CN116375870A

CN116375870A - Humanized antibody of anti-human CD40 protein, preparation method and application thereof

Info

Publication number: CN116375870A
Application number: CN202211730823.0A
Authority: CN
Inventors: 程昊; 娄阳; 陈嘉琛; 贤加欢; 肖航; 柳阳
Original assignee: Yourui Seth Wuhan Biotechnology Co ltd
Current assignee: Yourui Seth Wuhan Biotechnology Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-07-04
Anticipated expiration: 2042-12-30
Also published as: CN116375870B

Abstract

The application discloses humanized antibodies against human CD40 protein, a preparation method and applications thereof. The humanized antibody is obtained by humanized reconstruction of an anti-human CD40 rabbit source parent antibody, and the reconstruction method comprises the following steps: the amino acid sequences of the Framework Regions (FR) of the light chain variable region (VL) and the heavy chain variable region (VH) are substituted, added newly or removed to obtain the target sequence. The humanized antibody is capable of recognizing human CD40 protein, and the binding activity of human CD40 protein is equivalent to that of CD 40L; the affinity constant of the humanized antibody for binding to the recombinantly expressed human CD40 protein is 9.69×10 ^‑10 M, affinity constant (5.99X10) ^‑10 M) is higher.

Description

Humanized antibody of anti-human CD40 protein, preparation method and application thereof

Technical Field

The application relates to the field of biotechnology, in particular to a humanized antibody of an anti-human CD40 protein, a preparation method and application thereof.

Background

The human CD40 protein is a type I transmembrane glycoprotein, and consists of an N-terminal signal peptide (20 aa), an extracellular region (193 aa), a transmembrane region (22 aa) and a cytoplasmic region (62 aa). CD40 protein is widely expressed on the surface of immune cells, including B cells, T cells, monocytes, macrophages and dendritic cells, and is an important functional protein on the surface of cells, involved in the activation of various immune cells. And also expressed on the surface of a portion of non-immune cells such as epithelial cells. In addition, CD40 protein is also expressed on leukemia B cells and on the surface of certain cancer cells, such as epithelial cancer cells.

The ligand CD40L (also known as CD 154) of the human CD40 protein, CD40L, is predominantly expressed on the surface of activated cd4+ T lymphocytes, providing the co-stimulatory signals necessary for B lymphocyte activation. After the recipient cells are activated, CD40L expressed on the cell surface is then excised and flowed into the blood stream to produce a biologically active, soluble, trimeric fragment, also known as soluble CD40L.

The CD40-CD40L co-stimulatory pathway is important for thymus-dependent humoral immunity, and B cell activation, differentiation and immunological memory are all independent of the co-stimulatory pathway. And is also very important for activating Antigen Presenting Cells (APC), which is critical for T cell activation.

Other immune cells expressing CD40 protein can secrete various cytokines after being activated, and play different immune functions. For example, macrophages secrete TNF- α, dendritic cells and macrophages secrete interleukin 12, which are important for T cell and NK cell mediated antitumor immunity.

It is the important role played by the CD40 protein in the immune system that makes CD40 one of the most potential targets in immune cell costimulatory molecules. Pharmaceutical enterprises at home and abroad have the development of CD40 agonist antibodies, 7 in clinical second phase and 6 in clinical first phase at present, and the main indications are melanoma, non-small cell lung cancer, pancreatic cancer and the like, and the administration modes are single administration or combined administration with other treatment modes.

Development of high affinity rabbit monoclonal antibodies to CD40 helps to improve the accuracy of detection of CD40 protein. In addition, most of the existing CD40 agonist antibodies are derived from mouse monoclonal antibodies, have relatively weak affinity, and develop rabbit monoclonal antibodies with high affinity and high activation activity, so that new choices can be provided for therapeutic antibodies based on CD40 targets.

There are few drugs on the market for rabbit antibodies, only Broluzuzumab from North America is used to treat both age-related wet macular degeneration and Eptinezumab from North Danish is used to prevent adult migraine, there are few cases that can be referenced, and the longer CDR regions are more difficult to remodel than human murine antibodies.

Increasing the level of antibody humanization is a key to significantly reducing the immunogenicity of parent rabbit-derived monoclonal antibodies, however, increasing the level of humanization is highly susceptible to a decrease in antibody affinity and specificity. The maintenance of the level, affinity and specificity of antibody humanization is a technical difficulty in antibody humanization transformation, and is a main problem which plagues the clinical application of humanized antibodies.

Disclosure of Invention

In order to solve or alleviate the above part of the technical problems, the present application provides a humanized modified antibody of anti-human CD40 protein, wherein the humanized antibody is based on the amino acid sequence of a rabbit-derived monoclonal antibody, and the humanized antibody is a re-expressed antibody by substituting part or all of the humanized sequence by genetic engineering technology, and the antibody simultaneously retains the affinity and specificity of a parent rabbit-derived monoclonal antibody, reduces the heterology thereof, and is applicable to human clinical application. The variable region of an antibody is its functional region that maintains affinity and specificity, and in particular, the Complementarity Determining Regions (CDRs) of the antibody therein interact with epitope amino acids on the antigen molecule with the support of the Framework Regions (FR) of the antibody, determining the specificity and affinity of the antigen. After the FR region of the anti-human CD40 protein rabbit-derived monoclonal antibody is modified, the novel anti-human CD40 protein humanized antibody is obtained, and the specificity of the antibody are maintainedWhile affinity, the level of humanization was maximized. The humanized antibody provided by the application can recognize human CD40 protein, and the binding activity of the human CD40 protein is equivalent to that of CD 40L; the affinity constant of the humanized antibody for binding to the recombinantly expressed human CD40 protein is 9.69×10 ^-10 M, compared to the parent rabbit antibody affinity constant (5.99X10 ^-10 M) higher; the antibody can be combined with CD40 expressed on the surface of human B cells, and the B cells are activated, and the activation effect is equivalent to that of a parent rabbit antibody under the same experimental conditions. The specific technical scheme mainly comprises the following steps:

in a first aspect, the present application provides an engineered antibody of an anti-human CD40 protein rabbit-derived monoclonal antibody, which specifically binds to human CD40 protein, obtained by engineering an antibody Framework Region (FR) of the anti-human CD40 protein rabbit-derived monoclonal antibody, the engineering comprising substitution, addition or removal of bases; wherein the antibody comprises:

a light chain variable region (VL) defined according to the Kabat numbering system, said light chain variable region (VL) having three Complementarity Determining Regions (CDRs), respectively: VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 5 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with it; VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 6 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence; and VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence;

and/or

Comprising a heavy chain variable region (VH) defined according to the Kabat numbering system, said heavy chain variable region (VH) having three Complementarity Determining Regions (CDRs), respectively: a VH CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 8 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the amino acid sequence; a VH CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 9 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the amino acid sequence; and a VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 10 or a sequence having 1 to 3 amino acid substitutions, deletions or additions as compared with the amino acid sequence; preferably, the substitutions are conservative substitutions.

Further, the antibody comprises:

a light chain variable region (VL) comprising four framework regions (FR regions); the four framework regions of the light chain variable region (VL) are:

an L-FR1 region; the amino acid sequence is shown as SEQ ID NO. 11;

an L-FR2 region; the amino acid sequence is shown as SEQ ID NO. 12;

an L-FR3 region; the amino acid sequence is shown as SEQ ID NO. 13;

an L-FR4 region; the amino acid sequence is shown as SEQ ID NO. 14;

and/or

A heavy chain variable region (VH) comprising four framework regions (FR regions); the four framework regions of the heavy chain variable region (VH) are:

H-FR1 region; the amino acid sequence is shown as SEQ ID NO. 15;

H-FR2 region; the amino acid sequence is shown as SEQ ID NO. 16;

H-FR3 region; the amino acid sequence is shown as SEQ ID NO. 17;

H-FR4 region; the amino acid sequence is shown as SEQ ID NO. 18.

In a second aspect, the present application provides an antibody that specifically binds human CD40 protein, the antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH); the light chain variable region (VL) consists of an amino acid sequence shown in SEQ ID NO. 3; the heavy chain variable region (VH) consists of the amino acid sequence shown in SEQ ID NO. 4.

In a third aspect, the present application provides an antibody comprising a light chain and a heavy chain, said light chain consisting of the amino acid sequence shown in SEQ ID NO. 1; the heavy chain consists of an amino acid sequence shown in SEQ ID NO. 2.

In a fourth aspect, the present application provides a conjugate comprising an antibody of any one of the first to third aspects, and a detectable label attached to the antibody.

In a fifth aspect, the present application provides a kit for detecting human CD40 protein, the kit comprising an antibody according to any one of the first to third aspects or a conjugate according to the fourth aspect.

In a sixth aspect, the application discloses the use of an antibody according to any one of the first to third aspects or a kit according to the fifth aspect for detecting human CD40 protein.

Compared with the prior art, the application has the advantages and positive effects that at least:

the affinity of the obtained humanized antibody against human CD40 protein was measured by using Biacore instrument, and the affinity of the humanized antibody against human CD40 protein was 9.69×10 ^-10 M。

The humanized antibodies against human CD40 protein provided herein can be used to develop flow antibodies that detect human CD40 protein.

The humanized antibodies against human CD40 protein provided herein are capable of binding CD40L.

The humanized antibody of the anti-human CD40 protein provided by the application has the HPLC purity of 95 percent

The humanized antibody of the anti-human CD40 protein provided by the application has strong thermal stability.

Drawings

FIG. 1 is a graph showing the affinity of an anti-human CD40 protein antibody provided in the examples herein; wherein A is an affinity assay profile of a parent antibody derived from a rabbit anti-human CD40 protein and B is an affinity assay profile of a humanized anti-human CD40 protein.

FIG. 2 shows the results of the binding capacity (EC 50) of anti-human CD40 immune-derived parent antibody or anti-human CD40 humanized antibody provided in the examples of the present application to human CD40 protein.

FIG. 3 shows the binding activity of anti-human CD40 protein and human CD40 protein provided in the examples of the present application; wherein A is the binding activity of the immune source parent antibody and B is the binding activity of the humanized antibody.

FIG. 4 is a graph showing the peak flow pattern of anti-human CD 40-derived parent or anti-human CD 40-humanized antibodies against daudi cells (human B lymphoblastic cell line) provided in the examples of this application.

FIG. 5 is a HPLC method purity identification of anti-human CD40 protein antibodies provided in the examples herein; wherein A is the measurement result of the parent rabbit antibody, and B is the measurement result of the humanized antibody.

FIG. 6 is a graph showing the results of stability assays for anti-human CD40 protein antibodies provided in examples herein; wherein A is the stability measurement result of the parent rabbit antibody, and B is the stability measurement result of the humanized antibody.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

Interpretation of the terms

In the present application, the term "antibody" is to be interpreted in the broadest sense, having a variety of antibody structures, including but not limited to, Y-type antibodies, so-called full length antibodies, antigen binding portions of Y-type antibodies, and genetic or chemical modifications thereof. Wherein an "antigen binding portion" refers to one or more portions or fragments of a Y-type antibody that retains the ability of the antibody to specifically bind to human CD40 protein.

In this application, the term "monoclonal antibody" (mAb) includes a population of highly homogeneous antibodies having substantially identical antigenic determinants. That is, the individual antibodies are essentially identical in the population, except for the small number of mutations that may occur naturally. Monoclonal antibodies may exhibit a single binding specificity and affinity for a particular epitope on an antigen. Each monoclonal antibody may be directed against the same or substantially the same epitope on the antigen, as compared to a polyclonal antibody which typically comprises antibodies directed against different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring preparation by any particular method. The antibodies can be prepared by a variety of methods including, but not limited to, hybridoma methods, recombinant DNA methods, phage antibody libraries, and the like.

In this application, the modifier "rabbit" in the term "rabbit antibody" or "anti-human CD40 protein monoclonal antibody" or similar terms means that the Complementarity Determining Regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. In one embodiment, an anti-human CD40 protein rabbit monoclonal antibody may comprise CDRs and Framework Regions (FR) from an antibody of rabbit immunoglobulin sequence. In one embodiment, a rabbit antibody or rabbit monoclonal antibody against human CD40 protein may comprise CDRs from an antibody of rabbit immunoglobulin sequence. In one embodiment, the anti-human CD40 protein rabbit monoclonal antibody may be one in which the CDR regions are derived from rabbit immunoglobulin sequences and the FRs are derived from other mammalian germline immunoglobulin sequences (e.g., mouse or human). The term "anti-human CD40 protein rabbit monoclonal antibody" may also include antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, e.g., mutations introduced by random or point-specific mutations in vitro, or by somatic mutations in vivo. However, the term "anti-human CD40 protein rabbit monoclonal antibody" does not include antibodies in which the CDR regions are derived from the germline of other mammals (e.g., mice).

In this application, the term "antibody" refers to an immunoglobulin molecule consisting of four heterologous polypeptide chains, of which the two chains with the larger molecular weight are referred to as heavy chains (H) and the two chains with the smaller molecular weight are referred to as Light chains (L). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The heavy and light chains vary widely in about 110 amino acid sequences near the N-terminus, with the other portions of the amino acid sequences being relatively constant. Thus, the regions of the light and heavy chains that vary greatly near the N-terminal amino acid sequence are referred to as variable regions (V) and account for 1/4 and 1/2 of the heavy and light chains, respectively; the region of relatively stable amino acid sequence near the C-terminus is called constant region (C) and occupies 3/4 and 1/2 of the heavy and light chains, respectively.

The V chains of the heavy and light chains are referred to as VH and VL, respectively. Each of VH and VL contains a region of highly variable 3 amino acid composition and arrangement sequence, termed hypervariable region (hypervariable region, HVR) or complementarity determining region (complementarity determining region, CDR), including HVRl (CDRl), HVR2 (CDR 2) and HVR3 (CDR 3), wherein HVR3 (CDR 3) varies to a greater extent. The 3 CDRs of VH and VL together form the antigen-binding site of the antibody, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. In the V region, the amino acid composition and arrangement order of the regions outside the CDRs are relatively conserved, called Framework Regions (FR). VH or VL has four framework regions, denoted FR1, FR2, FR3 and FR4, respectively.

The C chains of the heavy and light chains are referred to as CH and CL, respectively. CL lengths of different classes (kappa or lambda) of Ig are substantially identical, but CH lengths of different classes of Ig are different, e.g., igG, igA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CHl, CH2, CH3, and CH4.

In the present application, the term "framework region" or "framework region" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

In the present application, the term "chimeric antibody" means that a portion of its light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which still retains binding activity to the antigen of interest.

In the present application, the term "humanized antibody" refers to a non-human antibody genetically engineered to have its amino acid sequence modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Typically, at least one or two, but typically all three, acceptor CDRs (of the heavy and/or light immunoglobulin chains) of the humanized antibody are replaced by donor CDRs. Immunoglobulins that provide CDRs are referred to as "donors" and immunoglobulins that provide frameworks are referred to as "acceptors". In one embodiment, the donor immunoglobulin is a non-human (e.g., rabbit) antibody, and the acceptor framework may be a naturally occurring human framework, or a sequence having about 85%, 90%, 95%, 99% or more identity thereto. Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

Chimeric or humanized antibodies of the present application can be prepared based on the sequence of a monoclonal antibody produced by immunization of an animal (e.g., rabbit). DNA encoding the heavy and light chains can be obtained from a hybridoma or specific B cell of interest from an immunized animal and engineered to contain human immunoglobulin sequences using standard molecular biology techniques.

In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In this application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

In the present application, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit, then the molecules are identical at that position. For example, if 8 out of 10 positions of two sequences match, then the two sequences have 80% identity.

In the present application, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, e.g., with a residue that is physically or functionally similar (of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue.

Antibodies to

An antibody disclosed in the examples of the present application, which specifically binds to human CD40 protein, comprising a light chain variable region (VL) defined according to the Kabat numbering system, said light chain variable region (VL) having three Complementarity Determining Regions (CDRs), respectively: VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 5 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with it; VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 6 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence; and VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence;

and/or

In certain embodiments, the antibodies are humanized by the following steps: the antibody Framework Region (FR) of the aforementioned antibody, namely, the anti-human CD40 protein rabbit-derived monoclonal antibody, is modified, wherein the modification comprises substitution, addition or removal of bases. The humanized antibodies obtained after the engineering include:

a light chain variable region (VL) comprising four framework regions (FR regions); the four framework regions of the light chain variable region (VL) are: an L-FR1 region; the amino acid sequence is shown as SEQ ID NO. 11; an L-FR2 region; the amino acid sequence is shown as SEQ ID NO. 12; an L-FR3 region; the amino acid sequence is shown as SEQ ID NO. 13; an L-FR4 region; the amino acid sequence is shown as SEQ ID NO. 14;

and/or

A heavy chain variable region (VH) comprising four framework regions (FR regions); heavy chain variable region (VH)

The four framework regions of (a) are respectively: H-FR1 region; the amino acid sequence is shown as SEQ ID NO. 15; H-FR2 region; the amino acid sequence is shown as SEQ ID NO. 16; H-FR3 region; the amino acid sequence is shown as SEQ ID NO. 17; H-FR4 region; the amino acid sequence is shown as SEQ ID NO. 18.

In certain embodiments, the light chain variable region (VL) of the antibody consists of the amino acid sequence set forth in SEQ ID NO. 3; the heavy chain variable region (VH) of the antibody consists of the amino acid sequence shown in SEQ ID NO. 4.

In certain embodiments, the light chain of the antibody consists of the amino acid sequence set forth in SEQ ID NO. 1; the heavy chain consists of an amino acid sequence shown in SEQ ID NO. 2.

In certain embodiments, the humanized antibody against human CD40 protein may have a Y-type molecular structure. In one embodiment, the humanized antibody against human CD40 protein may comprise a pair of heavy chains and a pair of light chains. The heavy chain may include one heavy chain variable region and one or more heavy chain constant regions. Mammalian antibodies generally comprise five types of heavy chains: antibodies of corresponding composition are termed IgG, igD, igA, igM and IgE five antibodies. The light chain may be a smaller polypeptide subunit relative to the heavy chain. The light chain may include a light chain variable region and a light chain constant region. VL is typically the N-terminal part of the light chain, exhibiting higher variability in amino acid sequence. VL between different antibodies has a specific amino acid sequence. In one embodiment, the heavy chain variable region VH and the light chain variable region VL may both be used to recognize and bind human CD40 protein. In one embodiment, the light chain constant region of the antibody is a kappa chain and the heavy chain constant region of the antibody is of the IgG1 type.

In certain embodiments, the CDRs located in the VL and VH regions can be separated from each other by a FR. FR is a conserved region in the sequence structure. FR can generally act as a scaffold to allow CDRs to form a three-dimensional structure that can specifically bind to an antigen (e.g., human CD40 protein). The three-dimensional structure of FR may be conserved among different antibodies. CDRs may be grafted between the FRs of another antibody from another species while retaining its ability to bind to human CD40 protein, forming a fusion antibody. In one embodiment, the CDRs of a Y-type rabbit monoclonal antibody are grafted between the FRs of a human antibody to form a humanized antibody against human CD40 protein. In certain embodiments, the antibody comprises an FR region derived from a human immunoglobulin, optionally comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 back mutations from a human residue to a corresponding rabbit residue.

In certain embodiments, the CDRs located in the VH and VL regions can be separated from each other by a FR. FR is a conserved region in the sequence structure. FR can generally act as a scaffold to allow CDRs to form a three-dimensional structure that can specifically bind to an antigen (e.g., human CD40 protein). The three-dimensional structure of FR may be conserved among different antibodies. The CDRs of a rabbit monoclonal antibody can be grafted between the FRs of another antibody from another species while retaining its ability to bind to human CD40 protein, forming a fusion antibody. In one embodiment, the CDRs of a Y-type rabbit monoclonal antibody are grafted between the FRs of a human antibody to form a humanized antibody against human CD40 protein.

The anti-human CD40 protein rabbit monoclonal antibodies provided in the embodiments of the present application may also include antigen binding portions thereof derived from the structures provided in the embodiments described above or obtained by genetic modification. In certain embodiments, anti-human CD40 protein rabbit monoclonal antibodies may have different transgenic antibody structures, including but not limited to humanized antibodies and chimeric antibodies. In one embodiment, the anti-human CD40 protein rabbit monoclonal antibody may be a humanized antibody having a protein sequence with high homology to that of a naturally occurring variant antibody adapted to humans. Wherein the protein sequence of the "humanized antibody" may be substantially identical to the protein sequence of a human variant antibody while maintaining the binding capacity of the rabbit-derived CDR regions to human CD40 protein. In one embodiment, the "humanized antibody" may be created by inserting CDR regions of a non-humanized antibody, e.g., a CDR region of a rabbit antibody into a humanized antibody scaffold to produce a humanized antibody. In one embodiment, the anti-human CD40 protein rabbit monoclonal antibody may be a chimeric antibody. In one embodiment, the chimeric antibody may be an antibody produced by grafting variable regions of heavy and light chains of Y-type antibodies of different sources to constant regions of another animal (e.g., human). In one embodiment, the anti-human CD40 protein rabbit monoclonal antibody may be a single chain Fv (scFv). Although the two domains of the Fv fragment, VL and VH, are encoded by two separate genes, the two separate encoding genes can be joined by recombinant methods to form one linker, thereby encoding an expressed scFv. In one of the embodiments, the relevant genetic modification and transgenic manipulation can be performed according to methods well known to those skilled in the art, and the transgenic antibody structure can be screened in the same manner as full length antibodies are screened.

Preparation of humanized antibodies against human CD40 protein

The present application discloses methods for preparing the above antibodies, and the monoclonal antibodies of the present application may be prepared by various methods known in the art, such as by genetic engineering recombinant techniques; DNA molecules encoding the heavy and light chain genes of the antibodies of the present application are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into an expression vector, then the host cells are transfected, and the transfected host cells are cultured under specific conditions and the antibodies of the present application are expressed.

The anti-human CD40 rabbit parent antibody used to prepare the humanized antibody herein is a recombinant antibody obtained by screening using a single B cell sorting technique. The anti-human CD40 rabbit parent antibody is recombinantly expressed in vitro by a mammalian expression system; the results prove that the polypeptide has the biological activity of activating human CD40 protein; the preparation method comprises the steps of obtaining variable region VH (variable region) of heavy chain and variable region VL (variable region) of a CD40 parent rabbit-derived parent antibody, using the amino acid sequences of the VL and the VH of the rabbit-derived parent antibody as probes to carry out sequence alignment on NCBI-Blastp tools, finding a human antibody sequence with highest similarity with the variable region VL and the VH on Genbank, using the human antibody sequence as a reference template for reconstruction of a humanized FR region, determining amino acid sites needing mutation, synthesizing a plurality of humanized antibody sequences, and respectively constructing an expression vector; through antibody affinity and activity test detection, the humanized modified amino acid sequence is obtained; the humanized antibody of the mutant obtained through total gene synthesis is subjected to recombinant antibody expression and purification.

1. Humanized modification of human CD40 rabbit parent antibody

In the embodiment of the application, the reconstruction method comprises the following steps: the antibody Framework Region (FR) of the aforementioned antibody, namely, the anti-human CD40 protein rabbit-derived monoclonal antibody, is modified, wherein the modification comprises substitution, addition or removal of bases. The light chain variable region (VL) of the humanized antibody obtained after modification comprises new four framework regions (FR regions); the method comprises the following steps of: an L-FR1 region; the amino acid sequence is shown as SEQ ID NO. 11; an L-FR2 region; the amino acid sequence is shown as SEQ ID NO. 12; an L-FR3 region; the amino acid sequence is shown as SEQ ID NO. 13; an L-FR4 region; the amino acid sequence is shown as SEQ ID NO. 14;

the heavy chain variable region (VH) of the modified humanized antibody comprises four new framework regions (FR regions); the method comprises the following steps of: H-FR1 region; the amino acid sequence is shown as SEQ ID NO. 15; H-FR2 region; the amino acid sequence is shown as SEQ ID NO. 16; H-FR3 region; the amino acid sequence is shown as SEQ ID NO. 17; H-FR4 region; the amino acid sequence is shown as SEQ ID NO. 18.

2. Construction of humanized antibody expression vectors:

a. entrusting the general biological company to synthesize humanized antibody heavy chain and light chain gene sequences aiming at the whole genes;

b. the heavy chain and light chain genes of the antibody are respectively loaded on the pCDN3.4 expression vector;

c. sequencing to verify whether the heavy chain and light chain genes of the antibody are correct;

d. and amplifying plasmids corresponding to the heavy chain and light chain genes of the antibody.

3. Production and purification of monoclonal antibodies:

a. transfecting the plasmid into 293F cells;

b. carrying out transfection for 72-96 hours to obtain a rabbit monoclonal antibody which contains recombinant human CD40 protein in a culture supernatant;

c. purifying recombinant humanized antibody recognizing human CD40 protein from the transfected culture medium supernatant by using protein A affinity gel resin;

d. and (5) after the antibody is identified, split charging and low-temperature storage at-20 ℃ for standby.

4. Results

The example obtained a humanized antibody that recognizes human CD40 protein, the light chain of which consists of the amino acid sequence shown in SEQ ID NO. 1 and the heavy chain of which consists of the amino acid sequence shown in SEQ ID NO. 2; the light chain variable region (VL) of the monoclonal antibody consists of an amino acid sequence shown in SEQ ID NO. 3; the heavy chain variable region (VH) consists of the amino acid sequence shown in SEQ ID NO. 4; the light chain variable region (VL) has three Complementarity Determining Regions (CDRs), respectively: VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 5; VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 6; and VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7; the heavy chain variable region (VH) has three Complementarity Determining Regions (CDRs), respectively: a VH CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 8; VH CDR2 consisting of the amino acid sequence shown in SEQ ID No. 9; and a VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 10; the light chain variable region (VL) of the humanized antibody comprises four framework regions (FR regions), respectively: an L-FR1 region; the amino acid sequence is shown as SEQ ID NO. 11; an L-FR2 region; the amino acid sequence is shown as SEQ ID NO. 12; an L-FR3 region; the amino acid sequence is shown as SEQ ID NO. 13; an L-FR4 region; the amino acid sequence is shown as SEQ ID NO. 14; the heavy chain variable region (VH) of the humanized antibody comprises four framework regions (FR regions), respectively: H-FR1 region; the amino acid sequence is shown as SEQ ID NO. 15; H-FR2 region; the amino acid sequence is shown as SEQ ID NO. 16; H-FR3 region; the amino acid sequence is shown as SEQ ID NO. 17; H-FR4 region; the amino acid sequence is shown as SEQ ID NO. 18; the light chain variable region (VL) of the humanized antibody consists of an amino acid sequence shown in SEQ ID NO. 3; the heavy chain variable region (VH) of the humanized antibody consists of an amino acid sequence shown in SEQ ID NO. 4; the light chain of the humanized antibody consists of an amino acid sequence shown in SEQ ID NO. 1; the heavy chain of the humanized antibody consists of an amino acid sequence shown in SEQ ID NO. 2.

Humanized antibody affinity detection with human CD40 protein

TABLE 1

Name of the name	Dissociation constant (1/s)	Binding constant (1/Ms)	Affinity (M)
				Parent antibody of anti-human CD40 rabbit source	1.37×10 ^-4	2.28×10 ⁵	5.99×10 ^-10
Humanized anti-human CD40 antibodies	1.50×10 ^-4	1.55×10 ⁵	9.69×10 ^-10

Note that: k (K) _off (dissociation rate constant) represents dissociation rate constants between molecules, representing how fast the molecules dissociate; k (K) _on (association rate constant) is the intermolecular binding rate constant, representing the speed of intermolecular binding

The affinity of the humanized antibody of the anti-human CD40 protein and the parent antibody of the rabbit source is accurately measured by using a Biacore 3000 biological molecular interaction analyzer of GE company; wherein the selected antibody is used at a concentration of 333nM and immobilized on a CM5 chip; then binding was performed with five concentrations of recombinant human CD40 protein of 64, 32, 16,8,4nm to obtain an affinity curve; finally, by curve fitting and calculation, the affinity curves are shown in fig. 1, and the fitting results are shown in table 1. The obtained humanized antibody against human CD40 protein has an affinity of 9.69×10 ^-10 Affinity of M compared to parent antibody of rabbit origin (5.99X10 ^-10 M) has a lift.

Human source of anti-human CD40 proteinUse of antibodies

1. ELISA method of humanized antibody and function detection

Human CD40 was diluted to a certain concentration (1. Mu.g/ml) with a coating solution (PBS pH 7.2) and coated on 384-well plates, 25. Mu.l/well, and left overnight at 2-8 ℃; removing the liquid in the well, washing with PBST for 5 times, spin-drying, adding 50 μl/well of blocking solution (1% BSA,5% skimmed milk powder PBST), standing at room temperature for 1hr, washing with PBST for 5 times, and spin-drying; diluting humanized antibody or parent antibody of rabbit origin with dilution (1% BSA, PBST) 3-fold from 1 μg/ml, loading into 384-well plate at 25 μl/Kong Fukong, incubating at 37deg.C for 1hr, discarding the solution, washing the plate 5 times, and spin-drying; d. diluting enzyme-linked antibody (HRP-goat anti-human IgG (H+L)) to a certain concentration (1:5000), reacting in 25 μl/Kong Jiazhi 384 well plate at room temperature for 1hr, discarding the solution, washing the plate for 5 times, and spin-drying; preparing a substrate mixed solution, adding 25 μl/well into a 384-well plate, and incubating for 4min at room temperature; the reaction was terminated by adding 10. Mu.l/well of a stop solution. The absorbance OD was read at 450nm and the binding capacity (EC 50) of the humanized antibody or parent antibody of rabbit origin to human CD40 was calculated as shown in figure 2.

Detection result: as shown in fig. 2, the binding activity of the anti-human CD40 humanized antibody and CD40 protein was substantially the same as the binding ability of the anti-human CD40 rabbit-derived parent antibody to CD40 protein.

2. ELISA binding Activity detection of humanized antibodies

The anti-human CD40 protein rabbit monoclonal antibodies and CD40L protein were coated with carbonate buffer (ph 9.4, 0.05M), incubated overnight at 4 degrees; washing the plate with a washing liquid; blocking with phosphate buffer (pH 7.2, 0.05M) containing 5% bovine serum albumin, 0.05% Tween-20; the biotinylated recombinant human CD40 protein is added into an ELISA plate after being diluted in a gradient way by a phosphate buffer of 0.05% Tween-20, incubated for 1 hour under normal temperature conditions, and then the plate is washed by a washing liquid; adding avidin-labeled horseradish peroxidase (HRP) diluted with phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20, incubating for 1 hour at normal temperature, and washing the plate with a washing solution; adding TMB color development liquid, developing at normal temperature for 10 minutes, adding oxalic acid to stop color development, and measuring light absorption values at 450nm and 630nm respectively, wherein OD450 is subtracted from OD630 to obtain a corrected light absorption value; the values of the human CD40 protein concentration (Log 10) are plotted on the abscissa and the corrected absorbance values (OD 450) are plotted on the ordinate.

Results: as shown in FIG. 3, the binding activity of the humanized antibody or the parent rabbit antibody to CD40 protein is substantially the same as that of CD40L, and the binding activity of the humanized antibody to CD40 protein is substantially the same as that of the parent rabbit antibody.

3. Detection of flow-through binding Activity of antibodies

Taking 1.5X10 ⁶ Individual daudi cells were divided into 3 aliquots and washed once with PBS +0.1% bsa buffer; 100. Mu.l PBS+0.1% BSA, 100. Mu.l Isotype mAb (10. Mu.g/ml), humanized or rabbit parent antibody (10. Mu.g/ml) were added, respectively, and incubated at 4℃for 30 min; 400g, centrifuged for 3 min, and the supernatant discarded, 100. Mu.l PBS+0.1% BSA, 100. Mu.l G, respectively&R_IgG_FITC (1:500 dilution), 100 μ l G&R_igg_fitc (1:500 dilution) resuspended cells and incubated at 4 ℃ for 30 min; 400g, centrifuged for 3 min, the supernatant discarded, and washed twice with 100. Mu.l PBS+0.1% BSA buffer; detecting fluorescent signals on the cell surface using an Agilent NovoCyte Advanteon analytical flow cytometer; the plots were plotted using Novexpress software.

Results: as shown in FIG. 4, the flow binding signals of either the humanized antibody against CD40 or the parent antibody of rabbit origin and positive cells each had 2 log transitions.

4. HPLC method purity identification of humanized antibodies

The humanized antibody or the rabbit parent antibody was diluted to 200. Mu.g/ml with mobile phase (pH 6.4 50mM PB,500mM NaCl), filtered through a 0.2 μm filter, and 10. Mu.l run, 0.3ml/min,30min on an Agilent 1100HPLC instrument using a TSKgel SuperSW3000,4 μm,4.6x 300mm column column.

Results: as shown in fig. 5, HPLC data showed that humanized antibodies were 95% pure and that parent rabbit antibodies were 96% pure.

5. Antibody thermal drift experiments

Preparing a fresh protein heat transfer staining solution, and diluting the fresh protein heat transfer staining solution by 8 times; the corresponding reaction plate or tube was placed on ice and then ready for the monoclonal antibody thawing reaction:

ensuring that the reaction arrangement in the reaction plate is completely consistent with the hole allocation in the experimental file; the reaction components and monoclonal antibodies were added to the plates in the order listed in table 2.

TABLE 2

Component parts	Volume of
		Protein heat transfer buffers	5.0μl
Water + protein + buffer and/or buffer	12.5μl
		Diluted protein heat transfer dye (8 x)	2.5μl
Total volume of each control reaction	20.0μl

Pipetting up and down for 10 times to mix each reaction uniformly; sealing with a MicroAmp "optical film" at 1000 rpm for 1 minute, and then placing on ice; using real-time PCR instrument software to open and establish an experiment operation file; the t program settings are shown in table 3 below:

TABLE 3 Table 3

Loading into a plate, and then starting the instrument to run.

Results: as shown in FIG. 6, the humanized antibody of the anti-CD 40 protein and the parent antibody of the rabbit source have close thermal stability and higher stability.

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims

1. An antibody that specifically binds human CD40 protein; the antibody:

comprising a light chain variable region (VL) defined according to the Kabat numbering system, said light chain variable region (VL) having three Complementarity Determining Regions (CDRs), respectively: VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 5 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with it; VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 6 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence; and VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence;

and/or

2. The antibody of claim 1, wherein the antibody comprises:

an L-FR1 region; the amino acid sequence is shown as SEQ ID NO. 11;

an L-FR2 region; the amino acid sequence is shown as SEQ ID NO. 12;

an L-FR3 region; the amino acid sequence is shown as SEQ ID NO. 13;

an L-FR4 region; the amino acid sequence is shown as SEQ ID NO. 14;

and/or

H-FR1 region; the amino acid sequence is shown as SEQ ID NO. 15;

H-FR2 region; the amino acid sequence is shown as SEQ ID NO. 16;

H-FR3 region; the amino acid sequence is shown as SEQ ID NO. 17;

H-FR4 region; the amino acid sequence is shown as SEQ ID NO. 18.

3. An antibody that specifically binds human CD40 protein, the antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH); the light chain variable region (VL) consists of an amino acid sequence shown in SEQ ID NO. 3; the heavy chain variable region (VH) consists of the amino acid sequence shown in SEQ ID NO. 4.

4. An antibody comprising a light chain and a heavy chain, said light chain consisting of the amino acid sequence set forth in SEQ ID No. 1; the heavy chain consists of an amino acid sequence shown in SEQ ID NO. 2.

5. A conjugate comprising the antibody of any one of claims 1-4, and a detectable label attached to the antibody.

6. A kit for detecting human CD40 protein, the kit comprising the antibody of any one of claims 1-4 or the conjugate of the fourth aspect.

7. Use of an antibody according to any one of claims 1 to 4 or a kit according to claim 6 for the detection of human CD40 protein.