CN117384290B - Human ROBO1 binding molecules and uses thereof - Google Patents

Human ROBO1 binding molecules and uses thereof Download PDF

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CN117384290B
CN117384290B CN202311685718.4A CN202311685718A CN117384290B CN 117384290 B CN117384290 B CN 117384290B CN 202311685718 A CN202311685718 A CN 202311685718A CN 117384290 B CN117384290 B CN 117384290B
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robo1
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human
amino acid
acid sequence
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CN117384290A (en
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李华顺
韩昆昆
韦治明
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Sichuan Asikeli Biotechnology Co ltd
Suzhou Inte Pharmaceutical Research And Development Co ltd
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Sichuan Asikeli Biotechnology Co ltd
Suzhou Inte Pharmaceutical Research And Development Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Abstract

The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a human ROBO1 binding molecule and application thereof. The specific nano antibody for resisting the ROBO1 target provided by the invention fills the gap of the detection of the ROBO1 target in the field of nano antibodies. It was verified that the invention also provides nanobodies with an affinity of VHH-12C of 8.27E-08 and an affinity of VHH-2H of 3.64E-08. And VHH-12C can specifically recognize human ROBO1, and does not bind to the ROBO1 of mice, rats and dogs; VHH-2H is capable of specifically recognizing human ROBO1, and does not bind to mouse, dog or rabbit ROBO 1. Meanwhile, the nanobody does not recognize other members ROBO2, ROBO3, ROBO4 in the human ROBO family, and shows superior specificity.

Description

Human ROBO1 binding molecules and uses thereof
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a human ROBO1 binding molecule and application thereof.
Background
During the development of the nervous system in vertebrates and invertebrates, several progressive and recursive processes driven primarily by axon guidance molecules, such as Slit and Robo, are involved. Slit proteins are highly conserved, secreted glycoproteins whose function is mediated by binding to transmembrane receptors; robo is a split receptor, an evolutionarily conserved transmembrane receptor IgG superfamily. Members of this family include ROBO1, ROBO2, ROBO3, ROBO4. The Slit/Robo signaling pathway was first established as an extracellular marker that directs axonal pathway discovery, promotes axonal branching, and controls neuronal migration. The interaction of Slit/Robo proteins is closely related to the development of important organs such as the mammary gland, lung, liver, kidney, eye and reproductive system. In recent years, more and more studies have shown that Slit/Robo signals also play a role in the formation of different types of cancers, while being involved in the regulation of oncogenic signaling pathways, including axon guidance, cell proliferation, cell motility, and angiogenesis, etc., the abnormal expression of which in cells can lead to the exacerbation and metastasis of cancer.
In recent years, the development of the immunocyte therapy industry in the field of biological medicine has been rapidly increased, and the therapy adopts immunocytes derived from a human body or a donor, and the immunocytes are infused into a patient body in vitro through a certain operation means, so that the purpose of tumor therapy is achieved. Among them, the engineering immunocyte therapies mainly represent different projects such as CAR-T, TCR-T, CAR-NK and CAR-M.
Meanwhile, nanobodies have the main advantages over conventional antibodies: 1. the molecular weight is small, and the blood brain barrier is easier to penetrate in vivo; 2. the specificity is strong, and the affinity is high; 3. high homology with human antibody sequences and relatively weak immunogenicity to human. Is also widely applied to the research and development of genetic engineering medicaments at present.
However, at present, a binding molecule of the target ROBO1, which can be simultaneously applied to immune cell preparation and nanobody preparation, has not been reported yet.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a binding molecule targeting human ROBO1, which is expected to be simultaneously used for preparing anti-ROBO 1 CAR cells and nanobodies so as to make up for the blank of the ROBO1 nanobodies, and is expected to have stronger killing effect on target cells by CAR cells containing the binding molecule.
In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:
in a first aspect, the present invention provides a human ROBO1 binding molecule comprising a module a or a module b that specifically binds to human ROBO1;
the module a comprises a first antigen binding domain comprising a domain fragment a1 having the amino acid sequence shown in SEQ ID No.1, a domain fragment a2 having the amino acid sequence shown in SEQ ID No.2 and a domain fragment a3 having the amino acid sequence shown in SEQ ID No. 3;
the module b comprises a second antigen binding domain comprising a domain fragment b1 having the amino acid sequence shown in SEQ ID No.4, a domain fragment b2 having the amino acid sequence shown in SEQ ID No.5 and a domain fragment b3 having the amino acid sequence shown in SEQ ID No. 6.
In alternative embodiments, the module a further comprises a first set of FR fragments for the linking domain fragments a1, a2 and a3;
the module b further comprises a second set of FR fragments for the linking domain fragments b1, b2 and b 3;
the first or second set of FR fragments are each selected from any one of the following sets:
first FR fragment: has the amino acid sequence shown in SEQ ID No.7,
second FR fragment: has the amino acid sequence shown in SEQ ID No.8,
third FR fragment: has the amino acid sequence shown in SEQ ID No.9,
fourth FR fragment: has an amino acid sequence shown as SEQ ID No. 10;
ii. Fifth FR fragment: has the amino acid sequence shown in SEQ ID No.11,
sixth FR fragment: has the amino acid sequence shown in SEQ ID No.12,
seventh FR fragment: has the amino acid sequence shown in SEQ ID No.13,
eighth FR fragment: has the amino acid sequence shown in SEQ ID No. 10.
In an alternative embodiment, the module a has the amino acid sequence shown in SEQ ID No. 14; the module b has an amino acid sequence shown as SEQ ID No. 15.
In an alternative embodiment, the human ROBO1 binding molecule is a nanobody that specifically binds to a human ROBO1 antigen.
The nanobody includes a monovalent nanobody, a bivalent or multivalent nanobody, or a bispecific nanobody.
In alternative embodiments, the human ROBO1 binding molecule is selected from the group consisting of scFv molecules, fv molecules, fab molecules, or intact antibody molecules that specifically bind to human ROBO1 antigen.
In a second aspect, the present invention provides the use of a human ROBO1 binding molecule according to any of the preceding embodiments for the preparation of a human ROBO1 assay product or for in vitro detection of human ROBO1 not for disease diagnosis or treatment purposes.
In a third aspect, the present invention provides a fusion protein comprising the nanobody and a tag protein of the previous embodiments.
In an alternative embodiment, the tag protein is an Fc tag which is a mouse igG1 Fc fragment having the amino acid sequence of SEQ ID No. 16.
In a fourth aspect, the present invention provides a biomaterial comprising:
a nucleic acid molecule comprising a first nucleotide sequence encoding a human ROBO1 binding molecule according to any of the preceding embodiments, or a second nucleotide sequence encoding a fusion protein according to any of the preceding embodiments;
a construct comprising a recombinant nucleic acid molecule obtained by ligating a third nucleotide sequence encoding a signal peptide to said nucleic acid molecule;
a recombinant vector obtained by ligating the construct to a primary plasmid vector, wherein the primary plasmid vector comprises pcDNA3.4;
a recombinant prokaryotic cell obtained by transforming the recombinant vector into bacteria;
a transformant obtained by transforming the recombinant vector into a eukaryotic host cell, wherein the eukaryotic host cell comprises an Expi-CHO.
In a fifth aspect, the present invention provides a method for producing a human ROBO 1-binding molecule according to any one of the preceding embodiments or a fusion protein according to any one of the preceding embodiments, comprising culturing the transformant according to the preceding embodiments, and recovering and purifying the human ROBO 1-binding molecule or the fusion protein from the supernatant obtained by the culture.
In a sixth aspect, the present invention provides a preparation or kit for in vitro detection of human ROBO1, comprising a human ROBO1 binding molecule according to any of the preceding embodiments or a fusion protein according to any of the preceding embodiments.
In a seventh aspect, the invention provides the use of a human ROBO1 binding molecule of any preceding claim or a nucleic acid molecule of i of any preceding claim in the preparation of a CAR cell which targets ROBO 1.
In an eighth aspect, the invention provides an anti-ROBO 1 CAR cell, the chimeric antigen receptor of which comprises a human ROBO1 binding molecule according to the previous embodiment.
The specific binding molecule of the anti-human ROBO1 target provided by the invention comprises the nano antibody, so that the gap of the detection of the ROBO1 target in the field of the nano antibody is filled. The verification proves that in the nanobody of the anti-human ROBO1 target, the affinity of the nanobody named VHH-12C in the module a is 8.27E-08, and the affinity of the nanobody named VHH-2H in the module b is 3.64E-08. And VHH-12C can specifically recognize human ROBO1, and does not bind to the ROBO1 of mice, rats and dogs; VHH-2H is capable of specifically recognizing human ROBO1, and does not bind to mouse, dog or rabbit ROBO 1. Meanwhile, the nanobody does not recognize other members ROBO2, ROBO3, ROBO4 in the human ROBO family, and shows superior specificity. Meanwhile, the CAR-NK cells prepared by integrating the two specific binding molecules into chimeric antigen receptors of the CAR-NK cells have remarkable targeted killing effect on target cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the construction of a pcDNA3.4 original plasmid vector used in the examples of the present invention;
FIG. 2 is a SDS-PAGE of nanobody purification according to an embodiment of the invention;
FIG. 3 is a diagram of a nanobody family specific reaction ELISA in an embodiment of the invention;
FIG. 4 is a cross-reactive ELISA diagram of nanobody species in an embodiment of the invention;
FIG. 5 is a CAR-NK cell construction flow chart;
FIG. 6 shows the results of target cell killing experiments on T47D target cells performed on two effector cells;
FIG. 7 shows the results of target cell killing experiments performed on T47D-2B1 target cells by two effector cells;
FIG. 8 shows the results of two effector cell lines killing MDA-MB-453 target cells;
FIG. 9 shows the results of two effector cell killing experiments on Hela target cells;
FIG. 10 shows the results of two effector cell killing experiments on HCT116-1C11 target cells;
FIG. 11 shows the results of affinity detection of six nanobodies in comparative example 1.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures. Furthermore, the terms "first," "second," "third," and the like are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
As used herein, the terms "VHH", "nanobody" and "nanobody" have the same meaning and are used interchangeably, and nanobody refers to a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function, obtained from the peripheral blood of camelids through antigen screening.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the specific binding of various specific antibodies to their specific antigens. 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 the Framework Regions (FR).
Each variable region contains 3 CDRs and 4 FRs, and the CDRs and FRs are alternately arranged to form the variable region.
The CDR regions of the antibodies of the invention are important. Thus, the invention includes those sequences that have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs of the invention.
In a first aspect, in a specific embodiment, the present invention provides a module a I that specifically binds to human ROBO1, said module a I comprising a domain fragment a1 having the amino acid sequence shown in SEQ ID No.1, a domain fragment a2 having the amino acid sequence shown in SEQ ID No.2 and a domain fragment a3 having the amino acid sequence shown in SEQ ID No. 3.
In another specific embodiment, the present invention provides a module b I that specifically binds to human ROBO1, comprising a domain fragment b1 having the amino acid sequence shown in SEQ ID No.4, a domain fragment b2 having the amino acid sequence shown in SEQ ID No.5 and a domain fragment b3 having the amino acid sequence shown in SEQ ID No. 6.
With reference to the first aspect, in a second aspect, in a specific embodiment of the present invention, there is provided a module aii that specifically binds to ROBO1 of human origin, the module aii comprising domain segments a1, a2 and a3 and a set of FR segments, connected in the relationship "first FR segment-domain segment a 1-second FR segment-domain segment a 2-third FR segment-domain segment a 3-fourth FR segment".
In another embodiment, the present invention provides a module aiii that specifically binds to ROBO1 of human origin, the module aiii comprising domain segments a1, a2 and a3 and another set of FR segments, linked in the relationship "fifth FR segment-domain segment a 1-sixth FR segment-domain segment a 2-seventh FR segment-domain segment a 3-eighth FR segment".
In another embodiment, the invention provides a module biI that specifically binds to human ROBO1, the module biI comprising domain segments b1, b2 and b3 and a set of FR segments linked in a "first FR segment-domain segment b 1-second FR segment-domain segment b 2-third FR segment-domain segment b 3-fourth FR segment".
In another embodiment, the invention provides a module BIII that specifically binds to human ROBO1, the module BIII comprising domain segments b1, b2 and b3 and another set of FR segments linked in the relationship of "fifth FR segment-domain segment b 1-sixth FR segment-domain segment b 2-seventh FR segment-domain segment b 3-eighth FR segment".
The amino acid sequence information of the FR fragment is as follows:
FR fragments Amino acid sequence
First FR fragment SEQ ID No.7
Second FR fragment SEQ ID No.8
Third FR fragment SEQ ID No.9
Fourth FR fragment SEQ ID No.10
Fifth FR fragment SEQ ID No.11
Sixth FR fragment SEQ ID No.12
Seventh FR fragment SEQ ID No.13
Eighth FR fragment SEQ ID No.10
With reference to the second aspect, the present invention uses the above provided modules ai to aiii and bii to biiii as monovalent nanobodies, respectively.
In another embodiment, the invention provides a bivalent nanobody obtained by linking the aI and aII.
In another specific embodiment, the trivalent nanobody is obtained by connecting the aI to aIII.
In another embodiment, the present invention provides a bivalent nanobody obtained by linking the above-mentioned BI and BII.
In another specific embodiment, the trivalent nanobody is obtained by connecting the BI to BIII.
In another specific embodiment, the bispecific nanobody is obtained by connecting any one of aI to aIII with any one of bI to bIII.
With reference to the modules of the second aspect, in a fourth aspect, the present invention provides a scFV molecule comprising a VH domain and a VL domain linked by a Linker; the VH domain is derived from module a or module b, and the amino acid residue composition and length of the VL domain and the elastic linking peptide can be adjusted by conventional means according to actual needs by those skilled in the art, wherein the amino acid residues of the elastic linking peptide include, but are not limited to, glycine (Gly) and serine (Ser) with a length of 15 to 25 amino acid residues.
In a fifth aspect, the invention provides Fv molecules comprising a VH domain and a VL domain linked by a short peptide; the VH domain is derived from module a or module b, and the amino acid residue composition and length for the VL domain and short peptide linkage can be adjusted by conventional means as required by the person skilled in the art. The short peptide comprises, but is not limited to, a short peptide chain consisting of 3-9 amino acid residues.
In a sixth aspect, the invention provides a Fab molecule comprising the VH domain, the VL domain, a light chain constant region (CL) and one heavy chain constant region (CH 1); the VH domain is derived from the module a or the module b, and for the VL domain, CH1 and CL, those skilled in the art can select according to actual needs, for example, the CH1 is selected from any one or several of IgG1, igG2, igG3, igG4, igA, igD, igE or IgM, and the CL is selected from a kappa chain or a lambda chain, and in addition, those skilled in the art can adjust the sequence and modification of the selected CH1 and CL by conventional means.
In combination with the foregoing embodiments, the present invention also provides an intact antibody molecule comprising 2 identical heavy chains comprising a VH domain and a heavy chain constant region and 2 identical light chains comprising a VL domain and a light chain constant region; for the specific sequence composition of the heavy chain constant region and the light chain constant region, those skilled in the art can select according to actual requirements, for example, the heavy chain constant region may be selected from any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE or IgM, the light chain constant region may be selected from a kappa chain or a lambda chain, and furthermore, the sequences or modifications of the above-selected heavy chain constant region and light chain constant region may be adjusted by conventional means according to actual requirements.
In an eighth aspect, the present invention provides the use of each module, nanobody, scFV molecule, fv molecule, fab molecule or whole antibody molecule according to any one of the preceding embodiments for the preparation of a product for the detection of human ROBO1 or for the in vitro detection of human ROBO1 not for the purpose of diagnosis or treatment of a disease.
In a ninth aspect, the present invention provides a fusion protein comprising the nanobody and Fc tag of the previous embodiments.
The addition of the Fc tag is mainly used for purification of Fc fusion proteins, and one skilled in the art can select from conventional Fc tags according to actual needs.
Preferably, the Fc tag comprises a mouse igG Fc fragment.
Further preferably, the mouse igG Fc fragment has the amino acid sequence shown as SEQ ID No. 16.
In a tenth aspect, the present invention provides a biomaterial comprising:
a nucleic acid molecule comprising a first nucleotide sequence encoding a human ROBO1 binding molecule according to the previous embodiment, or a second nucleotide sequence encoding a fusion protein according to the previous embodiment;
a construct comprising a recombinant nucleic acid molecule obtained by ligating a third nucleotide sequence encoding a signal peptide to said nucleic acid molecule;
a recombinant vector obtained by ligating the construct to a primary plasmid vector, wherein the primary plasmid vector comprises pcDNA3.4;
a recombinant prokaryotic cell obtained by transforming the recombinant vector into bacteria;
the transformant obtained by transforming the recombinant vector into a eukaryotic host cell, for which a person skilled in the art can routinely select according to the actual need.
The eukaryotic host cell includes an Expi-CHO.
In an eleventh aspect, the present invention provides a method for producing a human ROBO 1-binding molecule according to the previous embodiment or a fusion protein according to the previous embodiment, comprising culturing the transformant according to the previous embodiment, and recovering and purifying the human ROBO 1-binding molecule or the fusion protein from the supernatant obtained by the culture.
In a twelfth aspect, the present invention provides a preparation for in vitro detection of human ROBO1, said preparation comprising a human ROBO1 binding molecule according to the previous embodiment or a fusion protein according to the previous embodiment.
In a thirteenth aspect, the present invention provides a kit for in vitro detection of human ROBO1, which comprises the preparation for in vitro detection of human ROBO1 according to the foregoing embodiment and optionally consumable materials, and it is understood that the consumable materials comprise a kit composition conventional in the art, such as an orifice plate, a reaction vessel, a liquid extraction device, etc., and the dosage of the preparation and various consumable materials in the kit can be routinely selected by those skilled in the art according to actual needs.
In a fourteenth aspect, the present invention provides an anti-ROBO 1 CAR cell, the chimeric antigen receptor of which comprises a human ROBO1 binding molecule according to the previous embodiment. It is noted that, as is well known in the art, there are no technical hurdles to construct chimeric antigen receptors in immune cells, and therefore, those skilled in the art are able to routinely select the CAR cells from CAR-T cells, CAR-NK cells, and other immune cells as desired.
Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
The present example provides two expression methods for anti-ROBO 1 nanobodies:
experimental materials:
cell lines: expiocho (Thermo, cat No. a 29133);
transfection kit: expiFectamineTM CHO transfection kit (Thermo, cat No. a 29129);
culture medium: expiCHOTMExcompression Medium (Thermo, cat. No. A29100-01);
OptiPROTM SFM (Thermo, cat. No. 12309-050);
pcDNA3.4 original plasmid vector.
Culture conditions: 37 ℃,8% CO 2 Incubator, shake cultivation.
Two nanobody gene fragments, a signal peptide gene fragment consisting of 18 amino acid residues, which are known to those skilled in the art, a gene fragment comprising a first nanobody VHH-12C having a nucleotide sequence shown in SEQ ID No. 17 and a gene fragment comprising a second nanobody VHH-2H having a nucleotide sequence shown in SEQ ID No. 18, and an Fc-tagged gene fragment, which are human interleukin 10, are amplified by PCR. The amino acid sequence of the Fc tag mouse igG1 Fc fragment is shown as SEQ ID No. 16.
Then sequentially connecting the nanometer antibody gene fragment, the nucleotide sequence of the signal peptide and the nucleotide sequence of the Fc tag, connecting the connected fragment to a pcDNA3.4 original plasmid vector (the pcDNA3.4 original plasmid vector map is shown in figure 1) through enzyme digestion sites, converting the fragment into Top10 strain, and screening positive clones by ampicillin. Plasmids were extracted using a plasmid extraction kit for transfection of expcho cells.
ExpiCHO cells were cultured to a cell density of 6X 10 6 At this time, transfection was performed at 20. Mu.g plasmid/25 mL cells, and after 10 days the cell supernatant was collected. The supernatant was collected after high-speed centrifugation, and was used for protein purification after filtration.
It should be noted that, those skilled in the art can adjust or replace the signal peptide given in this embodiment according to actual needs, and the selection of the signal peptide given in this embodiment is only given as an example and should not be construed as limiting.
Example 2
In this example, the two anti-ROBO 1 nanobodies obtained in example 1 were purified as follows.
Experimental materials:
protein purification column (HiTrap rprotein A FF,1 mL).
Binding buffer: 20mM PB+2.5M NaCl,pH7.4.
Eluent: 0.1M citrate, pH3.0.
Neutralizing solution: 1M Tris HCl,pH9.0.
The AKTA instrument was started, the purification column was loaded onto AKTA, the column was rinsed with 5 column volumes of distilled water, and then the purification column was equilibrated with 5 column volumes of binding buffer. The cell supernatant filtered in example 1 was taken and passed through a purification column according to the instructions. The purification column was rinsed with 10 column volumes of binding buffer until baseline plateau. Finally, the target protein is eluted by the eluent gradient, and the elution peak is collected when the UV280 is more than 30 mAU.
After the collection of the proteins, the column is washed by using a 10-time column volume eluent, then salt ions are removed by using a 10kDa ultrafiltration tube, PBS is substituted for the washing solution to store two nanobodies, the obtained gel electrophoresis result of the two nanobodies is shown in figure 2, the molecular weight of the two nanobodies is 35 kDa-45 kDa, and the molecular weight of the two nanobodies is consistent with the molecular weight of fusion proteins containing the nanobodies, so that the successful expression of the nanobodies is proved.
Example 3
This example provides family-specific detection of two ROBO1 nanobodies obtained in example 2:
the two nanobodies obtained in example 2 were examined by ELISA for cross-reactivity with human-derived ROBO2, ROBO3, ROBO4.
2. Mu.g/mL human ROBO1, ROBO2, ROBO3, ROBO4 protein was added to the ELISA plate and coated overnight at 4deg.C, 100. Mu.L/well; after washing 3-5 times by PBST, adding 300 mu L of 5% BSA into each hole, and sealing for 2 hours at room temperature; after washing 3-5 times by PBST, respectively carrying out 3-time gradient dilution on the two nano antibodies obtained in the example 2 by taking 30 mug/mL as an initial point, adding 100 mug into each hole, and incubating at 37 ℃ for 1 hour; washing 3-5 times by using PBST, adding 0.1 mu g/mL HRP-labeled goat anti-mouse Fc antibody, and incubating at 37 ℃ for 1 hour in 100 mu L of each hole; washing with PBST for 5 times, adding color developing solution, and measuring the absorption value under the wavelength of 450nm by an enzyme-labeled instrument. The results are shown in FIG. 3, wherein the anti-ROBO 1 nanobody VHH-12C and VHH-2H have specific binding with ROBO1 and have no binding with ROBO 2-4.
Example 4
This example shows the species specificity of two ROBO1 nanobodies obtained in example 2.
The nanobody obtained in example 2 was examined by ELISA for cross-reactivity with ROBO1 derived from other species.
10. Mu.g/mL of human-ROBO 1, dog-ROBO 1, rabbit-ROBO 1, rat-ROBO 1, mouse-ROBO 1 protein were each added to the ELISA plate and coated overnight at 4℃at 100. Mu.L/well; after washing 3-5 times by PBST, adding 300 mu L of 5% BSA into each hole, and sealing for 2 hours at room temperature; after washing 3-5 times with PBST, the nanobody obtained in example 2 was subjected to 2-fold gradient dilution starting with 2. Mu.g/mL, and 100. Mu.L of each well was added for incubation at 37℃for 1 hour; washing 3-5 times by using PBST, adding 0.1 mu g/mL HRP-labeled goat anti-mouse Fc antibody, and incubating at 37 ℃ for 1 hour in 100 mu L of each hole; washing with PBST for 5 times, adding color developing solution, and measuring the absorption value under the wavelength of 450nm by an enzyme-labeled instrument. The results are shown in FIG. 4, wherein the anti-ROBO 1 nanobody VHH-12C specifically binds to only human-ROBO 1, but not to rat, dog, mouse-ROBO 1; nanobody VHH-2H specifically binds only to human-ROBO 1, but not to rabbit, dog, mouse-ROBO 1.
Example 5
The affinity of the antibodies for binding to ROBO1 antigen was determined using SPR techniques.
5.1 preparation of reagents
Running the reagent: 10 mM N- (2-hydroxyethyl) piperazine-N-2 sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediamine tetraacetic acid (EDTA), 0.005% (volume percent) Tween-20 (Tween-20), pH was adjusted to 7.4.
A murine IgG antibody capture kit (cat# BR-1008-38, GE) comprising: rabbit anti-mouse IgG antibody (1 mg/mL), fixative (10 mM sodium acetate, pH 5.0), rejuvenating agent (10 mM glycine HCl, pH 1.7).
Amino coupling kit (cat# BR100050, GE) comprising: 115 mg N-hydroxysuccinimide (NHS), 750 mg 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 10.5 mL 1M ethanolamine (pH 8.5). Adding 10 mL deionized water into each tube of EDC and NHS, respectively, packaging, and storing at-18deg.C to lower temperature for two months. (cf. GE amino coupling instruction Manual 22-0510-62 AG).
5.2 chip preparation
The rabbit anti-mouse IgG antibody was diluted to 30. Mu.g/mL with a fixative (10 mM sodium acetate, pH 5.0) and approximately 100. Mu.L of rabbit anti-mouse IgG antibody was used per chip channel. First, the surface of the CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10. Mu.L/min at 420 s. Next, 30. Mu.g/mL of a rabbit anti-mouse IgG antibody was injected into the experimental channel (FC 2) at a flow rate of 10. Mu.L/min at about 420 and s in a fixed amount of about 9000 to 14000 RU. Finally, the chip was blocked with 1M ethanolamine at 10. Mu.L/min at 420 s. The reference channel (FC 1) performs the same operation as the test channel (FC 2). (reference is made to the instruction manual for murine IgG antibody Capture kit for GE, 22-0648-97 AD).
5.3 Capture ligand
anti-ROBO 1-WT and anti-ROBO 1-CAR were diluted to 4 μg/mL with running reagent and injected sequentially into the experimental channel (FC 2) at a flow rate of 10 μl/min to capture about 120 RU. The reference channel (FC 1) does not require capture of the ligand.
5.4 Multi-cycle analysis of analytes
Robo1-His was diluted with running reagent from a 2-fold gradient at a concentration of 400 nM to 3.125 nM. The diluted Robo1-His was sequentially injected into the experimental and reference channels at a flow rate of 20. Mu.L/min for a binding time of 120 s and a dissociation time of 180 s. The binding dissociation steps are all performed in running reagents. After each concentration analysis, the chip was regenerated 180 s with 10 mM glycine-HCl (pH 1.7) at a flow rate of 20. Mu.L/min, removing the ligand and undissociated analyte.
The affinity assay results were as follows:
KD （M） kon（1/Ms） kdis（1/s）
VHH-12C 8.27E-08 6.04E+04 4.99E-03
VHH-2H 3.64E-08 7.59E+04 2.76E-03
as can be seen from the above table, VHH-12C and VHH-2H have good binding ability.
Example 6
In this example, starting from NK-92 cells, referring to the CAR-NK cell construction method provided in examples 1 to 3 in patent AU2018378395A1, the construction flow chart is shown in fig. 5, and a second binding molecule having the nucleotide sequence shown in SEQ ID No. 18 is used as ScFV fragment to replace the "Anti ROBO1-FN3" fragment in AU2018378395A1, so as to construct CAR-NK cells containing the second binding molecule, which is denoted ROBO1-CAR-NK92-VHH-2H cell line, and the positive rate of CAR expression is 99.62%.
Example 7
This example differs from example 6 only in that the second binding molecule of example 6 was replaced with a first binding molecule having the nucleotide sequence shown in SEQ ID No. 17, and the resulting CAR-NK cell was designated as ROBO1-CAR-NK92-VHH-12C cell line with a CAR expression positive rate of 99.64%.
Then, referring to the cancer cell killing test method described in example 5 of AU2018378395A1, target cells (T47D, T47D-2B1, MDA-MB-453, hela and HCT116-1C11, wherein T47D-2B1 is a ROBO1 knocked out on the basis of T47D, and then ROBO1 is overexpressed, and HCT116-1C11 is a ROBO1 directly overexpressed on HCT116 cells) were subjected to the two effector cells obtained by sorting in examples 6 and 7, with the CAR-NK cell ROBO1-CAR-NK92 of AU2018378395A1 as a control.
The killing test results are shown in fig. 6-10, and it can be seen from the figures that (1) the killing effect of the ROBO1-CAR-NK92-VHH-2H cells on the T47D target cells is increased along with the increase of the effective target ratio, and the killing effect is similar to that of the ROBO1-CAR-NK92 cells. ROBO1-CAR-NK92-VHH-12C cells had a killing effect on T47D target cells after an effective target ratio of 5:1. (2) The killing effect of the ROBO1-CAR-NK92-VHH-2H cells on the T47D-2B1 target cells is increased along with the increase of the effective target ratio, and the killing effect is similar to that of the ROBO1-CAR-NK92 cells. ROBO1-CAR-NK92-VHH-12C cells had a killing effect on T47D-KO-1C5-RF-2B1 target cells after an effective target ratio of 2.5:1. (3) ROBO1-CAR-NK92-VHH-12C cells kill target cells MDA-MB-453 after an effective target ratio of 2.5:1. The killing effect of ROBO1-CAR-NK92-VHH-2H cells on target cells MDA-MB-453 increases with the increase of the effective target ratio, and the killing effect is similar to that of ROBO1-CAR-NK92 cells. (4) The killing effect of ROBO1-CAR-NK92-VHH-2H cells on Hela target cells increases with the increase of the effective target ratio, and the ROBO1-CAR-NK92-VHH-12C cells have the killing effect on the Hela target cells after the effective target ratio is 1:1. (5) The killing of the HCT116-1C11 target cells by the ROBO1-CAR-NK92-VHH-2H cells increases with the increase of the effective target ratio, and the HCT116-1C11 target cells are killed by the ROBO1-CAR-NK92-VHH-12C cells more strongly after the effective target ratio is 10:1. (6) The target cells of the ROBO1-CAR-NK92-VHH-2H and the ROBO1-CAR-NK92-VHH-12C have different killing effects on T47D, T47D-2B1, MDA-MB-453, hela and HCT116-1C11 under different effective target ratios, and the targeted killing effect of the ROBO1-CAR-NK92-VHH-2H under the same effective target ratio is better than that of the ROBO 1-CAR-92-VHH-12C.
Example 8
In this example, referring to the method for constructing a CAR-T cell described in example 4 of AU2018378395A1, a CAR-T cell containing a first binding molecule was constructed by replacing the "Anti-ROBO 1-FN3" fragment of AU2018378395A1 with the first binding molecule having the nucleotide sequence shown in SEQ ID No. 17 as the ScFV fragment.
Example 9
This example differs from example 8 only in that the first binding molecule of example 8 is replaced by a second binding molecule having the nucleotide sequence shown in SEQ ID No. 18.
Comparative example 1
The comparative example provides four other anti-ROBO 1 nanobodies, the nucleotide sequences of which are respectively shown in SEQ ID NO. 19-SEQ ID NO.22 and respectively named VHH-3D, VHH-2F, VHH-10B, VHH-4F. The binding affinities of VHH-3D, VHH-2F, VHH-10B, VHH-4F and ROBO1 of VHH-2H and VHH-12C were compared using ELISA methods as follows:
(1) Coating antigen: the human-ROBO 1 antigen protein was diluted to 2. Mu.g/ml (optimal concentration 1-10. Mu.g/ml) with the coating solution, and the ELISA plate was added in the sample-adding configuration, and 100. Mu.l was added per well overnight at 4 ℃.
(2) Washing: removing the coating liquid, beating to dry, washing the plate 3 times with PBST washing liquid (PBST washing liquid: 0.05% Tween-20+1XPBS PH 7.4), and beating to dry for later use.
(3) Closing: adding 300 mu L of blocking solution (5% BSA by volume) into each well, blocking at 37 ℃, washing after 2 hours, washing the plate 4 times by using PBST washing solution, and beating for later use.
(4) The detection antibody is subjected to 2-time gradient dilution with 75 mug/ml as an initial, 100 mug/well is added according to the layout, and after incubation for 2 hours at 37 ℃ with a micro-oscillator at 750 rpm, the plate is washed 3 times with PBST washing liquid and is patted dry for standby.
(5) Adding 100 mu L of HRP enzyme-linked detection secondary antibody with the concentration of 0.15 mu g/ml into each hole, incubating for 2 hours at 37 ℃ with a micro-oscillator at 750 rpm, washing the plate for 4-6 times with PBST washing liquid, and beating for later use.
(6) 100 mu L of TMB color development solution is added into each hole, and the mixture is incubated for 10-30 min at 37 ℃ in a dark place.
(7) Adding a stop solution: the wells were terminated by adding 50. Mu.L of termination solution.
(8) The results were measured and recorded: the reaction was stopped by adding 50. Mu.L of ELISA stop solution, and absorbance was measured at 450nm within 30 minutes.
The results are shown in FIG. 11, and it can be seen that the binding capacity of the 6-strain nanobody to human-ROBO 1 protein is ranked as: VHH-12C > VHH-2H > VHH-4F > VHH-2F > VHH-10B > VHH-3D.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (13)

1. A human ROBO1 binding molecule comprising a module a or a module b that specifically binds to human ROBO1;
the module a comprises a first antigen binding domain comprising a domain fragment a1 having the amino acid sequence shown in SEQ ID No.1, a domain fragment a2 having the amino acid sequence shown in SEQ ID No.2 and a domain fragment a3 having the amino acid sequence shown in SEQ ID No. 3;
the module b comprises a second antigen binding domain comprising a domain fragment b1 having the amino acid sequence shown in SEQ ID No.4, a domain fragment b2 having the amino acid sequence shown in SEQ ID No.5 and a domain fragment b3 having the amino acid sequence shown in SEQ ID No. 6.
2. The human ROBO1 binding molecule according to claim 1 wherein module a further comprises a first set of FR fragments for the linking domain fragments a1, a2 and a3;
the module b further comprises a second set of FR fragments for the linking domain fragments b1, b2 and b 3;
the first or second set of FR fragments are each selected from any one of the following sets:
first FR fragment: has the amino acid sequence shown in SEQ ID No.7,
second FR fragment: has the amino acid sequence shown in SEQ ID No.8,
third FR fragment: has the amino acid sequence shown in SEQ ID No.9,
fourth FR fragment: has an amino acid sequence shown as SEQ ID No. 10;
ii. Fifth FR fragment: has the amino acid sequence shown in SEQ ID No.11,
sixth FR fragment: has the amino acid sequence shown in SEQ ID No.12,
seventh FR fragment: has the amino acid sequence shown in SEQ ID No.13,
eighth FR fragment: has the amino acid sequence shown in SEQ ID No. 10.
3. The human ROBO1 binding molecule according to claim 1 wherein said module a has the amino acid sequence shown in SEQ ID No. 14;
the module b has an amino acid sequence shown as SEQ ID No. 15.
4. The human-derived ROBO1 binding molecule according to any one of claims 1 to 3, wherein said human-derived ROBO1 binding molecule is a nanobody specifically binding to human-derived ROBO1 antigen;
the nanobody includes a monovalent nanobody, a bivalent or multivalent nanobody, or a bispecific nanobody.
5. The human ROBO1 binding molecule according to any of claims 1 to 3, wherein said human ROBO1 binding molecule is selected from scFv molecules, fv molecules, fab molecules or whole antibody molecules that specifically bind to human ROBO1 antigens.
6. The use of the human ROBO1 binding molecule according to any one of claims 1 to 5 for the preparation of a product for human ROBO1 detection or for in vitro detection of human ROBO1 not aimed at diagnosis or treatment of a disease.
7. A fusion protein comprising the nanobody and a tag protein according to claim 4.
8. The fusion protein of claim 7, wherein the tag protein is an Fc tag that is a mouse igG Fc fragment having the amino acid sequence of SEQ ID No. 16.
9. A biomaterial, comprising:
a nucleic acid molecule comprising a first nucleotide sequence encoding the human ROBO1 binding molecule of claim 4, or a second nucleotide sequence encoding the fusion protein of claim 7 or 8;
a construct comprising a recombinant nucleic acid molecule obtained by ligating a third nucleotide sequence encoding a signal peptide to said nucleic acid molecule;
a recombinant vector obtained by ligating the construct to a primary plasmid vector, wherein the primary plasmid vector comprises pcDNA3.4;
a recombinant prokaryotic cell obtained by transforming the recombinant vector into bacteria;
a transformant obtained by transforming the recombinant vector into a eukaryotic host cell, wherein the eukaryotic host cell comprises an Expi-CHO.
10. The method for producing a human ROBO1 binding molecule according to claim 4 or the fusion protein according to claim 7 or 8, characterized in that the transformant according to claim 9 is cultured, and the human ROBO1 binding molecule or the fusion protein is recovered and purified from the supernatant obtained by the culture.
11. A formulation or kit for in vitro detection of human ROBO1, characterized in that it comprises a human ROBO1 binding molecule according to claim 4 or a fusion protein according to claim 7 or 8.
12. Use of a human ROBO1 binding molecule of claim 4 or a nucleic acid molecule of claim 9 in the preparation of a CAR cell that targets ROBO 1.
13. An anti-ROBO 1 CAR cell, characterized in that the chimeric antigen receptor of the anti-ROBO 1 CAR cell comprises the humanized ROBO1 binding molecule of any one of claims 1 to 3.
CN202311685718.4A 2023-12-11 2023-12-11 Human ROBO1 binding molecules and uses thereof Active CN117384290B (en)

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