CN117586402A - NKp80 antibody and application thereof - Google Patents
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- CN117586402A CN117586402A CN202311373342.3A CN202311373342A CN117586402A CN 117586402 A CN117586402 A CN 117586402A CN 202311373342 A CN202311373342 A CN 202311373342A CN 117586402 A CN117586402 A CN 117586402A
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Abstract
The invention relates to the field of biotechnology, in particular to an NKp80 antibody and application thereof. The invention screens a murine anti-NKp 80 monoclonal antibody, which has higher binding activity with the extracellular section of the human or cynomolgus monkey NKP80 protein, can target and bind cells with high expression of NKp80, and can also bind human primary T cells and NK cells. In addition, bispecific binding molecules prepared using the anti-NKp 80 antibodies are also capable of specifically targeting binding to human NKp80 proteins and cynomolgus NKp80 proteins, and the bispecific binding molecules are capable of targeting binding to cells that highly express NKp80 and B7H6, for use in the treatment or prevention of diseases such as tumors (e.g. colorectal cancer, etc.).
Description
Technical Field
The invention relates to the field of biotechnology, in particular to an NKp80 antibody and application thereof. More specifically, the present invention relates to antibodies or antigen binding fragments thereof, bispecific binding molecules, nucleic acid molecules, expression vectors, recombinant cells, compositions, medicaments, and kits for detecting NKp80 capable of specifically recognizing NKp 80.
Background
T cells and natural killer cells (natural killer cells, NK cells) play an important role in direct killing of tumors as two types of immune cells important in anti-tumor immune responses. NK cells are important members that make up the innate immune system, unlike T cells, NK cells do not express antigen-specific receptors. NK cells themselves have broad-spectrum tumor killing capacity and play an important role in enhancing antibody and T cell responses. At present, tumor immunotherapy forms based on NK cells are various, and the adopted means are different.
The NKp80 protein is a C-type lectin-like receptor, which is normally expressed on the surface of peripheral blood NK cells and NKT cells, and is used as a surface marker for activated NK cells. NKp80 is usually expressed on the cell surface in dimeric form, lacking charged amino acids in its transmembrane region, and also lacks the usual ITAM activation motif in the cell (Alessandro Moretta et al, 2001). NKp80 has also been found to be expressed on the surface of a small population of CD 8T cells, which have effector memory phenotypes that promote T cell responses and thus better develop immune responses (Sabrina Kuttruff et al, 2009). The AICL protein is a ligand for NKp80, which is normally expressed on the surface of myeloid cells, which up-regulate AICL expression upon stimulation by toll-like receptors. Thus, NKp80 stimulates myeloid cells to release pro-inflammatory cytokines when NK cells interact with monocytes, which plays an important role in both initiation and maintenance of inflammation (Stefan Welte et al, 2006). Therefore, the NKp80 molecule can be used as a good potential tumor treatment target.
Bispecific antibodies are antibodies that can specifically bind to two antigenic sites simultaneously. Bispecific antibodies for tumor therapy can be classified into three classes according to their mechanism of action: redirecting effector cells; immunomodulation; targeting tumor cell receptor dual binding. Among them, antibodies for the redirecting function occupy the majority of them, and two antibodies for tumor treatment that have been currently marketed are also based on T cell redirecting. Depending on the nature of the bispecific antibody, it can also be well applied in other therapeutic systems, such as dual immunomodulation or targeting two molecules of the same cell membrane.
The NKp80 monoclonal antibody and the monoclonal antibody disclosed at present have fewer varieties, the varieties are required to be further enriched, and the specificity is required to be improved.
Disclosure of Invention
The present application is made based on the discovery and recognition by the inventors of the following facts and problems:
NKp80 is on the surface of peripheral blood NK cells and NKT cells, and is used as a surface marker of activated NK cells. When NK cells interact with monocytes, NKp80 stimulates the release of pro-inflammatory cytokines by cells of the myeloid lineage, which plays an important role in both initiation and maintenance of inflammation, and NKp80 molecules can be a good potential tumor therapeutic target.
The inventor successfully screens out a murine anti-NKp 80 monoclonal antibody, and the monoclonal antibody has higher binding activity with the extracellular segment of human or cynomolgus monkey NKp80 protein, can target cells which are combined with high expression NKp80, and can also combine with human primary T cells and NK cells (the cell surface expresses NKp80 protein).
In addition, bispecific binding molecules prepared using the anti-NKp 80 antibodies are also capable of specifically targeting binding to human NKp80 proteins and cynomolgus NKp80 proteins, and the bispecific binding molecules are capable of targeting binding to cells that highly express NKp80 and B7H6, thereby treating or preventing diseases such as tumors (e.g., colorectal cancer, etc.). For example, a B7H 6-targeting bispecific antibody containing a NKp80 scFv can promote PBMC to kill B7H6 specifically + Is a tumor cell of (a).
In a first aspect of the invention, the invention provides an antibody or antigen binding fragment. According to an embodiment of the invention, the antibody or antigen binding fragment comprises:
heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO 1, 2 and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NO 1, 2 and 3; and/or
Light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO 4, 5 and 6 or amino acid sequences having at least 80% identity to 4, 5 and 6, respectively. The antibody or antigen binding fragment according to the embodiments of the present invention is capable of specifically binding to human or cynomolgus monkey NKp80 protein and has a high affinity, and the use of the antibody or antigen binding fragment also provides a basis for NKp 80-based redirected multi-specific antibody development.
According to embodiments of the invention, the antibody or antigen binding fragment may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the antibody or antigen binding fragment is a murine antibody or antigen binding fragment.
According to an embodiment of the invention, the antibody or antigen binding fragment comprises:
A heavy chain variable region CDR1 sequence shown in SEQ ID NO. 1, a heavy chain variable region CDR2 shown in SEQ ID NO. 2, a heavy chain variable region CDR3 shown in SEQ ID NO. 3, a light chain variable region CDR1 shown in SEQ ID NO. 4, a light chain variable region CDR2 shown in SEQ ID NO. 5, and a light chain variable region CDR3 shown in SEQ ID NO. 6.
HCDR1 amino acid sequence: DYYMN SEQ ID NO 1
HCDR2 amino acid sequence: DIHPNNGGTSYNQKFKG SEQ ID NO 2
HCDR3 amino acid sequence: DYGGTYTNWAQNYFDY SEQ ID NO 3
LCDR1 amino acid sequence: RASQSVSTPSYSYMH SEQ ID NO 4
LCDR2 amino acid sequence: YASNLES SEQ ID NO 5
LCDR3 amino acid sequence: QHSWEIPWT SEQ ID NO. 6
According to an embodiment of the invention, the antibody or antigen binding fragment comprises at least one of a heavy chain FR region and a light chain FR region.
According to an embodiment of the invention, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a primate-source antibody and a murine antibody or a mutant thereof.
According to an embodiment of the invention, the light chain variable region of the antibody or antigen binding fragment further comprises a light chain FR region of a murine kappa chain or a murine kappa chain variant, or a murine lambda chain variant; wherein the antibody heavy chain variable region further comprises a heavy chain FR region of murine IgG1 or a variant thereof, or a heavy chain FR region of IgG2 or a variant thereof, or a heavy chain FR region of IgG3 or a variant thereof.
According to an embodiment of the invention, the antibody or antigen binding fragment comprises a heavy chain variable region as shown in SEQ ID NO. 7; and/or
The light chain variable region is shown as SEQ ID NO. 8.
SEQ ID NO:7：
EVQLQQSGPELVNPGASVKISCKTSGYTFTDYYMNWVKQSHGKSLEWIGDIHPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDYGGTYTNWAQNYFDYWGQGTTLTVSS
SEQ ID NO:8
DIVQTQSPASLAVSLGQRATISCRASQSVSTPSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPWTFGGGTKLEIK
According to an embodiment of the invention, the antibody or antigen binding fragment contains at least one of a heavy chain constant region and a light chain constant region, at least a portion of which is derived from at least one of a primates antibody and a murine antibody or a mutant thereof.
According to an embodiment of the invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.
According to an embodiment of the invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG1 antibody or a mutant thereof or a human IgG1 antibody or a mutant thereof.
According to an embodiment of the invention, the antibody or antigen binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO. 9 and a light chain with the amino acid sequence shown in SEQ ID NO. 10.
SEQ ID NO:9
EVQLQQSGPELVNPGASVKISCKTSGYTFTDYYMNWVKQSHGKSLEWIGDIHPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDYGGTYTNWAQNYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:10
DIVQTQSPASLAVSLGQRATISCRASQSVSTPSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPWTFGGGTKLEIKAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
According to an embodiment of the invention, the antibody or antigen binding fragment comprises a monoclonal antibody or a polyclonal antibody.
According to an embodiment of the invention, the monoclonal antibody comprises at least one of a full length antibody, fv antibody, single chain antibody, fab antibody, single domain antibody, and minimal recognition unit.
In a second aspect, the invention provides a multispecific binding molecule. According to an embodiment of the invention, the multispecific binding molecule comprises at least:
a first binding region comprising an antibody or antigen binding fragment of the first aspect; and
a second binding region capable of specifically binding to a target cell surface antigen.
Bispecific binding molecules according to embodiments of the invention not only specifically bind NKp80, but also have binding molecules specific for one or more other antigens. The multispecific molecules of the present invention include at least bispecific, trispecific, and tetraspecific molecules. Based on their multi-specificity, the binding molecules of the invention are capable of targeting NK cells and T cells to other antigens, eliminating cells producing such antigens by NK cell or T cell mediated cell killing or phagocytosis. The bispecific binding molecules according to one embodiment of the invention are capable of specific binding to human or cynomolgus NKp80 protein and B7H6 protein, which can be applied in scientific research or to kill B7H6 positive tumor cells effectively.
It will be appreciated by those skilled in the art that the binding activity of the second binding region is not particularly limited and may have other binding activities as long as the diabody has the antibody or antigen-binding fragment of the first aspect and both the antibody or antigen-binding fragment and the second binding region are capable of functioning effectively. In addition, more specific antibodies, such as trispecific, tetraspecific, penta, can be prepared using the antibodies or antigen-binding fragments of the invention, which are capable of targeting to the cell surface of highly expressed antigens, based on their multispecific properties, and eliminating cells producing such antigens by cell killing.
According to an embodiment of the invention, the bispecific binding molecule may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the multispecific binding molecule is a bispecific binding molecule.
According to an embodiment of the invention, the bispecific binding molecule comprises a symmetric bispecific binding molecule or an asymmetric bispecific binding molecule.
According to an embodiment of the invention, the bispecific binding molecule is a symmetric bispecific binding molecule.
According to an embodiment of the present invention, the first binding region comprises peptide chain 1 and peptide chain 2, wherein the peptide chain 1 comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID NO's 1, 2 and 3, respectively,
the peptide chain 2 comprises light chain variable region CDR1, CDR2 and CDR3 sequences shown as amino acid sequences of SEQ ID NO. 4, 5 and 6 respectively.
According to an embodiment of the invention, the peptide chain 1 comprises the heavy chain variable region shown in SEQ ID NO. 7 and the peptide chain 2 comprises the light chain variable region shown in SEQ ID NO. 8.
According to an embodiment of the invention, the first binding region further comprises a first connecting peptide, wherein the N-terminus of the first connecting peptide is linked to the C-terminus of the peptide chain 2 and the C-terminus of the first connecting peptide is linked to the N-terminus of the peptide chain 1; or the N end of the first connecting peptide is connected with the C end of the peptide chain 1, and the C end of the first connecting peptide is connected with the N end of the peptide chain 2.
According to an embodiment of the invention, the first connecting peptide has the amino acid sequence (GGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Those skilled in the art will appreciate that conventional linker peptides in the art, such as conventional flexible amino acid fragments or rigid amino acid fragments, may be used.
According to an embodiment of the invention, the first connecting peptide has the amino acid sequence shown in SEQ ID NO. 13.
According to an embodiment of the invention, the first binding region comprises a single chain antibody having the amino acid sequence shown in SEQ ID NO. 11.
According to an embodiment of the invention, the first binding region further comprises a first heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate-source antibody, and a murine antibody or a mutant thereof.
According to an embodiment of the invention, the first heavy chain constant region is derived from a human IgG antibody or a mutant thereof.
According to an embodiment of the invention, the first heavy chain constant region has the amino acid sequence shown in SEQ ID NO. 28.
According to an embodiment of the invention, the first binding region has the amino acid sequence shown as SEQ ID NO. 14.
According to an embodiment of the invention, the second binding region has B7H6 binding activity.
According to an embodiment of the invention, the second binding region comprises at least one of a full length antibody, fv antibody, single chain antibody, fab antibody, single domain antibody and minimal recognition unit having B7H6 binding activity.
According to an embodiment of the invention, the second binding region comprises an anti-B7H 6 single chain antibody.
According to an embodiment of the invention, the anti-B7H 6 single chain antibody comprises a light chain variable region of an anti-B7H 6 antibody and a heavy chain variable region of an anti-B7H 6 antibody.
According to an embodiment of the invention, the sequences of the CDR1, CDR2 and CDR3 of the heavy chain variable region of the anti-B7H 6 antibody are shown in SEQ ID NOS 30-32.
According to an embodiment of the invention, the sequences of the light chain variable regions CDR1, CDR2 and CDR3 of the anti-B7H 6 antibody are shown in SEQ ID NOS.33-35.
According to an embodiment of the invention, the heavy chain variable region of the anti-B7H 6 antibody has the amino acid sequence shown in SEQ ID NO. 36, and the light chain variable region of the anti-B7H 6 antibody has the amino acid sequence shown in SEQ ID NO. 37.
According to an embodiment of the invention, the anti-B7H 6 single chain antibody further comprises a second connecting peptide connecting the heavy chain variable region of the anti-B7H 6 antibody and the light chain variable region of the anti-B7H 6 antibody. According to an embodiment of the invention, the second connecting peptide has the amino acid sequence (GGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, further preferably 7. Those skilled in the art will appreciate that conventional linker peptides in the art, such as conventional flexible amino acid fragments or rigid amino acid fragments, may be used.
According to an embodiment of the invention, the second connecting peptide has the amino acid sequence shown in SEQ ID NO. 13.
Optionally, the anti-B7H 6 single chain antibody has an amino acid sequence as shown in SEQ ID NO. 12.
According to an embodiment of the present invention, the bispecific antibody provided by the present invention comprises a first antigen binding region comprising an antibody targeting NKp80 according to the first aspect of the present invention, said antibody or antigen binding fragment being an anti-NKp 80 single chain antibody, see SEQ ID No. 11, and a second binding region comprising an antibody targeting another target, comprising a single chain antibody against B7H6, see SEQ ID No. 12.
According to an embodiment of the invention, the second binding region further comprises a second heavy chain constant region, at least a portion of which is derived from at least one of a primate-source antibody and a murine antibody or a mutant thereof.
According to an embodiment of the invention, the second heavy chain constant region is derived from a human IgG antibody or a mutant thereof.
According to an embodiment of the invention, the second heavy chain constant region has the amino acid sequence shown in SEQ ID NO. 29.
According to an embodiment of the invention, the N-terminus of the second heavy chain constant region is linked to the C-terminus of the anti-B7H 6 single chain antibody.
According to an embodiment of the invention, the second binding region has the amino acid sequence shown in SEQ ID NO. 15.
According to an embodiment of the invention, the first heavy chain constant region and the second heavy chain constant region are linked by a knob-intoo-hole structure.
In a third aspect, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen binding fragment according to the first aspect or a multispecific binding molecule according to the second aspect.
According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen binding fragment according to the first aspect of the invention. Wherein the heavy chain coding sequence of the NKp80 antibody (chimeric antibody 53G 7) is SEQ ID NO:16 and light chain coding sequences are SEQ ID NOs: 17.
it should be noted that, for the nucleic acids mentioned herein, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, although only one strand is shown in most cases herein, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences of the present invention include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.
In a fourth aspect, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the third aspect.
The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host. The expression vector provided by the invention can efficiently and largely express the antibody or antigen binding fragment in a proper host cell, and can be effectively used for specific treatment or prevention of tumors. .
"operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. It will be appreciated by those skilled in the art that the nucleic acid molecules encoding the antibodies or antigen binding fragments may be inserted separately into different vectors, typically into the same vector. The usual vectors may be, for example, plasmids, phages and the like.
In a fifth aspect, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the nucleic acid molecule according to the third aspect, the expression vector according to the fourth aspect; or (b)
Capable of expressing an antibody or antigen binding fragment according to the first aspect or a multispecific binding molecule according to the second aspect.
According to an embodiment of the invention, the recombinant cell is obtained by introducing the expression vector of the fourth aspect into a host cell.
According to some embodiments of the invention, the antibody or antigen binding fragment or the bispecific binding molecule may be obtained in large amounts in vitro by recombinant cells.
According to some embodiments of the invention, the recombinant cells are not particularly limited, and either prokaryotic cells or eukaryotic cells may be used.
According to an embodiment of the invention, the recombinant cell is a eukaryotic cell.
According to an embodiment of the invention, the recombinant cell is a mammalian cell. According to some embodiments of the invention, the recombinant antibody is expressed more efficiently when the cell is a eukaryotic cell, such as a mammalian cell.
In a sixth aspect, the invention provides a composition. According to an embodiment of the invention, the composition comprises at least one of an antibody or antigen binding fragment according to the first aspect, a multispecific binding molecule according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect or a recombinant cell according to the fifth aspect.
The compositions of the invention may also be administered in combination with each other, or with one or more other therapeutic compounds, for example, with a chemotherapeutic agent. Thus, the composition may also contain a chemotherapeutic agent. The antibodies, or antigen-binding fragments thereof, or multispecific binding molecules of the invention may also be combined with a second therapeutic agent. It is noted that the compositions include combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.
A seventh aspect of the invention provides a medicament. According to an embodiment of the invention, the medicament comprises at least one of an antibody or antigen binding fragment according to the first aspect, a multispecific binding molecule according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect.
According to an embodiment of the invention, the medicament may further comprise a pharmaceutically acceptable carrier.
As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal.
The effective amount of the antibodies or antigen binding fragments or the multispecific binding molecules of the present invention may vary depending on the mode of administration and the severity of the disease being treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.
As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
The medicament of the invention contains safe and effective amount of the active ingredients of the invention and pharmaceutically acceptable carriers. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, wherein the administration mode can be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration or intraperitoneal administration, and the dosage form of the medicine is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent or sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The medicament is preferably manufactured under aseptic conditions. The antibody or antigen binding fragment may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
According to an eighth aspect of the invention there is provided the use of an antibody or antigen binding fragment according to the first aspect, a multispecific binding molecule according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect, a composition according to the sixth aspect in the manufacture of a medicament for the prevention and/or treatment of cancer,
The cancer includes at least one of the following: colorectal cancer, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, renal cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.
In a ninth aspect, the invention provides a kit for detecting NKp 80. According to an embodiment of the invention, the kit comprises at least one of an antibody or antigen binding fragment according to the first aspect, a multispecific binding molecule according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect.
As previously described, the antibodies or antigen binding fragments of some embodiments of the invention are effective to bind to human or cynomolgus NKp80 protein, the bispecific binding molecules are effective to bind to human or cynomolgus NKp80 protein and B7H6 protein, and therefore, kits comprising the antibodies or antigen binding fragments are effective to qualitatively or quantitatively detect human or cynomolgus NKp80 protein, and kits comprising the bispecific binding molecules are effective to qualitatively or quantitatively detect human or cynomolgus NKp80 protein and B7H6 protein. The kit provided by the invention can be used for detection of NKp80 and antibody specific binding performance of human or cynomolgus monkey, such as immunoblotting, immunoprecipitation and the like. These kits may comprise any one or more of the following: an antagonist, an anti-NKp 80 antibody, or a drug reference material; a protein purification column; immunoglobulin affinity purification buffers; cell assay diluent; instructions, literature, etc. The anti-NKp 80 antibody can be used for various types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins can be detected in vitro or in vivo, for example, the relevant diseases can be tested by detecting serum or blood of a subject, scientific research can be performed, and the kit can be used for detecting the NKp80 protein of a human or cynomolgus monkey, or the NKp80 protein and B7H6 protein of a human or cynomolgus monkey in a sample to be tested.
According to a tenth aspect of the present invention there is provided the use of an antibody or antigen binding fragment according to the first aspect, a multispecific binding molecule according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect in the preparation of a kit for the detection of NKp80.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows graphs of flow assays for 293T cells over-expressing human NKp80 and monkey NKp 80;
FIG. 2A shows a graph of ELISA results for chimeric NKp80 antibody (53G 7) binding to human NKp 80;
FIG. 2B shows a graph of ELISA results for chimeric NKp80 antibodies (53G 7) binding to cynomolgus monkey NKp 80;
FIG. 3A shows a graph of the results of chimeric NKp80 antibody (53G 7) binding to 293T cells overexpressing human NKp 80;
FIG. 3B shows a graph of the results of binding of chimeric NKp80 antibody (53G 7) to cynomolgus monkey NKp80 overexpressing 293T cells;
FIG. 4A shows that chimeric NKp80 antibody (53G 7) binds CD3 in human PBMC + A result map of cells;
FIG. 4B shows that chimeric NKp80 antibody (53G 7) binds CD56 in human PBMC + A result map of cells;
FIG. 5 shows the results of an in vitro cytotoxicity of NKp80 XB 7H6 bispecific binding protein against PBMC against HCT-15 cells.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
Antibodies or antigen binding fragments of the invention are typically prepared by biosynthetic methods. The coding nucleic acids according to the invention can be prepared by various known methods, conveniently by the person skilled in the art, based on the nucleotide sequences according to the invention. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR. Wherein the antibody or antigen fragment is numbered and defined using the Kabat numbering system.
Terminology
The term "antibody" is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind to an antigen (e.g., fab ', F' (ab) 2, fv, single chain antibodies, diabodies, fab), and recombinant peptides comprising the foregoing so long as they exhibit the desired biological activity. An "immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein composed of two heavy chains and two light chains (tetrapeptide chain structure linked by interchain disulfide bonds), each comprising a variable region and a constant region. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Immunoglobulins can be divided into five classes, igM, igD, igG, igA, igE. The same class of immunoglobulins can also be of different subclasses, e.g., igG1, igG2, igG3, igG4, depending on the amino acid composition. Immunoglobulin light chains are classified as either kappa chains or lambda chains depending on the constant region. In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4 or variants thereof of human or murine origin. In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin or variants thereof.
The term "antigen-binding fragment" as used herein refers to Fab fragments, fab 'fragments, F (ab') 2 fragments, and Fv fragments ScFv fragments that bind to human NKp 80; comprising one or more CDR regions selected from SEQ ID NO. 1 to SEQ ID NO. 6 of the antibody of the present invention. Fv fragments contain the antibody heavy and light chain variable regions, but no constant regions, and have the smallest antibody fragment with the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The variable regions of two antibodies can also be joined by different linkers into one polypeptide chain, known as a single chain antibody or single chain Fv.
Antibody fragments or antigen-binding portions include, inter alia, fab ', F (ab') 2, fv, domain antibodies (dabs), complementarity Determining Region (CDR) fragments, CDR-grafted antibodies, single chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies; chelating recombinant antibodies, tri-chain antibodies (tribodies) or diabodies (bibodies), intracellular antibodies, nanobodies, small Modular Immunopharmaceuticals (SMIPs), antigen binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies or variants or derivatives thereof, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide, such as one, two, three, four, five or six CDR sequences, so long as the antibody retains the desired biological activity.
As used herein, "heavy chain variable region" refers to a region of an antibody molecule that comprises at least one Complementarity Determining Region (CDR) of the antibody heavy chain variable domain. The heavy chain variable region may comprise one, two or three CDRs of the heavy chain of the antibody. The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.
The term "complementarity determining region" or "CDR sequence" refers to an amino acid sequence in an antibody responsible for antigen binding, e.g., generally comprising: amino acid residues in the light chain variable region in the vicinity of 23-34 (L1), 50-56 (L2) and 89-97 (L3), and in the vicinity of 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region (Kabat et al, sequencesofProteins ofImmunological Interest,5th Ed.Public Health Service,NationalInstitutes ofHealth,Bethesda,MD. (1991)); and/or from "hypervariable loops" (e.g., 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and amino acid residues near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and LeskJ. Mol. Biol.196:901-917 (1987)).
The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. Cloning variable region genes from mouse hybridoma cells, cloning constant region genes of human antibodies as required, connecting the mouse variable region genes and the human constant region genes into chimeric genes, inserting the chimeric genes into human vectors, and finally expressing chimeric antibody molecules in eukaryotic expression systems or prokaryotic expression systems. In a preferred embodiment of the present invention, the antibody light chain of said NKp80 chimeric antibody further comprises a light chain Fc region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the NKp80 chimeric antibody further comprises a heavy chain constant region of human IgG1, igG2, igG3, igG4, or variants thereof.
Herein, the "monoclonal antibody" refers to an antibody having a single antigen binding site.
Herein, the "diabody" refers to an antibody having two different antigen binding sites.
Herein, the term "single domain antibody" is a heavy chain antibody naturally lacking the light chain found in camel blood/alpaca blood, including only the heavy chain H chain of relatively heavy molecular weight. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable, and varies little, called constant region (C region). The V region of the H chain is called VH. Certain regions of amino acid composition and arrangement in the variable region have a higher degree of variation, known as hypervariable regions (Hypervariable region, HVR), which are the sites of antigen and antibody binding and are therefore also known as determinant-complementary-determining region (CDR). The heavy chain variable region has three CDR regions. CDR1 and CDR3 are slightly longer than human, CDR3 bulges outward in tertiary structure, and therefore, single domain antibodies are presumed to have higher antigen binding specificity and affinity than conventional antibodies.
The single domain antibody variable domain exhibits the same general structure of a relatively conserved Framework Region (FR) joined by three hypervariable regions or CDRs. From N-terminal to C-terminal, the heavy chain comprises the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid allocation of each domain is in accordance with the definition of the Kabat immunization-purpose protein sequence (Kabat Sequencesof Proteins of Immunological Interest) (National Institutes of Health, bethesda, md.) (1987 and 1991), or Chothia and Lesk, journal of molecular biology, 196:901-917,1987; chothia et al, nature, 342:878-883,1989).
The hypervariable region of an antibody refers to the CDR amino acid residues of the antibody that are responsible for antigen binding. Hypervariable regions comprise amino acid residues from the CDRs, e.g., 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (as described by Kabat et al, protein sequence for immunization (Sequences of Proteins of Immunological Interest), 5 th edition public health agency, national institutes of health, b.bescens, maryland (1991); and/or residues from hypervariable loops, e.g., 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (as described in Chothia et al, J. Mol. Biol. 196:901-917 (1987)).
As used herein, the term "mutant" or "variant" may refer to a molecule comprising a mutation of one or more nucleotides or amino acids of any naturally occurring or engineered molecule.
The term "murine antibody" is herein a monoclonal antibody to human NKp80 prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with an antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the present invention, the murine NKp80 antibody or antigen binding fragment thereof may further comprise a light chain constant region of murine kappa, lambda chain or variants thereof, or further comprise a heavy chain constant region of murine IgG1, igG2, igG3 or variants thereof.
Monoclonal antibodies refer to antibodies obtained from a substantially homogeneous population of antibodies, wherein all antibodies in a mixture have a single amino acid sequence derived from a single clone. Monoclonal antibodies are generally highly specific and are directed against a single antigenic site or epitope. In contrast, polyclonal antibody preparations typically include a mixture of antibodies with different amino acid sequences directed against the same or different determinants (epitopes). In addition to its specificity, a monoclonal antibody has the advantage that it is synthesized in homologous culture without contamination by other immunoglobulins with different specificities and characteristics.
As used herein, the term "Hinge" refers to the region between the CH1 and CH2 regions of an antibody heavy chain that includes an H-chain disulfide bond, is proline-rich, does not form an alpha helix, is prone to stretching and is distorted to some extent, facilitating complementary binding between the antigen binding site of the antibody and the epitope. The heavy chain variable region of the heavy chain single domain antibody of the present invention is linked to the Fc region by a hinge region.
As used herein, the term "Fc region" refers to a protein comprising heavy chain constant region 2 (CH 2) and heavy chain constant region 3 (CH 3) of an immunoglobulin, excluding heavy chain variable region, heavy chain constant region 1 (CH 1). In the present invention, the Fc fragment means to include not only a natural amino acid sequence but also a mutant sequence thereof. The immunoglobulin Fc region may be of human or animal origin, e.g., bovine, goat, porcine, mouse, rabbit, hamster, rat, or guinea pig.
As used herein, the term "constant region" refers to the carboxy-terminal (C-terminal) region of a polypeptide chain, the 3/4 or 4/5 of the H chain and the 1/2 segment of the L chain, which is relatively stable in amino acid number, type, order, configuration and sugar content, and is therefore referred to as the constant region, i.e., the C-region. The C regions of the H chain and L chain are denoted by CH and CL, respectively. The different classes of immunoglobulins have different CH lengths, and 3 CH groups of IgG, igA and IgD include CH1, CH2 and CH3; igM and IgE have 4 CH groups including CH1, CH2, CH3 and CH4. Each heavy chain consists of one variable region (VH) and first, second, third and fourth (optionally) constant regions (CH 1, CH2, CH3, CH4, respectively). Typically, a natural intact antibody is "Y" shaped, the stem of which is composed of the second and third constant regions of two heavy chains joined by disulfide bonds. Each arm of the "Y" type structure includes VH and CH1 (VH-CH 1) and light chain (VL-CL) of the heavy chain.
In this context, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see, ausubel et al, eds. (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing andWiley-Interscience, new York); and the ALIGN program (Dayhoff (1978), atlas ofProtein Sequence and Structure 5: support.3 (National BiomedicalResearch Foundation, washington, D.C.), there are many algorithms for alignment and determination of sequence identity, including homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, computer programs using these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs available in Peason et al (1988) Proc.Natl.Acad.Sci.85:2444, smith-Waterman algorithm (Meth.mol.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.mol.biol.215:403-410), and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs available in BLAST-5:35, and the programs available in the company of Witsin the open area, inc. 35, and the methods of the company, inc. 35, and the methods provided by the methods of the company, inc. Proc.Natl.Acad.Sci.85:2444, and the methods of the invention.
One skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequences of the invention to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the activity of the antibodies (retaining at least 95% of the activity). They are all considered to be included within the scope of the present invention. Such as substitution of amino acids with similar properties in the variable region. The variant sequences of the invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) to a reference sequence. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences mentioned in the present invention are all shown in N-terminal to C-terminal fashion.
As previously mentioned, a mab of the invention may be a full length antibody or may comprise only a functional fragment thereof (e.g., fab, F (ab') 2, or scFv fragment), or may be modified to affect function. The invention includes anti-B7H 6 antibodies with modified glycosylation patterns. In some applications, it may be useful to modify to remove undesired glycosylation sites, or antibodies in which no fucose moiety is present on the oligosaccharide chain, for example to enhance Antibody Dependent Cellular Cytotoxicity (ADCC) function. In other applications, galactosylation modifications may be made to alter Complement Dependent Cytotoxicity (CDC).
Herein, the "full length antibody" is a tetrapeptide chain structure formed by connecting two identical light chains and two identical heavy chains through inter-chain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin a (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). The same class of immunoglobulins can also be of different subclasses, e.g., igG1, igG2, igG3, igG4, depending on the amino acid composition. Immunoglobulin light chains are classified as either kappa chains or lambda chains depending on the constant region.
The term "functional fragment" as used herein refers in particular to an antibody fragment such as a CDR-grafted antibody, fab ', F (ab') 2, fv or scFv, nanobody, or any fragment which should be able to increase half-life by chemical modification, e.g. addition of a poly (alkylene) glycol such as polyethylene glycol ("pegylation, pegylation") (a pegylated fragment known as Fv-PEG, scFv-PEG, fab-PEG, F (ab ') 2-PEG or Fab' -PEG) ("PEG" is polyethylene glycol) which has B7H6 binding activity, or by incorporation into liposomes. Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy chain variable region or the light chain variable region of its source antibody, which partial sequence is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, preferably at least equal to 1/100, more preferably at least equal to 1/10, for B7H 6. Such functional fragments will comprise a minimum of 3 amino acids, preferably 5, 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.
Herein, the term "CDR-grafted antibody" refers to the grafting of CDRs of a species mab to antibody variable regions of another species. For example, the CDRs of a murine mab may be grafted to the variable regions of a human antibody in order to replace the CDRs of a human antibody, allowing the human antibody to acquire the antigen binding specificity of the murine mab while reducing its heterology.
In this context, the term "Fab antibody" or "Fab" generally refers to an antibody comprising only Fab molecules, consisting of VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond between the light and heavy chains.
In this context, the term "nanobody" (single domain antibody or VHH antibody), which was originally described as an antigen binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e. "antibody lacking a light chain") (Hamers-Casterman C, ataroucht, muydermans S, robinson G, hamers C, songa EB, bendahman N, hamers R.: "Naturallyoccurring antibodies devoid oflight chains"; nature 363,446-448 (1993)), comprises only a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind antigen via the heavy chain variable region.
As used herein, the term "Fv antibody" generally refers to an antibody consisting of only the light chain variable region (VL) and the heavy chain variable region (VH) joined by a non-covalent bond, which is the smallest functional fragment of an antibody that retains the intact antigen-binding site.
As used herein, the term "single chain antibody" or "scFv" is a fragment of an antibody in which the heavy and light chain variable regions are linked by a short peptide.
As used herein, a "Knob intonation hole structure" is a button (Knob) forming mutation in the CH3 region of the heavy chain constant region of an antibody that facilitates heavy chain occlusion to form a heterodimer, e.g., by mutating amino acids in the CH3 domain of the heavy chain constant region of a human IgG1 (in this application, the T366S, L368A, Y407V, Y349C mutation, i.e., "hole", in one chain and the T366W, S354C mutation, i.e., "Knob") in the other chain. Wherein the amino acid numbering herein is according to the Kabat numbering system, e.g. "T366S" means that the T amino acid at position 366 according to the Kabat numbering system is replaced by an S amino acid.
As used herein, the term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form, encompassing nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs). Nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
As used herein, the term "vector" refers to a vehicle into which a genetic element (e.g., a nucleic acid molecule as described above) may be operably inserted and which allows expression of the genetic element, e.g., the production of a protein, RNA or DNA encoded by the genetic element, or replication of the genetic element. Vectors may be used to transform, transduce or transfect host cells such that the genetic elements carried thereby are expressed within the host cells. For example, the carrier comprises: plasmids, phagemids, cosmids (cosmid), artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC), phages such as lambda phage or M13 phage, animal viruses, and the like. The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may also include components that assist in its entry into the cell, including, but not limited to, viral particles, liposomes, or protein shells. The vector may be an expression vector or a cloning vector. In some embodiments, the invention provides vectors (e.g., expression vectors) comprising a nucleic acid sequence encoding a fusion protein of the invention, at least one promoter (e.g., SV40, CMV, EF1 a) operably linked to the nucleic acid sequence, and at least one selectable marker.
As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and/or vector may or has been introduced. The host cell contains the vector, and the vector can be introduced into a mammalian cell to construct and obtain the host cell, and then the host cell is utilized to express the antibody or antigen binding fragment provided by the invention. The host cell is cultured to obtain the corresponding antibody or fusion protein. Useful mammalian cells may be CHO cells and the like.
As used herein, the term "composition" is in a form that allows for the biological activity of the active ingredient to be effective and does not include additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered.
As used herein, the term "treatment" refers to the temporary or permanent, partial or complete elimination, reduction, inhibition or amelioration of a clinical symptom, manifestation or progression of an event, disease or condition.
As used herein, the term "diagnosis" refers to the identification, revealing, ascertaining, and/or defining the localization of a pathological state, disease, or condition. In some embodiments, the pharmaceutical compositions of the invention, when administered to a subject or contacted with a sample from a subject, aid in diagnosing cancer, neoplasia or condition.
In many embodiments, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has already or is currently receiving any form of treatment. As used herein, the term "subject" or "patient" refers to a mammalian subject or patient. The terms "patient" or "subject" are used interchangeably herein unless indicated. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and/or an infection. "exogenous" refers to substances produced outside of an organism, cell or human body as the case may be. "endogenous" refers to substances produced in cells, organisms or humans, as the case may be.
The aspects of the present disclosure will be explained below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The sequence information according to the present invention is shown in the following table 1:
table 1:
example 1: construction of 293T cells overexpressing human and cynomolgus monkey NKp80
HEK293T cells were plated in T150 flasks and cultured in DMEM complete medium. After overnight incubation, the vector comprising psPAX2, pMD2.G and pCDH-CMV-NKp80-IRES-puro (pCDH-CMV-MCS-IRES-puro) with the coding sequence of human NKp80 protein (SEQ ID NO: 20) (SEQ ID NO: 21) or the coding sequence of cynomolgus monkey NKp80 protein (SEQ ID NO: 22) (SEQ ID NO: 23)) inserted between the multiple cloning sites was taken according to a sequence of 7:3:10 to 1.5mL of Opti-MEM (Gibco, cat. No. 31985070) and 100. Mu. L P3000 transfection reagent. mu.L of Lipofectamine 3000 (thermo, cat. L3000008) transfection reagent was added to 1.5mL of Opti-MEM medium and mixed well. DNA diluent and liposome diluent are mixed according to the volume ratio of 1:1, incubating for 5-10min at room temperature, adding into 293T cells, culturing for 48 hours, and harvesting virus supernatant. The virus supernatant was centrifuged at 2000g for 10min at 4℃and the supernatant was filtered through a 0.45 μm filter and added to PEG8000 solution (Shanghai chemical) and mixed well and allowed to stand overnight at 4 ℃.2200g was centrifuged for 90min, white pellet was placed at the bottom of the tube and virus was resuspended in sterile PBS buffer.
Polybrene (sigma, TR-1003) (final concentration 8. Mu.g/ml) was added to DMEM medium, and after mixing, an appropriate amount of virus solution was added. 293T cells of 2E5 were taken in 24-well plates, medium with virus was added, and fresh culture was changed after 8 hours in an incubator. Detecting the expression level of NKp80 on the surface of 293T cells in 48 hours by a flow cytometry, carrying out limited dilution after positive group cells appear, namely, after the cells are digested, diluting the density to 4 cells per milliliter, inoculating the cells into a 96-hole flat bottom plate, adding 200 mu l into each hole, culturing for 2 weeks until the cells become obvious single cell mass, detecting the expression level of NKp80 on the surface of each cloned cell by a flow cytometry, and obtaining the full positive cells which are 293T cells over-expressing human NKp80 or cynomolgus monkey NKp80 as required. The flow identification expression result is shown in figure 1.
Example 2 preparation of human and cynomolgus monkey NKp80 extracellular fragment proteins
The amino acid sequence of the extracellular segment of the human NKp80 is shown as SEQ ID NO. 24, and the nucleotide sequence is shown as SEQ ID NO. 25. The amino acid sequence of the extracellular segment of cynomolgus monkey NKp80 is shown as SEQ ID NO. 26, and the nucleotide sequence is shown as SEQ ID NO. 27.
Human or cynomolgus monkey NKp80 extracellular fragment proteins were prepared by transient transfection of expcho-S cells (Gibco, cat No. a 29127) with pcdna3.4 vector carrying the human or cynomolgus monkey NKp80 extracellular fragment nucleic acid sequence (with histidine tag). The day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4). Times.10 6 /ml，37℃，8％CO 2 The culture was continued overnight with shaking at 120 rpm. On the day of transfection, cells grew to 7X 10 6 -1×10 7 Per ml, with viability above 95% the cells were diluted to 6X 10 using freshly pre-warmed ExpiCHO medium (Gibco, cat. A2910002) 6 /ml, and taking the coding sequence of the extracellular segment containing human or cynomolgus monkey NKp80The plasmids listed were transfected into ExpiCHO-S cells using the ExpiFectamine CHO transfection reagent (Gibco, cat. No. A29129), 37℃and 8% CO 2 Shaking culture at 120 rpm. Mixing ExpiFectamine CHO Enhancer and ExpiCHO Feed for 18-22 hr, immediately adding transfected cells, mixing, and mixing at 32deg.C with 5% CO 2 Shaking culture at 120 rpm. On day 5 after transfection, 8ml of ExpiCHO Feed was added to the cells again, and culture was continued after mixing. The cell number and cell viability were observed daily and cells were harvested by centrifugation after the cell viability had fallen below 80% or after 10-14 days of culture. The expressed supernatant was filtered with a 0.22 μm filter membrane, histidine-tagged antibody was captured from the expressed supernatant using a HisTrap excel affinity column (cytova, cat# 17371206), the column was equilibrated with phosphate buffer at pH7.4, the supernatant was passed through the affinity column, eluted with phosphate elution buffer containing 500mM imidazole, and finally concentrated and displaced with PBS buffer, and purified human and cynomolgus monkey NKp80 extracellular proteins were identified to have a purity of 90% or more by SDS-PAGE, to thereby finally obtain human and cynomolgus monkey NKp80 extracellular proteins.
EXAMPLE 3 preparation of anti-human NKp80 hybridoma monoclonal antibodies
Anti-human NKp80 monoclonal antibodies were generated by immunizing mice. Balb/c mice used for experiments, females and 6 weeks old (Jiangsu Ji kang Biotechnology Co., ltd.) were immunized with human NKp80 extracellular domain protein prepared in example 2, were first immunized with Freund' S complete adjuvant (Sigma, F5881) and antigen mixed and emulsified, and were then immunized with the antigen mixed and emulsified, and then immunized with Ribi adjuvant system (Sigma, S6322).
Mice with higher antibody titer in serum were selected for spleen cell fusion, and the selected mice were immunized by sprint 72 hours prior to fusion, and were injected intraperitoneally. Spleen lymphocytes were fused with myeloma cells Sp2/0 cells (ATCC, CRL-8287) using an optimized PEG-mediated fusion procedure to give hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHAT and 1 XOPI) and plated in 96-well cell culture plates at 37℃and 5% CO 2 And (5) incubating. HAT complete medium, 50 ul/well, was added on day 5 post-fusion. On 7 th to 8 th day after fusion, according to finenessCell growth density, total medium, HT complete medium (RPMI-1640 medium with 20% FBS, 1 XHT and 1 XOPI), 200 ul/well.
Flow cytometry binding assays were performed on day 10-11 post-fusion, based on cell growth density. The positive wells were changed and expanded into 24 well plates in time according to cell density. The cell lines engrafted into 24-well plates were retested for seed protection and first subcloning. Seed protection is carried out when the first subclone screening is positive, and the second subclone is carried out. And performing seed conservation and protein expression on the positive secondary subcloning. Antibodies were further prepared by serum-free cell culture and purified by protein G affinity chromatography for subsequent functional activity detection.
EXAMPLE 4 hybridoma sequencing
The total number of candidate hybridoma cells was cultured to 10 by binding to the experimentally selected murine 53G7 antibody hybridoma 6 Cells were collected by centrifugation at 800rpm for 10 minutes and total RNA was extracted with Trizol kit (Invitrogen); the corresponding variable region nucleic acid sequences of hybridoma cells were PCR amplified using total RNA as template, reverse transcribed into a cDNA library (Invitrogen), and cDNA as template. The primer sequences used in the PCR amplification reactions are complementary to the antibody variable region first framework region or signal peptide region and constant region (Larrick, j.w., et al, (1990) scand.j.immunol.,32, 121128 and Coloma, j.j.et al, (1991) BioTechniques,11, 152156). In a 50. Mu.l reaction system, 2. Mu.L of cDNA, 5. Mu.L of 10 XPCR buffer, 2. Mu.L of upstream and downstream primers (5. Mu.M), 2. Mu.L of dNTP, 1. Mu.L of Taq enzyme (Takara, ex Taq), H were added, respectively 2 O38. Mu.L; pre-denaturation at 95 ℃ for 5min, and entering into temperature circulation for PCR amplification. The reaction conditions are as follows: denaturation at 94℃for 30S, annealing at 58℃for 45S, elongation at 72℃for 50S for 32 cycles, and then elongation at 72℃for 7min. And sequencing the amplified product to obtain the heavy chain and light chain variable region sequences of the murine monoclonal antibody.
Example 5 transient expression of anti-human NKp80 antibodies
The heavy chain variable region sequence of the anti-human NKp80 antibody 53G7 is shown as SEQ ID NO. 7, and the light chain variable region sequence is shown as SEQ ID NO. 8. The nucleic acid sequence (SEQ ID NO: 16) encoding the heavy chain amino acid sequence (SEQ ID NO: 9) and the nucleic acid sequence (SEQ ID NO: 17) encoding the light chain amino acid sequence (SEQ ID NO: 10) were recombined onto the pTT5 plasmid, respectively, by molecular biological techniques.
NKp80 antibodies were prepared by transient transfection of ExpiCHO-S cells (Gibco, cat. A29127) with pTT5 vector carrying anti-human NKp80 antibody light and heavy chains. The day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4). Times.10 6 /ml，37℃，8％CO 2 The culture was continued overnight with shaking at 120 rpm. On the day of transfection, cells grew to 7X 10 6 -1×10 7 Per ml, with viability above 95% the cells were diluted to 6X 10 using freshly pre-warmed ExpiCHO medium (Gibco, cat. A2910002) 6 Per ml, and taking plasmids containing anti-human NKp80 light chain and heavy chain according to a molar ratio of 2:1 transfection into ExpiCHO-S cells with ExpiFectamine CHO transfection reagent (Gibco, cat. No. A29129), 37℃and 8% CO 2 Shaking culture at 120 rpm. Mixing ExpiFectamine CHO Enhancer and ExpiCHO Feed for 18-22 hr, immediately adding transfected cells, mixing, and mixing at 32deg.C with 5% CO 2 Shaking culture at 120 rpm. On day 5 after transfection, 8ml of ExpiCHO Feed was added to the cells again, and culture was continued after mixing. The cell number and cell viability were observed daily and cells were harvested by centrifugation after the cell viability had fallen below 80% or after 10-14 days of culture. The expressed supernatant was filtered with a 0.22 μm filter membrane, an antibody having an Fc domain was captured from the expressed supernatant by using a Mabselect prism A affinity column (cytova, cat# 17549854), the column was equilibrated with a phosphate buffer of pH7.2, the supernatant was passed through the affinity column, eluted with an elution buffer (100 mM citric acid, pH 2.7), and finally concentrated and displaced with PBS buffer, and the purified antibody was identified to have a purity of 95% or more by SDS-PAGE, thereby obtaining a 53G7 antibody.
EXAMPLE 6NKp80 antibody ELISA binding assay
ELISA experiments were used to detect the binding properties of chimeric NKp80 antibody 53G 7. Human and cynomolgus monkey NKp80 extracellular fragment proteins were buffered with coating buffer (35 mM NaHCO 3 ，15mM Na 2 CO 3 pH 9.6) was diluted to 2. Mu.g/ml, 100. Mu.l per well was added to the ELISA plate, and the temperature was 4℃overnight. Thereafter, the cells were washed 3 times with PBST (0.05% Tween 20-PBS, pH 7.2). To the direction of To the plate, 300. Mu.l of blocking buffer (1% BSA,0.05% Tween20-PBS, pH 7.2) was added, and the mixture was allowed to stand at room temperature for 2 hours. The mixture was washed 3 times with PBST. Gradient concentrations of anti-NKp 80 antibody 53G7 were added to each well and incubated for 1 hour at room temperature. The mixture was washed 3 times with PBST. Mu.l of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-097) diluted with blocking buffer was added to each well and incubated for 1 hour at room temperature. Washing with PBST for 3 times, adding TMB into each hole, reacting at room temperature in a dark place for 2-5 minutes, stopping the reaction with 2M sulfuric acid in each hole, and finally reading the OD450 value with an ELISA reader.
The results are shown in fig. 2A and 2B, where fig. 2A shows that the 53G7 antibody of the present invention can bind to human NKp80, and fig. 2B shows that the 53G7 antibody of the present invention can bind to cynomolgus NKp80, hIgG1 as a control.
Example 7NKp80 antibody flow cytometry binding experiments
(1) NKp80 antibodies bind 293T cells overexpressing human and cynomolgus monkey NKp80 proteins
Diluting 293T cells overexpressing human NKp80 or cynomolgus monkey NKp80 to 2X 10 with PBS 6 Per mL, 100. Mu.L/tube was added to a 1.5mL EP tube, 10. Mu.L/tube goat serum was added thereto, and the mixture was blocked at 4℃for 30min. Gradient concentration of NKp80 antibody was added and incubated at 4℃for 30min. 1mL of PBS was added to the EP tube, centrifuged at 3500 rpm.times.5 min at 4℃and the supernatant was discarded and washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.l/tube PBS, to which 0.1. Mu.l/tube Alexa 647-labeled goat anti-mouse antibody secondary antibody (Jackson ImmunoResearch, 109-606-170) was added and incubated at 4℃for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.
The flow results are shown in fig. 3A and 3B, where the results in fig. 3A indicate that the 53G7 antibody can bind to 293T cells that overexpress human NKp80, and the results in fig. 3B indicate that the 53G7 antibody can bind to 293T cells that overexpress cynomolgus NKp80, indicating that the 53G7 antibody can bind to NKp80 protein on the cell surface.
(2) NKp80 antibodies bind human PBMC
Human Peripheral Blood Mononuclear Cells (PBMC) were adjusted to a cell density of 5X 10 7 Per mL, add to 100. Mu.L/tube volumeA gradient of NKp80 antibody was added to a 1.5ml EP tube and incubated at 4℃for 30min. 1mL of PBS was added to the EP tube, centrifuged at 3500 rpm.times.5 min at 4℃and the supernatant was discarded and washed once with PBS. Anti-human CD45 antibody (biolegend, B356107) was diluted with PBS; anti-human CD3 antibody (biolegend, B375999); anti-human CD56 antibody (biolegend, B371788); goat anti-mouse antibody secondary antibody (Jackson ImmunoResearch, 109-606-170), 100 μl per well was added to the cell wells, and incubated at 4deg.C for 30min after mixing. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.
FIG. 4A shows that 53G7 antibodies can bind CD3 in PBMC + T cells, FIG. 4B shows that 53G7 antibodies can bind CD56 in PBMC + NK cells, demonstrating that the 53G7 antibody can bind to both human primary T cells and NK cells.
Example 8 bispecific antibody targeting B7H6 containing NKp80 single chain antibody (scFv)
(1) Preparation of bispecific antibodies targeting B7H6 containing NKp80 scFv
A bispecific antibody was designed containing two monovalent units, one of which is the anti-NKp 80 scFv-Fc form, the variable region amino acid from the sequence of example 4 of the present invention and the other of which is the anti-B7H 6 scFv-Fc form (sequence from CN 116284397A), designated NKp80 XB 7H6. The bispecific antibody contains two polypeptide chains, one for each: heavy chain of scFv of NKp80 single-chain antibody (SEQ ID NO: 14), heavy chain of scFv of B7H6 single-chain antibody (SEQ ID NO: 15). Because of the special asymmetric structure of the molecule, different amino acid mutations are introduced in the constant regions of the two chains in order to reduce homodimer production. Also, in order to prevent activation of crosslinking by fcγ receptor, a (L234A/L235A) mutation was introduced in the heavy chain constant region.
Bispecific antibody recombinant proteins were prepared by one-step affinity purification using the procedure described in example 5 in combination with plasmid ratios (1:1 or other ratios). Table 2 below lists information for bispecific antibodies NKp80×B7H26.
TABLE 2
| NKp80 heavy chain | B7H6 heavy chain | |
| Amino acid sequence | SEQ ID NO：14 | SEQ ID NO：15 |
| Nucleotide sequence | SEQ ID NO：18 | SEQ ID NO：19 |
(2) Cell killing experiments
Colorectal cancer HCT-15 cells were counted after digestion and cell density was adjusted to 2X 10 5 /ml. The RTCA instrument (agilent) was turned on, the experimental mode was selected, and the cell information and drug information were filled in. Entering a schedule setting experiment step, adding 50 μl of fresh culture medium (89% RPMI 1640 culture medium+10% fetal bovine serum+1% green streptomycin) into the plate, putting the plate into an instrument, closing the plate, and clicking to start in the first step. After Done, the plate was removed, 100. Mu.l of cell suspension was added, left to stand at room temperature for 15-30min to prevent edge effects, placed in the instrument, and clicked on. After growth to log phase, suspension was continued, and 50. Mu.l of PBMC (4X 10) 6 /ml) and add a gradient of bispecific antibody, click on start, analysis after a period of time. FIG. 5 shows that the killing efficiency of PBMC against colorectal cancer HCT-15 cells gradually increases with increasing concentration of NKp80×B7H26 bispecific antibody, demonstrating that NKp80×B7H26 can promote PBMC to kill B7H6 specifically + Is a tumor cell of (a).
In the description of the present specification, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," "some embodiments," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (22)
1. An antibody or antigen-binding fragment comprising:
heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO 1, 2 and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NO 1, 2 and 3; and/or
Light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO 4, 5 and 6 or amino acid sequences having at least 80% identity to 4, 5 and 6, respectively.
2. The antibody or antigen-binding fragment of claim 1, comprising:
a heavy chain variable region CDR1 sequence shown in SEQ ID NO. 1, a heavy chain variable region CDR2 shown in SEQ ID NO. 2, a heavy chain variable region CDR3 shown in SEQ ID NO. 3, a light chain variable region CDR1 shown in SEQ ID NO. 4, a light chain variable region CDR2 shown in SEQ ID NO. 5, and a light chain variable region CDR3 shown in SEQ ID NO. 6.
3. The antibody or antigen-binding fragment of claim 1, comprising: at least one of a heavy chain FR region and a light chain FR region;
Optionally, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a primate-origin antibody and a murine-origin antibody or a mutant thereof.
4. An antibody or antigen-binding fragment according to claim 3, comprising:
a heavy chain variable region as shown in SEQ ID NO. 7; and/or
The light chain variable region is shown as SEQ ID NO. 8.
5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of which is derived from at least one of a primate-origin antibody and a murine-origin antibody or a mutant thereof;
optionally, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or mutant thereof or a human IgG antibody or mutant thereof;
optionally, the light chain constant region and the heavy chain constant region are both derived from a murine IgG1 antibody or a mutant thereof or a human IgG1 antibody or a mutant thereof.
6. The antibody or antigen-binding fragment of claim 5, wherein the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID No. 9 and a light chain of the amino acid sequence shown in SEQ ID No. 10.
7. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody;
optionally, the monoclonal antibody comprises at least one of a full length antibody, fv antibody, single chain antibody, fab antibody, single domain antibody, and minimal recognition unit.
8. A multispecific binding molecule comprising at least:
a first binding region comprising the antibody or antigen binding fragment of any one of claims 1-7; and
a second binding region capable of specifically binding to a target cell surface antigen.
9. The multispecific binding molecule of claim 8, wherein the multispecific binding molecule is a bispecific binding molecule;
optionally, the bispecific binding molecule comprises a symmetric bispecific binding molecule or an asymmetric bispecific binding molecule;
optionally, the bispecific binding molecule is a symmetric bispecific binding molecule.
10. The multi-specific binding molecule of claim 9, wherein the first binding region comprises peptide chain 1 and peptide chain 2, wherein peptide chain 1 comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID NO's 1, 2 and 3, respectively,
The peptide chain 2 comprises light chain variable region CDR1, CDR2 and CDR3 sequences shown as amino acid sequences of SEQ ID NO. 4, 5 and 6 respectively;
optionally, the peptide chain 1 comprises a heavy chain variable region shown in SEQ ID NO. 7, and the peptide chain 2 comprises a light chain variable region shown in SEQ ID NO. 8;
optionally, the first binding region further comprises a first linking peptide, wherein the N-terminus of the first linking peptide is linked to the C-terminus of the peptide chain 2 and the C-terminus of the first linking peptide is linked to the N-terminus of the peptide chain 1; or the N end of the first connecting peptide is connected with the C end of the peptide chain 1, and the C end of the first connecting peptide is connected with the N end of the peptide chain 2;
optionally, the first connecting peptide has an amino acid sequence (GGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
optionally, the first connecting peptide has an amino acid sequence shown in SEQ ID NO. 13;
optionally, the first binding region comprises a single chain antibody having the amino acid sequence shown in SEQ ID NO. 11.
11. The multispecific binding molecule of any one of claims 8 to 10, wherein the first binding region further comprises a first heavy chain constant region, at least a portion of the first heavy chain constant region being derived from at least one of a human antibody, a primate antibody, and a murine antibody or a mutant thereof;
Optionally, the first heavy chain constant region is from a human IgG antibody or mutant thereof;
optionally, the first heavy chain constant region has the amino acid sequence set forth in SEQ ID NO. 28;
optionally, the first binding region has the amino acid sequence shown as SEQ ID NO. 14.
12. The multispecific binding molecule of claim 11, wherein the second binding region has B7H6 binding activity;
optionally, the second binding region comprises at least one of a full length antibody, fv antibody, single chain antibody, fab antibody, single domain antibody, and minimal recognition unit having B7H6 binding activity;
optionally, the second binding region comprises an anti-B7H 6 single chain antibody;
optionally, the anti-B7H 6 single chain antibody comprises a light chain variable region of an anti-B7H 6 antibody and a heavy chain variable region of an anti-B7H 6 antibody,
optionally, the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the anti-B7H 6 antibody are shown in SEQ ID NO. 30-32;
optionally, the sequences of the CDR1, CDR2 and CDR3 of the light chain variable region of the anti-B7H 6 antibody are shown in SEQ ID NO. 33-35;
optionally, the heavy chain variable region of the anti-B7H 6 antibody has the amino acid sequence shown in SEQ ID NO. 36, and the light chain variable region of the anti-B7H 6 antibody has the amino acid sequence shown in SEQ ID NO. 37;
Optionally, the anti-B7H 6 single chain antibody further comprises a second connecting peptide linking the heavy chain variable region of the anti-B7H 6 antibody and the light chain variable region of the anti-B7H 6 antibody;
optionally, the second connecting peptide has an amino acid sequence (GGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
optionally, the second connecting peptide has an amino acid sequence shown in SEQ ID NO. 13;
optionally, the anti-B7H 6 single chain antibody has an amino acid sequence as shown in SEQ ID NO. 12.
13. The multispecific binding molecule of claim 12, wherein the second binding region further comprises a second heavy chain constant region, at least a portion of the second heavy chain constant region being derived from at least one of a primate-source antibody and a murine antibody or a mutant thereof;
optionally, the second heavy chain constant region is derived from a human IgG antibody or mutant thereof;
optionally, the second heavy chain constant region has the amino acid sequence shown in SEQ ID NO. 29;
optionally, the N-terminus of the second heavy chain constant region is linked to the C-terminus of the anti-B7H 6 single chain antibody;
optionally, the second binding region has the amino acid sequence shown in SEQ ID NO. 15;
Optionally, the first heavy chain constant region and the second heavy chain constant region are linked by a knob-intoo-hole structure.
14. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 7 or the multispecific binding molecule of any one of claims 8 to 13.
15. An expression vector carrying the nucleic acid molecule of claim 14.
16. A recombinant cell carrying the nucleic acid molecule of claim 14, the expression vector of claim 15; or (b)
An antibody or antigen binding fragment according to any one of claims 1 to 7 or a multispecific binding molecule according to any one of claims 8 to 13.
17. The recombinant cell according to claim 16, wherein the recombinant cell is obtained by introducing the expression vector of claim 15 into a host cell;
optionally, the recombinant cell is a eukaryotic cell;
optionally, the recombinant cell is a mammalian cell.
18. A composition comprising at least one of the antibody or antigen-binding fragment of any one of claims 1-7, the multispecific binding molecule of any one of claims 8-13, the nucleic acid molecule of claim 14, the expression vector of claim 15, or the recombinant cell of claim 16 or 17.
19. A medicament comprising at least one of the antibody or antigen-binding fragment of any one of claims 1 to 7, the multispecific binding molecule of any one of claims 8 to 13, the nucleic acid molecule of claim 14, the expression vector of claim 15, the recombinant cell of claim 16 or 17, or the composition of claim 18.
20. Use of an antibody or antigen binding fragment according to any one of claims 1 to 7, a multispecific binding molecule according to any one of claims 8 to 13, a nucleic acid molecule according to claim 14, an expression vector according to claim 15, a recombinant cell according to claim 16 or 17, a composition according to claim 18 for the preparation of a medicament for the prophylaxis and/or treatment of cancer,
the cancer includes at least one of the following: colorectal cancer, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, renal cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.
21. A kit for detecting NKp80, comprising at least one of the antibody or antigen-binding fragment of any one of claims 1-7, the multispecific binding molecule of any one of claims 8-13, the nucleic acid molecule of claim 14, the expression vector of claim 15, and the recombinant cell of claim 16 or 17.
22. Use of the antibody or antigen binding fragment of any one of claims 1 to 7, the multispecific binding molecule of any one of claims 8 to 13, the nucleic acid molecule of claim 14, the expression vector of claim 15, the recombinant cell of claim 16 or 17 in the preparation of a kit for detecting NKp80.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311373342.3A CN117586402A (en) | 2023-10-20 | 2023-10-20 | NKp80 antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311373342.3A CN117586402A (en) | 2023-10-20 | 2023-10-20 | NKp80 antibody and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117586402A true CN117586402A (en) | 2024-02-23 |
Family
ID=89910498
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311373342.3A Pending CN117586402A (en) | 2023-10-20 | 2023-10-20 | NKp80 antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117586402A (en) |
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2023
- 2023-10-20 CN CN202311373342.3A patent/CN117586402A/en active Pending
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