CN119751677B - Anti-idiotype antibody for Anti-CD19 CAR expression detection - Google Patents
Anti-idiotype antibody for Anti-CD19 CAR expression detectionInfo
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Abstract
本申请涉及抗体技术领域，具体涉及一种特异性结合CAR抗原结合位点的抗独特型抗体，该抗体具有高亲和力、高特异性和无非特异背景优势，更适于评估CAR转染阳性率以及给药后CAR阳性T细胞数量在受试体内的变化规律等方面。
This application relates to the field of antibody technology, specifically to an anti-idiotype antibody that specifically binds to the CAR antigen binding site. This antibody has the advantages of high affinity, high specificity, and no nonspecific background, making it more suitable for evaluating the CAR transfection positivity rate and the changes in the number of CAR-positive T cells in the test body after administration.
Description
The present application claims priority from the chinese patent application filed on 1 month 2 2024, filed on patent office application number 202410002320.4, entitled "Anti-idiotype antibody for Anti-CD19 CAR expression detection", the entire contents of which are incorporated herein by reference.
Technical Field
The application relates to the technical field of antibodies in the biotechnology field and the immune field, in particular to an Anti-uniqueness antibody for Anti-CD19 CAR expression detection and application thereof.
Background
CAR-T cell therapy is cancer immunotherapy based on the in vitro engineering of T cells to express on their surface receptor fragments that specifically recognize tumor surface antigens, the engineered T cells being infused into the patient without the aid of Antigen Presenting Cells (APCs), targeting cancer cells in vivo and exerting an immune killing effect. For CAR-T cells, the active ingredient that exerts a tumor killing effect is CAR positive T cells. The packaging specification and clinical dosage of the CAR-T cell product are expressed in terms of CAR-T positive cell number, and therefore, CAR transfection-positive rate is a necessary term in CAR-T cell development.
Currently, detection of CAR transfection positivity is generally performed by flow cytometry, and there are detection methods for different structural regions of CAR, including anti-Fab antibodies or Protein L proteins directed against the CAR antigen binding site, such as CD19 antigen, or against the light chain or hinge region. Among them, the CAR positive rate detection method for antigen binding sites is widely used because of its better specificity.
However, the existing CAR detection tool generally has the problems of low sensitivity, high non-specific background and the like. anti-Fab antibodies and Protein L belong to a universal CAR positive detection reagent, but they cannot stain individual CARs on a dual-target CAR cell, protein L specifically binds to antibodies containing kappa light chains, only to human kappa I, III, IV (not to vkappa II subtype and lambda light chain antibodies) and mouse kappa I light chain subtypes, are not applicable to other subtypes, and have high or low affinity for each subtype. Meanwhile, the Protein L is used for dyeing before and after being strictly washed, otherwise, serious nonspecific reaction occurs due to cross reaction with the non-CAR IgG-like Protein, and when the CAR transfection positive rate is detected by using the antigen, the activity and the use concentration of the antigen directly determine the detection accuracy. Furthermore, for CAR cell detection in patients, antigen proteins are highly unsuitable for CAR cell detection in vivo due to the cytosolic effect and the blocking effect of free antigen on CAR-scFv. In view of this, the present application has been proposed.
Summary of The Invention
Aiming at the technical problems, the application develops a series of anti-idiotype antibodies which specifically bind to the CAR antigen binding site through intensive research, and the antibodies can specifically evaluate the CAR transfection positive rate and the change rule of the CAR positive T cell number in the patient after the patient is dosed. Accordingly, the present application includes at least the following objects:
a first object of the application is to provide an antibody or antigen binding fragment thereof that specifically binds to a CAR antigen binding site;
The second object of the present application is to provide a polynucleotide encoding the above antibody or antigen-binding fragment thereof, a corresponding vector, a cell, or a kit comprising the same, etc.;
A third object of the present application is to provide the use of the above antibody or antigen binding fragment, including the use in detecting Anti-CD19 CAR expression, in assessing Anti-CD19 CAR transfection-positive rate, and in assessing the change in the number of Anti-CD19 CAR positive T cells in a subject following administration, etc.
The fourth object of the present application is to provide a detection method using the above antibody or antigen-binding fragment.
In order to achieve the above purpose, the present application adopts the following specific technical scheme.
In a first aspect the application provides an antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, which specifically binds to a CAR antigen binding site, comprising 3 CDRs in the heavy chain variable region amino acid sequence as set out in any one of SEQ ID nos. 4, 12, 20, 28 and 3 CDRs in the light chain variable region amino acid sequence as set out in any one of SEQ ID nos. 8, 16, 24, 32, or a variant having a single or multiple CDR with the light heavy chain CDR regions described above of no more than 2 amino acid changes per CDR region.
Further, the residue ranges of the CDRs are shown in the following table:
Further, when encoded by the IMGT encoding rules for antibodies HCDRs, the antibodies or antigen binding fragments comprise the following CDR sequences:
i. The amino acid sequence of the HCDR1 is shown in any one of SEQ ID NO.1, 9, 17 and 25;
The amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO.2, 10, 18 and 26;
the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO.3, 11, 19 and 27;
The amino acid sequence of the LCDR1 is shown in any one of SEQ ID NO.5, 13, 21 and 29;
v. the amino acid sequence of LCDR2 is shown in any one of SEQ ID NO.6, 14, 22, 30;
The amino acid sequence of the LCDR3 is shown in any one of SEQ ID NO.7, 15, 23 and 31;
Or a variant having a single or multiple CDR with the 6 CDR regions of i-vi described above that does not vary by more than 2 amino acids per CDR region.
Further, the combination of HCDR1/HCDR2/HCDR3 may comprise one or more groups selected from:
(1)HCDR1/HCDR2/HCDR3:
SEQ ID NO.1/SEQ ID NO.2/SEQ ID NO.3,
SEQ ID NO.9/SEQ ID NO.10/SEQ ID NO.11,
SEQ ID NO.17/SEQ ID NO.18/SEQ ID NO.19,
SEQ ID NO.25/SEQ ID NO.26/SEQ ID NO.27,
(2) Or an HCDR of the above (1) containing one or more amino acid substitutions, deletions or insertions of not more than 2 amino acids;
The LCDR1/LCDR2/LCDR3 combination comprises one or more groups selected from the group consisting of:
(1) LCDR1/LCDR2/LCDR3 is:
SEQ ID NO.5/SEQ ID NO.6/SEQ ID NO.7,
SEQ ID NO.13/SEQ ID NO.14/SEQ ID NO.15,
SEQ ID NO.21/SEQ ID NO.22/SEQ ID NO.23,
SEQ ID NO.29/SEQ ID NO.30/SEQ ID NO.31,
(2) Or an HCDR of the above (1) containing one or more amino acid substitutions, deletions or insertions of not more than 2 amino acids.
In some embodiments, an antibody or antigen binding fragment thereof of the application that specifically binds to a CAR antigen binding site further comprises a heavy chain variable region VH sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID No.4, 12, 20, 28, and a light chain variable region VL sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID No.8, 16, 24, 32.
Further, the antibody or antigen binding fragment may further comprise a coupling moiety attached to a polypeptide, the coupling moiety being selected from one or more of a radionuclide, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, a lipid, and biotin, wherein the polypeptide or antibody and the coupling moiety may be optionally attached via a linker, preferably the linker is a peptide or polypeptide.
Further, the antibody or antigen binding fragment may be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies, and human antibodies, more preferably, the antibody is selected from the group consisting of multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotype (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, fab fragments, F (ab') fragments, disulfide-linked bispecific Fv (sdFv), and intracellular antibodies.
In a second aspect of the application, there is also provided a nucleic acid encoding an antibody or antigen binding fragment thereof of the first aspect.
In a third aspect the application provides a vector (preferably a recombinant vector) comprising a nucleic acid of the second aspect, and optionally regulatory sequences;
Further, the vector may be a cloning vector or an expression vector, without limitation;
further, the regulatory sequence may be selected from a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal sequence, transcription terminator, or any combination thereof, without limitation.
In a fourth aspect the application provides a cell (preferably a host cell) comprising a nucleic acid of the second aspect or a vector of the third aspect.
Further, the cells include, but are not limited to, mammalian cells such as yeast cells, chinese hamster ovary cells, human embryonic kidney cells, or other cells suitable for use in the preparation of antibodies or antigen-binding fragments thereof.
In a sixth aspect the application provides a kit comprising one or more of an antibody or antigen binding fragment, polynucleotide, recombinant vector and host cell as described in any one of the preceding claims, in a suitable container.
In a seventh aspect the application provides a method of preparing an antibody or antigen binding fragment thereof of the first aspect that binds to a CAR antigen binding site, the method comprising expressing the vector of the third aspect in a host cell culture to produce the antibody, and recovering the antibody molecule from the cell culture.
An eighth aspect of the application provides the use of an antibody or antigen binding fragment thereof comprising the first aspect in any of the following:
a) Use in detecting Anti-CD19 CAR expression;
b) Use in assessing the transfection positive rate of an Anti-CD19 CAR;
c) Use of an Anti-CD19 CAR positive T cell number after administration in assessing the law of change in a subject, or use of an Anti-CD19 CAR positive T cell number after administration in the preparation of a reagent for assessing the law of change in a subject. It will be appreciated that a) and b) may be in vitro assays, the antibodies or antigen binding fragments of the application are a means of CAR detection, and that samples in practice may be ex vivo or in vitro, and when used for in vitro sample detection, the purpose may be for simple CAR detection or evaluation purposes, thus involving non-disease diagnostic applications.
In a ninth aspect, the application provides a method of detecting a sample to be detected using an antibody or antigen binding fragment as described in any of the preceding claims, or a kit as described above.
Further preferably, the detection method is used to evaluate whether the sample contains an Anti-CD19 CAR or an Anti-CD19 CAR expression level, etc., without limitation, as would be reasonably expected in the art.
Drawings
FIG. 1 partial results of FACS binding screening of positive clone supernatants.
FIG. 2 partial results of FACS blocking screening of positive clone supernatants.
FIG. 3 partial results of subclone blocking FACS detection.
FIG. 4 partial results of subclone blocking FACS detection.
FIG. 5 shows the SDS-PAGE identification of antibodies against FMC63 scFv of the present application.
FIG. 6 shows ELISA binding assay results for antibodies of the application against FMC63 scFv.
FIG. 7 shows the results of a competition ELISA assay for antibodies against FMC63 scFv of the present application.
FIG. 8 shows the SPR analysis results of antibodies against FMC63 scFv of the present application.
FIG. 9 shows the results of FACS binding analysis of an antibody against FMC63 scFv of the present application.
Detailed Description
The present application discloses an isolated antibody or antigen binding fragment thereof, and uses thereof will be made by those skilled in the art with reference to the disclosure herein, and it is specifically noted that all such substitutions and modifications as would be apparent to those skilled in the art are deemed to be included in the present application. While the present application has been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations and modifications can be made in the methods and applications of the present application, and that the techniques of the application can be implemented and practiced without departing from the spirit and scope of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The following terms or definitions are provided solely to aid in the understanding of the application. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the application are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.
As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The terms "about" and "substantially" in this application mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
The terms "or more," "at least," "exceeding," and the like, such as "at least one" should be understood to include, but not be limited to, at least 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or exceeding the stated values. But also any larger numbers or scores therebetween.
Conversely, the term "no more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100、99、98、97、96、95、94、93、92、91、90、89、88、87、86、85、84、83、82、81、80、79、78、77、76、75、74、73、72、71、70、69、68、67、66、65、64、63、62、61、60、59、58、57、56、55、54、53、52、51、50、49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2、1 and 0 nucleotides. But also any smaller numbers or scores therebetween.
The terms "plurality," "at least two," "two or more," "at least a second," and the like should be understood to include, but are not limited to, at least 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. But also any larger numbers or scores therebetween.
Definition of terms:
As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that is capable of non-covalently, reversibly and in a specific manner binding to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain CL. VH and VL regions can be further subdivided into regions of hypervariability known as Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). "antibodies" include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the present disclosure). Antibodies can be of any isotype/class (e.g., igG, igE, igM, igD, igA and IgY) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2).
Antibodies comprise globular regions of heavy or light chain polypeptides known as "domains". The domain may comprise peptide loops, typically 3 to 4 loops, which are stabilised, for example, by β -sheet and/or intrachain disulphide bonds. Based on the relative lack of sequence variation within the domains of the members of different classes in the case of "constant" domains, or significant variation within the domains of the members of different classes in the case of "variable" domains, the domains are often referred to as "constant" or "variable". An antibody or polypeptide "domain" is often interchangeably referred to in the art as an antibody or polypeptide "region".
Antibodies can be classified into 5 classes, igA, igD, igE, igG and IgM, and several isotypes in these classes can be further classified into subclasses, e.g., igG1, igG2, igG3, and IgA1, and IgA2, based on the amino acid sequence of the antibody heavy chain constant region. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Kappa and lambda can be distinguished by the difference in antibody light chain constant regions (CL). Within full length light and heavy chains, typically the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.
The term "monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, and does not relate to a method of preparation thereof. Monoclonal antibodies or immunologically active fragments thereof may be produced by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques, and the like, or other production techniques known in the art, and methods of the application involving monoclonal antibody production include in vitro culture of hybridoma cells or by DNA recombination techniques. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each monoclonal antibody is directed against a single determinant on the antigen.
The term "antigen" is an entity (e.g., a protein entity or peptide) to which an immunoglobulin or antibody (or antigen binding fragment thereof) specifically binds.
The term "fragment" refers to a portion or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with the portion when present in the intact antibody. Fragments may be obtained by chemical or enzymatic treatment of whole or complete antibodies or antibody chains. Fragments may also be obtained recombinantly.
The term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
"Complementarity determining domain" or "complementarity determining region" ("CDR") interchangeably refer to the hypervariable regions of VL and VH. CDRs are target protein binding sites of antibody chains carrying the specificity of such target proteins. Three CDRs (CDRs 1-3, numbered sequentially from the N-terminus) are present in each human VL or VH, totaling about 15-20% of the variable domains. CRD may be referred to by its region and sequence. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDRs are structurally complementary to epitopes of the target protein and are therefore directly responsible for binding specificity. The remaining stretches of VL or VH (so-called framework regions) exhibit little variation in amino acid sequence (Kuby, immunology, 4 th edition, chapter 4 w.h. frieman (w.h.freeman & co.), new york, 2000).
In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems including, for example, chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al ,"Standard conformations for the canonical structures of immunoglobulins",Journal of Molecular Biology,273,927-948(1997)), based on Kabat (Kabat et al Sequences of Proteins of Immunological Interest, 4 th edition, U.S. Department of HEALTH AND Human Services, national) based on the three-dimensional structure of the antibody and topology of the CDR loops
Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT), and North CDR definitions based on neighbor-propagated clusters (affinity propagation clustering) that utilize a large number of crystal structures.
However, it should be noted that the boundaries of CDRs of the variable regions of the same antibody obtained based on different assignment systems may differ. I.e. the CDR sequences of the same antibody variable region defined under different assignment systems are different. For example, the ranges of residues under different assignment system definitions for CDR regions using numbering Kabat, chothia et al are shown in the table below.
CDR residue ranges under different assignment system definitions
Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).
The CDRs of the antibodies of the application can be evaluated manually to determine boundaries according to any protocol or combination of protocols in the art. In the present application, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways.
Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelbody antibodies, affibodies, fab fragments, F (ab') 2 fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies), miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), and antigen binding fragments of any of the above.
The term "antigen binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen. Examples of binding fragments include, but are not limited to, single chain Fv (scFv), disulfide-linked Fv (sdFv), fab fragments, F (ab') fragments (i.e., monovalent fragments consisting of VL, VH, CL and CH1 domains), F (ab) 2 fragments (i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), fd fragments consisting of VH and CH1 domains, fv fragments consisting of the VL and VH domains of an antibody single arm, dAb fragments consisting of the VH domain (Ward et al, nature 341:544-546,1989), and isolated Complementarity Determining Regions (CDRs) or other epitope-binding fragments of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined, using recombinant methods, by a synthetic linker that enables the two domains to become a single protein chain in which the VL and VH pairs are used to form monovalent molecules, known as single chain Fv ("scFv"); see, e.g., bird et al Science 242:423-426,1988, and Huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883,1988). Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment". These antigen binding fragments are obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as whole antibodies.
Antigen binding fragments may also be incorporated into single domain antibodies, large antibodies, minibodies, nanobodies, intracellular antibodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFv (see, e.g., hollinger and Hudson, nature Biotechnology (Nature Biotechnology) 23:1126-1136,2005). Antigen binding fragments may be grafted into a scaffold based on a polypeptide such as fibronectin type III (Fn 3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monoclonal antibodies). Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH 1-VH-CH 1) that together with a complementary light chain polypeptide form a pair of antigen binding regions (Zapata et al, protein engineering (Protein Eng.)) 8:1057-1062,1995, and U.S. Pat. No. 5,641,870.
The term "chimeric antibody" refers to a heavy chain and/or a portion of a light chain (generally referred to as the variable region) that is derived from a particular species or from an antibody belonging to a particular antibody class or subclass that is identical or homologous to the corresponding sequence in the antibody, while the remainder of the chain (generally referred to as the constant region) is derived from an antibody from another species or from an antibody belonging to another antibody class or subclass that is identical or homologous to the corresponding sequence in the fragment of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies contemplated in the present application, such as heavy/light chain variable regions from murine antibodies, are grafted to the constant regions of the heavy/light chains of human antibodies by antibody engineering techniques, which exhibit similar biological activity.
The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to sequences of a non-human antibody and all or substantially all of the FRs correspond to sequences of a human antibody.
The term "affinity" refers to the strength of interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at a number of sites by weak non-covalent forces, the more interactions, the greater the affinity.
The term "competition" herein refers to competition between antigen binding proteins, as used in the context of antigen binding proteins (e.g. neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, as determined by an assay in which an antigen binding protein (e.g. antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g. reduces) specific binding of a reference antigen binding protein (e.g. ligand or reference antibody) to a common antigen.
As used herein, the term "variant" refers to a heavy chain variable region or a light chain variable region that has been modified by at least one, e.g., 1, 2, or 3 amino acid substitution, deletion, or addition, wherein a modified antigen binding protein comprising a heavy chain or light chain variant substantially retains the biological characteristics of the antigen binding protein prior to modification. In one embodiment, the antigen binding protein comprising a variable heavy chain variable region or light chain variable region sequence retains 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to modification. It will be appreciated that each heavy chain variable region or light chain variable region may be modified either alone or in combination with another heavy chain variable region or light chain variable region. The antigen binding proteins of the present disclosure comprise a heavy chain variable region amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a heavy chain variable region amino acid sequence described herein. The antigen binding proteins of the present disclosure include light chain variable region amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequences described herein. The percent homology may be over the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology may be limited to framework regions, while the sequences corresponding to CDRs have 100% identity to CDRs disclosed herein within the heavy chain variable region and/or the light chain variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified by at least one, e.g., 1, 2, or 3 amino acid substitution, deletion, or addition, wherein the modified antigen binding protein comprising the CDR variant substantially retains the biological characteristics of the pre-modified antigen binding protein. In one embodiment, the antigen binding protein comprising variant CDRs retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to modification. It will be appreciated that each CDR that may be modified alone or in combination with another CDR. In one embodiment, the modification is a substitution, particularly a conservative substitution.
The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked by transformation. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".
The term "host cell" refers to a cell that is foreign to nucleic acid, including the progeny of such a cell. And is capable of expressing the exogenous nucleic acid in a cell or cell membrane or released outside the cell.
The term "subject" encompasses both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, except when indicated.
The term "kit" is used to refer to a combination of reagents and other materials that facilitate analysis of a sample. In some embodiments, an immunoassay kit described herein includes a suitable antigen, a binding agent comprising a detectable moiety, and a detection reagent. The system for amplifying the signal generated by the detectable moiety may or may not also be included in the kit. Furthermore, in other embodiments, the kits include, but are not limited to, components such as devices for sample collection, sample tubes, racks, trays, shelves, plates, instructions for the kit user, solutions or other chemical reagents, and samples for normalization, and/or control.
1. The Anti-idiotypic antibodies or antigen binding fragments of the application directed against an Anti-CD19 CAR
The terms "Anti-unique antibody to Anti-CD19 CAR", "antibody specifically binding to the antigen binding site of Anti-CD19 CAR", "antigen binding fragment to Anti-CD19 CAR" are used interchangeably herein to refer to an antibody or antigen binding fragment of the application that is capable of binding to Anti-CD19 CAR with sufficient affinity and specificity such that the antibody can be used for detection of the CAR. In some embodiments, an antibody or antigen binding fragment of the application is capable of specifically binding to a CAR antigen binding site, which is capable of specifically binding to the antigen recognition site of an Anti-CD19 CAR of FMC63 origin, and has the property of high affinity, according to ELISA and SPR validation data. The antibody can detect the expression of the Anti-CD19 CAR from FMC63 through a flow cytometry method, and has the characteristics of high sensitivity and no non-specific background.
Antibodies and antigen binding fragments of the application specifically bind to an Anti-CD19 CAR with high affinity. In some embodiments, the antibodies or antigen binding fragments of the application are of a class of sequences as known from the different analytical methods in the definition above for CDRs:
It may comprise 3 CDRs in the heavy chain variable region amino acid sequence shown in any one of SEQ ID NO.4, 12, 20, 28 and 3 CDRs in the light chain variable region amino acid sequence shown in any one of SEQ ID NO.8, 16, 24, 32, or a variant having a single or multiple CDRs from the light and heavy chain CDR regions of not more than 2 amino acid changes per CDR region.
In some embodiments, the antibody or antigen binding fragment comprises the following CDR sequences when encoded against antibody HCDRs according to IMGT encoding rules:
i. The amino acid sequence of the HCDR1 is shown in any one of SEQ ID NO.1, 9, 17 and 25;
The amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO.2, 10, 18 and 26;
the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO.3, 11, 19 and 27;
The amino acid sequence of the LCDR1 is shown in any one of SEQ ID NO.5, 13, 21 and 29;
v. the amino acid sequence of LCDR2 is shown in any one of SEQ ID NO.6, 14, 22, 30;
The amino acid sequence of the LCDR3 is shown in any one of SEQ ID NO.7, 15, 23 and 31;
Or a variant having a single or multiple CDR with the 6 CDR regions of i-vi described above that does not vary by more than 2 amino acids per CDR region.
In some embodiments, the HCDR1/HCDR2/HCDR3 combination may comprise one or more groups selected from the group consisting of:
(1)HCDR1/HCDR2/HCDR3:
SEQ ID NO.1/SEQ ID NO.2/SEQ ID NO.3,
SEQ ID NO.9/SEQ ID NO.10/SEQ ID NO.11,
SEQ ID NO.17/SEQ ID NO.18/SEQ ID NO.19,
SEQ ID NO.25/SEQ ID NO.26/SEQ ID NO.27;
(2) Or an HCDR of the above (1) containing one or more amino acid substitutions, deletions or insertions of not more than 2 amino acids;
The LCDR1/LCDR2/LCDR3 combination comprises one or more groups selected from the group consisting of:
(1) LCDR1/LCDR2/LCDR3 is:
SEQ ID NO.5/SEQ ID NO.6/SEQ ID NO.7,
SEQ ID NO.13/SEQ ID NO.14/SEQ ID NO.15,
SEQ ID NO.21/SEQ ID NO.22/SEQ ID NO.23,
SEQ ID NO.29/SEQ ID NO.30/SEQ ID NO.31;
(2) Or an HCDR of the above (1) containing one or more amino acid substitutions, deletions or insertions of not more than 2 amino acids.
In some specific embodiments, the bindable antibody or antigen-binding fragment thereof of the application further comprises a heavy chain variable region VH sequence and a light chain variable region VL sequence. The heavy chain variable region VH sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID No.4, 12, 20, 28, and the light chain variable region VL sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID No.8, 16, 24, 32, preferably the amino acid change does not occur in the CDR region.
In some more specific embodiments, some specific antibody sequences of the application are shown in the following table (specific VH and VL sequences of an antibody or antigen binding fragment thereof).
In some embodiments, the antibodies or antigen binding fragments of the application further comprise an Fc region from an IgG, such as IgG1, igG2, igG3, or IgG4.
In some embodiments, the amino acid changes in amino acid homology described above include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table (amino acid substitutions).
In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FR). More preferably, the amino acid changes described in the present application occur in the Fc region.
In some embodiments, the application encodes the CDRs of an antibody according to the IMGT encoding rules, the CDRs sequences of a particular numbered antibody are shown in the following Table
In some embodiments, the antibody or antigen binding fragment may further comprise a coupling moiety attached to a polypeptide, the coupling moiety selected from one or more of a radionuclide, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, a lipid, and biotin, wherein the polypeptide or antibody and the coupling moiety may be selectively attached by a linker, preferably the linker is a peptide or polypeptide.
In some embodiments, the antibody or antigen binding fragment may be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies, and human antibodies, more preferably, the antibody is selected from the group consisting of multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotype (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, fab fragments, F (ab') fragments, disulfide-linked bispecific Fv (sdFv), and intracellular antibodies.
In some embodiments, the antibodies or antigen binding fragments thereof of the application may be produced by recombinant expression. The above-described nucleic acids encoding the light and heavy chain variable regions optionally linked to constant regions may be inserted into expression vectors. Vectors comprising nucleic acids encoding antibodies described herein are themselves an aspect of the application. The light and heavy chains may be cloned into the same or different expression vectors. The nucleic acids encoding the antibody chains described herein may be operably linked to one or more control sequences in an expression vector to ensure expression of the antibody chains. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Such vectors may be incorporated into a suitable host whereby the host is maintained under conditions suitable for high level expression of the nucleotide sequences and for collection and purification of the antibodies.
2. Nucleic acids of the application, vectors and host cells comprising the same
The application provides nucleic acids encoding any of the above antibodies or antigen binding fragments thereof or any of the chains thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is a recombinant vector, such as an expression vector or a cloning vector. In one embodiment, a cell, such as a host cell, comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for preparing an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.
The nucleic acid according to the present application is a nucleic acid encoding an antibody or antigen-binding fragment thereof or a VH or VL domain thereof, and it is understood that any nucleic acid capable of encoding an antibody or antigen-binding fragment thereof or a VH or VL domain thereof as described above is within the scope of the present application.
The vectors of the present application comprising one or more of the above-described nucleic acids encoding the antibodies described herein may be cloning vectors or expression vectors, and are not limited herein.
In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs), and the like.
In one embodiment, the vector comprises an optional regulatory sequence, which in some embodiments may be selected from a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal sequence, transcription terminator, or any combination thereof, without limitation.
The host cells of the application, such as host cells, comprising the expression vectors are yeast cells, mammalian cells or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof. In some embodiments, suitable host cells include prokaryotic microorganisms, such as E.coli. The host cell may also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, e.g., insect cells, etc. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV 1), african green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63, sp2/0, and the like. For a review of mammalian host cell lines suitable for the production of proteins see, e.g., yazaki and Wu, methods in Molecular Biology, vol.248 (b.K.C.Lo et al, humana Press, totowa, NJ), pages 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or 293 cell.
Vectors described herein comprising polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or virus-based transfection may be used for other cellular hosts. (see generally Green and Sambrook, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual) (Cold spring harbor Press, 4 th edition, 2012.) other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection (see generally Sambrook et al, supra.) to produce transgenic animals, the transgene may be microinjected into fertilized oocytes, or it may be integrated into the genome of embryonic stem cells, and the nuclei of these cells transferred into enucleated oocytes.
3. Preparation, production and purification of antibodies or antigen binding fragments of the application
The methods of making an Anti-unique antibody of an Anti-CD19 CAR, or an antigen-binding fragment thereof, of the present application may comprise expressing a vector described herein in a host cell culture to produce the antibody, and recovering the antibody from the cell culture.
In some embodiments, the method may comprise transferring a vector comprising one or more nucleic acids encoding an antibody or antigen-binding fragment thereof or antibody chain thereof as described above into a host cell as described herein, culturing the host cell culture under conditions allowing expression of the nucleic acids, and recovering the expressed corresponding antibody or antigen-binding fragment thereof. Any suitable method known in the art may be employed.
The application provides a method of Anti-idiomatic antibodies or antigen-binding fragments of an Anti-CD19 CAR, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the antibody or antigen-binding fragment, and optionally isolating the antibody or antigen-binding fragment. In a certain embodiment, the method further comprises recovering and purifying the corresponding antibody or antigen binding fragment from the host cell (or host cell culture medium).
In some embodiments, the antibodies or antigen binding fragments prepared as described herein may be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the antibodies of the application may be determined by any of a variety of well-known analytical methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
4. The detection method and application of the application
The antibodies or antigen binding fragments provided by the application can be used for detecting the presence or the content of the Anti-CD19 CAR in a biological sample, and further can be used for detection or evaluation purposes.
In some embodiments, the antibodies or antigen binding fragments thereof provided herein against an Anti-CD19 CAR can be conveniently used in a kit, and an Anti-CD19 CAR in a sample in vivo or in vitro can be detected by the antibodies or antigen binding fragments thereof provided herein with specificity, high sensitivity and no non-specific background. Thus, the antibodies or antigen binding fragments thereof of the application are particularly useful in assessing the rate of positive CAR transfection, the regularity of change in the number of CAR positive T cells in a subject following administration, and the like.
The term "detection" as used herein, including quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, SPR, and the like. In some embodiments, the protein antibodies or antigen binding fragments thereof of the present application may be conjugated to a detectable label such as luciferase, biotin, etc., for use in a direct or indirect immunoassay such as FACS, SPR, IHC, ELISA in a liquid or solid phase.
The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Examples
The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
EXAMPLE 1 preparation, screening and sequencing of monoclonal antibodies
1) Immunogen preparation an expression plasmid capable of expressing FMC63 scFv, his Tag was constructed by gene synthesis method, then transfected into HEK293 cells using Invitrogen Lipofectamine's transfection reagent, the culture supernatant was harvested 48 hours later, then FMC63 scFv, his Tag was purified by affinity chromatography method, then purified FMC63 scFv was coupled to KLH carrier protein (purchased from Sigma, cat. No. H7017).
2) Mice were immunized with FMC63 scFv conjugated KLH carrier protein as immunogen, 10 Balb/c mice were immunized with FMC63 scFv-KLH using conventional immunization. Conventional immunization schedules are shown in Table 1. After 7 days of each immunization, the immune response level was determined by detecting serum from the immunized animal by ELISA. After conventional immunization, cell fusion can be performed if the immunized animal is able to reach an immune response level against the immunogen (OD value >1.0, titer 1:8,000).
Table 1 immune list
| Step (a) | Time schedule | Dosage and route | Adjuvant |
| Blood collection before immunization | Day-4 | ||
| First immunization | Day 0 | 50 Micrograms/dose, subcutaneously | Freund's complete adjuvant |
| Second epidemic disease | Day 14 | 25 Μg/d, subcutaneous | Freund's incomplete adjuvant |
| Blood sampling test 1 | Day 21 | ||
| Third epidemic disease | Day 28 | 25 Μg/d, subcutaneous | Freund's incomplete adjuvant |
| Blood sampling test 2 | Day 35 | ||
| Terminal exempt | Day 50.+ -. 7 | 25 Μg/abdomen | |
| Cell fusion | Four days after final exemption |
3) Cell fusion and plating
Cell fusion was performed 2 times using electrofusion methods, and all cells fused at a time were plated into 96-well plates.
4) Screening assays
Primary screening, namely screening supernatant of the fused cells by ELISA method, and selecting supernatant positive against scFv. FMC63scFv and several other proteins were coated (as negative and isotype controls) and pre-immune serum and post-three immune antisera were tested (table 2). Titer was the highest dilution ratio at a signal/blank ratio value of 2.1 or more, NC was preimmune serum, and served as a negative control (table 3).
Table 2ELISA Primary screening protocol
TABLE 3ELISA method screening supernatant results of fused cells
Confirmation screening all positive maternal cell supernatants were screened by indirect ELISA, and counter screening was performed with total human IgG and irrelevant his protein. The positive maternal clone indirect ELISA screening protocol is shown in table 4. The results of ELISA screening of pre-immune serum and post-three immune antisera are shown in Table 5. Titer is the highest dilution ratio at a signal/blank ratio value of 2.1 or more, NC is preimmune serum, and serves as a negative control.
TABLE 4 Indirect ELISA screening protocol for Positive maternal clones
TABLE 5 ELISA screening results of preimmune serum and antiserum after three immunizations
FACS binding screening the screening experiments were carried out with FMC63 scFv HEK293,1X10 5 cells as positive cell lines, conventional HEK293,1X10 5 cells as negative cell lines, primary antibodies as a, 1:50 diluted pre-immune serum, b, 3ug/ml mouse IgG (isotype control), c, PBS as negative control, d, 1:50 diluted post-immune antisera, respectively. The secondary antibody is Alexa647AffiniPure Goat Anti-Mouse IgG,Fcγfragment specific(min X Hu,Bov,Hrs Sr Prot)(Jackson,115-605-071),1ug/ml.FACS binding The screening results are shown in FIG. 1. Black curve, negative cells +a/b/c/d+ secondary antibodies, gray curve positive cells +a/b/c/d+ secondary antibodies.
FACS blocking screening the cell lines used in the screening experiments were those expressing FMC63 scFv HEK293,1×105 cells. The primary antibodies are respectively a, pre-immune serum diluted by 1:50, b, 3ug/ml mouse IgG (isotype control), c, PBS (negative control), d, anti-serum diluted by 1:50 after three times of immunization, e, 1ug/ml human CD19 recombinant protein ligand with human Fc label. The secondary antibody was Goat Anti-Human IgG FC FRAGMENT SPECIFIC [ Alexa Fluor647] (Jackson, 109-605-098), and the results of the FACS blocking screen were shown in FIG. 2. The black curve is scFv HEK293+c+e+ secondary antibody, and the gray curve is scFv HEK293+a/b/c/d+e+ secondary antibody.
5) Clone expansion culture and cryopreservation
Positive master clone cells were transferred to 24 well plates for expansion culture for a maximum of 10 strains. 2 ml of supernatant was collected from each expanded clone for indirect ELISA and to block FACS detection. These specific positive clonal cells were cryopreserved to avoid clonal loss. Indirect connection
ELISA protocols are shown in Table 6 and test results are shown in Table 7.
TABLE 6 Indirect ELISA screening protocol for expanded culture supernatants of Positive master clone cells
TABLE 7 results of indirect ELISA screening of the supernatant of expanded culture of Positive blast cells
The cell line for blocking FACS detection was a cell line expressing FMC63 scFv HEK293,1x10 5 cells. The primary antibodies are respectively a, 1:50 diluted fusion serum (positive control), b, 3ug/ml mouse IgG (isotype control), c, PBS (negative control), d, 1ug/ml humanized CD19 recombinant protein ligand with humanized Fc label, and e, positive maternal clone cell expansion culture supernatant. The secondary antibody is Alexa647AffiniPure Goat Anti-Human IgG(H+L)(min X Bov,Hrs,Ms Sr Prot)(Jackson,1μg/ml)。
6) Subcloning
Positive master clones were subcloned by limiting dilution to ensure that these positive master clones were derived from individual master clone cells, respectively, and subcloned screening was performed by indirect ELISA or blocking FACS methods. The indirect ELISA assay protocol is shown in Table 8 and the assay results are shown in Table 9.
TABLE 8 subclone indirect ELISA screening protocol
TABLE 9 subclone indirect ELISA screening protocol results
The cell line for blocking FACS detection was a cell line expressing FMC63 scFv HEK293,1x10 5 cells. The primary antibodies are respectively a, 1:50 diluted fusion serum (positive control), b, 3ug/ml mouse IgG (isotype control), c, PBS (negative control), d, 1ug/ml humanized CD19 recombinant protein ligand with humanized Fc label, and e, subcloning supernatant. The secondary antibody is Alexa647AffiniPure Goat Anti-Human IgG (H+L) (min X Bov, hrs, ms Sr Prot) (Jackson, 1. Mu.g/ml). Blocking FACS detection results are shown in fig. 3 and 4. The black curve is scFv HEK293+c+d+ secondary antibody, and the gray curve is scFv HEK293+a/b/c/e+d+ secondary antibody.
Based on antigen recognition confirmation, 2 stable subcloned cell lines were selected from each master clone for cryopreservation. 5 ml of supernatant was collected for each subclone prior to cryopreservation and all subclones were subtype identified and stored.
7) Antibody production and purification
Hybridoma cells were grown in an expanded manner, the antibodies were purified by protein A/G affinity chromatography, and the purified antibodies were stored in Phosphate Buffer (PBS) by dialysis.
8) Hybridoma cell antibody Gene sequencing
Based on the screening data and results, 4 dominant subclones 24C1E4D8, 26A10C3E6, 23D4G9E9 and 16D5E8E7 are obtained, and the antibodies show more comprehensive activities such as affinity and blocking and can be used for subsequent detection purposes. Further, total RNA of the corresponding hybridoma cells was extracted, the RNA was reverse transcribed into cDNA by RT-PCR reaction, the antibody light chain and heavy chain sequences were cloned, the light chain and heavy chain sequences were constructed on a T vector, and DNA sequencing analysis was performed to obtain antibody gene sequences, with specific sequence results as shown in Table 10.
TABLE 10 amino acid sequences of dominant antibodies (IMGT rules determine CDR)
EXAMPLE 2 analytical identification and functional analysis of monoclonal antibodies
The above-screened dominant clones were further subjected to antibody identification and functional analysis, and some of the analysis results are exemplarily shown below.
ELISA detection by subclass kit showed that the subclasses of antibodies 24C1E4D8, 26A10C3E6, 23D4G9E9 and 16D5E8E7 were IgG1, kappa.
SDS-PAGE identification (FIG. 5) shows that, taking 16D5E8E7 antibody as an example, the molecular weight of the two bands of the reduction electrophoresis is 27kDa and 50kDa respectively, and the purity of the antibody is more than 99%.
ELISA binding data (FIG. 6) and competition ELISA data (FIG. 7) indicated that the 16D5E8E7 clone number antibody was able to very specifically recognize the antigen binding site of the Anti-CD19 (FMC 63) CAR.
SPR analysis data (FIG. 8) shows that 7 top-down fit lines represent the time dependence of affinity and dissociation of FMC63 scFv with 16D5E8E7 clone number antibodies at concentrations of 125nM, 62.5nM, 31.25nM, 15.625nM, 7.813nM, 3.906nM and 1.953nM, respectively. The results indicate that the affinity of the antibody for binding FMC63 scFv is as high as 1.1nM.
FACS binding data (fig. 9) demonstrate that the 16D5E8E7 clone number antibody can specifically bind to Anti-CD19 (FMC 63) CAR expressed on the cell surface, while having no non-specific binding signal to both 293 cells not transfected with CAR and PBMC cells transfected with CAR, exhibiting the advantages of good high specificity and no non-specific background.
The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the application and its practical application to thereby enable one skilled in the art to make and utilize the application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.
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