CN118221818A - Anti-CD 16A nano antibody and application thereof - Google Patents
Anti-CD 16A nano antibody and application thereof Download PDFInfo
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- CN118221818A CN118221818A CN202410527965.XA CN202410527965A CN118221818A CN 118221818 A CN118221818 A CN 118221818A CN 202410527965 A CN202410527965 A CN 202410527965A CN 118221818 A CN118221818 A CN 118221818A
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Abstract
The invention discloses an anti-CD 16A nano antibody and application thereof, wherein a heavy chain variable region of the nano antibody comprises a CDR1, a CDR2 and a CDR3, wherein the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3. The anti-CD 16A nanobody can specifically bind to a CD16A antigen, the affinity of the nanobody is 10 ‑9 M, and the anti-CD 16A nanobody belongs to a high-affinity nanobody. The hetero-dimeric bispecific antibody constructed by the anti-CD 16A nano-antibody and the anti-CD 22 nano-antibody still keeps good antigen binding activity on CD16A, has stronger killing activity on acute B lymphocyte leukemia, effectively improves the treatment effect, and can provide a novel method for high-efficiency and lasting anti-tumor.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to an anti-CD 16A nano antibody and application thereof.
Background
Natural killer cells (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. CD16 molecules are important markers on NK cell surfaces that activate the IgE NK cell receptor (FcεRIgamma) and the Immunoreceptor Tyrosine Activation Motif (ITAM) of CD3 zeta for ADCC. Thus, the CD16 target is preferentially selected among bispecific antibodies based on NK cell redirection.
Acute B-lymphoblastic leukemia (B cell-acute lymphoblastic leukemia, B-ALL) is one of the most common types of Acute Lymphoblastic Leukemia (ALL), originating from B-lineage lymphoprecursor cells, with an incidence of about 85% of ALL; B-ALL has a prevalence of about 80% in pediatric acute leukemia and about 20% in adult humans. The existing traditional treatment means can enable 80% of children to survive for a long time without diseases, but only 30% -40% of adults still have more than about 50% of patients to relapse. Therefore, in order to solve the problems of reduced curative effect in the treatment process, the development of safer, more effective and diverse new drugs is needed.
There are three major classes of human IgG Fc receptors: fcγri (CD 64), fcγrii (CD 32) and fcγriii (CD 16). Wherein the second class comprises three subclasses IIA/IIB/IIC and the third class comprises two subclasses IIIA/IIIB. The receptor most relevant to activating innate immune cells (e.g., NK cells) with killing function is IIIA, also known as CD16A. CD16A is expressed on NK cells, monocytes and macrophages, and also expressed in small amounts on DC cell surfaces, belonging to the activated receptor. Cell surface expressed Fc receptors can directly induce or regulate activation of cells, and their effects can include ADCC (antibody-dependent cell-mediated cell killing) and CDC (complement-dependent cytotoxicity). Antibody drugs targeted for CD16A action can be divided into two classes. One class is IgG-derived antibodies with an antibody constant region (Fc) that enhances cell killing in such a way that the Fc segment of the antibody binds to CD16A receptors, and in general, modifications to the Fc region of therapeutic antibodies can enhance their binding to CD16A.
Nanobody (Nb) is a class of naturally deleted light and heavy chain first constant region (CH 1) antibodies with antigen binding ability that are present in camelids. The molecular weight of the monoclonal antibody is small (15 kDa), is only 1/10 of that of the traditional monoclonal antibody, has the characteristics of good stability, solubility, affinity and specificity, flexibility of coupling with different protein molecules, easiness in transformation and the like, and has wide development and application values in the fields of disease treatment, detection and the like.
Bispecific antibodies (bispecific antibody, bsAb) refer to artificial antibodies that can target two antigens or different epitopes of the same antigen simultaneously. Since human CD16A is mainly expressed in NK cells, drugs targeting CD16A are mostly therapeutic by enhancing NK cell activity. Currently, a marketed antibody drug Margenza (Margetuximab), through engineering of the Fc region of a targeting Her-2 antibody (a proprietary Fc optimization platform from MacroGenetics), enhances the affinity of the antibody for CD16A and reduces the affinity for the inhibitory receptor CD32B, thereby enhancing the involvement of the innate immune system and better killing Her2 positive tumor cells. Therefore Margenza is also known as optimized version of Herceptin (trastuzumab, targeting Her 2).
With the in-depth understanding of tumor biology, it is becoming more and more apparent that personalized medical strategies are critical to improving therapeutic efficacy. Although a variety of therapeutic approaches (e.g., chemotherapy, radiation therapy, targeted therapy, etc.) have been used to treat acute B-lymphocyte leukemia, many patients eventually experience disease recurrence or resistance to existing therapies. Thus, new therapeutic strategies are urgently needed to improve the prognosis of these patients. Based on these factors, the development of bispecific antibodies against CD22 and NK cell surface molecule CD16A, which can direct NK cells to the vicinity of tumor cells, thus enhancing the recognition and killing ability of the immune system against B-ALL cells, can be considered an innovative therapeutic strategy aimed at improving survival and quality of life of B-ALL patients, especially those who react poorly to traditional therapeutic approaches. The research and development work not only can promote the progress of cancer treatment, but also can provide a new theoretical and practical basis for future NK cell immunotherapy.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an anti-CD 16A nano-antibody and application thereof, wherein the nano-antibody has higher affinity.
The invention screens the nano antibody with high affinity aiming at the CD16A target, the CD16A is a key factor in the intracellular signal transmission path, the structure and the function are relatively complex, and the difficulty of screening the specific nano antibody is great. According to the invention, a specific nano antibody is finally obtained through a specific screening method and large-scale screening, and meanwhile, the stability and the adaptability of the obtained nano antibody are identified; finally, the invention also provides a preparation method of the nano-antibody, and stable production of the anti-CD 16A nano-antibody can be realized according to the method.
The invention is realized in the following way:
in a first aspect, the present invention provides an anti-CD 16A nanobody, wherein the heavy chain variable region comprises a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3.
In a second aspect, the invention provides an antibody comprising a heavy chain variable region of an anti-CD 16A nanobody or an anti-CD 16A nanobody as described above.
In a third aspect, the invention provides a bispecific antibody comprising an anti-CD 16A nanobody and an anti-CD 22 nanobody as described above.
In a fourth aspect, the invention provides a nucleic acid encoding a nanobody, an antibody or a bispecific antibody against CD16A as described above.
In a fifth aspect, the present invention provides an expression vector comprising the nucleic acid described above.
In a sixth aspect, the invention provides a host cell comprising the above expression vector, or having the above nucleic acid integrated in the genome, or expressing the above nanobody, antibody or bispecific antibody against CD 16A.
In a seventh aspect, the present invention provides a method of preparing a nanobody, comprising: culturing the above-described host cell under conditions suitable for antibody production, thereby obtaining a culture containing the anti-CD 16A antibody, and isolating or recovering the anti-CD 16A antibody from the culture.
In an eighth aspect, the invention provides an immunoconjugate or pharmaceutical composition comprising the above-described anti-CD 16A nanobody, antibody or bispecific antibody.
In a ninth aspect, the present invention provides the use of the above-described anti-CD 16A nanobody, antibody, bispecific antibody, nucleic acid, expression vector, host cell or immunoconjugate or pharmaceutical composition for the preparation of a product for the prevention or treatment of a tumor.
The invention has the following beneficial effects:
The anti-CD 16A nanobody screened from the Bactrian camel VHH immune library can specifically bind to a CD16A antigen, has the nanobody affinity of 10 -9 M, and belongs to high-affinity nanobodies. In addition, the invention constructs the CD16A targeting nanobody and the CD22 resisting nanobody into hetero-dimeric bispecific antibody which still maintains good antigen binding activity to CD 16A; meanwhile, in vivo and in vitro experiments prove that the heterodimeric bispecific antibody has stronger killing activity on acute B lymphocyte leukemia, can effectively improve the treatment effect, provides a key material for the subsequent development of immunotherapy with high-efficiency and lasting anti-tumor activity, and has important clinical value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the binding specificity of CD16A nanobody to CD16A antigen by indirect ELISA in example 2 of the present invention;
FIG. 2 is a graph showing the binding capacity of CD16A nanobody to CD16A antigen by indirect ELISA in example 3 of the present invention;
FIG. 3 is a graph showing the effect of detecting recombinant nanobody binding to CD16A+ -HeLa cells using flow assay in example 4 of the present invention;
FIG. 4 is the construction of a bispecific antibody (CD 22X CD 16A) gene fragment according to example 6 of the present invention;
FIG. 5 is a graph showing the detection of killing efficiency of a CD22 positive cell line by a bispecific antibody of example 6 of the present invention;
FIG. 6 is a life cycle analysis of a B-ALL mouse transplantation model by a bispecific antibody of example 7 of the present invention.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (molecular μLar Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell culture (ANIMAL CELL C. Mu. Lture) (R.I. Freshney, 1987); the methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), the manual of experimental immunology (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), the gene transfer vector for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), the method of contemporary molecular Biology (Current Protocols in Molec. Mu.Lar Biology, inc., 1987), the PCR polymerase chain reaction (PCR: the Polymerase Chain Reaction) (M. Mu. Llis, etc., 1994), and the methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan, et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of a range should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
The invention provides a CD16A nano antibody, wherein a heavy chain variable region comprises a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3.
The nanobody of the present invention generally comprises 4 Framework Regions (FRs) and 3 Complementarity Determining Regions (CDRs), respectively referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, wherein the FRs and CDRs form a heavy chain variable region, and the heavy chain variable region has the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
In some embodiments, the nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific antibody, and a fusion nanobody.
Monovalent nanobody: the antigen-specific nanobody is obtained by screening specific antigen from a nanobody library, can maintain a strict monomer structure due to a large number of hydrophilic residues on the surface of the nanobody, and can be combined with the antigen with high specificity and high affinity only in a monomer form.
Multivalent nanobody: multivalent antibodies are polymers of monovalent antibodies that recognize the same epitope, with higher antigen affinity than the corresponding monovalent nanobody.
Multispecific antibodies are polymers of monovalent antibodies that recognize different epitopes, can bind to different targets or different epitopes of the same target, and have higher antigen recognition capabilities than monovalent antibodies.
Fusion nanobody: antibodies include, but are not limited to, enzymes, antimicrobial peptides, or imaging substances that bind to other structures (e.g., BSA, igG-Fc, etc.) by genetic engineering techniques to form novel fusion molecules, such as to extend their half-life.
In some embodiments, when the nanobody is a monovalent nanobody, the amino acid sequence is as set forth in SEQ ID NO: 4.
The invention also provides an antibody comprising the above-mentioned anti-CD 16A nanobody or the heavy chain variable region of the anti-CD 16A nanobody.
In some implementations, the antibody can be any of a full length antibody, a heavy chain antibody, a chimeric antibody, a multispecific antibody (e.g., bispecific antibody, trispecific antibody, tetraspecific antibody, etc.), a murine antibody, a humanized antibody, or an antigen-binding fragment.
The antigen binding fragment includes any one selected from the group consisting of F (ab ') 2, fab', fab, fv, and scFv of an antibody, so long as they exhibit the desired antigen binding activity.
Chimeric antibodies are antibodies in which the variable region of a non-human antibody is fused to the constant region or framework region of a human antibody, and thus can reduce the immune response induced by the non-human antibody.
The antigen binding fragments, i.e., functional fragments of antibodies, generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
The antigen binding fragments described above may also be obtained synthetically by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
The invention also provides a bispecific antibody comprising an anti-CD 16A nanobody and an anti-CD 22 nanobody.
In some embodiments, the bispecific antibody described above sequentially links the anti-CD 22 nanobody to the anti-CD 16A nanobody via a linking peptide.
In some embodiments, the above-described linker peptide is (G4S) n, wherein n is a non-0 integer.
In some embodiments, n is an integer between 1 and 20; specifically, n may be 1,2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
In some embodiments, n is 3 or 4.
In some embodiments, when the anti-CD 22 nanobody is a monovalent nanobody, the amino acid sequence of the heavy chain variable region thereof is as set forth in SEQ ID NO:5 (issued patent number: CN 202310419011.2).
The invention also provides a nucleic acid which codes for the above-mentioned anti-CD 16A nanobody, antibody or bispecific antibody.
Considering the degeneracy of codons, the sequence of the genes encoding the above antibodies may be modified in the coding region thereof without changing the amino acid sequence to obtain genes encoding the same antibodies; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.
The invention also provides an expression vector comprising the nucleic acid.
Expression vectors in the context of the present invention mean any recombinant polynucleotide construct which can be used for the direct introduction of a DNA fragment of interest into a host cell by transformation, transfection or transduction for the expression of the gene of interest.
The vector of the present invention may be selected from various vectors known in the art. For example, a commercially available vector is selected, and then the nucleotide sequence encoding the antibody of the present invention is operably linked to an expression control sequence to form an expression vector.
The invention also provides a host cell containing the expression vector, or the nucleic acid is integrated in the genome, or the host cell expresses the nano-antibody, the antibody or the bispecific antibody of the anti-CD 16A.
In some embodiments, the host cell is selected from at least one of a prokaryotic host cell, a eukaryotic host cell, and a phage.
In some embodiments, the eukaryotic host cell is an animal cell, a plant cell, or a fungus, wherein the animal cell comprises a mammalian cell.
In some embodiments, mammalian cells include 293 cells, 293T cells, 293FT cells, CHO cells, and Per6 cells, which are commonly used mammalian cells for the production of antibodies or recombinant proteins, as is well known to those of ordinary skill in the art.
The invention also provides a method for preparing the nano antibody, which comprises the following steps: culturing the above-described host cell under conditions suitable for antibody production, thereby obtaining a culture containing the anti-CD 16A antibody, and isolating or recovering the anti-CD 16A antibody from the culture. Specifically, the culture conditions for the host cells are not particularly limited in the present invention, and culture conditions capable of allowing the host cells to express and produce the antibody can be obtained based on conventional technical knowledge.
The invention also provides immunoconjugates or pharmaceutical compositions comprising the above-described anti-CD 16A nanobody, antibody or bispecific antibody.
In some embodiments, the immunoconjugate further comprises a therapeutic agent.
In some embodiments, the therapeutic agent comprises: at least one of a chemotherapeutic agent, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody to an inhibitory second signaling molecule, a PD-L1 inhibitor, and a PD-1/PD-L1 mab drug.
Relevant biomarkers for immune checkpoint inhibitor treatment include PD-L1, MSI/bMSI, TMB/bTMB, TNB, EGFR mutation, ALK fusion, TP53 mutation, KRAS mutation.
In some embodiments, the chemotherapeutic agent is selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, fraapine, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, darnusertib, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecine, and saquinavir.
The photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or a derivative thereof; (2) a photosensitive compound containing a tetrapyrrole ring; (3) a traditional Chinese medicine photosensitizer; or (4) ALA or a derivative thereof in combination with the compound of (2) or (3), respectively.
The photothermal agent is selected from the group consisting of IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, and IR-26.
The inhibitory second signal molecule may be PD-1; CTLA-4; PD-1 and CTLA-4.
In some embodiments, the PD-1/PD-L1 mab is selected from at least one of the following group: nivolumab (Nivolumab), pembrolizumab (Pembrolizumab), dermatitid (Pidilizumab), lanberrimab (Lambrolizumab), BMS-936559, atuzumab (Atezolizumab)、AMP-224、AMP224、AUNP12、BGB108、MCLA134、MEDI0680、PDROOl、REGN2810、SHR1210、STIAl lOX、STIAl l lO、TSR042,BMS-936558、BGB-A317、BCD-100, and JS001.
In some embodiments, the therapeutic agent further comprises a cytotoxic agent.
In some embodiments, the pharmaceutical composition comprises at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, and a pharmaceutically acceptable excipient.
Such pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (antibody or antigen binding portion thereof, conjugate/conjugate or bispecific molecule) may be coated in a substance to protect the active ingredient from acids and other natural conditions that may inactivate the active ingredient.
Pharmaceutical compositions must generally be sterile and stable under the conditions of manufacture and storage. The pharmaceutical compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof.
In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, fillers, lubricants, disintegrants, binders, glidants, and the like.
In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, one of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dibasic calcium phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, or a combination thereof.
The invention also provides application of the anti-CD 16A nano antibody, the bispecific antibody, the nucleic acid, the expression vector, the host cell or the immunoconjugate or the pharmaceutical composition in preparing products for preventing or treating tumors.
In some embodiments, the product comprises: at least one of immune cells, reagents, kits, medicaments and pharmaceutical compositions.
In some embodiments, the product is used for the prevention or treatment of at least one disease in acute B-lymphoblastic leukemia, non-hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, hairy cell leukemia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis.
In the present invention, the term "treating" includes curing, inhibiting or slowing the progression or progression of a disease, alleviating and/or stabilizing a disease state.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This example is the preparation of a CD16A recombinant protein, and is specifically as follows:
The purchased encoding human pCMV 3-tagged-CD 16A plasmid is used as a template for PCR amplification, the extracellular domain (ECD) fragment is obtained through agarose gel electrophoresis, and then the pcDNA3.1 vector is used as a framework, and the extracellular domain of the CD16A is cloned into a pcDNA3.1 expression vector with Fc or His tag at the C end. Wherein the Fc tag comprises human Fc (hFc) and murine Fc (mFc). Then, by transient transfection of 293FT, shake flask culture using FreeStyle TM serum free medium (Life Technologies) for 5-7 days, collecting supernatant, purifying the recombinant CD16A Protein carrying Fc or His tag by Protein A/G or nickel column affinity chromatography and molecular sieve chromatography column.
Example 2
This example is the construction of phage nanobody library, panning and ELISA primary screening, comprising the steps of:
(1) Bactrian camel immunity
Taking 2mg of the recombinant protein expressing the purified CD16A extracellular domain, adding 2mL of Freund's complete adjuvant into the recombinant protein, and fully emulsifying the recombinant protein by using an emulsifying instrument; the two-humped camel neck is subjected to subcutaneous multipoint injection immunization, then is immunized once every two weeks (2 mg of protein), and is immunized for 4 times by using Freund's incomplete adjuvant, and peripheral blood detection titers are collected after the last immunization. Bactrian camel peripheral blood was collected one week after the impact immunization to isolate lymphocytes.
(2) Nanobody library construction
And after the camel reaches a certain immune titer, performing final impact immunization on the camel, collecting 200mL of peripheral blood by using a blood collection bag after 7 days, and separating lymphocytes. The above-mentioned isolated lymphocytes were removed, and RNA extraction was performed according to the procedure of the RNA extraction kit of Promega. Immediately after RNA extraction of lymphocytes, cDNA was reverse transcribed using TaKaRa reverse transcription kit, followed by amplification of VHH gene using nested PCR; the amplified VHH gene was inserted into pMECS phage display vectors and TG1 competent cells were electrotransformed. And (3) taking the culture bacterial liquid after electrotransformation, performing double dilution (10 times dilution) by utilizing an LB/Amp-GLU culture medium, then taking 100 mu L of 10 -4、10-5、10-6、10-7 dilution liquid to be coated on an LB/Amp-GLU flat plate, performing inversion culture at 37 ℃, and counting colony numbers at different dilutions after culturing for 8 hours to calculate the library capacity of the antibody library, namely 6.56 multiplied by 10 9. Meanwhile, 50 colonies with similar morphology and size are randomly selected and cultured for 4-6 hours, and bacterial liquid PCR is carried out to identify the positive rate of the library, namely the insertion rate of the library reaches 97%.
(3) Screening for CD16A nanobodies
First, helper phage preparation, concentration, and phage library rescue were performed. Panning of nanobody phage library was performed as follows: ① Antigen coating: after diluting the CD16A-mFc recombinant protein with PBS, 20 mug (antigen coating amount of 10 mug/hole and 5 mug/hole of subsequent two rounds of panning respectively) of each hole is coated in a 96-hole ELISA plate, and the coating is carried out overnight at 4 ℃; ② washing: after overnight coating, the wells were discarded and each well was washed 5 times with 200 μl PBST; ③ Closing: 200 mu L of 5% skimmed milk powder is added into each hole, and the mixture is placed at 37 ℃ for sealing for 1 hour; ④ washing: the liquid in the wells was discarded and each well was washed 3 times with 200 μl PBST; ⑤ Incubation of recombinant phage: diluting recombinant phage to 5X 10 11 pfu/mL with 5% skimmed milk powder, adding 100 μl per well, and incubating at room temperature for 2h; ⑥ Washing: the liquid in the wells was discarded and each well was washed 15 times with 200. Mu.L of PBST. Adding 0.1M triethylamine prepared at present according to 100 mu L of each hole, standing at room temperature for 10min, sucking eluent into a 1.5mL centrifuge tube, and rapidly adding 1M Tris-HCl (pH=7.4) with equal volume for neutralization; ⑦ Recombinant phage titer assay: collecting the phage solution after neutralization, and measuring phage titer; Infecting 2mL of TG1 in logarithmic growth phase with the rest phage solution, and standing at 37 ℃ for 30min; 8mL of 2 XYT/Amp GLU medium is added and the culture is carried out at 220rpm at 37 ℃ until the logarithmic phase of growth; ⑧ rescue: 8mL of 2 XYT ampicillin-resistant medium was added, 4% glucose was added, and the mixture was incubated at 37℃and 220 rpm; ⑨ Concentrating phage; ⑩ Repeating the step ①-⑨, and performing a second and third screening and washing.
(4) Detection of enrichment of specific recombinant phages
Antigen coating: after dilution of both antigens with PBS, 400ng per well was coated in 96-well elisa plates at 4℃overnight. Washing: after overnight coating, the wells were discarded and each well was washed three times with 200 μl PBST. Closing: 200. Mu.L of 5% nonfat dry milk was added to each well and the wells were blocked at 37℃for 1 hour. Washing: the liquid in the wells was discarded and each well was washed three times with 200. Mu.L of PBST. Incubation of recombinant phage: phage concentrate (1:10) was diluted and 100. Mu.L per well was added and incubated for 1h at 37 ℃. Washing: the liquid in the wells was discarded and each well was washed three times with 200. Mu.L of PBST. And (2) secondary antibody: HRP-labeled murine anti-M13 secondary antibody 1:2000 was diluted, 100. Mu.L/well and incubated for 1h at 37 ℃. Washing: the liquid in the wells was discarded and each well was washed three times with 200. Mu.L of PBST. Color development: 100 mu L of TMB color development liquid is added into each hole, and the mixture is placed for 10 to 15 minutes at room temperature and in a dark place. Terminating and reading: after color development, 2M H 2SO4. Mu.L of each well was added to terminate the reaction; absorbance was read at 450 nm. The data is analyzed.
(5) Sequencing analysis of specific nanobodies
Clones with a value 3 times or more greater than the negative value were determined to be positive by ELISA detection, and were subjected to bacterial liquid sequencing and comparison analysis to finally obtain 1 CD16A nanobody sequence, designated Nb60 (# 1), the sequence of which is shown in Table 1 below.
TABLE 1 amino acid sequence of nanobodies
(6) Specificity analysis of anti-CD 16a protein nanobody
The recombinant supernatant of the nano antibody obtained by screening is primary antibody which is respectively incubated with CD16A recombinant protein and other irrelevant antigens (CD 16B, NKP30 and CD 22), and is detected by using HRP anti-M13 antibody, and the result is shown in figure 1, the obtained CD16A nano antibody can be combined with the CD16A recombinant protein and does not react with other irrelevant antigens, thus indicating that the nano antibody has good specific binding activity.
Example 3
The present example is an experiment for identifying the expression, purification and reactivity of a CD 16A-specific nanobody with an antigen, specifically as follows:
Recombinant nanobody expression platforms are constructed based on pcDNA3.1 eukaryotic expression vectors, and CD16A specific nanobody sequences are cloned to the pcDNA3.1 expression vectors respectively. And expressing and purifying the constructed expression vector by using an HEK293T eukaryotic protein expression system. SDS-PAGE results show that the recombinant nano antibody with higher purity is obtained after affinity chromatography purification, and the band sizes are all about 55 kDa.
To identify the reactivity of the recombinant nanobody with antigen, 200ng/well of CD16A recombinant protein was coated on an ELISA plate in advance, the plate was blocked after overnight at 4℃and different amounts of recombinant nanobody (dilution: 10 -6 -10. Mu.g/mL) were added, the reaction was washed, developed and stopped by adding a secondary antibody, and the binding capacity was determined using an optical density (OD 450) measurement at 450nm using an ELISA reader using a four-parameter nonlinear regression curve fitting.
The results in FIG. 2 show that the nanobody of CD16A can bind with higher specificity to the recombinant protein of CD 16A.
Example 4
The embodiment is a combination experiment for detecting the recombinant nanobody and CD16A + -Hela cells by flow cytometry, and the combination experiment is specifically as follows:
firstly, using slow virus containing CD16A full-length gene to infect Hela cells, obtaining high-purity CD16A + -Hela cells which stably express the CD16A gene through flow separation, incubating the recombinant nano antibody prepared in the example 3 with the CD16A + -Hela cells for 40min at 37 ℃, washing for 3 times by PBS, incubating with APC@coat anti-human secondary antibody, washing for 3 times by PBS, and detecting by a flow cytometer.
The results are shown in FIG. 3, which shows that recombinant nanobody Nb60 binds well to the cells.
Example 5
The present example is a CD16A nanobody affinity assay, specifically as follows:
Nanobodies of CD16A were validated for recombinant nanobody affinity by surface plasmon resonance and their binding kinetics constants (Kd) were determined. Using a GE Biacore TM K instrument, fixing an anti-mouse IgG antibody on the surface of a CM5 chip by using a coupling buffer solution in an amino coupling kit, and capturing CD16A (ECD) -mFc on the CM5 chip in 2-fold serial dilution; purified CD16A nanobody was then flowed over the surface of the chip and machine read Ka (1/Ms), KD (1/s), KD (M), i.e., the affinity of the recombinant nanobody was measured. The affinity measurement results show that the nano antibodies of the CD16A can specifically bind to the CD16A (ECD) -mFc protein, the affinity is on the order of 10 -9 M, the nano antibodies belong to high-affinity antibodies, and the specific data of detection are shown in Table 2.
Table 2 summary of affinity data for nanobodies
| Antibodies to | Ka(1/Ms) | kd(1/s) | KD(M) |
| Nb60 | 1.16E-03 | 7.33E+05 | 1.58E-09 |
Example 6
This example is the preparation of bispecific antibodies against CD22 and CD16A, specifically as follows:
The nanobody of the invention and the nanometer antibody of the CD22 molecule (named Nb513 (2 # in the embodiment), the sequence of which is shown as SEQ ID NO: 5) are constructed in series to a eukaryotic expression vector, HEK293T cells are transfected for expression, and Ni columns are used for purification. The construction is shown in fig. 4. After incubating NK cells and NALM-6-luciferases and Raji-luciferases, respectively, for 24 hours, the purified bispecific antibody was evaluated for killing activity by detecting fluorescence intensity.
The results are shown in FIG. 5, which shows that the killing ability of the bispecific antibody and NK cell group against tumor cells is significantly enhanced compared to the addition of NK cells alone. It was shown that bispecific antibodies can promote NK cell killing.
Example 7
The embodiment is an in vivo anti-tumor activity experiment for detecting bispecific antibody in B-ALL mouse transplantation model, and specifically comprises the following steps:
NALM-6 and Raji cells purchased from ATCC were prepared into cell lines NALM-6-mCherry. FfLuc and Raji-mCherry. FfLuc which stably expressed luciferases and inoculated into NCG mice of 6 to 8 weeks old through the tail vein in each of 2X 10 6 cells, respectively, and tumor growth was examined in a living body imager 10 days after inoculation and randomly divided into 8 groups of 5 cells each. On day 10 after tumor cell inoculation, PBS with the same volume as that of administration is set as a control group, the bispecific antibody is administered by tail vein, 30 μg/dose, 200 μl/dose of normal saline is injected into the tail vein of the control group, 3 times per week, and 1×10 7 NK cells are injected into the tail vein of the simple NK group 3 times per week; the CD22×CD16A group was treated continuously for 2 weeks with 1×10 7 NK cells/time, 3 times a week. The survival status of each mouse of the statistical experimental group (double antibody+nk), the control group (NK) and the blank group (PBS) was observed for a statistical time period of 80 days. Mice survival was plotted using the Kaplan-Meier method and the variability of survival for each group of mice was compared by log-rank (Mantel-Cox) test statistics.
The results are shown in fig. 6, where the bispecific antibody group survival was significantly longer than the negative control antibody and PBS group, indicating that the bispecific antibody effectively controlled proliferation and growth of B-ALL cells and significantly prolonged the mouse survival.
The experiments show that the anti-CD 16A nanobody obtained by screening can be specifically combined with a CD16A target, has high affinity, can be widely applied to the prevention and treatment of tumor or autoimmune diseases, and provides a new choice for the prevention or treatment of tumor or autoimmune diseases.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A nanobody against CD16A, wherein the heavy chain variable region of said nanobody comprises CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is as set forth in SEQ ID NO:1, the amino acid sequence of the CDR2 is shown as SEQ ID NO:2, the amino acid sequence of the CDR3 is shown as SEQ ID NO: 3.
2. The nanobody of claim 1, wherein the nanobody further comprises a framework region;
preferably, the nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific antibody, and a fusion nanobody;
preferably, when the nanobody is a monovalent nanobody, the amino acid sequence of the heavy chain variable region thereof is as set forth in SEQ ID NO: 4.
3. An antibody comprising the heavy chain variable region of the anti-CD 16A nanobody or anti-CD 16A nanobody of claim 1 or 2.
4. A bispecific antibody comprising the anti-CD 16A nanobody and the anti-CD 22 nanobody of claim 1 or 2;
preferably, the bispecific antibody sequentially links the anti-CD 22 nanobody and the anti-CD 16A nanobody through a linking peptide;
preferably, the linker peptide is (G4S) n, wherein n is a non-0 integer;
preferably, n is an integer between 1 and 20;
preferably, n is 3 or 4;
Preferably, when the anti-CD 22 nanobody is a monovalent nanobody, the amino acid sequence of the heavy chain variable region thereof is as shown in SEQ ID NO: shown at 5.
5. A nucleic acid encoding the anti-CD 16A nanobody of claim 1 or 2, the antibody of claim 3 or the bispecific antibody of claim 4.
6. An expression vector comprising the nucleic acid of claim 5;
preferably, the vectors include plasmids and viruses;
preferably, the virus is selected from any one of adenovirus, adeno-associated virus, retrovirus, lentivirus or oncolytic virus.
7. A host cell comprising the expression vector of claim 6, or having the nucleic acid of claim 5 integrated into the genome, or expressing the anti-CD 16A nanobody of claim 1 or 2, the antibody of claim 3, or the bispecific antibody of claim 4;
preferably, the host cell is selected from at least one of a prokaryotic host cell, a eukaryotic host cell, and a phage;
preferably, the eukaryotic host cell is an animal cell, including a mammalian cell, a plant cell, or a fungus;
preferably, the mammalian cells include 293 cells, 293T cells, 293FT cells, CHO cells and Per6 cells.
8. A method of producing nanobodies comprising: culturing the host cell of claim 7 under conditions suitable for antibody production, thereby obtaining a culture comprising said anti-CD 16A antibody, and isolating or recovering said anti-CD 16A antibody from said culture.
9. An immunoconjugate or pharmaceutical composition comprising the anti-CD 16A nanobody of claim 1 or 2, the antibody of claim 3 or the bispecific antibody of claim 4;
preferably, a therapeutic agent is also included in the immunoconjugate;
Preferably, the therapeutic agent comprises at least one of a chemotherapeutic drug, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody to an inhibitory second signaling molecule, a PD-L1 inhibitor, and a PD-1/PD-L1 mab drug;
preferably, the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable excipient, carrier and diluent.
10. Use of the nanobody of anti-CD 16A of claim 1 or 2, the antibody of claim 3, the bispecific antibody of claim 4, the nucleic acid of claim 5, the expression vector of claim 6, the host cell of claim 7 or the immunoconjugate or pharmaceutical composition of claim 9 for the preparation of a product for the prevention or treatment of a tumor;
preferably, the product comprises: at least one of immune cells, reagents, kits, medicaments and pharmaceutical compositions;
Preferably, the tumor is at least one of acute lymphoblastic leukemia including B cells, non-hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B cell lymphoma, hairy cell leukemia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200010547A1 (en) * | 2017-02-28 | 2020-01-09 | Affimed Gmbh | Combination of an anti-cd16a antibody with a cytokine |
| WO2023025215A1 (en) * | 2021-08-25 | 2023-03-02 | Nanjing GenScript Biotech Co., Ltd. | ANTIBODIES AND VARIANTS THEREOF AGAINST HUMAN CD16a |
| CN115960234A (en) * | 2022-10-27 | 2023-04-14 | 合肥天港免疫药物有限公司 | anti-CD 16A antibodies and uses thereof |
| WO2023078968A1 (en) * | 2021-11-03 | 2023-05-11 | Affimed Gmbh | Bispecific cd16a binders |
| WO2023079493A1 (en) * | 2021-11-03 | 2023-05-11 | Affimed Gmbh | Bispecific cd16a binders |
| CN116554326A (en) * | 2023-04-19 | 2023-08-08 | 四川大学 | Preparation and application of targeted CD22 allogeneic universal CAR-T cell |
| CN116745324A (en) * | 2021-01-27 | 2023-09-12 | 信达生物制药(苏州)有限公司 | Single domain antibodies against CD16A and uses thereof |
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- 2024-04-29 CN CN202410527965.XA patent/CN118221818A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200010547A1 (en) * | 2017-02-28 | 2020-01-09 | Affimed Gmbh | Combination of an anti-cd16a antibody with a cytokine |
| CN116745324A (en) * | 2021-01-27 | 2023-09-12 | 信达生物制药(苏州)有限公司 | Single domain antibodies against CD16A and uses thereof |
| WO2023025215A1 (en) * | 2021-08-25 | 2023-03-02 | Nanjing GenScript Biotech Co., Ltd. | ANTIBODIES AND VARIANTS THEREOF AGAINST HUMAN CD16a |
| WO2023078968A1 (en) * | 2021-11-03 | 2023-05-11 | Affimed Gmbh | Bispecific cd16a binders |
| WO2023079493A1 (en) * | 2021-11-03 | 2023-05-11 | Affimed Gmbh | Bispecific cd16a binders |
| CN115960234A (en) * | 2022-10-27 | 2023-04-14 | 合肥天港免疫药物有限公司 | anti-CD 16A antibodies and uses thereof |
| CN116554326A (en) * | 2023-04-19 | 2023-08-08 | 四川大学 | Preparation and application of targeted CD22 allogeneic universal CAR-T cell |
Non-Patent Citations (2)
| Title |
|---|
| SHAHRYAR KHOSHTINAT NIKKHOI等: "Bispecific killer cell engager with high affinity and specificity toward CD16a on NK cells for cancer immunotherapy.", FRONT.IMMUNOL., vol. 13, 6 January 2023 (2023-01-06) * |
| YINING ZHAO等: "Identification of anti-CD16A single domain antibodies and their application in bispecific antibodies.", CANCER BIOLOGY & THERAPY, vol. 21, no. 1, 29 September 2019 (2019-09-29) * |
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