TW202128772A - BCMA-binding protein, preparation method therefor, and application thereof - Google Patents

Abstract

Disclosed is a BCMA-binding protein, a preparation method therefor, and an application thereof. The BCMA-binding protein comprises a heavy chain variable region and a light chain variable region. The light chain variable region comprises LCDR1, LCDR2, and LCDR3. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3. The BCMA-binding protein has an excellent specific affinity and binds better to tumor cell lines compared with a control antibody. The BCMA-binding protein has an internalization effect superior to that of the control antibody. The BCMA-binding protein can serve as an ADC candidate for further studying. According to the ELISA test, the antibody has a partial-blocking function and may provide additional curative effects when being used as mAbs or CAR-T in treatment.

Description

A BCMA binding protein and its preparation method and application

This application claims the priority of the Chinese patent application 202010076287.1 with the filing date of 2020/1/23. This application quotes the full text of the aforementioned Chinese patent application.

The invention belongs to the field of biological macromolecules, and specifically relates to a BCMA binding protein and a preparation method and application thereof.

Multiple myeloma (MM) has always been characterized by uncontrolled proliferation of plasma cells derived from bone marrow, accompanied by abnormal secretion of immunoglobulins or free light chains. It ranks second among the most common hematological malignancies and accounts for approximately all hematological tumors. Of 10%. In the United States, there are more than 30,000 newly diagnosed patients and more than 12,000 deaths every year. Although there are new targeted therapies such as protease inhibitors (Bortezomb), it is generally believed that it is incurable, so more efforts are needed in drug development.

B cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a specific antigen that is only expressed in B cell lineages, especially terminally differentiated B cells. It is usually absent from initial and memory B cells and is highly expressed on multiple myeloma cells. When BCMA interacts with its ligands BAFF and APRIL, it can induce B cell maturation, proliferation and survival through the NF-kB and JNK pathways. As a type I transmembrane protein, it plays an important role in humoral immunity and is an ideal target for antibody-derived immunotherapy.

However, the anti-BCMA antibodies in the prior art have defects such as weak internalization and weak binding to 293T-huBCMA cells. Therefore, there is a great need for an anti-BCMA antibody that has sufficient drug affinity and excellent internalization.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the defects of weak internalization of the existing anti-BCMA antibody and weak binding with 293T-huBCMA cells in the prior art, and provide a BCMA binding protein and a preparation method and application thereof.

In order to solve the above technical problems, the present invention provides a BCMA binding protein, which includes a heavy chain variable region (VH) and a light chain variable region (VL). The light chain variable region includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO: 1 in the sequence listing or a variant thereof, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 2 in the sequence listing or a variant thereof The amino acid sequence of the LCDR3 is shown in SEQ ID NO: 3 in the sequence list or a variant thereof; the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the amino acid of the HCDR1 The sequence is shown in SEQ ID NO: 4 in the sequence listing or a variant thereof, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 5 in the sequence listing or a variant thereof, and the amine of the HCDR3 The base acid sequence is shown in SEQ ID NO: 6 in the sequence listing or a variant thereof. Wherein, the variants are amino acid sequences obtained after 3, 2 or 1 mutations are made on the basis of the amino acid sequences shown in SEQ ID NOs: 1 to 6, respectively.

Similar to "with 3, 2 or 1 amino acid mutations" in "amino acid mutations" means that compared with the original amino acid sequence, the variant sequence has amino acid mutations, including the amino acid mutation in the original amino acid sequence. The insertion, deletion or substitution of amino acids occurs on the basis of the acid sequence. An exemplary explanation is that the mutations to the CDRs can include 3, 2, or 1 amino acid mutations, and the same or different numbers of amino acid residues can optionally be selected for mutations between these CDRs, for example, One amino acid mutation was performed on CDR1, and no amino acid mutation was performed on CDR2 and CDR3.

The mutations described in the present invention may include mutations currently known to those skilled in the art, such as some mutations that may be made to the antibody during the production or application of the antibody, such as the transcription of the CDR regions that may exist. Potential post-translational modifications (PTMs) sites are mutated, including antibody aggregation, asparagine deamidation sites (NG, NS, and/or NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N-{P}S/T) sensitive sites and oxidation-sensitive sites and other related mutations.

Preferably, the BCMA binding protein described in the present invention also includes a framework region of an anti-BCMA antibody, and the framework region includes a heavy chain framework region (HFWR) and/or a light chain framework region (LFWR);

The heavy chain framework region is preferably a human heavy chain framework region, and the human heavy chain framework regions HFWR1~4 preferably contain amino acid sequences as shown in SEQ ID NO: 13~16 in the sequence listing, respectively, Or they are variants of the amino acid sequences shown in SEQ ID NOs: 13 to 16 respectively;

The light chain framework region is preferably a human light chain framework region, and the human light chain framework regions LFWR1~4 preferably respectively contain amino acid sequences as shown in SEQ ID NO: 17~20 in the sequence listing, respectively, Or, respectively, variants of the amino acid sequence shown in SEQ ID NO: 17-20.

In a preferred embodiment of the present invention, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing or a variant thereof, and/or, the light chain may The variable region includes the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing or a variant thereof. The variant is based on the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12 where one or more amino acid residues are deleted, substituted or added, and the mutated amino group The acid sequence has at least 85% sequence identity with the amino acid sequence of the VH and/or VL, and maintains or improves the binding of the BCMA binding protein to BCMA; the at least 85% sequence identity is preferably at least 90 % Sequence identity, more preferably at least 95% sequence identity, most preferably at least 99% sequence identity.

In a better embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11 in the sequence listing, and the amino acid sequence of the light chain variable region As shown in SEQ ID NO: 12 in the sequence listing.

In this application, the amino acid sequences of the CDRs listed above are all shown in accordance with the Kabat definition rules (the sequence in the patent application of the present invention is also shown in accordance with the Kabat definition rules). However, it is well known in the art that there are many methods in the art to define the CDR of an antibody, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Health Research Institute, Bethesda, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997). In this application, the combined definition rule including the Kabat definition and Chothia definition can also be used to determine the amino acid residues in the variable domain sequence. Among them, the combined definition rule is to combine the Kabat definition and the Chothia definition. Based on this, a larger scope is taken, as shown in Table 1-1. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region) should be understood to encompass the above-mentioned already described through the present invention. Complementary decision area defined by any one of the known schemes. Although the claimed scope of the patent application of the present invention is based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.

The BCMA binding protein described in the present invention preferably further includes a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3, hIgG4 or mutations thereof, The light chain constant region is selected from a kappa chain or a lambda chain or a mutation thereof; wherein the kappa chain or the lambda chain is preferably a human antibody light chain kappa chain or a lambda chain.

In a preferred embodiment of the present invention, the heavy chain constant region is hIgG1, and the light chain constant region is the light chain κ chain of a human antibody.

The BCMA binding protein described in the present invention can be a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single domain antibody, or by Monoclonal antibody or multi-strain antibody prepared by the above antibody. The monoclonal antibody can be developed by a variety of methods and technologies, including hybridoma technology, phage display technology, single lymphocyte gene transfer technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

In a specific embodiment of the present invention, the BCMA binding protein is a full-length antibody, the full-length antibody includes a heavy chain and a light chain, and the heavy chain includes the amino acid sequence shown in SEQ ID NO: 9, so The light chain includes the amino acid sequence shown in SEQ ID NO: 10.

The present invention also provides an isolated nucleic acid, which encodes the BCMA binding protein as described above.

The present invention also provides an expression vector containing the isolated nucleic acid as described above.

The present invention also provides a host cell comprising the expression vector as described above; preferably, the host cell is a prokaryotic cell or a eukaryotic cell. The preparation method of the host cell may be a conventional preparation method in the art, for example, it is prepared by transforming the above-mentioned expression vector into a host cell. The host cell is a variety of conventional host cells in the field, as long as it can make the above-mentioned expression vector stably replicate itself and the nucleic acid carried by it can be effectively expressed. Preferably, the host cell is E. coli TG1 or BL21 cell (expressing single-chain antibody or Fab antibody), or CHO-K1 cell (expressing full-length IgG antibody). The transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a thermal stimulation method or an electric transformation method.

The present invention also provides a method for preparing a BCMA binding protein, which comprises culturing the host cell as described above, and obtaining the BCMA binding protein from the culture.

The present invention also provides an immunoconjugate comprising a cytotoxic agent and the BCMA binding protein as described above.

The present invention also provides a pharmaceutical composition, which comprises the above-mentioned BCMA binding protein or the above-mentioned immunoconjugate.

The present invention also provides the application of the above-mentioned BCMA binding protein, the above-mentioned immunoconjugate and the above-mentioned pharmaceutical composition in the preparation of a medicine for the treatment and/or prevention of cancer; preferably, the cancer is bone marrow Tumor, colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer, or ovarian cancer.

In addition, in order to solve the above technical problems, the present invention also provides a kit combination, which includes a kit A and a kit B; the kit A includes the BCMA binding protein of the invention, the host cell, and the The immunoconjugate and the pharmaceutical composition; The kit B contains other target or the same target antibody, bispecific antibody, genetically modified cell or pharmaceutical composition. The use of the medicine box A and the medicine box B is in no particular order, or the medicine box A is used first and then the medicine box B, or the medicine box B is used first and then the medicine box A is used.

The BCMA binding protein, the immunoconjugate and the pharmaceutical composition or the kit combination of the present invention can be administered to patients for the treatment of related tumors.

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, definitions and explanations of related terms are provided below.

The three-letter code and one-letter code of the amino acid used in the present invention are known to those skilled in the art or described in J. Biol. Chem, 243, p3558 (1968).

As used herein, the term "comprising" or "comprising" is intended to mean that the composition and method include the described elements but not exclude other elements, but according to the context, it also includes the case where "consisting of" is also included.

The term "antibody" in the present invention includes immunoglobulin (Ig), which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are µ chain, δ chain, γ chain, Alpha chain and epsilon chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of its hinge area and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain is divided into kappa chain or lambda chain through the difference of constant region. Each of the five types of Ig can have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region, and the light chain constant region comprises human-derived kappa, lambda chains or variants thereof. In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human IgG1, 2, 3, 4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 complementarity determining regions (CDR) and 4 framework regions (FWR). The sequence from the amino end to the carboxyl end is : FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The 3 CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The term "mutation" includes substitutions, additions and/or deletions of amino acids or nucleotides. "Substitutions of amino acids" are those in which the amino acid residue is replaced with another amino acid residue and has a similar side chain. Replacement of amino acid residues.

The term "vector" or "expression vector" as used herein is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plastids, and viruses. Therefore, the term "vector" includes autonomously replicating plastids or viruses. The term should also be interpreted to include aplastic and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion , Lipofection, protoplast fusion, retroviral infection and biolistics.

The preparation method of the immunoconjugate can be conventional in the art, preferably the preparation method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124. Preferably, the preparation method produces antibody-drug conjugates with a minimum low conjugation fraction (LCF) of less than 10%. The immunoconjugate can exist in any physical form known in the art, and is preferably a clear solution.

As used in the present invention, the terms "cancer", "cancer" and "tumor" are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type Or what is the aggressiveness stage. Examples include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effects of the present invention are: The present invention immunoscreens out a fully human antibody with good specific affinity with hBCMA, which binds better than the control antibody on tumor cell lines. The antibody has an internalization effect and is better than the control, so it can be further developed as an ADC candidate. According to the ELISA test, the antibody has partial blocking function, and may obtain additional curative effect when used as mAbs or CAR-T treatment.

Detailed description of the invention

The following further describes the present invention by way of examples, but the present invention is not limited to the scope of the described examples. In the following examples, the experimental methods without specific conditions are selected according to conventional methods and conditions, or according to the product specification.

The positive control (also referred to as the control antibody) used in the following examples is the GSK clinical phase II ADC drug GSK2857916 (ie, the CA8 J6M0 humanized antibody in the US patent application US9273141B2). The amino acid sequence of each functional region of the control antibody is shown in the following table (according to the Kabat numbering rule): Functional area Amino acid sequence VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 21) HCDR1 NYWMH (SEQ ID NO: 22) HCDR2 ATYRGHSDTYYNQKFKG (SEQ ID NO: 23) HCDR3 GAIYDGYDVLDN (SEQ ID NO: 24) VL DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK (SEQ ID NO: 25) LCDR1 SASQDISNYLN (SEQ ID NO: 26) LCDR2 YTSNLHS (SEQ ID NO: 27) LCDR3 QQYRKLPWT (SEQ ID NO: 28)

Heavy chain amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)

Light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8 ) of the detailed description of the preferred embodiments Example Example 1. Preparation of antigen and immunization of mice and preparation of hybridoma embodiments 1. Preparation of antigen (ACRO cat.no: BCA- C52H7) supplier name NCBI ID Cat# Acrobio huBCMA-ECD-Fc Q02223 BCA-H522y Acrobio BCMA-His BCA-C52H7 Acrobio CynoBCMA-Fc G7NPN8 BCA-C5253 Acrobio CynoBCMA-His BCA-C5225 Acrobio BAFF-his-avitag Q9Y275 BAF-H82Q2 2. Immunization

Fully human anti-BCMA antibodies were identified from hybridomas produced by H2L2 mice (and Platinum Medicine, EP2379727B1) immunized with BCMA-ECD-Fc protein. The first injection of 50 μg of the above fusion protein was immunized with CFA as an immune adjuvant, and then 25 μg of protein and Ribi adjuvant (Sigma-Aldrich; Sigma Adjuvant System ; Catalog Number S6322) strengthen 7 times. Blood was collected on the 50th, 78th and 107th days, and the binding affinity of mouse serum was tested by FACS using HEK293T cells or tumor cell lines expressing human BCMA (HEK293T-BCMA, purchased from kyinno or Capan-2 cells, purchased from ATCC) At the same time, BCMA-His protein was used in parallel for ELISA detection. According to the results of the serum titer of the immunized mice, mice were selected for hybridoma fusion. Three days before the fusion, that is, the 132th day, the final boost was performed with 25 μg protein and Ribi adjuvant. 3. Integration

Hybridomas are produced and colonized by conventional methods, that is, through electrofusion, mouse spleen and lymph nodes are extracted, single cells are crushed and extracted, red blood cells are lysed and washed and mixed with sp2/0 cells. The cell suspension is placed in an electrofusion tank for electric shock fusion, and after standing still, it is replaced with 20% FBS HT medium, and then replaced with HAT medium for culture.

The collected spleen B cells and the mouse myeloma cell line Sp2/0 were mixed at a ratio of 2:1 (cell number ratio), and the mixed cells were fused with an electric fusion instrument (BTX ECM2001). The fused cells Spread it on a 96-well cell culture dish, culture it in a carbon dioxide incubator at 37°C for 10 days, and then conduct a preliminary screening of hybridomas. After overnight recovery, the fused cells were seeded into 96-well plates by limiting dilution and screened with hypoxanthine-aminopterin-thymidine. The presence of anti-BCMA antibodies in the culture supernatant of hybridomas was detected by ELISA test and flow cytometry. Example 2. Antibody screening and sequencing 1. ELISA screening

A 96-well plate (Corning 9018) was added with newly prepared hBCMA ECD-Fc protein or hFc at 1 μg/ml in PBS, coated overnight at 4°C, then discarded and washed with PBST 3 times. Block the culture plate with 5% milk at room temperature for 2 hours, and wash it with PBST 3 times. 100 μl/well of hybridoma supernatant was added and incubated at room temperature for 1 hour, and then washed with PBST 3 times. Add 100 μl/well of the secondary antibody and incubate at room temperature for 1 hour, followed by washing. Add 100 μl/well of TMB to the culture plate and incubate at room temperature for 15 minutes, then stop and read. 2. FACS screening

For flow cytometry screening, digest the adherent cells with TypLE (CAT#12605010, Gibco) at 37°C for 3 minutes, and then terminate the digestion with a complete medium containing 10% FBS. Wash the cells with FACS buffer (CAT#14190250, Gibco) and count them, then dilute to a ^{density of 3~5×10 6} /ml. Add 100 μl/well of cells to a 96-well plate (Corning 3894). After blocking for 3 to 4 minutes, add 100 μl/well of hybridoma supernatant and incubate at 4°C for 1 hour. After washing, add secondary antibody and incubate at 4°C for one hour. The cells were then washed and analyzed by FACS. 3. Sub-selection and selection

The limiting dilution method was used to perform the blocking test by the ELISA method.

Block screening: For the blocking enzyme binding immunosorbent assay (ELISA), the culture plate was coated with hBCMA-ECD-FC at 2 μg/ml in PBS overnight, and the washing, blocking and incubation were the same as the above steps. Then add 6 ng/ml biotinylated BAFF to the culture plate and incubate at room temperature for 1 hour. The subsequent steps of cultivation and detection of the second antibody are the same as above. 4. Sequencing

Select positive single transgenic plants and extract total RNA. RT-PCR produces cDNA, and then the heavy and light chains are amplified by PCR respectively (RT-PCR comes from Thermo Fisher’s SuperScript® first-strand synthesis system, Cat#: 11904018, see the product manual for specific operations. NEB is used for PCR High-fidelity DNA polymerase, Cat#: M0530L, see the product manual for specific operations). Then the PCR product was constructed on the T vector, and sequenced (Beijing Kinco Biotechnology Co., Ltd. Shanghai Branch), and the antibody subtype was determined at the same time. 5. Antibody sequence information

The sequencing results are as follows (according to the Kabat numbering rules): The amino acid sequences of LCDR1~3 in the light chain variable region are as shown in SEQ ID NO: 1~3 in the sequence listing; The amino acid sequences of HCDR1~3 are shown in SEQ ID NO: 4~6 in the sequence table, respectively. The specific amino acid sequence is shown in the following table: Functional area Amino acid sequence SEQ ID NO: LCDR1 RASQSISSWLA 1 LCDR2 KASSLES 2 LCDR3 QQYNSYLFT 3 HCDR1 SYGMY 4 HCDR2 AIWNDGSNNYYADSVKG 5 HCDR3 DRLPMASLRYFDWLGVMDA 6 Example 3. Antibody production, purification and verification 1. Recombinant antibody production and purification

After obtaining the light and heavy chain variable domain sequences encoding antibody molecules, conventional recombinant DNA technology can be used to combine the light and heavy chain variable domain sequences and the corresponding human antibody light and heavy chain constant domain sequences. Fusion performance to obtain recombinant antibody molecules. In this example, the antibody heavy chain variable domain sequence (VH) was synthesized by gene and cloned into the mammalian cell expression plastid vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the full length of the IgG1 antibody. Heavy chain. The antibody light chain variable domain sequence (VL) is synthesized by gene and transgenic into mammalian cell expression plastid vector encoding the human antibody Igκ light chain constant domain sequence to encode the full-length light chain of the antibody. In this example, since the sequence of the variable domain of the monoclonal antibody molecule obtained from the immunized Harbour H2L2 mouse is a human antibody sequence, this example also obtained a fully human anti-BCMA recombinant IgG1 antibody.

For example, the present invention is a humanized antibody heavy chain sequence of: QVQLVESGGGVVQPGRSLRLSCAATGFTFSSYGMYWVRQAPGKGLEWVAAIWNDGSNNYYADSVKGRFTISRDDSKNTLNLQMNSLRAEDTAMYYCARDRLPMASLRYFDWLGVMDAWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9)

The light chain sequence of the humanized antibody of the present invention is: DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQTDDFATYYCQQYNSYLFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALSSYPREAKVQWKVDNALLIYKASSLESGVPVSSQSGSNTK IDVSSVSEQIDQSLQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALSSGESFGEPVVTSVSSQSGVTSVTEQSLGENOGSL:

The amino acid sequence of the heavy chain variable region of the humanized antibody of the present invention is: QVQLVESGGGVVQPGRSLRLSCAATGFTFSSYGMYWVRQAPGKGLEWVAAIWNDGSNNYYADSVKGRFTISRDDSKNTLNLQMNSLRAEDTAMYYCARDRLPMASLRYFDWLGVMDAWGQGTSVTVSS (SEQ ID NO: 11)

The amino acid sequence of the light chain variable region of the humanized antibody of the present invention is: DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQTDDFATYYCQQYNSYLFTFGQGTKLEIK (SEQ ID NO: 12)

Transfect the plastid encoding the antibody heavy chain (Genscript US) and the plastid encoding the antibody light chain (Genscript US) simultaneously into mammalian host cells (such as human embryonic kidney cells HEK293), using conventional recombinant protein expression and purification techniques, A purified recombinant antibody with the correct pairing and assembly of light and heavy chains can be obtained. Specifically, HEK293 cells were expanded and cultured in FreeStyle™ F17 Expression Medium medium (Thermo, Cat#: A1383504). Before the transient transfection starts, adjust the cell concentration to 6-8×10 ⁵ cells/ml and incubate in a shaker at 37°C 8% CO ₂ for 24 hours. The cell concentration is 1.2×10 ⁶ cells/ml. Prepare 30 ml of cultured cells. The above-mentioned plastids encoding antibody heavy chains and plastids encoding antibody light chains were mixed in a ratio of 2:3 (quality ratio), a total of 30 μg plastids were dissolved in 1.5 ml Opti-MEM reduced serum medium (Thermo, Cat#: 31985088) ), and filter and sterilize with a 0.22 µm filter membrane. Take 1.5 ml of Opti-MEM and dissolve it in 120 µl of 1 mg/ml PEI (Polysciences, Cat#: 23966-2), and let it stand for 5 minutes. Slowly add PEI to the plastid, incubate at room temperature for 10 minutes, slowly drop the plastid PEI mixed solution while shaking the culture flask, and incubate for 5 days _{in a 37°C 8% CO 2 shaker.} The cell viability was observed after 5 days. Collect the culture, centrifuge at 3300 g for 10 minutes and take the supernatant; then centrifuge the supernatant at high speed to remove impurities. Equilibrate the gravity column (Bio-Rad, Cat#: 7311550) containing MabSelect ™ (GE Healthcare Life Science, Cat#: 71-5020-91 AE) with PBS (pH7.4), wash 2-5 times the column volume . Pass the supernatant sample through the column; wash the column with 5-10 times the column volume of PBS, then elute the target protein with 0.1M glycine acid at pH 3.5, then adjust to neutral with Tris-HCl at pH 8.0, and finally Use an ultrafiltration tube (Millipore, Cat#: UFC901024) to concentrate and change the solution to PBS buffer to obtain a purified antibody solution. Finally, use NanoDrop (Thermo Scientific™ NanoDrop™ One) to determine the concentration, aliquot and store for later use.

Appropriate amounts of the above-mentioned purified samples were applied to the analytical SEC column TSKgel G3000SWxl (HPLC instrument model: Agilent 1260 Infinity II), and the purity of the samples was checked to ensure that the purity of the homogeneous samples was above 95%. The mobile phase of this method is 1X PBS, pH7.4 (Shenggong, Cat#: E607016), room temperature, flow rate 1.0 ml/min, sample concentration 1 mg/ml, injection volume 20 μl, and detection wavelength 280 nm. After collection, use ChemStation software to integrate the chromatogram and calculate the relevant data. 2. Recombinant antibody verification

FACS identification: Recombinant antibodies were diluted to 9 different concentrations in sequence, and incubated on 293T-huBCMA, 293T-cynoBCMA and NCI-H929 cell lines for 1 hour, and then the secondary antibody was incubated for 1 hour and then tested. The verification results are shown in Figure 1. Shown. In Figure 1a, PR000892 binds to 293T-huBCMA cells [purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.; Item No.: CAT#KC-0233] has significant binding, and the affinity is better than the control antibody; Figure 1b , PR000892 binds to 293T-cynoBCMA [purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.; item number: CAT#KC-0979] has the same affinity as the control antibody; in Figure 1c, PR000892 and tumor cell line NCI-H929 Its affinity is comparable to that of the control antibody.

Blocking: Recombinant antibodies are serially diluted to 7 different concentrations and tested on Corning 9018 discs pre-coated with 2 μg/ml huBCMA-ECD-FC. The subsequent steps are the same as above. As shown in Figure 2, PR000892 can partially block the binding of BAFF and BCMA under in vitro conditions.

Internalization: Count 293T-huBCMA cells or NCI-H929 cells, and seed 5000 or 10000 cells/well to 96-well black-walled and transparent-bottomed culture plates (PE6005225). Recombinant antibodies were serially diluted to 9 different concentrations and added, with the final concentration starting from 100 nM. Add a-hFc-MMAF to make the final concentration 1 μg/ml. The culture plate was incubated at 37°C and 5% CO ₂ for 72 hours, then lysed with a CTG kit, and luminescence was detected with Enspire. As shown in Figure 3a, the internalization effect of PR000892 in the 293T-huBCMA cell line is comparable to that of the control antibody; as shown in Figure 3b, the internalization effect of PR000892 in the NCI-H929 cell line is significantly better than that of the control antibody.

In addition: It has been verified that the antibody of the present invention and the control antibody have the same affinity with the BCMA antigen (Cat. BCA-H522y).

Figure 1 shows the test results of the binding strength of the recombinant antibody to the cell line expressing BCMA; a. The binding of the recombinant antibody to the 293T-huBCMA cell line b. The binding of the recombinant antibody to the 293T-cynoBCMA cell line, c. The recombinant antibody and NCI- Combination of H929 cell line.

Figure 2 shows the test of BCMA recombinant antibody blocking the binding of BAFF and BCMA.

Figure 3 shows the determination of the internalization of recombinant antibodies into BCMA cell lines. a. Recombinant antibody is internalized into 293T-huBCMA cell line, b. Recombinant antibody is internalized into NCI-H929 cell line.

Claims (12)

A BCMA binding protein, characterized in that it comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequence of LCDR1 is shown in the sequence table In SEQ ID NO: 1, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 3 in the sequence listing. ； The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 4 in the sequence list, and the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4 in the sequence list. ID NO: 5, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 6 in the sequence listing. The BCMA binding protein according to claim 1, wherein the BCMA binding protein further includes a framework region of an anti-BCMA antibody, and the framework region includes a heavy chain framework region and/or a light chain framework region; The heavy chain framework region is preferably a human heavy chain framework region, and the human heavy chain framework regions HFWR1~4 comprise amino acid sequences as shown in SEQ ID NO: 13~16 in the sequence listing, respectively; The light chain framework region is preferably a human light chain framework region, and the human light chain framework regions LFWR1~4 contain amino acid sequences as shown in SEQ ID NO: 17~20 in the sequence listing, respectively. The BCMA binding protein according to claim 2, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing, and the light chain variable region Including the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing. The BCMA binding protein according to any one of claims 1 to 3, which is characterized in that it further comprises a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4 or a mutation thereof, and the light chain constant region is selected from a kappa chain or a lambda chain or a mutation thereof. The BCMA binding protein according to any one of claims 1 to 4, characterized in that it is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody , Single-domain antibodies or single-domain antibodies, or monoclonal antibodies or multi-strain antibodies made from the above-mentioned antibodies. The BCMA binding protein according to claim 5, wherein the full-length antibody includes a heavy chain and a light chain, the heavy chain includes the amino acid sequence shown in SEQ ID NO: 9, and the light chain includes The amino acid sequence shown in SEQ ID NO: 10. An isolated nucleic acid encoding the BCMA binding protein according to any one of claims 1 to 6. An expression vector comprising the isolated nucleic acid according to claim 7. A host cell comprising the expression vector according to claim 8; preferably, the host cell is a prokaryotic cell or a eukaryotic cell. A method for preparing a BCMA binding protein, which comprises culturing the host cell according to claim 9 and obtaining the BCMA binding protein from the culture. A pharmaceutical composition comprising the BCMA binding protein according to any one of claims 1 to 6. The application of the BCMA binding protein according to any one of claims 1 to 6 and the pharmaceutical composition according to claim 11 in the preparation of drugs for the treatment and/or prevention of cancer; preferably, the cancer is myeloma, Colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer, or ovarian cancer.

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