An anti-fda0128 antibody, its preparation method and application

Abstract

The application discloses an anti-FDA0128 antibody and application thereof. The amino acid sequences of a heavy chain variable region and a light chain variable region of the FDA0128 are shown in SEQ ID NO: 3 and 4 respectively; the anti-FDA0128 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1 of an amino acid sequence shown in SEQ ID NO: 17 or 22, HCDR2 of an amino acid sequence shown in SEQ ID NO: 18 or 23 and HCDR3 of an amino acid sequence shown in SEQ ID NO: 19 or 24; and the light chain variable region comprises LCDR1 of an amino acid sequence shown in SEQ ID NO: 20 or 25, LCDR2 of an amino acid sequence shown in YAY or LAS and LCDR3 of an amino acid sequence shown in SEQ ID NO: 21 or 26. The anti-FDA0128 antibody of the application has strong binding force with the FDA0128, and can realize rapid and accurate pharmacokinetic analysis on the FDA0128 antibody and the FDA0128 antibody conjugated drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibody detection, and particularly relates to an anti-FDA0128 antibody, a nucleic acid encoding the same, a recombinant expression vector comprising the nucleic acid, a transformant comprising the recombinant expression vector, and a preparation method thereof, and an antibody drug conjugate comprising the anti-FDA0128 antibody, a detection reagent and a kit, and further relates to a method for detecting FDA0128 and applications of the anti-FDA0128 antibody. BACKGROUND

[0002] The analysis and characterization of pharmacokinetics and absorption, distribution, metabolism and excretion of antibody-drug conjugates (ADC) reflect the dynamic interaction between biological systems and ADC, and provide key information for early screening and clinical development of drugs. The pharmacokinetics and pharmacodynamics of ADC can be applied to target selection, antibody design, linker-drug selection and optimization of drug-to-antibody ratio (DAR value), which is beneficial to the development of safe and effective ADC. Due to the heterogeneity of its structure, the DAR value presents dynamic changes in vivo, and the PK characteristics of each component of ADC, including total antibody (including conjugated antibody and free antibody), conjugated antibody and free small molecule drug, should be determined during the development process. The determination of total antibody can be used to investigate whether its PK characteristics meet the general antibody PK characteristics, and will not change due to the conjugation of small molecule drugs. The total antibody and ADC concentration in vivo after administration needs to be monitored in clinical and non-clinical trials to determine the administration dose. Common quantitative analysis includes enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS).

[0003] Generally, an anti-idiotypic antibody is an antibody that can recognize the variable region of an antibody and produce specific binding. Anti-idiotypic antibodies are widely used in drug development for immunogenicity analysis of antibody drugs, preclinical research of therapeutic antibody drugs, clinical development of anti-drug antibodies, ligand neutralization test and antibody blocking test, and pharmacokinetic (PK) and pharmacodynamic (PD) analysis of antibody drugs. Compared with other monoclonal antibody drug concentration determination methods based on the same ELISA principle, such as antigen capture antibody method, antibody capture antigen method (bridge method), anti-species antibody capture and detection method and competitive ELISA detection, the use of anti-idiotypic monoclonal antibodies for antibody pharmacokinetic determination has the following advantages: high specificity, i.e. when detecting the total antibody of antibody-drug conjugates in serum, the interference of serum is small; using anti-idiotypic antibodies in experimental design, the antibody and antibody-drug conjugates in serum can be detected, which are free, partially bound to antigen or completely bound to antigen.

[0004] Rabbit monoclonal antibodies have rich antibody expression profiles, stable batches and advantages such as controllable large-scale recombinant production, and are increasingly recognized in the fields of preclinical and clinical drug development, companion diagnostics and cell therapy. The development of anti-idiotype antibodies of rabbit antibodies is simpler than the structure of traditional mouse antibodies, only IgA, IgG, IgE, IgM, no IgD, and only one subtype of IgG; the unique antibody B cell library generation mechanism of rabbits can produce diverse antibodies; rabbit antibodies have a double maturation mechanism, and the specificity of the monoclonal antibodies produced is strong, and the affinity is higher than that of mouse monoclonal antibodies of the same target.

[0005] FDA0128 is a humanized monoclonal anti-Trop-2 antibody. The anti-idiotype antibody developed for FDA0128 can be used in the development process of FDA0128 and FDA0128 antibody drug conjugates to detect antibodies and antibody conjugate drugs in serum free, partially bound antigens or completely bound antigens. SUMMARY

[0006] The technical problem to be solved by the present application is to solve the defect that there is no anti-idiotype antibody for FDA0128 in the prior art, and to provide an antibody that can effectively bind to FDA0128 antibody. The anti-FDA0128 antibody obtained has strong binding force with FDA0128, and can realize rapid and accurate pharmacokinetic analysis of FDA0128 antibody and FDA0128 antibody conjugate drugs.

[0007] To solve the above technical problems, the present application provides an anti-FDA0128 antibody, the amino acid sequences of the heavy chain variable region and the light chain variable region of the FDA0128 are shown in SEQ ID NO: 3 and 4 respectively; the anti-FDA0128 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 17 or 22, HCDR2 of the amino acid sequence shown in SEQ ID NO: 18 or 23 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 19 or 24; the light chain variable region comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 20 or 25, LCDR2 of the amino acid sequence shown in YAY or LAS and LCDR3 of the amino acid sequence shown in SEQ ID NO: 21 or 26.

[0008] In one embodiment of the application, the nucleotide sequence encoding the amino acid sequences set forth in SEQ ID NO: 17-20, YAY, SEQ ID NO: 21-25, LAS and SEQ ID NO: 26 are set forth in SEQ: ID: NO: 5 at positions 73-96, SEQ: ID: NO: 5 at positions 148-168, SEQ: ID: NO: 5 at positions 280-327 (P2C7 HCDR1\2\3), SEQ: ID: NO: 6 at positions 79-96, SEQ: ID: NO: 6 at positions 148-156, SEQ: ID: NO: 6 at positions 265-300 (P2C7 LCDR1\2\3), SEQ: ID: NO: 9 at positions 73-96, SEQ: ID: NO: 9 at positions 148-168, SEQ: ID: NO: 9 at positions 277-321 (P1C11 HCDR1\2\3), SEQ: ID: NO: 10 at positions 79-96, SEQ: ID: NO: 10 at positions 160-168 and SEQ: ID: NO: 10 at positions 265-300 (P1C11 LCDR1\2\3), respectively.

[0009] Preferably, the VH comprises HCDR1, HCDR2 and HCDR3 amino acid sequences set forth in SEQ ID NO: 17-19, respectively, or the VH comprises HCDR1, HCDR2 and HCDR3 amino acid sequences set forth in SEQ ID NO: 22-24, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 amino acid sequences set forth in SEQ ID NO: 20, YAY and SEQ ID NO: 21, respectively, or the VL comprises LCDR1, LCDR2 and LCDR3 amino acid sequences set forth in SEQ ID NO: 25, LAS and SEQ ID NO: 26, respectively.

[0010] More preferably, the VH comprises HCDR1, HCDR2 and HCDR3 amino acid sequences set forth in SEQ ID NO: 17-19, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 amino acid sequences set forth in SEQ ID NO: 20, YAY and SEQ ID NO: 21, respectively; or, the VH comprises HCDR1, HCDR2 and HCDR3 amino acid sequences set forth in SEQ ID NO: 22-24, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 amino acid sequences set forth in SEQ ID NO: 25, LAS and SEQ ID NO: 26, respectively, see Table 1.

[0011] Table 1. Specific combinations of HCDR amino acid sequences and LCDR amino acid sequences of antibodies

[0012]

[0013] In a preferred embodiment of the present application, the VH further comprises a heavy chain variable region framework region (HFR), and / or the VL further comprises a light chain variable region framework region (LFR); wherein the HFR is a heavy chain variable region framework region of a human or rabbit antibody, and the LFR is a light chain variable region framework region of a human or rabbit antibody. More preferably, the VH comprises a HFR1 of the amino acid sequence set forth in SEQ ID NO: 27 or 35, a HFR2 of the amino acid sequence set forth in SEQ ID NO: 28 or 36, a HFR3 of the amino acid sequence set forth in SEQ ID NO: 29 or 37, and a HFR4 of the amino acid sequence set forth in SEQ ID NO: 30 or 38; and the VL comprises a LFR1 of the amino acid sequence set forth in SEQ ID NO: 31 or 39, a LFR2 of the amino acid sequence set forth in SEQ ID NO: 32 or 40, a LFR3 of the amino acid sequence set forth in SEQ ID NO: 33 or 41, and a LFR4 of the amino acid sequence set forth in SEQ ID NO: 34. Still more preferably, the VH comprises a HFR1 of the amino acid sequence set forth in SEQ ID NO: 27, a HFR2 of the amino acid sequence set forth in SEQ ID NO: 28, a HFR3 of the amino acid sequence set forth in SEQ ID NO: 29, and a HFR4 of the amino acid sequence set forth in SEQ ID NO: 30, or the VH comprises a HFR1 of the amino acid sequence set forth in SEQ ID NO: 35, a HFR2 of the amino acid sequence set forth in SEQ ID NO: 36, a HFR3 of the amino acid sequence set forth in SEQ ID NO: 37, and a HFR4 of the amino acid sequence set forth in SEQ ID NO: 38; and the VL comprises a LFR1 of the amino acid sequence set forth in SEQ ID NO: 31, a LFR2 of the amino acid sequence set forth in SEQ ID NO: 32, a LFR3 of the amino acid sequence set forth in SEQ ID NO: 33, and a LFR4 of the amino acid sequence set forth in SEQ ID NO: 34, or the VL comprises a LFR1 of the amino acid sequence set forth in SEQ ID NO: 39, a LFR2 of the amino acid sequence set forth in SEQ ID NO: 40, a LFR3 of the amino acid sequence set forth in SEQ ID NO: 41, and a LFR4 of the amino acid sequence set forth in SEQ ID NO: 34. In a specific embodiment of the present application, the combinations of HFR and LFR are shown in Table 2.

[0014] Table 2. Specific combinations of HFR amino acid sequences and LFR amino acid sequences of antibodies

[0015]

[0016] In a preferred embodiment of the present application, the VH comprises an amino acid sequence as shown in SEQ ID NO: 13 or 15, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 14 or 16, see Table 3 below.

[0017] Table 3. Amino acid sequences of SEQ ID NO: 13-16

[0018]

[0019] In a specific embodiment of the present application, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 13-16 are as shown in SEQ: ID: NO: 5 (1st-360th position, P2C7 VH), SEQ: ID: NO: 6 (1st-330th position, P2C7 VL), SEQ: ID: NO: 9 (1st-354th position, P1C11 VH), and SEQ: ID: NO: 10 (1st-330th position, P1C11 VL), respectively.

[0020] More preferably, the VH comprises an amino acid sequence as shown in SEQ ID NO: 13, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 14; or, the VH comprises an amino acid sequence as shown in SEQ ID NO: 15, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 16.

[0021] In the present application, the amino acid sequences of the above-listed CDRs are shown according to the IMGT definition rule (the sequences shown in the technical solutions of the present application are also according to the IMGT definition rule, which can be analyzed at http: / / www.imgt.org / ). Although the scope of protection claimed in the technical solutions of the present application is based on the sequences shown according to the IMGT definition rule, the amino acid sequences corresponding to the CDRs according to other definition rules should also fall within the scope of protection of the present application (these definition rules can also be referred to at https: / / qinqianshan.com / biology / antibody / antibody-numbering-description / ).

[0022] Preferably, the anti-FDA0128 antibody satisfies one or more of the following three conditions:

[0023] (1) the antibody is a full-length antibody, Fab, Fab', F(ab')2, or Fv, and the Fv is preferably an scFv;

[0024] (2) the antibody is a mono-specific antibody, a bi-specific antibody or a multi-specific antibody;

[0025] (3) the antibody is a monoclonal antibody or a polyclonal antibody prepared from the above antibody.

[0026] In a preferred embodiment of the present application, the anti-FDA0128 antibody is a full-length antibody, which comprises a heavy chain and a light chain; the heavy chain comprises a heavy chain constant region, which is preferably a rabbit-derived antibody heavy chain constant region; the light chain comprises a light chain constant region, which is preferably a rabbit-derived antibody light chain constant region. More preferably, the amino acid sequence of the rabbit-derived antibody heavy chain constant region is shown in SEQ ID NO: 7, positions 121-443, and the amino acid sequence of the rabbit-derived antibody light chain constant region is shown in SEQ ID NO: 8, positions 111-214.

[0027] In a preferred embodiment of the present application, the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 7 or 11, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 8 or 12, as shown in Table 4 below.

[0028] Table 4. Amino acid sequences of antibody heavy and light chains

[0029]

[0030] In a specific embodiment of the present application, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 7, 8, 11 and 12 are shown in SEQ ID NO: 5, 6, 9 and 10, respectively.

[0031] More preferably, the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 7, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 8; or, the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 11, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 12.

[0032] In another aspect of the present application, there is provided an isolated nucleic acid encoding the anti-FDA0128 antibody as described above.

[0033] Preferably, the isolated nucleic acid comprises a nucleic acid encoding the heavy chain variable region, and / or a nucleic acid encoding the light chain variable region; the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is shown in SEQ ID NO: 7 or 11, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is shown in SEQ ID NO: 8 or 12.

[0034] More preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as set forth in positions 1 to 360 of SEQ ID: NO: 5 or positions 1 to 354 of SEQ ID: NO: 9, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as set forth in positions 1 to 330 of SEQ ID: NO: 6 or positions 1 to 330 of SEQ ID: NO: 10.

[0035] The present application also provides, in some aspects, a recombinant expression vector comprising the isolated nucleic acid as described above. Preferably, the recombinant expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector. More preferably, the eukaryotic cell expression vector is pV81.

[0036] The present application also provides, in some aspects, a transformant comprising the recombinant expression vector as described above. Preferably, the host cell of the transformant is a prokaryotic cell, preferably an E. coli cell such as TG1, BL21 cell, and / or a eukaryotic cell, preferably a HEK293 cell or a CHO cell.

[0037] The present application also provides, in some aspects, a method for preparing an anti-FDA0128 antibody, the method comprising the steps of culturing the transformant as described above, and obtaining the anti-FDA0128 antibody from the culture.

[0038] The present application also provides, in some aspects, an antibody drug conjugate comprising a tag and the anti-FDA0128 antibody as described above. Preferably, the tag is biotin.

[0039] The present application also provides, in some aspects, a detection reagent comprising the anti-FDA0128 antibody as described above and / or the antibody drug conjugate as described above, and a pharmaceutically acceptable carrier. Preferably, the detection reagent is in a liquid dosage form, a gaseous dosage form, a solid dosage form, and a semi-solid dosage form. More preferably, the detection reagent further comprises a secondary antibody, Trop-2, or biotinylated Trop-2, such as an antibody conjugated horseradish peroxidase against human IgG and an antibody conjugated biotin protein against human IgG.

[0040] The present application also provides, in some aspects, a kit comprising the anti-FDA0128 antibody as described above, and / or the antibody drug conjugate as described above, and / or the detection reagent as described above; and optionally, an instruction.

[0041] The application also provides, in one aspect, the use of an anti-FDA0128 antibody as described above, an antibody drug conjugate as described above, a detection reagent as described above and / or a kit as described above for the detection of FDA0128, an antibody drug conjugate comprising FDA0128 and / or a chimeric antigen receptor cell comprising FDA0128, or for the detection of an antibody having 90% or more sequence identity to FDA0128. The FDA0128 conjugate is, for example, an antibody drug conjugate targeting Trop-2 as disclosed in WO2010 / 093395, preferably IMMU-132. When Trop-2 is present in the subject to be detected, an anti-FDA0128 antibody comprising light and heavy chain variable regions having the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively, is preferably used.

[0042] The application also provides, in one aspect, a method for detecting FDA0128 in a sample, comprising the step of detecting said sample using an anti-FDA0128 antibody as described above. Preferably, the method is for non-diagnostic and / or therapeutic purposes. The sample is, for example, a blood sample (e.g. a whole blood sample and a serum sample), a reagent comprising FDA0128, an antibody drug conjugate comprising FDA0128 or a chimeric antigen receptor cell comprising FDA0128.

[0043] The application also provides an antibody panel (comprising molecules comprising or consisting of antibody fragments or variants), wherein the members of the panel correspond to one, two, three, four, five, or more different antibodies [e.g. whole antibodies, Fabs, F(ab)2 fragments, and scFvs, etc.] of the application.

[0044] The antibodies of the application can be prepared using techniques well known in the art, such as the hybridoma method, recombinant DNA technology, phage display technology, synthetic technology or a combination of these technologies, or other techniques known in the art.

[0045] The term "anti-idiotypic antibody" as used herein refers to an antibody which is capable of recognizing and specifically binding to the variable region of an antibody. The anti-idiotypic antibody described in the embodiments of the application is the anti-FDA0128 antibody obtained in the application.

[0046] An "antibody molecule" or "antibody" as described herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also fragments thereof and variants (including derivatives) of the same. The term antibody molecule as described herein includes, for example, but is not limited to, single chain Fv (scFv), Fab fragments, Fab' fragments, F(ab')2, disulfide linked Fv (sdFv), Fv, and intact antibodies or full-length antibodies. The term "single chain Fv" or "scFv" refers to a polypeptide that comprises the VL domain of an antibody linked to the VH domain of an antibody. Antibodies that immunospecifically bind to FDA0128 can cross-react with other antigens. Preferably, antibodies that immunospecifically bind to FDA0128 do not cross-react with other antigens. Antibodies that immunospecifically bind to FDA0128 can be identified, for example, by immunoassays or other methods known to those of skill in the art. An "intact antibody" or "full-length antibody" refers to a protein comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, comprising: (1) for the heavy chain, a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains, CH1, CH2, and CH3; and (2) for the light chain, a light chain variable region (abbreviated herein as "VL") and a light chain constant region comprising one domain, CL. Antibodies of the application include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the application), and epitope binding fragments of any of the above. Immunoglobulin molecules of the application can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or isotype. Antibodies of the application can be a monoclonal antibody or a polyclonal antibody, with the monoclonal antibody preferably being a murine anti-human monoclonal antibody. Antibodies of the application can be a hyperhumanized antibody or a diabody.

[0047] In the present application, the "heavy chain antibody" refers to an antibody comprising only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as HCAbs.

[0048] "Single domain antibody", also known as "nanobody", refers to a VHH structure cloned from a heavy chain antibody, which is the smallest unit known to bind to a target antigen.

[0049] The amino acid sequence having 90%, 95%, 98%, or 99% or more homology described in the present application is obtained by insertion, deletion, or substitution of the amino acid sequence shown in the aforementioned sequence listing, and the substitution can be, for example, analysis of the sequence by computer structure modeling, analysis of potential post-translational modifications (PTMs) sites, particularly in the CDR region, including analysis and substitution of sites susceptible to aggregation, asparagine deamidation (sites (NG, NS, NH, etc.), aspartate isomerization (DG, DP), N-glycosylation (N-{P}S / T), and oxidation.

[0050] As known in the art, "polynucleotide" or "nucleic acid" used interchangeably in the present application means a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by the action of a DNA or RNA polymerase.

[0051] As used herein, "vector" means a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or bacteriophage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0052] The term "host cell" in the present application can include cells into which foreign nucleic acid has been introduced, including the progeny of those cells. Host cells include "transformants" and "transformed cells" and include the primary transformed cells as well as progeny that are not identical to the parent cell in nucleic acid content, but can contain mutations. The present application includes mutated progeny that have the same function or biological activity as the cells originally selected or screened in the initial transformation.

[0053] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, i.e., to obtain each preferred example of the present application.

[0054] The reagents and raw materials used in the present application are commercially available.

[0055] The positive progress effect of the present application is that:

[0056] The FDA0128 specific antibody of the present application has high specificity in binding with FDA0128, and can specifically bind with the variable region of FDA0128, and can be used in the research and development of FDA0128 antibody and FDA0128 antibody drug conjugate, and can be used for detecting the antibody and antibody conjugate drug in serum free, partially bound antigen or completely bound antigen. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 The FDA0128 antibody light chain expression vector. DETAILED DESCRIPTION

[0058] The abbreviations of the reagents used in the present application are shown in Table 5.

[0059] Table 5. Explanation of abbreviations

[0060] PBST phosphate buffered saline with tween BSA bovine serum albumin EDA diethanolamine PNPP paranitrophenyl phosphate disodium MRD minimum dilution ratio TMB 3,3',5,5'-tetramethylbenzidine

[0061] The present application is further illustrated by the following examples, but the present application is not limited in the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to the conventional methods and conditions, or according to the product instructions.

[0062] Example 1 Preparation of immunogen

[0063] The monoclonal antibody FDA0128 specifically targeting Trop-2 has heavy chain amino acid as shown in SEQ ID NO: 1, and light chain amino acid as shown in SEQ ID NO: 2. The FDA0128 light chain and heavy chain nucleotide sequences are obtained by full gene synthesis (Suzhou Jinyizhi), and are separately constructed into pV81 vector by EcoR I (purchased from NEB, R3104S) and Hind III (purchased from NEB, R3101S) double enzyme digestion (such as Figure 1), and the clones were picked and subjected to PCR identification and sequencing confirmation. The positive clones were cultured and expanded, and the plasmids were extracted to obtain the antibody light chain eukaryotic expression plasmid FDA0128-L / pV81 and the antibody heavy chain eukaryotic expression plasmid FDA0128-H / pV81. The two plasmids were linearized by Xba I (purchased from Takara, 1093S), and the light chain and heavy chain eukaryotic expression plasmids were in a ratio of 1.5 / 1. The linearized plasmids were transformed into CHO cells (purchased from ATCC) adapted for suspension growth by electroporation. The cells after electroporation were seeded into a 96-well plate at a density of 2000-5000 cells per well, and the expression amount was determined by HTRF method (homogeneous time-resolved fluorescence) after 3 weeks of culture. The cell pool with the highest expression amount was selected and expanded, and then stored in a frozen state. One cell pool was thawed and inoculated into a 125 mL shake flask (culture volume 30 mL) for culture at 37°C, 5.0% CO2, and 130 rpm shaking. After 3 days of culture, the culture was expanded into a 1000 mL shake flask (culture volume 300 mL) for culture at 37°C, 5.0% CO2, and 130 rpm shaking. Starting from the fourth day, the feed medium was added at a flow rate of 5-8% of the initial culture volume every other day. The culture was terminated at 10-12 days, and the harvest was centrifuged at 9500 rpm for 15 min to remove the cell pellet. The supernatant was collected and filtered through a 0.22 μm filter membrane. The treated sample was purified by MabSelect affinity chromatography column (purchased from GE Company) to prepare the antibody FDA0128.

[0064] The FDA0128 antibody was digested with papain, and the Fab fragment of the antibody was separated by magnetic beads to remove the constant region (Fc) of the antibody to obtain the variable region protein (Fab) of the antibody. The Fab fragment of the antibody was prepared according to the instructions of the kit (Thermo, item number 20341), and finally 17.6 mg of the Fab fragment of the FDA0128 antibody was obtained. The amino acid sequence of the heavy chain variable region of the FDA0128 antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. The sequence information is shown in Table 6.

[0065] Table 6. Sequence information of FDA0128

[0066]

[0067] Example 2: Animal immunization and serum titer detection

[0068] Three rabbits (No. A200, A201 and A202) were immunized. Blood was collected before immunization for later use. Immunization was performed on day 0, day 21, day 35, day 49 and day 63, a total of 5 rounds of immunization (immunization process was referred to Spiekerpolet H, Pc. Y, KI. K, et al. Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas [J]. Proceedings of the National Academy of Sciences, 1995, 92(20): 9348-9352.). Blood was collected from the rabbits on day 12 after the fourth round of immunization. ELISA was used to detect the serum of the immunized animals to determine the level of immune response. Cell fusion was performed after the fifth round of immunization.

[0069] ELISA method was used to detect serum titer on day 12 after the fourth round of immunization. The Fab fragment of FDA0128 antibody (derived from Example 1), FDA0128 antibody (derived from Example 1) and hIgG1 (abcam, ab90283), hIgG (sigma, I4506-10MG) were diluted to 1 μg / mL, 100 μL per well was added to the 96-well enzyme-labeled plate, and was placed at 2-8°C overnight. The next day, after discarding the coating solution, the plate was washed with PBST (10.14 mmol / L sodium phosphate dibasic, 1.76 mmol / L potassium dihydrogen phosphate, 137 mmol / L sodium chloride, 2.68 mmol / L potassium chloride, pH 7.4, 0.05% Tween 20) for 3 times, and was dried. 200 μL of blocking solution (containing 3% BSA in PBST) was added to each well, and was placed at room temperature for 2 hours. The plate was washed with PBST for 3 times, and was dried. The serum of the three immunized rabbits was diluted with diluent (containing 0.5% BSA in PBST) to 250, 1000, 4000, 16000, 64000 and 256000 times, and 100 μL per well was added to the enzyme-labeled plate, and was combined with the FDA0128 antibody Fab fragment, FDA0128 antibody and hIgG1, hIgG was incubated at room temperature for 1.5 hours, washed with PBST, and repeated 3 times. The plates were then blotted dry. 50 μL of diluted alkaline phosphatase-labeled goat anti-rabbit ELISA (Jackson, catalog number 111-055-006, dilution 1:20000) was added to each well. The plates were incubated at room temperature with shaking at 200 rpm for 1 hour, followed by PBST washing, and repeated 4 times. The plates were then blotted dry. PNPP substrate reaction buffer was added, and the plates were developed at room temperature for 15 minutes. The reaction was terminated with 3N NaOH. OD values ​​were then read using a microplate reader. 405nm Absorption value. OD 405nm The absorbance was measured, and the results are shown in Table 7. The results showed that the serum titers of the three rabbits were all high after immunization. Two rabbits, A200 and A202, were selected for hybridoma cell fusion.

[0070] Table 7. Summary of ELISA serum titer OD values

[0071]

[0072] Example 3: Initial Screening of Cell Fusion and Clonal Culture

[0073] according to The patented technology (US 7429487) involves electroporation fusion of spleen cells and myeloma cells from immunized rabbits (A200 and A202). After fusion, monoclonal cells are picked and cultured in 96-well plates (10 plates in total). The supernatant from the monoclonal cell culture in the 96-well plates is then screened using ELISA.

[0074] Dilute FDA0128 antibody and carbonate-coated buffer. Add hIgG1 to 1 μg / mL, then add 100 μL to each well of a 96-well ELISA plate and incubate overnight at 2-8°C. The next day, discard the coating solution, wash the plate with PBST three times, blot dry, add 200 μL of blocking buffer (PBST containing 3% BSA) to each well, and incubate at room temperature for 2 hours. Wash the plate with PBST three times, blot dry, add 100 μL of 20-fold diluted rabbit hybridoma clone supernatant to each well, and vortex at 200 rpm for 1 hour at room temperature. Wash the plate with PBST three times, blot dry, add 50 μL of diluted alkaline phosphatase-labeled goat anti-rabbit ELISA (1:20000 dilution), and incubate at 200 rpm for 1 hour at room temperature. Wash the plate with PBST four times, blot dry, add PNPP substrate reaction buffer, and develop color at room temperature for 15 min. Terminate the reaction with 3N NaOH (sodium hydroxide), and read the OD value on the ELISA reader. 405nmAbsorbance value. The higher the OD value of the binding with FDA0128, the higher the concentration of the anti-idiotypic antibody produced. 180 hybridoma cell positive clones were tested for the binding activity with FDA0128 by ELISA, OD≥1, 164 clones; OD≥2, 40 clones; the binding signal with IgG1 control was all lower than 0.2. The ELISA test results of the supernatant of rabbit hybridoma clones are shown in Table 8.

[0075] Table 8. Rabbit hybridoma clones were tested for the binding activity with FDA0128, hIgG1 by ELISA

[0076]

[0077]

[0078] The binding activity of the clones with FDA0128 antibody was tested by competitive ELISA. Trop-2 protein (Sino Biological. 10428-H08H) was diluted to 0.25 μg / mL in carbonate coating buffer and 100 μL was added to each well of a 96-well enzyme-linked plate, which was then placed at 2-8°C overnight. The next day, after the coating solution was discarded, the plate was washed with PBST three times, and then dried, 200 μL of blocking solution (containing 3% BSA in PBST) was added to each well, and the plate was placed at room temperature for 2 hours. The plate was washed with PBST three times, and then dried, 100 μL of equal volume of hybridoma supernatant and 15 ng / mL of biotinylated FDA0128 antibody was added to each well, the biotinylated FDA0128 antibody was prepared according to the operation instruction of the biotin coupling kit (abcam, ab201795), the antibody in the supernatant of the clones competed with Trop-2 coated on the enzyme-linked plate strip to bind to the biotinylated FDA0128, after washing, 100 μL of horseradish peroxidase-labeled streptavidin enzyme-linked secondary antibody (Thermo, item number 21134) diluted to 1:400 was added to each well, and the plate was incubated at room temperature for 1 hour, TMB color developing solution was added, and the plate was reacted at room temperature for 20 min, 50 μL of 0.2 M H2SO4 was added to each well to stop the reaction. The lower the color developing signal value, the stronger the activity of the hybridoma clones in competing with the binding of FDA0128 antibody. The results are shown in Table 9.

[0079] Table 9. ELISA detection value (OD) of clones in competition 405 )

[0080]

[0081]

[0082] Example 4 Culture of clones for re-screening

[0083] According to the screening results, the clones were selected according to the following criteria: the top 40 clones with the highest OD value in the competition ELISA were selected as non-competitive anti-antibody candidates; 4 clones with OD value less than 0.08 in the competition ELISA and OD value greater than 2.15 in the FDA0128 binding ELISA were selected as competitive anti-antibody candidate molecules.

[0084] The antibody protein of these clones P1A5, P1A6, P1A10, P1B1, P1C9, P1C11, P1D4, P1D12, P1E10, P1F3, P1F4, P1F7, P1G1, P1G6, P1H3, P1H6, P1H12, P2B1, P2B9, P2C1, P2C3, P2C7, P2D2, P2D3, P2D4, P2D5, P2D10, P2E1, P2E3, P2E10, P2E11, P2F3, P2F12, P2G1, P2G5, P2G7, P2G9, P2G11, P2H1, P2H2, P2H6, P2H9, P2H11 and P2H12 was quantified and rescreened. The results are shown in Table 10.

[0085] Table 10. Summary of antibody quantification results in supernatant of clones

[0086]

[0087]

[0088] After quantification, the clones were subjected to competitive binding activity detection. The Trop-2 protein was diluted to 0.25 μg / mL with carbonate coating buffer, 100 μL was added to each well of a 96-well enzyme-labeled plate, and the plate was placed at 2-8°C overnight. The next day, after the coating solution was discarded, the plate was washed with PBST for 3 times, and then dried. 200 μL of blocking solution (containing 3% BSA in PBST) was added to each well, and the plate was placed at room temperature for 2 hours. The plate was washed with PBST for 3 times, and then dried. 100 μL of hybridoma supernatant with a final concentration of 0.64 μg / mL, 0.16 μg / mL, 0.04 μg / mL and 0.01 μg / mL, and an equal volume of 40 ng / mL biotinylated FDA0128 were added to each well. The anti-idiotype antibody in the clone supernatant competed with the Trop-2 coated on the enzyme-labeled plate strip for the binding of the biotinylated FDA0128 antibody. After washing, 100 μL of horseradish peroxidase-labeled streptavidin enzyme-labeled secondary antibody diluted to 1:400 was added to each well, and the plate was incubated at room temperature for 1 hour. TMB color developing solution was added, and the plate was reacted at room temperature for 20 min. 50 μL of 2M H2SO4 was added to each well to stop the reaction. The lower the color developing reaction signal value, the stronger the activity of the hybridoma clone in competing for the binding of the FDA0128 antibody. The results are shown in Table 11. Among them, the P1C11, P2H9, P2E10 and P2E11 clones are competitive anti-idiotype antibodies, and the competition activity increases with the increase of the antibody concentration, and the OD signal value decreases. The non-competitive clones include P1C9, P1A5, P2H11, P2G7 and P2C7.

[0089] Table 11. OD value results of competitive binding activity detection of the screened antibodies

[0090]

[0091]

[0092] Example 5: Determination of the sequences of the monoclonal antibodies

[0093] The hybridoma clones P2C7 and P1C11 were sequenced. After the hybridoma cells were recovered, the cell pellets were freshly collected, and the cell count reached 5 x 10 6 above, 1 mL of TriZol Reagent (Thermo) was added for lysis, and total RNA was extracted. The total RNA concentration was not less than 100 ng / μL, the total volume was 20 μL, and the RNA band was clear without obvious degradation after agarose gel electrophoresis. cDNA was obtained by reverse transcription, and the variable region sequences of the heavy and light chains were amplified and cloned into the target vector for sequencing of the light and heavy chains. The sequence information of P2C7 and P1C11 is shown in Tables 12-15.

[0094] Table 12. HCDR amino acid sequences and LCDR amino acid sequences of the anti-idiotype antibodies

[0095]

[0096] Table 13. HFR amino acid sequences and LFRs amino acid sequences of anti-idiotype antibodies

[0097]

[0098] Table 14. Light and heavy chain variable region amino acid sequences of anti-idiotype antibodies

[0099]

[0100] Table 15. Light and heavy chain nucleic acid sequences and amino acid sequences of anti-idiotype antibodies

[0101]

[0102]

[0103]

[0104] Example 6. Recombinant construction, expression and identification of antibodies

[0105] The light and heavy chain nucleotide sequences of monoclonal antibodies P1C11 and P2C7 (see SEQ ID NOs: 5, 6, 9 and 10) were synthesized entirely (Suzhou Jinyuzhi) and constructed into pV81 vectors by EcoR I (NEB, Cat No. R3104S) and Hind III (NEB, Cat No. R3101S) double digestion, respectively, and transformed into Trans 1-T1 competent cells (Quanta Bio, Cat No. CD501) by ligation, from which clones were picked for sequencing confirmation, and positive clones were cultured for plasmid mid-volume extraction, obtaining 80 μg of antibody light chain eukaryotic expression plasmid P1C11-L / pV81 and antibody heavy chain eukaryotic expression plasmid P1C11-H / pV81, which were co-transfected into ExpiCHOS cells. 6 cells / mL, 100 mL total volume, placed in a shaker (Bio Scientific, Model Kuhner Climo-Shaker ISF4 XC), culture conditions: CO2: 5%, rotation speed: 130 rpm, temperature: 37°C, overnight culture. On the 8th day after transfection, the cell supernatant was collected by centrifugation (7000 rpm, 10 min, 4°C). The 0.22 μm filter membrane was filtered and treated, and the antibody was purified by Protein A / G affinity chromatography, and the purified antibody was preserved in phosphate buffer (PBS) by dialysis. ELISA was used to detect the titer, and competitive ELISA was used for identification. The P2C7 monoclonal antibody was harvested according to the same experimental method.

[0106] ELISA assay: P1C11 and P2C7 recombinant expressed antibodies were diluted with carbonate buffer (pH 9.6) to 1 μg / mL, 100 μL per well was added to 96-well enzyme-linked plates, and coated overnight at 2-8 °C. The next day, after the coating solution was discarded, the plate was washed with PBST for 3 times, and then dried by tapping. 200 μL of blocking solution (PBST containing 3% BSA) was added to each well, and incubated at room temperature for 2 hours. The plate was washed with PBST for 3 times, and then dried by tapping. 100 μL of FDA0128 antibody and FDA0128-ADC (structure and preparation method see hRS7-16 in J. Med. Chem. 2008, 51, 6916-6926) of different concentrations (gradient concentrations were 1000 ng / mL, 250 ng / mL, 62.5 ng / mL, 15.625 ng / mL, 3.906 ng / mL, 0.977 ng / mL, 0.244 ng / mL and 0.024 ng / mL, respectively) diluted with PBST containing 0.5% BSA was added to each well, and incubated at room temperature with 200 rpm shaking for 1 hour. The plate was discarded with washing solution PBST, washed for 3 times and dried by tapping. 100 μL of enzyme-linked secondary antibody goat anti-human IgG Fab labeled with horseradish peroxidase (dilution 1:40000) was added to each well, and TMB color development was reacted at room temperature for 20 min. 50 μL of 2M H2SO4 was added to each well to stop the reaction. The 96-well enzyme-linked plate was placed in an enzyme-linked instrument, and the wavelength of 450 nm was used as the detection wavelength, and the wavelength of 650 nm was used as the reference wavelength for double-wavelength reading. The results obtained are shown in Tables 16 and 17.

[0107] Table 16. OD value summary of anti-idiotype antibody binding ELISA assay with FDA0128 antibody

[0108]

[0109] Table 17. OD value summary of anti-idiotype antibody binding ELISA assay with FDA0128-ADC

[0110]

[0111] It can be seen that the two idiotype antibodies obtained by the present application have good binding activity with FDA0128 and FDA0128-ADC.

[0112] Competitive ELISA assay: 80 ng / mL of Trop-2 protein diluted in carbonate buffer was added to each well of the plate strip, and the plate strip was coated overnight at 2-8°C. The next day, the coating solution was discarded, and the plate strip was washed with PBST for 3 times and patted dry. Then, 200 μL of blocking solution (PBST containing 3% BSA) was added to each well, and the plate strip was incubated at room temperature for 2 hours. The plate strip was washed with PBST for 3 times and patted dry. 100 μL of a mixture of 40 ng / mL of biotinylated FDA0128 antibody and gradient concentrations of P1C11 or P2C7 antibody (antibody concentration range: 1000-0.061 ng / mL) was added to each well. The Trop-2 protein coated on the plate strip and the P1C11 or P2C7 antibody added competed with the biotinylated FDA0128 antibody for binding to Trop-2. The OD value decreased as the concentration of the anti-idiotypic antibody added increased, indicating that the P1C11 had stronger activity in competing with FDA0128 for binding to Trop-2. The results are shown in Table 18.

[0113] Table 18. Summary of OD values of anti-idiotypic antibody competing with FDA0128 antibody in ELISA assay

[0114]

[0115] The results showed that P1C11 competed with FDA0128 for binding to Trop-2, while P2C7 did not have the activity of competing for binding.

[0116] Example 7. Detection in human serum samples

[0117] The healthy Chinese serum of different sources was used for investigation, and Trop-2 protein was diluted with phosphate buffer to 0.3 μg / mL, 100 μL per well was added to a 96-well enzyme-labeled plate, and was coated at 2-8°C overnight. The next day, after the coating solution was discarded, the plate was washed with PBST for 3 times, and was dried, 300 μL of blocking solution (PBST containing 5% skim milk) was added to each well, and was placed at room temperature for 2 hours. The plate was washed with PBST for 3 times, and was dried, and human serum was mixed and diluted FDA0128-ADC (structure and preparation method refer to hRS7-16 in J. Med. Chem. 2008, 51, 6916-6926), the minimum dilution ratio (MRD) was 1:200, and the final concentrations were 500000 ng / mL, 20000 ng / mL, 5000 ng / mL, 1250 ng / mL, 312.5 ng / mL and 78.125 ng / mL respectively, 100 μL per well was added, and was incubated at room temperature at 200 rpm for 1 hour. The plate was discarded and washed with washing solution PBST for 3 times and dried. 100 μL of P2C7 antibody diluted to 0.3 μg / mL was added to each well for detecting FDA0128-ADC captured on the enzyme-labeled plate, and was incubated at room temperature at 200 rpm for 1 hour. The plate was discarded and washed with washing solution PBST for 3 times and dried. Then 100 μL of enzyme-labeled secondary antibody goat anti-rabbit IgG (Yiqiao, product number SSA003, dilution ratio 1:20000) was added to each well, TMB color development was reacted at room temperature for 20 min, and 50 μL of 2M H2SO4 was added to each well to stop. The 96-well enzyme-labeled plate was placed in an enzyme-labeled instrument, and was read at 450 nm as the detection wavelength and 620 nm as the reference wavelength.

[0118] Table 19. ELISA detection OD value of anti-idiotype antibody P2C7 detecting FDA0128-ADC in serum

[0119]

[0120]

[0121] The detection results show that the anti-idiotype antibody of the application can be used for detecting FDA0128-ADC in a biological sample. SEQUENCE LISTING <110> Shanghai Fudan Zhangjiang Biomedicine Co., Ltd. <120> Anti-FDA0128 antibody, preparation method and application thereof <130> P21018103C <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> FDA0128 HC <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> FDA0128 LC <400> 2 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> FDA0128 VH <400> 3 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gin Ala Pro Gly Gin Gly Leu Lys Trp Met 35 40 45 Gly Trp He Asn Thr Tyr Thr Gly Gin Pro Thr Tyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gin He Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> FDA0128 VL <400> 4 Asp He Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser He Thr Cys Lys Ala Ser Gin Asp Val Ser He Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu He 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 1332 <212> DNA <213> Artificial Sequence <220> <223> P2C7 HC <400> 5 cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggaatcga cctcagtatg tatgcaatgg gctgggtccg ccagactcca 120 gggaaggggc tggaatacat cggaatcatt gataaaggtg gtaggacata ctacgcgagc 180 tgggcgaaag gccgattcac catctccaga acctcgtcga ccacggtgga tctggaaatc 240 accagtccga caaccgagga cacggccacc tgtttctgtg ccagaatacc tgactatgct 300 acttatggtg atgctatatt tgacttgtgg ggccaaggca ccctggccac cgtctcctca 360 gggcaaccta aggctccatc agtcttccca ctggccccct gctgcgggga cacacccagc 420 tccacggtga ccctgggctg cctggtcaaa gggtacctcc cggagccagt gaccgtgacc 480 tggaactcgg gcaccctcac caatggggta cgcaccttcc cgtccgtccg gcagtcctca 540 ggcctctact cgctgagcag cgtggtgagc gtgacctcaa gcagccagcc cgtcacctgc 600 aacgtggccc acccagccac caacaccaaa gtggacaaga ccgttgcgcc ctcgacatgc 660 agcaagccca cgtgcccacc ccctgaactc ctggggggac cgtctgtctt catcttcccc 720 ccaaaaccca aggacaccct catgatctca cgcacccccg aggtcacatg cgtggtggtg 780 gacgtgagcc aggatgaccc cgaggtgcag ttcacatggt acataaacaa cgagcaggtg 840 cgcaccgccc ggccgccgct acgggagcag cagttcaaca gcacgatccg cgtggtcagc 900 accctcccca tcgcgcacca ggactggctg aggggcaagg agttcaagtg caaagtccac 960 aacaaggcac tcccggcccc catcgagaaa accatctcca aagccagagg gcagcccctg 1020 gagccgaagg tctacaccat gggccctccc cgggaggagc tgagcagcag gtcggtcagc 1080 ctgacctgca tgatcaacgg cttctaccct tccgacatct cggtggagtg ggagaagaac 1140 gggaaggcag aggacaacta caagaccacg ccggccgtgc tggacagcga cggctcctac 1200 ttcctctaca gcaagctctc agtgcccacg agtgagtggc agcggggcga cgtcttcacc 1260 tgctccgtga tgcacgaggc cttgcacaac cactacacgc agaagtccat ctcccgctct 1320 ccgggtaaat ga 1332 <210> 6 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> P2C7 LC <400> 6 gatgttgtga tgacccagac tccagcctcc gtgtctgcag ctgtgggagg cacagtcacc 60 atcaagtgcc aggccagtca gagcattggt aatagcttag cctggtatca gcagaaacca 120 gggcagcgtc ccaagctcct gatttattat gcatacactc tggcatctgg ggtcccatcg 180 cggttcagcg gcagtggatc tgggacagag ttcactctca ccatcaccga cctggagtgt 240 gccgatgctg ccacttatta ttgtcaatgt acttattgtg gtagtgatta tgtgaatgct 300 ttcggcggag ggaccgaggt ggtggtcaaa ggtgatccag ttgcacctac tgtcctcatc 360 ttcccaccag ctgctgatca ggtggcaact ggaacagtca ccatcgtgtg tgtggcgaat 420 aaatactttc ccgatgtcac cgtcacctgg gaggtggatg gcaccaccca aaactggc 480 atcgagaaca gtaaaacc gcagaattct gcagattgta cctacaacct cagcagcact 540 ctgacactga ccagcacaca gtacaacagc cacaaagagt acacctgcaa ggtgacccag 600 ggcacgacct cagtcgtcca gagcttcaat aggggtgact gttag 645 <210> 7 <211> 443 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HC <400> 7 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Met Tyr Ala 20 25 30 Met Gly Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Asp Lys Gly Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Thr Ser Ser Thr Thr Val Asp Leu Glu Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Cys Phe Cys Ala Arg Ile 85 90 95 Pro Asp Tyr Ala Thr Tyr Gly Asp Ala Ile Phe Asp Leu Trp Gly Gln 100 105 110 Gly Thr Leu Ala Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr 145 150 155 160 Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val 165 170 175 Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr 180 185 190 Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr 210 215 220 Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr 260 265 270 Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg 275 280 285 Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile 290 295 300 Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg 325 330 335 Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu 340 345 350 Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu 370 375 380 Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr 385 390 395 400 Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly 405 410 415 Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 435 440 <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> P2C7 LC <400> 8 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Gly Asn Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Tyr Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Thr Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Tyr Cys Gly Ser Asp 85 90 95 Tyr Val Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp 100 105 110 Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val 115 120 125 Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro 130 135 140 Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly 145 150 155 160 Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn 165 170 175 Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys 180 185 190 Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser 195 200 205 Phe Asn Arg Gly Asp Cys 210 <210> 9 <211> 1326 <212> DNA <213> Artificial Sequence <220> <223> P1C11 HC <400> 9 cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagcct ctggattctc cctcagtagc tactacatga gctgggtccg ccaggctcca 120 gggaaggggc tggaatggat cggaatcatc tttcctggtg gttatatata ctacgcgagc 180 tgggcgagag gccgattcgc catctccaaa acctcgacca cggtggatct gaaaatcacc 240 agtccgacaa ccgaggacac ggccacctat ttctgtgccg gggatcagtt tacttggagt 300 actactacgg atttaaacgt gtggggccaa ggcaccctgg tcaccgtctc ctcagggcaa 360 cctaaggctc catcagtctt cccactggcc ccctgctgcg gggacacacc cagctccacg 420 gtgaccctgg gctgcctggt caaagggtac ctcccggagc cagtgaccgt gacctggaac 480 tcgggcaccc tcaccaatgg ggtacgcacc ttcccgtccg tccggcagtc ctcaggcctc 540 tactcgctga gcagcgtggt gagcgtgacc tcaagcagcc agcccgtcac ctgcaacgtg 600 gcccacccag ccaccaacac caaagtggac aagaccgttg cgccctcgac atgcagcaag 660 cccacgtgcc caccccctga actcctgggg ggaccgtctg tcttcatctt ccccccaaaa 720 cccaaggaca ccctcatgat ctcacgcacc cccgaggtca catgcgtggt ggtggacgtg 780 agccaggatg accccgaggt gcagttcaca tggtacataa acaacgagca ggtgcgcacc 840 gcccggccgc cgctacggga gcagcagttc aacagcacga tccgcgtggt cagcaccctc 900 cccatcgcgc accaggactg gctgaggggc aaggagttca agtgcaaagt ccacaacaag 960 gcactcccgg cccccatcga gaaaaccatc tccaaagcca gagggcagcc cctggagccg 1020 aaggtctaca ccatgggccc tccccgggag gagctgagca gcaggtcggt cagcctgacc 1080 tgcatgatca acggcttcta cccttccgac atctcggtgg agtgggagaa gaacgggaag 1140 gcagaggaca actacaagac cacgccggcc gtgctggaca gcgacggctc ctacttcctc 1200 tacagcaagc tctcagtgcc cacgagtgag tggcagcggg gcgacgtctt cacctgctcc 1260 gtgatgcacg aggccttgca caaccactac acgcagaagt ccatctcccg ctctccgggt 1320 aaatga 1326 <210> 10 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> P1C11 LC <400> 10 gatgtcgtga tgacccagac tccagcctcc gtggaggcag ctgtgggagg cacagtcacc 60 atcaagtgcc aggccagtga gagcattggt agttacttag cctggtatca gcagaaaaca 120 gggcagcctc ccaagcgcct gatctatctg gcatccactc tggcatctgg ggtctcatcg 180 cggttcaaag gcagtggatc tgggacagag ttcactctca ccatcagcga cctggagtgt 240 gccgatgctg ccacttacta ctgtcaaagc gctaatgcta ttcctagtat tggtgccact 300 ttcggcggag ggaccgaggt ggtggttaaa ggtgatccag ttgcacctac tgtcctcatc 360 ttcccaccag ctgctgatca ggtggcaact ggaacagtca ccatcgtgtg tgtggcgaat 420 aaatactttc ccgatgtcac cgtcacctgg gaggtggatg gcaccaccca aacaactggc 480 atcgagaaca gtaaaacacc gcagaattct gcagattgta cctacaacct cagcagcact 540 ctgacactga ccagcacaca gtacaacagc cacaaagagt acacctgcaa ggtgacccag 600 ggcacgacct cagtcgtcca gagcttcaat aggggtgact gttag 645 <210> 11 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HC <400> 11 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Tyr 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Phe Pro Gly Gly Tyr Ile Tyr Tyr Ala Ser Trp Ala Arg Gly 50 55 60 Arg Phe Ala Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Gly Asp Gln 85 90 95 Phe Thr Trp Ser Thr Thr Thr Asp Leu Asn Val Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gin Pro Lys Ala Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Leu Pro Gin Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gin 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser 180 185 190 Ser Gin Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys 195 200 205 Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro 210 215 220 Pro Pro Gin Leu Leu Gly Gly Pro Ser Val Phe He Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Gin Val Thr Cys Val 245 250 255 Val Val Asp Val Ser Gin Asp Asp Pro Gin Val Gin Phe Thr Trp Tyr 260 265 270 Ile Asn Asn Glu Gin Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gin 275 280 285 Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His 290 295 300 Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys 305 310 315 320 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gin 325 330 335 Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu 340 345 350 Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn 370 375 380 Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu 385 390 395 400 Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gin Arg Gly Asp Val 405 410 415 Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 420 425 430 Lys Ser Ile Ser Arg Ser Pro Gly Lys 435 440 <210> 12 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> P1C11 LC <400> 12 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Gly Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Thr Gly Gln Pro Pro Lys Arg Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Ala Asn Ala Ile Pro Ser 85 90 95 Ile Gly Ala Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp 100 105 110 Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val 115 120 125 Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro 130 135 140 Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly 145 150 155 160 Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn 165 170 175 Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys 180 185 190 Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser 195 200 205 Phe Asn Arg Gly Asp Cys 210 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> P2C7 VH <400> 13 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Met Tyr Ala 20 25 30 Met Gly Trp Val Arg Gin Thr Pro Gly Lys Gly Leu Glu Tyr He Gly 35 40 45 He He Asp Lys Gly Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr He Ser Arg Thr Ser Ser Thr Thr Val Asp Leu Glu He 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Cys Phe Cys Ala Arg He 85 90 95 Pro Asp Tyr Ala Thr Tyr Gly Asp Ala He Phe Asp Leu Trp Gly Gin 100 105 110 Gly Thr Leu Ala Thr Val Ser Ser 115 120 <210> 14 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> P2C7 VL <400> 14 Asp Val Val Met Thr Gin Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr He Lys Cys Gin Ala Ser Gin Ser He Gly Asn Ser 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Arg Pro Lys Leu Leu lie 35 40 45 Tyr Tyr Ala Tyr Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr lie Thr Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gin Cys Thr Tyr Cys Gly Ser Asp 85 90 95 Tyr Val Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> P1C11 VH <400> 15 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Tyr 20 25 30 Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp lie Gly 35 40 45 Ile Ile Phe Pro Gly Gly Tyr Ile Tyr Tyr Ala Ser Trp Ala Arg Gly 50 55 60 Arg Phe Ala Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Gly Asp Gln 85 90 95 Phe Thr Trp Ser Thr Thr Thr Asp Leu Asn Val Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 16 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> P1C11 VL <400> 16 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Gly Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Thr Gly Gln Pro Pro Lys Arg Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Ala Asn Ala Ile Pro Ser 85 90 95 Ile Gly Ala Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HCDR1 <400> 17 Gly Ile Asp Leu Ser Met Tyr Ala 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HCDR2 <400> 18 Ile Asp Lys Gly Gly Arg Thr 1 5 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HCDR3 <400> 19 Ala Arg Ile Pro Asp Tyr Ala Thr Tyr Gly Asp Ala Ile Phe Asp Leu 1 5 10 15 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> P2C7 LCDR1 <400> 20 Gln Ser Ile Gly Asn Ser 1 5 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P2C7 LCDR3 <400> 21 Gln Cys Thr Tyr Cys Gly Ser Asp Tyr Val Asn Ala 1 5 10 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HCDR1 <400> 22 Gly Phe Ser Leu Ser Ser Tyr Tyr 1 5 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HCDR2 <400> 23 Ile Phe Pro Gly Gly Tyr Ile 1 5 <210> 24 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HCDR3 <400> 24 Ala Gly Asp Gln Phe Thr Trp Ser Thr Thr Thr Asp Leu Asn Val 1 5 10 15 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> P1C11 LCDR1 <400> 25 Glu Ser Ile Gly Ser Tyr 1 5 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P1C11 LCDR3 <400> 26 Gln Ser Ala Asn Ala Ile Pro Ser Ile Gly Ala Thr 1 5 10 <210> 27 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HFR1 <400> 27 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser 20 <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HFR2 <400> 28 Met Gly Trp Val Arg Gin Thr Pro Gly Lys Gly Leu Glu Tyr He Gly 1 5 10 15 He <210> 29 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HFR3 <400> 29 Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr He Ser Arg Thr Ser 1 5 10 15 Ser Thr Thr Val Asp Leu Glu He Thr Ser Pro Thr Thr Glu Asp Thr 20 25 30 Ala Thr Cys Phe Cys 35 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> P2C7 HFR4 <400> 30 Trp Gly Gin Gly Thr Leu Ala Thr Val Ser Ser 1 5 10 <210> 31 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> P2C7 LFR1 <400> 31 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 20 25 <210> 32 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> P2C7 LFR2 <400> 32 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 33 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> P2C7 LFR3 <400> 33 Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Thr Leu Thr Ile Thr Asp Leu Glu Cys Ala Asp Ala Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> P2C7 & P1C11 LFR4 <400> 34 Phe Gly Gly Gly Thr Glu Val Val Val Lys 1 5 10 <210> 35 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HFR1 <400> 35 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser 20 <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HFR2 <400> 36 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 15 Ile <210> 37 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HFR3 <400> 37 Tyr Tyr Ala Ser Trp Ala Arg Gly Arg Phe Ala Ile Ser Lys Thr Ser 1 5 10 15 Thr Thr Val Asp Leu Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala 20 25 30 Thr Tyr Phe Cys 35 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> P1C11 HFR4 <400> 38 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 39 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> P1C11 LFR1 <400> 39 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 20 25 <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> P1C11 LFR2 <400> 40 Leu Ala Trp Tyr Gin Gin Lys Thr Gly Gin Pro Pro Lys Arg Leu He 1 5 10 15 Tyr <210> 41 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> P1C11 LFR3 <400> 41 Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr He Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys 20 25 30

Claims

1. An anti-FDA0128 antibody, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the FDA0128 are shown in SEQ ID NO: 3 and 4, respectively; the anti-FDA0128 antibody comprises a heavy chain variable region (VH) of the anti-FDA0128 antibody and a light chain variable region (VL) of the anti-FDA0128 antibody, the VH of the anti-FDA0128 antibody comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 17-19, respectively, and the VL of the anti-FDA0128 antibody comprises the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 20, YAY and SEQ ID NO: 21, respectively; or, the VH of the anti-FDA0128 antibody comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 22-24, respectively, and the VL of the anti-FDA0128 antibody comprises the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 25, LAS and SEQ ID NO: 26, respectively.

2. The anti-FDA0128 antibody of claim 1, wherein, The VH of the anti-FDA0128 antibody comprises the amino acid sequence shown in SEQ ID NO: 13, and the VL of the anti-FDA0128 antibody comprises the amino acid sequence shown in SEQ ID NO: 14; or, the VH of the anti-FDA0128 antibody comprises the amino acid sequence shown in SEQ ID NO: 15, and the VL of the anti-FDA0128 antibody comprises the amino acid sequence shown in SEQ ID NO:

16.

3. The anti-FDA0128 antibody of claim 2, wherein, The anti-FDA0128 antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv or scFv.

4. The anti-FDA0128 antibody of claim 3, wherein The anti-FDA0128 antibody is a full-length antibody, which comprises a heavy chain and a light chain; the heavy chain comprises a heavy chain constant region; and the light chain comprises a light chain constant region.

5. The anti-FDA0128 antibody of claim 4, wherein The heavy chain constant region is a rabbit-derived antibody heavy chain constant region; and / or, the light chain constant region is a rabbit-derived antibody light chain constant region.

6. The anti-FDA0128 antibody of claim 5, wherein, The amino acid sequence of the rabbit-derived antibody heavy chain constant region is shown in positions 121-443 of SEQ ID NO: 7, and the amino acid sequence of the rabbit-derived antibody light chain constant region is shown in positions 111-214 of SEQ ID NO:

8.

7. The anti-FDA0128 antibody according to any one of claims 1 to 6, characterized in that, The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 8; or, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 11, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

12. 8.An isolated nucleic acid encoding the anti-FDA0128 antibody of any one of claims 1-7. 9.A recombinant expression vector comprising the isolated nucleic acid of claim 8.

10. The recombinant expression vector of claim 9, wherein, The recombinant expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

11. The recombinant expression vector of claim 10, wherein, The eukaryotic cell expression vector is pV81.

12. A transformant comprising the recombinant expression vector of any one of claims 9-11.

13. The transformant of claim 12, wherein, The host cell of the transformant is a prokaryotic cell and / or a eukaryotic cell.

14. The transformant according to claim 13, characterized by, The prokaryotic cell is an E. coli cell, and the eukaryotic cell is a HEK293 cell or a CHO cell.

15. The transformant of claim 14, wherein, The E. coli cell is a TG1 or BL21 cell.

16. A method of making an anti-FDA0128 antibody, the method comprising the steps of: The transformant of any one of claims 12-15 is cultured, and an anti-FDA0128 antibody is obtained from the culture.

17. An antibody drug conjugate comprising a tag and the anti-FDA0128 antibody of any one of claims 1-7.

18. The antibody drug conjugate of claim 17, wherein, The tag is biotin.

19. A detection reagent comprising the anti-FDA0128 antibody of any one of claims 1-7 and / or the antibody drug conjugate of claim 17 or 18, and a pharmaceutically acceptable carrier.

20. The detection reagent of claim 19, wherein the antibody is a monoclonal antibody. The detection reagent is in a liquid dosage form.

21. The test reagent of claim 19 or 20, wherein the antibody is a monoclonal antibody. The detection reagent further comprises a secondary antibody, Trop-2, or biotinylated Trop-2.

22. A kit comprising the anti-FDA0128 antibody of any one of claims 1-7, the antibody drug conjugate of claim 17 or 18, and / or the detection reagent of any one of claims 19-21.

23. The kit of claim 22, wherein The kit comprises an instruction.

24. Use of the anti-FDA0128 antibody of any one of claims 1-7, the antibody drug conjugate of claim 17 or 18, the detection reagent of any one of claims 19-21, and / or the kit of claim 22 or 23 in detecting FDA0128, an antibody drug conjugate comprising FDA0128, and / or a chimeric antigen receptor cell comprising FDA0128.

25. The use of claim 24, wherein, The antibody drug conjugate of FDA0128 is an antibody conjugated drug targeting Trop-2 disclosed in WO2010 / 093395.

26. Use according to claim 24 or 25, wherein The antibody drug conjugate of FDA0128 is IMMU-132.

27. The use of claim 26, wherein, The anti-FDA0128 antibody comprising the amino acid sequences of the heavy and light chain variable regions shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, is used when Trop-2 is present in the detection object.

28. A method of detecting FDA0128 in a sample, comprising the step of detecting the sample using the anti-FDA0128 antibody of any one of claims 1-7, which method is for non-diagnostic and / or therapeutic purposes.

29. The method of claim 28, wherein, The sample is a blood sample, a reagent comprising FDA0128, an antibody drug conjugate comprising FDA0128, or a chimeric antigen receptor cell comprising FDA0128.

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