Humanized antibody design device and method based on CDR transplantation and computer readable storage medium

Abstract

The invention discloses a device and a method for designing a humanized antibody based on CDR transplantation, and a computer readable storage medium. The method comprises the following steps: replacing an antigen complementarity determining region sequence of a humanized antibody sequence with an antigen complementarity determining region sequence of a target antigen heterologous antibody to obtain a CDR region transplantation antibody; the method comprises the following steps: performing single amino acid reverse mutation on different amino acid sites of a CDR region transplantation antibody and a heterologous antibody, and comparing structural difference scores of protein three-dimensional structures before and after mutation; retaining the single amino acid reverse mutation site with the structural difference score less than or equal to 1, and finally obtaining the CDR transplanted humanized antibody of the target antigen. According to the method, the similarity of heterologous antibody and human antibody sequences can be increased, the immunogenicity of the target antigen human antibody is reduced, and the affinity is maintained.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioinformatics, and in particular relates to a device, a method and a computer-readable storage medium for designing humanized antibodies based on CDR transplantation. Background Art

[0002] Injection of heterologous antibodies into the human body can cause ADA. To solve this problem, common methods include chimeric antibodies, CDR grafting, SDR transplantation technology, fully human immunization, and immune site removal technology.

[0003] Modified antibodies are also called CDR grafted antibodies. The CDR in the variable region of an antibody is the region where the antibody recognizes and binds to the antigen, which directly determines the specificity of the antibody. The CDR of a heterologous monoclonal antibody is transplanted into the variable region of a human antibody to replace the CDR of a human antibody, so that the human antibody obtains the antigen binding specificity of the heterologous monoclonal antibody and reduces its heterology. However, although the antigen mainly contacts the CDR of the antibody, the FR region often participates in the action and affects the spatial configuration of the CDR. Therefore, after replacing the human FR region, the V region in which the heterologous CDR and the human FR are embedded may change the CDR configuration of the single antigen, and the ability to bind to the antigen will decrease or even decrease significantly. Although it is possible to molecularly design antibodies and introduce certain key amino acid residues in the heterologous FR region into the human FR region, if properly configured, its affinity can be equivalent to that of the original mouse antibody, but humanized antibodies often fail to reach the affinity of the original heterologous monoclonal antibody.

[0004] A common method for humanizing non-humanized antibodies is complementary determining region (CDR) transplantation, that is, the CDR region of a non-human antibody is transplanted onto the human antibody framework region. Usually, the human antibody framework region with the highest homology to the non-human antibody framework region is selected as the receptor for CDR transplantation. The main problem with this method is that the affinity for binding to a specific target will be reduced or even lost. Directly transplanting the CDR loop of a mouse antibody onto the human antibody framework will not affect the antibody affinity in some cases, but in most cases it will significantly reduce the affinity. Some residues in the framework region of mouse antibodies have been shown to affect the conformation of the CDR loop and the affinity of the antibody, and are called vernier region residues. These residues are located near the β-fold of the CDR region. Therefore, after selecting the desired human antibody framework region, these residues need to be reverse mutated to retain them in the humanized antibody. In addition, mutations of amino acid residues outside the variable region have also been used to give new properties to humanized antibodies. It is difficult to find suitable residue back-mutation sites in the prior art.

[0005] The concept of B-factor temperature factor originated from crystal research. It is mainly used to reflect the ambiguity of the atomic conformational state in the crystal. In fact, it reflects the conformational state of the protein molecule in the crystal. The higher the B-factor, the greater the ambiguity, and the corresponding part is unstable or unstable. In crystallographic data, B-factor is generally given in atoms, and it is usually converted into the B-factor value of the corresponding amino acid residue, so that the conceptual stability or flexibility of the amino acid residue can be analyzed. Studies have found that the thermal stability of some mutants can be greatly improved after mutating the amino acids with higher B-factor values ​​in the protein structure. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to transform heterologous antibodies to obtain humanized antibodies and / or how to reduce the immunogenicity of heterologous antibodies and / or how to reduce the immunogenicity of heterologous antibodies while maintaining their affinity to antigens.

[0007] In order to solve the above technical problems, the present invention first provides a device for obtaining (designing) a humanized antibody based on CDR region transplantation, and the device may include the following modules:

[0008] A1) Humanized framework region selection module: used to obtain the humanized antibody sequence of a heterologous antibody specific to a target antigen using sequence alignment software;

[0009] A2) CDR region transplantation module: used to annotate the antigen complementary determining region sequence of the heterologous antibody using the antibody sequence CDR annotation system to obtain the heterologous antibody CDR sequence, and to annotate the humanized sequence to obtain the humanized antibody CDR sequence; and to replace the humanized antibody CDR sequence in the humanized antibody sequence with the heterologous antibody CDR sequence to obtain the CDR region transplanted antibody sequence of the target antigen;

[0010] A3) A module for selecting back mutation sites and obtaining humanized antibodies: used to compare the sequence of the CDR region-grafted antibody with the sequence of the heterologous antibody to obtain differential amino acid sites, and to perform back mutation on each of the differential amino acid sites to obtain single amino acid back mutation CDR region-grafted antibodies, and to perform structural prediction on all the single amino acid back mutation CDR region-grafted antibodies using protein structure prediction software to obtain the three-dimensional structures of all the single amino acid back mutation CDR region-grafted antibodies, and to predict the three-dimensional structure of the heterologous antibody CDR sequence using the protein structure prediction software to obtain the three-dimensional structure of the heterologous antibody;

[0011] The three-dimensional structure of the single amino acid back-mutated CDR region-grafted antibody and the three-dimensional structure of the heterologous antibody are scored for structural difference using the following formula 1 to obtain a structural difference score;

[0012] score=important_score×exposed_area×3D_smiliarity Formula 1;

[0013] In Formula 1, score represents the structural difference score between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody; important_score is the value obtained by scoring the similarity between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody based on the B-factor and hydrophilicity of each amino acid residue in the sequence; exposed_area represents the difference in the size of the exposed area of ​​the three-dimensional structure of the heterologous antibody and the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody; 3D_similarity is the structural similarity score between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody (representing the similarity of the three-dimensional structure of the protein before and after the single amino acid site reversion mutation);

[0014] The amino acid mutation sites in the single amino acid back mutation CDR region transplanted antibody with a structural difference score less than 1 are selected and merged to obtain back mutation combination sites, and the amino acid residues at the back mutation combination site positions in the sequence of the CDR region transplanted antibody are replaced with the amino acid residues at the back mutation combination site positions corresponding to the heterologous antibody to obtain a humanized antibody sequence for the target antigen.

[0015] The complementary determining region sequences include sequences of CDR1, CDR2 and CDR3 regions.

[0016] In the above device, the heterologous antibody may be a non-human antibody.

[0017] In the above device, in formula 1 of A3), the important_score can be calculated using the abnumber package.

[0018] In formula 1 of A3), the exposed_area can be calculated using the colabfold package. In formula 1 of A3), the 3D_similarity can be calculated using the pymol package.

[0019] In the above device, the antibody sequence CDR annotation system is a CDR annotation system with joint numbering of Kabat and Chothia.

[0020] In order to solve the above technical problems, the present invention also provides a method for obtaining (designing) a humanized antibody based on CDR region transplantation, which may include the following steps:

[0021] B1) Selection of humanized framework regions: using sequence alignment software to obtain the humanized antibody sequence of the target antigen heterologous antibody;

[0022] B2) CDR region transplantation: using an antibody sequence CDR annotation system to annotate the antigen complementary determining region sequence of the heterologous antibody to obtain a heterologous antibody CDR sequence, and annotating the humanized antibody sequence to obtain a humanized antibody CDR sequence; replacing the humanized antibody CDR sequence in the humanized antibody sequence with the heterologous antibody CDR sequence to obtain a CDR region transplanted antibody sequence of the target antigen;

[0023] B3) Selection of back mutation sites and acquisition of humanized antibodies: comparing the sequence of the CDR region-grafted antibody with the sequence of the heterologous antibody to obtain differential amino acid sites, performing back mutation on each of the differential amino acid sites to obtain single amino acid back mutation CDR region-grafted antibodies, using protein structure prediction software to perform structural prediction on all of the single amino acid back mutation CDR region-grafted antibodies to obtain the three-dimensional structures of all of the single amino acid back mutation CDR region-grafted antibodies, and using the protein structure prediction software to predict the three-dimensional structure of the heterologous antibody CDR sequence to obtain the three-dimensional structure of the heterologous antibody;

[0024] The three-dimensional structure of the single amino acid back-mutated CDR region-grafted antibody and the three-dimensional structure of the heterologous antibody are scored for structural difference using the following formula 1 to obtain a structural difference score;

[0025] score=important_score×exposed_area×3D_smiliarity Formula 1;

[0026] In Formula 1, score represents the structural difference score between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody; important_score is the value obtained by scoring the similarity between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody based on the B-factor and hydrophilicity of each amino acid residue in the sequence; exposed_area represents the difference in the size of the exposed area of ​​the three-dimensional structure of the heterologous antibody and the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody; 3D_similarity is the structural similarity score between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody (representing the similarity of the three-dimensional structure of the protein before and after the single amino acid site reversion mutation);

[0027] The amino acid mutation sites in the single amino acid back mutation CDR region transplanted antibody with a structural difference score less than 1 are selected and merged to obtain back mutation combination sites, and the amino acid residues at the back mutation combination site positions in the sequence of the CDR region transplanted antibody are replaced with the amino acid residues at the back mutation combination site positions corresponding to the heterologous antibody to obtain a humanized antibody sequence for the target antigen.

[0028] The heterologous antibodies mentioned above are non-human antibodies.

[0029] The complementary determining region sequences mentioned above include sequences of the CDR1, CDR2 and CDR3 regions.

[0030] In the above method, the antibody sequence CDR annotation system can be a CDR annotation system with combined numbering of Kabat and Chothia.

[0031] In the above method, in formula 1 of B3), the important_score can be calculated using the abnumber program package; in formula 1 of B3), the exposed_area can be calculated using the colabfold program package; in formula 1 of B3), the 3D_similarity can be calculated using the pymol program package.

[0032] In order to solve the above technical problem, the present invention further provides a computer-readable storage medium storing a computer program, wherein the computer program can enable a computer to execute the steps of the method described above.

[0033] In order to solve the above technical problem, the present invention further provides a computer device, including a memory, a processor, and a computer program stored in the memory, wherein the processor can execute the computer program to implement the following steps:

[0034] B1) Selection of humanized framework regions: using sequence alignment software to obtain the humanized antibody sequence of the target antigen heterologous antibody;

[0035] B2) CDR region transplantation: using an antibody sequence CDR annotation system to annotate the antigen complementary determining region sequence of the heterologous antibody to obtain a heterologous antibody CDR sequence, and annotating the humanized antibody sequence to obtain a humanized antibody CDR sequence; replacing the humanized antibody CDR sequence in the humanized antibody sequence with the heterologous antibody CDR sequence to obtain a CDR region transplanted antibody sequence of the target antigen;

[0036] B3) Selection of back mutation sites and acquisition of humanized antibodies: comparing the sequence of the CDR region-grafted antibody with the sequence of the heterologous antibody to obtain differential amino acid sites, performing back mutation on each of the differential amino acid sites to obtain single amino acid back mutation CDR region-grafted antibodies, using protein structure prediction software to perform structural prediction on all of the single amino acid back mutation CDR region-grafted antibodies to obtain the three-dimensional structures of all of the single amino acid back mutation CDR region-grafted antibodies, and using the protein structure prediction software to predict the three-dimensional structure of the heterologous antibody CDR sequence to obtain the three-dimensional structure of the heterologous antibody;

[0037] The three-dimensional structure of the single amino acid back-mutated CDR region-grafted antibody and the three-dimensional structure of the heterologous antibody are scored for structural difference using the following formula 1 to obtain a structural difference score;

[0038] score=important_score×exposed_area×3D_smiliarity Formula 1;

[0039] In Formula 1, score represents the structural difference score between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody; important_score is the value obtained by scoring the similarity of the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody based on the B-factor and hydrophilicity of each amino acid residue in the sequence; exposed_area represents the difference value of the size of the exposed area of ​​the three-dimensional structure of the heterologous antibody and the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody; 3D_similarity is the structural similarity score between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody;

[0040] The amino acid mutation sites in the single amino acid back mutation CDR region transplanted antibody with a structural difference score less than 1 are selected and merged to obtain back mutation combination sites, and the amino acid residues at the back mutation combination site positions in the sequence of the CDR region transplanted antibody are replaced with the amino acid residues at the back mutation combination site positions corresponding to the heterologous antibody to obtain a humanized antibody sequence for the target antigen.

[0041] In order to solve the above technical problem, the present invention further provides a computer device, including a memory, a processor and a computer program stored in the memory, wherein the processor executes the computer program to implement the steps of the above method.

[0042] The heterologous source mentioned above may be non-human.

[0043] The present invention increases the similarity between heterologous antibodies and human antibody sequences through CDR grafting, reduces immunogenicity and maintains affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the process of the present invention.

[0045] Figure 2 These are the Elisa EC50 test results of the CDR-grafted protein sequence thera_52 and the parent CD28 antibody after site-reverse mutation in Example 1 and CD28, respectively.

[0046] Figure 3 These are the affinity test results of hu_PD-L1 and PD-L1 antibody parent with PD-L1 in Example 2, respectively. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0049] The antibody expression in the embodiment of the present invention is synthesized and prepared by genetic engineering technology using the protein synthesis system of eukaryotic or prokaryotic host cells, and the method is as follows:

[0050] Primer design: Design and synthesize specific primers for the antibody gene and add appropriate restriction endonuclease recognition sequences to the 5' end of the primer.

[0051] PCR amplification: Using designed primers and template DNA, antibody gene fragments are amplified by PCR technology.

[0052] Restriction digestion reaction: The PCR product and the cloning vector are digested with the same restriction endonuclease to produce sticky ends.

[0053] Ligation reaction: connect the digested antibody gene fragment to the vector using DNA ligase.

[0054] Transformation: Transform the ligation product into competent E. coli.

[0055] Screening and identification: Positive clones were screened by antibiotics, and colony PCR and sequence analysis were performed to confirm the correctness of the inserted fragment.

[0056] Expression and purification: The verified plasmid is transformed into a eukaryotic or prokaryotic expression system to induce the expression of antibody protein, and the protein is purified by affinity chromatography and other methods.

[0057] The method for antibody affinity testing in the embodiment of the present invention is as follows:

[0058] The antibody affinity was tested by SPR using a biomolecular interaction instrument (Biacore 8K).

[0059] 1) Chip activation: Use NHS (N-hydroxysuccinimide) to activate the chip surface.

[0060] 2) Antibody fixation: The purified target antibody protein is diluted to an appropriate concentration and injected into the activated chip channel to fix it on the surface.

[0061] 3) Blocking: Inject a high concentration of blocking agent (bovine serum albumin) into the chip to reduce nonspecific binding.

[0062] 4) Baseline correction: Ensure that the baseline of the chip is stable before injecting the antigen.

[0063] 5) Injection of antigen:

[0064] Prepare a series of antigen solutions with different concentrations and inject them into the channel with the antibody fixed in order from high to low concentrations. Record the response units (RU) during the binding and dissociation process.

[0065] 6) Data collection and analysis: After collecting the data, use BIACORE software for kinetic analysis. Use a 1:1 Langmuir binding model or other suitable model to fit the data. Calculate the KD value, i.e. the dissociation constant, which is a key parameter for measuring affinity.

[0066] The ELISA method for detecting the antigen binding activity of antibodies in the embodiment of the present invention is as follows:

[0067] Antigen preparation: Prepare a series of antigen solutions at different concentrations (10 concentrations) covering the expected EC50 values.

[0068] Antibody preparation: Prepare antibodies at known concentrations.

[0069] ELISA plate: Use 96-well ELISA plate.

[0070] Reagents: Prepare enzyme-labeled secondary antibody (horseradish peroxidase-labeled anti-antibody), substrate solution, stop solution, etc.

[0071] Experimental steps:

[0072] 1) Coating: Add appropriate concentration of antibody to each well of the ELISA plate and coat overnight at 4°C or room temperature.

[0073] 2) Blocking: Remove the coating solution, wash and block the wells with PBS or PBS containing 1-5% bovine serum albumin (BSA) or skimmed milk powder to reduce nonspecific binding.

[0074] 3) Sample addition: Add different concentrations of antigen solution to each well, including negative control and positive control.

[0075] 4) Incubation: Incubate the plate at room temperature for 1 hour to allow the antigen to bind to the antibody.

[0076] 5) Washing: Wash the wells with PBS or PBS containing 0.05% Tween 20 (PBST) to remove unbound substances.

[0077] 6) Add enzyme-labeled secondary antibody: Add enzyme-labeled secondary antibody to each well and incubate for 1 hour.

[0078] 7) Wash again: Wash the wells to remove unbound secondary antibody.

[0079] 8) Add substrate: Add substrate solution to each well to produce color changes according to the activity of the enzyme.

[0080] 9) Color development: Color development for a certain period of time at room temperature and away from light.

[0081] 10) Termination reaction: Add stop solution to stop the substrate reaction.

[0082] 11) Plate reading: Use a microplate reader to read the absorbance (OD) value at an appropriate wavelength (450 nm).

[0083] 12) Plotting curves: Use GraphPad Prism software to plot the antigen concentration and the corresponding average OD value into a dose-response curve.

[0084] 13) Fitting model: A four-parameter logistic regression (4PL) model was used to fit the data.

[0085] 14) Calculate EC50: Extract the EC50 value, i.e., the antigen concentration that achieves 50% of the maximum response, from the fitted curve.

[0086] Example 1. Process for humanizing CD28 nanobody

[0087] In this example, T cell activating factor-CD28 nanobody (VHH) is humanized, and the similarity between the heterologous antibody and the human antibody sequence is increased by CDRgrafting, thereby reducing the immunogenicity of the CD28 nanobody and maintaining its affinity to the APC antigen.

[0088] 1. Antibody framework region selection

[0089] The framework selection in this embodiment is based on NCBI's blastp to search the human germline and therapeutic structural antibody database (Thera-SAbDab) of heterologous antibodies, and the template for selecting the humanized antibody framework region is based on the sequence similarity score result (TOP5) of blastp (https: / / ftp.ncbi.nlm.nih.gov / blast / executables / blast+ / LATEST / ).

[0090] By using blastp to search for human germline on the amino acid sequence of CD28 nanobody (sequence 1 in the sequence list), the sequence corresponding to the most similar germline is shown in sequence 2-sequence 6 in the sequence list. Among them, sequence 2-sequence 5 is the germline of the selected antibody variable region (V region), and sequence 6 is the selected antibody connecting region (J region) germline. Finally, the sequence with the highest blastp score is selected as the human germline sequence of CD28 nanobody for CDR region transplantation (grafting). The blastp score is the result of summing the scores of each pair of amino acid residues by using a scoring matrix to score the similarity of the matching fragments. Generally speaking, the longer the matching fragment and the higher the similarity, the greater the score and the more reliable the result. The scoring results are shown in (Table 1). The thera_heavy_v_52 sequence shown in sequence 2 in the sequence list has the highest score, which is the highest scoring V region. The highest scoring thera_heavy_v_52 (sequence 2) and the highest scoring J region thera_heavy_j_30 (sequence 6) were spliced ​​to obtain sequence 7, which was selected for CDR region transplantation.

[0091] Sequence 1:

[0092] QVQLVESGGGLVQAGGSLRLSCTAS ERTFRTYTMA WFRQAPGKEREFVG YITWYGGSTYYGDSVKG RFTISRDNSENTVYLEMNRLESEDTAVYYCAA GGLVSRDPGDFSS WGQGTQVTVSS.

[0093] In Sequence 1, the underlined sequence is the CDR region sequence of the CD28 nanobody, wherein the CDR1 region corresponds to amino acids 26-35 of Sequence 1, the CDR2 region corresponds to amino acids 50-66 of Sequence 1, and the CDR3 region corresponds to amino acids 99-111 of Sequence 1.

[0094] Sequence 2 (thera_heavy_v_52(Envafolimab)):

[0095] QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA;

[0096] Sequence 3 (IGHV3-23*03):

[0097] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK;

[0098] Sequence 4 (IGHV3-23*04):

[0099] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK;

[0100] Sequence 5 (IGHV3-64*04):

[0101] QVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR;

[0102] Sequence 6 (thera_heavy_j_30(Caplacizumab)):

[0103] GRVRTLPSEYTFWGQGTQVTVSS.

[0104] Sequence 7 (thera_heavy_v_52+thera_heavy_j_30):

[0105] QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGRVRTLPSEYTFWGQGTQVTVSS.

[0106] Table 1. Blastp scoring results for the germline most similar to the CD28 antibody sequence

[0107]

[0108] 2. Selection of sequence CDR annotations and CDR region transplantation

[0109] This example uses the kabat and chothia joint numbering method to annotate the CDR regions in the CD28 nanobody (sequence 1) and the human germline antibody (sequence 7), and obtains the CDR regions (including CDR1-CDR3) of the CD28 nanobody (VHH). Then the CDR regions of the CD28 nanobody (VHH) are replaced with the human germline: the CDR1-CDR3 regions of the CD28 nanobody (VHH) are replaced with the CDR1-CDR3 regions (including CDR1-CDR3) in the human germline antibody (sequence 7), specifically using the abnumber package (https: / / abnumber.readthedocs.io / en / latest / ).

[0110] 2.1 Antibody sequence CDR annotation

[0111] The CDR annotation information of the human germline shown in sequence 7 obtained by the joint numbering method of Kabat and Chothia includes: the positions of the complementary determining region 1 (CDR1) sequence region of the heavy chain variable region, the antibody framework region 2 (FR2) sequence region, the complementary determining region 2 (CDR2) sequence region of the heavy chain variable region, the antibody framework region 3 (FR3) sequence region, the complementary determining region 3 (CDR3) sequence region of the heavy chain variable region and the antibody framework region 4 (FR4) sequence region.

[0112] 2.2 CDR Region Transplantation

[0113] The sequence of the CDR1-CDR3 region of the CD28 nanobody shown in sequence 1 (the CDR1 region corresponds to amino acids 26-35 of sequence 1, the CDR2 region corresponds to amino acids 50-66 of sequence 1, and the CDR3 region corresponds to amino acids 99-111 of sequence 1) was transplanted to the CDR1-CDR3 region of sequence 2 obtained by CDR annotation in step 2.1 to obtain the sequence after CDR region transplantation (sequence 8 in the sequence table). The similarity score of sequence 8 and sequence 1 after CDR region transplantation was scored using blastp. The results showed that the similarity parameters of the two sequences were as follows: identities = 113 / 122 (93%) (identities = 100% represents exactly the same), positives = 116 / 122 (95%) (representing similar amino acid attributes (blastp internal matrix score)), gaps = 0 / 122 (0%) (representing the difference in spaces after align).

[0114] Sequence 8:

[0115] QVQLVESGGGLVQPGGSLRLSCAASERTFRTYTMAWFRQAPGKERERVAYITWYGGSTYYGDSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGGLVSRDPGDFSSWGQGTQVTVSS.

[0116] 3. Selection of reverse mutation sites

[0117] The affinity sites of antibodies and antigens may occur in more than just the CDR region, so for humanization of antibodies in which the antigen-antibody binding region is in the antibody framework region (FR region), it is necessary to determine the affinity-related amino acid sites in the specific FR region. The present invention can easily find suitable sites based on the combined scoring of biophysical and biochemical properties.

[0118] In this example, single-point back mutations were performed on different points in the FR region of the CDR region grafted sequence obtained based on the nanoantibody parent (alpaca) and humanized grafting (nine different amino acid points in this example, see Table 2), and then the open source software colabfold was used for modeling. Then, a custom physicochemical function (based on the definition of the V region and the size of the exposed area, the consistency of the CDR region and the parent after back mutation was scored) was used to score the structural differences of the 3D structures of the CD28 nanoantibody and the 9 mutated CDR grafted protein sequences obtained by 9 different single-point back mutations.

[0119] 3.1 Sequence-to-Structure Modeling

[0120] Based on the existing sequence-to-structure tool colabfold (https: / / github.com / sokrypton / ColabFold), the 9 mutated CDR-grafted protein sequences were converted to obtain the (3D) three-dimensional structures of the mutated CDR-grafted proteins. Colabfold was also used to obtain the three-dimensional structure of the CD28 nanobody.

[0121] 3.2 Selection of reverse mutation sites

[0122] Humanized sequences that are only transplanted often encounter the problem of decreased affinity, and back mutation is required to maintain affinity consistent with the parental sequence.

[0123] The three-dimensional structure of the CD28 nanobody and the three-dimensional structure of the 9 mutated CDR-grafted protein sequences obtained by 9 different single-point back mutations were scored for structural difference using the following formula 1 to obtain a structural difference score:

[0124] score=important_score×exposed_area×3D_smiliarity Formula 1;

[0125] In formula 1, important_score is the numerical value obtained by scoring the similarity of the three-dimensional structure of the CD28 nanobody and the three-dimensional structure of the CDR-grafted protein sequence after mutation based on the B-factor and hydrophilicity of each amino acid residue in the sequence, which is calculated using the abnumber program package (https: / / abnumber,readthedocs.io / en / latest / ); exposed_area represents the difference in the size of the exposed area between the three-dimensional structure of the CD28 nanobody and the three-dimensional structure of the CDR-grafted protein sequence after mutation, which is calculated using the colabfold program package (https: / / github.com / sokrypton / ColabFold); 3D_similarity is the structural similarity score between the three-dimensional structure of the CD28 nanobody and the three-dimensional structure of the CDR-grafted protein sequence after mutation (representing the similarity of the three-dimensional structure of the protein before and after the single amino acid site back mutation), which is calculated using the pymol program package (https: / / www.pymo1.org / ).

[0126] Table 2. Structural difference scores of CD28 nanobody after humanized grafting and antibody back mutation

[0127] sc reverse mutation site Score structural difference score hu_1(P14A) 1.245 hu_2(A23T) 1.267 hu_3(R47F) 0.843 hu_4(A49G) 1.276 hu_5(K76E) 1.3068 hu_6(Q82E) 1.229 hu_7(S85R) 1.3146 hu_8(R87E) 1.27 hu_9(A88S) 1.28

[0128] Note: The mutation information in the name of the back mutation site is as follows: For the back mutation site hu_1 (P13A), the P in P13A means that the 13th amino acid residue in the humanized sequence mutates from P to A (i.e., the 13th amino acid residue in sequence 8 mutates from P to A).

[0129] Through calculation, it was obtained that the 3D structure of the CDR-grafted protein sequence after the hu_3 (R46F) site reversion mutation was less than 0.85 with the CD28 antibody structure difference score, and the secondary structure difference was only two different framework sheets, with high similarity. Therefore, the CDR-grafted protein sequence (sequence 9) after the hu_3 (R47F) site reversion mutation (i.e., the 47th amino acid residue of sequence 8 mutated from R to F) of the CDR-grafted protein sequence shown in sequence 8 can be prepared to detect the affinity of the obtained humanized antibody to the antigen.

[0130] Sequence 9:

[0131] QVQLVESGGGLVQPGGSLRLSCAASERTFRTYTMAWFRQAPGKEREFVAYITWYGGSTYYGDSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAGGLVSRDPGDFSSWGQGTQVTVSS.

[0132] 4. Evaluation of humanized antibodies

[0133] The affinity between CD28 nanobody (sequence 1) and the CDR-grafted protein sequence thera_52 (sequence 9) after site reversion mutation of hu_3 (R46F) was evaluated respectively.

[0134] like Figure 2 The EC50 of the parent CD28 nanobody and CD28 is 0.31 ( Figure 2 CD28-base_1 represents the CD28-base_1 in the sample); and the EC50 of thera_52 and CD28 is 0.277 ( Figure 2 CD28-base_1_hu_1 represents the middle), which is in line with expectations.

[0135] Example 2. Process for humanizing PD-L1 VHH

[0136] In this example, the nanobody against PD-L1 is humanized, and the similarity between the heterologous antibody and the human antibody sequence is increased by CDRgrafting, thereby reducing the immunogenicity of PD-L1 VHH and maintaining its affinity to the APC antigen.

[0137] 1. Antibody framework region selection

[0138] The framework selection of the present invention is based on NCBI's blastp to search the human germline of heterologous antibodies and the Therapeutic Structural Antibody Database (Thera-SAbDab), and the template for selecting the humanized antibody framework region is based on the scoring result (TOP5) of blastp (https: / / ftp.ncbi.nlm.nih.gov / blast / executables / blast+ / LATEST / ).

[0139] Using the same process as in Example 1, the human germline was searched using blastp for the amino acid sequence of the PD-L1 nanobody (sequence 10 in the sequence list), and the sequence similarity score was obtained, and the sequence corresponding to the most similar germline, such as sequence 11 in the sequence list, was used as the highest-scoring V region. The highest-scoring IGHV3-48*03 (sequence 11) and the highest-scoring J region were spliced ​​to obtain a spliced ​​sequence, and the spliced ​​sequence was selected as the human germline for CDR region transplantation to obtain a CDR region transplanted sequence (sequence 12 in the sequence list).

[0140] Sequence 10:

[0141] QVQLVESGGGLVQPGGSLRLSCAAS GSIFSSGTQFSDSKID WYRQAPGKQRDWIA GIFSTGSTIYEDS VKG RFAISRDNAKNMGYLQMNSLKPEDTAVYYC RVIGRGILA WGQGTQVTVSS.

[0142] In sequence 10, the underlined sequence is the sequence of the variable region CDR1-CDR3 of the PD-L1 nanobody.

[0143] Sequence 11:

[0144] IGHV3-48*03

[0145] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR;

[0146] Sequence 12:

[0147] (PD-L1_grafted_vhh)

[0148] EVQLVESGGGLVQPGGSLRLSCAASGSIFSSGTQFSDSKIDWVRQAPGKGLEWVSGIFSTGSTIYEDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCRVIGRGILAWGQGTQVTVSS;

[0149] 2. Selection of sequence CDR annotations and CDR region transplantation

[0150] The same method as in Example 1 was used to perform CDR annotation and CDR region transplantation of human germline sequences to obtain a CDR region transplanted sequence (sequence 12 in the sequence list). The CDR region transplanted sequence (sequence 12 in the sequence list) and the parent PD-L1 nanobody sequence (sequence 10) were scored for similarity using blastp, and the results showed that the similarity parameters of the two sequences were as follows: identities = 109 / 121 (90%) (identities = 100% represents exactly the same)), positives = 114 / 121 (94%) (representing similar amino acid attributes (blastp internal matrix scoring)), gaps = 0 / 121 (0%) (representing the difference in spaces after align).

[0151] 3. Selection of reverse mutation sites

[0152] The same method as step 3 in Example 1 was used to select the reverse mutation site, specifically, sequence 12 and sequence 10 were aligned to obtain 12 different amino acid sites between the CDR-grafted protein sequence and the parent PD-L1 nanobody sequence. Colabfold was used to perform structural prediction on the 12 mutated CDR-grafted protein sequences obtained by performing 12 different single-point reverse mutations on the CDR-grafted protein sequence (sequence 12) to obtain the 3D structures of the 12 mutated CDR-grafted proteins.

[0153] Then, using the custom physicochemical function in Formula 1 in the example (based on the definition of the V region and the size of the exposed area, the consistency of the CDR region and the parent after back-mutation is scored), the 3D structures of the 12 mutated CDR-grafted proteins and the 3D structure of the PD-L1 nanobody sequence were scored for structural differences to obtain a structural difference score.

[0154] The results in Table 3 show that the five mutation sites of the CDR region grafted antibody shown in sequence 12 were reverse mutated (V42Y, G49Q, L50R, T73A and T82M) to obtain the five mutated CDR grafted proteins. The structural difference scores (scores) compared with the 3D structure of the PD-L1 nanoantibody sequence were small (less than 1), which were 0.967, 0.843, 0.976, 0.91 and 0.92 respectively.

[0155] Table 3. Structural difference scores of PD-L1 nanobody after humanized grafting and antibody reversion mutation

[0156] Scanning mutation sites Score hu_1(E1Q) 1.045 hu_2(V43Y) 0.967 hu_3(G50Q) 0.843 hu_4(L51R) 0.976 hu_5(E52D) 1.3068 hu_6(V54I) 1.229 hu_7(S55A) 1.3146 hu_8(T74A) 0.91 hu_10(T83M) 0.92 hu_11(L84G) 1.27 hu_12(R92K) 1.27 hu_13(A93P) 1.28

[0157] The combination antibody hu_PD-L1 (sequence 13) with five reverse mutation sites (V43Y, G50Q, L51R, T74A, T83M) with a score within 1 was selected, and the antibody hu_PD-L1 with the CDR-grafted protein sequence after the corresponding five site reverse mutations was prepared using the same method in Example 1, and the affinity of hu_PD-L1 and the PD-L1 nanoantibody mother to PD-L1 was tested respectively.

[0158] Sequence 13:

[0159] EVQLVESGGGLVQPGGSLRLSCAASGSIFSSGTQFSDSKIDWYRQAPGKQREWVSGIFSTGSTIYEDSVKGRFAISRDNSKNMLYLQMNSLRAEDTAVYYCRVIGRGILAWGQGTQVTVSS(hu_PD-L1).

[0160] The results are as follows Figure 3 As shown in the SPR (surface plasmon resonance) full kinetic analysis, the affinity determination results of the PD-L1 nanobody parent showed that its binding constant (Kd) with the PD-L1 protein was 4.04E-9 (as shown in Figure 3 In contrast, the SPR test results of the hu_PD-L1 nanobody showed that its binding constant (Kd) with PD-L1 was 1.44E-8 (as shown in Figure 3 Based on this data, we can observe that the affinity of hu_PD-L1 nanobody to PD-L1 is slightly lower than that of the parent nanobody, while retaining the high affinity of the parent antibody, which is in line with the expectations of humanized antibodies.

[0161] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A device obtained based on a humanized antibody with CDR region transplantation, characterized in that: The device comprises the following modules: A1) Humanized framework region selection module: used to obtain the humanized antibody sequence of a heterologous antibody specific to a target antigen using sequence alignment software; A2) CDR region transplantation module: used to annotate the antigen complementary determining region sequence of the heterologous antibody using the antibody sequence CDR annotation system to obtain the heterologous antibody CDR sequence, and to annotate the humanized antibody sequence to obtain the humanized antibody CDR sequence; and to replace the humanized antibody CDR sequence in the humanized antibody sequence with the heterologous antibody CDR sequence to obtain the CDR region transplantation antibody sequence of the target antigen; A3) A module for selecting back mutation sites and obtaining humanized antibodies: used to compare the sequence of the CDR region-grafted antibody with the sequence of the heterologous antibody to obtain differential amino acid sites, and to perform back mutation on each of the differential amino acid sites to obtain single amino acid back mutation CDR region-grafted antibodies, and to perform structural prediction on all the single amino acid back mutation CDR region-grafted antibodies using protein structure prediction software to obtain the three-dimensional structures of all the single amino acid back mutation CDR region-grafted antibodies, and to predict the three-dimensional structure of the heterologous antibody using the protein structure prediction software to obtain the three-dimensional structure of the heterologous antibody; The three-dimensional structure of the single amino acid back-mutated CDR region-grafted antibody and the three-dimensional structure of the heterologous antibody are scored for structural difference using the following formula 1 to obtain a structural difference score; score=important_score ×exposed_area ×3D_smiliarity Equation 1; In Formula 1, score represents the structural difference score between the three-dimensional structure of the single amino acid back-mutated CDR region-grafted antibody and the three-dimensional structure of the heterologous antibody; important_score is the value obtained by scoring the similarity between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody based on the B-factor and hydrophilicity of each amino acid residue in the sequence; exposed_area represents the difference in the size of the exposed area of ​​the three-dimensional structure of the heterologous antibody and the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody; 3D_similarity is the structural similarity score of the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody; The amino acid mutation sites in the single amino acid back mutation CDR region transplanted antibody with a structural difference score less than 1 are selected and merged to obtain back mutation combination sites, and the amino acid residues at the back mutation combination site positions in the CDR region transplanted antibody sequence are replaced with the amino acid residues at the back mutation combination site positions corresponding to the heterologous antibody to obtain a humanized antibody sequence for the target antigen.

2. The device according to claim 1, characterized in that: The heterologous antibody is a non-human antibody.

3. The device according to claim 1 or 2, characterized in that: A3) in Formula 1, the important_score is calculated using the abnumber package; the exposed_area is calculated using the colabfold package; and the 3D_similarity is calculated using the pymol package.

4. The device according to any one of claims 1 to 3, characterized in that: The antibody sequence CDR annotation system is a CDR annotation system with joint numbering of Kabat and Chothia.

5. A method for obtaining a humanized antibody based on CDR region transplantation, characterized in that: The method comprises the following steps: B1) Selection of humanized framework regions: using sequence alignment software to obtain the humanized antibody sequence of the target antigen heterologous antibody; B2) CDR region transplantation: using an antibody sequence CDR annotation system to annotate the antigen complementary determining region sequence of the heterologous antibody to obtain a heterologous antibody CDR sequence, and annotating the humanized antibody sequence to obtain a humanized antibody CDR sequence; replacing the humanized antibody CDR sequence in the humanized antibody sequence with the heterologous antibody CDR sequence to obtain a CDR region transplanted antibody sequence of the target antigen; B3) Selection of back mutation sites and acquisition of humanized antibodies: comparing the sequence of the CDR region-grafted antibody with the sequence of the heterologous antibody to obtain differential amino acid sites, performing back mutation on each of the differential amino acid sites to obtain single amino acid back mutation CDR region-grafted antibodies, using protein structure prediction software to perform structural prediction on all of the single amino acid back mutation CDR region-grafted antibodies to obtain the three-dimensional structures of all of the single amino acid back mutation CDR region-grafted antibodies, and using the protein structure prediction software to predict the three-dimensional structure of the heterologous antibody to obtain the three-dimensional structure of the heterologous antibody; The three-dimensional structure of the single amino acid back-mutated CDR region-grafted antibody and the three-dimensional structure of the heterologous antibody are scored for structural difference using the following formula 1 to obtain a structural difference score; score=important_score ×exposed_area ×3D_smiliarity Equation 1; In Formula 1, score represents the structural difference score between the three-dimensional structure of the single amino acid back-mutated CDR region-grafted antibody and the three-dimensional structure of the heterologous antibody; important_score is the value obtained by scoring the similarity between the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody based on the B-factor and hydrophilicity of each amino acid residue in the sequence; exposed_area represents the difference in the size of the exposed area of ​​the three-dimensional structure of the heterologous antibody and the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody; 3D_similarity is the structural similarity score of the three-dimensional structure of the single amino acid reversion mutation CDR region transplanted antibody and the three-dimensional structure of the heterologous antibody; The amino acid mutation sites in the single amino acid back mutation CDR region transplanted antibody with a structural difference score less than 1 are selected and merged to obtain back mutation combination sites, and the amino acid residues at the back mutation combination site positions in the sequence of the CDR region transplanted antibody are replaced with the amino acid residues at the back mutation combination site positions corresponding to the heterologous antibody to obtain a humanized antibody sequence for the target antigen.

6. The method according to claim 5, characterized in that: The heterologous antibody is a non-human antibody.

7. The method according to claim 5 or 6, characterized in that: The antibody sequence CDR annotation system is a CDR annotation system with joint numbering of Kabat and Chothia.

8. The method according to any one of claims 5 to 7, characterized in that: In formula 1 of B3), the important_score is calculated using the abnumber package; in formula 1 of B3), the exposed_area is calculated using the colabfold package; in formula 1 of B3), the 3D_similarity is calculated using the pymol package.

9. A computer-readable storage medium storing a computer program, characterized in that: The computer program enables a computer to execute the steps of the method according to any one of claims 5 to 8.

10. A computer device comprising a memory, a processor and a computer program stored in the memory, characterized in that: The processor executes the computer program to implement the steps of the method according to any one of claims 5 to 8.

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