Anti-cdh3 nanobodies and uses

Abstract

The application belongs to the field of biological medicine, and relates to an anti-CDH3 nanobody and application. The anti-CDH3 nanobody comprises three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:5; the amino acid sequence of CDR2 is one of the sequences shown in SEQ ID NO:6 to SEQ ID NO:10; and the amino acid sequence of CDR3 is one of the sequences shown in SEQ ID NO:11 to SEQ ID NO:15. Compared with conventional antibodies, the anti-CDH3 nanobody of the application has high expression efficiency, simple purification, small molecular weight, high affinity, good specificity, high solubility, strong tolerance, and can be widely applied in the fields of scientific research, diagnosis and treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and in particular, relates to an anti-CDH3 nanobody and application. BACKGROUND

[0002] Immunotherapy based on specific targets in the mutual interaction between cancer cells and their microenvironment has become a research hotspot in tumor treatment. At present, immune checkpoint inhibitors including anti-CTLA-4, anti-PD-1, anti-PD-L1, and CAR-T cells have been approved for clinical use and have achieved good results. At the same time, more new target drugs are being developed. However, traditional monoclonal antibodies are difficult to cope with tumor heterogeneity and drug resistance due to their single structure and limited mechanism. Therefore, developing new antibody technologies with diverse structures and programmable functions is the key to breaking through the current research bottleneck.

[0003] Cadherin 3 (CDH3) is one of the genes encoding members of the classical cadherin family. It is encoded by a gene located at 16q22.1. The CDH3 promoter contains a CAAT box, two AP2 binding motifs, and a GC box of the putative Sp1 binding site. The transcription start site is located at 68, 678, 739 bp of the DNA forward strand, and the translation start site is located 553 bp downstream of the transcription start site. The P-cad protein encoded by CDH3 is a single-pass transmembrane glycoprotein with a molecular weight of 118 kDa. Its extracellular region contains 5 cadherin repeat sequences, which can mediate homophilic cis / trans interactions. The intracellular region is connected to the actin cytoskeleton through p120-catenin, forming the core structure of intercellular adhesion junctions.

[0004] CDH3 is highly expressed in placental basement membrane and then physiologically distributed in stratified epithelial tissues such as skin and breast duct epithelium, maintaining tissue polarity and barrier function. In normal adult tissues, the expression of CDH3 is usually limited, mainly existing in the epithelial layer of certain specific tissues such as breast, prostate, and skin. Its physiological function mainly reflects its participation in regulating cell movement, differentiation, and proliferation, thereby promoting tissue development and homeostasis maintenance. However, under pathological conditions, CDH3 is abnormally expressed in various malignant tumors and solid tumors, especially in non-small cell lung cancer, breast cancer, ovarian cancer, bladder cancer, cholangiocarcinoma, pancreatic cancer, and thyroid cancer. In addition, high expression of CDH3 has been confirmed to be closely related to poor prognosis and short survival of patients.

[0005] Currently, the development of targeted drugs for CDH3 mainly focuses on antibody conjugate drugs (ADC) and bispecific antibodies, and some candidate drugs have been discontinued due to efficacy or safety problems. The preparation process of traditional monoclonal antibodies is relatively complex, the production cost is high, and the molecular weight is large, and the tissue penetration ability is poor. Nanobody is the smallest fragment that can bind to antigen known at present, only 1 / 10 of the size of monoclonal antibody, has the structural stability and binding activity comparable to the original heavy chain antibody. Compared with traditional antibodies, nanobodies have a variety of unique advantages, such as good tissue penetration ability, rapid clearance, easy production and modification, high stability, lower immunogenicity, etc., and are a very promising new type of antibody molecule.

[0006] There is no report and clinical application of nanobodies targeting CDH3, and there is an urgent need in the art to develop new specific nanobodies targeting CDH3. SUMMARY

[0007] DEFINITIONS

[0008] Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by the skilled person. Furthermore, all methods, steps, techniques and procedures not specifically detailed can be and have been carried out in a manner known per se, which will be understood by the skilled person, unless otherwise indicated.

[0009] Unless otherwise indicated, the terms "antibody" or "immunoglobulin" are used interchangeably herein, whether referring to heavy chain antibodies or to conventional four-chain antibodies, as a general term to include full-length antibodies, individual chains thereof, and all portions, domains or fragments thereof, including but not limited to antigen binding domains or fragments, such as VHH domains or VH / VL domains, respectively. Furthermore, the term "sequence" as used herein (e.g. in the terms "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is to be understood generally to include both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding said amino acid sequence, unless a more defined interpretation is required herein.

[0010] The technical problem to be solved by the present application is to provide a VHH chain of anti-CDH3 nanobody which can block the binding of CDH3 to its ligand, and further derivatives and applications thereof.

[0011] To achieve the above-mentioned object, the first aspect of the present application provides an anti-CDH3 nanobody comprising three complementarity determining regions CDR1, CDR2 and CDR3; wherein,

[0012] the amino acid sequence of CDR1 is a sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5;

[0013] the amino acid sequence of CDR2 is a sequence shown in any one of SEQ ID NO: 6 to SEQ ID NO: 10;

[0014] the amino acid sequence of CDR3 is a sequence shown in any one of SEQ ID NO: 11 to SEQ ID NO: 15.

[0015] The position of CDR in the sequence of antibody or nanobody can be determined by those skilled in the art using the prior art. Generally, the CDR can be identified by sequencing the DNA of the antibody or nanobody, and then analyzing the sequence thus obtained using a special database (for example, the international ImMunoGeneTics database or IMGT).

[0016] It is well known to those skilled in the art that the CDR of antibody can be defined by various methods in the art, and in the sequence provided in the present application, the CDR is according to IMGT mapping (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results).

[0017] According to a preferred embodiment of the present application, the anti-CDH3 nanobody has the CDR sequence characteristics of any one of the following:

[0018] (1) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 11;

[0019] (2) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 12;

[0020] (3) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 3, SEQ ID NO: 8 and SEQ ID NO: 13;

[0021] (4) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 4, SEQ ID NO: 9 and SEQ ID NO: 14;

[0022] (5) the amino acid sequences of CDR1, CDR2, CDR3 are respectively the sequences shown in SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15.

[0023] According to the present application, in addition to the above-mentioned complementarity determining regions, the anti-CDH3 nanobody further comprises four framework regions FR1, FR2, FR3 and FR4 arranged alternately with the three complementarity determining regions, wherein,

[0024] the amino acid sequence of FR1 is the sequence shown in one of SEQ ID NO: 16~SEQ ID NO: 20;

[0025] the amino acid sequence of FR2 is the sequence shown in one of SEQ ID NO: 21~SEQ ID NO: 25;

[0026] the amino acid sequence of FR3 is the sequence shown in one of SEQ ID NO: 26~SEQ ID NO: 30;

[0027] the amino acid sequence of FR4 is the sequence shown in one of SEQ ID NO: 31~SEQ ID NO: 32.

[0028] According to a preferred embodiment of the present application, the anti-CDH3 nanobody has the following FR sequence characteristics of any one of:

[0029] (a) the amino acid sequences of FR1, FR2, FR3, FR4 are respectively the sequences shown in SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31;

[0030] (b) the amino acid sequences of FR1, FR2, FR3, FR4 are respectively the sequences shown in SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID NO: 31;

[0031] (c) the amino acid sequences of FR1, FR2, FR3, FR4 are respectively the sequences shown in SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 31;

[0032] (d) the amino acid sequences of FR1, FR2, FR3, FR4 are respectively the sequences shown in SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 32;

[0033] (e) the amino acid sequences of FR1, FR2, FR3, FR4 are respectively SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31.

[0034] The present application includes all sequences satisfying the above sequence characteristics, and specifically preferably,

[0035] The nanobody comprises one or more of the following sequences:

[0036] (i) an amino acid sequence as shown in one of SEQ ID NO: 33~SEQ ID NO: 37;

[0037] (ii) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in one of SEQ ID NO: 33~SEQ ID NO: 37 and functionally equivalent; the meaning of "functionally equivalent" is capable of binding to CDH3 protein.

[0038] ph-2-D03:

[0039] QVKLEESEEGLVQAGGSLRLSCTGSGGIFNGKVVAWYRQAPGSQREAIARITGPGLGISYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVVNPNGLPAFASWGQGTQVTVSS (SEQ ID NO: 33).

[0040] ph-5-F06:

[0041] EVQLVESGGGLVQAGGSPTLSCAVSGSIFSISTMYWYRQAPGKQRELVAFISRGGTTSYADSVKGRCTISRDNAKNTTSLQIDSLKSEDTGIYYCVAVINQRVYWGQGTQVTVSS (SEQ ID NO: 34).

[0042] ph-4-C09:

[0043] QVKLEESGEGLVQAGGSLTLSCSAPKSVQIMGWYRQTPGKQRELVARFAPHGGHIIYGDSMKGRVTISRDVAEGAVNLQMHSLRPDDTGVYYCFVPSWGQDAWGQGTQVTVSS (SEQ ID NO: 35).

[0044] ph-12-B11:

[0045] AVQLVESGGGLVQAGGSLRLSCVASGFTFDDYAIGWFRQAPGKEREGVSCISGSDSSTWYKDSVEGRFTFSTDNAKNTVYLQMNSLKVEDTAVYYCAAHPDYQRKVCGTYDYGYGMHFRGKGTLVTVSS (SEQ ID NO: 36)

[0046] ph-12-G08:

[0047] QVKLEESGGGLVQAGGSLRLSCVASGSVFNIKSMGWYRQPLGKQRELVAFITSGGISKYADSVKGRFGISRDNAKKSVYLQMNALKPEDTAVYYCYVVERSVSWGQGTQVTVSS (SEQ ID NO: 37).

[0048] The second aspect of the present application provides a nucleic acid molecule encoding the anti-CDH3 Nanobody described above.

[0049] The third aspect of the present application provides a vector comprising the nucleic acid molecule of the second aspect described above.

[0050] The fourth aspect of the present application provides a host cell containing the vector of the third aspect described above or the nucleic acid molecule of the second aspect described above.

[0051] The host cell includes, but is not limited to, a bacterial cell, a fungal cell, an animal cell, or a plant cell.

[0052] The anti-CDH3 Nanobody of the present application can be obtained by the following method:

[0053] (1) immunizing a llama with human, mouse CDH3 protein to prepare a phage display library.

[0054] (2) affinity screening the phage display library with human, monkey, mouse CDH3 protein;

[0055] (3) ELISA, FACS to identify positive clones;

[0056] (4) expression and purification of CDH3 Nanobody.

[0057] The fifth aspect of the present application provides a method for engineering production of anti-CDH3 Nanobody, comprising the following steps:

[0058] (a) culturing the host cell as described in the fourth aspect of the present application under conditions suitable for production of the nanobody, thereby obtaining a culture comprising said anti-CDH3 nanobody; and

[0059] (b) isolating and / or recovering said anti-CDH3 nanobody from said culture; and optionally

[0060] (c) purifying and / or modifying the anti-CDH3 nanobody obtained in step (b).

[0061] In the present application, the modification includes various conventional nanobody modification methods, and the purpose of modification is, for example, easier purification, easier connection with other structures, fusion expression.

[0062] In some embodiments, the modification includes connecting a His tag sequence to the anti-CDH3 nanobody. The His tag sequence is a short sequence containing consecutive histidine residues, such as 6 His, 8 His, and 10 His, etc.

[0063] In some embodiments, the modification includes modifying Cys at the C-terminus or N-terminus of the anti-CDH3 nanobody.

[0064] The sixth aspect of the present application provides an antibody conjugate drug, comprising the anti-CDH3 nanobody and an effector, and optionally a linker; the linker is an intermediate structure connecting the nanobody and the effector, which can be optionally set according to the structural characteristics of the effector.

[0065] The effector of the present application can include at least one of a nuclide, a cytotoxic agent, a fluorescent group, an enzyme catalyzing the development of a substrate, a chemiluminescent reagent, and a nanoparticle-based label. The nuclide can be a diagnostic radionuclide or a therapeutic radionuclide, and the diagnostic radionuclide is preferably 11 C、 13 N、 14 C、 15 O、 18 F、 24 Na、 32 P、 33 P、 35 S、 42 K、 43 Sc、 44 Sc、 45 Ti、 47 Sc、 51 Cr、 51 Mn、 52 Mn、 52 Fe、 55 Co、 57 Co、 58m Co、 59 Fe、60 Cu, 61 Cu, 62 Cu, 63 Zn, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 75 Br, 76 Br, 77 Br, 77 As, 81m Kr, 82 Rb, 86 Y, 87 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 97 Ru, 99 Tc, 99m Tc, 99 Mo, 101m Rh, 103m Rh, 105 Pd, 105 Rh, 111 Ag, 111 ln, 117m Sn, 119 Sb, 123 I, 124 I, 125 I, 131 I, 133 Xe, 137 Cs, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 152 Tb, 153 Sm, 154-159 Gd, 161 Tb, 165 Dy, 166 Dy, 166 Ho, 169 Er, 169 Yb, 175 Yb, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 191m Pt, 193m Pt, 195m Pt, 194 lr, 197 Pt,198 Au、 199 Au、 201 Tl、 203 Pb, 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra、 224 Ra and 225 At least one of Ac; the therapeutic radionuclide is preferably... 32 P, 47 Sc、 51 Cr 57 Co、 58m Co、 60 Co、 61 Cu、 67 As、 89 Sr、 90 Y、 103 Pd, 103m Rh、 105 Rh、 106 Ru、 117m Sn、 119 Sb, 124 I, 125 I, 131 I, 131 Cs、 137 Cs、 149 Tb, 149 Pm, 161 Ho、 165 Dy、 177 Lu、 177 Yb、 186 Re、 188 Re、 192 Ir、 193m Pt, 195m Pt, 197 Pt, 199 Au、 201 Tl、 203 Pb, 211 As、 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 224 Ra、 225 Ac、 226 Th、 227 Th and 229at least one of the group consisting of DOTA, NOTA, NODA, NODAGA, DOTP, TETA, RESCA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A, NOTAM, CB-DO2A, Cyclen, SHNH, NOTA-AA, NETA, HETA, TRITA, DAR, DO3A, DO3AP, HYNIC, MAG2, MAS3, MAG3, TPEN, m-MeATE, AAZTA, DOTAGA, NOTAGA, DOTMA, DOTAM, BAPTA-AM, TRAP, CB-TE2A, Cyclam, DADT, CE-DTS, NS3, CP256, PCTA, PTSM, NODA-MPAA, HBED, THP, Macropa, isonitrile, porphyrin, polyamine, crown ether, dithiosemicarbazone, or oxime, and derivatives thereof. The bifunctional chelator can be covalently linked to the nanobody by various methods conventional in the art, which are not particularly limited in the present application.

[0066] The seventh aspect of the present application provides a pharmaceutical composition comprising the anti-CDH3 nanobody or the antibody-drug conjugate described above.

[0067] The eighth aspect of the present application provides at least one use of the anti-CDH3 nanobody, the antibody-drug conjugate, or the pharmaceutical composition described above:

[0068] (i) preparing a reagent for detecting a CDH3-related disease in a human; the reagent is preferably a kit for detecting a CDH3-related disease;

[0069] (ii) preparing a medicament for treating a CDH3-related disease;

[0070] (iii) detecting the expression level of CDH3 in a sample to be tested for a non-diagnostic purpose.

[0071] The anti-CDH3 nanobody described above can also be used for treating a CDH3-related disease.

[0072] The CDH3-related disease includes but is not limited to a tumor, and the "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer", "cancerous", "proliferative disorder" and "tumor" are not mutually exclusive when mentioned in the present application. In some embodiments, the tumor includes but is not limited to at least one of non-small cell lung cancer, breast cancer, ovarian cancer, bladder cancer, cholangiocarcinoma, pancreatic cancer and thyroid cancer. The CDH3-targeted nanobody can be used as an immune checkpoint inhibitor, as a cancer treatment drug alone, or in combination with other anticancer drugs, so the active components of the drug include the CDH3-targeted nanobody and optionally other anticancer drugs.

[0073] In the present application, the application scenarios for "non-diagnostic purposes" include but are not limited to, for example, in vitro detection of the presence or absence of antigens (proteins containing CDH3 extracellular region) in the laboratory; or as a positive antibody to screen other CDH3-targeted antibodies; or to detect whether there is competition between antibodies by competing with other CDH3-targeted antibodies, that is, whether the antigen epitopes are the same or similar, etc.

[0074] Compared with conventional antibodies, the anti-CDH3 nanobody of the present application has high expression efficiency, simple purification, small molecular weight (about 15 kDa), high affinity, good specificity, high solubility, strong tolerance, and can be widely used in scientific research, diagnosis and treatment fields. In addition, nanobodies are more likely to recognize antigens that cannot be captured by traditional antibodies, have better tissue penetration, can enter tumor tissues and pass through the blood-brain barrier, and can be developed as immune checkpoint inhibitors to provide solutions for tumor imaging and treatment.

[0075] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0076] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

[0077] Figure 1 Reduced SDS-PAGE detection gel electrophoresis map of recombinant protein

[0078] Figures 2-3 The amplification process of the VHH fragment of the llama yeast display library of 47-66# and 48-49# is shown respectively.

[0079] Figure 4 The QC results of the llama yeast display library are shown.

[0080] Figure 5The results of the FACS detection of the binding of the overexpressing cell lines to the target protein are shown.

[0081] Figures 6-10 The results of the ELISA detection of the binding of the candidate clones to the target protein are shown.

[0082] Figures 11-16 The results of the FACS detection of the binding of the candidate clones to the target protein are shown.

[0083] Figures 17-19 The results of the FACS detection of the binding of the first batch of candidate antibodies to the target protein are shown.

[0084] Figures 20-22 The results of the FACS detection of the binding of the second batch of candidate antibodies to the target protein are shown. DETAILED DESCRIPTION

[0085] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0086] Examples

[0087] 1. Antigen preparation

[0088] According to the amino acid sequence information of Human CDH3, Cyno CDH3 and Mouse CDH3 proteins (SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, respectively) in the uniprot database, the antigen-encoding nucleic acid sequence was synthesized by codon optimization according to the codon bias of mammals. The nucleic acid sequence was subcloned into the pCDNA3.4 vector with a HisTag added at the C-terminus to construct a eukaryotic expression vector. The three plasmids were transfected into 293F cells, respectively, and the supernatant was collected for nickel column purification of the target protein. SDS-PAGE was performed to detect the purity of the protein.

[0089] The purity of the recombinant protein was detected by SDS-PAGE, and the results are shown in Figure 1 Figure 1 wherein M is a Protein marker, Lane 1: Human CDH3(108-654)-His, Lane 2: Cyno CDH3(108-654)-His, Lane 3: Mouse CDH3(100-647)-His. The results show that the purity of Human CDH3, Cyno CDH3 and Mouse CDH3 proteins is >90%, which can be used for immunization and subsequent panning work. ​

[0090] 2. Detection of ELISA binding activity of recombinant protein antigen with positive antibody

[0091] Using sterile CBS to dilute CDH3-His recombinant protein to a final concentration of 1 μg / mL, take a new 96-well plate, add 100 μL per well, 4 ℃ coating overnight; remove the antigen coating solution, wash 3 times with PBST (containing 0.5% Tween); add 200 μL / well of 3% MPBS 37 ℃ blocking for 2 hours; after removing the blocking buffer, wash the plate 3 times with PBST; positive control antibody 194-g09 (expressed by Aikangde Biotechnology Co., Ltd.) is diluted with PBS to 10 μg / mL, 3-fold dilution of 7 points, add 100 μL / well to the enzyme-labeled plate, incubate at room temperature for 1 hour, and the control wells are PBS; remove the liquid in the wells, and wash 3 times with PBST; add secondary antibody HRP-ProteinA (1:10000 dilution), add 100 μL / well to the enzyme-labeled plate, incubate at room temperature for 1 hour; after removing the liquid in the wells, wash the plate 3 times with PBST; add 100 μL / well TMB color developing solution; incubate at room temperature for 15 minutes in the dark; add 50 μL / well of stop solution (2 M HCl); use a microplate reader to read the OD 450 values in the wells. The results are shown in Table 1 below:

[0092] Table 1

[0093]

[0094] The results show that the proteins of the three species have good binding activity with the positive control antibody, and can be used for subsequent experiments.

[0095] 3. Immunization of alpaca

[0096] Human CDH3-His recombinant protein and Mouse CDH3-His recombinant protein were used to immunize alpacas (Alpaca), with alpaca numbers 48-49# and 47-66#, respectively, with an immunization interval of 14 days. Peripheral blood was collected seven days after each immunization to monitor the titer of immune serum. The immunization schedule is shown in Table 2 below:

[0097] Table 2

[0098]

[0099] 4. Detection of immune titer

[0100] The immune titer detection steps include:

[0101] After immunization, 5 mL of peripheral blood was collected, and the centrifuge tube containing the blood sample was placed in a 37 °C incubator for 1 hour. Then the blood sample was transferred to 4 °C overnight. The centrifuge tube containing the blood sample was placed in a centrifuge, and centrifuged at 5000 rpm for 20 min. The upper serum was separated, and the serum was transferred to a new sterile centrifuge tube. The immune serum was collected. The target recombinant protein was diluted to a final concentration of 1 pg / mL using sterile CBS (carbonate buffer). A new 96-well enzyme-labeled plate was taken, and 100 pL / well of 4 °C coating overnight was added. The antigen coating liquid was removed, and PBST (containing 0.05% Tween 20) was used for washing 5 times. 200 pL / well of 3% MPBS was added, and 37 °C was blocked for 2 hours. After removing the blocking buffer, the plate was washed 5 times with PBST. 100 pL of gradient-diluted serum (100 pL / well) was added, and the control well was PBS. The liquid in the well was removed, and PBST was used for washing 5 times. 100 pL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) was added, and incubated at room temperature for 1 hour. After removing the liquid in the well, the plate was washed 5 times with PBST. 100 pL / well of TMB color developing liquid was added. Incubate at room temperature for 10-15 minutes in the dark. Add 50 pL / well of stop solution. The OD 450 value in the well was read using an enzyme-labeled instrument.

[0102] The immune titer detection results are shown in Tables 3-6, and the sample is tested in parallel for 2 groups.

[0103] Table 3

[0104]

[0105] Table 4

[0106]

[0107] Table 5

[0108]

[0109] Table 6

[0110]

[0111] The serum was separated from the immunized llama, and the dilution gradient was performed according to the table. ELISA detection was performed with the pre-coated 96-well plate. According to the ELISA detection results, the immune serum can bind to Human CDH3 and Mouse CDH3 recombinant proteins, and the OD value changes with the gradient dilution of the immune serum. The immune titer reaches 1:64K, which meets the standard of blood collection (OD450 is 3 times the negative hole PBS), and the blood collection library is arranged.

[0112] 5. Construction and screening of single-domain antibody display library targeting CDH3

[0113] 48-49# alpaca and 47-66# alpaca, the amplification process of the VHH fragment of the yeast display library is shown in Figure 2 and 3 : peripheral blood is collected to separate PBMC cells, and after RNA extraction, a cDNA library is prepared using a reverse transcription kit, Figure 2 (a) and Figure 3 (a) are electrophoretograms of RNA extracted from separated PBMC cells; 1 round of PCR is performed using single-domain antibody amplification primers, and the results of agarose gel electrophoresis of the PCR products are shown; about 1000 bp and 750 bp PCR bands are obtained in the first round of PCR, and the 750 bp fragment is recovered from the gel as a template for the second round of PCR; about 500 bp bands are obtained in the second round of PCR, which are VHH fragments Figure 2 (b) and Figure 3 (b), after the VHH fragments are connected with linearized phage display vectors (vector electrophoretograms are shown in Figure 2 (c) and Figure 3 (c)), SS320 is electrotransformed, and the phage library of 47-66# alpaca has a capacity of 1.34 10 9 ( Figure 2 (d), after dilution by 2 10 6 times, the plates are coated), and the yeast library of 48-49# alpaca has a capacity of 1.22 10 9 ( Figure 3 (d), after dilution by 2 10 6 times, the plates are coated). The specific steps are as follows:

[0114] 5.1 PBMC separation and VHH antibody fragment cloning

[0115] 5.1.1 100 mL of peripheral blood is collected, and lymphocyte separation medium is used to separate PBMC.

[0116] 5.1.2 Extraction of RNA, reverse transcription using PrimeScript™ II 1st Strand cDNA Synthesis Kit to prepare cDNA: prepare the following reaction mixture Mixl in 200 μL PCR: Oligo dT Primer (50 μM) 8 μL, dNTP Mixture (10 mM each) 8 μL, total RNA sample 20 μg, RNase-Free water to 80 μL; 65 °C for 5 min, then quickly cool on ice; prepare the following reaction in the above PCR tube: the above denatured reaction 80 μL, 5x PrimeScript II Buffer 32 μL, RNase Inhibitor (40 Μ / μL) 4 μL, PrimeScript II RTase (200 Μ / μL) 8 μL, RNase-Free water, 36 μL; mix well after blowing, 80 μL / tube, 42 °C for 1 hour in PCR instrument, 70 °C for 15 min, finally store the cDNA sample on ice or at -20 °C.

[0117] 5.1.3 Amplification of VHH fragments

[0118] (1) Prepare the first round of PCR reaction system (50 μL / tube): upstream primer (5 μM) 2 μL, downstream primer (10 μM) 1 μL, NuHi Power mix (2x) 25 μL, cDNA template, 2 μL, sterile water 20 μL, the upstream primer is combined with the signal peptide, and the downstream primer is combined with the CH2 region. After preparing the PCR reaction system, set the PCR instrument according to the following program: pre-denaturation 95 °C for 10 min, denaturation 95 °C for 15 s, annealing 55 °C for 30 s, extension 68 °C for 1 min, and finally extension 68 °C for 10 min.

[0119] (2) Agarose electrophoresis of PCR products: use 1% agarose for electrophoretic analysis of PCR products, and separate fragments with a molecular weight of about 750 bp. Use a gel recovery kit to recover the PCR products, and use NanoDrop to determine the concentration.

[0120] (3) Configuration of two-round PCR reaction system (50 μL / tube): 2nd F primer 2 μL, 2nd R primer 2 μL, NuHiPower mix (2x) 25 μL, one-round PCR recovery product 200 ng, sterile water to 50 μL, upstream primer binding in antibody FR1 region, downstream primer binding in antibody Hinge and FR4 region, enzyme cutting site Sfil. After the PCR reaction system is configured, the PCR instrument is set according to the following program: pre-denaturation 95 °C 10 min, denaturation 95 °C 15 s, annealing 55 °C 30 s, extension 68 °C 1 min, and finally extension 68 °C 10 min.

[0121] (4) Agarose electrophoresis analysis of two-round PCR product: 1% agarose is used for electrophoresis analysis of PCR product, and VHH fragment with a molecular weight of about 400 bp is separated; VHH PCR product is recovered using a gel recovery kit, and the concentration is measured using NanoDrop.

[0122] (5) PCR product precipitation: 200 μL of recovered two-round PCR product in each 1.5 mL centrifuge tube is added with 1 / 10 volume (20 μL) of 3 M sodium acetate, 1 μg / μL glycogen (Glycogen), mixed well by blowing and sucking, 880 μL of anhydrous ethanol is added, mixed well by inverting, and stored at -80 °C.

[0123] 5.2 Construction of phage display library

[0124] 5.2.1. Construction of single-domain antibody phage display vector

[0125] The pDisplay vector and the VHH PCR gel recovery product obtained above are respectively cut with Sfil, and the enzyme cutting system is as follows: pDisplay 140 μg, 10xCutSmart® Buffer 200 μL, Sfil 1200 U (60 μL), sterile water to 2000 μL, the above enzyme cutting system is divided into 100 μL / tube, and is placed in a PCR instrument for enzyme cutting at 50 °C overnight. The second-round PCR purification product 10 μg, 10xCutSmart® Buffer 20 μL, Sfil 360 U (18 μL), sterile water to 200 μL, the above enzyme cutting system is divided into 100 μL / tube, and is placed in a PCR instrument for enzyme cutting at 50 °C overnight. The pDisplay vector fragment is separated using a 1% agarose gel, and the 5000 bp vector fragment is cut and recovered. At the same time, the DNA fragment recovery kit is used to purify the PCR enzyme cutting product, and the concentration is measured using NanoDrop. The pDisplay vector and the VHH fragment cut with enzyme are connected using T4 ligase, and are connected at 16 °C overnight.

[0126] 5.2.2. Bacteriophage ligation product electrotransformation of E. coli

[0127] Prepare the electrotransformation cup, ligation product and electrotransformation competent cells, and place them on ice for precooling; take the pre-cooled library ligation product and add it to the electrotransformation competent cells, place them on ice for 1 min, add 300 μL of DNA / competent cell mixture to each electrotransformation cup, and place the electrotransformation cup on ice; perform electrotransformation at 2500 V, 5 ms; immediately after the end of the electric shock, resuspend the bacteria in SOC medium equilibrated to room temperature, and incubate them in a shaking incubator at 37°C for 1 h; take 15 mL of the bacterial solution and directly perform bacteriophage rescue, and the remaining 5 mL of the electrotransformation product is added to an equal volume of 50% glycerol, mixed uniformly, and stored at -80°C; additionally, take 20 μL of the bacterial solution, dilute it in 980 μL of 2YT medium, take 100 μL of the diluted product, dilute it again in 900 μL of 2YT medium, take 50 μL and uniformly spread it on an LB plate containing ampicillin, and incubate it at 37°C overnight; the next day, take out the plate, calculate the number of clones that can be produced by each ligation, and calculate the library capacity; at the same time, pick 20 single clones from the plate and inoculate them in 2YT medium containing ampicillin, and incubate them at 37°C for about 6-8 h, send the bacterial solution for sequencing (universal primer M13R), and calculate the diversity of the library.

[0128] 5.2.3 Preparation of bacteriophage library and bacteriophage precipitation

[0129] Dilute the electrotransformation product with 2YT to adjust the OD 600 to about 0.2, add ampicillin at a final concentration of 100 ug / mL, and incubate in a constant-temperature shaking incubator at 37°C, 225 rpm, until the OD 600 is 0.5; add M13KO7, shake well, and incubate at 37°C for 30 min, then incubate at 37°C, 225 rpm, for 1 h; the volume of M13KO7 = 10 x volume x OD 600 x 5 x 10 8 / M13KO7 titer; Centrifuge the bacterial culture at 6000 rpm for 10 min, resuspend in 2YT-AK medium, and incubate overnight at 25°C and 200 rpm; Centrifuge the bacterial culture at 10000 rpm for 15 min; Discard the precipitate, transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG / NaCl to the tube, mix well, and incubate at 4°C for 2 h; Centrifuge the precipitated phage supernatant at 10000 rpm and 4°C for 30 min; Discard the supernatant, resuspend the precipitate (phage) in 1 mL sterile PBS for each 50 mL centrifuge tube; Transfer the resuspended phage to a 1.5 mL EP tube, centrifuge at 12000 g and 4°C for 5 min; Transfer the supernatant to a new 1.5 mL EP tube. Add 250 μL of PEG / NaCl to each EP tube, mix well, and incubate at 4°C for 10 min. Centrifuge at 12000g for 10 min, discard the supernatant, and resuspend in 1 mL of PBS. Centrifuge at 12000g for 5 min, discard the precipitate, and transfer the supernatant to a new 1.5 mL EP tube. Centrifuge at 12000g for 5 min, and transfer the supernatant to a new 1.5 mL EP tube to obtain the original phage library. Add 10 μL of the precipitate to 90 μL of 2YT medium, and label this as 10. -1 Dilute 10 times to 10 in succession. -9 Take 10 -7 10 -8 10 -9 Three gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD. 600 ER2738 was 0.5, mixed well, and placed in a 37°C water bath for 10 minutes. 100 μL was spread onto one LB-AMP solid plate and incubated overnight at 37°C. The spots were counted the next day to determine the titer. Titer calculation: Plates with 30-300 spots were selected, and the average value of two plates was taken. The number of spots was multiplied by the dilution factor and then multiplied by 100 to obtain the titer.

[0130] Results: The yeast display library QC for alpacas was as follows: Figure 4 As shown: Randomly selected single clones were sequenced and analyzed. The peak-stacking stop codon sequences were removed. There were no empty or repetitive sequences, and the sequences showed great differences, indicating good library diversity.

[0131] 5.2.4 Selection of Phage Display Libraries

[0132] Recombinant proteins were panned, and then the phage display library was incubated with the recombinant proteins. The recombinant phages bound to the target antigen were eluted using TEA and amplified. After 3-4 rounds of panning, single clones were selected for sequencing.

[0133] Take approximately 6 × 10 11150 μL of Pfu Phage library was diluted to approximately 1 mL with 1% PBSA and added to an ELISA plate coated with the target protein. The plate was incubated at 4°C for 1 h. The bound phage was eluted with 600 μL of 1×TEA for 10 min. The eluted product was transferred to a pre-blocked EP tube and neutralized with 300 μL of Tris-HCl. 10 μL of the output product was added to 90 μL of 2YT medium (labeled as 100), and this was subsequently diluted 10-fold to 10⁻⁶. -2 Take 10 1 10 0 10 -1 10 -2 Four gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD. 600 ER2738 (10) with a value of 0.5 1 The undiluted product (20 μL) was directly added to ER2738, mixed well, and placed in a 37°C water bath for 10 min. Each 100 μL was spread onto an LB-AMP solid plate and incubated overnight at 37°C. The spots were counted the next day to determine the titer. Titer calculation: Plates with 30-300 spots were selected, and the average value of two plates was taken. The number of spots was multiplied by the dilution factor and then by the elution volume.

[0134] The selection results are as follows:

[0135] First round of selection:

[0136] Cell selection: CHO-K1-Human CDH3 cells / CHO-K1-Mouse CDH3 cells / CHO-K1-Cyno CDH3 cells were selected in 1 / 2 / 3 rounds respectively; negative screening cells: CHO-K1 cells.

[0137]

[0138] Second round of selection:

[0139] The recombinant protein of the target antigen was co-incubated with a single-domain antibody phage display library to enrich phages that specifically bind to the target antigen. Four enrichment cycles were performed using alternating Human-CDH3 and Mouse-CDH3 antigens. The input and output of each round were statistically analyzed, and the enrichment factor for each round was calculated.

[0140]

[0141] Third round of selection:

[0142]

[0143] Figure 5The third round of output products for cell panning; this FACS uses CHO-K1-CDH3 cell lines for detection; select clones with non-specific binding to CHO-K1 cells Pick G4, H4, F4, F11 for testing.

[0144] 5.2.5 Phage ELISA

[0145] Subdivide 2YT-Amp medium into 96-well deep well plates, 500 μL per well, pick single colonies from the output plate, 37°C, 225 rpm culture to OD 600 0.5; where E12 and F12 two holes do not pick clones, only put culture medium as blank control; G12 and H12 two holes do not pick clones, detect antigen and positive antibody binding as positive control; at the same time, coat the antigen with CBS to the ELISA plate, concentration 1 μg / mL, 100 μL / well, 37°C, coat for 2h; take another 96-well deep well plate, subdivide 2YT-A medium, 500 μL per well, use the gun to sequentially suck OD 600 0.5 bacteria liquid 10 μL to a newly subdivided 96-well plate, place at 37°C, 225 rpm culture overnight, which is the sample for sequencing bacteria liquid; to OD 600 0.5 bacteria liquid, add M13KO7, mix well and place at 37°C for 15 min; M13KO7 volume = 10 x volume x OD 600 x 5 x 10 8 / M13KO7 titer; place the bacteria liquid after the end of the infection on a shaker, 37°C, 225 rpm culture for 45 min; centrifuge the bacteria liquid in a centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend with 2YT-AK medium, 800 μL per well, reset on the shaker, 30°C, 210 rpm overnight culture; at the same time, ELISA plate is discarded antigen, washed with PBST three times, then blocked with 3% MPBS, 250 μL / well, 4°C overnight; and an additional blank plate is blocked as BLANK.

[0146] The next day, place the 96-well deep well plate in a centrifuge at 4000 rpm for 10 min; discard the milk in the ELISA plate and wash with 200 μL PBST four times; add 50 μL PBST to each well first, then add 50 μL of the centrifuged phage supernatant one by one, incubate at 4°C for 1 h; discard the supernatant and wash with PBST five times; dilute HRP-Anti M13 secondary antibody with PBST, 100 μL per well, incubate at 4°C for 45 min, then wash away the secondary antibody, wash with PBST five times, develop with TMB at room temperature for 10 min, stop with hydrochloric acid, read, and select clones with S / N ratio greater than 6, using the preserved bacteria liquid for testing.

[0147] Figure 6 : No.2 plate, the plate protein panning the output product of the 4th round; the ELISA adopts the coated CDH3 protein scheme, wherein A-D are parallel test groups, E, F are negative controls (PBS control), G, H are positive controls (immune serum 1:1K); pick human mouse CDH3 binding clones for testing. Figure 7 : No.3 plate, the plate protein panning the output product of the 3rd round; the ELISA adopts the coated CDH3 protein scheme, wherein A-D are parallel test groups, E, F are negative controls (PBS control), G, H are positive controls (immune serum 1:1K); no testing. Figure 8 : No.4 plate, the plate protein panning the output product of the 4th round; the ELISA adopts the coated CDH3 protein scheme, wherein A-D are parallel test groups, E, F are negative controls (PBS control), G, H are positive controls (immune serum 1:1K); pick human mouse CDH3 binding clones for testing; the positive rate of panning 4th round clones is low, increase the protein amount and re-screen. Figure 9 : No.5 plate, the plate protein panning the output product of the 3rd round; the ELISA adopts the coated CDH3 protein scheme, wherein A-D are parallel test groups, E, F are negative controls (PBS control), G, H are positive controls (immune serum 1:1K); pick human mouse CDH3 binding clones for testing. Figure 10 : No.6 plate, the plate protein panning the output product of the 4th round; the ELISA adopts the coated CDH3 protein scheme, wherein A-D are parallel test groups, E, F are negative controls (PBS control), G, H are positive controls (immune serum 1:1K); pick human mouse CDH3 binding clones for testing.

[0148] Take the bacterial liquid of the target clone for PCR amplification, enzyme digestion and connection to construct a eukaryotic expression vector, transiently transfect 293F cells, collect the antibody expression supernatant for FACS detection, and the transfection supernatant detection of candidate clones is as shown in Figures 11-16 According to the experimental data, it can be known that the clones ph-2-D3, ph-5-F06, ph-4-C09, ph-12-B11 and ph-12-G08 cross bind with CHO-K1-PLVX-Human-CDH3(Full)-puro, CHO-K1-PLVX-Mouse-CDH3(Full)-puro and CHO-K1-PLVX-CYNO-CDH3(Full) cells.

[0149] 6. Detection of binding of recombinant antibody to target protein by FACS

[0150] ph-2-D3, ph-5-F06, ph-4-C09, ph-12-B11, ph-12-G08 sequences were linked to human IgG1 Fc (containing hinge region) fragment (the sequence is shown in SEQ ID NO: 41) respectively, to construct recombinant VHH-Fc candidate antibodies. The CHO-K1, CHO-K1-CDH3 cell strains were recovered from liquid nitrogen, and the cell state was adjusted to the logarithmic growth phase; the cells were divided into several parts, and the number of cells in each part was 2 x 105cells; the candidate antibodies or transfection supernatant were incubated with target cells, and after fully mixing, they were incubated at room temperature for 1 hour; 800 x g centrifugation at room temperature for 3 minutes, remove the supernatant containing the antibody, and wash the cells with PBS 3 times; add secondary antibody PE anti human IgG (1:1000 dilution), mix well, and incubate at room temperature for 30 minutes; 800 x g centrifugation at room temperature for 3 minutes, remove the supernatant containing the secondary antibody, and wash the cells with PBS 3 times; resuspend the cells with 500 μL PBS, and perform flow cytometry analysis. 5

[0151] Figure 17 The FACS binding detection results of the first batch of candidate antibodies are shown: cells: CHO-K1-Human CDH3, 2 10^5cells / well; primary antibody: antibody (20 μg / mL, 5-fold dilution of 8 points); secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0152] Figure 18 The FACS binding detection results of the first batch of candidate antibodies are shown: cells: CHO-K1-Human CDH3, 2 10^5cells / well; primary antibody: antibody (20 μg / mL, 5-fold dilution of 8 points); secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0153] Figure 19 The FACS binding detection results of the first batch of candidate antibodies are shown: cells: CHO-K1-Human CDH3, 2 10^5cells / well; primary antibody: antibody (20 μg / mL, 5-fold dilution of 8 points); secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0154] Figure 20 ​FACS binding assay results of the second batch of candidate antibodies: cells: CHO-K1-Human CDH3, 2 10^5 cells / well; primary antibody: antibody (20 μg / mL, 5-fold dilution for 8 points); secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0155] Figure 21 FACS binding assay results of the second batch of candidate antibodies: cells: CHO-K1-Human CDH3, 2 10^5 cells / well; primary antibody: antibody (20 μg / mL, 5-fold dilution for 8 points); secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0156] Figure 22 FACS binding assay results of the second batch of candidate antibodies: cells: CHO-K1-Human CDH3, 2 10^5 cells / well; primary antibody: antibody (20 μg / mL, 5-fold dilution for 8 points); secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0157] As can be seen from the above results, ph-2-D3, ph-5-F06, ph-4-C09, ph-12-B11, and ph-12-G08 have good binding ability to at least one of human, monkey, and mouse CDH3 proteins, and ph-5-F06 has excellent binding activity to all three CDH3 proteins.

Claims

1. An anti-CDH3 nanobody, characterized in that, It contains three complementary determinant regions, CDR1, CDR2, and CDR3; among them, The amino acid sequence of CDR1 is shown in SEQ ID NO: 4; The amino acid sequence of CDR2 is shown in SEQ ID NO: 9; The amino acid sequence of CDR3 is shown in SEQ ID NO:

14.

2. The anti-CDH3 nanobody according to claim 1, characterized in that, It also includes four frame regions FR1, FR2, FR3, and FR4, which are alternately set with the three complementary determinant regions. The amino acid sequence of FR1 is shown in SEQ ID NO: 19; The amino acid sequence of FR2 is shown in SEQ ID NO: 24; The amino acid sequence of FR3 is shown in SEQ ID NO: 29; The amino acid sequence of FR4 is shown in SEQ ID NO:

32.

3. The anti-CDH3 nanobody according to claim 1, characterized in that, The nanobody sequence is shown in SEQ ID NO:

36.

4. A nucleic acid molecule, characterized in that, Its encoding is the anti-CDH3 nanobody as described in any one of claims 1-3.

5. A carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It contains the nucleic acid molecule as described in claim 4 or the vector as described in claim 5.

7. A method for generating anti-CDH3 nanobodies, characterized in that, Includes the following steps: (a) Under conditions suitable for the production of nanobodies, the host cells as described in claim 6 are cultured to obtain a culture containing the anti-CDH3 nanobodies; (b) Isolating and / or recovering the anti-CDH3 nanobody from the culture; and optionally, (c) Purification and / or modification of the anti-CDH3 nanobody obtained in step (b).

8. An antibody-drug conjugate, characterized in that, It includes the anti-CDH3 nanobody and effector as described in any one of claims 1-3, and optionally a linker; the effector is selected from at least one of radionuclides, cytotoxic agents, fluorescent groups, enzymes that catalyze substrate color development, chemiluminescent reagents, and nanoparticle-based markers.

9. A pharmaceutical composition, characterized in that, Contains the anti-CDH3 nanobody as described in any one of claims 1-3, or the antibody-drug conjugate as described in claim 8.

10. The use of at least one of the following: the anti-CDH3 nanobody according to any one of claims 1-3, the antibody-drug conjugate according to claim 8, or the pharmaceutical composition according to claim 9: (i) Prepare a reagent for detecting CDH3-related diseases in humans; wherein the CDH3-related diseases are selected from at least one of non-small cell lung cancer, breast cancer, ovarian cancer, bladder cancer, bile duct cancer, pancreatic cancer, and thyroid cancer; (ii) Prepare a medicament for treating CDH3-related diseases; wherein the CDH3-related diseases are selected from at least one of non-small cell lung cancer, breast cancer, ovarian cancer, bladder cancer, bile duct cancer, pancreatic cancer, and thyroid cancer; (iii) Detection of CDH3 expression level in test samples for non-diagnostic purposes.

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