CN120554516A

CN120554516A - Anti-CD45 antibodies and their applications

Info

Publication number: CN120554516A
Application number: CN202511056484.6A
Authority: CN
Inventors: 胡钰; 高欣瑶; 刀丽威; 张维; 苏香; 王媚娘; 郑越
Original assignee: Hangzhou Huada Life Science Research Institute
Current assignee: Hangzhou Huada Life Science Research Institute
Priority date: 2025-07-30
Filing date: 2025-07-30
Publication date: 2025-08-29
Anticipated expiration: 2045-07-30
Also published as: CN120554516B

Abstract

The invention relates to the technical field of antibodies, and provides an anti-CD 45 antibody and application thereof, wherein the antibody comprises at least one CDR selected from the group consisting of a heavy chain variable region CDR, a light chain variable region CDR and a light chain variable region CDR, wherein the amino acid sequence is 1-3 of SEQ ID NO or a conservatively modified form of the heavy chain variable region CDR, and the amino acid sequence is 4-6 of the light chain variable region CDR or a conservatively modified form of the light chain variable region CDR. The antibodies or antigen binding fragments thereof are capable of efficiently and specifically recognizing CD45, and are useful for detecting CD45, and for example, for immunohistochemistry.

Description

Anti-CD 45 antibody and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-CD 45 antibody and application thereof.

Background

CD45 (leukocyte common antigen ) is a vital transmembrane protein tyrosine phosphatase in the immune system and is widely expressed on the surface of all nucleated leukocytes including T cells, B cells, natural killer cells (NK cells), macrophages and dendritic cells, and the like. As a core regulatory molecule for immune cell signaling, CD45 mediates the immune response of lymphocytes, playing a key role in immune cell development, activation, differentiation and tolerance. Its structural diversity, functional complexity and association with disease make it an important target for immunological research and clinical transformation.

Functionally, CD45 releases its self-inhibited state by dephosphorylating the inhibitory tyrosine residues (e.g., tyr505 of Lck) at the C-terminus of Src family kinases (e.g., lck and Fyn), thereby activating downstream signaling pathways. This process is critical for T Cell Receptor (TCR) and B Cell Receptor (BCR) mediated immune responses, regulating T/B cell activation, proliferation and differentiation. In addition, CD45 is involved in positive and negative selection of thymocytes, affects T cell maturation, and plays a role in cytokine production and modulation of immune response intensity. Studies have shown that CD45 deficiency can lead to severe immune dysfunction, and its abnormal expression in autoimmune diseases and leukemia also suggests its importance as a potential diagnostic marker or therapeutic target. The function of CD45 is environmentally dependent, possibly exhibiting a complex regulatory network through different isoforms or interacting molecules. Therefore, the development of CD45 antibodies is of great importance for both immunological research and clinical diagnosis.

At present, commercial CD45 antibodies have remarkable limitations, mainstream products depend on the traditional mouse hybridoma technology, the production process is limited to animal-derived systems (such as ascites preparation), and risks of large batch-to-batch differences, animal ethical disputes, potential immunogenicity and the like exist. Therefore, there is a need to break through the technical barriers by innovative development strategies (such as non-animal source recombination technology, single B cell sequencing technology), shorten the development time, and screen CD45 antibodies with high affinity, high specificity and low batch-to-batch variation.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent.

Thus, in a first aspect of the invention, the invention provides an antibody or antigen binding fragment thereof. According to an embodiment of the invention, the antibody or antigen binding fragment thereof has a heavy chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO. 1 or a conservatively modified form thereof, a heavy chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO. 2 or a conservatively modified form thereof, a heavy chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO. 3 or a conservatively modified form thereof, a light chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO. 4 or a conservatively modified form thereof, a light chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO. 5 or a conservatively modified form thereof, and a light chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO. 6 or a conservatively modified form thereof. The antibody or antigen binding fragment thereof according to the embodiment of the present invention is a high affinity (pM-grade) rabbit recombinant antibody developed by single B cell sequencing and non-animal source recombinant technology, which is capable of recognizing CD45 efficiently and specifically, detecting CD45 efficiently and diagnosing diseases associated with abnormal expression of CD 45.

In a second aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen binding fragment thereof as described previously. The antibodies or antigen-binding fragments thereof encoded by the nucleic acid molecules according to embodiments of the invention are capable of efficiently and specifically recognizing CD45, detecting CD45, e.g., performing IHC detection, and also are effective in detecting CD45 and diagnosing diseases associated with abnormal expression of CD 45.

In a third aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. Thus, the expression of the antibody or the antigen binding fragment thereof is effectively realized, and the in vitro mass acquisition of the antibody or the antigen binding fragment thereof is further realized.

In a fourth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises a nucleic acid molecule or expression vector carrying the foregoing or an antibody or antigen binding fragment thereof expressing the foregoing. The recombinant cell can be used to efficiently express the aforementioned antibody or antigen-binding fragment thereof in a cell under appropriate conditions.

In a fifth aspect of the invention, the invention provides a method of preparing an antibody or antigen binding fragment thereof according to the first aspect. According to an embodiment of the invention, the method comprises culturing the recombinant cell of the fourth aspect.

In a sixth aspect of the invention, the invention provides an antibody conjugate. According to an embodiment of the invention, the antibody conjugate comprises an antibody or antigen binding fragment thereof as described above, and a coupling moiety coupled thereto, said coupling moiety being linked to said antibody or antigen binding fragment thereof. The antibody conjugate of the invention can specifically recognize CD45, detect CD45, such as IHC detection, and can also effectively detect CD45 and diagnose diseases related to abnormal expression of CD 45.

In a seventh aspect of the invention, the invention provides a reagent or kit. According to an embodiment of the invention, the reagent or kit comprises the aforementioned antibody or antigen-binding fragment thereof or the aforementioned antibody conjugate. The kit provided by the invention can be specifically combined with CD45, and can be used for effectively detecting CD45.

In an eighth aspect of the invention, the invention provides the use of an antibody or antigen binding fragment thereof, an antibody conjugate or a reagent or kit as defined above for the detection of CD45, for the manufacture of a product for the detection of CD45 or for the diagnosis of a CD45 related disease. As described above, the antibody or antigen-binding fragment thereof according to the embodiment of the present invention is a high affinity (pM-grade) rabbit recombinant antibody developed by single B cell sequencing and non-animal source recombinant technology, which can effectively detect CD45, for example, IHC detection.

In a ninth aspect of the invention, the invention provides a method of detecting CD45 in a test sample. According to an embodiment of the invention, the method comprises contacting the aforementioned antibody or antigen binding fragment thereof, antibody conjugate or reagent or kit with CD45 antigen in the sample to be detected, forming an immune complex.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of an animal immunization protocol and serum titers after four immunizations according to an embodiment of the present invention, wherein:

A in figure 1 is the animal immunization protocol flow,

B in fig. 1 is a graph showing the results of serum titer detection after four immunizations of experimental animals;

FIG. 2 is A graph showing the results of flow sorting B cells according to an embodiment of the present invention, wherein P1 represents the selection of A main cell population by forward scattered light (FSC-A) and side scattered light (SSC-A), the removal of cell debris, P2 and P3 represent the removal of adherent cells by areA (A) and height (H) of FSC and SSC, P4 represents the selection of FITC channel positive lgG+ cells, and P5 represents the selection of APC channel positive Ag+lgG+ cells;

FIG. 3 is a diagram showing SDS-PAGE detection result of a recombinant antibody according to an embodiment of the present invention, wherein M represents a protein marker, +DTT represents that a protein loading buffer contains a reducing agent DTT, and-DTT represents that a protein loading buffer does not contain a reducing agent DTT;

FIG. 4 is a graph of binding antibody affinity assay results according to an embodiment of the present invention, wherein the abscissa represents time(s) and the ordinate represents relative displacement height (nm);

FIG. 5 is a graph showing IHC detection of CD45-58 rabbit recombinant monoclonal antibodies on various tissue sections according to an embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

As used herein, the term "fragment" refers to a protein or polypeptide of interest, as well as a protein or polypeptide of interest having an N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and/or internal deletion.

In this context, the terms "identity", "homology" or "similarity" are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see Ausubel et al, editions (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); there are many algorithms for alignment and determination of sequence identity with ALIGN program (Dayhoff(1978),Atlas of Protein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation,Washington,D.C.)., including, the homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, the local homology algorithms of Smith et al (1981) adv.appl.Math.2:482, the similarity search method of Pearson et al (1988) Proc.Natl.Acad.Sci.85:2444, the computer programs using the Smith-Waterman algorithm (Meth.mol.biol.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.mol.biol.215:403-410), and including but not limited to ALIGN or Megalign (DNASTAR) software, or WU-BLASH-2 (Altschul et al, enym.266:460-480 (1996) or TFA, and/or more specifically, FIG. 35, and the programs available in Table 35, inc. 35, U.S. 35:35, and Witsbook, witsin the United states, magnomon et al.

The term "at least 80% identity" as used herein refers to at least 80% identity, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to each reference sequence.

In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The expression vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors having a plurality of the above functions. The expression vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the expression vector will produce the desired expression product by culturing a suitable host cell containing the expression vector.

As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined with genetic material of a host cell using genetic engineering techniques or cell fusion techniques to obtain a unique trait that is stably inherited. Wherein the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of a nucleic acid (e.g., vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the invention and can be used for expression and/or secretion of a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, chinese Hamster Ovary (CHO) cells, heLa cells, cap cells (human amniotic fluid derived cells) and CoS cells.

An antibody or antigen binding fragment thereof

In some embodiments, the invention provides an antibody or antigen binding fragment thereof comprising a CDR selected from at least one of the amino acid sequences of SEQ ID NOs 1-3 or conservatively modified forms thereof and the amino acid sequences of light chain variable region CDRs 4-6 or conservatively modified forms thereof. The antibody or antigen binding fragment thereof according to the embodiment of the present invention is a high affinity (pM-grade) rabbit recombinant antibody developed by single B cell sequencing and non-animal source recombinant technology, which can efficiently and specifically recognize CD45, detect CD45, for example, perform IHC detection, and can also efficiently detect CD45 and diagnose diseases associated with abnormal expression of CD 45.

As used herein, the term "antibody" is used in its broadest sense and may include full length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited so long as they exhibit the desired biological activity. It generally comprises a light chain of relatively light molecular weight and a heavy chain of relatively heavy molecular weight, the heavy chain (H chain) and the light chain (L chain) being linked by disulfide bonds to form an antibody molecule. Wherein the amino terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called variable region (V region), and the carboxy terminal (C-terminal) is relatively stable, and varies little, called constant region (C region). The V chains of the L chain and H chain are referred to as VL and VH, respectively. As used herein, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions comprising one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or functional fragment thereof to an antigen or epitope recognized by the antibody or fragment thereof. In particular embodiments of the present disclosure, CDRs refer to the highly variable regions of the heavy and light chains of the antibodies.

As used herein, the term "antigen binding fragment" is a fragment comprising a portion or all of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen, e.g., the fragment may comprise a portion or all of the CDRs of an antibody. Such fragments are biologically active in that they bind to an antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, fv, scFv or single domain antibodies. Such fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

In this context, a "conservatively modified form of an amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications may be introduced into the antibodies of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies of the invention may be replaced with other amino acid residues from the same side chain family, and the altered antibodies tested for retention function using the functional assay methods described herein. Preferably, the conservative modifications are not more than 1 or 2 in number.

According to some embodiments of the invention, the above-described antibody or antigen binding fragment thereof may further comprise at least one of the following additional technical features:

According to some embodiments of the invention, the antibody or antigen binding fragment thereof comprises:

A heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO. 1 or a conservatively modified version thereof;

a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO. 2 or a conservatively modified version thereof;

a heavy chain variable region CDR3 having the amino acid sequence depicted in SEQ ID No.3 or a conservatively modified version thereof;

a light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO. 4 or a conservatively modified version thereof;

a light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO. 5 or a conservatively modified version thereof, and

A light chain variable region CDR3 having the amino acid sequence depicted in SEQ ID No. 6 or a conservatively modified version thereof.

According to some embodiments of the invention, the heavy chain variable regions CDR1, CDR2, CDR3 and light chain variable regions CDR1, CDR2, CDR3 are defined by either of Kabat, chothia, IMGT or AbM systems. CDR numbering schemes commonly used in the art include Kabat numbering, chothia numbering, IMGT numbering, martin numbering and AHo numbering. CDR definition schemes include Kabat definition, chothia definition, IMGT definition and AbM definition. As used herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described by Kabat et al, U.S. Dept.of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR.

According to some embodiments of the invention, the antibody or antigen binding fragment thereof comprises a heavy chain framework region and/or a light chain framework region.

According to some embodiments of the invention, at least a portion of the heavy chain framework region and/or the light chain framework region is derived from at least one of a rabbit, murine, human, primates, sheep, dogs, cats, and alpaca antibody and mutants thereof.

According to some embodiments of the invention, at least a portion of the heavy chain framework region and/or the light chain framework region is derived from a rabbit antibody.

According to some embodiments of the invention, the heavy chain framework region and/or the light chain framework region comprises an FR selected from at least one of the following:

The heavy chain variable region FR is the amino acid sequence of SEQ ID NO 7-10 or a conservatively modified form thereof;

The light chain variable region FR is the amino acid sequence of SEQ ID NO. 11-14 or a conservatively modified form thereof.

a heavy chain variable region FR1 having the amino acid sequence shown in SEQ ID NO. 7 or a conservatively modified version thereof;

A heavy chain variable region FR2 having the amino acid sequence shown in SEQ ID NO. 8 or a conservatively modified version thereof;

A heavy chain variable region FR3 having the amino acid sequence shown in SEQ ID NO. 9 or a conservatively modified version thereof;

a heavy chain variable region FR4 having the amino acid sequence shown in SEQ ID NO. 10 or a conservatively modified version thereof;

a light chain variable region FR1 having the amino acid sequence shown in SEQ ID NO. 11 or a conservatively modified version thereof;

A light chain variable region FR2 having the amino acid sequence shown in SEQ ID NO. 12 or a conservatively modified version thereof;

A light chain variable region FR3 having the amino acid sequence shown in SEQ ID NO. 13 or a conservatively modified version thereof, and

A light chain variable region FR4 having the amino acid sequence shown in SEQ ID NO. 14 or a conservatively modified version thereof.

According to some embodiments of the invention, the heavy chain variable regions FR1, FR2, FR3, FR4 and the light chain variable regions FR1, FR2, FR3, FR4 are defined by either the Kabat, chothia, IMGT or AbM system.

Likewise, it will be appreciated by those skilled in the art that given the variable region amino acid sequence of an antibody, those skilled in the art can routinely determine which residues comprise a particular FR.

According to some embodiments of the invention, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO. 15 or an amino acid sequence having at least 80% identity thereto, and/or a light chain variable region of an amino acid sequence as set forth in SEQ ID NO. 16 or an amino acid sequence having at least 80% identity thereto.

According to some embodiments of the invention, the antibody or antigen binding fragment thereof further comprises a constant region.

According to some embodiments of the invention, the constant region comprises a heavy chain constant region and/or a light chain constant region.

According to some embodiments of the invention, at least a portion of the heavy and/or light chain constant regions is derived from at least one of rabbit, murine, human, primates, sheep, dogs, cats and alpacas antibodies and mutants thereof.

According to some embodiments of the invention, at least a portion of the heavy chain constant region and/or the light chain constant region is derived from at least one of a rabbit, murine, human, and primate-derived antibody.

According to some embodiments of the invention, the heavy chain constant region comprises a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD, or the light chain constant region comprises a light chain constant region selected from the group consisting of kappa type or lambda type.

According to some embodiments of the invention, the N-terminus of the heavy chain constant region is linked to the C-terminus of the heavy chain variable region and/or the N-terminus of the light chain constant region is linked to the N-terminus of the light chain variable region.

According to some embodiments of the invention, the antibody comprises at least one selected from the group consisting of a full length monoclonal antibody, a Fab 'antibody, a F (ab') 2 antibody, a Fv antibody, a single chain antibody, a single domain antibody, and a minimal recognition unit, or the antigen-binding fragment comprises at least one selected from the group consisting of a F (ab ') 2 fragment, a Fab' fragment, a Fab fragment, a F (ab) 2 fragment, an Fv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, and a minimal recognition unit.

As used herein, the term "full length antibody", "full length monoclonal antibody" or "full length monoclonal antibody" is made up of at least two identical light chains and at least two identical heavy chains joined by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

The terms "single domain antibody," "nanobody," and "VHH antibody" are used interchangeably herein, and are initially described as an antigen-binding immunoglobulin (variable) domain (Hamers-Casterman C ,AtarhouchT ,Muyldermans S ,Robinson G ,Hamers C ,Songa EB,Bendahman N ,Hamers R .:"Naturallyoccurring antibodies devoid of light chains";Nature 363 ,446-448(1993)), of a "heavy chain antibody" (i.e., an "antibody lacking a light chain") comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigen protein (e.g., CD 45) via the heavy chain variable region.

In this context, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment comprising only Fab molecules, consisting of VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond between the light and heavy chains.

As used herein, the term "F (ab ') 2 antibody" or "F (ab ') 2 fragment" has two antigen binding F (ab ') moieties linked together by disulfide bonds.

As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting of only the light chain variable region (VL) and the heavy chain variable region (VH) joined by a non-covalent bond, which is the smallest functional fragment of an antibody that retains the intact antigen-binding site.

As used herein, the terms "single chain antibody", "scFv fragment" are antibodies or fragments that are made up of antibody heavy and light chain variable regions linked by short peptides.

As used herein, the terms "minimal recognition unit" and "MRU" refer to antibodies or fragments consisting of only one CDR, which have a molecular weight that is sufficiently small to account for only about 1% of the total antibody.

Nucleic acid molecules, expression vectors and recombinant cells

In some embodiments, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen binding fragment thereof as described previously. The antibodies or antigen-binding fragments thereof encoded by the nucleic acid molecules according to embodiments of the invention are capable of efficiently and specifically recognizing CD45, detecting CD45, e.g., performing IHC detection, and also are effective in detecting CD45 and diagnosing diseases associated with abnormal expression of CD 45.

According to some embodiments of the invention, the nucleic acid molecule is DNA.

It is noted that, for the nucleic acid molecules mentioned herein, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences of the present application include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.

In some embodiments, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. Of course, the nucleic acid molecule may be operably linked to a control element. "operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the antibody or antigen-binding fragment thereof can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the antibody or antigen-binding fragment thereof.

According to some embodiments of the invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

According to some embodiments of the invention, the expression vector is a plasmid expression vector.

In some embodiments, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises a nucleic acid molecule or expression vector carrying the foregoing or an antibody or antigen binding fragment thereof expressing the foregoing. The recombinant cell can be used to efficiently express the aforementioned antibody or antigen-binding fragment thereof in a cell under appropriate conditions.

The term "suitable conditions" as used herein refers to conditions suitable for expression of the antibodies or antigen-binding fragments thereof of the present application. Those skilled in the art will readily appreciate that conditions suitable for expression of the antibody or antigen-binding fragment thereof include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and one skilled in the art can optimize the conditions for optimal expression of the antibody or antigen-binding fragment thereof according to the specific environment of the laboratory.

According to some embodiments of the invention, the recombinant cell is a eukaryotic cell

According to some embodiments of the invention, the recombinant cell is a mammalian cell.

Conjugates and kits

In some embodiments, the invention provides an antibody conjugate. According to an embodiment of the invention, the antibody conjugate comprises an antibody or antigen binding fragment thereof as described previously, and a coupling moiety coupled thereto. The antibody conjugate of the invention can specifically identify CD45, can detect CD45 protein, and can effectively diagnose diseases related to abnormal expression of CD 45.

According to some embodiments of the invention, the coupling moiety is selected from a purification tag or label.

According to some embodiments of the invention, the coupling moiety comprises at least one selected from the group consisting of colloidal gold, a radiolabel, a phosphorescent chemical agent, a chemiluminescent agent, fluorescein, an enzyme, a native toxin, a nucleic acid, and an affinity label.

According to some embodiments of the invention, the coupling moiety comprises a radioisotope.

According to some embodiments of the invention, the coupling moiety comprises at least one selected from phycoerythrin, fluorophore, rhodamine, luciferase, fluorescein isothiocyanate, green fluorescent protein, blue fluorescent protein, red fluorescent protein.

According to some embodiments of the invention, the coupling moiety comprises at least one selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase.

According to some embodiments of the invention, the coupling moiety comprises at least one selected from biotin and avidin.

According to some embodiments of the invention, the coupling moiety comprises at least one member selected from the group consisting of magnetic beads, magnetic microspheres, plastic microparticles, microplates, nylon and nitrocellulose membranes.

Herein, the coupling moiety may be a substance capable of being suspended or dispersed in a liquid phase (e.g., a solid phase carrier such as particles or magnetic beads), or a solid phase capable of housing or carrying a liquid phase (e.g., a support such as a plate, a membrane, a test tube, and a well plate, a microfluidic channel, a glass capillary, a nanopillar, a monolithic column, or the like), or a labeling carrier for labeling an antibody or an antigen-binding fragment thereof, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase (luciferin), β -galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of nucleic acids of a sense strand and an antisense strand complementary to each other), a fluorescent substance (e.g., fluorescein isothiocyanate, rhodamine, a green fluorescent protein, a red fluorescent protein), a luminescent substance (e.g., luciferin, aequorin (Aequorin), acridine, tris (2, 2' -bipyridine) ruthenium, luminol), a radioactive substance (e.g., 3H, 14C, P, 32, S125, and the like).

According to some embodiments of the invention, the conjugation moiety may be a protein tag including, but not limited to, his tag, flag tag, GST tag, MBP tag, SUMO tag, C-Myc tag, and the like.

The method for binding the coupling moiety to the antibody or antigen-binding fragment thereof may be any method known in the art. Examples thereof include physical adsorption, covalent bonding, and methods using affinity substances (e.g., biotin and streptavidin), and ion bonding.

In some embodiments, the invention provides a reagent or kit. According to an embodiment of the invention, the reagent or kit comprises the aforementioned antibody or antigen binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell or antibody conjugate. It will be appreciated from the foregoing that the antibodies or antigen binding fragments thereof described above bind to CD45, and that the antibodies or antigen binding fragments thereof described above bind specifically to CD45, and that, under appropriate conditions, the nucleic acid molecule, expression vector, recombinant cell or antibody conjugate is capable of expressing the antibodies or antigen binding fragments thereof, and further that a kit comprising the same is capable of binding efficiently to CD45 and can be used to detect CD45 efficiently. The kit can be used for scientific research, such as qualitative or quantitative detection of CD45 in a biological sample, and can also be used for judging the state of a subject, such as judging whether the CD45 level of the subject is higher or lower than the normal level after the CD45 level of the subject is obtained, wherein the biological sample can be cells, tissues, blood and the like.

Use of the same

In some embodiments, the invention provides the use of the aforementioned antibodies or antigen-binding fragments thereof, the aforementioned antibody conjugates or reagents or kits for detecting CD45, for preparing a product for detecting CD45, or for diagnosing a CD 45-related disorder.

Method of

In some embodiments, the invention provides a method of detecting CD45 in a test sample, the method comprising contacting a sample to be detected with an antibody or antigen-binding fragment thereof, an antibody conjugate, or a reagent or kit as described above to form an immunocomplex.

According to some embodiments of the invention, the test sample comprises cells, tissue, blood, etc.

According to some embodiments of the invention, it is determined whether the sample to be tested contains CD45 or the content of CD45 based on the signal of the immunocomplexes.

According to some embodiments of the invention, the immune complex further comprises a second antibody, which binds to the antibody or antigen binding fragment thereof.

According to some embodiments of the invention, the immune complex further comprises a second antibody, which binds to CD 45.

The nucleic acid and amino acid sequences to which the present application relates are shown in Table 1.

TABLE 1

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are fully explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I.Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.3425 vector for MAMMALIAN CELLS), and PCR methods (J.M.Miller and M.P.Calos, 1987), and PCR methods (J.3456) are incorporated by reference in each of 2011, and by reference.

In the present embodiment, the nucleotide sequence used for preparing the expression vector may be obtained by a conventional method or a conventional software (e.g., an on-line program Vectorbuilder, geneOptimizer on-line program, etc.) according to the amino acid sequence thereof.

Example 1 Gene synthesis and protein expression

This example prepares a CD45 recombinant protein (its sequence number is uniprot P08575) as an immunogen, whereby a CD45_pcDNA3.4 expression vector (gene synthesis is carried out by New production technology Co., ltd.) was constructed based on the coding nucleic acid of the CD45 recombinant protein, the 3' end of the CD45 protein (G25-S576) coding region in the expression vector was tagged with a Tain-Strep-Tag, codon optimized according to the codon preference of the Homo sapiens species, and cloned into pcDNA3.4 expression vector by XbaI and HindIII restriction enzyme sites. Plasmid CD45_pcDNA3.4 was transformed into E.coli DH 5. Alpha. (purchased from TIANGEN) followed by bulk extraction of the plasmid, which was then transfected into an Expi293FTM cell (purchased from Thermo) by PEI (purchased from POLYSCIENCE) for protein expression. Cell supernatants containing the target protein CD45 recombinant protein were harvested after day 5 of transfection, and purified by Strep column affinity chromatography (Strep-Tactin XT column, IBA) and gel filtration chromatography (SuperoseTM Increate 10/300GL, GE) to obtain a purer target protein.

EXAMPLE 2 animal immunization

In this example, the immunogen CD45 recombinant protein obtained in example 1 was used for animal immunization, and the specific procedures are as follows:

The immunogen CD45 recombinant protein is mixed with an adjuvant and injected into New Zealand white rabbits for immunization, wherein, freund's complete adjuvant (Freund's complete adjuvant: sigma, cat F5881) is used for the first time, freund's incomplete adjuvant (sigma, cat F5506) is used for the subsequent immunization, and the mixing ratio is that the immunogen and the adjuvant are mixed in a ratio of 1:1. After a plurality of rounds of immunization, the rabbits are subjected to blood sampling from ears, serum is separated, antibody titer detection is carried out in the serum through ELISA, B cell sorting is carried out after the serum titer is qualified, the operation flow is shown in a graph 1A, the specific experimental result is shown in a graph 1, wherein the graph 1B is an ELISA result of serum titer detection after four-immunity, the first column of data represents the reciprocal of 11 dilutions of the diluted serum, the second column of data and the third column of data represent absorbance values of two parallel compound holes at an OD450 wavelength, and the result shows that the four-immunity serum titer reaches more than 78W, and B cell sorting can be started.

EXAMPLE 3B cell sorting of immunized rabbits

10ML of whole rabbit blood that was immunized as described in example 2 was collected and used to isolate PBMCs. PBMC were first labeled with biotin-T-lymphocyte antibody (from Bio-Rad), biotin-IgM antibody (from BD Pharmingen), biotin-CD11B antibody (from STEMCELL Technologies), then streptavidin beads (from Miltenyi Biotec) were added, incubated on ice for 15min, and finally negative-sorted by magnetic column to give B cells.

1X10≡6B cells were taken, incubated on ice for 30 min after addition of CD45 recombinant protein, washed 3 times with PBS after incubation was completed, then AF488 Donkey Anti-Rabbit IgG H & L (purchased from Biolegend), strepMAB-Immo DY-649 (purchased from IBA) antibodies were added, incubated on ice for 30 min times with PBS, finally CD45+ IgG+ B cells were sorted into 15mL by flow sorter FACSARIATM II for subsequent single cell sequencing, the primary cell population was first selected by forward scattered light (FSC-A) and side scattered light (SSC-A), cell debris was removed (P1), then adherent cells were removed by areA (A) and height (H) of FSC and SSC, respectively, then FITC channel positive lgG+ cells (P4) were selected, and finally channel positive Ag+ lgG+ cells (P5) were further selected.

Example 4 Single B cell sequencing and antibody expression vector construction

Single cell sequencing was performed on single B cells that were sorted. Firstly, 300g of the sorted cells are centrifuged for 10min, counted after being resuspended, and pass through a microfluidic chip together with an oil phase to generate water-in-oil droplets through the microfluidic chip, each droplet wraps a single cell, a lysis reagent and magnetic beads carrying unique molecular labels, and then cell lysis, mRNA capture and cDNA reverse transcription reactions are completed in the droplets. Then demulsification is carried out to recover cDNA products, BCR enrichment is carried out, finally, cDNA library and BCR library are established, on-machine sequencing is carried out, single cell data information is obtained, and information analysis is carried out

After transcriptome data are obtained, immune group library BCR analysis is carried out to obtain antibody sequences, and the antibody sequences matched with light and heavy chains are selected and sent to a new generation technology limited company of Huada, so as to synthesize antibody expression vector plasmids.

EXAMPLE 5 expression and purification of recombinant antibodies

Recombinant antibody expression the antibody sequence was synthesized into pcdna3.4 (+) expression vectors, expression plasmids containing the specific antibody light and heavy chain encoding genes obtained in example 3 were extracted in large amounts, and then the light and heavy chain vectors were co-transfected with 293F at a molar ratio of 3:2. The mixing ratio of plasmid and PEI (POLYSCIENCE) is 1:3, the mixture is stood for half an hour at room temperature after mixing, the mixture is added into cells dropwise, SMS293-SUPI feed liquid (Sino Biological) is added for 24 hours and 72 hours after transfection, and cell supernatant is collected after 5 days.

Recombinant antibody purification, namely collecting cell supernatant, adding Protein A filler (Yiqiao Shenzhou) 1mL, incubating for half an hour at room temperature, taking out the filler, loading the filler into a purification column, flushing 20 mL by adding PBS solution, then adding 10 mL of 100 mM glycine solution with pH of 3.0 for eluting, and adding the eluent into 1M Tris (pH of 9.0) solution with the neutralization solution to pH of 7.0. The neutralized eluate was concentrated to 1mL, dialyzed into PBS, the protein concentration was measured and purity was confirmed by SDS-PAGE of a part of representative antibodies, and as a result, as shown in FIG. 3, the purity of the antibodies was high and the bands were single.

EXAMPLE 6 BLI Primary screening binding antibodies and identification of antibody affinity

Antibodies binding to CD45 recombinant proteins were primary screened by biofilm interference technology (BLI). Biofilm interference technology (BLI) allows real-time monitoring of intermolecular interactions, with molecular change reactions shown in the relative shift intensities (nm) of the interference spectrum. The experiment used protein A probe capture antibody (capture amount greater than 0.2 nM), flow antigen, PBST (0.2% Tween) as buffer. The antibody was diluted to 5ug/mL and the antigen diluted to 200nM,Gator Primer sequentially flowed PBST, antibody, PBST, antigen and PBST for binding and dissociation. PBST was added as a control for association for each set of experiments to subtract background changes in relative shift of the interference spectrum during dissociation. Kinetic parameter calculation was performed using a 1:1 Binding model of analytical software to determine whether the antibody bound to the antigen and to determine the affinity KD of the antibody, and the experimental results are shown in FIG. 4, wherein the affinity K _D (M) of the CD45-58 Binding antibody (SEQ ID NO:15, 16) according to the present invention is less than 1.00E-12, and the affinity reaches the picomolar (pM) scale.

EXAMPLE 7 IHC test with binding antibody

Firstly, preparing a formalin-fixed paraffin-embedded tissue sample into paraffin sections with the thickness of 3 microns, flattening the paraffin sections by water temperature of 40 ℃, baking the sections in a 60 ℃ oven for 1h, dewaxing the sections by xylene dewaxing and alcohol gradient treatment, thermally repairing antigens by EDTA microwaves, sealing tissue endoperoxidase by endogenous peroxidase blocking liquid, then dripping sheep serum to seal the tissue, incubating at 37 ℃ for 30 minutes, removing redundant liquid, adding primary antibodies with different concentrations (namely antibodies CD45-58 (SEQ ID NO:15 and 16) discovered by the invention), incubating at normal temperature, and incubating at 1 h-2 h, wherein the NC group uses PBS instead of the antibodies. After the primary antibody incubation is finished, PBST washes the excess primary antibody, adds secondary antibody Polymer HRP Goat anti-mouse/rabit lgG (Ready-to-Use) (purchased from Chongqing New production technology Co., ltd., cat LS-PA-03015T) and incubates at normal temperature for 30 min, and PBST washes the excess secondary antibody. And then preparing DAB chromogenic solution for chromogenic, observing under a mirror, controlling chromogenic time, dyeing cell nuclei through hematoxylin after water washing termination reaction, and carrying out blue returning treatment. Finally, alcohol gradient dehydration and xylene transparent treatment are carried out, neutral resin is used for sealing the piece after the drying, and the dyeing result is observed and photographed by a high-power mirror. As shown in FIG. 5, the CD45-58 rabbit recombinant monoclonal antibody of the invention shows high positive rate and strong specific membrane localization in multi-tissue paraffin sections such as tonsils, spleens and the like, and the staining mode (clear edge and continuous signals of cell membranes) is consistent with the result of a widely verified commercial positive control antibody PC (Zhonghua gold bridge ZM-0183). Under the same experimental condition, compared with a positive control antibody, the rabbit recombinant monoclonal antibody of CD45-58 has higher staining intensity at high concentration, has no cytoplasm or interstitial nonspecific background, and still maintains the staining intensity equivalent to that of the positive control at low working concentration (0.1 mug/mL), thus indicating that the CD45-58 has the core advantages of high specificity and low background interference in IHC detection.

In the description of the present specification, reference to the term "one embodiment," "some embodiments," "an embodiment," or "a particular embodiment," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments described in this specification, as well as the features of the various embodiments, can be combined and combined by one skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. An antibody or antigen-binding fragment thereof comprising

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain framework region and/or a light chain framework region, at least a portion of which is derived from at least one of a rabbit, murine, human, primates, sheep, dog, cat, and alpaca antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

4. The antibody or antigen-binding fragment thereof of claim 1, comprising:

A heavy chain variable region of the amino acid sequence shown as SEQ ID NO. 15 or an amino acid sequence having at least 80% identity thereto, and/or

The amino acid sequence shown as SEQ ID NO. 16 or an amino acid sequence having at least 80% identity thereto.

5. The antibody or antigen-binding fragment thereof of claim 1, further comprising a heavy chain constant region and/or a light chain constant region, at least a portion of which is derived from at least one of a rabbit, murine, human, primates, sheep, dogs, cats, and alpaca antibody.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises at least one member selected from the group consisting of full length monoclonal antibodies, fab 'antibodies, F (ab') 2 antibodies, fv antibodies, single chain antibodies, single domain antibodies, and minimal recognition units, or wherein the antigen-binding fragment comprises at least one member selected from the group consisting of F (ab ') 2 fragments, fab' fragments, fab fragments, F (ab) 2 fragments, fv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, and minimal recognition units.

7. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A recombinant cell comprising the nucleic acid molecule of claim 7, the expression vector of claim 8, or the antibody or antigen-binding fragment thereof of any one of claims 1-6.

10. A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising culturing the recombinant cell of claim 9.

11. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6 and a conjugate moiety coupled thereto.

12. The antibody conjugate of claim 11, wherein the conjugate moiety is selected from a purification tag or label.

13. The antibody conjugate of claim 12, wherein the conjugate moiety comprises at least one member selected from the group consisting of colloidal gold, radiolabel, phosphorescent chemistry, chemiluminescent, fluorescein, enzyme, native toxin, nucleic acid, polypeptide, and affinity label, and/or

The coupling moiety comprises at least one member selected from the group consisting of magnetic beads, magnetic microspheres, plastic microparticles, microwell plates, nylon and nitrocellulose membranes.

14. A reagent or kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or the antibody conjugate according to any one of claims 11 to 13.

15. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 6, an antibody conjugate according to any one of claims 11 to 13, or a reagent or kit according to claim 14 for the detection of CD45, for the preparation of a product for the detection of CD45 or for the diagnosis of a CD45 related disease.

16. A method of detecting CD45 in a test sample, the method comprising contacting the antibody or antigen-binding fragment thereof of any one of claims 1-6, the antibody conjugate of any one of claims 11-13, or the reagent or kit of claim 14 with CD45 antigen in the sample to be detected to form an immune complex.