WO2024140863A1 - Antibody targeting crtam and pd-l1 and use thereof - Google Patents
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- WO2024140863A1 WO2024140863A1 PCT/CN2023/142554 CN2023142554W WO2024140863A1 WO 2024140863 A1 WO2024140863 A1 WO 2024140863A1 CN 2023142554 W CN2023142554 W CN 2023142554W WO 2024140863 A1 WO2024140863 A1 WO 2024140863A1
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- amino acid
- antibody
- crtam
- acid sequence
- variable region
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
Definitions
- the present invention relates to the field of antibody drugs, and in particular to anti-CRTAM/anti-PD-L1 antibodies, pharmaceutical compositions comprising anti-CRTAM/anti-PD-L1 antibodies, and uses thereof.
- upregulated PD-L1 binds to PD-1 on the surface of T cells, inhibits T cell activation to prevent autoimmune attack, and maintains cellular immune tolerance.
- PD-L1 is highly expressed in a variety of solid tumors, including non-small cell lung cancer, colorectal cancer, ovarian cancer, and renal cell carcinoma. Tumors use the signaling axis of PD-L1 and PD-1 to inhibit the activity and proliferation of CD8 + T cells, induce T cell anergy and exhaustion, build a local immune-tolerant tumor microenvironment, and promote tumor immune escape.
- the present invention provides an anti-CRTAM/anti-PD-L1 antibody, which includes an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region comprises a complementarity determining region 1 (H1CDR1) of the first heavy chain variable region, a complementarity determining region 2 (H1CDR2) of the first heavy chain variable region and/or a complementarity determining region 3 (H1CDR3) of the first heavy chain variable region, and the first light chain variable region comprises a complementarity determining region 1 (L1CDR1) of the first light chain variable region, a complementarity determining region 2 (L1CDR2) of the first light chain variable region and/or a complementarity determining region 3 (L1CDR3) of the first light chain variable region; and the anti-
- the present invention provides an anti-CRTAM/anti-PD-L1 antibody, comprising an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
- the first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NO: 4, 5 and 6, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 4, 5 and 6.
- (A2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A1) and having the same or similar functions as the amino acid sequence shown in (A1);
- the present invention provides an anti-CRTAM/anti-PD-L1 humanized antibody, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the light chain comprises a light chain constant region of human ⁇ , ⁇ chain or a variant thereof.
- the scFv contained in the bispecific antibody may contain the heavy chain variable region and the light chain variable region in any order.
- the scFv contained in the bispecific antibody may contain the heavy chain variable region and the light chain variable region in the direction from the N-terminus to the C-terminus and optionally a peptide linker therebetween, or alternatively, the scFv contained in the bispecific antibody may contain the light chain variable region and the heavy chain variable region in the direction from the N-terminus to the C-terminus and optionally a peptide linker therebetween.
- the peptide linker can include, for example, Gly, Asn and/or Ser residues, and can also include neutral amino acids, such as Thr and/or Ala.
- the amino acid sequence applicable to the peptide linker can be those known in the relevant art.
- the length of the peptide linker can be determined differently within such limits that the fusion protein function is not affected.
- the peptide linker can be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 groups selected from by Gly, Asn, Ser, Thr and Ala composition.
- the peptide linker can be expressed as (GmSl)n (m, l and n are independently about 1 to about 10 integers, particularly about 2 to about 5 integers).
- the full-length antibody may be in the form of a full-length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment may be a scFv.
- the antibodies described herein may contain a flexible linker sequence, or may be modified to add a functional moiety (eg, PEG, a drug, a toxin, or a label).
- a functional moiety eg, PEG, a drug, a toxin, or a label.
- the structure of the anti-PD-L1 antibody or its antigen-binding fragment is (VL-CL)-peptide linker-(VH)-IgG4CH, and the structure of the anti-CRTAM antibody or its antigen-binding fragment is self-assembled by (VL-CL) and (VH)-IgG4CH.
- the peptide linker is in the form of (GGGGS)n, wherein n is 1-12, preferably 3-10, and more preferably 6-8, for example 6, 7, or 8 GGGGS repeats.
- the IgG4CH in the (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 portion is an IgG4CH segment containing S228P, L235E, T366W, and S354C mutations to form a "Knob" structure
- the IgG4CH in the (VH)-IgG4CH targeting the CRTAM portion is an IgG4CH segment containing S228P, L235E, Y349C, T366S, L368A, and Y407V mutations to form a "Hole" structure.
- Another aspect of the present invention provides a pharmaceutical composition comprising an anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention and a pharmaceutically acceptable carrier.
- the present invention provides a pharmaceutical composition comprising an anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention, and further comprising other active components, such as other antibodies, targeted drugs, etc.
- the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions and surfactants.
- the pharmaceutically acceptable carrier is a buffered aqueous solution.
- the pharmaceutically acceptable carrier is in the form of liposomes.
- the second scFv of VH and VL against PD-L1 antigen has H2CDR1, H2DR2 and H2CDR3 of the second heavy chain variable region and L2CDR1, L2CDR2 and L2CDR3 of the second light chain variable region described in the above embodiment.
- Another aspect of the present invention provides the use of the anti-CRTAM/anti-PD-L1 bispecific antibody or anti-CRTAM antibody of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating and/or preventing a disease associated with CRTAM, PD-L1 or both.
- the disease associated with CRTAM, PD-L1 or both includes hematological tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and/or melanoma.
- the tumor can be any tumor expressing CRTAM protein, such as bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer, etc.), breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, etc.
- the tumor can be a primary or metastatic tumor.
- the present invention provides the use of the above-mentioned anti-CRTAM/anti-PD-L1 bispecific antibody or the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs, for example, the tumor is selected from blood tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.
- the anti-CRTAM/anti-PD-L1 bispecific antibody provided by the present invention has a significant anti-tumor effect and can significantly inhibit tumor growth.
- the immunogenicity of the humanized antibody is greatly reduced, and the rejection reaction of the human immune system to exogenous monoclonal antibodies is effectively eliminated. It can be used in the preparation of drugs for the treatment of various tumor diseases and has broad market prospects.
- the term "at least 80% sequence identity” refers to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
- the term “at least 85% sequence identity” refers to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
- sequence identity described in the present invention can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and identity percentage determination between two sequences can be performed by BLASTN/BLASTP algorithm on the website of National Center For Biotechnology Instutute.
- three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface.
- the antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity determining regions" or "CDRs".
- CDRs complementarity determining regions
- the “antibody” of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
- the antibody can be a complete antibody and any antigen-binding fragment or single chain thereof.
- the "antibody” of the present invention includes any protein or peptide that contains at least a portion of an Ig molecule that has a biological activity of binding to an antigen. Examples of the “antibody” of the present invention include, but are not limited to, a heavy chain or light chain CDR or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region or any portion thereof.
- the "antigen-binding fragment" described in the present invention includes Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to human CRTAM or PD-L1 with antigen binding activity.
- the Fv fragment contains the second heavy chain variable region and the second light chain variable region of the antibody, but has no constant region, and is the smallest antibody fragment with all antigen binding sites.
- the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.
- the two antibody variable regions can also be connected into a polypeptide chain using different connectors, which is called a single-chain antibody or single-chain Fv (scFv).
- the anti-CRTAM or anti-PD-L1 antibody of the present invention may be a single-chain variable region fragment (scFv), which is derived from a single-chain polypeptide of an antibody and retains the ability to bind to an antigen.
- scFv include antibody polypeptides formed by recombinant DNA technology, in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are connected via spacer sequences.
- H chain immunoglobulin heavy chain
- L chain light chain
- the antibody of the present invention refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., a molecule comprising an antigen binding site that specifically binds to (immunoreacts with) an antigen.
- "Specific binding” means that the antibody reacts with one or more antigenic determinants of an antigen but does not react with other polypeptides or binds to other polypeptides with very low affinity (Kd> 10-6 ).
- Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibodies), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv and Fab expression libraries.
- Monoclonal antibodies are antibodies obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage clonal cell lines.
- Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, and synthetic technology such as CDR grafting or other existing technologies.
- the "peptide linkers" described in the present invention may be those including 1 to 10, particularly 2 to 50, any amino acids, and may include any kind of amino acids without any limitation.
- FIG1 is a schematic diagram of the structure of the bispecific ScFab (HuPL721-C110-HK).
- a gene fragment encoding the full length of human CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO: 26.
- the nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pT-hCRTAM.
- a gene fragment encoding the full-length monkey CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO: 27.
- the nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pT-cCRTAM.
- the amino acid sequence of the fused human CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 28. After codon optimization of the human CRTAM protein extracellular region sequence, the nucleotide sequence of hCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-hCRTAM-mFc.
- amino acid sequences of the fused extracellular region of human CRTAM protein and hIgG1-Fc or His tag are shown in SEQ ID NO:29 and SEQ ID NO:30.
- the nucleotide sequences of hCRTAM-hFc and hCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-hCRTAM-hFc and pHR-hCRTAM-His.
- the amino acid sequence of the fused monkey CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 31. After codon optimization of the monkey CRTAM protein extracellular region sequence, the nucleotide sequence of cCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-cCRTAM-mFc.
- amino acid sequences of the fused monkey CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO:32 and SEQ ID NO:33.
- the nucleotide sequences of cCRTAM-hFc and cCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-cCRTAM-hFc and pHR-cCRTAM-His.
- the amino acid sequence of the fused mouse CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 34. After codon optimization of the mouse CRTAM protein extracellular region sequence, the nucleotide sequence of mCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-mCRTAM-mFc.
- amino acid sequences of the fused mouse CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO:35 and SEQ ID NO:36.
- the nucleotide sequences of mCRTAM-hFc and mCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-mCRTAM-hFc and pHR-mCRTAM-His.
- the amino acid sequence of the fused human CADM1 protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 37.
- the above amino acid sequence was codon-optimized to synthesize the nucleotide sequence of CADM1-mFc with the tag. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CADM1-mFc.
- amino acid sequences of the fused extracellular region of human CADM1 protein and hIgG1-Fc or His tag are shown in SEQ ID NO:38 and SEQ ID NO:39.
- the nucleotide sequences of CADM1-hFc and CADM1-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-CADM1-hFc and pHR-CADM1-His.
- the humanized antibody 5A11 disclosed in PCT application WO2019/086878 (hereinafter referred to as 5A11) was used as a positive control antibody.
- 5A11 was prepared according to the method disclosed in WO2019/086878.
- the amino acid sequence of 5A11 is as follows:
- 5A11 heavy chain amino acid sequence SEQ ID NO:40;
- the codons of the amino acid sequence corresponding to the 5A11 antibody were artificially optimized, and its light and heavy chain gene fragments were cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the heavy chain eukaryotic expression plasmid pHR-5A11-hG1m and the light chain expression plasmid pHR-5A11-h ⁇ of 5A11.
- the cell culture was centrifuged at 2000rpm for 5min, the supernatant was collected, and then centrifuged at 6000rpm for 20min, the supernatant was collected, and filtered using 0.45 ⁇ m and 0.22 ⁇ m filter cups, respectively, and the collected filtrate was stored in a 4°C refrigerator for purification.
- CHO-K1-hCRTAM cells were resuspended in PBS, and mice were immunized at 1 ⁇ 10 7 cells/mouse; SD rats were immunized at 2 ⁇ 10 7 cells/mouse.
- the cell suspension was added to a 9mL electrotransfer fusion tank for fusion. After fusion, transfer the cell suspension to 15mL RPMI 1640 complete medium containing 20% FBS and place at room temperature for 20 minutes. Resuspend the fused cells in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, and 20% FBS. Add the cell suspension to several 96-well cell culture plates at 100 ⁇ L/well to ensure that the cell volume per well is about 4 ⁇ 10 4 cells/well, and culture in a 37°C cell culture incubator. After 5 days, add 100 ⁇ L/well RPMI 1640 complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3).
- the monoclonal antibody cell line was determined based on the results of the subclone supernatant activity analysis and expanded.
- the culture conditions were 1640 medium containing 10% FBS, 1 ⁇ NAEE, 1 ⁇ sodium pyruvate, and 1% penicillin-streptomycin double antibody. When the cell confluence was greater than >80%, the cells were subcultured and expanded. When the culture reached about 50 mL, the supernatant was collected and the antibody was purified. The obtained antibody was determined to have good purity by SDS-PAGE gel electrophoresis.
- Universal primers were designed based on the conserved sequences at both ends of the antibody variable region (the 5' end contained a homology arm sequence for homologous recombination with a eukaryotic expression vector), and cDNA was used as a template for PCR amplification of the antibody variable region gene, thereby obtaining the gene fragments of the mouse antibody light chain and heavy chain variable regions respectively; primers were designed (references: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201: 35-55; 2.
- the amino acid sequence of the heavy chain variable region of mouse anti-C110 is SEQ ID NO: 42, and the amino acid sequence of the light chain variable region is SEQ ID NO: 43.
- the chimeric antibody C110CHI was transformed into a humanized antibody.
- the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid culture medium containing ampicillin antibiotics and cultured at 37°C with shaking for more than 12 hours.
- the bacteria were then collected for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids.
- the concentration and purity of the plasmids were detected using a nucleic acid quantitative analyzer.
- the plasmid was transfected into HEK293E cells, and a large amount of antibodies were expressed and purified, and then purity, activity and affinity tests were performed.
- the complete humanized antibody sequence is shown in Table 2.
- the experimental results show that the anti-CRTAM antibody of the present invention has a good ability to bind to human CRTAM.
- Example 6 Determination of the binding activity of anti-CRTAM antibody to cynomolgus monkey CRTAM on the cell surface (FACS)
- the binding activity of the antibody to cCRTAM on the surface of CHO-K1-cCRTAM cells was analyzed by FACS. After digestion, CHO-K1-cCRTAM cells were resuspended in 2% FBS-PBS solution and counted. The above cells were plated on a cell plate at a density of 1.5x10 5 cells per well.
- the anti-CRTAM antibody of the present invention was used as a primary antibody starting from 20 ⁇ g/ml and was added to the cell plate in a 5-fold gradient dilution, with a total of 8 concentrations, namely 20000 ng/mL, 4000 ng/mL, 800 ng/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, 1.28 ng/mL, and 0.26 ng/mL, and incubated at 4°C for 1.5 h; after washing the cells three times with PBS, the secondary antibody was incubated at 4°C for 1 h using PE-Anti-Human IgG (Biolegend, Cat. No. 409303, 1.25 ⁇ l/well); after washing with PBS three times, the fluorescence intensity generated by the binding of the antibody to the cell surface was detected by flow cytometry, and the results are shown in Table 4.
- Example 7 Determination of epitope competition activity of anti-CRTAM antibody and 5A11 on human CRTAM (ELISA)
- the blocked ELISA plate was washed 3 times with 1x PBST, and then HuC110-biotin solution with a concentration of 1 ⁇ g/mL was prepared respectively.
- the primary antibody was diluted with the above solution as the diluent.
- the anti-CRTAM antibody of the present invention (competitive positive control) and 5A11 were used as the primary antibody starting from 100 ⁇ g/mL, and 5-fold gradient dilution was added to the ELISA plate (50 ⁇ L/well), with a total of 8 concentrations, namely 100000 ng/mL, 20000 ng/mL, 4000 ng/mL, 800 ng/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, and 1.28 ng/mL, and incubated at 37°C for 1.5 h; after washing 3 times with 1x PBST, patted dry, Streptavidin-HRP (BD, 554066, 1:10000, 50 ⁇ L/well)
- Example 8 Affinity determination of anti-CRTAM antibodies and human CRTAM protein
- the affinity of the humanized anti-CRTAM antibody of the present invention to the antigen hCRTAM-His was measured using Fortebio Octet.
- the anti-CRTAM antibody was diluted to a concentration of 10 ⁇ g/mL with SD buffer (PBS+0.02% Tween20+0.1% BSA), and the antigen hCRTAM-His was diluted 4 times with SD buffer to a concentration of 12 ⁇ g/mL, 3 ⁇ g/mL, 0.75 ⁇ g/mL, and 0 ⁇ g/mL.
- the AHC sensor was used to solidify the antibody, and the affinity was measured according to the operating procedures of Fortebio Octet RED96. The specific parameters and experimental results are shown in Table 5.
- the experimental results show that the affinity of the humanized anti-CRTAM antibody of the present invention is significantly better than that of 5A11, showing a more persistent ability to bind to the human CRTAM protein.
- Resuscitate PBMC according to the required cell amount, add to 8-9 ml of IMDM complete medium, centrifuge at 900 g for 10 min, and discard the supernatant; resuspend with an appropriate amount of medium, count with a hemocytometer, and resuspend to 1 M/mL, and add 100 ⁇ L/well to a 96-well plate; prepare the antibody and Isotype to be tested with IMDM complete medium at 4 times the concentration (i.e., 40 ⁇ g/mL), 50 ⁇ L/well, mark, and vortex; add the antibody solution to the 96-well plate, add 50 ⁇ L/well of medium to the control group, place the 96-well plate in a 37°C incubator, and incubate the cells and antibodies for 1 hour; prepare SEB solution with IMDM complete medium at 4 times the concentration (i.e., 200 ng/mL), and add 50 ⁇ L/well to the corresponding wells; after incubating the 96-well plate
- Anti-CRTAM antibodies promote IFN- ⁇ release from PBMC
- HCC827 (del19) cells were purchased from the American Type Culture Collection (ATCC);
- the anti-CRTAM antibody of the present invention can better inhibit the growth of HCC827 (del19) human non-small cell lung cancer tumors.
- S228P/L235E/S354C/T366W mutations are added to the CH3domain of the anti-PD-L1 antibody single chain, and Y349C/T366S/L368A/Y407V mutations are added to the CH3domain of the anti-CRTAM antibody single chain.
- the ScFabHuPL7-21Ks, HuC110HHs and HuC110L nucleotide fragments were co-transformed into E. coli DH5 ⁇ competent cells with the linearized eukaryotic expression plasmid pHR, and the competent cells transformed with the plasmids were evenly spread on the surface of agar plates containing corresponding antibiotics, and several single colonies were picked for DNA sequencing after overnight culture in a 37°C constant temperature incubator.
- the positive clones with correct sequencing were subjected to plasmid extraction to obtain ScFabHuPL7-21Ks, HuC110HHs and HuC110L expression vectors.
- (GGGGS)8 flexible linker peptide with 8 GGGGS repeats
- the experimental results showed that the anti-PD-L1//anti-CRTAM bispecific antibody HuPL721-C110-HK has a good effect in promoting SEB to activate PBMC to release cytokine IFN- ⁇ .
- Example 16 Anti-CRTAM/anti-PD-L1 bispecific antibody induced PBMC to kill HCC827 cells
- the experimental results showed that the anti-CRTAM/anti-PD-L1 bispecific antibody HuPL721-C110-HK has a good ability to induce PBMC to kill small cell lung cancer cells.
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Abstract
The present invention relates to the field of antibody drugs, and particularly relates to an anti-CRTAM/anti-PD-L1 antibody, a drug combination comprising same and the use thereof. The anti-CRTAM/anti-PD-L1 antibody has significant anti-tumor activity and can be used in the preparation of anti-tumor drugs.
Description
本发明涉及抗体药物领域,尤其涉及抗CRTAM/抗PD-L1抗体,包含抗CRTAM/抗PD-L1抗体的药物组合物及其应用。The present invention relates to the field of antibody drugs, and in particular to anti-CRTAM/anti-PD-L1 antibodies, pharmaceutical compositions comprising anti-CRTAM/anti-PD-L1 antibodies, and uses thereof.
程序性死亡配体1(PD-L1,Programed death-ligand 1),也称为B7-H1或CD274,属于免疫球蛋白超家族的I型跨膜蛋白,分子量为40kDa,包含一个Ig-V和Ig-C样细胞外结构域、一个跨膜结构域和一个不包含典型信号基序的短胞浆尾。PD-L1在不同类型的细胞上广泛表达,包括肝细胞、血管内皮细胞以及B细胞、巨噬细胞等免疫细胞,是参与免疫抑制的PD-1配体之一。IFN-γ可诱导PD-L1的表达上调,通常情况下,上调的PD-L1与T细胞表面的PD-1结合,抑制T细胞激活以防止自身免疫攻击,维持细胞的免疫耐受。PD-L1在多种实体瘤中高表达,包括非小细胞肺癌、结直肠癌、卵巢癌和肾细胞癌等。肿瘤利用PD-L1和PD-1的信号轴,抑制CD8+T细胞的活性和增殖,诱导T细胞无能与竭耗,筑建局部免疫耐受的肿瘤微环境,促进肿瘤免疫逃逸。Programmed death-ligand 1 (PD-L1), also known as B7-H1 or CD274, is a type I transmembrane protein belonging to the immunoglobulin superfamily. It has a molecular weight of 40 kDa and contains an Ig-V and Ig-C-like extracellular domain, a transmembrane domain, and a short cytoplasmic tail that does not contain a typical signal motif. PD-L1 is widely expressed on different types of cells, including hepatocytes, vascular endothelial cells, and immune cells such as B cells and macrophages. It is one of the PD-1 ligands involved in immunosuppression. IFN-γ can induce upregulation of PD-L1 expression. Normally, upregulated PD-L1 binds to PD-1 on the surface of T cells, inhibits T cell activation to prevent autoimmune attack, and maintains cellular immune tolerance. PD-L1 is highly expressed in a variety of solid tumors, including non-small cell lung cancer, colorectal cancer, ovarian cancer, and renal cell carcinoma. Tumors use the signaling axis of PD-L1 and PD-1 to inhibit the activity and proliferation of CD8 + T cells, induce T cell anergy and exhaustion, build a local immune-tolerant tumor microenvironment, and promote tumor immune escape.
PD-L1在人非小细胞肺癌中表达,促进了癌细胞增殖;PD-L1的敲除则抑制了HCC827和PC9细胞系中肿瘤细胞的增殖并诱导其凋亡。PD-L1 is expressed in human non-small cell lung cancer and promotes cancer cell proliferation; knockout of PD-L1 inhibits tumor cell proliferation and induces apoptosis in HCC827 and PC9 cell lines.
CRTAM(Class-I-restricted T cell associated molecule),或称为CD355,是免疫球蛋白超家族的一员。CRTAM是由393氨基酸组成的I型跨膜蛋白,分子量为42.7kDa。CRTAM的表达受到NK细胞激活受体和T细胞受体(TCR)激活的严格调控,限制性的表达于激活的免疫细胞上,如活化的CD8+T细胞和NK细胞,而在正常人的PBMC中低表达,在哮喘病人的PBMC中高表达。CRTAM基因启动子受到了AP-1转录因子的正调控,TCR激活信号通过AP-1调控元件控制CRTAM在CD8+T细胞上的表达。CRTAM的配体Necl-2属于Nectin家族的黏附分子,又被称为TSLC1(The tumor suppressor in lung cancer-1)或CADM1。Necl-2与CRTAM结合作用能在体外促进NK细胞的细胞毒作用和CD8+T细胞释放IFN-γ,并在体内促进NK细胞对肿瘤的排斥反应。现有文献表明,Necl-2在肿瘤细胞表达会抑制肿瘤细胞的生长和增殖,但肿瘤细胞的Necl-2的表达会逐渐减少至沉默,为癌细胞逃脱免疫系统的监测提供了一种可能机制。CRTAM (Class-I-restricted T cell associated molecule), or CD355, is a member of the immunoglobulin superfamily. CRTAM is a type I transmembrane protein composed of 393 amino acids with a molecular weight of 42.7 kDa. The expression of CRTAM is strictly regulated by NK cell activation receptors and T cell receptor (TCR) activation, and is restricted to activated immune cells, such as activated CD8 + T cells and NK cells. It is lowly expressed in PBMCs of normal people and highly expressed in PBMCs of asthmatic patients. The CRTAM gene promoter is positively regulated by AP-1 transcription factors, and TCR activation signals control the expression of CRTAM on CD8 + T cells through AP-1 regulatory elements. CRTAM's ligand Necl-2 belongs to the Nectin family of adhesion molecules, also known as TSLC1 (The tumor suppressor in lung cancer-1) or CADM1. The binding of Necl-2 to CRTAM can promote the cytotoxicity of NK cells and the release of IFN-γ by CD8 + T cells in vitro, and promote the rejection of NK cells to tumors in vivo. Existing literature shows that the expression of Necl-2 in tumor cells will inhibit the growth and proliferation of tumor cells, but the expression of Necl-2 in tumor cells will gradually decrease to silence, providing a possible mechanism for cancer cells to escape the surveillance of the immune system.
Necl-2和CRTAM通路与PD-1-PD-L1通路具有不同的信号机制且具备互补的作用,通过开发一种靶向CRTAM与PD-L1的双功能抗体,该双特异性抗体包含PD-L1结合部分和CRTAM结合部分,既能阻断PD-L1与PD-1的结合,又能激发CRTAM,解除靶细胞对T细胞的抑制作用,并激活T细胞,促进IFN-γ的释放,增强免疫细胞对肿瘤细胞的杀伤作用。能双重阻断肿瘤的免疫逃逸机制,重新肿瘤微环境中发生免疫耐受免疫细胞,从而具备弥补PD-L1低表达肿瘤患者治疗不佳的优势,具有更好的抗肿瘤活性、靶向特异性和/或更高的安全性。Necl-2 and CRTAM pathways have different signaling mechanisms from the PD-1-PD-L1 pathway and have complementary effects. By developing a bifunctional antibody targeting CRTAM and PD-L1, the bispecific antibody contains a PD-L1 binding portion and a CRTAM binding portion, which can not only block the binding of PD-L1 to PD-1, but also stimulate CRTAM, relieve the inhibitory effect of target cells on T cells, activate T cells, promote the release of IFN-γ, and enhance the killing effect of immune cells on tumor cells. It can double block the immune escape mechanism of tumors and regenerate immune-tolerant immune cells in the tumor microenvironment, thus making up for the poor treatment of patients with low PD-L1 expression tumors, and has better anti-tumor activity, targeting specificity and/or higher safety.
发明内容Summary of the invention
本发明提供一种抗CRTAM/抗PD-L1抗体,其包括抗CRTAM抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段,其中所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中所述第一重链可变区包含第一重链可变区的互补决定区1(H1CDR1)、第一重链可变区的互补决定区2(H1CDR2)和/或第一重链可变区的互补决定区3(H1CDR3),所述第一轻链可变区包含第一轻链可变区的互补决定区1(L1CDR1)、第一轻链可变区的互补决定区2(L1CDR2)和/或第一轻链可变区的互补决定区3(L1CDR3);和所述抗PD-L1抗体或其抗原结合片段为特异性结合PD-L1的抗体或其抗原结合片段。The present invention provides an anti-CRTAM/anti-PD-L1 antibody, which includes an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region comprises a complementarity determining region 1 (H1CDR1) of the first heavy chain variable region, a complementarity determining region 2 (H1CDR2) of the first heavy chain variable region and/or a complementarity determining region 3 (H1CDR3) of the first heavy chain variable region, and the first light chain variable region comprises a complementarity determining region 1 (L1CDR1) of the first light chain variable region, a complementarity determining region 2 (L1CDR2) of the first light chain variable region and/or a complementarity determining region 3 (L1CDR3) of the first light chain variable region; and the anti-PD-L1 antibody or the antigen-binding fragment thereof is an antibody or the antigen-binding fragment thereof that specifically binds to PD-L1.
在一些实施方案中,本发明提供一种抗CRTAM/抗PD-L1抗体,其包括抗CRTAM抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段,其中所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中:In some embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, comprising an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
(1)所述第一重链可变区包含H1CDR1、H1CDR2和H1CDR3,其氨基酸序列分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85%序列同一性的氨基酸序列;和(1) the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3, whose amino acid sequences are SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; and
(2)所述第一轻链可变区包含L1CDR1、L1CDR2和L1CDR3,其氨基酸序列分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85%序列同一性的氨基酸序列。(2) The first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NO: 4, 5 and 6, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 4, 5 and 6.
在一些实施方案中,根据本发明的抗CRTAM/抗PD-L1抗体,所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中:In some embodiments, according to the anti-CRTAM/anti-PD-L1 antibody of the present invention, the anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
(1)所述第一重链可变区的氨基酸序列选自:(1) The amino acid sequence of the first heavy chain variable region is selected from:
(A1)如SEQ ID NO:17所示的氨基酸序列;(A1) the amino acid sequence shown in SEQ ID NO:17;
(A2)(A1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(A1)所示的氨基酸序列功能相同或相似的氨基酸序列;和(A2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A1) and having the same or similar functions as the amino acid sequence shown in (A1); and
(A3)与(A1)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和(A3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A1); and
(2)所述第一轻链可变区的氨基酸序列选自:(2) The amino acid sequence of the first light chain variable region is selected from:
(A4)如SEQ ID NO:19所示的氨基酸序列;(A4) the amino acid sequence shown in SEQ ID NO:19;
(A5)(A4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(A4)所示的氨基酸序列功能相同或相似的氨基酸序列;和(A5) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A4), and having the same or similar functions as the amino acid sequence shown in (A4); and
(A6)与(A4)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列。(A6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A4).
在一些实施方案中,根据本发明的抗CRTAM/抗PD-L1抗体,所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中:In some embodiments, according to the anti-CRTAM/anti-PD-L1 antibody of the present invention, the anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
所述第一重链可变区的氨基酸序列为SEQ ID NO:17所示的氨基酸序列,SEQ ID NO:17经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:17功能相同的氨基酸序列或与SEQ ID NO:17具有至少85%,或至少90%,或至少95%,或至少98%序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和3所示的氨基酸序列,且所述第一轻链可变区的氨基酸序列为SEQ ID NO:19,SEQ ID NO:19经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:19功能相同的氨基酸序列或与SEQ ID NO:19具有至少85%,或至少90%,或至少95%,或至少98%序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:4、5和6所示的氨基酸序列。The amino acid sequence of the first heavy chain variable region is the amino acid sequence shown in SEQ ID NO:17, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:17 and having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:17, and the H1CDR1, H1CDR2 and H1CDR3 are the amino acid sequences shown in SEQ ID NO:1, 2 and 3, and the amino acid sequence of the first light chain variable region is SEQ ID NO:19, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:19 and having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:19, and the L1CDR1, L1CDR2 and L1CDR3 are the amino acid sequences shown in SEQ ID NO:4, 5 and 6.
在一些实施方案中,本发明提供一种抗CRTAM/抗PD-L1抗体,其包括抗CRTAM抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段,其中所述抗CRTAM抗体或其抗原结合片段如以上实施方案中所限定;和所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和/或第二轻链可变区,其中所述第二重链可变区包含第二重链可变区的互补决定区1(H2CDR1)、第二重链可变区的互补决定区2(H2CDR2)和/或第二重链可变区的互补决定区3(H2CDR3),所述第二轻链可变区包含第二轻链可变区的互补决定区1(L2CDR1)、第二轻链可变区的互补决定区2(L2CDR2)和/或第二轻链可变区的互补决定区3(L2CDR3)。In some embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, which includes an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or its antigen-binding fragment is as defined in the above embodiments; and the anti-PD-L1 antibody or its antigen-binding fragment comprises a second heavy chain variable region and/or a second light chain variable region, wherein the second heavy chain variable region comprises a complementarity determining region 1 (H2CDR1) of a second heavy chain variable region, a complementarity determining region 2 (H2CDR2) of a second heavy chain variable region, and/or a complementarity determining region 3 (H2CDR3) of a second heavy chain variable region, and the second light chain variable region comprises a complementarity determining region 1 (L2CDR1) of a second light chain variable region, a complementarity determining region 2 (L2CDR2) of a second light chain variable region, and/or a complementarity determining region 3 (L2CDR3) of a second light chain variable region.
在一些具体的实施方案中,本发明提供一种抗CRTAM/抗PD-L1抗体,其包括抗CRTAM抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段,其中所述抗CRTAM抗体或其抗原结合片段如以上实施方案中所限定,和所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区,其中:In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, which comprises an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or an antigen-binding fragment thereof is as defined in the above embodiments, and the anti-PD-L1 antibody or an antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
(1)所述第二重链可变区包含H2CDR1、H2DR2和H2CDR3,其氨基酸序列分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85%序列同一性的氨基酸序列;和(1) the second heavy chain variable region comprises H2CDR1, H2DR2 and H2CDR3, whose amino acid sequences are SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9; and
(2)所述第二轻链可变区包含L2CDR1、L2CDR2和L2CDR3,其氨基酸序列分别为SEQ ID NO:10、11和12或与SEQ ID NO:10、11和12所示的氨基酸序列具有至少85%序列同一性的氨基酸序列。(2) The second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3, whose amino acid sequences are SEQ ID NO: 10, 11 and 12, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.
在一些具体的实施方案中,本发明提供一种抗CRTAM/抗PD-L1抗体,其包括抗CRTAM抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段,其中:In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, which includes an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:
所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中:The anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
(1)所述第一重链可变区包含H1CDR1、H1CDR2和H1CDR3,其氨基酸序列分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85%序列同一性的氨基酸序列;(1) the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3, whose amino acid sequences are SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3;
(2)所述第一轻链可变区包含L1CDR1、L1CDR2和L1CDR3,其氨基酸序列分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85%序列同一性的氨基酸序列;和(2) the first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6; and
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区,其中:The anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
(1)所述第二重链可变区包含H2CDR1、H2DR2和H2CDR3,其氨基酸序列分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85%序列同一性的氨基酸序列;(1) the second heavy chain variable region comprises H2CDR1, H2DR2 and H2CDR3, whose amino acid sequences are SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9;
(2)所述第二轻链可变区包含L2CDR1、L2CDR2和L2CDR3,其氨基酸序列分别为SEQ ID NO:10、11和12或与SEQ ID NO:10、11和12所示的氨基酸序列具有至少85%序列同一性的氨基酸序列。(2) The second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3, whose amino acid sequences are SEQ ID NO: 10, 11 and 12, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.
在一些具体的实施方案中,本发明提供一种抗CRTAM/抗PD-L1抗体,其中所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中:In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, wherein the anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
(1)所述第一重链可变区的氨基酸序列选自:(1) The amino acid sequence of the first heavy chain variable region is selected from:
(A1)如SEQ ID NO:17所示的氨基酸序列;(A1) the amino acid sequence shown in SEQ ID NO:17;
(A2)(A1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(A1)所示的氨基酸序列功能相同或相似的氨基酸序列;和(A2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A1) and having the same or similar functions as the amino acid sequence shown in (A1); and
(A3)与(A1)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和(A3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A1); and
(2)所述第一轻链可变区的氨基酸序列选自:(2) The amino acid sequence of the first light chain variable region is selected from:
(A4)如SEQ ID NO:19所示的氨基酸序列;(A4) the amino acid sequence shown in SEQ ID NO:19;
(A5)(A4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(A4)所示的氨基酸序列功能相同或相似的氨基酸序列;和(A5) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A4), and having the same or similar functions as the amino acid sequence shown in (A4); and
(A6)与(A4)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和(A6) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A4); and
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区,其中:The anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
(1)所述第二重链可变区的氨基酸序列选自:(1) The amino acid sequence of the second heavy chain variable region is selected from:
(B1)如SEQ ID NO:13所示的氨基酸序列;(B1) the amino acid sequence shown in SEQ ID NO:13;
(B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列;和(B2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B1) and having the same or similar functions as the amino acid sequence shown in (B1); and
(B3)与(B1)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和(B3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B1); and
(2)所述第二轻链可变区的氨基酸序列选自:(2) The amino acid sequence of the second light chain variable region is selected from:
(B4)如SEQ ID NO:15所示的氨基酸序列;(B4) the amino acid sequence shown in SEQ ID NO:15;
(B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列;和(B5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B4) and having the same or similar functions as the amino acid sequence shown in (B4); and
(B6)与(B4)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列。(B6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B4).
在一些具体的实施方案中,本发明提供一种抗CRTAM/抗PD-L1抗体,其中所述第一重链可变区的氨基酸序列为SEQ ID NO:17,且所述第一轻链可变区的氨基酸序列为SEQ ID NO:19;和所述第二重链可变区的氨基酸序列为SEQ ID NO:13,且所述第二轻链可变区的氨基酸序列为SEQ ID NO:15。In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:17, and the amino acid sequence of the first light chain variable region is SEQ ID NO:19; and the amino acid sequence of the second heavy chain variable region is SEQ ID NO:13, and the amino acid sequence of the second light chain variable region is SEQ ID NO:15.
在一些实施方案中,本发明提供一种抗CRTAM/抗PD-L1人源化抗体,其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区,所述轻链包含人源的κ、λ链或其变体的轻链恒定区。In some embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 humanized antibody, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the light chain comprises a light chain constant region of human κ, λ chain or a variant thereof.
在一些具体的实施方案中,根据本发明的抗CRTAM/抗PD-L1抗体,所述抗CRTAM人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区,和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中,本发明的抗CRTAM人源化抗体或其抗原结合片段还包含人源IgG4或其变体的重链恒定区,和人源κ链或其变体的轻链恒定区。In some specific embodiments, according to the anti-CRTAM/anti-PD-L1 antibody of the present invention, the anti-CRTAM humanized antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and a light chain constant region of human κ, λ chain or a variant thereof. In some preferred embodiments, the anti-CRTAM humanized antibody or its antigen-binding fragment of the present invention further comprises a heavy chain constant region of human IgG4 or a variant thereof, and a light chain constant region of human κ chain or a variant thereof.
在一些具体的实施方案中,根据本发明的抗CRTAM/抗PD-L1抗体,所述抗PD-L1人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区,和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中,本发明的抗PD-L1人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG4或其变体的重链恒定区,和人源κ链或其变体的轻链恒定区。In some specific embodiments, according to the anti-CRTAM/anti-PD-L1 antibody of the present invention, the anti-PD-L1 humanized antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and a light chain constant region of human κ, λ chain or a variant thereof. In some preferred embodiments, the anti-PD-L1 humanized antibody or its antigen-binding fragment of the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof, and a light chain constant region of human κ chain or a variant thereof.
在一些实施方案中,本发明提供抗CRTAM/抗PD-L1抗体,其中所述的抗CRTAM抗体或其抗原结合片段以及抗PD-L1抗体或其抗原结合片段分别为Fab、Fv、sFv或F(ab)2。在一个具体的实施方案中,本发明提供的抗CRTAM/抗PD-L1抗体为scF(ab)2。In some embodiments, the present invention provides anti-CRTAM/anti-PD-L1 antibodies, wherein the anti-CRTAM antibody or antigen-binding fragment thereof and the anti-PD-L1 antibody or antigen-binding fragment thereof are Fab, Fv, sFv or F(ab) 2 , respectively. In a specific embodiment, the anti-CRTAM/anti-PD-L1 antibody provided by the present invention is scF(ab) 2 .
优选地,本发明以上实施方案中的抗CRTAM/抗PD-L1抗体是抗CRTAM/抗PD-L1双特异性抗体。在一些实施方案中,所述双特异性抗体是人抗体或人源化抗体。在一些实施方案中,所述结合特异性之一是针对CRTAM,而另一个结合特异性是针对任何其它抗原。在一些实施方案中,所述结合特异性之一是针对PD-L1,而另一个结合特异性是针对CRTAM。可将本发明的双特异性抗体制备成全长抗体或抗体片段(例如F(ab')2双特异性抗体)。Preferably, the anti-CRTAM/anti-PD-L1 antibody in the above embodiments of the present invention is an anti-CRTAM/anti-PD-L1 bispecific antibody. In some embodiments, the bispecific antibody is a human antibody or a humanized antibody. In some embodiments, one of the binding specificities is for CRTAM, and the other binding specificity is for any other antigen. In some embodiments, one of the binding specificities is for PD-L1, and the other binding specificity is for CRTAM. The bispecific antibody of the present invention can be prepared as a full-length antibody or an antibody fragment (e.g., F(ab') 2 bispecific antibody).
制备双特异性抗体的方法是本领域已知的。传统上,双特异性抗体的重组制备基于两个免疫球蛋白重链-轻链对的共表达,其中两个重链具有不同的特异性(Millstein and Cuello,Nature 305:537(1983))。由于免疫球蛋白重链和轻链的随机分配,这些杂交瘤(quadroma))可能产生10种不同抗体分子的混合物,其中只有一种分子具有正确的双特异性结构。该正确分子的纯化通常通过亲和层析步骤进行,相当麻烦且产物产量低。类似的方法在WO93/08829及Trauneckeretal.,EMBOJ.10:3655(1991)中有公开。Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant preparation of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, in which the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random distribution of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, of which only one molecule has the correct bispecific structure. The purification of the correct molecule is usually carried out by affinity chromatography steps, which is quite cumbersome and the product yield is low. Similar methods are disclosed in WO93/08829 and Traunecker et al., EMBO J. 10:3655 (1991).
根据一种不同的方法,具有期望结合特异性(抗体-抗原结合位点)的抗体可变区与免疫球蛋白恒定区序列融合。在一些实施方案中,与包含至少部分铰链、CH2和CH3区的免疫球蛋白重链恒定区进行融合。在一些实施方案中,包含与轻链结合所必需位点的第二重链恒定区(CH1)存在于该融合的至少一部分中。将编码免疫球蛋白重链融合片段以及免疫球蛋白轻链(如果需要)的DNA插入不同的表达载体中并共转染入合适的宿主生物体。在用于构建的三种多肽链比例不等时提供最佳产量的实施方案中,这为调整三种多肽片段的相互比例提供极大的灵活性。不过,在至少两种多肽链以相同比例表达产生高产量时或比例没有特别意义时,有可能将两种或所有三种多肽链的编码序列插入一个表达载体中。According to a different approach, an antibody variable region with a desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. In some embodiments, the constant region of an immunoglobulin heavy chain comprising at least a portion of the hinge, CH2, and CH3 regions is fused. In some embodiments, a second heavy chain constant region (CH1) comprising a site necessary for binding to a light chain is present in at least a portion of the fusion. DNA encoding the immunoglobulin heavy chain fusion fragment and the immunoglobulin light chain (if necessary) is inserted into different expression vectors and co-transfected into a suitable host organism. In embodiments where the optimal yield is provided when the three polypeptide chains used to construct are not equal in ratio, this provides great flexibility for adjusting the mutual ratio of the three polypeptide fragments. However, when at least two polypeptide chains are expressed in the same ratio to produce high yields or when the ratio is not particularly meaningful, it is possible to insert the coding sequence of two or all three polypeptide chains into one expression vector.
在该方法的一个实施方案中,所述双特异性抗体由一个臂中具有第一结合特异性的杂合免疫球蛋白重链和另一个臂中的杂合免疫球蛋白重链-轻链对(提供第二结合特异性)组成。由于免疫球蛋白轻链仅在该双特异性分子的一半中存在提供了便利的分离途径,因此发现该不对称性结构便于将期望的双特异性物质与不想要的免疫球蛋白链组合物分开。该方法在WO94/04690中公开。关于产生双特异性抗体的进一步信息参见例如Sureshetal.,Methods in Enzymology 121:210(1986)。In one embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. Since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a convenient separation pathway, it was found that the asymmetric structure facilitates separation of the desired bispecific substance from the unwanted immunoglobulin chain composition. The method is disclosed in WO94/04690. For further information on the generation of bispecific antibodies, see, for example, Sureshetal., Methods in Enzymology 121:210 (1986).
根据另一种方法,可改造一对抗体分子间的界面以使从重组细胞培养物回收的异二聚体的百分比最大化。该界面包含抗体恒定区CH3结构域的至少一部分。在该方法中,将第一抗体分子界面的一个或多个小氨基酸侧链用较大侧链(例如酪氨酸或色氨酸)替换。通过将大氨基酸侧链用较小氨基酸侧链(例如丙氨酸或苏氨酸)替换,在第二抗体分子的界面上产生与大侧链相同或相似大小的补偿性“空腔”。这提供了提高异二聚体相比于其它不想要的终产物诸如同二聚体的产量的机制。According to another method, the interface between a pair of antibody molecules can be transformed so that the percentage of the heterodimer recovered from the recombinant cell culture is maximized. The interface comprises at least a portion of the antibody constant region CH3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (for example tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (for example alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule. This provides a mechanism to improve the output of heterodimers compared to other undesirable end products such as homodimers.
双特异性抗体包括交联或“异源缀合”抗体。例如,一种异源缀合抗体可以与亲合素偶联,另一种异源缀合抗体可以与生物素偶联。可使用任何便利的交联方法来制备异源缀合抗体。合适的交联剂是本领域众所周知的,连同许多交联技术一起在美国专利No.4,676,980中公开。Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one heteroconjugate antibody can be coupled to avidin and the other heteroconjugate antibody can be coupled to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
本发明的双特异性抗体可以由抗体片段生成。例如,可使用化学连接技术来制备双特异性抗体。Brennanetal.,Science 229:81(1985)描述了通过蛋白水解切割完整抗体以生成F(ab')2片段的方法。将这些片段在存在二硫醇络合剂亚砷酸钠的情况下(用以稳定邻近的二硫醇和防止分子间二硫键的形成)分解。然后将产生的Fab'片段转变为硫代硝基苯甲酸酯(TNB)衍生物。然后将Fab'-TNB衍生物之一通过巯基乙胺的还原重新恢复成Fab'-硫醇,并与等摩尔量的另一种Fab'-TNB衍生物混合,以形成双特异性抗体。The bispecific antibodies of the present invention can be generated by antibody fragments. For example, chemical connection techniques can be used to prepare bispecific antibodies. Brennen et al., Science 229: 81 (1985) describes a method for generating F(ab') 2 fragments by proteolytic cleavage of intact antibodies. These fragments are decomposed in the presence of a dithiol complexing agent, sodium arsenite (to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds). The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then restored to Fab'-thiol by reduction of mercaptoethylamine and mixed with another Fab'-TNB derivative in an equimolar amount to form a bispecific antibody.
可以从大肠杆菌直接回收Fab'-SH片段,这些片段可化学偶联以形成双特异性抗体。Shalaby et al.,J.Exp.Med.175:217-225(1992)描述了完全人源化的双特异性抗体F(ab')2分子的生成。每个Fab'片段由大肠杆菌单独分泌,并在体外进行定向化学偶联以形成双特异性抗体。Fab'-SH fragments can be directly recovered from E. coli, and these fragments can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the generation of fully humanized bispecific antibody F(ab') 2 molecules. Each Fab' fragment is secreted separately by E. coli and directed chemically coupled in vitro to form bispecific antibodies.
在一些实施方案中,本发明的双特异性抗体片段可以直接从重组细胞培养物生成和分离。例如,可以使用亮氨酸拉链生成双特异性抗体(Kostelnyetal.,J.Immunol.148(5):1547-1553(1992))。将来自Fos和Jun蛋白的亮氨酸拉链肽通过基因融合与两种不同抗体的Fab'部分连接。抗体同二聚体在铰链区分解以形成单体,然后重新氧化以形成抗体异二聚体。该方法也可用于生成抗体同二聚体。双抗体技术提供了制备双特异性抗体片段的其他机制。所述双特异性抗体片段包含通过接头相连的重链可变区(VH)和轻链可变区(VL),所述接头太短以使得同一条链上的两个结构域之间不能配对。因此,迫使一个片段上的VH和VL结构域与另一个片段上的互补VL和VH结构域配对,由此形成两个抗原结合位点。在另一实施方案中,可以通过使用单链Fv(sFv)二聚体构建双特异性抗体片段。In some embodiments, the bispecific antibody fragments of the present invention can be generated and isolated directly from recombinant cell culture. For example, bispecific antibodies can be generated using leucine zippers (Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992)). Leucine zipper peptides from Fos and Jun proteins are connected to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are decomposed in the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The bispecific antibody technology provides other mechanisms for preparing bispecific antibody fragments. The bispecific antibody fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker, and the linker is too short to allow the two domains on the same chain to be paired. Therefore, the VH and VL domains on one fragment are forced to pair with the complementary VL and VH domains on another fragment, thereby forming two antigen binding sites. In another embodiment, bispecific antibody fragments can be constructed through the use of single-chain Fv (sFv) dimers.
本发明涵盖具有超过两价的多价抗体,例如,可制备三特异性抗体。多价抗体可以比二价抗体更快的受到表达该抗体结合的抗原的细胞的内在化(和/或异化)。本发明的抗体可以是可容易地通过编码抗体多肽链的核酸的重组表达而生成的、具有三个或更多抗原结合位点(例如四价抗体)的多价抗体。多价抗体可包含二聚化结构域和三个或更多抗原结合位点。在一些实施方案中,二聚化结构域包含(或由其组成)Fc区或铰链区。在这种情况中,抗体会包含Fc区及Fc区氨基末端的三个或更多抗原结合位点。在一些实施方案中,多价抗体包含(或由其组成)三个至大约八个抗原结合位点。在一些实施方案中,多价抗体包含四个抗原结合位点。多价抗体包含至少一条多肽链(例如两条多肽链),其中所述多肽链包含两个或更多可变区。本发明的多价抗体可进一步包含至少两条(例如四条)轻链可变区多肽。本发明的多价抗体可包含例如约两条至约八条轻链可变区多肽。本发明的轻链可变区多肽包含轻链可变区,且任选进一步包含CL结构域。The present invention encompasses multivalent antibodies with more than two valencies, for example, trispecific antibodies can be prepared. Multivalent antibodies can be internalized (and/or catabolized) by cells expressing antigens to which the antibodies bind faster than bivalent antibodies. The antibodies of the present invention can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies) that can be easily generated by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. In some embodiments, the multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In some embodiments, the multivalent antibody comprises four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable regions. The multivalent antibody of the present invention may further comprise at least two (e.g., four) light chain variable region polypeptides. The multivalent antibody of the present invention may comprise, for example, about two to about eight light chain variable region polypeptides.The light chain variable region polypeptide of the present invention comprises a light chain variable region, and optionally further comprises a CL domain.
在包含CRTAM靶向部分和PD-L1靶向部分的双特异性抗体中,CRTAM靶向部分和PD-L1靶向部分之一可以是全长抗体,并且另一个可以是包含重链CDR、轻链CDR或其组合的抗原结合片段(例如scFv)。靶向CRTAM和PD-L1蛋白之一的全长抗体和靶向另一蛋白的抗原结合片段可以直接或通过肽接头以化学方式连接(例如共价连接)。抗原结合片段(例如scFv)可以直接或通过肽接头与全长抗体的N-末端(例如全长抗体的轻链或重链的N-末端)、全长抗体的C-末端(例如全长抗体的重链(或Fc或CH3结构域)的C-末端)或两者连接。In a bispecific antibody comprising a CRTAM targeting portion and a PD-L1 targeting portion, one of the CRTAM targeting portion and the PD-L1 targeting portion can be a full-length antibody, and the other can be an antigen-binding fragment (e.g., scFv) comprising a heavy chain CDR, a light chain CDR, or a combination thereof. A full-length antibody targeting one of the CRTAM and PD-L1 proteins and an antigen-binding fragment targeting another protein can be chemically linked (e.g., covalently linked) directly or through a peptide linker. An antigen-binding fragment (e.g., scFv) can be directly or through a peptide linker to the N-terminus of a full-length antibody (e.g., the N-terminus of a light chain or a heavy chain of a full-length antibody), the C-terminus of a full-length antibody (e.g., the C-terminus of a heavy chain (or Fc or CH3 domain) of a full-length antibody), or both.
在一个实施方案中,双特异性抗体可以包含全长抗CRTAM抗体、抗PD-L1抗体的抗原结合片段(例如scFab、scFv)以及它们之间的肽接头。在其他实施方案中,双特异性抗体可以包含全长抗CRTAM抗体、抗PD-L1抗体的抗原结合片段(例如scFab、scFv)以及它们之间的肽接头。In one embodiment, the bispecific antibody may comprise a full-length anti-CRTAM antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker therebetween. In other embodiments, the bispecific antibody may comprise a full-length anti-CRTAM antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker therebetween.
在一个实施方案中,双特异性抗体中包含的scFv可以按任何顺序包含重链可变区和轻链可变区。例如,双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包含重链可变区和轻链可变区以及任选地在它们之间的肽接头,或者可替代地,双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包含轻链可变区和重链可变区以及任选地在它们之间的肽接头In one embodiment, the scFv contained in the bispecific antibody may contain the heavy chain variable region and the light chain variable region in any order. For example, the scFv contained in the bispecific antibody may contain the heavy chain variable region and the light chain variable region in the direction from the N-terminus to the C-terminus and optionally a peptide linker therebetween, or alternatively, the scFv contained in the bispecific antibody may contain the light chain variable region and the heavy chain variable region in the direction from the N-terminus to the C-terminus and optionally a peptide linker therebetween.
在一些实施方案中,所述肽接头可包括例如Gly、Asn和/或Ser残基,并且还可以包括中性氨基酸,例如Thr和/或Ala。适用于肽接头的氨基酸序列可以是相关领域中已知的那些。同时,可以在使得融合蛋白功能不受影响的这样的限度内不同地确定肽接头的长度。例如,肽接头可以通过包括总共约1至约100、约2至约50、或约5至约25个选自由Gly、Asn、Ser、Thr、和Ala组成的组的一种或多种来形成。在一个实施方案中,肽接头可以表示为(GmSl)n(m、l和n独立地是约1至约10的整数,特别是约2至约5的整数)。In some embodiments, the peptide linker can include, for example, Gly, Asn and/or Ser residues, and can also include neutral amino acids, such as Thr and/or Ala. The amino acid sequence applicable to the peptide linker can be those known in the relevant art. Meanwhile, the length of the peptide linker can be determined differently within such limits that the fusion protein function is not affected. For example, the peptide linker can be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 groups selected from by Gly, Asn, Ser, Thr and Ala composition. In one embodiment, the peptide linker can be expressed as (GmSl)n (m, l and n are independently about 1 to about 10 integers, particularly about 2 to about 5 integers).
在另一个实施方案中,CRTAM靶向部分和PD-L1靶向部分可以均是全长抗体或包含重链CDR、轻链CDR或其组合的抗原结合片段。In another embodiment, the CRTAM targeting moiety and the PD-L1 targeting moiety can both be full-length antibodies or antigen-binding fragments comprising heavy chain CDRs, light chain CDRs, or a combination thereof.
在另一个实施方案中,双特异性抗体可以是异二聚体形式,其包含第一臂和第二臂,该第一臂包括靶向CRTAM和PD-L1之一的一对第二重链和第二轻链,该第二臂包括靶向另一者的一对第一重链和第一轻链。In another embodiment, the bispecific antibody can be in the form of a heterodimer comprising a first arm including a pair of a second heavy chain and a second light chain targeting one of CRTAM and PD-L1, and a second arm including a pair of a first heavy chain and a first light chain targeting the other.
在一个实施方案中,全长抗体可以是全长免疫球蛋白形式(例如IgG、IgM、IgA、IgE或IgD,例如人IgG、人IgM、人IgA、人IgE或人IgD),并且抗原结合片段可以选自由Fab、Fab’、F(ab’)2、Fd、Fv、scFv、scFab、单链抗体、sdFv等组成的组。例如,全长抗体可以是全长人IgG(人IgG1、人IgG2、人IgG3或人IgG4)形式,并且抗原结合片段可以是scFv。In one embodiment, the full-length antibody may be in the form of a full-length immunoglobulin (e.g., IgG, IgM, IgA, IgE, or IgD, such as human IgG, human IgM, human IgA, human IgE, or human IgD), and the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, scFv, scFab, single-chain antibody, sdFv, etc. For example, the full-length antibody may be in the form of a full-length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment may be a scFv.
例如,本文描述的抗体可以包含柔性接头序列,或者可以被修饰以添加功能部分(例如PEG、药物、毒素或标记)。For example, the antibodies described herein may contain a flexible linker sequence, or may be modified to add a functional moiety (eg, PEG, a drug, a toxin, or a label).
在一些具体的实施方案中,根据本发明的抗CRTAM/抗PD-L1双特异性抗体,所述抗PD-L1抗体或其抗原结合片段的结构为(VL-CL)-肽接头-(VH)-IgG4CH,所述抗CRTAM抗体或其抗原结合片段的结构为(VL-CL)和(VH)-IgG4CH自组装。在一些具体的实施方案中,所述肽接头为(GGGGS)n形式,其中n为1-12,优选为3-10,更优选为6-8,例如6、7、8个GGGGS重复序列。在另一些具体的实施方案中,靶向PD-L1部分的(VL-CL)-肽接头-(VH)-IgG4CH中的IgG4CH为含有S228P、L235E、T366W、S354C突变形成“Knob”结构的IgG4CH段,靶向CRTAM部分的(VH)-IgG4CH中的IgG4CH为含有S228P、L235E、Y349C、T366S、L368A、Y407V突变形成“Hole”结构的IgG4CH段。在一些具体的实施方案中,靶向PD-L1部分的(VL-CL)-肽接头-(VH)-IgG4CH中的VL的氨基酸序列为SEQ ID NO:15,CL的氨基酸序列为SEQ ID NO:16,VH的氨基酸序列为SEQ ID NO:13,IgG4CH的氨基酸序列为SEQ ID NO:14;和/或靶向CRTAM部分的(VL-CL)和(VH)-IgG4CH中的VL的氨基酸序列为SEQ ID NO:19,CL的氨基酸序列为SEQ ID NO:16,VH的氨基酸序列为SEQ ID NO:17,IgG4CH的氨基酸序列为SEQ ID NO:18。In some specific embodiments, according to the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention, the structure of the anti-PD-L1 antibody or its antigen-binding fragment is (VL-CL)-peptide linker-(VH)-IgG4CH, and the structure of the anti-CRTAM antibody or its antigen-binding fragment is self-assembled by (VL-CL) and (VH)-IgG4CH. In some specific embodiments, the peptide linker is in the form of (GGGGS)n, wherein n is 1-12, preferably 3-10, and more preferably 6-8, for example 6, 7, or 8 GGGGS repeats. In other specific embodiments, the IgG4CH in the (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 portion is an IgG4CH segment containing S228P, L235E, T366W, and S354C mutations to form a "Knob" structure, and the IgG4CH in the (VH)-IgG4CH targeting the CRTAM portion is an IgG4CH segment containing S228P, L235E, Y349C, T366S, L368A, and Y407V mutations to form a "Hole" structure. In some specific embodiments, the amino acid sequence of VL in the (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 portion is SEQ ID NO: 15, the amino acid sequence of CL is SEQ ID NO: 16, the amino acid sequence of VH is SEQ ID NO: 13, and the amino acid sequence of IgG4CH is SEQ ID NO: 14; and/or the amino acid sequence of VL in the (VL-CL) and (VH)-IgG4CH targeting the CRTAM portion is SEQ ID NO: 19, the amino acid sequence of CL is SEQ ID NO: 16, the amino acid sequence of VH is SEQ ID NO: 17, and the amino acid sequence of IgG4CH is SEQ ID NO: 18.
在一些实施方案中,根据本发明的抗PD-L1抗体或其抗原结合片段,其中所述抗体是人源化抗体或完全人抗体。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody is a humanized antibody or a fully human antibody.
本发明的另一方面提供分离的核酸。在一些实施方案中,根据本发明的分离的核酸编码本发明的抗PD-L1/抗CRTAM抗体。在一些实施方案中,根据本发明的分离的核酸编码本发明的抗CRTAM抗体或其抗原结合片段。在另一些实施方案中,根据本发明的分离的核酸编码本发明的抗PD-L1抗体或其抗原结合片段。Another aspect of the present invention provides isolated nucleic acids. In some embodiments, the isolated nucleic acids according to the present invention encode the anti-PD-L1/anti-CRTAM antibodies of the present invention. In some embodiments, the isolated nucleic acids according to the present invention encode the anti-CRTAM antibodies of the present invention or their antigen-binding fragments. In other embodiments, the isolated nucleic acids according to the present invention encode the anti-PD-L1 antibodies of the present invention or their antigen-binding fragments.
在一个具体的实施方案中,根据本发明的分离的核酸,编码第一重链可变区SEQ ID NO:17的核苷酸序列如SEQ ID NO:22所示,且编码第一轻链可变区SEQ ID NO:19的核苷酸序列如SEQ ID NO:23所示。在另一个具体的实施方案中,根据本发明的分离的核酸,编码第二重链可变区SEQ ID NO:13的核苷酸序列如SEQ ID NO:20所示,且编码第二轻链可变区SEQ ID NO:15的核苷酸序列如SEQ ID NO:21所示。In a specific embodiment, according to the isolated nucleic acid of the present invention, the nucleotide sequence encoding the first heavy chain variable region SEQ ID NO: 17 is shown as SEQ ID NO: 22, and the nucleotide sequence encoding the first light chain variable region SEQ ID NO: 19 is shown as SEQ ID NO: 23. In another specific embodiment, according to the isolated nucleic acid of the present invention, the nucleotide sequence encoding the second heavy chain variable region SEQ ID NO: 13 is shown as SEQ ID NO: 20, and the nucleotide sequence encoding the second light chain variable region SEQ ID NO: 15 is shown as SEQ ID NO: 21.
本发明的另一方面提供表达载体。在一些实施方案中,本发明的表达载体表达本发明的抗CRTAM/抗PD-L1双特异性抗体。在一些实施方案中,本发明的表达载体表达本发明的抗CRTAM抗体或其抗原结合片段。在另一些实施方案中,本发明的表达载体表达本发明的抗PD-L1抗体或其抗原结合片段。在一些实施方案中,根据本发明的表达载体,表达本发明的抗CRTAM抗体或其抗原结合片段的载体和表达抗PD-L1抗体或其抗原结合片段的载体是同种表达载体。根据本发明的表达载体其包含本发明的分离的核酸分子。Another aspect of the present invention provides an expression vector. In some embodiments, the expression vector of the present invention expresses the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention. In some embodiments, the expression vector of the present invention expresses the anti-CRTAM antibody of the present invention or its antigen-binding fragment. In other embodiments, the expression vector of the present invention expresses the anti-PD-L1 antibody of the present invention or its antigen-binding fragment. In some embodiments, according to the expression vector of the present invention, the vector expressing the anti-CRTAM antibody of the present invention or its antigen-binding fragment and the vector expressing the anti-PD-L1 antibody or its antigen-binding fragment are the same expression vectors. According to the expression vector of the present invention, it comprises an isolated nucleic acid molecule of the present invention.
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
在一些实施方案中,根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中,所述的宿主细胞为细菌,优选为大肠杆菌。在另一个优选的实施方案中,所述的宿主细胞为哺乳动物细胞。In some embodiments, the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is a bacterium, preferably Escherichia coli. In another preferred embodiment, the host cell is a mammalian cell.
本发明的另一方面提供制备本发明的抗CRTAM/抗PD-L1双特异性抗体的方法,包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。Another aspect of the present invention provides a method for preparing the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
本发明的另一方面提供一种药物组合物,其包含本发明的抗CRTAM/抗PD-L1双特异性抗体和药学可接受的载体。在一些实施方案中,本发明提供药物组合物,其包含本发明的抗CRTAM/抗PD-L1双特异性抗体,还包含其他活性组分,如其他抗体、靶向药物等。在一些实施方案中,所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中,所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中,所述药学可接受的载体为脂质体的形式。Another aspect of the present invention provides a pharmaceutical composition comprising an anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a pharmaceutical composition comprising an anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention, and further comprising other active components, such as other antibodies, targeted drugs, etc. In some embodiments, the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions and surfactants. In a specific embodiment, the pharmaceutically acceptable carrier is a buffered aqueous solution. In another specific embodiment, the pharmaceutically acceptable carrier is in the form of liposomes.
本发明的另一方面提供一种嵌合抗原受体(CAR)融合蛋白,其包含本发明的抗CRTAM抗体或其抗原结合片段和/或抗PD-L1抗体或其抗原结合片段。在一些实施方案中,所述嵌合抗原受体融合蛋白包含本发明的抗CRTAM抗体或其抗原结合片段,其为针对CRTAM抗原的VH和VL的单链可变片段(scFv)。在另一些实施方案中,所述嵌合抗原受体融合蛋白包含本发明的抗PD-L1抗体或其抗原结合片段,其为针对PD-L1抗原的VH和VL的单链可变片段(scFv)。在另一些实施方案中,所述嵌合抗原受体融合蛋白包含针对CRTAM抗原的VH和VL的第一单链可变片段(scFv)和针对PD-L1抗原的VH和VL的第二单链可变片段(scFv)。所述针对CRTAM抗原的VH和VL的第一scFv具有以上实施方案中描述的第一重链可变区的H1CDR1、H1CDR2和H1CDR3和第一轻链可变区的L1CDR1、L1CDR2和L1CDR3。所述针对PD-L1抗原的VH和VL的第二scFv具有以上实施方案中描述的第二重链可变区的H2CDR1、H2DR2和H2CDR3和第二轻链可变区的L2CDR1、L2CDR2和L2CDR3。Another aspect of the present invention provides a chimeric antigen receptor (CAR) fusion protein, which comprises an anti-CRTAM antibody or antigen binding fragment thereof and/or an anti-PD-L1 antibody or antigen binding fragment thereof of the present invention. In some embodiments, the chimeric antigen receptor fusion protein comprises an anti-CRTAM antibody or antigen binding fragment thereof of the present invention, which is a single-chain variable fragment (scFv) of V H and V L for CRTAM antigen. In other embodiments, the chimeric antigen receptor fusion protein comprises an anti-PD-L1 antibody or antigen binding fragment thereof of the present invention, which is a single-chain variable fragment (scFv) of V H and V L for PD-L1 antigen. In other embodiments, the chimeric antigen receptor fusion protein comprises a first single-chain variable fragment (scFv) of V H and V L for CRTAM antigen and a second single-chain variable fragment (scFv) of V H and V L for PD-L1 antigen. The first scFv of VH and VL against CRTAM antigen has H1CDR1, H1CDR2 and H1CDR3 of the first heavy chain variable region and L1CDR1, L1CDR2 and L1CDR3 of the first light chain variable region described in the above embodiment. The second scFv of VH and VL against PD-L1 antigen has H2CDR1, H2DR2 and H2CDR3 of the second heavy chain variable region and L2CDR1, L2CDR2 and L2CDR3 of the second light chain variable region described in the above embodiment.
可以将本发明的抗CRTAM/抗PD-L1双特异性抗体与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂,以适合于经口或胃肠外给药。给药方法包括,但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用,例如通过输注或推注,通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备,且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。The anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration. The method of administration includes, but is not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes. The preparation can be administered by any route, such as by infusion or push injection, and by a route of absorption through the epithelium or skin mucosa (such as oral mucosa or rectum, etc.). Administration can be systemic or local. The preparation can be prepared by methods known in the art and includes carriers, diluents or excipients conventionally used in the field of pharmaceutical preparations.
本发明的另一方面提供治疗和/或预防与CRTAM、PD-L1或两者相关的疾病的方法,所述方法包括向有此需要的个体施用本发明的抗CRTAM/抗PD-L1双特异性抗体或本发明的药物组合物。Another aspect of the present invention provides a method for treating and/or preventing a disease associated with CRTAM, PD-L1 or both, comprising administering the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.
本发明的另一方面提供本发明的抗CRTAM/抗PD-L1双特异性抗体或抗CRTAM抗体或本发明的药物组合物在制备治疗和/或预防与CRTAM、PD-L1或两者相关的疾病的药物中的应用。在一些实施方案中,所述与CRTAM、PD-L1或两者相关的疾病包括血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。所述肿瘤可以是任何表达CRTAM蛋白的肿瘤,例如膀胱癌、肝癌、结肠癌、直肠癌、子宫内膜癌、白血病、淋巴瘤、胰腺癌、肺癌(例如小细胞肺癌、非小细胞肺癌等)、乳腺癌、尿道癌、头颈癌、胃肠癌、胃癌、食道癌、卵巢癌、肾癌、黑素瘤、前列腺癌、甲状腺癌等。所述肿瘤可以是原发性或转移性肿瘤。在一些实施方案中,本发明提供上述抗CRTAM/抗PD-L1双特异性抗体或本发明的药物组合物在制备抗肿瘤的药物中的应用,例如所述肿瘤选自血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和黑素瘤。Another aspect of the present invention provides the use of the anti-CRTAM/anti-PD-L1 bispecific antibody or anti-CRTAM antibody of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating and/or preventing a disease associated with CRTAM, PD-L1 or both. In some embodiments, the disease associated with CRTAM, PD-L1 or both includes hematological tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and/or melanoma. The tumor can be any tumor expressing CRTAM protein, such as bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer, etc.), breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, etc. The tumor can be a primary or metastatic tumor. In some embodiments, the present invention provides the use of the above-mentioned anti-CRTAM/anti-PD-L1 bispecific antibody or the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs, for example, the tumor is selected from blood tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.
本发明提供的抗CRTAM/抗PD-L1双特异性抗体具有显著的抗肿瘤作用,可明显抑制肿瘤生长,人源化后的抗体免疫原性大大降低,有效消除人体免疫系统对外源性单抗的排异反应,可在制备用于治疗各类肿瘤疾病的药物中应用,具有广阔的市场前景。The anti-CRTAM/anti-PD-L1 bispecific antibody provided by the present invention has a significant anti-tumor effect and can significantly inhibit tumor growth. The immunogenicity of the humanized antibody is greatly reduced, and the rejection reaction of the human immune system to exogenous monoclonal antibodies is effectively eliminated. It can be used in the preparation of drugs for the treatment of various tumor diseases and has broad market prospects.
定义definition
除非另有定义,本文中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。本文中所述的细胞和组织培养、分子生物学以及蛋白质和寡或多核苷酸化学及杂交中使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和组织培养与转化(如电穿孔、脂质转染),使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等,Molecular Cloning:A Laboratory Manual)(第2版,Cold Spring Harbor Laboratory Press,纽约冷泉港(1989))。本文所述的分析化学、合成有机化学以及医学和药学化学中使用的命名以及实验室方法和技术是本领域公知且普遍使用的。Unless otherwise defined, the meaning of the scientific and technical terms used herein is the meaning commonly understood by those skilled in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. For recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (such as electroporation, lipofection), standard techniques are used. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or methods commonly used in the art or described herein. The aforementioned techniques and methods are generally used as described in a number of comprehensive and more specific documents that are well known in the art and cited and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). The nomenclature and laboratory methods and techniques used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art.
在本发明中,术语“至少80%序列同一性”是指至少80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,100%的序列同一性。在本发明中,术语“至少85%序列同一性”是指至少85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,100%的序列同一性。在一些优选的实施方案中,本发明所述的序列同一性可以至少为90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,100%。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Instutute网站上的BLASTN/BLASTP算法来进行。In the present invention, the term "at least 80% sequence identity" refers to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In the present invention, the term "at least 85% sequence identity" refers to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In some preferred embodiments, the sequence identity described in the present invention can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and identity percentage determination between two sequences can be performed by BLASTN/BLASTP algorithm on the website of National Center For Biotechnology Instutute.
在抗体分子中,轻链的三个高变区和重链的三个高变区在三维空间中以相对彼此的位置排列以形成抗原结合表面。抗原结合表面与所结合抗原的三维表面互补,且每条重链和轻链的三个高变区均被称作“互补决定区”或“CDR”。氨基酸向每个结构域的分配是根据Kabat《免疫学感兴趣的蛋白质的序列》(国立卫生研究院,马里兰州贝塞斯达(1987和1991))或Chothia和Lesk,J.Mol.Biol.196:901-917(1987),Chothia等,Nature 342:878-883(1989)定义。In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity determining regions" or "CDRs". The assignment of amino acids to each domain is based on Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989).
本发明的“抗体”是指特异性地识别并结合抗原的多肽或多肽复合物。抗体可以是完整抗体和其任何抗原结合片段或单链。本发明的“抗体”包括含有Ig分子的具有结合抗原的生物活性的至少一部分的任何蛋白质或肽。本发明“抗体”的实例包括但不限于重链或轻链的CDR或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分。The "antibody" of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. The "antibody" of the present invention includes any protein or peptide that contains at least a portion of an Ig molecule that has a biological activity of binding to an antigen. Examples of the "antibody" of the present invention include, but are not limited to, a heavy chain or light chain CDR or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region or any portion thereof.
本发明所述的“抗原结合片段”包括具有抗原结合活性的Fab片段、Fab’片段、F(ab’)2片段及与人CRTAM或PD-L1结合的Fv片段、scFv片段。Fv片段含有抗体第二重链可变区和第二轻链可变区,但没有恒定区,并具有全部抗原结合位点的最小抗体片段。一般地,Fv抗体还包含在VH和VL结构域之间的多肽接头,且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链,称为单链抗体或单链Fv(scFv)。本发明的抗CRTAM或抗PD-L1抗体可以是单链可变区片段(scFv),其源自抗体的单链多肽,保留了结合抗原的能力。scFv的实例包括通过重组DNA技术形成的抗体多肽,其中免疫球蛋白重链(H链)和轻链(L链)片段的Fv区经由间隔序列连接。制备scFv的各种方法是本领域技术人员所熟知的。The "antigen-binding fragment" described in the present invention includes Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to human CRTAM or PD-L1 with antigen binding activity. The Fv fragment contains the second heavy chain variable region and the second light chain variable region of the antibody, but has no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding. The two antibody variable regions can also be connected into a polypeptide chain using different connectors, which is called a single-chain antibody or single-chain Fv (scFv). The anti-CRTAM or anti-PD-L1 antibody of the present invention may be a single-chain variable region fragment (scFv), which is derived from a single-chain polypeptide of an antibody and retains the ability to bind to an antigen. Examples of scFv include antibody polypeptides formed by recombinant DNA technology, in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are connected via spacer sequences. Various methods for preparing scFv are well known to those skilled in the art.
本发明所述的抗体指免疫球蛋白分子或其免疫活性部分,即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10-6)结合其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体,所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。The antibody of the present invention refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., a molecule comprising an antigen binding site that specifically binds to (immunoreacts with) an antigen. "Specific binding" means that the antibody reacts with one or more antigenic determinants of an antigen but does not react with other polypeptides or binds to other polypeptides with very low affinity (Kd> 10-6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibodies), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv and Fab expression libraries. Monoclonal antibodies (mAbs) are antibodies obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, and synthetic technology such as CDR grafting or other existing technologies.
本发明所述的“双特异性抗体”指对至少两种不同抗原具有结合特异性的单克隆抗体。The "bispecific antibody" described in the present invention refers to a monoclonal antibody that has binding specificity for at least two different antigens.
本发明所述的“肽接头”可以是包括1至10,特别是2至50个任何氨基酸的那些,并且可以包括任何种类的氨基酸而没有任何限制。The "peptide linkers" described in the present invention may be those including 1 to 10, particularly 2 to 50, any amino acids, and may include any kind of amino acids without any limitation.
图1为双抗ScFab(HuPL721-C110-HK)的结构示意图。FIG1 is a schematic diagram of the structure of the bispecific ScFab (HuPL721-C110-HK).
下面代表性的实施例是为了更好地说明本发明,而非用于限制本发明的保护范围。以下实施例中未注明条件的实验方法通常按照常规条件,如冷泉港的抗体技术实验手册、分子克隆手册等,或按照原料或商品制造厂商所建议的条件进行。实施例中使用的材料、试剂如无特殊说明均为商购获得。The following representative examples are intended to better illustrate the present invention, but are not intended to limit the scope of protection of the present invention. The experimental methods in the following examples where the conditions are not specified are usually carried out under conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual, Molecular Cloning Manual, etc., or under the conditions recommended by the raw material or commodity manufacturer. The materials and reagents used in the examples were commercially available unless otherwise specified.
实施例1:CRTAM抗体相关抗原蛋白及阳性对照抗体的制备Example 1: Preparation of CRTAM antibody-related antigen protein and positive control antibody
1、抗原蛋白及阳性对照抗体的表达载体构建1. Construction of expression vectors for antigen protein and positive control antibody
(1)抗原蛋白的表达载体构建(1) Construction of expression vector for antigen protein
合成编码人源CRTAM蛋白全长的基因片段,氨基酸序列设计如SEQ ID NO:26所示。将其核苷酸序列克隆至真核表达质粒pTargetT上,获得其表达质粒pT-hCRTAM。A gene fragment encoding the full length of human CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO: 26. The nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pT-hCRTAM.
合成编码猴源CRTAM蛋白全长的基因片段,氨基酸序列设计如SEQ ID NO:27所示。将其核苷酸序列克隆至真核表达质粒pTargetT上,获得其表达质粒pT-cCRTAM。A gene fragment encoding the full-length monkey CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO: 27. The nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pT-cCRTAM.
融合的人源CRTAM蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:28所示。对人源CRTAM蛋白胞外区序列进行密码子优化后,合成带有标签的hCRTAM-mFc的核苷酸序列,并将克隆至真核表达质粒pHR上,获得其表达质粒pHR-hCRTAM-mFc。The amino acid sequence of the fused human CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 28. After codon optimization of the human CRTAM protein extracellular region sequence, the nucleotide sequence of hCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-hCRTAM-mFc.
融合的人源CRTAM蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:29和SEQ ID NO:30所示,对上述氨基酸序列进行密码子优化后合成带有标签的hCRTAM-hFc和hCRTAM-His的核苷酸序列,并将其分别克隆至真核表达质粒pHR上,获得其表达质粒pHR-hCRTAM-hFc、pHR-hCRTAM-His。The amino acid sequences of the fused extracellular region of human CRTAM protein and hIgG1-Fc or His tag are shown in SEQ ID NO:29 and SEQ ID NO:30. After codon optimization of the above amino acid sequences, the nucleotide sequences of hCRTAM-hFc and hCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-hCRTAM-hFc and pHR-hCRTAM-His.
融合的猴源CRTAM蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:31所示。对猴源CRTAM蛋白胞外区序列进行密码子优化后,合成带有标签的cCRTAM-mFc的核苷酸序列,并将克隆至真核表达质粒pHR上,获得其表达质粒pHR-cCRTAM-mFc。The amino acid sequence of the fused monkey CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 31. After codon optimization of the monkey CRTAM protein extracellular region sequence, the nucleotide sequence of cCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-cCRTAM-mFc.
融合的猴源CRTAM蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:32和SEQ ID NO:33所示,对上述氨基酸序列进行密码子优化后合成带有标签的cCRTAM-hFc和cCRTAM-His的核苷酸序列,并将其分别克隆至真核表达质粒pHR上,获得其表达质粒pHR-cCRTAM-hFc、pHR-cCRTAM-His。The amino acid sequences of the fused monkey CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO:32 and SEQ ID NO:33. After codon optimization of the above amino acid sequences, the nucleotide sequences of cCRTAM-hFc and cCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-cCRTAM-hFc and pHR-cCRTAM-His.
融合的鼠源CRTAM蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:34所示。对鼠源CRTAM蛋白胞外区序列进行密码子优化后,合成带有标签的mCRTAM-mFc的核苷酸序列,并将克隆至真核表达质粒pHR上,获得其表达质粒pHR-mCRTAM-mFc。The amino acid sequence of the fused mouse CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 34. After codon optimization of the mouse CRTAM protein extracellular region sequence, the nucleotide sequence of mCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-mCRTAM-mFc.
融合的鼠源CRTAM蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:35和SEQ ID NO:36所示,对上述氨基酸序列进行密码子优化后合成带有标签的mCRTAM-hFc和mCRTAM-His的核苷酸序列,并将其分别克隆至真核表达质粒pHR上,获得其表达质粒pHR-mCRTAM-hFc、pHR-mCRTAM-His。The amino acid sequences of the fused mouse CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO:35 and SEQ ID NO:36. After codon optimization of the above amino acid sequences, the nucleotide sequences of mCRTAM-hFc and mCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-mCRTAM-hFc and pHR-mCRTAM-His.
(2)配体蛋白的表达载体构建(2) Construction of expression vector for ligand protein
融合的人源CADM1蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:37所示,对上述氨基酸序列进行密码子优化后合成带有标签的CADM1-mFc的核苷酸序列。将其克隆至真核表达质粒pHR上,获得其表达质粒pHR-CADM1-mFc。The amino acid sequence of the fused human CADM1 protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 37. The above amino acid sequence was codon-optimized to synthesize the nucleotide sequence of CADM1-mFc with the tag. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CADM1-mFc.
融合的人源CADM1蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:38和SEQ ID NO:39所示,对上述氨基酸序列进行密码子优化后合成带有标签的CADM1-hFc和CADM1-His的核苷酸序列,并将其分别克隆至真核表达质粒pHR上,获得其表达质粒pHR-CADM1-hFc、pHR-CADM1-His。The amino acid sequences of the fused extracellular region of human CADM1 protein and hIgG1-Fc or His tag are shown in SEQ ID NO:38 and SEQ ID NO:39. After codon optimization of the above amino acid sequences, the nucleotide sequences of CADM1-hFc and CADM1-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-CADM1-hFc and pHR-CADM1-His.
(3)阳性对照抗体的表达载体构建(3) Construction of expression vector for positive control antibody
使用PCT申请WO2019/086878中公开的人源化抗体5A11(本文简称5A11)作为阳性对照抗体。参照WO2019/086878中公开的方法制备5A11。5A11的氨基酸序列如下所示:The humanized antibody 5A11 disclosed in PCT application WO2019/086878 (hereinafter referred to as 5A11) was used as a positive control antibody. 5A11 was prepared according to the method disclosed in WO2019/086878. The amino acid sequence of 5A11 is as follows:
5A11重链氨基酸序列:SEQ ID NO:40;5A11 heavy chain amino acid sequence: SEQ ID NO:40;
5A11轻链氨基酸序列:SEQ ID NO:41;5A11 light chain amino acid sequence: SEQ ID NO:41;
对5A11抗体所对应的氨基酸序列进行密码子人工优化,将其轻、重链基因片段分别克隆到真核表达质粒pHR上,获得5A11的重链真核表达质粒pHR-5A11-hG1m,轻链表达质粒pHR-5A11-hλ。The codons of the amino acid sequence corresponding to the 5A11 antibody were artificially optimized, and its light and heavy chain gene fragments were cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the heavy chain eukaryotic expression plasmid pHR-5A11-hG1m and the light chain expression plasmid pHR-5A11-hλ of 5A11.
2、抗原蛋白的表达与纯化2. Expression and purification of antigen protein
(1)表达抗原蛋白的稳定转染细胞株构建(1) Construction of stable transfected cell lines expressing antigenic proteins
将真核表达质粒pT-hCRTAM在160V电压,15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所),置于37℃,5%CO2浓度的培养箱中培养。24h后采用含1000μg/mL G418(Gibco,#10131-027)的培养基进行加压培养。转染16天后采用流式细胞术检测转染pool的阳性率,将阳性率较高的pool的细胞进行铺板(按照1×106个/mL的细胞密度,100μL/孔,铺96孔板),采用5A11抗体和Goat pAb to Hu IgG(PE)(Abcam,ab98596)抗体与细胞孵育,以流式细胞仪(ACEABIO,Novocyte 2060R)检测392nm波长下mean值,使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆,挑选出高表达hCRTAM蛋白的克隆化的CHO-K1细胞株,命名为CHO-K1-hCRTAM。The eukaryotic expression plasmid pT-hCRTAM was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) at 160V voltage and 15msec square pulses, and cultured in an incubator at 37°C and 5% CO 2 concentration. After 24h, the cells were cultured under pressure in a medium containing 1000μg/mL G418 (Gibco, #10131-027). 16 days after transfection, the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to a cell density of 1×10 6 /mL, 100μL/well, 96-well plate), and the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, and the mean value at a wavelength of 392nm was detected by flow cytometry (ACEABIO, Novocyte 2060R), and data analysis was generated using GraphPad. The positive cell lines were subcloned, and the cloned CHO-K1 cell line with high expression of hCRTAM protein was selected and named CHO-K1-hCRTAM.
将真核表达质粒pT-cCRTAM在160V电压,15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所),置于37℃,5%CO2浓度的培养箱中培养。24h后采用含1000μg/mL G418(Gibco,#10131-027)的培养基进行加压培养。转染16天后采用流式细胞术检测转染pool的阳性率,将阳性率较高的pool的细胞进行铺板(按照1×106个/mL的细胞密度,100μL/孔,铺96孔板),采用5A11抗体和Goat pAb to Hu IgG(PE)(Abcam,ab98596)抗体与细胞孵育,以流式细胞仪(ACEABIO,Novocyte 2060R)检测392nm波长下mean值,使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆,挑选出高表达cCRTAM蛋白的克隆化的CHO-K1细胞株,命名为CHO-K1-cCRTAM。The eukaryotic expression plasmid pT-cCRTAM was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) at 160V voltage and 15msec square pulses, and cultured in an incubator at 37°C and 5% CO 2 concentration. After 24h, the cells were cultured under pressure in a medium containing 1000μg/mL G418 (Gibco, #10131-027). 16 days after transfection, the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to a cell density of 1×10 6 /mL, 100μL/well, 96-well plate), and the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, and the mean value at a wavelength of 392nm was detected by flow cytometry (ACEABIO, Novocyte 2060R), and data analysis was generated using GraphPad. The positive cell lines were subcloned, and the cloned CHO-K1 cell line with high expression of cCRTAM protein was selected and named CHO-K1-cCRTAM.
(2)标签抗原蛋白的表达(2) Expression of tagged antigen protein
在1L细胞培养瓶中接种密度为1×106个/ml的293E细胞(来源于ATCC),加入新鲜的预热的FreeStyle293培养基,使接种后总体积达到250mL,置37℃,8%CO2,加湿的细胞摇床中100rpm培养过夜。取7.5mL FreeStyle293培养基,加入1mg/mL的PEI溶液500μL,混合均匀,静置5min,同时取250μg待转染质粒加入8mL FreeStyle293培养基中,混合均匀,静置5min,其中标签抗原蛋白质粒pHR-hCRTAM-mFc、pHR-hCRTAM-hFc、pHR-hCRTAM-His、pHR-cCRTAM-mFc、pHR-cCRTAM-hFc、pHR-cCRTAM-His、pHR-mCRTAM-mFc、pHR-mCRTAM-hFc、pHR-mCRTAM-His、pHR-CADM1-mFc、pHR-CADM1-hFc、pHR-CADM1-His分别转染,阳性对照抗体5A11重链质粒pHR-5A11-hG1m和轻链质粒pHR-5A11-hλ按照质量比1.1:1共同转染。将PEI与FreeStyle293培养基的混合溶液加入到质粒中,混合均匀,室温静置20min,然后加入细胞培养物中,置37℃,8%CO2,加湿的细胞摇床中100rpm培养。在细胞转染后第1天和第3天对细胞进行补料,每瓶加入12.5mL的OPM-CHO PFF05(上海奥浦迈生物科技有限公司,F81279)、5mL的葡萄糖(母液浓度为180g/L)和2.5mL的谷氨酰胺(母液浓度为200mM)。当细胞活力降至75%时,收集细胞上清。将细胞培养物2000rpm离心5min,收集上清,再6000rpm离心20min,收集上清,分别使用0.45μm和0.22μm的滤杯过滤,收集滤液保存于4℃冰箱待纯化。In a 1L cell culture flask, inoculate 293E cells (from ATCC) at a density of 1×10 6 cells/ml, add fresh preheated FreeStyle293 medium to a total volume of 250 mL, and culture overnight at 37°C, 8% CO 2 , and 100 rpm in a humidified cell shaker. Take 7.5 mL of FreeStyle293 medium, add 500 μL of 1 mg/mL PEI solution, mix well, let stand for 5 minutes, and take 250 μg of the plasmid to be transfected and add 8 mL The cells were mixed evenly in FreeStyle293 culture medium and allowed to stand for 5 minutes. The label antigen protein plasmids pHR-hCRTAM-mFc, pHR-hCRTAM-hFc, pHR-hCRTAM-His, pHR-cCRTAM-mFc, pHR-cCRTAM-hFc, pHR-cCRTAM-His, pHR-mCRTAM-mFc, pHR-mCRTAM-hFc, pHR-mCRTAM-His, pHR-CADM1-mFc, pHR-CADM1-hFc, and pHR-CADM1-His were transfected respectively, and the positive control antibody 5A11 heavy chain plasmid pHR-5A11-hG1m and light chain plasmid pHR-5A11-hλ were co-transfected at a mass ratio of 1.1:1. The mixed solution of PEI and FreeStyle293 medium was added to the plasmid, mixed evenly, and allowed to stand at room temperature for 20 minutes, then added to the cell culture, and cultured at 37°C, 8% CO2 , and 100rpm in a humidified cell shaker. On the first and third days after cell transfection, the cells were fed, and 12.5mL of OPM-CHO PFF05 (Shanghai Aopuma Biotechnology Co., Ltd., F81279), 5mL of glucose (mother liquor concentration of 180g/L) and 2.5mL of glutamine (mother liquor concentration of 200mM) were added to each bottle. When the cell viability dropped to 75%, the cell supernatant was collected. The cell culture was centrifuged at 2000rpm for 5min, the supernatant was collected, and then centrifuged at 6000rpm for 20min, the supernatant was collected, and filtered using 0.45μm and 0.22μm filter cups, respectively, and the collected filtrate was stored in a 4°C refrigerator for purification.
(3)亲和层析柱纯化(3) Affinity chromatography column purification
利用AKTA(GE,AKTA pure-150)根据蛋白性质采用亲和层析柱进行纯化。Use AKTA (GE, AKTA pure-150) to purify the protein using affinity chromatography column according to the properties of the protein.
实施例2:抗CRTAM单克隆抗体的制备Example 2: Preparation of anti-CRTAM monoclonal antibodies
1、杂交瘤单克隆的制备1. Preparation of hybridoma monoclones
(1)动物免疫(1) Animal immunization
采用不同标签的hCRTAM抗原蛋白与佐剂共同免疫实验动物,或采用CHO-K1-hCRTAM细胞株进行细胞免疫。实验动物包括Balb/c品系小鼠、SJL品系小鼠和SD大鼠。蛋白免疫:小鼠免疫按照首次免疫50μg抗原免疫一只小鼠,后期均使用抗原蛋白25μg/只;SD大鼠免疫按照首次免疫100μg抗原免疫一只大鼠,后期均使用抗原蛋白50μg/只。免疫佐剂可以是弗氏佐剂(Sigma)或Quick Antibody-Mouse5W(Q5W,北京博奥龙免疫技术有限公司)。采用弗氏佐剂乳化抗原,将不同标签的hCRTAM抗原蛋白样品逐滴加入到佐剂溶液中,边滴加边涡旋以充分混合,佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫。采用Quick Antibody-Mouse5W作为佐剂,将不同标签的hCRTAM抗原蛋白样品与Quick Antibody-Mouse5W按照1:1的体积比进行混合,混匀后即采用肌肉注射的方式,免疫SD大鼠。细胞免疫:CHO-K1-hCRTAM细胞使用PBS重悬,小鼠免疫按照1×107个细胞/只;SD大鼠免疫按照2×107个细胞/只。The experimental animals were immunized with hCRTAM antigen proteins with different labels and adjuvants, or CHO-K1-hCRTAM cell lines were used for cell immunization. The experimental animals included Balb/c mice, SJL mice and SD rats. Protein immunization: For mouse immunization, 50 μg of antigen was used to immunize one mouse for the first immunization, and 25 μg of antigen protein was used per mouse in the later period; for SD rat immunization, 100 μg of antigen was used to immunize one rat for the first immunization, and 50 μg of antigen protein was used per rat in the later period. The immune adjuvant can be Freund's adjuvant (Sigma) or Quick Antibody-Mouse5W (Q5W, Beijing Biolong Immunotechnology Co., Ltd.). Freund's adjuvant was used to emulsify the antigen, and the hCRTAM antigen protein samples with different labels were added dropwise to the adjuvant solution, and vortexed while adding to mix thoroughly. The dosage of the adjuvant was based on the instructions. After mixing evenly to form an oil-in-water emulsion, immunization was performed. Quick Antibody-Mouse5W was used as an adjuvant, and hCRTAM antigen protein samples with different labels were mixed with Quick Antibody-Mouse5W at a volume ratio of 1:1. After mixing, SD rats were immunized by intramuscular injection. Cellular immunization: CHO-K1-hCRTAM cells were resuspended in PBS, and mice were immunized at 1×10 7 cells/mouse; SD rats were immunized at 2×10 7 cells/mouse.
(2)杂交瘤融合(2) Hybridoma fusion
脾细胞的获取和制备:将加强免疫后的小鼠/大鼠处死后浸泡75%的酒精中,解剖取出脾脏,用研磨棒研磨后,经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min,弃上清。加入2mL红细胞裂解液,室温裂解红细胞2min,加入PBS至20mL,1500rpm离心7min,弃上清,重悬后进行活细胞计数。收集培养瓶中的Sp2/0细胞,1000rpm离心5min后弃上清,重悬后进行活细胞计数。按脾细胞:Sp2/0细胞=1.6:1的比例混合细胞,1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞,1500rpm离心7min。弃上清,重复一次。分别用适量电转缓冲液重悬细胞,保证细胞浓度2×107个细胞/mL左右。把细胞悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20%FBS的15mL RPMI 1640完全培养基中,室温放置20min。用含1×HAT、1×BIOMYC3、20%FBS的RPMI 1640培养基重悬融合细胞。按100μL/孔将细胞悬液加到若干块96孔细胞培养板中,保证每孔细胞量约为4×104个细胞/孔,置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20%FBS,1×HAT,1×BIOMYC-3)。Obtaining and preparing spleen cells: After boosting immunization, mice/rats were killed and soaked in 75% alcohol. The spleen was dissected and removed. After grinding with a grinding rod, it was filtered through a cell screen to prepare a single cell suspension. The spleen cell suspension was centrifuged at 2000rpm for 5min and the supernatant was discarded. 2mL of red blood cell lysis buffer was added to lyse the red blood cells at room temperature for 2min, PBS was added to 20mL, and centrifuged at 1500rpm for 7min, the supernatant was discarded, and the viable cells were counted after resuspending. Sp2/0 cells in the culture flask were collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the viable cells were counted after resuspending. The cells were mixed at a ratio of spleen cells: Sp2/0 cells = 1.6:1, and the supernatant was discarded after centrifugation at 1500rpm for 7min. The cells were resuspended with 20mL of electrotransfer buffer and centrifuged at 1500rpm for 7min. The supernatant was discarded and repeated once. The cells were resuspended with an appropriate amount of electrotransfer buffer to ensure that the cell concentration was about 2×10 7 cells/mL. The cell suspension was added to a 9mL electrotransfer fusion tank for fusion. After fusion, transfer the cell suspension to 15mL RPMI 1640 complete medium containing 20% FBS and place at room temperature for 20 minutes. Resuspend the fused cells in RPMI 1640 medium containing 1×HAT, 1×BIOMYC3, and 20% FBS. Add the cell suspension to several 96-well cell culture plates at 100μL/well to ensure that the cell volume per well is about 4×10 4 cells/well, and culture in a 37℃ cell culture incubator. After 5 days, add 100μL/well RPMI 1640 complete medium (containing 20% FBS, 1×HAT, 1×BIOMYC-3).
(3)杂交瘤及亚克隆筛选(3) Hybridoma and subclone screening
融合一周后,取杂交瘤母克隆的细胞培养上清,通过ELISA筛选结合hCRTAM蛋白和cCRTAM蛋白的杂交瘤母克隆,进一步通过流式细胞术筛选出能结合CHO-K1-hCRTAM细胞株的母克隆。One week after fusion, the cell culture supernatant of the hybridoma mother clone was taken, and the hybridoma mother clones binding to hCRTAM protein and cCRTAM protein were screened by ELISA, and the mother clones that could bind to CHO-K1-hCRTAM cell line were further screened by flow cytometry.
通过ELISA和FACS筛选出结合能力较强的母克隆,利用有限稀释法将阳性母克隆进行亚克隆,培养一周后利用ELISA检测亚克隆上清与hCRTAM蛋白的结合活性,进而获得分泌抗hCRTAM抗体的单克隆细胞株。选取较好的一个单克隆细胞株,标记为C110。The mother clones with strong binding ability were screened by ELISA and FACS, and the positive mother clones were subcloned by limiting dilution method. After one week of culture, the binding activity of the subclone supernatant with hCRTAM protein was detected by ELISA to obtain the monoclonal cell line secreting anti-hCRTAM antibody. A better monoclonal cell line was selected and marked as C110.
2、单克隆抗体的制备2. Preparation of monoclonal antibodies
根据亚克隆上清活性分析结果确定单克隆抗体细胞株,将其扩大培养。培养条件是含有10%FBS、1×NAEE、1×丙酮酸钠、1%青链霉素双抗的1640培养基。待细胞汇合度大于>80%时,进将细胞传代扩培,待培养至约50mL时收集上清,纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。The monoclonal antibody cell line was determined based on the results of the subclone supernatant activity analysis and expanded. The culture conditions were 1640 medium containing 10% FBS, 1×NAEE, 1× sodium pyruvate, and 1% penicillin-streptomycin double antibody. When the cell confluence was greater than >80%, the cells were subcultured and expanded. When the culture reached about 50 mL, the supernatant was collected and the antibody was purified. The obtained antibody was determined to have good purity by SDS-PAGE gel electrophoresis.
3、单克隆抗体测序3. Monoclonal Antibody Sequencing
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养,取适量细胞按RNeasy Plus Mini Kit(Qiagen,74134)试剂盒说明书提取总RNA,利用Prime Script 1st strand cDNA Synthesis Kit(Takara,6110A)反转录试剂盒合成cDNA第一条链。The positive hybridoma cells after subcloning were expanded and cultured. An appropriate amount of cells were taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134) kit, and the first strand of cDNA was synthesized using the Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) reverse transcription kit.
根据抗体可变区两端保守序列设计通用引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列),以cDNA为模板进行抗体可变区基因的PCR扩增,从而分别获得鼠抗轻链与重链可变区的基因片段;设计引物(参考文献:1.Anke Krebber,Susanne Bornhauser,Jorg Burmester et al.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35-55;2.SimonAntibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078),进行DNA测序获得序列。鼠抗C110的重链可变区氨基酸序列为SEQ ID NO:42,轻链可变区氨基酸序列SEQ ID NO:43。Universal primers were designed based on the conserved sequences at both ends of the antibody variable region (the 5' end contained a homology arm sequence for homologous recombination with a eukaryotic expression vector), and cDNA was used as a template for PCR amplification of the antibody variable region gene, thereby obtaining the gene fragments of the mouse antibody light chain and heavy chain variable regions respectively; primers were designed (references: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201: 35-55; 2. Simon Antibody variable-region sequencing as a method for hybridoma cell-line authentication, 2008, 78: 1071-1078), DNA sequencing was performed to obtain the sequence. The amino acid sequence of the heavy chain variable region of mouse anti-C110 is SEQ ID NO: 42, and the amino acid sequence of the light chain variable region is SEQ ID NO: 43.
实施例3:抗CRTAM嵌合抗体的构建Example 3: Construction of anti-CRTAM chimeric antibody
将纯化后的小鼠抗体重链与轻链可变区基因片段分别与线性化的含有人抗体重链或轻链恒定区的真核表达质粒pHR-hG1m/pHR-hλ共转化大肠杆菌DH5α感受态细胞。将混合液均匀涂布于含有氨苄抗生素的琼脂平板表面,于37℃恒温培养箱倒置过夜培养后分别挑取若干单菌落进行DNA测序。将测序正确的嵌合抗体标记为C110CHI。抗体C110CHI的重链可变区氨基酸序列和轻链可变区氨基酸序列与相应的鼠源抗体C110相同。The purified mouse antibody heavy chain and light chain variable region gene fragments were co-transformed with the linearized eukaryotic expression plasmid pHR-hG1m/pHR-hλ containing the human antibody heavy chain or light chain constant region into E. coli DH5α competent cells. The mixture was evenly spread on the surface of an agar plate containing ampicillin antibiotics, and several single colonies were picked for DNA sequencing after inverted overnight culture in a 37°C constant temperature incubator. The correctly sequenced chimeric antibody was labeled C110CHI. The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the antibody C110CHI are the same as those of the corresponding mouse antibody C110.
将嵌合抗体重轻链质粒共转染HEK293E细胞,表达纯化获得嵌合抗体,然后进行纯度检测、活性分析及亲和力测定。The chimeric antibody heavy and light chain plasmids were co-transfected into HEK293E cells, and the chimeric antibody was expressed and purified, and then subjected to purity detection, activity analysis and affinity determination.
实施例4:抗CRTAM人源化抗体的构建及生产Example 4: Construction and production of anti-CRTAM humanized antibodies
根据嵌合抗体的免疫活性分析,对嵌合抗体C110CHI进行人源化抗体改造。Based on the immunological activity analysis of the chimeric antibody, the chimeric antibody C110CHI was transformed into a humanized antibody.
抗体的人源化改造,首先是通过与免疫基因数据库(IMGT)中的小鼠抗体序列进行比对,确认C110CHI抗体可变区的鼠源种系,经过同源比对,C110CHI抗体的重链可变区序列的FR区与小鼠抗体种系基因(IGHV1-85*01)最为相似;抗体轻链可变区的FR序列则与小鼠抗体(IGKV1-110*01)最为相似。以C110CHI抗体框架区序列FR1-FR3作为模板,在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代C110CHI的FR1-FR3序列,重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析,综合考虑抗原性和3D结构相似度,选择C110CHI的4条人源化重链可变区和5条人源化轻链可变区进行下一步优化。C110CHI的人源化抗体非CDR区序列均达到95%以上人源化。The humanization of antibodies was first confirmed by comparing with the mouse antibody sequence in the immune gene database (IMGT) to confirm the mouse germline of the variable region of the C110CHI antibody. After homology comparison, the FR region of the heavy chain variable region sequence of the C110CHI antibody was most similar to the mouse antibody germline gene (IGHV1-85*01); the FR sequence of the antibody light chain variable region was most similar to the mouse antibody (IGKV1-110*01). Using the C110CHI antibody framework region sequence FR1-FR3 as a template, a full human framework with similar 3D structure but lower immunogenicity was searched in the human framework region library to replace the FR1-FR3 sequence of C110CHI. The full-length sequence of the heavy chain/light chain was 3D modeled and structurally compared with the heavy chain/light chain sequence of the original antibody. Taking into account the antigenicity and 3D structural similarity, 4 humanized heavy chain variable regions and 5 humanized light chain variable regions of C110CHI were selected for the next step of optimization. The non-CDR region sequences of C110CHI's humanized antibodies were more than 95% humanized.
挑选纯度、活性和亲和力均较好的人源化抗体,标记为HuC110A。利用定点突变的方法,将人源化抗体HuC110A轻链可变区第34位G基因突变为K(参见SEQ ID NO:19),提高抗体稳定性,标记为HuC110。序列见表1。Humanized antibodies with good purity, activity and affinity were selected and labeled as HuC110A. The 34th G gene of the light chain variable region of the humanized antibody HuC110A was mutated to K (see SEQ ID NO: 19) by site-directed mutagenesis to improve the stability of the antibody, which was labeled as HuC110. The sequence is shown in Table 1.
表1抗CRTAM人源化抗体序列
Table 1 Anti-CRTAM humanized antibody sequences
考虑到抗体的功能与稳定性,选择合适的抗体亚型骨架与优化后的人源化抗体可变区进行匹配,构建完整的人源化抗体。将人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列,根据表达宿主进行密码子优化,并生成相邻片段之间含有互补序列的寡核苷酸片段,通过Overlap PCR将寡核苷酸片段退火后连接起来,再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段;将纯化后的轻链可变区核苷酸片段与线性化的含有人κ轻链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞,将纯化后的重链可变区核苷酸片段与人IgG重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞,分别将转化质粒的感受态细胞均匀涂布于含有氨苄抗生素的琼脂平板表面,于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。Considering the function and stability of the antibody, the appropriate antibody subtype skeleton was selected to match the optimized humanized antibody variable region to construct a complete humanized antibody. The amino acid sequences of the light chain and heavy chain variable regions of the humanized antibody were reverse transcribed into corresponding nucleotide sequences, codons were optimized according to the expression host, and oligonucleotide fragments containing complementary sequences between adjacent fragments were generated. The oligonucleotide fragments were annealed and connected by Overlap PCR, and then the complete light chain and heavy chain variable region nucleotide fragments were amplified using specific primers (the 5' end contained a homologous arm sequence for homologous recombination with the eukaryotic expression vector); the purified light chain variable region nucleotide fragments and the linearized eukaryotic expression plasmid containing the human κ light chain constant region were co-transformed into Escherichia coli DH5α competent cells, and the purified heavy chain variable region nucleotide fragments and the eukaryotic expression plasmid containing the human IgG heavy chain constant region were co-transformed into Escherichia coli DH5α competent cells, and the competent cells of the transformed plasmids were evenly spread on the surface of agar plates containing ampicillin antibiotics, and several single colonies were picked for DNA sequencing after overnight culture in a 37°C constant temperature incubator.
将测序正确的阳性克隆接种于含有氨苄抗生素的2×YT液体培养基中,于37℃振荡培养12小时以上,然后收集菌体进行质粒提取,从而获得人源化抗体轻链与重链表达质粒,使用核酸定量分析仪检测质粒的浓度与纯度。The positive clones with correct sequencing were inoculated into 2×YT liquid culture medium containing ampicillin antibiotics and cultured at 37°C with shaking for more than 12 hours. The bacteria were then collected for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids. The concentration and purity of the plasmids were detected using a nucleic acid quantitative analyzer.
将质粒转染HEK293E细胞,表达纯化获得大量抗体,进行纯度检测、活性分析及亲和力的检测。完整的人源化抗体序列见表2。The plasmid was transfected into HEK293E cells, and a large amount of antibodies were expressed and purified, and then purity, activity and affinity tests were performed. The complete humanized antibody sequence is shown in Table 2.
表2抗CRTAM人源化抗体完整序列
Table 2 Complete sequence of anti-CRTAM humanized antibody
实施例5:抗CRTAM抗体与人CRTAM结合活性测定(ELISA)Example 5: Anti-CRTAM antibody binding activity assay for human CRTAM (ELISA)
采用ELISA分析抗体的结合活性。将hCRTAM-His蛋白(1.5μg/mL,50μL/孔,实施例1中制得)包被到96孔酶标板,于37℃孵育2h。用1x PBST清洗3次后用含5%脱脂牛奶的PBST 37℃封闭2h。用1x PBST清洗3次后,本发明的抗CRTAM抗体作为一抗从10μg/mL开始,5倍梯度稀释加入酶标板(50μL/孔),共8个浓度,浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL,37℃孵育1.5h,对照抗体为5A11(实施例1中制得);用1x PBST清洗3次后拍干,二抗使用Anti-Human IgG HRP(Jackson,109-035-003,1:5000,50μL/孔),37℃孵育1h。用1xPBST清洗5次后拍干,加入单组分TMB显色液(索莱宝,PR1200,50μL/孔),避光显色约5min,加入2M硫酸(50μL/孔)终止,使用酶标仪(thermo,Multiskan FC)读取OD450值。使用GraphPad生成EC50,结果如表3所示。The binding activity of the antibody was analyzed by ELISA. The hCRTAM-His protein (1.5 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well ELISA plate and incubated at 37°C for 2 h. After washing three times with 1x PBST, the plate was blocked with PBST containing 5% skim milk at 37°C for 2 h. After washing three times with 1x PBST, the anti-CRTAM antibody of the present invention was used as the primary antibody starting from 10 μg/mL, and was added to the ELISA plate (50 μL/well) with a 5-fold gradient dilution, with a total of 8 concentrations, namely 10000 ng/mL, 2000 ng/mL, 400 ng/mL, 80 ng/mL, 16 ng/mL, 3.2 ng/mL, 0.64 ng/mL, and 0.128 ng/mL, and incubated at 37°C for 1.5 h. The control antibody was 5A11 (prepared in Example 1). After washing three times with 1x PBST, the plate was patted dry, and Anti-Human IgG HRP (Jackson, 109-035-003, 1:5000, 50 μL/well) was used as the secondary antibody, and the plate was incubated at 37°C for 1 h. After washing with 1xPBST for 5 times, pat dry, add single-component TMB colorimetric solution (Solebol, PR1200, 50μL/well), color development for about 5 minutes in the dark, add 2M sulfuric acid (50μL/well) to terminate, and read OD450 value using a microplate reader (thermo, Multiskan FC). EC50 was generated using GraphPad, and the results are shown in Table 3.
表3抗CRTAM人源化抗体与人CRTAM结合活性
Table 3 Binding activity of anti-CRTAM humanized antibodies to human CRTAM
实验结果显示,本发明的抗CRTAM抗体具有较好的与人CRTAM结合的能力。The experimental results show that the anti-CRTAM antibody of the present invention has a good ability to bind to human CRTAM.
实施例6:抗CRTAM抗体与细胞表面食蟹猴CRTAM结合活性测定(FACS)Example 6: Determination of the binding activity of anti-CRTAM antibody to cynomolgus monkey CRTAM on the cell surface (FACS)
采用FACS分析抗体与CHO-K1-cCRTAM细胞表面的cCRTAM的结合活性。CHO-K1-cCRTAM细胞消化后,用2%FBS-PBS的溶液重悬,计数。将上述细胞按照每孔1.5x105个细胞的方式铺细胞板,本发明的抗CRTAM抗体作为一抗从20μg/ml开始,5倍梯度稀释加入细胞板,共8个浓度,浓度分别为20000ng/mL、4000ng/mL、800ng/mL、160ng/mL、32ng/mL、6.4ng/mL、1.28ng/mL、0.26ng/mL,4℃条件下孵育1.5h;使用PBS清洗三次细胞后,二抗使用PE-Anti-Human IgG(Biolegend,Cat.No.409303,1.25μl/孔)4℃条件下孵育1h;PBS三次洗涤后使用流式细胞仪检测抗体与细胞表面结合产生的荧光强度,结果如表4所示。The binding activity of the antibody to cCRTAM on the surface of CHO-K1-cCRTAM cells was analyzed by FACS. After digestion, CHO-K1-cCRTAM cells were resuspended in 2% FBS-PBS solution and counted. The above cells were plated on a cell plate at a density of 1.5x10 5 cells per well. The anti-CRTAM antibody of the present invention was used as a primary antibody starting from 20 μg/ml and was added to the cell plate in a 5-fold gradient dilution, with a total of 8 concentrations, namely 20000 ng/mL, 4000 ng/mL, 800 ng/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, 1.28 ng/mL, and 0.26 ng/mL, and incubated at 4°C for 1.5 h; after washing the cells three times with PBS, the secondary antibody was incubated at 4°C for 1 h using PE-Anti-Human IgG (Biolegend, Cat. No. 409303, 1.25 μl/well); after washing with PBS three times, the fluorescence intensity generated by the binding of the antibody to the cell surface was detected by flow cytometry, and the results are shown in Table 4.
表4抗CRTAM抗体与细胞表面食蟹猴CRTAM结合活性
Table 4 Binding activity of anti-CRTAM antibodies to cynomolgus monkey CRTAM on cell surface
实验结果显示,本发明的人源化抗CRTAM抗体具有较好的与细胞表面CRTAM结合能力。The experimental results show that the humanized anti-CRTAM antibody of the present invention has a good ability to bind to CRTAM on the cell surface.
实施例7:抗CRTAM抗体与5A11对人CRTAM的表位竞争活性测定(ELISA)Example 7: Determination of epitope competition activity of anti-CRTAM antibody and 5A11 on human CRTAM (ELISA)
采用ELISA法分析本发明的抗CRTAM抗体与5A11对人CRTAM的表位竞争活性。将hCRTAM-His蛋白(2μg/mL,50μL/孔,实施例1中制得)包被到96孔酶标板,于37℃孵育2h。用1x PBST清洗3次后用含5%脱脂牛奶的PBST37℃封闭2h。同时用Biotin-NHS esterTM(Biovsion公司,2347-50)标记本发明的抗CRTAM抗体,标记好的抗体命名为HuC110-biotin。封闭好的酶标板用1x PBST清洗3次后,分别配置浓度为1μg/mL的HuC110-biotin溶液,以上述溶液为稀释液分别稀释一抗,本发明的抗CRTAM抗体(竞争阳性对照)与5A11作为一抗从100μg/mL开始,5倍梯度稀释加入酶标板(50μL/孔),共8个浓度,浓度分别为100000ng/mL、20000ng/mL、4000ng/mL、800ng/mL、160ng/mL、32ng/mL、6.4ng/mL、1.28ng/mL,37℃孵育1.5h;用1x PBST清洗3次后拍干,二抗使用Streptavidin-HRP(BD,554066,1:10000,50μL/孔),37℃孵育1h。用1xPBST清洗5次后拍干,加入单组分TMB显色液(索莱宝,PR1200,50μL/孔),避光显色约5min,加入2M硫酸(50μL/孔)终止,使用酶标仪(thermo,Multiskan FC)读取OD450值。使用GraphPad生成IC50。The ELISA method was used to analyze the epitope competitive activity of the anti-CRTAM antibody of the present invention and 5A11 on human CRTAM. The hCRTAM-His protein (2 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well ELISA plate and incubated at 37°C for 2 h. After washing 3 times with 1x PBST, the plate was blocked with PBST containing 5% skim milk at 37°C for 2 h. At the same time, the anti-CRTAM antibody of the present invention was labeled with Biotin-NHS ester TM (Biovsion, 2347-50), and the labeled antibody was named HuC110-biotin. The blocked ELISA plate was washed 3 times with 1x PBST, and then HuC110-biotin solution with a concentration of 1 μg/mL was prepared respectively. The primary antibody was diluted with the above solution as the diluent. The anti-CRTAM antibody of the present invention (competitive positive control) and 5A11 were used as the primary antibody starting from 100 μg/mL, and 5-fold gradient dilution was added to the ELISA plate (50 μL/well), with a total of 8 concentrations, namely 100000 ng/mL, 20000 ng/mL, 4000 ng/mL, 800 ng/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, and 1.28 ng/mL, and incubated at 37°C for 1.5 h; after washing 3 times with 1x PBST, patted dry, Streptavidin-HRP (BD, 554066, 1:10000, 50 μL/well) was used as the secondary antibody, and incubated at 37°C for 1 h. After washing with 1xPBST for 5 times, pat dry, add single-component TMB colorimetric solution (Solebol, PR1200, 50 μL/well), color development for about 5 min in the dark, add 2M sulfuric acid (50 μL/well) to terminate, and read OD450 value using a microplate reader (thermo, Multiskan FC). IC50 was generated using GraphPad.
实验结果显示,本发明的人源化抗CRTAM抗体HuC110与5A11几乎不存在表位竞争,不阻断5A11与人CRTAM的结合,表明本发明的人源化抗CRTAM抗体与5A11对CRTAM的结合表位完全不同。The experimental results show that there is almost no epitope competition between the humanized anti-CRTAM antibody HuC110 of the present invention and 5A11, and it does not block the binding of 5A11 to human CRTAM, indicating that the binding epitopes of the humanized anti-CRTAM antibody of the present invention and 5A11 to CRTAM are completely different.
实施例8:抗CRTAM抗体与人CRTAM蛋白的亲和力测定Example 8: Affinity determination of anti-CRTAM antibodies and human CRTAM protein
利用Fortebio Octet对本发明的人源化抗CRTAM抗体结合抗原hCRTAM-His(实施例1中制得)的亲和力进行测定。将抗CRTAM抗体用SD缓冲液(PBS+0.02%Tween20+0.1%BSA)稀释到浓度10μg/mL,抗原hCRTAM-His用SD缓冲液4倍浓度梯度稀释,使其浓度为12μg/mL、3μg/mL、0.75μg/mL、0μg/mL,选用AHC传感器固化抗体,按Fortebio Octet RED96的操作规程进行亲和力测定,具体参数及实验结果如表5所示。The affinity of the humanized anti-CRTAM antibody of the present invention to the antigen hCRTAM-His (prepared in Example 1) was measured using Fortebio Octet. The anti-CRTAM antibody was diluted to a concentration of 10 μg/mL with SD buffer (PBS+0.02% Tween20+0.1% BSA), and the antigen hCRTAM-His was diluted 4 times with SD buffer to a concentration of 12 μg/mL, 3 μg/mL, 0.75 μg/mL, and 0 μg/mL. The AHC sensor was used to solidify the antibody, and the affinity was measured according to the operating procedures of Fortebio Octet RED96. The specific parameters and experimental results are shown in Table 5.
表5抗CRTAM抗体与人CRTAM蛋白的亲和力测定
Table 5 Affinity determination of anti-CRTAM antibodies and human CRTAM protein
实验结果显示,本发明的人源化抗CRTAM抗体的亲和力显著优于5A11,显示出更持久的与人CRTAM蛋白结合能力。The experimental results show that the affinity of the humanized anti-CRTAM antibody of the present invention is significantly better than that of 5A11, showing a more persistent ability to bind to the human CRTAM protein.
实施例9:抗CRTAM抗体的体外药效检测Example 9: In vitro efficacy testing of anti-CRTAM antibodies
采用SEB(葡萄球菌肠毒素B)刺激健康人PBMC的方法来检测抗CRTAM抗体的体外药效。The in vitro efficacy of anti-CRTAM antibodies was detected by stimulating healthy human PBMC with SEB (Staphylococcal enterotoxin B).
按所需细胞量复苏PBMC,加到8-9ml的IMDM完全培养基中,900g离心10min,弃上清;用适量培养基重悬,用血球计数板计数,并重悬为1M/mL,100μL/孔加入96孔板中;按4倍浓度(即40μg/mL),50μL/孔,用IMDM完全培养基配制待检测抗体和Isotype,做好标记,涡旋;将抗体溶液加入96孔板中,对照组加入50μL/孔的培养基,96孔板置于37℃孵箱中,细胞和抗体孵育1h;按4倍浓度(即200ng/mL)用IMDM完全培养基配制SEB溶液,50μL/孔加入对应孔中;96孔板置于37℃,5%CO2孵箱中孵育72h后,收集无细胞上清150μL,按一定比例稀释后,按照hIFN-γELISA检测试剂盒(R&DELISA试剂盒,R&D system Cat:DY285B)说明书检测上清中IFN-γ的浓度,结果如表6所示。Resuscitate PBMC according to the required cell amount, add to 8-9 ml of IMDM complete medium, centrifuge at 900 g for 10 min, and discard the supernatant; resuspend with an appropriate amount of medium, count with a hemocytometer, and resuspend to 1 M/mL, and add 100 μL/well to a 96-well plate; prepare the antibody and Isotype to be tested with IMDM complete medium at 4 times the concentration (i.e., 40 μg/mL), 50 μL/well, mark, and vortex; add the antibody solution to the 96-well plate, add 50 μL/well of medium to the control group, place the 96-well plate in a 37°C incubator, and incubate the cells and antibodies for 1 hour; prepare SEB solution with IMDM complete medium at 4 times the concentration (i.e., 200 ng/mL), and add 50 μL/well to the corresponding wells; after incubating the 96-well plate in a 37°C, 5% CO 2 incubator for 72 hours, collect 150 μL of cell-free supernatant, dilute it according to a certain ratio, and use the hIFN-γ ELISA detection kit (R&D The concentration of IFN-γ in the supernatant was detected according to the instructions of ELISA kit, R&D system Cat: DY285B. The results are shown in Table 6.
表6抗CRTAM抗体促进PBMC释放IFN-γ
Table 6 Anti-CRTAM antibodies promote IFN-γ release from PBMC
实验结果显示,与5A11相比,本发明的抗CRTAM抗体具有更好地促IFN-γ释放能力。The experimental results show that compared with 5A11, the anti-CRTAM antibody of the present invention has a better ability to promote the release of IFN-γ.
实施例10:HCC827(del19)人非小细胞肺癌细胞PBMC重建模型抗肿瘤实验Example 10: Anti-tumor experiment of HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model
1、实验材料1. Experimental Materials
(1)实验细胞及动物(1) Experimental cells and animals
HCC827(del19)细胞购自美国典型培养物保藏中心(ATCC);HCC827 (del19) cells were purchased from the American Type Culture Collection (ATCC);
NSG小鼠,雌性,6-8周龄,体重18-20克,购自百奥赛图(江苏)基因生物技术有限公司;NSG mice, female, 6-8 weeks old, weighing 18-20 g, were purchased from Biocytogen (Jiangsu) Gene Biotechnology Co., Ltd.;
(2)供试品及对照品(2) Test and control samples
对照品5A11由实施例1制得,用作阳性对照;对照品PBS用作阴性对照;试验前,将本发明的抗CRTAM抗体用PBS配制为1mg/mL。The reference substance 5A11 was prepared in Example 1 and used as a positive control; the reference substance PBS was used as a negative control; before the test, the anti-CRTAM antibody of the present invention was prepared with PBS to 1 mg/mL.
2、实验方法2. Experimental methods
HCC827(del19)人非小细胞肺癌细胞PBMC重建模型HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model
将含有1×107个HCC827(del19)细胞的50μL PBS与50μL基质胶混合后接种于小鼠的右后肢皮下,待肿瘤生长至平均体积达100mm3时开始分组。PBMC来自健康献血者的新鲜血液,于DAY1分组后给抗体前1小时进行尾静脉注射(每只小鼠注射10M的PBMC细胞)。尾静脉给药每周2次,每周两次用游标卡尺测量肿瘤直径,计算肿瘤体积,肿瘤体积的计算公式为:V=0.5a×A2,a和b分别表示肿瘤的长径和短径,计算相对肿瘤增殖率(T/C)。肿瘤生长抑制率用下列公式计算:TGI(%)=[1-(Ti-T0)/(Vi-V0)]×100,其中Ti为某一天某给药组的平均肿瘤体积,T0为此给药组在开始给药时的平均肿瘤体积;Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积,V0为溶媒对照组在给开始药时的平均肿瘤体积。记录连续给药22天测量的肿瘤体积,用GraphPad Prism绘制瘤体积生长曲线。在实验结束后检测肿瘤重量,并计算各组的肿瘤抑制率(TGI)%,结果如表7所示。50 μL PBS containing 1×10 7 HCC827 (del19) cells was mixed with 50 μL matrix gel and inoculated subcutaneously in the right hind limb of mice. Grouping began when the tumor grew to an average volume of 100 mm 3. PBMCs were fresh blood from healthy blood donors and were injected into the tail vein 1 hour before antibody administration after grouping on DAY1 (10M PBMC cells were injected into each mouse). Tail vein administration was performed twice a week, and the tumor diameter was measured with a vernier caliper twice a week to calculate the tumor volume. The calculation formula for tumor volume was: V = 0.5a × A2, a and b represent the long diameter and short diameter of the tumor, respectively, and the relative tumor proliferation rate (T/C) was calculated. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(Ti-T0)/(Vi-V0)] × 100, where Ti is the average tumor volume of a certain dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the vehicle control group at the start of drug administration. The tumor volume measured for 22 consecutive days of dosing was recorded, and the tumor volume growth curve was drawn using GraphPad Prism. After the experiment, the tumor weight was detected, and the tumor inhibition rate (TGI)% of each group was calculated. The results are shown in Table 7.
表7 HCC827(del19)人非小细胞肺癌细胞PBMC重建模型抗肿瘤结果
Table 7 Anti-tumor results of HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model
实验结果显示,相比于5A11,本发明的抗CRTAM抗体能够更好地抑制HCC827(del19)人非小细胞肺癌肿瘤生长。The experimental results show that compared with 5A11, the anti-CRTAM antibody of the present invention can better inhibit the growth of HCC827 (del19) human non-small cell lung cancer tumors.
实施例11:抗CRTAM/抗PD-L1双特异性抗体的构建、表达及纯化Example 11: Construction, expression and purification of anti-CRTAM/anti-PD-L1 bispecific antibodies
1.抗CRTAM/抗PD-L1双特异性抗体表达载体的构建1. Construction of anti-CRTAM/anti-PD-L1 bispecific antibody expression vector
(1)运用基因工程手段构建抗CRTAM/抗PD-L1的双特异性抗体,其结构形式如图1所示。该双特异性抗体是由两条分别抗CRTAM/抗PD-L1的抗体单链通过异源二聚体而形成,其中抗PD-L1抗体的轻链通过额外添加的柔性连接肽连接至抗体重链N端,该连接肽是含甘氨酸(G)和丝氨酸(S)残基的GGGGS重复序列,包含8个GGGGS的重复序列,抗CRTAM抗体分别由一条重链和一条轻链通过链间二硫键配对而成。此外,为促进异源二聚体的形成,在抗PD-L1抗体单链的CH3domain增加S228P/L235E/S354C/T366W突变,在抗CRTAM抗体单链的CH3domain增加Y349C/T366S/L368A/Y407V突变。根据该结构形式,利用抗PD-L1抗体HuPL7-21(亦称HuPL721,轻链可变区SEQ ID NO:15和恒定区SEQ ID NO:16)序列,通过基因合成获得抗PD-L1的抗体单链的核苷酸片段,即ScFabHuPL7-21Ks(参见WO2021/170082);利用抗CRTAM抗体HuC110(轻链可变区SEQ ID NO:19和恒定区SEQ ID NO:16)序列,通过基因合成获得抗CRTAM的抗体单链的轻链核苷酸片段,即HuC110L;利用抗CRTAM抗体HuC110(重链可变区SEQ ID NO:17)序列,通过基因合成获得抗CRTAM的抗体单链的重链核苷酸片段,即HuC110HHs。将该ScFabHuPL7-21Ks、HuC110HHs和HuC110L核苷酸片段(上下游均含有适当长度的同源臂)分别与线性化的真核表达质粒pHR共转化大肠杆菌DH5α感受态细胞,分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面,于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。将测序正确的阳性克隆进行质粒提取,从而获得ScFabHuPL7-21Ks、HuC110HHs和HuC110L表达载体。(1) Genetic engineering was used to construct an anti-CRTAM/anti-PD-L1 bispecific antibody, the structure of which is shown in Figure 1. The bispecific antibody is formed by two anti-CRTAM/anti-PD-L1 antibody single chains through a heterodimer, wherein the light chain of the anti-PD-L1 antibody is connected to the N-terminus of the antibody heavy chain through an additional flexible connecting peptide, and the connecting peptide is a GGGGS repeating sequence containing glycine (G) and serine (S) residues, containing 8 GGGGS repeating sequences, and the anti-CRTAM antibody is composed of a heavy chain and a light chain paired through an interchain disulfide bond. In addition, to promote the formation of heterodimers, S228P/L235E/S354C/T366W mutations are added to the CH3domain of the anti-PD-L1 antibody single chain, and Y349C/T366S/L368A/Y407V mutations are added to the CH3domain of the anti-CRTAM antibody single chain. According to this structural form, the anti-PD-L1 antibody HuPL7-21 (also known as HuPL721, light chain variable region SEQ ID NO: 15 and constant region SEQ ID NO: 16) sequence is used to obtain a single-chain nucleotide fragment of the anti-PD-L1 antibody, namely ScFabHuPL7-21Ks (see WO2021/170082) by gene synthesis; the anti-CRTAM antibody HuC110 (light chain variable region SEQ ID NO: 19 and constant region SEQ ID NO: 16) sequence is used to obtain a single-chain light chain nucleotide fragment of the anti-CRTAM antibody, namely HuC110L, by gene synthesis; the anti-CRTAM antibody HuC110 (heavy chain variable region SEQ ID NO: 17) sequence is used to obtain a single-chain heavy chain nucleotide fragment of the anti-CRTAM antibody, namely HuC110HHs, by gene synthesis. The ScFabHuPL7-21Ks, HuC110HHs and HuC110L nucleotide fragments (with homology arms of appropriate lengths in both upstream and downstream) were co-transformed into E. coli DH5α competent cells with the linearized eukaryotic expression plasmid pHR, and the competent cells transformed with the plasmids were evenly spread on the surface of agar plates containing corresponding antibiotics, and several single colonies were picked for DNA sequencing after overnight culture in a 37°C constant temperature incubator. The positive clones with correct sequencing were subjected to plasmid extraction to obtain ScFabHuPL7-21Ks, HuC110HHs and HuC110L expression vectors.
ScFabHuPL7-21Ks序列示意结构如下:
The schematic structure of the ScFabHuPL7-21Ks sequence is as follows:
其中HuPL721VL-CL:人源化PD-L1单抗HuPL7-21轻链;Among them, HuPL721VL-CL: humanized PD-L1 monoclonal antibody HuPL7-21 light chain;
(GGGGS)8:8个GGGGS重复序列的柔性连接肽;(GGGGS)8: flexible linker peptide with 8 GGGGS repeats;
HuPL721-VH:人源化PD-L1单抗HuPL7-21重链可变区;HuPL721-VH: heavy chain variable region of humanized PD-L1 monoclonal antibody HuPL7-21;
IgG4CH/Ks:含有S228P/L235E/S354C/T366W突变形成“Knobs”结构的IgG4重链恒定区(具体序列如SEQ ID NO:14);IgG4CH/Ks: IgG4 heavy chain constant region containing S228P/L235E/S354C/T366W mutations forming a "Knobs" structure (specific sequence is shown in SEQ ID NO: 14);
HuC110HHs序列示意结构如下:
The schematic structure of the HuC110HHs sequence is as follows:
其中HuC110-VH:人源化CRTAM单抗HuC110重链可变区;;Wherein HuC110-VH: heavy chain variable region of humanized CRTAM monoclonal antibody HuC110;
IgG4CH/Hs:含有S228P/L235E/Y349C/T366S/L368A/Y407V突变形成“HoLe”结构的IgG4重链恒定区(具体序列如SEQ ID NO:18);IgG4CH/Hs: IgG4 heavy chain constant region containing S228P/L235E/Y349C/T366S/L368A/Y407V mutations forming a "HoLe" structure (specific sequence is shown in SEQ ID NO: 18);
HuC110L序列示意结构如下:
The schematic structure of the HuC110L sequence is as follows:
人源化CRTAM抗体HuC110轻链。Humanized CRTAM antibody HuC110 light chain.
2.抗CRTAM/抗PD-L1双特异性抗体在Expi-CHO细胞中的瞬时表达2. Transient expression of anti-CRTAM/anti-PD-L1 bispecific antibody in Expi-CHO cells
利用Expi-Fectamine CHO Transfection Kit质粒转染试剂盒将上述的ScFabHuPL7-21Ks、HuC110HHs和HuC110L三个表达载体共重组质粒转染Expi-CHO细胞,在无血清培养基中培养12天后收集Expi-CHO细胞上清液,免疫印迹检测双特异性抗体的表达情况。其中,将由ScFabHuPL7-21Ks、HuC110HHs和HuC110L三个单链二聚化形成的双特异性抗体命名为HuPL721-C110-HK。The above-mentioned ScFabHuPL7-21Ks, HuC110HHs and HuC110L expression vectors were co-recombinantly transfected into Expi-CHO cells using the Expi-Fectamine CHO Transfection Kit plasmid transfection kit. After 12 days of culture in serum-free medium, the supernatant of Expi-CHO cells was collected and the expression of bispecific antibodies was detected by immunoblotting. Among them, the bispecific antibody formed by dimerization of three single chains of ScFabHuPL7-21Ks, HuC110HHs and HuC110L was named HuPL721-C110-HK.
3.抗CRTAM/抗PD-L1双特异性抗体的纯化3. Purification of anti-CRTAM/anti-PD-L1 bispecific antibodies
本发明的双特异性抗体在Expi-CHO细胞表达并分泌后,采用Protein A亲和层析的方法对其进行纯化,具体方法如下:Protein A亲和层析柱用缓冲液平衡后,将超滤器浓缩过的Expi-CHO细胞培养液上清液进行进样,并以A280(nm)进行监测,用清洗液洗至未结合的蛋白全部被洗脱,然后用洗脱液进行洗脱,以获得相应的双特异性抗体。纯化后的双特异性抗体SEC-HPLC检测纯度进行质量鉴定后用于后续的药学研究。SEC-HPLC鉴定结果表明,双抗HuPL721-C110-HK的纯度均达到95%以上。After the bispecific antibody of the present invention is expressed and secreted by Expi-CHO cells, it is purified by Protein A affinity chromatography. The specific method is as follows: After the Protein A affinity chromatography column is equilibrated with a buffer, the supernatant of the Expi-CHO cell culture fluid concentrated by the ultrafilter is injected, and monitored by A280 (nm), washed with a cleaning solution until all unbound proteins are eluted, and then eluted with an eluent to obtain the corresponding bispecific antibody. The purified bispecific antibody is used for subsequent pharmaceutical research after SEC-HPLC purity detection and quality identification. The SEC-HPLC identification results show that the purity of the bispecific antibody HuPL721-C110-HK is more than 95%.
实施例12:高表达hPD-L1、hCRTAM稳转细胞株的构建Example 12: Construction of a stable cell line with high expression of hPD-L1 and hCRTAM
将hPD-L1(人PD-L1)序列290个氨基酸(Primary accession:Q9NZQ7,SEQ ID NO:24)的真核表达质粒pTargeT-hPD-L1以电转的方式转染到CHO-K1细胞(来自中国科学院上海细胞生物学研究所)中,置37℃,5%CO2的培养箱中培养。48h后采用含500ug/ml G418的培养基加压培养。12天后采用FACS检测pool阳性率,将电转质粒后的细胞铺板(1x106个/ml的细胞密度,100uL/孔),将FITC anti-human PD-L1抗体(SINO BIOLOGICAL,10084-MMB6-F)与细胞在4℃孵育60min,以流式细胞仪读取FITC通道下mean值,对结果进行数据分析后,挑出阳性细胞株进行亚克隆,挑选出克隆化的CHO-K1细胞株,该细胞株高水平表达PD-L1分子,命名为CHO-K1-hPD-L1。The eukaryotic expression plasmid pTargeT-hPD-L1 of the hPD-L1 (human PD-L1) sequence of 290 amino acids (Primary accession: Q9NZQ7, SEQ ID NO: 24) was transfected into CHO-K1 cells (from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation and cultured in an incubator at 37°C and 5% CO 2. After 48 hours, the cells were cultured under pressure in a medium containing 500ug/ml G418. After 12 days, FACS was used to detect the pool positivity rate. The cells after electroporation of the plasmid were plated (cell density of 1x106 cells/ml, 100uL/well). FITC anti-human PD-L1 antibody (SINO BIOLOGICAL, 10084-MMB6-F) was incubated with the cells at 4°C for 60 min. The mean value under the FITC channel was read by flow cytometer. After data analysis of the results, the positive cell lines were selected for subcloning, and the cloned CHO-K1 cell line was selected. This cell line expressed PD-L1 molecules at a high level and was named CHO-K1-hPD-L1.
将含hCRTAM(人CRTAM)序列393个氨基酸(Primary accession:O95727,SEQ ID NO:25)的真核表达质粒pTargeT-hCRTAM以电转的方式转染到CHO-K1细胞(来自中国科学院上海细胞生物学研究所)中,置37℃,5%CO2的培养箱中培养。48h后采用含500ug/ml G418的培养基加压培养。12天后采用FACS检测pool阳性率,将电转质粒后的细胞铺板(1x106个/ml的细胞密度,100uL/孔),将PE anti-human CRTAM抗体(SINO BIOLOGICAL,11975-MM12-P)与细胞在4℃孵育60min,以流式细胞仪读取PE通道下mean值,对结果进行数据分析后,挑出阳性细胞株进行亚克隆,挑选出克隆化的CHO-K1细胞株,该细胞株高水平表达CRTAM分子,命名为CHO-K1-hCRTAM。The eukaryotic expression plasmid pTargeT-hCRTAM containing 393 amino acids of the hCRTAM (human CRTAM) sequence (Primary accession: O95727, SEQ ID NO: 25) was transfected into CHO-K1 cells (from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation and cultured in an incubator at 37°C and 5% CO 2. After 48 hours, the cells were cultured under pressure in a medium containing 500ug/ml G418. After 12 days, the pool positivity rate was detected by FACS, and the cells after electroporation of the plasmid were plated (cell density of 1x10 6 cells/ml, 100uL/well). PE anti-human CRTAM antibody (SINO BIOLOGICAL, 11975-MM12-P) was incubated with the cells at 4°C for 60 min, and the mean value under the PE channel was read by flow cytometer. After data analysis of the results, the positive cell lines were selected for subcloning, and the cloned CHO-K1 cell line was selected. This cell line expressed CRTAM molecules at a high level and was named CHO-K1-hCRTAM.
实施例13:抗CRTAM/抗PD-L1双特异性抗体ELISA体外结合与阻断实验Example 13: Anti-CRTAM/anti-PD-L1 bispecific antibody ELISA in vitro binding and blocking experiments
1.抗CRTAM/抗PD-L1双特异性抗体PD-L1结合ELISA实验1. Anti-CRTAM/anti-PD-L1 bispecific antibody PD-L1 binding ELISA experiment
将人PD-L1-His蛋白(1μg/mL,50μL/孔)包被到96孔酶标板,37℃孵育2h。用1xPBST清洗3次后用5%的脱脂牛奶4℃封闭过夜,用1xPBST清洗3次。双抗浓度从133nmoL/L开始,5倍梯度稀释加入酶标板,37℃孵1.5h,对照抗体为单抗HuPL7-21;用1xPBST清洗5次后,加入HRP-Anti-Human IgG二抗(Jackson,109-035-003,1:10000),37℃孵育40min。用1xPBST清洗5次,加入显色液TMB,终止后利用酶标仪(thermo,MuLtiskan FC)读取OD450值,EC50结果见表8。Human PD-L1-His protein (1 μg/mL, 50 μL/well) was coated onto a 96-well ELISA plate and incubated at 37°C for 2 h. After washing 3 times with 1xPBST, the plate was blocked with 5% skim milk at 4°C overnight and washed 3 times with 1xPBST. The concentration of the double antibody started from 133nmoL/L, and a 5-fold gradient dilution was added to the ELISA plate, incubated at 37°C for 1.5 h. The control antibody was the monoclonal antibody HuPL7-21; after washing 5 times with 1xPBST, HRP-Anti-Human IgG secondary antibody (Jackson, 109-035-003, 1:10000) was added and incubated at 37°C for 40 min. Wash 5 times with 1xPBST, add the colorimetric solution TMB, and read the OD450 value using an ELISA reader (thermo, MuLtiskan FC) after termination. The EC 50 results are shown in Table 8.
表8双抗与人PD-L1结合活性
Table 8 Binding activity of bispecific antibodies to human PD-L1
实验结果显示,双特异性抗体HuPL721-C110-HK具有与人PD-L1结合的能力,且结合活性与单抗HuPL7-21相当。Experimental results showed that the bispecific antibody HuPL721-C110-HK has the ability to bind to human PD-L1, and its binding activity is comparable to that of the monoclonal antibody HuPL7-21.
2.抗CRTAM/抗PD-L1双特异性抗体CRTAM结合ELISA实验2. Anti-CRTAM/anti-PD-L1 bispecific antibody CRTAM binding ELISA experiment
将人CRTAM蛋白(2μg/mL,100μL/孔)包被到96孔酶标板,37℃孵育2h。用1xPBST清洗3次后用5%的脱脂牛奶4℃封闭过夜,用1xPBST清洗3次。双抗浓度从200nmoL/L开始,5倍梯度稀释加入酶标板,37℃孵1.5h,对照抗体为5A11(专利WO2016154341);用1xPBST清洗5次后,加入HRP-Anti-Human IgG二抗(Jackson,109-035-003,1:10000),37℃孵育40min。用1xPBST清洗5次,加入显色液TMB,终止后利用酶标仪(thermo,MuLtiskan FC)读取OD450值。Human CRTAM protein (2μg/mL, 100μL/well) was coated onto a 96-well ELISA plate and incubated at 37°C for 2h. After washing with 1xPBST 3 times, the plate was blocked with 5% skim milk at 4°C overnight and washed with 1xPBST 3 times. The double antibody concentration started from 200nmoL/L, and was diluted 5-fold to add to the ELISA plate, incubated at 37°C for 1.5h. The control antibody was 5A11 (patent WO2016154341); after washing with 1xPBST 5 times, HRP-Anti-Human IgG secondary antibody (Jackson, 109-035-003, 1:10000) was added and incubated at 37°C for 40min. After washing with 1xPBST 5 times, the color developing solution TMB was added, and the OD450 value was read using an ELISA reader (thermo, MuLtiskan FC) after termination.
实验表明,双特异抗体HuPL721-C110-HK与CRTAM结合活性与单抗HuC110相当。Experiments showed that the binding activity of the bispecific antibody HuPL721-C110-HK to CRTAM was comparable to that of the monoclonal antibody HuC110.
实施例14:抗CRTAM/抗PD-L1双特异性抗体与人PD-L1和CRTAM亲和力的测定(Fortebio)Example 14: Determination of the affinity of anti-CRTAM/anti-PD-L1 bispecific antibodies to human PD-L1 and CRTAM (Fortebio)
1.抗CRTAM/抗PD-L1双特异性抗体与人PD-L1亲和力测定实验1. Anti-CRTAM/anti-PD-L1 bispecific antibody affinity test for human PD-L1
将人PD-L1-His用SD缓冲液(0.02%Tween20+0.1%BSA溶液)3倍浓度梯度稀释,使其浓度为3ug/ml、1ug/ml、0.33ug/ml、0ug/ml,将所述双特异性抗体HuPL721-C110-HK用SD缓冲液稀释到浓度10ug/ml,选用AHC传感器固化抗体蛋白,按fortebio Octet RED96的操作规程进行亲和力测定,具体参数及实验结果如表10所示。The human PD-L1-His was diluted 3 times with SD buffer (0.02% Tween20 + 0.1% BSA solution) to a concentration of 3ug/ml, 1ug/ml, 0.33ug/ml, and 0ug/ml. The bispecific antibody HuPL721-C110-HK was diluted to a concentration of 10ug/ml with SD buffer. The AHC sensor was used to solidify the antibody protein, and the affinity was measured according to the operating procedures of fortebio Octet RED96. The specific parameters and experimental results are shown in Table 10.
[根据细则26改正 11.01.2024]
表10双抗与人PD-L1蛋白结合的亲和力测定 [Corrected 11.01.2024 in accordance with Rule 26]
Table 10 Affinity determination of bispecific antibodies binding to human PD-L1 protein
表10双抗与人PD-L1蛋白结合的亲和力测定 [Corrected 11.01.2024 in accordance with Rule 26]
Table 10 Affinity determination of bispecific antibodies binding to human PD-L1 protein
实验结果表明,双特异抗体HuPL721-C110-HK与PD-L1具有较好的亲和力。2.抗CRTAM/抗PD-L1双特异性抗体与人CRTAM亲和力测定实验The experimental results show that the bispecific antibody HuPL721-C110-HK has a good affinity with PD-L1. 2. Anti-CRTAM/anti-PD-L1 bispecific antibody and human CRTAM affinity test experiment
参照实施例8的方法,利用Fortebio Octet对本发明的双特异抗体HuPL721-C110-HK结合抗原hCRTAM-His(实施例1中制得)的亲和力进行测定。按Fortebio Octet RED96的操作规程进行亲和力测定,具体参数及实验结果如表11所示。Referring to the method of Example 8, the affinity of the bispecific antibody HuPL721-C110-HK of the present invention to the antigen hCRTAM-His (prepared in Example 1) was measured using Fortebio Octet. The affinity was measured according to the operating procedures of Fortebio Octet RED96, and the specific parameters and experimental results are shown in Table 11.
[根据细则26改正 11.01.2024]
表11双抗与人CRTAM蛋白结合的亲和力测定 [Corrected 11.01.2024 in accordance with Rule 26]
Table 11 Affinity determination of the bispecific antibody binding to human CRTAM protein
表11双抗与人CRTAM蛋白结合的亲和力测定 [Corrected 11.01.2024 in accordance with Rule 26]
Table 11 Affinity determination of the bispecific antibody binding to human CRTAM protein
实验结果表明,双特异抗体HuPL721-C110-HK与CRTAM具有较好的亲和力。The experimental results showed that the bispecific antibody HuPL721-C110-HK has a good affinity with CRTAM.
实施例15:抗CRTAM/抗PD-L1双特异抗体SEB激活PBMC实验Example 15: Anti-CRTAM/anti-PD-L1 bispecific antibody SEB activation PBMC experiment
复苏PBMC,用适量IMDM培养基重悬计数后,按1M/mL,100μL/孔加入96孔板中;本发明提供的抗CRTAM/抗PD-L1双特异性抗体,初始浓度为40μg/mL(终浓度降低4倍),10倍梯度稀释,共3个浓度,按50μL/孔加入铺有PBMC的96孔板,37℃培养箱中孵育1h;用培养基配置400ng/mL SEB,50μL/孔加入96孔板中,37℃,5%CO2培养箱中孵育72h后,取无细胞上清120μL进行ELISA检测细胞因子IFN-γ的分泌。按ELISA检测试剂盒操作说明进行操作,随即利用酶标仪(thermo,Multiskan FC)读取OD450值。结果如表12所示。Resuscitate PBMC, resuspend and count with an appropriate amount of IMDM medium, and add 1M/mL, 100μL/well to a 96-well plate; the anti-CRTAM/anti-PD-L1 bispecific antibody provided by the present invention has an initial concentration of 40μg/mL (the final concentration is reduced by 4 times), 10-fold gradient dilution, a total of 3 concentrations, and 50μL/well is added to a 96-well plate with PBMC, and incubated in a 37°C incubator for 1h; 400ng/mL SEB is configured with culture medium, 50μL/well is added to a 96-well plate, and after incubation in a 37°C, 5% CO2 incubator for 72h, 120μL of cell-free supernatant is taken for ELISA detection of cytokine IFN-γ secretion. Operate according to the operating instructions of the ELISA detection kit, and then read the OD450 value using an enzyme reader (thermo, Multiskan FC). The results are shown in Table 12.
表12抗CRTAM/抗PD-L1双特异抗体促进PBMC释放IFN-γ
Table 12 Anti-CRTAM/anti-PD-L1 bispecific antibodies promote PBMC release of IFN-γ
实验结果显示,抗PD-L1//抗CRTAM双特异性抗体HuPL721-C110-HK具有很好的促进SEB激活PBMC释放细胞因子IFN-γ。The experimental results showed that the anti-PD-L1//anti-CRTAM bispecific antibody HuPL721-C110-HK has a good effect in promoting SEB to activate PBMC to release cytokine IFN-γ.
实施例16:抗CRTAM/抗PD-L1双特异性抗体诱导PBMC杀伤HCC827细胞实验Example 16: Anti-CRTAM/anti-PD-L1 bispecific antibody induced PBMC to kill HCC827 cells
第一天:取HCC827细胞用1640培养基重悬计数后,按1×104个/孔铺于96孔细胞培养板,过夜培养。复苏PBMC,计数后分瓶过夜培养,每瓶<10M,培养基:1640+10%FBS+IL-2(10ng/mL)+1%P/S。第二天:弃去孔中旧培养基,每孔加入50μl新鲜培养基,对照孔平行操作。培养基:1640+2%FBS(灭活)+1%P/S。本发明的抗CRTAM/抗PD-L1双特异性抗体及对照抗体Atezolizumab(Sino Biological,Cat:68049-H001,简写Ate),初始浓度为800nmol/L(终浓度降低4倍),10倍梯度稀释,共7个浓度,按50μl/孔加入预先铺有HCC827的细胞板。取第一天复苏的PBMC细胞,用1640+2%FBS(灭活)+1%P/S调整细胞为4×106个/mL,按照效靶比为40:1,每孔加入100ul,充分混匀后,于37℃培养箱中孵育48h。按LDH试剂盒操作说明进行操作,取细胞上清120μl与60μl LDH工作液混匀,于室温避光孵育30min,随即利用酶标仪(thermo,Multiskan FC)读取OD490值。计算各组的杀伤活性(细胞毒)%,结果如表13所示。Day 1: HCC827 cells were resuspended and counted in 1640 medium, and then plated on a 96-well cell culture plate at 1×10 4 cells/well for overnight culture. PBMCs were revived, counted, and cultured overnight in flasks, with <10M per flask, medium: 1640+10% FBS+IL-2 (10 ng/mL)+1% P/S. Day 2: The old medium in the wells was discarded, 50 μl of fresh medium was added to each well, and the control wells were operated in parallel. Medium: 1640+2% FBS (inactivated)+1% P/S. The anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention and the control antibody Atezolizumab (Sino Biological, Cat: 68049-H001, abbreviated as Ate) were prepared at an initial concentration of 800 nmol/L (the final concentration was reduced by 4 times), 10-fold gradient dilution, a total of 7 concentrations, and added to a cell plate pre-plated with HCC827 at 50 μl/well. Take the PBMC cells resuscitated on the first day, adjust the cells to 4×10 6 /mL with 1640+2% FBS (inactivated)+1% P/S, add 100ul to each well according to the effect-target ratio of 40:1, mix thoroughly, and incubate in a 37°C incubator for 48h. According to the LDH kit operating instructions, take 120μl of the cell supernatant and mix with 60μl of LDH working solution, incubate at room temperature in the dark for 30min, and then use an enzyme reader (thermo, Multiskan FC) to read the OD490 value. Calculate the killing activity (cytotoxicity)% of each group, and the results are shown in Table 13.
表13双抗诱导PBMC杀伤HCC827细胞
Table 13 Dual antibodies induce PBMC to kill HCC827 cells
实验结果显示,抗CRTAM/抗PD-L1双特异性抗体HuPL721-C110-HK具有较好的诱导PBMC杀伤小细胞肺癌细胞的能力。The experimental results showed that the anti-CRTAM/anti-PD-L1 bispecific antibody HuPL721-C110-HK has a good ability to induce PBMC to kill small cell lung cancer cells.
尽管以上已经对本发明作了详细描述,但是本领域技术人员理解,在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述,而应归属于权利要求书。Although the present invention has been described in detail above, it is understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of the present invention is not limited to the detailed description above, but should be attributed to the claims.
Claims (13)
- 一种抗CRTAM/抗PD-L1抗体,其包括抗CRTAM抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段,其中:An anti-CRTAM/anti-PD-L1 antibody, comprising an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中:The anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:(1)所述第一重链可变区包含H1CDR1、H1CDR2和H1CDR3,其氨基酸序列分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85%序列同一性的氨基酸序列;(1) the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3, whose amino acid sequences are SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3;(2)所述第一轻链可变区包含L1CDR1、L1CDR2和L1CDR3,其氨基酸序列分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85%序列同一性的氨基酸序列。(2) The first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NO: 4, 5 and 6, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 4, 5 and 6.
- 如权利要求1所述的抗CRTAM/抗PD-L1抗体,其中The anti-CRTAM/anti-PD-L1 antibody according to claim 1, wherein所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区,其中:The anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:(1)所述第二重链可变区包含H2CDR1、H2DR2和H2CDR3,其氨基酸序列分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85%序列同一性的氨基酸序列;和(1) the second heavy chain variable region comprises H2CDR1, H2DR2 and H2CDR3, whose amino acid sequences are SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9; and(2)所述第二轻链可变区包含L2CDR1、L2CDR2和L2CDR3,其氨基酸序列分别为SEQ ID NO:10、11和12或与SEQ ID NO:10、11和12所示的氨基酸序列具有至少85%序列同一性的氨基酸序列。(2) The second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3, whose amino acid sequences are SEQ ID NO: 10, 11 and 12, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.
- 如权利要求1或2所述的抗CRTAM/抗PD-L1抗体,其中:The anti-CRTAM/anti-PD-L1 antibody according to claim 1 or 2, wherein:所述抗CRTAM抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区,其中:The anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:(1)所述第一重链可变区的氨基酸序列选自:(1) The amino acid sequence of the first heavy chain variable region is selected from:(A1)如SEQ ID NO:17所示的氨基酸序列;(A1) the amino acid sequence shown in SEQ ID NO:17;(A2)(A1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(A1)所示的氨基酸序列功能相同或相似的氨基酸序列;和(A2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A1), and having the same or similar functions as the amino acid sequence shown in (A1); and(A3)与(A1)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和 (A3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A1); and(2)所述第一轻链可变区的氨基酸序列选自:(2) The amino acid sequence of the first light chain variable region is selected from:(A4)如SEQ ID NO:19所示的氨基酸序列;(A4) the amino acid sequence shown in SEQ ID NO:19;(A5)(A4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(A4)所示的氨基酸序列功能相同或相似的氨基酸序列;和(A5) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A4), and having the same or similar functions as the amino acid sequence shown in (A4); and(A6)与(A4)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列。(A6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A4).
- 如权利要求1-3之任一项所述的抗CRTAM/抗PD-L1抗体,其中所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区,其中:The anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 3, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:(1)所述第二重链可变区的氨基酸序列选自:(1) The amino acid sequence of the second heavy chain variable region is selected from:(B1)如SEQ ID NO:13所示的氨基酸序列;(B1) the amino acid sequence shown in SEQ ID NO:13;(B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列;和(B2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B1) and having the same or similar functions as the amino acid sequence shown in (B1); and(B3)与(B1)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和(B3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B1); and(2)所述第二轻链可变区的氨基酸序列选自:(2) The amino acid sequence of the second light chain variable region is selected from:(B4)如SEQ ID NO:15所示的氨基酸序列;(B4) the amino acid sequence shown in SEQ ID NO:15;(B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列;和(B5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B4) and having the same or similar functions as the amino acid sequence shown in (B4); and(B6)与(B4)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列。(B6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B4).
- 如权利要求1-4之任一项所述的抗CRTAM/抗PD-L1抗体,其中所述抗体是人源化抗体或完全人抗体。The anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 4, wherein the antibody is a humanized antibody or a fully human antibody.
- 如权利要求1-5之任一项所述的抗CRTAM/抗PD-L1抗体,其中所述抗体是双特异性抗体。The anti-CRTAM/anti-PD-L1 antibody of any one of claims 1 to 5, wherein the antibody is a bispecific antibody.
- 一种分离的核酸,其编码权利要求1-6之任一项所述的抗CRTAM/抗PD-L1抗体。An isolated nucleic acid encoding the anti-CRTAM/anti-PD-L1 antibody of any one of claims 1-6.
- 如权利要求7所述的核酸,其中:The nucleic acid of claim 7, wherein:(1)编码所述第一重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:22所示;(1) the nucleotide sequence encoding the amino acid sequence of the first heavy chain variable region is shown in SEQ ID NO: 22;(2)编码所述第一轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:23所示;(2) the nucleotide sequence encoding the amino acid sequence of the first light chain variable region is shown in SEQ ID NO: 23;(3)编码所述第二重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:20所 示;和(3) The nucleotide sequence encoding the amino acid sequence of the second heavy chain variable region is as shown in SEQ ID NO: 20 show; and(4)编码所述第二轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:21所示。(4) The nucleotide sequence encoding the amino acid sequence of the second light chain variable region is shown in SEQ ID NO:21.
- 一种表达载体,其包含如权利要求7或8所述的核酸。An expression vector comprising the nucleic acid according to claim 7 or 8.
- 一种宿主细胞,其转化如权利要求9所述的表达载体,所述宿主细胞选自原核细胞和真核细胞,优先为哺乳动物细胞。A host cell transformed with the expression vector according to claim 9, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably mammalian cells.
- 制备权利要求1-6任一项所述的抗CRTAM/抗PD-L1抗体的方法,包括在如权利要求10所述的宿主细胞中表达抗体,以及从宿主细胞中分离所述抗体的步骤。A method for preparing the anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 6, comprising the steps of expressing the antibody in the host cell according to claim 10, and isolating the antibody from the host cell.
- 一种药物组合物,其包含权利要求1-6之任一项所述的抗CRTAM/抗PD-L1抗体和药学可接受的载体。A pharmaceutical composition comprising the anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
- 如权利要求1-6之任一项所述的抗CRTAM/抗PD-L1抗体或如权利要求12的药物组合物在制备用于抑制PD-L1活性和/或激发CRTAM活性的药物中的应用,优选所述药物用于治疗血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。 Use of the anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 12 in the preparation of a medicament for inhibiting PD-L1 activity and/or stimulating CRTAM activity, preferably the medicament is used to treat blood tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and/or melanoma.
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