Nanobodies against nkg2d and methods of making and using the same

Abstract

The application discloses a nano antibody against NKG2D and a preparation method and application thereof, and relates to the technical field of antibodies.The nano antibody or antigen binding fragment thereof has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.The nano antibody or antigen binding fragment thereof has good affinity and binding properties for NKD2G protein, has strong tissue penetration capacity, can effectively enhance NK cell activation and infiltration, and reverses an immunosuppressive microenvironment.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to anti-NKG2D nanobodies, their preparation methods, and applications. Background Technology

[0002] Natural killer (NK) cells are considered important effector cells in solid tumor immunotherapy due to their killing mechanism independent of the major histocompatibility complex (MHC) and low risk of cytokine release. The NK cell surface activation receptor NKG2D is a key molecule for initiating its killing function; however, NKG2D signaling is often suppressed in the tumor microenvironment, leading to NK cell inactivation. Currently, immune cell adaptors are the mainstream strategy for activating immune cells at tumor sites. Therefore, there is a need to provide a nanobody that can target NKG2D. Summary of the Invention

[0003] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a nanobody targeting NKG2D or its antigen-binding fragment.

[0004] The present invention also provides a bispecific antibody or a multispecific antibody.

[0005] The present invention also provides biomaterials related to the above-mentioned nanobodies or their antigen-binding fragments, or bispecific antibodies or multispecific antibodies.

[0006] The present invention also provides a coupling agent.

[0007] The present invention also provides applications of the above-mentioned nanobody or its antigen-binding fragment, the above-mentioned bispecific antibody or multispecific antibody, the above-mentioned biomaterial or the above-mentioned conjugate.

[0008] The present invention also provides a product comprising the above-mentioned nanobody or its antigen-binding fragment, the above-mentioned bispecific antibody or multispecific antibody, the above-mentioned biomaterial or the above-mentioned conjugate.

[0009] The present invention also provides a method for preparing the above-mentioned nanobody or its antigen-binding fragment, or bispecific antibody or multispecific antibody.

[0010] According to a first aspect of the present invention, an anti-NKG2D nanobody or antigen-binding fragment thereof has three complementary determinant clusters CDR1, CDR2 and CDR3; The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0011] The nanobodies or antigen-binding fragments thereof according to embodiments of the present invention have at least the following beneficial effects: The nanobodies or their antigen-binding fragments in this embodiment exhibit excellent affinity and binding properties to the NKD2G protein, strong tissue penetration, and can effectively enhance NK cell activation and infiltration, reverse the immunosuppressive microenvironment, and serve as a module for constructing multispecific antibody targeting, expanding synergistic anti-tumor strategies, and improving tumor efficacy when combined with existing immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors). Furthermore, they can be efficiently expressed in prokaryotic expression systems such as *E. coli*, eliminating the need for complex eukaryotic cell culture and resulting in low production costs.

[0012] According to some embodiments of the present invention, the nanobody or its antigen-binding fragment is as follows: A1) or A2). A1) A protein with the amino acid sequence shown in SEQ ID NO: 5; A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 5.

[0013] According to some embodiments of the present invention, the protein tag refers to a polypeptide or protein fused together with the target protein for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a His tag, Flag tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, the Fc fragment of immunoglobulin G, etc. The nanobody or its antigen-binding fragment may contain one or more protein tags; multiple protein tags may be a combination of multiple identical protein tags or a combination of multiple different protein tags.

[0014] A bispecific antibody or multispecific antibody according to a second aspect of the present invention comprises the nanobody or its antigen-binding fragment described in the first aspect embodiment.

[0015] According to some embodiments of the present invention, the bispecific antibody further includes a nanobody or its antigen-binding fragment that specifically binds to a membrane surface protein highly expressed by cancer cells.

[0016] According to some embodiments of the present invention, the multispecific antibody further includes two or more nanobodies or antigen-binding fragments thereof that specifically bind to membrane surface proteins highly expressed by cancer cells.

[0017] According to some embodiments of the present invention, the cancer cells specifically highly express membrane surface proteins including at least one of CEACAM5, PD-L1, HER2, MUC1, CDH17, Claudin18.2, B7H3, EGFR, and GRP78.

[0018] According to some embodiments of the present invention, the nanobody or its antigen-binding fragment described in the first aspect embodiment is linked to the nanobody or its antigen-binding fragment that specifically binds to the membrane surface protein highly expressed by cancer cells via a linker peptide.

[0019] According to some embodiments of the present invention, the linker peptide is a flexible linker peptide.

[0020] According to some embodiments of the present invention, the flexible linker peptide is a peptide linker comprising at least one of glycine, serine, proline, and lysine residues. The peptide linker consists of 1-40 amino acids. For example, the flexible linker peptide includes, but is not limited to, at least one of (G)n, (S)n, (GxS)n, (SxG)n, (GSSGG)n, (GGSGG)n, (GSGGSG)n, (GSGSGS)n, and (GGQGG)n. Wherein: n is any integer between 1 and 10; x is any integer between 1 and 6.

[0021] According to a third aspect of the present invention, the biomaterial is any one of B1) to B5) related to the nanobody or antigen-binding fragment thereof described in the first aspect embodiment, or the bispecific antibody or multispecific antibody described in the second aspect embodiment; B1) A nucleic acid molecule encoding the nanobody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody; B2) An expression cassette containing the nucleic acid molecules described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), the expression cassettes described in B2), or the recombinant vectors described in B3); B5) A transgenic cell line containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0022] According to some embodiments of the present invention, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0023] According to some embodiments of the present invention, the nucleic acid molecule is a DNA molecule encoding the nanobody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody.

[0024] According to some embodiments of the present invention, the nucleic acid molecule is any one of C1) to C2): C1) A DNA molecule with a nucleic acid sequence as shown in SEQ ID NO: 6; The DNA sequence defined in C2) has at least 80% homology with the DNA sequence defined in C1) and encodes the DNA molecule of the nanobody or its antigen-binding fragment.

[0025] Those skilled in the art can mutate nucleotide sequences using known methods (such as directed evolution and point mutation). Nucleotide sequences that are artificially modified and have a certain degree of homology with the nanobodies or antigen-binding fragments described in this invention, as long as they encode the nanobodies or their antigen-binding fragments or variants and have the same function as the nanobodies or their antigen-binding fragments, are all derived from and equivalent to the nucleotide sequences of this invention. In this document, the term "homology" refers to the similarity between amino acid sequences or nucleotide sequences. Homology can be evaluated using computer software. The at least 80% homology can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0026] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the nanobody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody, in host cells. This DNA may include not only a promoter to initiate transcription of the DNA molecule encoding it, but also a terminator to terminate transcription of the DNA molecule encoding it. Furthermore, the expression cassette may also include a replication initiation site, a transcription initiation sequence, an enhancer sequence, a selection element, or a reporter gene.

[0027] According to some embodiments of the present invention, the recombinant vector may be a plasmid, bacteriophage, lentivirus, adenovirus, or adeno-associated virus. The recombinant vector is used to achieve the replication, integration, amplification, and / or expression of the exogenous target gene in recipient cells; it may be a cloning vector or an expression vector.

[0028] According to some embodiments of the present invention, the recombinant microorganism may be bacteria (such as Escherichia coli or Bacillus subtilis) or fungi (such as yeast or Aspergillus). For example, the recombinant microorganism in B4 may specifically be Escherichia coli.

[0029] According to some embodiments of the present invention, the transgenic cell line may be insect cells (such as S2 Drosophila cells or Sf9 cells), animal cell lines (such as 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells), or plant cells (such as Arabidopsis thaliana or tobacco). All transgenic cell lines may be non-reproductive materials.

[0030] A conjugate according to a fourth aspect of the present invention comprises: the nanobody or its antigen-binding fragment described in the first aspect embodiment, or the bispecific antibody or multispecific antibody described in the second aspect embodiment; And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a radionuclide, and an enzyme.

[0031] According to some embodiments of the present invention, the detectable marker is selected from at least one of radioactive isotopes, fluorescent substances, chemiluminescent substances, and colored substances.

[0032] The use of the nanobody or antigen-binding fragment thereof described in the first aspect embodiment of the fifth aspect embodiment of the present invention, the bispecific antibody or multispecific antibody described in the second aspect embodiment, the biomaterial described in the third aspect embodiment, or the conjugate described in the fourth aspect embodiment in any one of D1) to D3); D1) Preparation of drugs for treating tumors; D2) Prepare products for detecting NKG2D; D3) to prepare products combined with NKG2D.

[0033] According to some embodiments of the present invention, the amino acid sequence of the NKG2D is shown in SEQ ID NO: 1.

[0034] According to some embodiments of the present invention, the product includes at least one of reagents, reagent kits, chips, and test strips.

[0035] A kit according to a sixth aspect of the present invention includes a nanobody or its antigen-binding fragment as described in the first aspect embodiment, a bispecific antibody or multispecific antibody as described in the second aspect embodiment, a biomaterial as described in the third aspect embodiment, or a conjugate as described in the fourth aspect embodiment.

[0036] The method for preparing the nanobody or its antigen-binding fragment as described in the first aspect embodiment of the seventh aspect embodiment of the present invention, or the bispecific antibody or multispecific antibody as described in the second aspect embodiment, includes the following steps: The encoding gene of the nanobody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody, is introduced into a biological cell to express the encoding gene.

[0037] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0038] Figure 1The enrichment factor of the phage display library during the three rounds of biological screening.

[0039] Figure 2 The purification and binding activity verification of nanobody E3 are shown in Figure A: SDS-PAGE analysis of purified nanobody E3; Figure B: ELISA results of binding activity of nanobody E3 to mNKG2D protein.

[0040] Figure 3 The following data were used to construct, purify, and validate the binding activity of the bispecific nanobody E3-B12. A: SDS-PAGE analysis results of the purified bispecific nanobody E3-B12 and the irrelevant control (Control); B: Western blotting results of the bispecific nanobody E3-B12 and the irrelevant control (Control); C: ELISA results of the binding activity of the bispecific nanobody E3-B12 to CEACAM5 protein; D: ELISA results of the binding of the bispecific nanobody E3-B12 to mNKG2D protein.

[0041] Figure 4 The results show the in vitro binding evaluation of the bispecific nanobody E3-B12. A: Binding of E3-B12 to PC3-OE cells (left: fluorescence image; right: fluorescence intensity quantification); B: Binding of E3-B12 to HT29 cells (left: fluorescence image; right: fluorescence intensity quantification); C: Binding of E3-B12 to MKN45 cells (left: fluorescence image; right: fluorescence intensity quantification); D: Binding of E3-B12 to CHO-NKG2D cells (left: fluorescence image; right: fluorescence intensity quantification); E: Binding of E3-B12 to CHO cells (left: fluorescence image; right: fluorescence intensity quantification).

[0042] Figure 5 In vitro cytotoxicity analysis of the bispecific nanobody E3-B12. A: Killing rate of NK cells against PC3-OE cells under different concentrations of E3-B12 treatment; B: Comparison of E3-B12-mediated cytotoxicity under different effector-to-target ratios (1:1, 1:5, 1:10); C: Detection of granzyme B release in co-culture supernatant after E3-B12 treatment under different effector-to-target ratios (1:1, 1:5, 1:10). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no significant difference.

[0043] Figure 6To evaluate the antitumor activity of the bispecific nanobody E3-B12 in various CEACAM5-positive cell line subcutaneous tumor models. A: Schematic diagram of in vivo administration regimens; B: Tumor volume growth curves of each group in the PC3-OE subcutaneous tumor model; C: Survival curves of mice in each treatment group corresponding to B; D: Tumor volume growth curves of each group in the HT29 subcutaneous tumor model; E: Survival curves of mice in each treatment group corresponding to D; F: Tumor volume growth curves of each group in the MKN45 subcutaneous tumor model; G: Survival curves of mice in each treatment group corresponding to F. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no significant difference.

[0044] Figure 7 To evaluate the antitumor activity of the bispecific nanobody E3-B12 in various patient-derived xenograft models (PDX models) with high CEACAM5 positivity. A. Schematic diagram of dosing schedule; B: Tumor volume growth curves of each group in the prostate cancer PDX model; C: Survival curves of mice in each treatment group corresponding to B; D: Tumor volume growth curves of each group in the colorectal cancer PDX-80 model; E: Survival curves of mice in each treatment group corresponding to D; F: Tumor volume growth curves of each group in the colorectal cancer PDX-1425 model; G: Survival curves of mice in each treatment group corresponding to F. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no significant difference. Detailed Implementation

[0045] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0046] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0047] Unless otherwise specified, "room temperature" in this application means (25±5)℃.

[0048] Unless otherwise specified, "amino acid" in this application refers to the basic unit that constitutes a protein, giving the protein a specific molecular structure and morphology, and enabling its molecules to possess biochemical activity. For example, the "amino acid" used in this invention includes the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0049] Unless otherwise specified, the amino acid sequences in this application are from the N-terminus to the C-terminus; the nucleic acid sequences are from the 5' end to the 3' end.

[0050] Unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0051] In the following examples, some experimental materials and reagents are described below: The amino acid sequence of mouse-derived NKG2D (mNKG2D) is NKEVPVSSREGYCGPCPNNWICHRNNCYQFFNEEKTWNQSQASCLSQNSSLLKIYSKEEQDFLKLVKSYHWMGLVQIPANGSWQWEDGSSLSYNQLTLVEIPKGSCAVYGSSFKAYTEDCANLNTYICMKRAV (SEQ ID NO: 1). It was artificially prepared by a biotechnology company.

[0052] Example 1 1. Nanobody screening: The immunotube method was used to screen natural alpaca-derived phage-displaying nanobody libraries. The selected phage display libraries had a capacity of 2 × 10⁻⁶. 9 The screening steps are as follows: 1) The target protein mNKG2D was coated onto an immunoassay tube at a concentration of 100 μg / mL and subjected to three rounds of enrichment screening.

[0053] like Figure 1 As shown, the nanobody library was enriched by approximately 25-fold after three rounds of screening.

[0054] 2) Using the third round of phage elution buffer for plating, 192 single clones were randomly selected for ELISA validation. A positive criterion was defined as an ELISA reading greater than twice the corresponding BSA reading. A total of 48 potential positive clones were identified.

[0055] 3) Positive monoclonal antibodies that have undergone two phage ELISA tests will be sent to the company for sequencing to determine their sequence information; 4) Design and synthesize the screened nanobodies based on sequencing information, and then express and purify them using E. coli; 5) The affinity of nanobodies was initially identified using ELISA affinity assays, and nanobodies with better affinity, such as E3, were selected for further research.

[0056] The amino acid sequences of CDR1, CDR2, and CDR3 of nanobody E3 are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. The amino acid sequence of nanobody E3 is shown in SEQ ID NO: 5.

[0057] GSTFRIRTIS (SEQ ID NO: 2).

[0058] SITAGGSTN (SEQ ID NO: 3).

[0059] SGLFLR (SEQ ID NO: 4).

[0060] MAVQLVESGGGLVRAGGSLRLSCTAP GSTFRIRTIS WYRQAPEKQGELVA SITAGGSTN YADSVKGRFTISRDNANNTVYLQMNSLKPEDTAVYYC SGLFLR RGQGTQVTVSS (SEQ ID NO: 5); The underscore marker sequences are CDR1, CDR2, and CDR3, respectively.

[0061] 2. Purification and expression of nanobodies: The positive nanobody E3 gene sequence obtained from screening (as shown in SEQ ID NO: 6) and the irrelevant control nanobody Control (the mCherry-targeting nanobody LAM4, amino acid sequence shown in SEQ ID NO: 7, and nucleic acid sequence encoding it shown in SEQ ID NO: 8) were cloned into the pET-15b vector and fused with an HA tag for subsequent detection. The expression vectors pET-15b / E3 and pET-15b / LAM4 were obtained, respectively.

[0062] ATGGCGGTGCAATTAGTTGAGAGTGGCGGCGGTCTGGTGCGCGCGGGTGGTAGCCTGCGCCTGAGTTGCACCGCGCCGGGCAGCACCTTTCGCATTCGCACCATTAGCTGGTATCGCCAAGCGCCGGAAAAACAAGGCGAACTGGTGGCGAGCATTACCGCGGGTGGCAGTACCAACTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAACAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCAGCGGCCTGTTTCTGCGCCGCGGCCAAGGCACCCAAGTGACCGTGAGCAGC(SEQ ID NO: 6)。

[0063] MAQVQLVESGGSLVQPGGSLRLSCAASGRFAESSSMGWFRQAPGKEREFVAAISWSGGATNYADSAKGRFTLSRDNTKNTVYLQMNSLKPDDTAVYYCAANLGNYISSNQRLYGYWGQGTQVTVSS(SEQ ID NO: 7)。

[0064] ATGGCACAAGTTCAATTAGTAGAAAGTGGAGGATCGTTAGTACAACCGGGGGGATCATTAAGGTTAAGTTGCGCAGCATCAGGAAGATTCGCAGAAAGCAGTTCAATGGGATGGTTTAGACAAGCACCAGGTAAAGAACGTGAGTTCGTCGCAGCGATTTCTTGGAGTGGGGGCGCCACAAACTACGCGGACTCCGCTAAGGGCCGCTTTACGTTATCGCGTGATAACACTAAAAATACCGTGTATCTGCAGATGAACAGCCTGAAACCGGATGACACCGCGGTTTATTACTGCGCCGCAAATTTGGGTAACTATATCTCAAGCAATCAGAGATTATACGGATATTGGGGACAAGGAACACAAGTTACAGTAAGCTCA(SEQ ID NO: 8)。

[0065] The constructed expression vectors pET-15b / E3 and pET-15b / LAM4 were transformed into E. coli DE3 competent cells, and positive clones were screened. Expression was induced at 16°C using 0.5 mM IPTG, and the cells were lysed using a high-pressure autoclave at 1000 W. The cells were then centrifuged at 17000×g at 4°C for 30 min, and the supernatant was incubated with Ni packing material at 4°C for 1 hour. A gradient wash with phosphate buffer containing 10 mM and 20 mM imidazole was used to remove contaminating proteins, and the target protein was eluted with a high-concentration 250 mM imidazole buffer. The purified product was separated by 15% SDS-PAGE and identified by Coomassie brilliant blue staining and Western blotting.

[0066] mNKG2D protein was coated onto an ELISA plate, blocked with 3% BSA, and then different concentrations of the target nanobodies were added. The plate was incubated at room temperature for 1 hour, followed by three washes with PBST. Anti-HA tag antibody (primary antibody) was then added and incubated at room temperature for 1 hour. After washing, horseradish peroxidase-labeled secondary antibody was added for signal amplification. Finally, TMB was used for color development to terminate the reaction, and the absorbance values ​​were read. An irrelevant control nanobodies (Control) were also included as a negative control.

[0067] like Figure 2 As shown, nanobody E3 was successfully purified with high purity and a molecular weight of approximately 15 kD. It was verified that it exhibits good binding activity to mNKG2D.

[0068] Example 2 Nanobody E3 was fused with HA-tagged CEACAM5-targeting nanobody B12 via a flexible linker to obtain the bispecific nanobody E3-B12 (amino acid sequence shown in SEQ ID NO: 9, nucleic acid sequence encoding it shown in SEQ ID NO: 10). The encoding gene of the bispecific nanobody E3-B12 was cloned into the vector pET-15 to construct the bispecific nanobody expression vector pET-15b-B12-NKG2D-Nb. Simultaneously, the expression vector pET-15b-LAM4-LAM4 was constructed for the unrelated control bispecific nanobody Lam4-Lam4 (amino acid sequence shown in SEQ ID NO: 11, nucleic acid sequence encoding it shown in SEQ ID NO: 12).

[0069] MAVQLVESGGGLVRAGGSLRLSCTAPGSTFRIRTISWYRQAPEKQGELVASITAGGSTNYADSVKGRFTISRDNANNTVYLQMNSLKPEDTAVYYCSGLFLRRGQGTQVTVSSGAYPYDVPDYAGGGGSGGGGSGGGGSMAVQLVESGGGLVQPGESLRLSCAASGVTFSTYGMGWARQVPGKGLEWVCGTYSDGSTYCADSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYCTAPKHEYGTNWYERTIYSNELDYWGQGTQVTVSSGAYPYDVPDYAS(SEQ ID NO: 9)。

[0070] ATGGCGGTGCAATTAGTTGAGAGTGGCGGCGGTCTGGTGCGCGCGGGTGGTAGCCTGCGCCTGAGTTGCACCGCGCCGGGCAGCACCTTTCGCATTCGCACCATTAGCTGGTATCGCCAAGCGCCGGAAAAACAAGGCGAACTGGTGGCGAGCATTACCGCGGGTGGCAGTACCAACTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAACAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCAGCGGCCTGTTTCTGCGCCGCGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGCGCGTATCCGTATGATGTGCCGGATTATGCGGGTGGCGGTGGCAGTGGTGGTGGCGGCAGCGGCGGTGGTGGCAGCATGGCGGTGCAGCTGGTGGAAAGCGGCGGTGGTTTAGTTCAGCCGGGCGAGAGCCTGCGTCTGAGCTGCGCGGCGAGCGGCGTGACCTTTAGCACCTATGGCATGGGCTGGGCGCGCCAAGTGCCGGGCAAAGGCCTGGAATGGGTGTGCGGCACCTATAGCGATGGCAGCACCTATTGCGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGACGAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCACCGCGCCGAAACATGAATATGGCACCAACTGGTATGAACGCACCATTTATAGCAACGAACTGGATTATTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGCGCGTATCCGTATGATGTGCCGGATTATGCGAGC(SEQ ID NO: 10)。

[0071] MAQVQLVESGGSLVQPGGSLRLSCAASGRFAESSSMGWFRQAPGKEREFVAAISWSGGATNYADSAKGRFTLSRDNTKNTVYLQMNSLKPDDTAVYYCAANLGNYISSNQRLYGYWGQGTQVTVSSGGGGSYPYDVPDYAGGGGSGGGGSGGGGSMAQVQLVESGGSLVQPGGSLRLSCAASGRFAESSSMGWFRQAPGKEREFVAAISWSGGATNYADSAKGRFTLSRDNTKNTVYLQMNSLKPDDTAVYYCAANLGNYISSNQRLYGYWGQGTQVTVSSGAYPYDVPDYAS(SEQID NO: 11)。

[0072] ATGGCACAAGTTCAATTAGTAGAAAGTGGAGGATCGTTAGTACAACCGGGGGGATCATTAAGGTTAAGTTGCGCAGCATCAGGAAGATTCGCAGAAAGCAGTTCAATGGGATGGTTTAGACAAGCACCAGGTAAAGAACGTGAGTTCGTCGCAGCGATTTCTTGGAGTGGGGGCGCCACAAACTACGCGGACTCCGCTAAGGGCCGCTTTACGTTATCGCGTGATAACACTAAAAATACCGTGTATCTGCAGATGAACAGCCTGAAACCGGATGACACCGCGGTTTATTACTGCGCCGCAAATTTGGGTAACTATATCTCAAGCAATCAGAGATTATACGGATATTGGGGACAAGGAACACAAGTTACAGTAAGCTCAGGGGGAGGTGGTTCATATCCGTACGACGTACCAGATTACGCAGGAGGGGGAGGAAGTGGGGGAGGGGGAAGTGGGGGTGGAGGGAGTATGGCACAAGTTCAATTAGTAGAAAGTGGGGGTTCATTAGTACAGCCGGGGGGGTCATTAAGATTATCATGTGCAGCGAGTGGGCGCTTTGCCGAATCATCCAGCATGGGTTGGTTCCGTCAAGCTCCGGGCAAAGAGCGGGAATTTGTCGCAGCGATCAGCTGGAGTGGGGGCGCCACTAATTACGCCGACTCCGCAAAAGGCCGTTTCACATTATCTCGCGATAACACCAAAAATACGGTTTATCTGCAGATGAACAGCTTGAAGCCAGACGATACCGCGGTGTATTACTGCGCGGCTAACCTGGGTAACTATATTTCTTCGAATCAGAGACTATACGGATATTGGGGGCAAGGGACACAAGTGACAGTTAGCAGTGGCGCGTATCCGTATGATGTGCCGGATTATGCGAGC(SEQ ID NO: 12)。

[0073] The recombinant plasmids were transformed into E. coli DE3 competent cells, and positive clones were screened. After IPTG induction, the bacterial cells were lysed using a high-pressure sterilizer at 1000 W; then centrifuged at 17000×g, 4℃ for 30 min, and the supernatant was incubated with Ni packing material at 4℃ for 1 hour; the cells were washed with a gradient of phosphate buffer containing 10 mM and 20 mM imidazole to remove impurities, and finally eluted with a high concentration of 250 mM imidazole buffer. The purified product was separated by 15% SDS-PAGE and identified by Coomassie brilliant blue staining and Western blotting.

[0074] like Figure 3 As shown in the figure. After expression and purification, SDS-PAGE results showed that the molecular weight of both the bispecific nanobody E3-B12 and the irrelevant control bispecific nanobody Lam4-Lam4 was approximately 30 kDa, indicating good purity. Western blotting (WB) using anti-HA antibody (11846S, CST) and anti-VHH antibody (A02361, GenScript) further confirmed the correct expression of the bispecific nanobody. ELISA results showed that the bispecific nanobody E3-B12 specifically bound to CEACAM5 and mNKG2D proteins, while no binding signal was detected in the irrelevant control bispecific nanobody.

[0075] Detection Example 1 CEACAM5-positive cells (PC3 cells overexpressing CEACAM5 (PC3-OE), HT29, and MKN45), CHO cells, and mNKG2D-overexpressing cells (CHO-NKG2D)) were fixed in 96-well plates with 4% paraformaldehyde and blocked with blocking buffer (3% BSA) for 1 hour. The nanobodies to be tested were then diluted with blocking buffer to prepare gradient working solutions (up to 400 nM) and incubated with the cells at room temperature for 1 hour. The cells were washed three times with PBST. Anti-HA-tagged fluorescent antibody (362607, BioLegend) was added and the cells were incubated at room temperature for 1 hour. The cells were washed three times with PBST. The binding of the nanobodies to the cell surface was detected using an automated electrophoresis fluorescence immunoassay analyzer.

[0076] like Figure 4 As shown.

[0077] The bispecific nanobody E3-B12 specifically recognized CEACAM5-positive cell lines PC3-OE, HT29, and MKN45, exhibiting excellent binding ability, while no binding signal was detected for the unrelated bispecific nanobody Control (LAM4-LAM4). Furthermore, the bispecific nanobody E3-B12 also showed high binding ability to CHO cells overexpressing NKG2D (CHO-NKG2D), but no significant binding signal was observed for parental CHO cells. These results indicate that the bispecific nanobody E3-B12 possesses specific in vitro binding ability to simultaneously target both CEACAM5 and NKG2D.

[0078] Detection Example 2 The in vitro cytotoxic activity of NK cells mediated by the bispecific nanobody E3-B12 against PC3-OE cells was detected using the lactate dehydrogenase (LDH) release assay. The effector-to-target ratio was set at 5:1. Purified NK cells (effect cells) and PC3-OE cells (target cells) were co-cultured in 96-well plates, with different concentration gradients of the bispecific nanobody E3-B12 and the irrelevant bispecific nanobody LAM4-LAM4 added simultaneously. After 4 hours of incubation, the supernatant was collected, and the LDH release level was measured. The following control wells were set up: High Control: containing only target cells, with lysis buffer added to completely lyse the target cells, and the maximum LDH release was measured; Low Control: containing only target cells, without lysis buffer, and the spontaneous LDH release (background toxicity) was measured; Background Control: containing only cell culture medium, without cells, to subtract the absorbance interference of the culture medium itself. Specific cytotoxicity was calculated using the following formula: Specific cytotoxicity = (OD value of experimental group - OD value of low control group) / (OD value of high control group - OD value of low control group) × 100%.

[0079] After determining the optimal concentration (1000 nM), the effects of different effector-target ratios (1:1, 5:1, 10:1) on cytotoxicity were further compared, and the co-culture supernatant was collected simultaneously to detect granzyme release levels in order to comprehensively evaluate the activation level of NK cells.

[0080] like Figure 5 As shown.

[0081] The bispecific nanobody E3-B12 dose-dependently enhanced the killing effect of NK cells on PC3-OE cells. Further comparisons under different effector-to-target ratios revealed that at an effector-to-target ratio of 1:10, E3-B12 induced a near 100% target cell killing rate. Furthermore, the release of granzyme B in the supernatant of the E3-B12-treated group was significantly increased.

[0082] Detection Example 3 Subcutaneous xenograft tumor models were established using CEACAM5-high expressing human tumor cell lines PC3-OE, HT29, and MKN45 to evaluate the in vivo antitumor activity of bispecific nanobodies. A schematic diagram of the in vivo dosing regimen is shown below. Figure 6 As shown in Figure A (NKG2D-Nb-B12 refers to the bispecific nanobody E3-B12).

[0083] Six-week-old CB17 immunodeficient mice (with preserved innate immune function) were selected and subcutaneously inoculated with 5 × 10⁶ mice. 6 PC3-OE, 2×10 6 One HT29 or 3×10 6 MKN45 cells were collected. Once the tumor volume reached approximately 50 mm³, the cells were randomly divided into three groups: a PBS control group, a CON-B12 nanobody control group (its amino acid sequence is shown in SEQ ID NO: 13), and a bispecific E3-B12 nanobody group (E3-B12), with 5–6 cells in each group. The antibody was administered once daily via tail vein at a dose of 150 μg / cell for 10 consecutive days. Tumor diameter was measured periodically, and tumor volume was calculated (tumor volume = (long diameter × short diameter)). 2 (2) Monitor tumor growth. The tumor volume should reach 1500 mm². 3 The endpoint is to record survival time and assess the survival benefit of treatment.

[0084] MAVQLVESGGGLVQPGESLRLSCAASGVTFSTYGMGWARQVPGKGLEWVCGTYSDGSTYCADSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYCTAPKHEYGTNWYERTIYSNELDYWGQGTQVTVSS (SEQ ID NO: 13).

[0085] like Figure 6 As shown.

[0086] In the PC3-OE subcutaneous tumor model, the E3-B12 treatment group showed a significant tumor growth inhibition effect compared with the PBS control group and the nanobody B12 control group; furthermore, the survival time of mice in the E3-B12 treatment group was significantly prolonged. In the HT29 and MKN45 subcutaneous tumor models, the E3-B12 treatment group also showed a clear trend of tumor growth inhibition, accompanied by a significant prolongation of mouse survival. In summary, E3-B12 has consistently demonstrated significant in vivo antitumor efficacy in multiple CEACAM5-positive tumor models.

[0087] Detection Example 4 A schematic diagram of the in vivo drug delivery regimen is shown below. Figure 7 As shown in Figure A (NKG2D-Nb-B12 refers to the bispecific nanobody E3-B12).

[0088] Tumor tissues from CEACAM5-positive tumor patients (CRPC PDX-159 prostate cancer tumor tissue and PDX-80 and PDX-1425 colon cancer tumor tissues; all samples used were ethically reviewed) were harvested and cut into 3×3×3 mm tissue blocks. These tissue blocks were subcutaneously inoculated into the right forelimb of CB17 immunodeficient mice (preserving innate immune function) for passage expansion. The tumors were allowed to grow to approximately 800–1000 mm² in volume. 3 At that time, tumor tissue was collected, recut into small tissue blocks, and then reinoculated into new-generation CB17 immunodeficient mice for expansion. The tumor volume to be transplanted reached approximately 50 mm. 3 Subsequently, animals were randomly divided into three groups: a PBS control group, a nanobody B12 control group (CON-B12), and a bispecific nanobody E3-B12 group (E3-B12), with 5-6 animals in each group. The antibody was administered once daily via tail vein at a dose of 150 μg per animal for 10 consecutive days, with tumor volume changes monitored periodically. The endpoint was defined as a tumor volume reaching 1500 mm³, and survival time was recorded to assess efficacy.

[0089] like Figure 7 As shown.

[0090] In a prostate cancer PDX model, the E3-B12 treatment group showed significant tumor growth inhibition and significantly prolonged the survival of tumor-bearing mice.

[0091] To verify the generalizability of the therapeutic effect, parallel experiments were conducted in two colorectal cancer PDX models (PDX-80 and PDX-1425) from different sources that highly expressed CEACAM5. The results showed that the bispecific nanobody E3-B12 effectively inhibited tumor growth in both colorectal cancer PDX models, and a significant prolongation of survival was consistently observed.

[0092] In summary, the bispecific nanobody E3-B12 demonstrated stable and significant antitumor activity in models that preserved patient tumor heterogeneity, further confirming its broad therapeutic potential against CEACAM5-positive solid tumors.

[0093] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A nanobody against NKG2D or its antigen-binding fragment, characterized in that, The nanobody or its antigen-binding fragment has three complementary determinant clusters CDR1, CDR2 and CDR3; The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The nanobody or its antigen-binding fragment is either A1) or A2) below. A1) A protein with the amino acid sequence shown in SEQ ID NO: 5; A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:

5.

3. A bispecific antibody or a multispecific antibody, characterized in that, It comprises the nanobody or its antigen-binding fragment as described in claim 1 or 2.

4. The bispecific antibody or multispecific antibody according to claim 3, characterized in that, The bispecific antibody further includes a nanobody or its antigen-binding fragment that targets and binds to a membrane surface protein specifically expressed by cancer cells; preferably, the membrane surface protein specifically expressed by cancer cells includes at least one of CEACAM5, PD-L1, HER2, and MUC1.

5. Biomaterials related to the nanobody or its antigen-binding fragment as described in claim 1 or 2, or the bispecific antibody or multispecific antibody as described in claim 3 or 4, characterized in that, The biomaterial is any one of B1) to B5); B1) A nucleic acid molecule encoding the nanobody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody; B2) An expression cassette containing the nucleic acid molecules described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), the expression cassettes described in B2), or the recombinant vectors described in B3); B5) A transgenic cell line containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

6. The biomaterial according to claim 5, characterized in that, The nucleic acid molecule is any one of C1) to C2): C1) A DNA molecule with a nucleic acid sequence as shown in SEQ ID NO: 6; The DNA sequence defined in C2) has at least 80% homology with the DNA sequence defined in C1) and encodes the DNA molecule of the nanobody or its antigen-binding fragment.

7. A coupling agent, characterized in that, This includes the nanobody or its antigen-binding fragment as described in claim 1 or 2, or the bispecific antibody or multispecific antibody as described in claim 3 or 4; And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a radionuclide, and an enzyme.

8. The use of the nanobody or antigen-binding fragment of claim 1 or 2, the bispecific antibody or multispecific antibody of claim 3 or 4, the biomaterial of claim 5 or 6, or the conjugate of claim 7 in any one of D1) to D3); D1) Preparation of drugs for treating tumors; D2) Prepare products for detecting NKG2D; D3) to prepare products combined with NKG2D.

9. A reagent kit, characterized in that, This includes the nanobody or its antigen-binding fragment as described in claim 1 or 2, the bispecific antibody or multispecific antibody as described in claim 3 or 4, the biomaterial as described in claim 5 or 6, or the conjugate as described in claim 7.

10. A method for preparing the nanobody or its antigen-binding fragment according to claim 1 or 2, or the bispecific antibody or multispecific antibody according to claim 3 or 4, characterized in that, Includes the following steps: The encoding gene of the nanobody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody, is introduced into a biological cell to express the encoding gene.

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