CN115010809A - anti-CD 20 antibody, preparation method and application thereof - Google Patents

anti-CD 20 antibody, preparation method and application thereof Download PDF

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CN115010809A
CN115010809A CN202110244873.7A CN202110244873A CN115010809A CN 115010809 A CN115010809 A CN 115010809A CN 202110244873 A CN202110244873 A CN 202110244873A CN 115010809 A CN115010809 A CN 115010809A
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antibody
antigen
binding fragment
seq
sequence
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石剑
方丽娟
严永祥
张敬
华珊
周鹏飞
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Wuhan Youzhiyou Biopharmaceutical Co ltd
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Wuhan Youzhiyou Biopharmaceutical Co ltd
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Abstract

The present invention relates to an anti-CD 20 antibody or antigen-binding fragment thereof, characterized in that it comprises or consists of a heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID No. 9 or a mutant thereof; the light chain variable region comprises or consists of an amino acid sequence shown in SEQ ID NO. 10 or a mutant thereof; more preferably, the anti-CD 20 antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO.11 and the light chain variable region of SEQ ID NO. 12.

Description

anti-CD 20 antibody and preparation method and application thereof
Technical Field
The invention relates to the field of medicines, in particular to an anti-CD 20 humanized antibody and a preparation method and application thereof.
Background
Leukocyte differentiation antigen (CD) refers to a cell surface marker of blood cells during differentiation and maturation. CD20, expressed on the surface of B cells at various stages of developmental differentiation except plasma cells (immunoglobulin-secreting B cells), may act directly on B cells by modulating transmembrane calcium ion flux, playing an important regulatory role in B cell proliferation and differentiation. The CD20 antigen is a B cell differentiation antigen, located only on pre-B cells and mature B cells, and is expressed in more than 95% of B cell lymphomas, but not in hematopoietic stem cells, plasma cells and other normal tissues.
Due to the importance of CD20, Rituximab (Rituximab, trade name Rituxan, mabther) which is the first chimeric mab drug approved by the FDA for the treatment of lymphoma targeted against CD20 has been listed as the global prime for the treatment of rheumatoid arthritis and B-cell type non-hodgkin lymphoma since its approval to be marketed in the united states in 1997.
Disclosure of Invention
The invention aims to provide an anti-CD 20 humanized antibody and a preparation method and application thereof.
Specifically, the present invention relates to the following:
1. an anti-CD 20 antibody or antigen-binding fragment thereof, comprising or consisting of a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID No. 9 or a mutant thereof; preferably, the mutant comprises one or more site mutations selected from the group consisting of: based on the sequence indicated in SEQ ID NO 9, the methionine (Met) at position 48 is replaced by isoleucine (Ile), leucine (Leu), glycine (Gly), valine (Val) or alanine (Ala), preferably by isoleucine (Ile); glutamic acid (Glu) at position 74 is substituted with lysine (Lys), arginine (Arg), glutamine (gin), or asparagine (Asn), preferably with lysine (Lys);
the light chain variable region comprises or consists of an amino acid sequence shown in SEQ ID NO. 10 or a mutant thereof; preferably, the mutant comprises one or more site mutations selected from the group consisting of: on the basis of the sequence shown in SEQ ID NO. 10, glutamic acid (Glu) at position 1 is substituted with glutamine (Gln) or asparagine (Asn), preferably with glutamine (Gln); tyrosine (Tyr) at position 35 is substituted with phenylalanine (Phe) or tryptophan (Trp), preferably with phenylalanine (Phe); substitution of alanine (Ala) at position 42 with serine (Ser) or threonine (Thr), preferably with serine (Ser); leucine (Leu) at position 45 is replaced with proline (Pro); a substitution of leucine (Leu) at position 46 with tryptophan (Trp) or phenylalanine (Phe), preferably with tryptophan (Trp); aspartic acid (Asp) at position 69 is substituted with serine (Ser) or threonine (Thr), preferably with serine (Ser); the phenylalanine (Phe) at position 70 is substituted with tyrosine (Tyr) or tryptophan (Trp), preferably with tyrosine (Tyr).
2. The anti-CD 20 antibody or antigen-binding fragment thereof according to item 1, which comprises the heavy chain variable region shown by SEQ ID NO.11 and the light chain variable region shown by SEQ ID NO. 12.
3. The anti-CD 20 antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the antibody is a monoclonal antibody, a partially or fully humanized antibody, a chimeric antibody, a single chain antibody, a multispecific antibody (e.g., bispecific antibody), preferably wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab') 2 、F(ab) 2 Fd, Fv, dAb, Fab/c, scFv multimer, disulfide bond stability Fv (dsFv), (dsFv) 2 Bispecific dsFvs (dsFv-dsFvs'), diabodies (diabodies), disulfide stabilized diabodies (ds-diabodies), single domain antibodies (sdabs), nanobodies, domain antibodies or bivalent domain antibodies.
4. An isolated polypeptide or variant thereof selected from the group consisting of:
(1) an isolated polypeptide or variant thereof comprising the sequence shown in SEQ ID No.11, wherein said polypeptide specifically binds to CD20 as part of an anti-CD 20 antibody further comprising the sequence shown in SEQ ID No. 12;
(2) an isolated polypeptide or variant thereof comprising the sequence set forth in SEQ ID No. 12, wherein the polypeptide specifically binds CD20 as part of an anti-CD 20 antibody further comprising the sequence set forth in SEQ ID No. 11;
wherein the variant is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding sequence indicated by the sequence number and retaining binding affinity to CD20, or an amino acid sequence variable region having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the corresponding sequence indicated by the sequence number and retaining binding affinity to CD 20.
5. A nucleic acid molecule encoding the anti-CD 20 antibody or antigen-binding fragment thereof of any one of items 1-3, or the isolated polypeptide or variant thereof of item 4.
6. A vector comprising the nucleic acid molecule of item 5.
7. A host cell comprising the nucleic acid molecule of item 5, or the vector of item 6.
8. A conjugate comprising an anti-CD 20 antibody or antigen-binding fragment thereof according to any one of items 1-3, and a conjugating moiety, wherein the conjugating moiety is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof; preferably, the coupling moiety is a radioisotope, fluorescent substance, chemiluminescent substance, colored substance, chemotherapeutic agent, biotoxin, polyethylene glycol or enzyme.
9. A fusion protein or a multispecific antibody (preferably a bispecific antibody) comprising the anti-CD 20 antibody or antigen-binding fragment thereof according to any one of items 1 to 3.
10. A kit comprising an anti-CD 20 antibody or antigen-binding fragment thereof according to any one of items 1-3, a conjugate according to item 8, or a fusion protein or multispecific antibody according to item 9; preferably, the kit further comprises a second antibody that specifically recognizes the antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme; preferably, the kit is for detecting the presence or level of CD20 in a sample; or
The kit comprises (1) the anti-CD 20 antibody or antigen-binding fragment thereof according to any one of items 1-3, the conjugate according to item 8, or the fusion protein or multispecific antibody according to item 9, and (2) an antibody or antigen-binding fragment thereof against another antigen, and/or a cytotoxic agent, and optionally, instructions for use.
11. A pharmaceutical composition comprising an anti-CD 20 antibody or antigen-binding fragment thereof according to any one of items 1-3, a conjugate according to item 8, or a fusion protein or multispecific antibody according to item 9; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.
12. Use of the anti-CD 20 antibody or antigen-binding fragment thereof according to any one of items 1-3, the conjugate according to item 8, or the fusion protein or multispecific antibody according to item 9 for the treatment and/or prevention of a tumor (e.g., treatment of a solid tumor such as lymphoma, rheumatoid arthritis, or B-cell type non-hodgkin's lymphoma), or for the manufacture of a medicament for the treatment and/or prevention of a tumor (e.g., a solid tumor such as lymphoma, rheumatoid arthritis, or B-cell type non-hodgkin's lymphoma), or for the manufacture of a medicament for the diagnosis of a tumor (e.g., a solid tumor such as lymphoma, rheumatoid arthritis, or B-cell type non-hodgkin's lymphoma).
13. A method of treating or preventing a tumor (e.g., a solid tumor such as lymphoma, rheumatoid arthritis, or B-cell non-hodgkin's lymphoma), comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of items 1-3, the conjugate of item 8, or the fusion protein or multispecific antibody of item 9.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
The terms referred to in the present invention have the conventional meanings as understood by those skilled in the art. Where a term has two or more definitions, as used and/or acceptable in the art, the definitions of the term as used herein are intended to include all meanings.
It will be appreciated by those of ordinary skill in the art that the CDR regions of an antibody are responsible for the binding specificity of the antibody for an antigen. Given the known sequences of the heavy and light chain variable regions of antibodies, there are several methods for determining the CDR regions of antibodies, including the Kabat, IMGT, Chothia and AbM numbering systems. However, every use of the definition with respect to the CDRs of an antibody or variant thereof is intended to be within the scope of the terms defined and used herein. Given the variable region amino acid sequence of the antibody, one skilled in the art can generally determine the particular CDRs without relying on any experimental data other than the sequence itself.
As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules including monoclonal or polyclonal human, humanized, composite and chimeric antibodies and antibody fragments. The term "antibody" thus includes any protein or peptide containing a molecule comprising at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen. Examples of such cases include, but are not limited to, Complementarity Determining Regions (CDRs) of a heavy or light chain or ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a Framework (FR) region or any portion thereof, or at least a portion of a binding protein. In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using 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 prepared using recombinant DNA techniques well known in the art. The immunoglobulin or antibody molecules of the present application can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.
The term "antibody fragment" or "antigen-binding fragment" includes, but is not limited to, F (ab') 2 、F(ab) 2 Fab', Fab, Fv, Fd, dAb, Fab/c, Complementarity Determining Region (CDR) fragments, single chain Fvs (scFv), Disulfide-stabilized Fv (dsFv), (dsFv) 2 Bispecific dsFv (dsFv-dsFv'), diabodies (diabodies), disulfide stabilized diabodies (ds-diabodies), scFv multimers (e.g., scFv dimers, scFv trimers), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, nanobodies, single domain antibodies (sdabs), domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not comprise a complete antibody structure. Regardless of structure, an antigen-binding fragment includes any polypeptide or polypeptide complex capable of binding the same antigen to which the parent antibody or parent antibody fragment binds. Mao C S et al, "Disulf stabilized Fv Fragments (dsFv): a New Type of Engineering Antibody Fragments". Progress in Biochemistry and Biophysics,1998,25(6):525-526, describe the structure of dsFvs. Holt et al "Domain antibodies: proteins for therapy "Trends in Biotechnology (2003): vol.21, No. 11: 484-490 reviewed antigen binding fragments referred to as "domain antibodies" or dAbs, which contained only the VH or VL domain of the antibody and were therefore smaller than, for example, Fab and scFv. dAbs are the smallest known antigen-binding fragments of antibodies, from 11kDa to 15 kDa. The term "antibody fragment" includes aptamers, aptamer enantiomers (spiegelmers) and diabodies (diabodies). The term "antibody fragment" also includes any synthetic or genetically engineered protein that, like antibodies, binds to a particular antigen to form a complex. In general, antibody fragments have the instant inventionThe antibody has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
Embodiments of the present application provide humanized anti-CD 20 antibodies. The antigen binding domain that binds the CD20 antigen is a Fab, or ScFv, or a non-covalent pairing (Fv) between the heavy chain variable region (VH) -the light chain variable region (VL). Any of the above antibodies or polypeptides may also include additional polypeptides, e.g., a signal peptide at the N-terminus of the antibody, which is used to direct secretion, or other heterologous polypeptides as described herein. The invention not only comprises the complete antibody, but also comprises the antibody fragment with immunological activity or fusion protein formed by the antibody and other sequences. The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, most preferably 96%, 97%, 98% or more than 99% homologous to the variable regions of the heavy and light chains of the antibody of the invention. Thus, the invention includes those molecules having light and heavy chain variable regions of monoclonal antibodies with CDRs that are more than 90% (preferably more than 95%, most preferably more than 96%, 97%, 98% or 99%) homologous to the CDRs of the invention.
The invention also includes fragments, variants, derivatives and analogs of the antibodies. The antibodies, antigen-binding fragments, variants or derivatives thereof of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), human antibodies, antibodies of animal origin, humanized antibodies, primatized (primatized) antibodies, or chimeric antibodies, CDR grafted and/or modified antibodies, single chain antibodies (e.g., scFv), diabodies, epitope-binding fragments, e.g., Fab 'and F (ab') 2 Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide linked Fvs (dsFv), fragments comprising a VL domain or a VH domain, produced by a Fab expression libraryCrude fragments, and anti-idiotypic (anti-Id) antibodies. An antibody fragment, antigen-binding fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6 His-tag). Such fragments, derivatives and analogs are well within the skill of those in the art in light of the teachings herein.
The antibody of the present invention refers to a polypeptide having a binding activity to human CD20, including the CDR regions described above. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids that are similar or analogous in performance do not typically alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention. Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
For the purpose of comparing two or more amino acid sequences, the percentage of "sequence homology" (also referred to herein as "amino acid homology") between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [ the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence ] by [ the total number of amino acid residues in the first amino acid sequence ] and multiplying by [ 100% ], wherein each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence-as compared to the first amino acid sequence-is considered a difference in a single amino acid residue (position), i.e., as an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as NCBI Multiple Alignment (https:// www.ncbi.nlm.nih.gov/tools/cobalt. Some other techniques, computer algorithms and arrangements for determining the degree of sequence identity are described, for example, in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768-A. Typically, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation methods set forth above, the amino acid sequence with the greatest number of amino acid residues is considered the "first" amino acid sequence, and the other amino acid sequence is considered the "second" amino acid sequence.
Furthermore, in determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure and which have little or no effect on the function, activity or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, from WO 04/037999, GB-A-3357768, WO 98/49185, WO 00/46383 and WO 01/09300; and such alternative (preferred) types and/or combinations may be selected based on the relevant teachings of WO 04/037999 and WO 98/49185 and other references cited therein.
It will be appreciated by those of ordinary skill in the art that amino acids within the 20 basic amino acids that are structurally close to each other may be conservatively substituted. For example, the leucine amino group may be substituted with isoleucine, alanine, glycine, valine, or the like.
In some embodiments, the antibodies of the invention may bind to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, an agent, or PEG. The antibodies of the invention may be linked or fused to a therapeutic agent which may include a detectable label such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or toxin, an ultrasound enhancing agent, a nonradioactive label, combinations thereof and other such art-known ingredients.
In some embodiments, the anti-CD 20 antibodies of the invention, for example, have one or more of the following advantages:
(a) the antibody has excellent biological activity and specificity, high affinity with CD20 positive cells, no non-specific combination with CD20 negative cells and no obvious potential toxic side effect.
(b) The antibody of the invention is humanized and modified, still maintains high affinity and reduces immunogenicity.
(c) The antibody of the invention has good stability, and particularly, the stability of the antibody still keeps good under the conditions of acid environment and heat treatment.
Drawings
FIG. 1 shows FACS detection of specific binding of the humanized antibody, rituximab, (huritu mAb) and the positive control antibody, rituximab, (ritu mAb), to CD20 positive cells.
FIG. 2 shows FACS detection of non-specific binding of the humanized antibody, rituximab, (ritu mAb), and the positive control antibody, rituximab, (ritu mAb), to CD20 negative cells.
Figure 3 shows competition FACS detection of inhibition of binding of the humanized antibody rituximab (huritu mab) to the positive control antibody rituximab (ritu mab) to CD20 positive cells.
Figure 4 shows that the humanized antibody, rituximab (huritu mab) and the positive control antibody, rituximab (ritu mab), mediate ADCC effects of effector cells NK92MI-CD16a on target cells.
Detailed Description
The present invention is described in detail below by way of examples. It will be understood by those of ordinary skill in the art that the following examples are for illustrative purposes only. The spirit and scope of the present invention are defined by the appended claims. The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.
Example 1 monoclonal antibody Rituximab humanized variable region sequence design
The variable region humanized sequence of the invention is derived from the monoclonal antibody rituximab, the variable region sequence of the monoclonal antibody rituximab is as follows, and the underlined parts are CDR regions:
>VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEW IGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARST YYGGDWYFNVWGAGTTVTVSA(SEQ ID NO:1)
the sequence of HCDR1 is as follows:GYTFTSYNMH(SEQ IDNO:2)
the sequence of HCDR2 is as follows:AIYPGNGDTSYNQKFKG(SEQ ID NO:3)
the sequence of HCDR3 is as follows:STYYGGDWYFNV(SEQ IDNO:4)
>VL
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KR(SEQ ID NO:5)
the sequence of LCDR1 is as follows:RASSSVSYIH(SEQ ID NO:6)
the sequence of LCDR2 is as follows:ATSNLAS(SEQ IDNO:7)
the sequence of LCDR3 is as follows:QQWTSNPPT(SEQIDNO:8)
the variable region sequence after grafting the CDRs of the parent sequence into the human Germline sequence was determined by using Germline (Germline) of human IgG as a template, the constant region was unchanged (no marker), the underlined amino acids were replaced with the amino acids at the corresponding positions of the parent antibody molecule:
>CDR graft VH
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEW MGAIYPGNGDTSYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARST YYGGDWYFNVWGQGTLVTVSS(SEQ ID NO:9)
>CDR graft VL
EIVLTQSPATLSLSPGERATLSCRASSSVSYIHWYQQKPGQAPRLLIYATSN LASGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQWTSNPPTFGGGTKVEIK(SEQ ID NO:10)
the amino acids that may affect the activity (boxes) are back-mutated, i.e., replaced with amino acids at the corresponding positions of the original parent sequence or similar amino acids (as will be understood by those of ordinary skill in the art, based on the similarity in structure and properties of amino acids, back-mutated to amino acids that are similar in structure and/or properties to the amino acids at the corresponding positions of the parent sequence)
>humanized VH
Figure BDA0002963732070000081
>humanized VL
Figure BDA0002963732070000082
All DNA sequences were obtained by reverse translation of the above amino acid sequences, and DNA fragments were synthesized by Wuhan Oakco.
Example 2: plasmid preparation of antibodies
Heavy and light chains of the anti-CD 20 humanized antibody, hurituximab, were constructed by ligating the cDNA of the VH and VL regions of the PCR-cloned monoclonal antibody with the coding DNA of the heavy chain constant region of human IgG1 (GenBank No. AK303185.1) and the light chain constant region of kappa (GenBank No. MG815648.1), respectively, to obtain expression plasmids for the heavy and light chains of the anti-CD 20 humanized antibody, typically pcDNA3.1(-) (purchased from Invitrogen) or other eukaryotic expression vectors. The sequences of the heavy chain constant region and the light chain constant region of anti-CD 20 monoclonal antibody rituximab were the same as those of rituximab injection purchased from roche ltd, and the heavy chain and the light chain of the antibody were similarly constructed to obtain expression plasmids of the heavy chain and the light chain of anti-CD 20 monoclonal antibody rituximab.
Example 3: expression and purification of antibodies
Plasmid extraction was performed using a endotoxin-free bulk extraction plasmid kit (Qiagen, cat # 12391) following the manufacturer's instructions. CHO-S cell culture in CD CHO Medium (Gibco, cat # 10743- 2 After the cells were prepared by culturing in a cell incubator, plasmids containing heavy/light chain sequences were co-transfected together into CHO-S cells, and the two plasmids were co-transfected to express monoclonal antibodies against CD20, respectively. On the next day after transfection, the culture temperature was adjusted to 32 ℃ and supplemented with 3.5% 2 XEFC + (Gibco, cat # A2503105) daily, and after 14 days of culture, the expression supernatant was harvested by centrifugation at 800 Xg. And filtered through a 0.22 μm filter. Purifying the culture supernatant by protein A affinity chromatography and cation exchange chromatography or a molecular sieve of Superdex 200 to obtain the anti-CD 20 monoclonal antibody rituximab and the anti-CD 20 humanized antibody hurituximab. The purified antibody concentration was determined by ultraviolet absorbance at 280nm (UV280) and the corresponding extinction coefficient for each protein. The purity and homogeneity of the antibodies were assessed by SDS-PAGE and SEC-HPLC.
Example 4: acid stability study of humanized antibodies
Acid stability test method: in the case of protein A affinity chromatography of antibody molecules, the eluted antibody solution was neutralized by adding 1/10 volumes of 1M Tris-HCl (pH8.0) at 0h and 1h in an acid elution step (using a citric acid buffer solution of pH3.5), and subjected to high performance liquid chromatography (HPLC-SEC) detection of the sample.
TABLE 1 SEC purities of citric acid buffer treatment of antibodies 0h (T0) and 1h (T1) at pH3.5
Sample(s) T0 SEC T1(pH3.5)SEC
rituximab 91.58％ 91.80％
hurituximab 99.31％ 99.26％
T0 SEC and T1(pH3.5) SEC indicate the purity of the antibody detected by HPLC-SEC after 0h treatment with citrate buffer and 1h treatment with antibody, respectively. The result shows that the purity change amplitude of the antibody after acid treatment is equivalent to that of the maternal antibody, and the hurituximab has good acid stability.
Example 5: evaluation of thermal stability of humanized antibody
The humanized antibody was evaluated according to the following thermal stability evaluation method. The antibody molecule has no aggregation or degradation phenomenon after being treated at 40 ℃ for 14 days, the purity change is less than 5 percent, which indicates that the antibody molecule can keep stability in the environment of 40 ℃, the treatment at 40 ℃ is carried out for 0 day and 14 days, and the purity is detected by a molecular sieve chromatographic column (HPLC-SEC) of high performance liquid chromatography.
The method comprises the following steps:
firstly, protein purification is carried out to obtain high-purity antibody. Protein purification was performed as described in example 3.
The method comprises the following steps of (1) detecting the thermal acceleration stability at 40 ℃:
and subpackaging the purified antibody into 500 mu L of each tube, sealing the tubes, placing the tubes in a water bath at 40 ℃, sampling every 24h, and carrying out HPLC-SEC detection for 14 days in total.
TABLE 240 ℃ purity of antibody by HPLC-SEC detection for 0 and 14 days
Sample (I) D0 SEC D14 SEC
rituximab 93.45％ 91.15％
hurituximab 98.17％ 98.55％
D0 SEC and D14 SEC represent the antibody purity as determined by HPLC-SEC at day 0 and day 14 of 40 ℃ treatment, respectively. The results show that the range of the change in the purity of the antibody after heat treatment is lower than that of the parent antibody, and the hurituximab has good thermal stability and smaller change in the purity.
Example 6: the humanized antibody can specifically bind to CD20 positive cells
The cell affinity detection method comprises the following steps:
1. the Daudi (CCTCC, GDC067)/293(CCTCC, GDC187) cells with good growth state are digested with pancreatin to prepare single cell suspension. After cell counting, at 2X 10 5 Individual cells/well were plated into 96-well plates;
2. adding antibodies to be detected into each hole according to experimental design, wherein the highest concentration of the antibodies is 1000nM, sequentially diluting the antibodies by 3 times, setting 10 concentration gradients in total, and setting Blank control (Blank, namely no antibodies to be detected is added);
3. after incubating the cells at room temperature for 30min, washing the cells in each well for 3 times, resuspending, adding a fluorescence-labeled secondary antibody PE-anti-human IgG Fc (Biolegend, 409304), and incubating at room temperature in a dark place for 30 min;
4. the cells in each well were washed 3 times, resuspended and then tested on a flow machine.
The results are shown in fig. 1 and fig. 2, the affinity of the humanized CD20 monoclonal antibody hurituximab to CD20 positive cells is substantially consistent with that of the maternal monoclonal antibody at the cellular level, and hurituximab does not bind non-specifically to CD20 negative cells at higher concentrations, indicating that the humanized monoclonal antibody hurituximab has higher specificity for the target.
Example 7: the humanized antibody can compete with the maternal antibody for binding to CD20 positive cells
Cell competition detection method:
1. daudi cells with good growth status were prepared as single cell suspension. After cell counting, at 2X 10 5 Individual cells/well were plated into 96-well plates;
2. performing Biotin labeling on the antibody, respectively fixing the concentrations of rituximab-Biotin and hurituximab-Biotin to be 5nM/4nM according to cell affinity, respectively adding the antibody to be detected into each hole according to experimental design, sequentially diluting the antibody by 3 times with the highest concentration of 1000nM, setting 10 concentration gradients in total, and setting blank control;
3. after incubating the cells at room temperature for 30min, washing the cells in each well for 3 times, resuspending, adding a fluorescence-labeled secondary antibody PE Streptavidin (Biolegend, 405204), and incubating for 30min at room temperature in the dark;
4. the cells in each well were washed 3 times, resuspended and then tested on a flow machine.
The results are shown in figure 3, and the antibody of hurituximab and rituximab compete with each other through competition experiments, which shows that the humanized monoclonal antibody hurituximab and the parent antibody rituximab bind to the same epitope and the affinity is kept consistent.
Example 8: the humanized antibody can mediate ADCC (antibody-mediated isothermal amplification) effect of NK cells on CD20 positive cells
ADCC experimental method:
1. taking out Daudi cells from the incubator, and digesting with pancreatinThen preparing into single cell suspension, after counting cells, adjusting cell density to 8 × 10 6 PermL, stained with 2.5. mu.M CFSE solution in PBS at 37 ℃ for 15 min. Excess CFSE was washed away by adding 10mL of complete medium. Cells were resuspended in complete medium and counted at 2X 10 4 Laying 96-well plate at 37 deg.C and 5% CO 2 The incubator was used for overnight culture.
2. NK92MI-CD16a (ImmuneOnco, YMAK-C0003) cells were removed as effector cells in the day incubator and plated in 96-well plates at an effective target ratio of 3:1 after cell counting. Adding the antibodies to be detected in gradient dilution according to the experimental design, wherein the highest concentration of each antibody is 10 mu g/ml, sequentially carrying out gradient dilution by 5 times, setting 9 concentration gradients, setting 2 repeated holes in each group, and setting a Blank group (without adding the antibodies).
3. Placing 96-well culture plate at 37 deg.C and 5% CO 2 Incubating for 6h in a cell incubator, taking out the culture plate, adding a PI solution with the final concentration of 1 mu g/ml into each hole, detecting on a flow cytometer after 10min,
4. and analyzing the proportion of the CFSE/PI double-positive cells to the CFSE positive cells, namely the death proportion of the target cells, namely the ADCC (antibody-mediated ADCC) effect of the NK92MI-CD16a cells on the target cells.
The results are shown in figure 4, where the ADCC in vitro activities of the hurituximab and rituximab were substantially similar, and the activity of the humanized antibody hurituximab was slightly higher, as measured by cell killing.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (12)

1. An anti-CD 20 antibody or antigen-binding fragment thereof, characterized in that it comprises or consists of a heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID No. 9 or a mutant thereof; preferably, the mutant comprises one or more site mutations selected from the group consisting of: 9, methionine (Met) at position 48 is substituted with isoleucine (Ile), leucine (Leu), glycine (Gly), valine (Val) or alanine (Ala), preferably with isoleucine (Ile); glutamic acid (Glu) at position 74 is substituted with lysine (Lys), arginine (Arg), glutamine (gin), or asparagine (Asn), preferably with lysine (Lys);
the light chain variable region comprises or consists of an amino acid sequence shown in SEQ ID NO. 10 or a mutant thereof; preferably, the mutant comprises one or more site mutations selected from the group consisting of: 10, wherein glutamic acid (Glu) at position 1 is substituted with glutamine (Gln) or asparagine (Asn), preferably glutamine (Gln); tyrosine (Tyr) at position 35 is substituted with phenylalanine (Phe) or tryptophan (Trp), preferably with phenylalanine (Phe); substitution of alanine (Ala) at position 42 with serine (Ser) or threonine (Thr), preferably with serine (Ser); leucine (Leu) at position 45 is replaced with proline (Pro); a substitution of leucine (Leu) at position 46 with tryptophan (Trp) or phenylalanine (Phe), preferably with tryptophan (Trp); aspartic acid (Asp) at position 69 substituted with serine (Ser) or threonine (Thr), preferably with serine (Ser); phenylalanine (Phe) at position 70 is substituted with tyrosine (Tyr) or tryptophan (Trp), preferably with tyrosine (Tyr).
2. The anti-CD 20 antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain variable region shown by SEQ ID No.11 and the light chain variable region shown by SEQ ID No. 12.
3. The anti-CD 20 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody is a monoclonal antibody, a partially or fully humanized antibody, a chimeric antibody, a single chain antibody, a multispecific antibody (e.g. bispecific antibody), preferably the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab') 2 、F(ab) 2 Fd, Fv, dAb, Fab/c, scFv multimerDisulfide bond stability Fv (dsFv), (dsFv) 2 Bispecific dsFvs (dsFv-dsFvs'), diabodies (diabodies), disulfide stabilized diabodies (ds-diabodies), single domain antibodies (sdabs), nanobodies, domain antibodies or bivalent domain antibodies.
4. An isolated polypeptide or variant thereof selected from the group consisting of:
(1) an isolated polypeptide or variant thereof comprising the sequence shown in SEQ ID No.11, wherein said polypeptide specifically binds to CD20 as part of an anti-CD 20 antibody further comprising the sequence shown in SEQ ID No. 12;
(2) an isolated polypeptide or variant thereof comprising the sequence shown in SEQ ID No. 12, wherein said polypeptide specifically binds to CD20 as part of an anti-CD 20 antibody further comprising the sequence shown in SEQ ID No. 11;
wherein the variant is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding sequence as set forth in sequence No. and retaining binding affinity to CD20, or an amino acid sequence variable region having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) as compared to the corresponding sequence as set forth in sequence No. and retaining binding affinity to CD 20.
5. A nucleic acid molecule encoding the anti-CD 20 antibody or antigen-binding fragment thereof of any one of claims 1-3, or the isolated polypeptide or variant thereof of claim 4.
6. A vector comprising the nucleic acid molecule of claim 5.
7. A host cell comprising the nucleic acid molecule of claim 5, or the vector of claim 6.
8. A conjugate comprising the anti-CD 20 antibody or antigen-binding fragment thereof of any one of claims 1-3 and a conjugate moiety, wherein the conjugate moiety is a purification tag (e.g., a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof; preferably, the coupling moiety is a radioisotope, fluorescent substance, chemiluminescent substance, colored substance, chemotherapeutic agent, biotoxin, polyethylene glycol or enzyme.
9. A fusion protein or multispecific antibody (preferably a bispecific antibody) comprising the anti-CD 20 antibody or antigen-binding fragment thereof of any one of claims 1-3.
10. A kit comprising the anti-CD 20 antibody or antigen-binding fragment thereof of any one of claims 1-3, the conjugate of claim 8, or the fusion protein or multispecific antibody of claim 9; preferably, the kit further comprises a second antibody that specifically recognizes the antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme; preferably, the kit is for detecting the presence or level of CD20 in a sample; or
The kit comprises (1) the anti-CD 20 antibody or antigen-binding fragment thereof of any one of claims 1-3, the conjugate of claim 8, or the fusion protein or multispecific antibody of claim 9, and (2) an antibody or antigen-binding fragment thereof to another antigen, and/or a cytotoxic agent, and optionally, instructions for use.
11. A pharmaceutical composition comprising the anti-CD 20 antibody or antigen-binding fragment thereof of any one of claims 1-3, the conjugate of claim 8, or the fusion protein or multispecific antibody of claim 9; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.
12. Use of the anti-CD 20 antibody or antigen-binding fragment thereof according to any one of claims 1-3, the conjugate according to claim 8, or the fusion protein or multispecific antibody according to claim 9 for the treatment and/or prevention of a tumour (e.g. the treatment of a solid tumour, such as lymphoma, rheumatoid arthritis or B-cell type non-hodgkin's lymphoma), or for the manufacture of a medicament for the treatment and/or prevention of a tumour (e.g. a solid tumour, such as lymphoma, rheumatoid arthritis or B-cell type non-hodgkin's lymphoma), or for the manufacture of a medicament for the diagnosis of a tumour (e.g. a solid tumour, such as lymphoma, rheumatoid arthritis or B-cell type non-hodgkin's lymphoma).
CN202110244873.7A 2021-03-05 2021-03-05 anti-CD 20 antibody, preparation method and application thereof Pending CN115010809A (en)

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