a reactive group before being linked to an antibody.
[0318] Useful drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors; typical cytotoxic drugs include, for example, auristatins, camptothecins, docamycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g. DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g. pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), and vinca alkaloids.
[0319] In the present invention, a drug-linker can be used to form an ADC in a simple step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step process. For example, a cysteine residue is reacted with the reactive moiety of a linker in a first step, and then the functional group on the linker is reacted with a drug in the subsequent step, so as to form an ADC.
[0320] In general, the functional group on a linker is selected so that it can specifically react with the suitable reactive group on a drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with the reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1,3-dipolar cycloaddition between the azide and alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reacting with hydrazides and alkoxyamines), phosphines (suitable for reacting with azides); isocyanates and isothiocyanates (suitable for reacting with amines and alcohols); and activated esters, for example, N-hydroxysuccinimide esters (suitable for reacting with amines and alcohols). These and other linkage strategies, for example, those described in “Bioconjugation Technology” (2nd Edition (Elsevier)), are well known to those skilled in the art. Those skilled in the art could understand that when a complementary pair of reactive functional groups are selected for a selective reaction between a drug moiety and a linker, each member of the complementary pair can be used for the linker, and can also be used for the drug.
[0321] The present invention further provides a method for preparing an ADC, which may further comprise: under conditions sufficient to form an antibody-drug conjugate (ADC), binding an antibody to a drug-linker compound.
[0322] In certain embodiments, the method according to the present invention comprises: under conditions sufficient to form an antibody-linker conjugate, binding an antibody to a bifunctional linker compound. In these embodiments, the method according to the present invention further comprises: under conditions sufficient to covalently link the drug moiety to the antibody via a linker, binding the antibody-linker conjugate to the drug moiety.
[0323] In some embodiments, an antibody-drug conjugate (ADC) has a formula as follows:
##STR00001##
[0324] wherein:
[0325] Ab is an antibody,
[0326] LU is a linker;
[0327] D is a drug;
[0328] And the subscript p is a value selected from 1 to 8.
[0329] Application
[0330] The present invention also provides use of the antibody, the antibody conjugate ADC, the recombinant protein, and/or immune cell of the present invention, for example for the preparation of diagnostic preparations or the preparation of drugs.
[0331] Preferably, the drug is for prevention and/or treatment of diseases associated with abnormal IL-5 expression or function.
[0332] In the present invention, the diseases associated with abnormal IL-5 expression or function are conventional diseases associated with abnormal IL-5 expression or function in the art. Preferably, the disease associated with abnormal IL-5 expression or function is asthma, atopic dermatitis, or inflammation diseases such as chronic obstructive pulmonary disease.
[0333] In the present invention, the asthma is a conventional asthma in the art, preferably eosinophilic asthma. In the invention, the specific dermatitis disease is a conventional specific dermatitis disease in the art, preferably an exogenous specific dermatitis or an endogenous specific dermatitis. In the invention, the chronic obstructive pulmonary disease is a conventional chronic obstructive pulmonary disease in the art, preferably chronic bronchitis.
[0334] Uses of the antibody, the ADC, the recombinant protein, and/or the immune cell of the present invention include (but are not limited to):
[0335] (i) diagnosis, prevention and/or treatment of occurrence and/or development of asthma, especially asthma with high IL-5 expression. The asthma includes (but is not limited to) eosinophilic asthma.
[0336] (ii) diagnosis, prevention and/or treatment of specific dermatitis including (but not limited to)exogenous specific dermatitis or endogenous specific dermatitis.
[0337] (iii) diagnosis, prevention and/or treatment of chronic obstructive pulmonary disease including (but not limited to) chronic bronchitis.
[0338] Use for Detection and Kit
[0339] The antibody or ADC thereof of the present invention can be used for detection, for example, for detecting samples, thereby providing diagnostic information.
[0340] In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term “biopsy” used in the present invention shall include all kinds of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, body fluid samples of patients with asthma, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
[0341] The samples used in the present invention include fixed or preserved cell or tissue samples.
[0342] The present invention also provides a kit comprising the antibody (or a fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction for use, buffer, and the like. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.
[0343] Pharmaceutical Composition
[0344] The invention further provides a composition. In the preferred examples, the composition is a pharmaceutical composition comprising the antibody, or an active fragment, a fusion protein or an ADC thereof, or a corresponding immune cell, and a pharmaceutically acceptable carrier. In general, these substances may be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, pH is about 6-8, though the pH value may be varied depending on the nature of the substances to be formulated and the condition to be treated.
[0345] The formulated pharmaceutical composition may be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the administration route of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in one of a variety of conventional dosage forms in the art, preferably in solid, semi-solid or liquid form, and can be an aqueous solution, a non-aqueous solution or a suspension, and more preferably tablets, capsules, granules, injection or infusion, etc.
[0346] The antibody of the present invention can also be used for cell therapy by expressing the nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
[0347] The pharmaceutical composition of the present invention is a pharmaceutical composition for prevention and/or treatment of diseases associated with abnormal IL-5 expression or function.
[0348] The pharmaceutical composition according to the present invention can be directly used for binding to an IL-5 protein molecule, and thus can be used for preventing and treating diseases such as asthma.
[0349] The pharmaceutical composition according to the present invention comprises a safe and effective amount (e.g. 0.001-99 wt %, preferably 0.01-90 wt %, preferably 0.1-80 wt %) of the monoclonal antibody according to the present invention (or a conjugate thereof) and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, glucose, water, glycerin, ethanol and a combination thereof. The pharmaceutical preparation should be matched to the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions. The dosage of active ingredient is therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day. Further, the polypeptide of the present invention can also be used in combination with the other therapeutic agents.
[0350] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, and preferably includes pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutically acceptable carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.
[0351] In the present invention, preferably, the administration amount of the pharmaceutical composition is an effective amount, and the effective amount is an amount that can alleviate or delay the progression of the disease, and the degenerative or traumatic condition. The effective amount can be determined on an individual basis and will be partly based on consideration of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above-mentioned factors such as individual basis and using no more than conventional experiments.
[0352] When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is about 10 μg/kg body weight to about 20 mg/kg body weight. Of course, the particular dose should also depend on various factors, such as the route of administration, patient healthy status, which are well within the skills of an experienced physician.
[0353] The present invention provides use of the above-mentioned pharmaceutical composition in the preparation of a medicine for preventing and/or treating diseases associated with abnormal IL-5 expression or function. Preferably, the disease associated with abnormal IL-5 expression or function is asthma, atopic dermatitis, or inflammation diseases such as chronic obstructive pulmonary disease.
[0354] Method and Composition for Detecting IL-5 Protein in Sample
[0355] The present invention also provides a method for detecting IL-5 protein in a sample (for example, detecting cells over-expressing IL-5), which comprises the following steps: contacting the above-mentioned antibody with a sample to be tested in vitro, and detecting whether the above-mentioned antibody binds to the sample to be tested, to form an antigen-antibody complex.
[0356] The meaning of overexpression is conventional in the art, which refers to the overexpression of RNA or protein of IL-5 protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and protein degradation changes), and local overexpression and increased functional activity (such as in the case of increased enzymatic hydrolysis of the substrate) due to changes in protein transport mode (increased nuclear localization).
[0357] In the present invention, the detection method for detecting whether an antigen-antibody complex is formed is a conventional detection method in the art, preferably a flow cytometry (FACS) detection.
[0358] The present invention provides a composition for detecting IL-5 protein in a sample, which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, and a combination thereof as an active ingredient. Preferably, it also comprises a compound composed of the functional fragments of the above-mentioned antibody as an active ingredient.
[0359] On the basis of conforming to common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present invention.
[0360] The Main Advantages of the Invention are:
[0361] According to the invention, a group of mouse-derived full-length antibodies combined with IL-5 are screened out using hybridoma technology. The variable regions of these antibodies can be sequenced for amino acids and DNA by conventional molecular biological methods. All variable regions contain three complementary determining regions or hypervariable regions, CDR1, CDR2 and CDR3. The murine antibody obtained from hybridoma can be combined with the constant region of human antibody to form mouse-human chimeric antibody. This group of antibodies or antigen binding fragments has a series of excellent characteristics, including high affinity and specificity for binding human IL-5 protein, effectively blocking the binding of IL-5 to its receptor, neutralizing the proliferation of CTLL-2 and TF-1 stimulated by IL-5, etc. Specific features include:
[0362] (1) having high affinity with human IL-5 protein, and its dissociation constant is usually less than 5×10.sup.−10;
[0363] (2) having strong binding ability with human and monkey (cynomolgus macaques) IL-5; some antibodies also have strong binding ability with mouse IL-5.
[0364] (3) can obviously block the binding of IL-5 to its receptor, which is verified by receptor ligand binding block assay;
[0365] (4) can obviously neutralize the activity of IL-5 and inhibit the proliferation of CTLL-2 cells in the neutralization assay of antibody on CTLL-2 proliferation stimulated by IL-5; wherein the antibody has obvious inhibitory effect at the concentration of 10.sup.−11M.
[0366] (5) can obviously neutralize the activity of IL-5 and inhibit the proliferation of TF-1 cells in the neutralization assay of antibody on TF-1 proliferation stimulated by IL-5.
[0367] The invention is further illustrated by the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The experimental methods without detailed conditions in the following examples are generally in accordance with the conditions described in the conventional conditions such as Sambrook. J et al. “Guide to Molecular Cloning Laboratory” (translated by Huang Peitang et al., Beijing: Science Press, 2002), or in accordance with the conditions recommended by the manufacturer (for example, product manuals). Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples are commercially available unless otherwise specified.
[0368] The room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30° C.
EXAMPLE 1
Expression and Purification of Recombinant Human IL-5
[0369] DNA encoding six histidines was added to the 3′end of the DNA fragment encoding Met1-Ser134 in the amino acid sequence of human IL-5 protein (NP_000870.1) by PCR, and the obtained DNA fragment encoding his-tagged recombinant human IL-5 protein was cloned into the expression vector by molecular biological methods. The plasmid was amplified by Escherichia coli and purified by alkaline lysis. The expression plasmid was transfected instantaneously and the recombinant protein was expressed by insect cell SF21. After 5-7 days of culture, centrifugal filtration was carried out to collect cell culture supernatant. The recombinant human IL-5 protein with His tag was purified by Ni-NTA affinity chromatography, and then further purified by molecular sieve column to remove impurities such as macromolecular polymers. The purified protein was stored in PBS buffer, filtered aseptically by 0.22 micron filter, then packed separately and stored at −80° C. The protein samples must undergo a series of quality control tests before use, including protein concentration, purity, molecular weight, biological activity, and so on. The biological activity of recombinant human IL-5 protein was detected by experiments. Part of the experimental results are shown in Table 1 and
TABLE-US-00008 TABLE 1 Biological activity of recombinant human IL-5 protein was examined by TF-1 cell proliferation assay Relative light unit IL-5 concentration (ng/mL) IL-5(Peprotech) IL-5(CP-20161104) 1000.000 11993.51 13032.22 11139.83 11753.8 200.000 14546.12 13873.27 13120.53 13692.45 40.000 13545.26 14987.68 13435.92 12973.34 8.000 13831.22 13646.19 13217.25 13915.33 1.600 13015.39 13784.96 13456.95 13709.27 0.320 11265.99 13890.1 12149.1 11934.63 0.064 11413.18 11320.66 11615.03 10008.61 0.013 9970.76 10315.59 10008.61 9642.746 0.003 8725.992 9802.548 8801.687 7922.78 0.001 7910.164 8410.594 8339.104 7670.462
[0370] Table 1 shows that the recombinant human IL-5 protein can stimulate the proliferation of TF-1 cells, and the biological activity of recombinant human IL-5 protein is basically consistent with that of commercial protein. Among them, IL-5 (Peprotech) refers to commercial IL-5 protein purchased from Peprotech as positive control; IL-5 (CP-20161104) is the above mentioned recombinant human IL-5 protein.
EXAMPLE 2
Preparation of Anti-Human IL-5 Antibody Using Hybridoma Technique
[0371] 2.1 Mice were Immunized with Recombinant Human IL-5 Protein
[0372] Balb/c and SJL/J mice aged 6-8 weeks (provided by Shanghai slake) were used for protein immunization. Mice were fed under SPF conditions after receiving. The primary immunization dose was 50 micrograms of recombinant human IL-5 protein per mouse. The protein was emulsified with Freund's complete adjuvant, and then injected subcutaneously into the tail with 0.25 ml. Two weeks after the primary immunization, booster immunization was preformed. Recombinant human IL-5 protein (25 microgram protein per mouse) was emulsified with Freund's incomplete adjuvant and then intraperitoneally injected with 0.25 ml. After that, the interval of each booster immunization was 3 weeks. Serum samples were collected one week after each booster immunization, and the antibody titer in serum was detected by ELISA and the antibody activity in serum was detected by receptor ligand binding block test. Mice with higher serum titer and better blocking on IL-5 binding to receptor would be preferentially selected for cell fusion and hybridoma cell preparation, and the remaining mice would continue to booster immunization for standby. Part of the experimental results are shown in
TABLE-US-00009 TABLE 2 Detection of serum antibody titer of mice immunized with immunogen of recombinant human IL-5 protein by ELISA Batch OD.sub.450nm 3386 3387 3388 3389 3390 Serum dilution (TB2) (TB2) (TB2) (TB2) (TB2) 1:100 3.67 3.82 4 3.81 3.77 1:1000 3.77 3.71 3.6 3.8 3.69 1:10k 2.96 2.79 2.09 2.6 2.12 1:100k 0.72 0.73 0.46 0.59 0.5 1:1000k 0.13 0.15 0.1 0.12 0.28 1:10000k 0.07 0.06 0.06 0.05 0.05 Blank control 0.06 0.06 0.06 0.07 0.06
[0373] Table 2 shows that the serum of mice immunized with recombinant human IL-5 protein has different degrees of binding to recombinant human IL-5 protein, showing antigen-antibody reaction. Among them, the highest dilution (i.e. dilution multiple) of serum was about 100,000. Among them, the blank control was 1% (w/w) BSA, and the batch TB2 referred to the mice serum on the seventh day after the second booster immunization. The data in the table is the value of OD450 nm.
[0374] 2.2 Preparation of Hybridoma Cells and Screening of Antibody
[0375] Usually, the titer of most mice can reach more than 1:1000 after 2-3 immunizations, which can be used to collect lymphocytes for cell fusion and hybridoma preparation. Before cell fusion, mice were intraperitoneally injected with 50-100 micrograms of recombinant human IL-5 protein per mouse, for the last immunization. After 3-5 days, the mice were sacrificed and splenocytes were collected. NH.sub.4OH was added to a final concentration of 1% to lyse the red blood cells in the spleen cell suspension. Cells were washed 2-3 times by centrifugation with DMEM basal medium, and then mixed with mouse myeloma cells SP2/0 at a ratio of 5:1. Cell fusion was performed using traditional PEG cell fusion method or high-efficiency electrofusion method. The fused cells were diluted into DMEM selective medium containing 20% fetal bovine serum and 1×HAT, and added to a 96-well cell culture plate at 1×10.sup.5/20 microliters per well, and placed in a 5% CO.sub.2, 37° C. incubator. After 10-14 days, the supernatant of cell fusion plate was screened by ELISA. The positive clones were amplified into a 24-well plate for expansion culture. After 2-3 days, the supernatant of 24-well plate was re-examined, including detection of the binding activity of antibody in the supernatant to human IL-5 protein by ELISA, detection of the blocking of antibody in the supernatant on the binding activity of human IL-5 to its receptor, and detection of neutralization of antibody in the supernatant against CTLL-2 proliferation experiment stimulated by IL-5, to test its biological activity with human IL-5.
[0376] According to the secondary screening results of 24-well plate samples, positive clones were selected for subcloning on a 96-well plate by limited dilution method. 7-10 days after subcloning, ELISA were used for preliminary screening, and 3-4 positive monoclones were selected and expanded to a 24-well plate for continue culture. After 2-3 days, the supernatant was re-examined according to the test methods between subclones, including ELISA, receptor ligand binding blocking test, neutralization of antibody in the supernatant against CTLL-2 proliferation test stimulated by IL-5. According to the results of 24-well plate samples, the best subclones were selected for expanded culture, liquid nitrogen cryopreservation, antibody production and purification.
[0377] 2.3 Preliminary Production and Purification of Hybridoma Antibody
[0378] The hybridoma cells was expanded and domesticated with a production medium (Hybridoma serum free medium, Invitrogen) in the present invention, and then inoculated into a spinner flask for cell culture. 200-500 ml of production medium was added to each 2-liter culture spinner flask, and the inoculation cell density was 0.5−1.0×10.sup.5/ml. The inoculation cell density sometimes needs to be properly adjusted according to the growth state of cells. After inoculating cells aseptically, the spinner flask was placed at a spinner flask machine in a 37° C. incubator. After 10-14 days of continuous rotating culture, cell culture solution was collected, cells was removed and the culture was filtered to clarify. The treated cell culture supernatant can be purified immediately or cryopreserved at −30° C.
[0379] Monoclonal antibodies in the supernatant of hybridoma cell culture can be purified by Protein G affinity chromatography column. According to the size of the sample, the chromatographic column was prepared with corresponding volume. For small volume purification of 200-300 ml, 1-2 ml protein G column was needed. The protein G column was first equilibrated with equilibration buffer (PBS, pH7.2), and then the culture supernatant was loaded onto the chromatography column. After loading, the chromatography column was washed with equilibration buffer to remove other unbound proteins. The antibody bound to the column was eluted with 0.1 M, pH 2.5 glycine hydrochloride buffer, and the eluted antibody (UV absorption peak) was collected. 10% volume of 1.0 M Tris-HCl buffer was added to neutralize pH and then dialysis with PBS was immediately performed. The dialyzed antibodies were filtered and sterilized with a 0.22 micron filter, and then aseptically packaged and stored. Small samples were taken for detection and analysis of protein concentration, purity, and internal toxicity. Endotoxin (LPS) is a component in the cell wall of gram-negative bacteria, which is a common impurity in the process of antibody purification, and has potential influence on many biological experiments in vitro and in vivo. Therefore, it is necessary to strictly control the contamination of endotoxin in the antibody purification process, and the concentration of endotoxin in the final product should be controlled within 1.0 EU/mg as much as possible. Part of the results are shown in Table 3.
TABLE-US-00010 TABLE 3 Antibody detection and analysis Protein concentration Antibody Endotoxin Clone number (mg/ml) purity (EU/mg) 69F2F3 0.65 100% 0.74 102A2E11B6 0.52 100% 1.74 100F4E11B3 0.15 100% 6.31 90F9G10H2 0.59 100% 1.06 136E11F2 0.63 100% 0.18 147D7H2 0.1 100% <1.202 151G3B11 0.93 100% <0.134 154F10G8 0.41 100% <0.305 128G11B3 0.74 100% <0.168
EXAMPLE 3
Detection of Lead Antibody
[0380] 3.1 Antigen Binding Assay:
[0381] The titer of antibody reacting with human IL-5 protein and the cross reaction with mouse and monkey IL-5 protein were detected and analyzed by enzyme-linked immunosorbent assay (ELISA). Streptavidin was diluted with PBS to a final concentration of 1.0 μg/ml, and then added to a 96-well enzyme-labeled plate as 100 microliters per well. The plate was incubated at 4° C. overnight. On the second day, the plate was washed twice with washing solution (PBS+0.01% Tween20). The blocking solution (PBS+0.05% Tween20+2% BSA) was added for blocking at 37° C. for 1-2 hours. Then the blocking solution was discarded. The biotin-labeled human IL-5 was diluted with sample diluent (PBS+0.05% Tween20+0.2% BSA) to 0.5 μg/ml, and added to an enzyme-labeled plate at 50-100 microliters per well, and incubated at 37° C. for 1 hour. The plate was washed with plate washing solution (PBS+0.05% Tween 20) for 2-3 times. 50-100 microliters of antibody samples to be tested were added to each well, and incubated at 37° C. for 1 hour. Then the plate was washed with plate washing solution (PBS+0.05% Tween20) for 2-3 times. The horseradish peroxidase labeled secondary antibody against human or mouse IgG was added, and reacted at 37° C. for 1 hour. The plate was washed with plate washing solution (PBS+0.05% Tween 20) for 2-3 times. 100 microliters of TMB substrate was added to each well. After incubation at room temperature for 15 minutes, 50 microliters of termination solution (1.0 N HCl) was added to each well. The value of O.D. 450 nm was read by ELISA plate reader (SpectraMax M5e, Molecular Device). Part of the experimental results are shown in
TABLE-US-00011 TABLE 4 Detection of binding activity of lead antibody to human IL-5 by ELISA OD.sub.450 nm Antibody Clone number Concentration mIgG (nM) 154F10G8 90F9G10H2 136E11F2 147D7H2 151G3B11 128G11B3 control 13.3333 3.298 3.491 2.514 2.667 3.506 3.468 3.208 3.518 4.069 4.043 3.95 4.041 0.108 0.115 2.6667 3.226 3.168 2.303 2.34 3.355 3.124 3.087 3.199 4.068 3.803 4.017 4.085 0.079 0.079 0.5333 2.763 2.69 1.84 1.972 2.896 2.68 2.759 2.764 4.03 3.75 3.958 4.089 0.08 0.077 0.1067 1.694 1.705 1.046 1.085 1.558 1.602 1.42 1.473 3.503 3.399 3.538 3.579 0.08 0.079 0.0213 0.595 0.64 0.393 0.368 0.534 0.613 0.438 0.475 2.018 2.106 2.042 2.116 0.074 0.079 0.0043 0.205 0.193 0.14 0.137 0.187 0.175 0.158 0.159 0.625 0.609 0.666 0.711 0.078 0.072 0.0009 0.093 0.092 0.09 0.087 0.092 0.095 0.088 0.09 0.185 0.186 0.204 0.2 0.075 0.075 0.0002 0.079 0.07 0.081 0.076 0.075 0.07 0.073 0.074 0.103 0.094 0.106 0.103 0.076 0.075
TABLE-US-00012 TABLE 5 Detection of binding activity of lead antibody to monkey IL-5 by ELISA OD.sub.450 nm Antibody Clone number Concentration (nM) 154F10G8 90F9G10H2 136E11F2 147D7H2 151G3B11 128G11B3 mIgG control 13.3333 2.914 2.727 2.408 2.447 3.526 3.321 2.413 2.304 3.918 3.939 4.217 4.287 0.092 0.089 2.6667 2.565 2.547 2.184 2.318 3.126 3.101 2.118 2.023 3.923 3.882 4.229 4.218 0.073 0.072 0.5333 2.083 2.066 2.007 2.058 2.777 2.632 1.445 1.344 3.773 3.781 4.051 4.109 0.057 0.056 0.1067 1.181 1.053 1.127 1.15 1.425 1.38 0.644 0.516 3.047 3.27 3.453 3.442 0.056 0.056 0.0213 0.581 0.555 0.368 0.35 0.765 0.528 0.208 0.203 1.719 1.746 1.883 1.864 0.057 0.056 0.0043 0.202 0.165 0.15 0.139 0.236 0.192 0.095 0.088 0.574 0.598 0.71 0.645 0.055 0.055 0.0009 0.087 0.081 0.076 0.073 0.093 0.091 0.04 0.069 0.163 0.174 0.228 0.22 0.054 0.053 0.0002 0.079 0.068 0.063 0.061 0.067 0.079 0.075 0.074 0.11 0.125 0.114 0.109 0.055 0.056
TABLE-US-00013 TABLE 6 Detection of binding activity of lead antibody to mouse IL-5 by ELISA OD.sub.450 nm Antibody Clone number Concentration (nM) 69F2F3 102A2E11B6 100F4E11B3 154F10G8 mIgG control 13.3333 2.821 2.809 1.809 1.988 0.587 0.551 4.006 4.039 0.052 0.054 2.6667 2.452 2.588 1.45 1.512 0.33 0.27 3.973 3.97 0.059 0.06 0.5333 1.824 1.71 1.025 1.193 0.189 0.176 3.706 3.632 0.053 0.052 0.1067 1.048 0.789 0.461 0.479 0.107 0.102 2.61 2.589 0.05 0.05 0.0213 0.265 0.256 0.139 0.138 0.065 0.063 1.114 1.134 0.05 0.332 0.0043 0.244 0.086 0.068 0.067 0.05 0.05 0.412 0.392 0.046 0.048 0.0009 0.059 0.058 0.051 0.052 0.05 0.049 0.205 0.216 0.049 0.049 0.0002 0.049 0.049 0.046 0.047 0.048 0.048 0.17 0.152 0.046 0.048
[0382] Tables 4-6 show that the lead antibodies bind to recombinant human IL-5 protein and recombinant monkey IL-5 at ELISA level. Some lead antibodies bind to mouse IL-5 at ELISA level. Wherein, the mIgG control is mouse IgG, and the data in the table are OD.sub.450 nm values.
[0383] 3.2 Biological Function Analysis
[0384] 3.2.1 Construction of Stably Expressed Cell Lines:
[0385] The full-length sequences of human IL-5Ra and CSF2RB chains were cloned into pIRES expression vector and packaged into lentivirus (Shanghai Jima). CHOK1 cells were infected with lentivirus containing genes expressing human IL-5Ra and CSF2RB chains, and then cultured in culture medium containing screening antibiotics. After 2 weeks, the infected cells were subcloned into a 96-well culture plate by limited dilution method. After the clones grew up, cells of the monoclonal well were expanded into 6-well plates or culture flasks. The receptor expression level of the expanded clones and their binding ability to ligand IL-5 protein were detected by flow cytometry with specific antibodies against each receptor. Monoclonal cell lines with better growth, higher expression level and strong binding were selected for further expanding culture and cryopreserved in liquid nitrogen. CTLL-2 cells were infected with virus particles expressing human IL-5Ra and CSF2RB chains, and monoclonal cell lines with better growth, higher expression level and strong binding were selected with the same method for expanding culture and preserved in liquid nitrogen. Some results are shown in
TABLE-US-00014 TABLE 7 Expression level of IL-5Ra protein in CHOK1 cell line overexpressing full-length human IL-5Ra detected by flow cytometry IL-5Ra antibody IgG subtype control Mean Mean fluorescence Positive fluorescence Positive Sample intensity cells (%) intensity cells (%) Transfected 5,651 99.52 28 1.58 mother cells
[0386] The results in Table 7 show that CHOK1 cells express IL-5Ra receptor, and the expression level of receptor on the cell surface is high, which can be used for follow-up test.
[0387] 3.2.2 Receptor Ligand Binding Block Assay Using Flow Cytometry:
[0388] CHOK1 cell line overexpressing full-length human IL-5Ra/CSF2RB was expanded to a confluence of 75-90% in a T-175 cell culture flask, and the culture medium was discarded. The cells were washed with PBS for 1-2 times, and then digested with recombinant enzyme cell dissociation solution (TrypLE: Life technology). The cells were collected and washed with PBS buffer solution for 1-2 times. Then the cells were counted and diluted to 1−2×10.sup.6 cells per milliliter with blocking solution (PBS, 2% fetal bovine serum), incubated on ice for 20-30 minutes, and then washed twice with a blocking solution (PBS, 2% fetal bovine serum). The collected cells were suspended with a blocking solution (PBS, 2% fetal bovine serum) to 1×10.sup.6 cells/ml, and added to a 96-well FACS reaction plate (1×10.sup.5 cells per well) as 100 microliters per well. The gradient diluted antibody sample or culture supernatant was mixed with biotin labeled IL-5 (the final concentration is 30 ng/ml for hIL-5Ra1/hIL-4Ra; 20 ng/ml for HIL-5Ra2) and then was added to the cells as 100 microliters per well and incubated on ice for 1-2 hours. The plate was washed twice with blocking solution (PBS, 2% fetal calf serum) by centrifugation, added with 100 microliters of streptavidin labeled with fluorescence (Alexa 488) per well, and incubated on ice for 0.5-1.0 hours. The plate was washed 2-3 times by centrifugation with blocking solution (PBS, 2% fetal calf serum), added with 100 microliters of PBS per well to suspend cells, and detected using FACS (FACS Verse, BD) and the results were analyzed. Some results are shown in
TABLE-US-00015 TABLE 8 Blocking of lead antibody on the binding of IL-5 to cell surface receptor IL- 5Ra detected by FACS Fluorescence intensity Clone number Antibody concentration mIgG (nM) 90F9G10H2 136E11F2 147D7H2 151G3B11 128G11B3 154F10G8 control 33.3333 25 31 31 30 29 34 80 6.6667 25 36 36 34 32 43 85 1.3333 26 47 55 46 39 66 83 0.2667 38 69 74 69 64 86 84 0.0533 66 83 90 84 86 92 83 0.0107 79 84 88 87 87 91 83 0.0021 85 87 93 86 89 91 81 0.0004 92 93 96 93 92 90 86
[0389] Table 8 shows that the binding of IL-5 antibody to human IL-5 can block the binding of human IL-5 to the cell surface receptor IL-5Ra, wherein the mIgG control is mouse IgG, and the data in the table is average fluorescence intensity.
[0390] 3.2.3 Antibody Neutralization Against CTLL-2 Proliferation Experiment Stimulated by IL-5.
[0391] CTLL-2 cells (ATCC) overexpressing full-length human IL-5Ra/CSF2RB were cultured in 1640 medium containing 10% fetal bovine serum (Gibco) (RPMI1640+10% FBS+2 mM L-Glutamine+1 mM Sodium Pyruvate+10 ng/ml IL2+1.5 ug/ml puromycin+200 ug/ml Hygromycin). When the cells were expanded to 75-90% confluence in a T-175 cell culture flask, the culture medium was discarded by centrifugation and the cells were collected. The collected cells were re-suspended in 1640 medium (Gibco) (RPMI1640+2% FBS+2 mM L-Glutamine+1 mM Sodium Pyruvate) containing 2% fetal bovine serum. After counting, the cells were diluted to 2×10.sup.5 cells per milliliter and added to a 96-well cell culture plate as 50 microliters per well (1×10.sup.4 cells per well). The culture plate was placed in a 37° C., 5% CO.sub.2 incubator. The gradient diluted antibody or culture supernatant was mixed with IL-5 (final concentration was 5 ng/ml), then added to the cell culture plate and cultured in a 37° C., 5% CO.sub.2 incubator. After 48 hours, 50 microliters of Cell Titer-Glo reagent (Promega) was added to each well of the cell culture plate, and the relative luminescence intensity was detected by microplate reader. Part of the results are shown in
TABLE-US-00016 TABLE 9 Neutralization of lead antibody against CTLL-2 cell proliferation stimulated by IL-5 RLU Clone number Ab conc. (nM) 154F10G8 90F9G10H2 136E11F2 147D7H2 66.66666 11496.64 10928.79 7665.881 9552.866 6216.864 6365.508 10743.41 11275.3 13.33333 16170.42 14728.97 10369.69 12012.06 7580.901 9080.469 12936.93 13482.01 2.666667 19725.99 17122.65 13418.57 16602.86 10623.75 10247.77 13653.45 14449.09 0.5333334 21525.62 20158.43 16240.31 16170.42 13325.6 13198.81 16726.13 15191.99 0.1066667 36119.19 30003.95 18843.65 21468.84 15664.57 15074.36 26537.62 26309.04 0.02133333 56867.3 52272.14 44130.14 41946.13 45411.09 41764.91 49039.88 45162.76 0.004266667 64244.89 64349.72 47790.54 46563.13 56192.23 59051.46 50591.61 55932.54 0.000853333 64755.95 54526.04 55535.05 56011.16 65338.27 54745.13 56402.89 57290.85 0.000170667 57924.36 56343.13 54578.45 51429.11 55383.42 61482.25 56772.14 61031.7 3.41333E-05 58714.97 61274.63 58688.76 58435.42 61753.3 61871.34 58943.68 59783.28 RLU Clone number Ab conc. (nM) 151G3B11 128G11B3 mInG control 66.66666 6497.037 5235.433 6647.966 6312.923 43168.49 34361.45 13.33333 7411.369 6598.14 8168.888 8909.51 45781.71 46324.49 2.666667 8721.326 8840.014 11462.01 10060.12 51170.11 46188.8 0.5333334 12506.14 11719.28 15694.14 14058.6 56199.58 50465.38 0.1066667 13956.76 12211.61 16862.38 16064.45 58199.99 53498.81 0.02133333 39325.09 38823.97 36475.65 35355.89 59780.18 55928.2 0.004266667 53589.56 50916.89 50975.09 52002.27 57862.94 48241.73 0.000853333 56504 56293 51455.61 55295.39 58589.57 53792.09 0.000170667 58029.35 54684.12 47906.8 48845.8 57433.97 51016.91 3.41333E-05 60640.47 48587.11 46429.96 48127.22 58602.7 55004.59
[0392] Table 9 shows that by binding to human IL-5, the lead antibody can neutralize proliferation of CTLL-2 cells stimulated by IL-5. The data in Table 9 are the relative light unit values of ATP measured in the cell culture plate, wherein the mIgG control is mouse IgG.
[0393] 3.2.4 Antibody Neutralization Against TF-1 Proliferation Experiment Stimulated by IL-5.
[0394] TF-1 cells (ATCC) expressing endogenous human IL-5Ra/CSF2RB were cultured in 1640 medium (Gibco) containing 10% fetal bovine serum (RPMI1640+10% FBS). When the cells were expanded to 75-90% confluence in a T-175 cell culture flask, the culture medium was discarded by centrifugation and the cells were collected. The cells were washed with PBS for 3 times. The collected cells were re-suspended in 1640 medium (Gibco) (RPMI1640+2% FBS) containing 2% fetal bovine serum. After counting, the cells were diluted to 2×10.sup.5 cells per milliliter and added to a 96-well cell culture plate as 50 microliters per well (1×10.sup.4 cells per well). The culture plate was placed in a 37° C., 5% CO.sub.2 incubator. The gradient diluted antibody or culture supernatant was mixed with IL-5 (final concentration was 5 ng/ml), then added to the cell culture plate and cultured in a 37° C., 5% CO.sub.2 incubator. After 48 hours, 50 microliters of Cell Titer-Glo reagent (Promega) was added to each well of the cell culture plate, and the relative luminescence intensity was detected by microplate reader. Part of the results are shown in
TABLE-US-00017 TABLE 10 Neutralization of lead antibody against TF-1 cell proliferation stimulated by IL-5 RLU Clone number Ab conc. (nM) 154F10G8 90F9G10H2 136E11F2 147D7H2 13.33333 33824.62 35341.05 34596.45 37960.76 35048.18 37362 35483.73 37477 2.66667 34492.03 41969.77 34936.97 41043.56 36121 34129.93 39860.75 38103.59 0.53333 40625.86 42342.07 38087.88 39722.36 37034.7 39771.28 36173.89 39406.7 0.10667 42142.3 51540.55 43558.84 44344.3 42626.05 40412.24 42317.14 43633.87 0.02133 54782.26 57451.91 52398.64 54764.1 53690.55 43603.39 72166.16 55893.13 0.00427 76094.04 79662.66 73637.78 75903.35 61918.46 60872.93 90250.83 86341.48 0.00085 99335.41 103299 93074.46 97178.8 95507.44 89957.01 104857.5 105370.6 0.00017 114127.4 108275.1 101723.6 102708.8 105544.6 101853.4 116058.8 111718.1 0.00003 110018.5 114540.6 107112.8 109900.5 108035.7 106121.9 111100.6 112748.8 0.00001 108275.1 112869.8 109110.5 114817.5 108172.1 112204.2 110900.9 111550.1 RLU Clone number Ab conc. (nM) 151G3B11 128G11B3 mIgG control 13.33333 35338.44 36564.37 39002.46 33243.16 100840.8 101667.4 2.66667 37894.72 34280.51 36513.43 34810.16 101199.6 105936.7 0.53333 35138.66 37749.43 35259.82 31430.89 103434.2 106463.5 0.10667 40401.06 40437.38 44184.92 41218.98 108035 114497.9 0.02133 49854.31 47207.22 55708.07 54854.17 108062.2 130830.1 0.00427 51102.94 54685.36 68502.98 65087.38 116092.1 126837.9 0.00085 72729.17 70704.13 89441.77 87552.28 114520.6 117368.3 0.00017 86473.16 91331.45 93161.7 96313.87 112204.3 120642.9 0.00003 95354.31 98251.13 98153.4 101918.7 112208.9 116055.7 0.00001 103599.8 105465.9 94288.13 101260.1 113362.5 117922.4
[0395] Table 10 shows that the lead antibody can neutralize proliferation of TF-1 cells stimulated by IL-5 by binding to human IL-5. The data in Table 10 are the relative light unit values of ATP measured in the cell culture plate, wherein the mIgG control is mouse IgG.
EXAMPLE 4
Determination of Amino Acid Sequences of Light and Heavy Chain Variable Regions
[0396] Total RNA extraction: The hybridoma cell lines corresponding to the selected lead antibodies were resuscitated and cultured, and 1−5×10.sup.7 cells were collected by centrifugation. 1 ml Trizol was added to the cell precipitation, blew and sucked repeatedly to lyse the cells. The cell lysate was transfered to a 1.5 ml centrifuge tube, and placed at room temperature for 5 minutes. 0.2 ml of chloroform was added, shaked for 15 seconds, placed for 2 minutes, and then centrifuged at 12000 g at 4° C. for 10 minutes. The supernatant aqueous liquid was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, mixed well, and placed at room temperature for 10 minutes, and then centrifuged at 12000 g at 4° C. for 15 minutes. The precipitate was collected, washed gently with 1 ml of 75% ethanol. After centrifugation, the supernatant was carefully aspirated, and the precipitate was dried. A proper amount of DEPC-treated H.sub.2O was used to dissolve (55° C. water bath to promote dissolution for 10 minutes) the RNA precipitate. The RNA concentration was determined by light absorption method.
[0397] Reverse transcription and PCR: 1 μg of total RNA was taken, and a 20 μl reaction system was prepared by adding dNTP, buffer and reverse transcriptase, etc, and reacted at 42° C. for 60 minutes, and then reacted at 70° C. for 10 minutes to inactivate the reverse transcriptase. 1 microliter of reverse transcriptate product (cDNA) was taken, and a 50 microliter PCR system was prepared by adding 25 pmol of primer, 1 microliter of DNA polymerase, matching buffer system and 250 μmol dNTPs. PCR program was set, comprising pre-denaturation at 95° C. for 3 minutes, denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, extension at 72° C. for 35 seconds, and further extension at 72° C. for 5 min after 35 cycles.
[0398] Cloning and sequencing: 5 μl of PCR product was taken for agarose gel electrophoresis detection. Column recovery kit was used to purify the positive samples. Ligation reaction were preformed at 16° C. for half an hour in a 10 μl reaction system containing sample 50 ng, T vector 50 ng, ligase 0.54 and buffer 1 μl. 5 μl of the ligation product was taken and added to 100 μl of competent cells. Ice bathed for 5 minutes, then heat shock in a 42° C. water bath was performed for 1 minute, and cells were put back on ice for 1 minute, and added with 650 μl antibiotic-free SOC medium. The cells were resuscitated on a shaker at 37° C. at 200 RPM for 30 minutes. Then 200 μl of the culture was taken out, spreaded on LB solid medium containing antibiotics and incubated overnight at 37° C. in an incubator. On the next day, primers M13F and M13R on the T vector were used to configure a 30 μl PCR system to perform a colony PCR. A pipette tip was used to dip the colony into the PCR reaction system and pipette, and 0.5 μl was aspirated onto another LB solid petri dish containing antibiotics to preserve the strain. After the PCR reaction was over, 5 μl of the reaction solution was take out for agar glycogel electrophoresis detection, and the positive samples were sequenced.
[0399] The sequencing results are shown in the sequence information of the present invention in the appendix.
EXAMPLE 5
Antibody Affinity Test
[0400] The human antigen IL-5 was immobilized on the surface of CMS chip to 150-200 RU and the process was as follows: a freshly prepared mixture of 50 mM NHS and 200 mM EDC (1:1) was used to activate for 200 sec, the antigen IL-5 was diluted to 1 ug/ml with 10 mM NaOAc (pH 5.0), and captured on the chip at a flow rate of 10 ul/min for about 1 min. The remaining activated sites were blocked with 1M ethanolamine. Then the antibody to be tested was diluted to different concentrations (0, 12.5, 25 nM), and then flowed through the chip surface at a flow rate of 30 ul/min, respectively. The binding time was 180 s and the dissociation time was 1200 s. At the end of each cycle, the chip surface was regenerated with 10 mM Glycine at pH 1.5. The kinetic rate constant needed to be subtracted the blank control, and the data was fitted with the global fit analysis method 1:1 combination model. The dissociation equilibrium rate constant (KD) was calculated according to the following formula: KD=kd/ka. Part of the results are shown in Table 11 and
TABLE-US-00018 TABLE 11 Detection results of affinity between antibodies and human IL-5 Antibody Ka (1/Ms) Kd (1/s) KD (M) 69F2F3 3.678E+05 8.143E−06 2.214E−11 102A2E11B6 3.691E+05 2.324E−05 6.297E−11 100F4E11B3 1.259E+05 1.237E−04 9.824E−10 144D3C4A5 4.262E+05 1.429E−05 3.35E−11 90F9G10H2 1.601E+05 <1 E−05 <1/1.601 E−10 136E11F2 3.655E+05 <1 E−05 <1/3.655 E−10 147D7H2 1.985E+05 <1 E−05 <1/1.985 E−10 151G3B11 4.864E+05 9.037E−06 1.858E−11 128G11B3 2.869E+05 <1 E−05 <1/2.869 E−10 154F10G8 4.221E+05 6.650E−05 1.576E-10
[0401] Table 11 shows that the lead antibody has strong affinity with human IL-5.
EXAMPLE 6
Construction of Mouse-Human Chimeric Antibody, Production and Purification of the Antibody
[0402] Plasmid construction and preparation: The hybridoma antibody heavy chain variable region sequence was cloned into the pCP expression vector containing the signal peptide and human heavy chain antibody IgG4 constant region, and the light chain variable region was recombined to the pCP vector containing the signal peptide and human antibody light chain kappa constant region, and the plasmid was verified by sequencing. Plasmids were extracted using alkaline lysis kit to increase the purity, filtered through a 0.22 μm filter membrane for transfection.
[0403] Cell transfection: Freestyle 293F cells were used, and the medium was Freestyle 293 expression medium, added with 10% F68 to a final concentration of 0.1% when used. During transfection, the cell density was cultured to 1−1.5×10.sup.6 cells per milliliter; and the shaker was set to 37° C., 130RPM, with a CO.sub.2 concentration of 8%. 5 ml of medium was taken and mixed well with PEI (200 μg/ml). 5 ml of medium was taken and mixed well with a certain amount of plasmids (the amount of plasmids was 100 μg/ml). After 5 minutes, the two solutions were combined and mixed well, let stand for 15 minutes. The mixture was added slowly into the cells, being shaken while added, to avoid excessive concentration of PEI. And the mixed solution was cultured in a shaker. On the next day, it was added with peptone (sigma) to a final concentration of 0.5%. On the 5-7 day, the antibody titer of the culture medium was tested. On the 6-7 day, it was centrifuged (3500 RPM, 30 min) and filtered to collect the supernatant for purification.
[0404] Antibody purification: for continuously used endotoxin-free chromatography columns and Protein A fillers, 0.1M NaOH was used for treatment for 30 minutes, or 5 column volumes of 0.5M NaOH was used for washing. For long-term unused column materials and chromatography columns, at least 1M NaOH was used for soaking for 1 hour, and non-endotoxic water was used for rinsing to neutrality, and the column material was washed with 10 column volumes of 1% Triton X100. 5 column volumes of PBS were used for equilibrate, and the filtered cell supernatant was loaded on the column, and the flow-through was collectd if necessary. After the samples were loaded, the column was washed with 5 column volumes of PBS. Elution was carried out with 5 column volumes of 0.1M Glycine-HCl with pH3.0, and the eluate was collected, and neutralized with 1/10 volume of 1M Tris-HCl (1.5M NaCl) with pH8.5. After the antibodies were harvested, they were dialyzed overnight in 1× PBS to avoid endotoxin contamination. After dialysis, spectrophotometry or a kit was used to determine the concentration, and HPLC-SEC was used to determine the purity of the antibody, and an endotoxin detection kit was used to detect the content of antibody endotoxin. Part of the results are shown in Table 12.
TABLE-US-00019 TABLE 12 Detection and analysis of chimeric antibody Protein concentration Clone number (mg/ml) Antibody purity Endotoxin (EU/mg) 69F2F3 0.573 >90% 1.71 102A2E11B6 1.193 >90% 1.49 100F4E11B3 1.317 >90% 1.03 144D3C4A5 0.67 >90% 0.22 90F9G10H2 1.101 >90% 0.75 136E11F2 1.156 >90% 0.74 147D7H2 1.073 >90% 0.63 151G3B11 0.721 >90% 0.96 128G11B3 1.149 >90% 0.53 154F10G8 0.51 >90% 1.96
EXAMPLE 7
Assay of Mouse-Human Chimeric Antibody
[0405] 7.1 Antigen Binding Assay
[0406] The titer of chimeric antibody reacting with human IL-5 protein and the cross reaction with mouse and monkey IL-5 protein were detected and analyzed by enzyme-linked immunosorbent assay (ELISA). The experimental process was the same as that of Example 4.1, and see 3.1 for details. Part of the experimental results are shown in
TABLE-US-00020 TABLE 13 Detection of binding activity of chimeric antibody to human IL-5 by ELISA OD.sub.450 nm Clone number Ab conc. (nM) 154F10G8 90F9G10H2 136E11F2 147D7H2 151G3B11 128G11B3 hIgG control 13.3333 3.701 3.707 2.821 2.903 3.795 3.662 3.44 3.718 3.704 3.525 3.621 3.655 0.172 0.07 2.6667 2.959 3.011 2.365 2.375 3.353 3.383 3.221 3.347 3.468 3.503 3.359 3.579 0.09 0.088 0.5333 1.933 1.999 1.902 1.812 2.673 2.714 2.741 2.909 3.032 2.928 3.11 3.201 0.079 0.078 0.1067 0.716 0.77 1.056 1.002 1.454 1.473 1.255 1.3 1.872 1.919 1.703 1.845 0.078 0.078 0.0213 0.224 0.221 0.395 0.402 0.467 0.507 0.406 0.385 0.622 0.678 0.622 0.603 0.079 0.076 0.0043 0.108 0.106 0.168 0.173 0.17 0.171 0.172 0.156 0.194 0.244 0.204 0.211 0.074 0.077 0.0009 0.081 0.087 0.128 0.134 0.108 0.115 0.102 0.104 0.127 0.114 0.118 0112 0.08 0.078 0.0002 0.083 0.098 0.123 0.135 0.108 0.085 0.103 0.101 0.098 0.127 0.096 0.106 0.082 0.08
TABLE-US-00021 TABLE 14 Detection of binding activity of lead antibody to monkey IL-5 by ELISA OD.sub.450 nm Clone number Ab conc. (nM) 154F10G8 90F9G10H2 136E11F2 147D7H2 151G3B11 128G11B3 hIgG control 13.3333 2.301 1.965 1.873 1.645 2.543 2.297 1.576 1.735 2.152 2.208 2.522 2.839 0.092 0.092 2.6667 1.606 1.608 1.547 1.711 2.061 1.888 1.435 1.344 1.876 1.677 2.398 2.221 0.082 0.075 0.5333 0.988 0.997 1.661 1.364 1.678 1.623 1.031 0.983 1.533 1.815 1.769 1.9 0.065 0.063 0.1067 0.399 0.409 1.013 0.677 0.862 0.781 0.405 0.448 0.947 0.301 1.068 1.036 0.068 0.073 0.0213 0.154 0.157 0.415 0.233 0.309 0.308 0.168 0.173 0.291 0.3 0.404 0.386 0.072 0.062 0.0043 0.096 0.092 0.163 0.12 0.203 0.173 0.092 0.092 0.146 0.16 0.167 0.16 0.066 0.064 0.0009 0.071 0.067 0.096 0.081 0.089 0.098 0.071 0.082 0.095 0.128 0.095 0.09 0.063 0.064 0.0002 0.073 0.072 0.073 0.074 0.092 0.093 0.093 0.093 0.098 0.116 0.099 0.097 0.067 0.064
TABLE-US-00022 TABLE 15 Detection of binding activity of lead antibody to mouse IL-5 by ELISA OD.sub.450 nm Clone number Ab conc. (nM) 69F2F3 102A2E11B6 100F4E11B3 154F10G8 hIgG control 13.3333 2.659 2.631 1.77 1.866 0.611 0.566 4.276 4.173 0.05 0.049 2.6667 2.074 2.124 1.304 1.336 0.294 0.368 4.152 4.196 0.06 0.058 0.5333 1.161 1.129 0.99 0.77 0.156 0.184 3.059 3.157 0.047 0.047 0.1067 0.535 0.601 0.41 0.452 0.091 0.094 1.346 1.318 0.047 0.052 0.0213 0.178 0.193 0.147 0.158 0.062 0.057 0.419 0.444 0.046 0.046 0.0043 0.083 0.076 0.069 0.069 0.051 0.048 0.166 0.158 0.044 0.045 0.0009 0.06 0.054 0.052 0.053 0.049 0.048 0.117 0.109 0.045 0.047 0.0002 0.079 0.048 0.046 0.046 0.047 0.049 0.097 0.106 0.045 0.046
[0407] Tables 13-15 show that the chimeric antibodies bind to recombinant human IL-5 protein and recombinant monkey IL-5 at ELISA level. Some chimeric antibodies bind to mouse IL-5 at ELISA level. Wherein, the hIgG control is human IgG, and the data in the table is OD.sub.450 nm values.
[0408] 7.2 Biological Function Analysis
[0409] 7.2.1 Receptor Ligand Binding Blocking Assay
[0410] In this experiment, flow cytometry was used to detect the blocking of IL-5 chimeric antibody on the binding of biotinylated IL-5 to the receptor IL-5Ra heterodimer on the surface of CHOK1 cell line overexpressing full-length human IL-5Ra/CSF2RB. The detailed steps were the same as those in 3.2.2. Part of the results are shown in
TABLE-US-00023 TABLE 16 Blocking of lead antibody on the binding of IL-5 to cell surface receptor IL- 5Ra detected by FACS Fluorescence intensity Clone number Antibody hIgG concentration (nM) 90F9G10H2 136E11F2 147D7H2 151G3B11 128G11B3 154F10G8 control 33.3333 26 28 31 28 28 35 84 6.6667 28 32 36 33 31 39 83 1.3333 33 44 52 41 36 46 84 0.2667 56 66 76 68 51 67 88 0.0533 77 83 94 82 73 87 86 0.0107 81 85 86 85 81 88 85 0.0021 84 88 88 90 88 88 83 0.0004 94 88 94 95 93 91 88
[0411] Table 16 shows that the binding of human-mouse chimeric antibody to human IL-5 can block the binding of human IL-5 to the cell surface receptor IL-5Ra, wherein the hIgG control is human IgG, and the data in the table is average fluorescence intensity.
[0412] 7.2.2 Neutralization Experiment of Chimeric Antibody Against CTLL-2 Proliferation Stimulated by IL-5
[0413] The neutralization experiment of chimeric antibody against CTLL-2 proliferation stimulated by IL-5 was that IL-5 chimeric antibody blocked the binding of IL-5 to its receptor and neutralized CTLL-2 cell line overexpres sing full-length human IL-5Ra/CSF2RB stimulated by IL-5. See 4.2. 3 for detailed process. Part of the results are shown in
TABLE-US-00024 TABLE 17 Neutralization of chimeric antibody against CTLL-2 cell proliferation stimulated by IL-5 RLU Clone number Ab conc. (nM) 154F10G8 90F9G10H2 136E11F2 147D7H2 66.66666 12178.05 12440.13 5687.862 6461.691 3685.525 3956.583 5226.642 5239.829 13.33333 16166.05 16969.77 9500.172 10072.89 5788.416 5154.488 7305.869 6686.057 2.666667 27981.56 25408.79 14694.01 14221.84 8088.043 7445.372 8620.222 8163.056 0.5333334 44890.18 38172.15 21278.11 20381.87 11952.82 12083.97 9569.721 10444.49 0.1066667 54779.38 51966.38 30249.28 29742.14 16044.93 14396.72 12717.13 13187.49 0.02133333 62087.08 59317.76 57962.86 63952.38 23000.65 22987.53 33751.18 36441.43 0.004266667 64354.09 61348.89 64704.35 68879.53 44348.71 45756.46 41355.96 48226.65 0.000853333 59837.55 56731.89 59982.68 67200.71 50233.31 52646.61 50780.63 50723.48 0.000170667 59946.75 54888.58 58417.54 57975.97 45953.2 52336.2 50661.94 51743.31 3.41333E-05 60728.63 50197.32 55964.89 61915.06 58544.32 66868.45 56055.62 52165.31 RLU Clone number Ab conc. (nM) 151G3B11 128G11B3 hIgG control 66.66666 3689.886 4029.338 8371.677 9777.978 43168.49 34361.45 13.33333 5748.64 5814.767 12158.55 14164.4 45781.71 46324.49 2.666667 6581.84 7939.647 18771.25 20944.62 51170.11 46188.8 0.5333334 8367.268 8248.239 25855.65 25595.55 56199.58 50465.38 0.1066667 10880.09 10289.36 37590.99 35519.01 58199.99 53498.81 0.02133333 41651.19 43934.77 46328.57 48867.84 59780.18 55928.2 0.004266667 51217.55 57980.14 51759.8 58174.11 57862.94 48241.73 0.000853333 54329.93 62961.71 50075.77 60554.68 58589.57 53792.09 0.000170667 47893.57 63164.5 45587.95 59602.46 57433.97 51016.91 3.41333E-05 46817.91 55030.88 48876.66 64517.9 58602.7 55004.59
[0414] Table 17 shows that by binding to human IL-5, the chimeric antibody can neutralize the proliferation of CTLL-2 cells induced by IL-5 stimulation. The data in Table 17 are the relative light unit values of ATP measured in cell culture plate, wherein the hIgG control is human IgG.
[0415] 7.2.3 Neutralization Experiment of Chimeric Antibody Against TF1 Proliferation Stimulated by IL-5
[0416] The neutralization experiment of chimeric antibody against TF1 proliferation stimulated by IL-5 was that IL-5 chimeric antibody bound to IL-5, blocked the binding of IL-5 to its receptor on the surface of TF-1 cells, and then neutralized IL-5-induced TF-1 cell proliferation. See 3.2.4 for detailed process. Part of the results are shown in
TABLE-US-00025 TABLE 18 Neutralization of chimeric antibody against TF-1 cell proliferation stimulated by IL-5 RLU Clone number Ab conc. (nM)) 154F10G8 90F9G10H2 136E11F2 147D7H2 13.33333 38491.96 39962.99 40225.86 33989.01 37675.66 36448.29 35692.59 39025.3 2.66667 45161.54 42628.1 44326.18 35820.97 35470.95 38489.36 34766.34 41236.5 0.53333 60398.51 56811.74 50222.09 39957.66 35120.92 37180.17 36441.77 40124.09 0.10667 81392.48 82781.78 62023.01 50958.51 40430.42 39562.17 38975.35 38662.06 0.02133 99081.16 101206 103044.4 70696.41 45535.36 45430.81 45259.36 43919.92 0.00427 109287.6 114295.4 128887.3 90493.41 50235.73 51954.05 56687.71 54812.5 0.00085 108878.9 108302.3 129887.4 107172 67978.02 67109.77 89215.6 82945.22 0.00017 110586.1 111330.7 129973.8 113108.8 81724.55 80679.02 96425.86 88816.04 0.00003 111308 108842.6 132674 110263.1 92175.36 95857.47 99962.88 95000.16 0.00001 110413.5 113028.7 125096.1 111663.3 100485.1 107631.1 99499.76 100598.5 RLU Clone number Ab conc. (nM)) 151G3B11 128G11B3 hIgG control 13.33333 36181.86 33488.43 34960.05 34469.51 100840.8 101667.4 2.66667 38398.37 36772.32 36390.79 35595.93 101199.6 105936.7 0.53333 36408.96 36790.49 43485.45 48045.66 103434.2 106463.5 0.10667 41595.96 38802.61 59673.27 63897.36 108035 114497.9 0.02133 38570.97 44112.25 94905.84 93293.42 108062.2 130830.1 0.00427 41546 45988.11 108386.6 105865.8 116092.1 126837.9 0.00085 66250.13 74216.87 105370.7 106115.6 114520.6 117368.3 0.00017 76565.1 80444 104825.6 109167.8 112204.3 120642.9 0.00003 89809.68 92616.65 107437.3 101573.5 112208.9 116055.7 0.00001 95537.18 93897.51 107092.1 100533.4 113362.5 117922.4
[0417] Table 18 shows that by binding to human IL-5, the chimeric antibody can neutralize proliferation of TF-1 cells induced by IL-5 stimulation. The data in Table 18 are the relative light unit values of ATP measured in cell culture plate, wherein the hIgG control is human IgG.
EXAMPLE 8
Chimeric Antibody Affinity Test
[0418] The human antigen IL-5 was immobilized on the surface of CM5 chip to 150-200 RU and the process was as follows: a freshly prepared mixture of 50 mM NHS and 200 mM EDC (1:1) was used to activate for 200 sec, the antigen IL-5 was diluted to lug/ml with 10 mM NaOAc (pH 5.0), and captured on the chip at a flow rate of 10 ul/min for about 1 min. The remaining activated sites were blocked with 1M ethanolamine. Then the antibody to be tested was diluted to different concentrations (0, 3.125, 6.25, 12.5, 25, 50 nM), and then flowed through the chip surface at a flow rate of 30 ul/min, respectively. The binding time was 180 s and the dissociation time was 1200 s. At the end of each cycle, the chip surface was regenerated with 10 mM Glycine at pH 1.5. The kinetic rate constant needed to be subtracted the blank control, and the data was fitted with the global fit analysis method 1:1 combination model. The dissociation equilibrium rate constant (KD) was calculated according to the following formula: KD=kd/ka. Part of the results are shown in
TABLE-US-00026 TABLE 19 Detection results of affinity between chimeric antibodies and human IL-5 Antibody Ka (1/Ms) Kd (1/s) KD (M) 69F2F3 8.19E+04 <1E−05 <1.2E−10 102A2E11B6 1.24E+05 2.73E−05 2.21E−10 100F4E11B3 3.11E+05 1.16E−04 3.71E−10 144D3C4A5 2.69E+05 <1E−05 <3.7E−10 90F9G10H2 9.44E+04 <1E−05 <1.06E−10 136E11F2 1.05E+05 <1E−05 <9.5E−11 147D7H2 1.57E+05 <1E−05 <6.37E−11 151G3B11 1.57E+05 <1E−05 <6.37E−11 128G11B3 1.93E+05 3.76E−05 1.94E−10 154F10G8 2.79E+05 5.24E−05 1.88E−10
[0419] Table 19 shows that the chimeric antibody has high affinity with human IL-5. The data in Table 19 are ka, kd and KD values when the chimeric antibody binds to IL-5, and the hIgG control is human IgG.
EXAMPLE 9
Antibody of the Present Invention Can More Effectively Neutralize TF1 Proliferation Experiment Induced by IL-5
[0420] Compared with the comparative antibody, the antibody in the present invention can more effectively neutralize the TF1 proliferation experiment induced by IL-5. The results are shown in Table 20
TABLE-US-00027 Antibody TF1 (IC50, nM) Mepolizumab 0.045 0.050 0.036 0.028 0.048 Amgen IL-5 antibody 0.046 0.026 0.031 0.016 0.040 102A2E11B6 0.006 0.001 147D7H2 0.002 0.002 151G3B11 0.000 0.001
[0421] Table 20 shows that IC50 of the antibodies in the present invention (102A2E11B6, 147D7H2, and 151G3B11) in this experiment are at the level of 10E.sup.−12 M, and the comparative antibodies (Mepolizumab, Amgen IL-5 antibody) are at the level of 10E.sup.−11M.
Discussion
[0422] The technical solution of the invention: the present invention uses traditional hybridoma technology to prepare monoclonal antibodies.
[0423] Therapeutic monoclonal antibodies can be developed by various technologies and approaches, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology and so on. However, the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology is still the mainstream of the preparation methods of therapeutic monoclonal antibodies. According to the latest advances in monoclonal antibody technology, the present invention adopts optimized hybridoma technology to prepare the required anti-IL-5 antibody.
[0424] The traditional hybridoma preparation technology was established by Kohler and Milstein 40 years ago (Kohler and Milstein 1975, Nature 256: 495), and has now been widely used in the preparation and production of many related monoclonal antibodies in scientific research, diagnosis, and treatment. Although the basic method is still in use today, there have been changes, improvements and innovations in many aspects, including the use of different strains of animals such as genetically modified animals, the introduction of electrofusion technology, and the application of high-efficiency screening technology equipment such as ClonePix equipment, which make the application of tumor technology more diverse and efficient. Monoclonal antibodies prepared from conventional animals such as mice can be cloned by conventional molecular biology methods to clone the antibody heavy chain variable region and light chain variable region genes, and the variable region genes can be grafted to human antibody constant region genes to form human-mouse chimeric antibody to greatly reduce the immunogenicity of the human body. In addition, the CDR domains of the variable region of the mouse antibody can be grafted onto the framework of the human antibody, thereby reducing the composition of the mouse antibody to less than 5%, greatly increasing the safety of the antibody used in human body. Antibodies obtained through this approach are called humanized antibodies and are currently the main product in the antibody drug market.
[0425] Appendix
[0426] Sequence information of the present invention (protein and gene (DNA) sequences of antibody products of the present invention):
TABLE-US-00028 TABLE 21 Protein (amino acid) and gene (nucleotide) sequence numbers of IL-5 antibody Heavy chain protein Light chain protein Variable Variable region region Amino Amino Clone acid Nucleotide VH- VH- VH- acid Nucleotide VL- VL- VL- number sequence sequence CDR1 CDR2 CDR3 sequence sequence CDR1 CDR2 CDR3 147D7H2 1 2 3 4 5 6 7 8 9 10 102A2E11B6 11 12 13 14 15 16 17 18 19 20 69F2F3 21 22 23 24 25 26 27 28 29 30 154F10G8 31 32 33 34 35 36 37 38 39 40 151G3B11 41 42 43 44 45 46 47 48 49 50
TABLE-US-00029 TABLE 22 CDR region sequences and the amino acid sequence numbers (SEQ ID NO:) of IL-5 antibody are as follows: Clone Amino acid sequence number CDR information SEQ ID NO. 147D7H2 VH- DYFIH 3 CDR1 VH- RIDTEDGETKYAPKFQG 4 CDR2 VH- YGNYVAMDY 5 CDR3 VL- SASSSVSYMF 8 CDR1 VL- LTSKLAS 9 CDR2 VL- QQWNSNPYT 10 CDR3 102A2E11B6 VH- SYGVH 13 CDR1 VH- VIWSGGSTDYNAVFMS 14 CDR2 VH- NRYVLDY 15 CDR3 VL- KSSQSLLTSGDQKNHLA 18 CDR1 VL- GASTRES 19 CDR2 VL- QNDHSFPLT 20 CDR3 69F2F3 VH- NYGVH 23 CDR1 VH- VIWSGGSTDYNAAFKS 24 CDR2 VH- NKDFLDY 25 CDR3 VL- KSSQGLLNSGNQKNYLT 28 CDR1 VL- WASTRES 29 CDR2 VL- QNDYHFPLT 30 CDR3 154F10G8 VH- SYWIT 33 CDR1 VH- DIYPGSGSTFYNEKFKS 34 CDR2 VH- ETTLGY 35 CDR3 VL- RSSQSIVHNNGNTYLE 38 CDR1 VL- KVSNRFS 39 CDR2 VL- FQGSHVPFT 40 CDR3 151G3B11 VH- DYYMH 43 CDR1 VH- RIDPEDGETKYAPKFQG 44 CDR2 VH- YGNFPDY 45 CDR3 VL- SASSSVSYMY 48 CDRI VL- LTSKLAS 49 CDR2 VL- OQWNSNPYT 50 CDR3 Note: VH-CDR1 is heavy chain variable region-CDR1, VH-CDR2 is heavy chain variable region-CDR2, and VH-CDR3 is heavy chain variable region-CDR3; wherein, VL-CDR1 is light chain variable region-CDR1, VL-CDR2 is light chain variable region-CDR2, and VL-CDR3 is light chain variable region-CDR3.
Protein (amino acid) and gene (nucleotide) sequences of five monoclonal antibodies of the invention
TABLE-US-00030 Protein and gene sequences of heavy chain variable region of IL-5 antibody 147D7H2 Amino acid sequence 118aa SEQ ID NO. 1 EVQLQQSGAELVKPGASVKLSCTASGFNIKDYFIH WVKQRTEQGLEWIGRIDTEDGETKYAPKFQGKATI TIDTSSNTAYLYFSSLTSEDTAMYYCARYGNYVAM DYWGQGTSVTVSS Gene sequence SEQ ID NO. 2 GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGT GAAACCAGGGGCCTCAGTCAAGTTGTCCTGCACAG CTTCTGGCTTCAACATTAAAGACTACTTTATACAC TGGGTGAAGCAGAGGACTGAACAGGGCCTGGAGTG GATTGGAAGGATTGATACTGAGGATGGTGAAACTA AATATGCCCCGAAATTCCAGGGCAAGGCCACTATA ACAATAGACACATCCTCCAATACAGCCTACCTGTA TTTCAGCAGCCTGACATCTGAGGACACTGCCATGT ATTACTGTGCTAGATATGGTAATTACGTCGCTATG GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTC CTCA Protein and gene sequences of light chain variable region of IL-5 antibody 147D7H2 Amino acid sequence 106aa SEQ ID NO. 6 QIVLTQSPALMSASPGEKVTMTCSASSSVSYMFWY QQKPSSSPKPWIYLTSKLASGVPARFSGSGSGTSY SLTISSVEAEDAATYYCQQWNSNPYTFGGGTKLEI K Gene sequence SEQ ID NO. 7 CAAATTGTTCTCACCCAGTCTCCAGCACTCATGTC TGCATCTCCAGGGGAGAAGGTCACCATGACCTGCA GTGCCAGCTCAAGTGTAAGTTACATGTTCTGGTAC CAGCAGAAGCCAAGTTCCTCCCCCAAACCCTGGAT TTATCTCACATCCAAGCTGGCTTCTGGAGTCCCTG CTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTAC TCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGC TGCCACTTATTACTGTCAGCAGTGGAACAGTAACC CGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA AAA Protein and gene sequences of heavy chain variable region of IL-5 antibody 102A2E11B6 Amino acid sequence 115aa SEQ ID NO. 11 QVQLRQSGPGLVQPSQSLSITCTVSGFSLTSYGVH WVRQSPGKGLEWLGVIWSGGSTDYNAVFMSRLSIS KDSSKSQVFFKMNSLQTDDTAIYYCARNRYVLDYW GQGTSVTVSS Gene sequence SEQ ID NO. 12 CAGGTGCAGCTGAGGCAGTCAGGACCTGGCCTGGT GCAGCCCTCACAGAGCCTGTCCATCACCTGCACAG TCTCTGGTTTCTCATTAACTAGCTATGGTGTACAC TGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTG GCTGGGAGTGATATGGAGTGGTGGAAGCACAGACT ATAATGCAGTTTTCATGTCCAGACTGAGCATCAGC AAGGACAGTTCCAAGAGCCAAGTTTTCTTTAAAAT GAACAGTCTGCAAACTGATGACACAGCCATATATT ACTGTGCCAGAAACCGTTACGTTTTGGACTACTGG GGTCAAGGAACCTCAGTCACCGTCTCCTCA Protein and gene sequences of light chain variable region of IL-5 antibody 102A2E11B6 Amino acid sequence 113aa SEQ ID NO. 16 DIVMTQSPSSLSVSAGERVTMRCKSSQSLLTSGDQ KNHLAWYQQKPGQPPKLLIYGASTRESGVPDRFTG SGSGTDFTLTITSVQAEDLAVYYCQNDHSFPLTFG AGTKLELK Gene sequence SEQ ID NO. 17 GACATTGTGATGACACAGTCGCCATCCTCCCTGAG TGTGTCAGCAGGAGAGAGGGTCACTATGAGGTGCA AGTCCAGTCAGAGTCTGTTAACCAGTGGAGATCAA AAGAACCACTTGGCCTGGTACCAGCAGAAACCAGG GCAGCCTCCTAAACTGTTGATCTACGGGGCATCCA CTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGC AGTGGATCTGGAACCGATTTCACTCTTACCATCAC CAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACT GTCAGAATGATCATAGTTTTCCGCTCACGTTCGGT GCTGGGACCAAGCTGGAGCTGAAA Protein and gene sequences of heavy chain variable region of IL-5 antibody 69F2F3 Amino acid sequence 115aa SEQ ID NO. 21 QVQLKQSGPGLVQPSQSLSITCTVSGFSLSNYGVH WVRQSPGKGLEWLGVIWSGGSTDYNAAFKSRLSIT KDNSKSQVVFRMNSLQTDDTATYYCARNKDFLDYW GQGTKFTVSS Gene sequence SEQ ID NO. 22 CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGT GCAGCCCTCACAGAGCCTGTCCATCACCTGCACAG TCTCTGGTTTCTCATTAAGTAACTATGGTGTACAC TGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTG GCTGGGAGTGATATGGAGTGGTGGAAGCACAGACT ATAATGCAGCTTTCAAATCCAGACTGAGCATCACC AAGGACAATTCCAAGAGCCAAGTTGTCTTTAGAAT GAACAGTCTGCAAACTGATGACACAGCCACTTATT ACTGTGCCAGAAATAAGGACTTCTTGGACTACTGG GGCCAAGGCACCAAGTTCACAGTCTCCTCA Protein and gene sequences of light chain variable region of IL-5 antibody 69F2F3 Amino acid sequence 113aa SEQ ID NO. 26 DIVMTQSPSSLTVTAGEKVTMTCKSSQGLLNSGNQ KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTG SGSGTDFTLTISSVQAEDLAVYYCQNDYHFPLTFG AGTKLELK Gene sequence SEQ ID NO. 27 GACATTGTGATGACACAGTCTCCATCCTCCCTGACT GTGACAGCAGGAGAGAAGGTCACTATGACCTGCAA GTCCAGTCAGGGTCTGTTAAACAGTGGAAATCAAA AGAACTACTTGACCTGGTACCAGCAGAAACCAGGG CAGCCTCCTAAACTGTTGATCTACTGGGCATCCAC CAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCA GTGGATCTGGAACAGATTTCACTCTCACCATCAGC AGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTG TCAGAATGATTATCATTTTCCGCTCACGTTCGGAG CTGGGACCAAGCTGGAACTGAAA Protein and gene sequences of heavy chain variable region of IL-5 antibody 154F10G8 Amino acid sequence 115aa SEQ ID NO. 31 QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWIT WVKQRPGQGLEWIGDIYPGSGSTFYNEKFKSEATL TVDTSSSTAYMQLSSLTSEDSAVFYCSTETTLGYW GQGTTLTVSS Gene sequence SEQ ID NO. 32 CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGT GAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGG CTTCTGGCTACACCTTCACCAGCTACTGGATAACC TGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTG GATTGGAGATATTTATCCTGGTAGTGGTAGTACTT TCTACAATGAGAAGTTCAAGAGCGAGGCCACACTG ACTGTAGACACATCCTCCAGCACAGCCTACATGCA GCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCT TTTACTGTTCAACCGAGACTACCCTGGGCTACTGG GGCCAAGGCACCACTCTCACAGTCTCCTCA Protein and gene sequences of light chain variable region of IL-5 antibody 154F10G8 Amino acid sequence 112aa SEQ ID NO. 36 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHNNGN TYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGS GTKLEIK Gene sequence SEQ ID NO. 37 GATGTTTTGATGACCCAAACTCCACTCTCCCTGCC TGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCA GATCTAGTCAGAGCATTGTACATAATAATGGAAAC ACCTATTTAGAATGGTACCTGCAGAAACCAGGCCA GTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACC GATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGT GGATCAGGGACAGATTTCACACTCAAGATCAGCAG AGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCT TTCAAGGTTCACATGTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA Protein and gene sequences of heavy chain variable region of IL-5 antibody 151G3B11 Amino acid sequence 116aa SEQ ID NO. 41 EVHLQQSGAELVKPGASVKLSCTASGFNIKDYYMH WVKQRTEQGLEWIGRIDPEDGETKYAPKFQGQATI TADTSSNTANLQLSSLTSEDTAVYYCARYGNFPDY WGPGTTLTVSS Gene sequence SEQ ID NO. 42 GAGGTTCACCTGCAGCAGTCTGGGGCAGAGCTTGT GAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAG CTTCTGGCTTCAACATTAAAGACTACTATATGCAC TGGGTGAAGCAGAGGACTGAACAGGGCCTGGAGTG GATTGGAAGGATTGATCCTGAGGATGGTGAAACTA AATATGCCCCGAAATTCCAGGGCCAGGCCACTATA ACAGCGGACACATCCTCCAACACTGCCAACCTGCA GCTCAGCAGCCTGACATCTGAGGACACTGCCGTCT ATTACTGTGCGAGATATGGTAACTTCCCTGACTAC TGGGGCCCAGGCACCACTCTCACAGTCTCCTCA Protein and gene sequences of light chain variable region of IL-5 antibody 151G3B11 Amino acid sequence 106aa SEQ ID NO. 46 QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWY QQKPRSSPKPWIYLTSKLASGVPARFSGSGSGTSY SLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEI K Gene sequence SEQ ID NO. 47 CAAATTGTTCTCACCCAGTCTCCAGCACTCATGTC TGCATCTCCAGGGGAGAAGGTCACCATGACCTGCA GTGCCAGCTCAAGTGTAAGTTACATGTACTGGTAC CAGCAGAAGCCAAGATCCTCACCCAAACCCTGGAT TTATCTCACATCCAAACTGGCTTCTGGAGTCCCTG CTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTAC TCTCTCACAATCAGCAGCATGGAGGCTGAAGATGC TGCCACTTATTACTGCCAGCAGTGGAATAGTAACC CGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA AAA