Anti-vhh domain antibodies and uses thereof

Abstract

This invention provides a group of anti-VHH domain antibodies and their uses. This invention further provides applications of the above-mentioned antibodies in the development, screening, and purification of nanobodies, and their applications in the field of immunotherapy.

Description

Technical Field

[0001] This invention relates to a group of antibodies against the VHH domain. The invention also relates to methods for preparing and obtaining these antibodies. Furthermore, this invention relates to the application of these antibodies in the development, screening, and purification of nanobodies. This invention further relates to the application of these antibodies in the field of immunotherapy. Background Technology

[0002] Camel-derived nanobodies, also known as single-domain antibodies or variable domain of heavy chain of HCAb (VHH) antibodies, consist only of the variable domain fragment of camel-derived heavy chain antibodies. They are only about 15 kDa in size and can bind to antigens with high affinity and high specificity. They have very important applications in antibody drug development and immunotherapy.

[0003] In 1993, Hamers-Casterman et al. first discovered heavy-chain antibodies (HCAbs) lacking the light chain in camels [C. Hamers, et al., Naturally occurring antibodies devoid of light chains. Nature, 1993. Vol 363: p 466-468]. These antibodies contain only the heavy chain variable region and the CH2 and CH3 constant regions. The VHH variable region expressed through molecular cloning exhibits excellent stability and affinity, and is currently the smallest known antibody unit. Ablynx has successfully developed and marketed its first therapeutic nanobody drug, caplacizumab, based on nanobody development technology, for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) [https: / / www.ablynx.com / rd-portfolio / clinical-programmes / caplacizumab / ].

[0004] Meanwhile, camel-derived nanobodies also have significant applications in the field of immunocellular therapy. Nanjing Legend Biotech's LCAR-B38M is the first cell therapy in China to pass the CFDA clinical trial application, and also the first project to achieve dual approval in both China and the US. The publicly available data shows remarkable therapeutic effects, holding significant importance in the development of immunocellular therapy in China. This project employs a unique nanobody design to chimeric antigen receptors in its CAR design, avoiding the poor stability and low affinity of scFv in conventional technologies. With the development of immunocellular therapy technology, nanobodies are increasingly favored by researchers.

[0005] While nanobodies hold significant importance in antibody drug development and immunotherapy, a specific antibody is currently lacking to recognize camel-derived nanobodies, hindering their development and optimizing the identification, sorting, and magnetic separation of CAR-T cells in immunotherapy. This invention develops a group of high-affinity, high-specificity, and high-functionality antibodies targeting camel-derived nanobodies, effectively addressing the aforementioned issues and meeting the needs of various application areas. Invention Overview

[0007] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to a VHH domain. In one embodiment, the VHH domain is the VHH domain of a camel-derived antibody. In one embodiment, the camel-derived antibody is a single-domain antibody or heavy-chain antibody derived from a dromedary camel (Camelus dromedarius), a Bactrian camel (Camelus bactrianus), a llama (Vicugna pacos), or a lama (Lama glama).

[0008] In another aspect, the present invention provides an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment disclosed herein contains a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein (A) the heavy chain variable region includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, (a) the HCDR1 sequence is selected from the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above sequences, and (b) the HCDR2 sequence is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid sequences shown in NO:16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences described above, and (c) the HCDR3 sequence is selected from SEQ ID NO:16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and (c) the HCDR3 sequence is selected from SEQ ID NO:16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and (d ...0, 26, 27, 2 NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the above sequences; (d) the heavy chain complementarity-determining region HCDR contains one or more substitutions of no more than three amino acids, deletions or insertions of amino acid sequences (a), (b) and (c);(B) The light chain variable region includes light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3; (e) The LCDR1 sequence is selected from the amino acid sequences shown in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above sequences; (f) The LCDR2 sequence is selected from SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the above sequences, (g) the LCDR3 sequence is selected from SEQ ID. The amino acid sequences shown in NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences mentioned above, and (h) the light chain complementarity-determining region (LCDR) contains one or more substitutions of no more than three amino acids, deletions, or insertions of amino acid sequences in (e), (f), and (g).

[0009] In a further embodiment, the antibody or its antigen-binding fragment disclosed in this invention contains (1) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:16, HCDR3 shown in SEQ ID NO:31, LCDR1 shown in SEQ ID NO:46, LCDR2 shown in SEQ ID NO:61, and LCDR3 shown in SEQ ID NO:76; (2) HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:17, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:47, LCDR2 shown in SEQ ID NO:62, and LCDR3 shown in SEQ ID NO:77; (3) HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:18, HCDR3 shown in SEQ ID NO:33, LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:63, and LCDR3 shown in SEQ ID NO:78; (4) HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:48, and LCDR2 shown in SEQ ID NO:78. (5) HCDR1 shown in SEQ ID NO:34, LCDR1 shown in SEQ ID NO:49, LCDR2 shown in SEQ ID NO:64, and LCDR3 shown in SEQ ID NO:79; (6) HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:20, HCDR3 shown in SEQ ID NO:35, LCDR1 shown in SEQ ID NO:50, LCDR2 shown in SEQ ID NO:65, and LCDR3 shown in SEQ ID NO:80; (7) HCDR1 shown in SEQ ID NO:6, HCDR2 shown in SEQ ID NO:21, HCDR3 shown in SEQ ID NO:36, LCDR1 shown in SEQ ID NO:51, LCDR2 shown in SEQ ID NO:66, and LCDR3 shown in SEQ ID NO:81; (8) HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:22, HCDR3 shown in SEQ ID NO:37, LCDR1 shown in SEQ ID NO:52, and LCDR2 shown in SEQ ID NO:67, and LCDR3 shown in SEQ ID NO:79. LCDR3 shown in NO:82; (8) HCDR1 shown in SEQ ID NO:8, HCDR2 shown in SEQ ID NO:23, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:53, LCDR2 shown in SEQ ID NO:68 and LCDR3 shown in SEQ ID NO:83;(9) HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:24, HCDR3 shown in SEQ ID NO:39, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:69, and LCDR3 shown in SEQ ID NO:84; (10) HCDR1 shown in SEQ ID NO:10, HCDR2 shown in SEQ ID NO:25, HCDR3 shown in SEQ ID NO:40, LCDR1 shown in SEQ ID NO:55, LCDR2 shown in SEQ ID NO:70, and LCDR3 shown in SEQ ID NO:85; (11) HCDR1 shown in SEQ ID NO:11, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:41, LCDR1 shown in SEQ ID NO:56, LCDR2 shown in SEQ ID NO:71, and LCDR3 shown in SEQ ID NO:86; (12) HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:27, HCDR3 shown in SEQ ID NO:42, SEQ ID NO:9, HCDR2 shown in SEQ ID NO:27, HCDR3 shown in SEQ ID NO:42, HCDR2 shown in SEQ ID NO:27, HCDR2 shown in SEQ ID NO:42, HCDR2 shown in SEQ ID NO:27, HCDR2 shown in SEQ ID NO:42, HCDR2 shown in SEQ ID NO:27, HCDR2 shown in SEQ ID NO:49, HCDR2 shown in SEQ ID NO:27, HCDR2 shown in SEQ ID NO:49, HCDR2 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:69, and LCDR3 shown in SEQ ID NO:85; (13) LCDR1 shown in SEQ ID NO:57, LCDR2 shown in SEQ ID NO:72, and LCDR3 shown in SEQ ID NO:87; (14) HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:44, LCDR1 shown in SEQ ID NO:59, LCDR2 shown in SEQ ID NO:74, and LCDR3 shown in SEQ ID NO:89; or (15) HCDR1 shown in SEQ ID NO:15, HCDR2 shown in SEQ ID NO:30, HCDR3 shown in SEQ ID NO:45, LCDR1 shown in SEQ ID NO:60, LCDR2 shown in SEQ ID NO:75, and LCDR3 shown in SEQ ID NO:90.

[0010] In some embodiments, the antibody or antigen-binding fragment disclosed in this invention comprises a heavy chain variable region (HCVR) whose amino acid sequence is selected from the amino acid sequences shown in SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the above sequences.

[0011] In some embodiments, the antibody or antigen-binding fragment disclosed in this invention comprises a light chain variable region (LCVR) whose amino acid sequence is selected from the amino acid sequences of SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the above sequences.

[0012] In a further embodiment, the antibody or its antigen-binding fragment disclosed in this invention contains (1) the heavy chain variable region shown in SEQ ID NO: 91 and the light chain variable region shown in SEQ ID NO: 106; (2) the heavy chain variable region shown in SEQ ID NO: 92 and the light chain variable region shown in SEQ ID NO: 107; (3) the heavy chain variable region shown in SEQ ID NO: 93 and the light chain variable region shown in SEQ ID NO: 108; (4) the heavy chain variable region shown in SEQ ID NO: 94 and the light chain variable region shown in SEQ ID NO: 109; (5) the heavy chain variable region shown in SEQ ID NO: 95 and the light chain variable region shown in SEQ ID NO: 110; (6) the heavy chain variable region shown in SEQ ID NO: 96 and the light chain variable region shown in SEQ ID NO: 111; (7) the heavy chain variable region shown in SEQ ID NO: 97 and the light chain variable region shown in SEQ ID NO: 112; (8) the heavy chain variable region shown in SEQ ID NO: 98 and the light chain variable region shown in SEQ ID NO: 113; (9) ... the heavy chain variable region shown in SEQ ID NO: 94 and the light chain variable region shown in SEQ ID NO: (10) The heavy chain variable region shown in SEQ ID NO:99 and the light chain variable region shown in SEQ ID NO:114; (11) The heavy chain variable region shown in SEQ ID NO:100 and the light chain variable region shown in SEQ ID NO:115; (12) The heavy chain variable region shown in SEQ ID NO:101 and the light chain variable region shown in SEQ ID NO:116; (13) The heavy chain variable region shown in SEQ ID NO:102 and the light chain variable region shown in SEQ ID NO:117; (14) The heavy chain variable region shown in SEQ ID NO:103 and the light chain variable region shown in SEQ ID NO:118; (15) The heavy chain variable region shown in SEQ ID NO:105 and the light chain variable region shown in SEQ ID NO:120.

[0013] In any of the above embodiments, the antibody or its antigen-binding fragment of the present invention specifically binds to the VHH domain. In one embodiment, the VHH domain is the VHH domain of a camel-derived antibody. That is, the antibody or its antigen-binding fragment specified in the above sequence has good binding ability to camel-derived single-domain antibodies or camel-derived heavy-chain antibodies. In one embodiment, the camel-derived antibody is a single-domain antibody or heavy-chain antibody derived from dromedary camels, Bactrian camels, llamas, or llamas.

[0014] In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention binds at the frame region of the VHH domain. In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention binds at a conformational epitope of the VHH domain. In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention binds at a conformational epitope of the frame region of the VHH domain.

[0015] In one embodiment of the foregoing aspects, the antibody or its antigen-binding fragment of the present invention specifically binds to any of the VHH domains shown in SEQ ID NO: 241 and 246-255, or to any of the VHH domains of SEQ ID NO: 241 and 246-255 having at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity, or to a common sequence of SEQ ID NO: 241 and 246-255. In one embodiment of the foregoing aspects, the antibody or its antigen-binding fragment of the present invention specifically binds to the frame region of the VHH domain shown in any of SEQ ID NO: 241 and 246-255, or the frame region having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the frame region of the VHH domain shown in any of SEQ ID NO: 241 and 246-255, or the shared frame sequence of SEQ ID NO: 241 and 246-255.

[0016] In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention is K DA binding affinity of 10 nM to 1 pM, for example, 1 nM to 10 pM, for example, 1 pM to 10 pM, for example, 1 pM to 5 pM, specifically binding to a VHH domain, such as any of the VHH domains shown in SEQ ID NO: 241 and 246-255, or a VHH domain having at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any of the VHH domains shown in SEQ ID NO: 241 and 246-255, or a shared sequence with SEQ ID NO: 241 and 246-255, or a frame region containing any of the VHH domains shown in SEQ ID NO: 241 and 246-255, or a VHH domain with at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any of the VHH domains shown in SEQ ID NO: 241 and 246-255, or a frame region containing any of the VHH domains shown in SEQ ID NO: 241 and 246-255, or a VHH domain with at least 35%, at least 40%, at least 45%, at least 5 The VHH domain of any of the VHH domains shown in SEQ ID NO: 241 and 246-255 has a frame region with at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity, or a shared frame sequence of SEQ ID NO: 241 and 246-255. In one embodiment, binding affinity is determined by surface plasmon resonance (SPR) technology.

[0017] In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention binds to a VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 3-4, 6-10, 13, 15, 17-18, 20-22, 25-26, 36, 38, 41, 46, 48, 66-67, 69-70, 72, 75, 79-80, 82C, 85-88, 90, 92-93, 105-107, 109, and 111-112 in a Kabat numbering manner; or with SEQ IDs in a sequential numbering manner. NO: 241 positions 3-4, 6-10, 13, 15, 17-18, 20-22, 25-26, 36, 38, 41, 46, 48, 66-67, 69-70, 72, 75, 79-80, 85, 88-91, 93, 95-96, 107-109, 111 and 113-114 corresponding positions (see) Figure 11 ).

[0018] In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention binds to the VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 3-10, 13, 15, 17-22, 25-26, 36-43, 45-46, 48-49, 66-70, 72-73, 75-82, 82B-88, 90-94, and 103-113 according to Kabat numbering; or positions corresponding to positions 3-10, 13, 15, 17-22, 25-26, 36-43, 45-46, 48-49, 66-70, 72-73, 75-82, 84-91, 93-97, and 105-115 according to sequential numbering of SEQ ID NO: 248 and / or 255.

[0019] In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention binds to a VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 3-10, 13, 15, 17-22, 25-26, 36-38, 40-41, 43, 45-46, 48-49, 66-70, 72-73, 75-77, 79-82, 82C-88, 90-94, 103-109, and 111-113 according to Kabat numbering; or with SEQ IDs in sequential numbering. NO: Positions 248 and / or 255, corresponding to positions 3-10, 13, 15, 17-22, 25-26, 36-38, 40-41, 43, 45-46, 48-49, 66-70, 72-73, 75-77, 79-82, 85-91, 93-97, 105-111, and 113-115.

[0020] In one embodiment of the foregoing aspects, the antibody or antigen-binding fragment of the present invention binds to the VHH domain at an epitope comprising one or more positions selected from the group consisting of: positions 39, 42, 78, 82B and 110 in accordance with Kabat numbering; or positions corresponding to positions 39, 42, 78, 84 and 112 in accordance with consecutive numbering of SEQ ID NO: 248 and / or 255.

[0021] In another aspect, the present invention covers an antibody or an antigen-binding fragment thereof that binds to the same epitope as the aforementioned antibody or antigen-binding fragment thereof. In yet another aspect, the present invention covers an antibody or an antigen-binding fragment thereof that competes for binding with the aforementioned antibody or antigen-binding fragment thereof.

[0022] In one aspect, the antibody or antigen-binding fragment thereof of the present invention is a naked antibody or antigen-binding fragment thereof.

[0023] In one aspect, the present invention provides one or more polynucleotides encoding the antibodies of the present invention or antigen-binding fragments thereof. In one aspect, the present invention provides one or more vectors comprising the polynucleotides of the present invention. In one embodiment, the vector is selected from cloning vectors and expression vectors. In one aspect, the present invention provides a host cell comprising the polynucleotides or vectors of the present invention. In one embodiment, the host cell is selected from prokaryotic cells, yeast cells, insect cells, and mammalian cells. In one aspect, the present invention provides a method for generating antibodies or antigen-binding fragments thereof, comprising culturing the host cell of the present invention under conditions suitable for antibody generation to express the antibody or antigen-binding fragment. In one embodiment, the method further comprises recovering the antibody or antigen-binding fragment.

[0024] In one aspect, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the antibody or antigen-binding fragment thereof is conjugated to a fluorescein, biotin, an enzyme, agarose resin, magnetic beads, or a biochip.

[0025] In one aspect, the present invention provides a kit comprising a container containing an antibody or antigen-binding fragment thereof, or a conjugate thereof.

[0026] In one aspect, the present invention provides a method for detecting a VHH domain, comprising adding an antibody or antigen-binding fragment or conjugate as described in the present invention to a sample known or suspected to contain a VHH domain, and detecting a complex formed between the antibody or antigen-binding fragment or conjugate and the VHH domain.

[0027] In one aspect, the present invention provides a method for isolating a VHH domain, comprising adding an antibody of the present invention or an antigen-binding fragment or conjugate thereof to a sample known or suspected to contain a VHH domain, and isolating the complex formed between the antibody or the antigen-binding fragment or the conjugate and the VHH domain.

[0028] In one embodiment, the VHH domain is in a camel-derived antibody. In one embodiment, the camel-derived antibody is a single-domain antibody or heavy-chain antibody derived from dromedary, Bactrian, llama, or llama. In one embodiment, the VHH domain is in a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor is on an immune cell. In one embodiment, the immune cell is selected from PBMCs, T cells, NK cells, or macrophages. In one embodiment, the method is performed using FACS or MCS. Attached Figure Description

[0029] Figure 1 This diagram shows the camel-derived nanobody VHH-His used in animal immunization and its amino acid sequence.

[0030] Figure 2 This demonstrates the effectiveness of ELISA testing in the serum of fusion animals.

[0031] Figure 3 Display of ELISA titer of supernatant from maternal clone isolates.

[0032] Figure 4 Display of ELISA titer of subcloned strain supernatant.

[0033] Figure 5a , 5b The image shows the purity of 15 purified antibodies as determined by PAGE.

[0034] Figure 6a , 6b The results show the affinity assays of 15 strains of this antibody with 10 different camel-derived nanobodies EC50.

[0035] Figure 7 The amino acid sequence alignment of the framework region of the camel-derived nanobody is shown.

[0036] Figure 8 Affinity testing of clone R166.C5 is shown.

[0037] Figure 9 Displaying CART cell flow cytometry detection.

[0038] Figure 10 Display of magnetically separated CART cell flow cytometry detection.

[0039] Figure 11 This shows the Kabat numbering method and residue occurrence frequency of the camel-derived nanobodies used in animal immunization.

[0040] Figure 12 The image shows the FACS diagram of five PE-labeled purified antibodies used in the flow cytometry analysis of camel PBMCs.

[0041] Figure 13 This shows SDS-PAGE images of the antibody-conjugated magnetic beads before and after purification of camel serum heavy chain antibody. Invention Details

[0043] I. Definition

[0044] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, a reference to “a molecule” optionally includes a combination of two or more such molecules, and so on.

[0045] As used herein, the term "about" refers to a typical range of error for a corresponding value that is readily known to those skilled in the art. References to "about" in this document include (and describe) embodiments involving that value or parameter itself.

[0046] It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments that are “comprising,” “composed of,” and “substantially composed of.”

[0047] The terms "anti-VHH domain antibody," "VHH domain-binding antibody," and "VHH domain-specific antibody" refer to antibodies capable of binding to the VHH domain with sufficient affinity. In one embodiment, the extent to which the anti-VHH domain antibody binds to unrelated, non-VHH domain proteins is less than about 10% of the antibody's binding to the VHH domain, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the VHH domain-binding antibody has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or less, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (Kd) of M). In some embodiments, anti-VHH domain antibodies bind to conserved VHH domain epitopes in VHH domains from different species.

[0048] The term "heavy chain antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in 4-chain antibodies. Camels (such as dromedary camels, Bactrian camels, llamas, or lama) are known to produce HCAbs.

[0049] The term "single-domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). A single sdAb is capable of binding an antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camel HCAbs, and their heavy chain variable domains are referred to herein as "VHHs" (heavy chain variable domains of heavy chain antibodies). A camel sdAb is the smallest known antigen-binding antibody fragment. The basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to frame regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0050] For the purposes of this document, "recipient human frame" refers to a frame containing the amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from the human immunoglobulin frame or the human common frame as defined below. A recipient human frame "derived" from the human immunoglobulin frame or the human common frame may contain the same amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL recipient human frame is sequence-identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

[0051] “Affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed as a dissociation constant (Kd). Affinity can be measured by methods commonly known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0052] "Affinity-mature" antibodies are those that have one or more alterations in one or more hypervariable regions (HVRs), which result in improved affinity of the antibody for the antigen compared to parental antibodies that do not have such alterations.

[0053] The term “antibody” is used in the broadest sense in this article and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0054] An "antibody fragment" refers to a molecule distinct from the intact antibody that contains a portion of the intact antibody and binds to the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For example, an antigen-binding fragment may contain a heavy chain variable domain and / or a light chain variable domain of the antibody.

[0055] "Antibody that binds to the same epitope as the reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of that antibody to its antigen by 50% or more in a competitive assay. Illustrative competitive assays are provided herein.

[0056] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0057] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0058] "Frame" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. Generally, the variable domain FR consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences in VH (or VL) generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0059] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document to refer to an antibody that has a structure substantially similar to that of a natural antibody or that has a heavy chain containing an Fc region as defined herein.

[0060] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells that have been introduced with exogenous nucleic acids, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny regardless of passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0061] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody derived from a non-human source using the coding sequences of the complete set of human antibodies or other human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0062] "Human common framework" refers to the framework representing the most frequently occurring amino acid residues in the human immunoglobulin VL or VH framework sequence selection. Typically, the human immunoglobulin VL or VH sequence selection is derived from a variable domain sequence subgroup. Typically, this subgroup is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIHP Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is as described in Kabat et al., see subgroup κI above. In one embodiment, for VH, the subgroup is as described in Kabat et al., see subgroup III above.

[0063] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody may contain at least one, typically two, substantially entire variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of the non-human antibody, and all or substantially all FRs correspond to those of the human antibody. Optionally, a humanized antibody may contain at least a portion of an antibody constant region derived from a human antibody. The term “humanized form” of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0064] As used herein, the term “hypervariant region” or “HVR” refers to each region in the antibody variable domain that is sequence-hypervariant (“complementarity-determining region” or “CDR”) and / or forms a structurally defined loop (“hypervariant loop”) and / or contains antigen contact residues (“antigen contact”). Generally, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs in this document include:

[0065] (a) High-variability rings, present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J.Mol.Biol.196:901-917(1987));

[0066] (b) CDR, present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0067] (c) Antigen contact, present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

[0068] Combinations of (d)(a), (b), and / or (c), including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).

[0069] Unless otherwise stated, HVR residues and other residues in the variable domain (e.g., FR residues) are referenced in this paper to Kabat et al., see above numbering.

[0070] "Immune conjugate" refers to an antibody conjugated with one or more heterologous molecules, including but not limited to cytotoxic agents, such as conjugates containing the antibodies of this invention.

[0071] "Isolated nucleic acid encoding anti-VHH domain antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single or different vectors, and such nucleic acid molecules present at one or more locations in the host cell.

[0072] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, meaning that the individual antibodies constituting the group are identical and / or bind to the same epitope, except for possible variant antibodies, such as those containing naturally occurring mutations or those that may occur during the production of the monoclonal antibody preparation, which are generally present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic that the antibody is obtained from a group of substantially homogeneous antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci, such methods and other illustrative methods for producing monoclonal antibodies are described herein.

[0073] "Naked antibody" refers to an antibody that is not conjugated to a heterologous module (such as a cytotoxic module) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0074] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfides. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain.

[0075] Based on their constant domain amino acid sequences, antibody light chains can be classified into one of two types, called Kappa (κ) and Lambda (λ).

[0076] The "percentage (%) amino acid sequence identity" of a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after alignment and, where necessary, introducing gaps to obtain the maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithm required to obtain maximum alignment across the full length of the compared sequences. However, for the purposes of this invention, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, along with user documentation, has been submitted to the US Copyright Office (Washington DC, 20559), where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is available to the public from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0077] In the case of using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A relative to, with, or against a given amino acid sequence B (or can be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows: fraction X / Y multiplied by 100

[0078] Where X is the number of amino acid residues that are identified as identical matches in the A and B alignments by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It should be understood that if the lengths of amino acid sequences A and B are not equal, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. Unless otherwise explicitly stated, all % amino acid sequence identity values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0079] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to specific antigens can be isolated by screening libraries of complementary VL or VH domains, using the VH or VL domains of the antibody binding the antigen separately. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0080] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures and vectors incorporated into the genome of the host cell to which they are introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0081] “Expression” generally refers to the process by which information (e.g., gene-encoded and / or epigenetic) is transformed into structures that are present and function within the cell. Therefore, as used herein, “expression” can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotide fragments, translated polypeptide fragments, or polynucleotide and / or polypeptide modified fragments (e.g., post-translational modifications of a polypeptide) should also be considered expressed, regardless of whether they originate from transcripts generated through alternative splicing or from degraded transcripts, or from post-translational processing of polypeptides (e.g., through protein hydrolysis). “Expressed genes” include genes transcribed into polynucleotides (e.g., mRNA) and then translated into polypeptides, as well as genes transcribed into RNA but not translated into polypeptides (e.g., transport and ribosomal RNA).

[0082] II. Composition and Usage

[0083] This invention provides a set of anti-VHH domain antibodies or their antigen-binding fragments, which specifically recognize conformational epitopes of the camel-derived VHH domain framework region. This invention reveals that these antibodies possess high affinity, high specificity, and high functionality. Furthermore, this invention provides a method for preparing these antibodies and their applications in the development of camel-derived nanobodies and immunocellular therapy.

[0084] In some embodiments, the antibodies and methods disclosed in this invention are used to detect nanobodies or heavy chain antibodies derived from dromedary camels. In other embodiments, the antibodies and methods disclosed in this invention are used to detect nanobodies or heavy chain antibodies derived from Bactrian camels. In still other embodiments, the antibodies and methods disclosed in this invention are used to detect nanobodies or heavy chain antibodies derived from llamas. In a preferred embodiment, the antibodies and methods disclosed in this invention are used to detect nanobodies or heavy chain antibodies derived from llamas.

[0085] In one embodiment, the antibody and method disclosed in this invention are used for the isolation of camel-derived heavy chain antibody-specific PBMCs; the antibody is also applicable to the isolation of other cell types. In another embodiment, the antibody is used for the binding analysis of nanobodies. In a preferred embodiment, the antibody is used for affinity purification of VHH class antibodies via coupling to a solid-phase carrier medium.

[0086] In one embodiment, the antibody disclosed in this invention is used for flow cytometry identification of a type of CAR-T cell. In another embodiment, the antibody is used for flow cytometry sorting of CAR-T cells. In a preferred embodiment, the antibody is used for MACS separation and purification of CAR-T cells.

[0087] A. Exemplary anti-VHH domain antibodies

[0088] In one aspect, the present invention provides an anti-VHH domain antibody or an antigen-binding fragment thereof.

[0089] In one embodiment, the antibody of the present invention, or its antigen-binding fragment, specifically binds to the VHH domain. In one embodiment, the VHH domain is the VHH domain of a camel-derived antibody. In one embodiment, the camel-derived antibody is a single-domain antibody or heavy-chain antibody derived from dromedary camels, Bactrian camels, llamas, or llamas.

[0090] In one embodiment, the antibody or antigen-binding fragment of the present invention binds at a frame region of the VHH domain. In one embodiment, the antibody or antigen-binding fragment of the present invention binds at a conformational epitope of the VHH domain. In one embodiment, the antibody or antigen-binding fragment of the present invention binds at a conformational epitope of the frame region of the VHH domain.

[0091] In one embodiment, the antibody or its antigen-binding fragment of the present invention specifically binds to any of the VHH domains shown in SEQ ID NO: 241 and 246-255, or to any of the VHH domains of SEQ ID NO: 241 and 246-255 having amino acid sequence identity of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or to a common sequence of SEQ ID NO: 241 and 246-255. In one embodiment, the antibody or its antigen-binding fragment of the present invention specifically binds to the frame region of the VHH domain shown in any of SEQ ID NO: 241 and 246-255, or the frame region having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the frame region of the VHH domain shown in any of SEQ ID NO: 241 and 246-255, or the frame sequence shared by SEQ ID NO: 241 and 246-255.

[0092] In another aspect, the present invention provides an anti-VHH domain antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45; (d) comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. HVR-L1 containing the amino acid sequence SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60; (e) HVR-L2 containing the amino acid sequence SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75; and (f) HVR-L3 containing the amino acid sequence SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90.

[0093] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three of the following VH HVR sequences: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

[0094] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three of the following VL HVR sequences: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90.

[0095] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three selected from the VH HVR sequences below: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45; and (b) a VL domain comprising at least one, at least two, or all three selected from the VL HVR sequences below: (i) comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45; and (b) a VL domain comprising at least one, at least two, or all three selected from the VL HVR sequences below: (i) comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, HVR-L1 containing the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60; (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75; and (iii) HVR-L3 containing the amino acid sequence of SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90.

[0096] In another aspect, the anti-VHH domain antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 amino acid sequences with sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-VHH domain antibody containing this sequence retains its ability to bind to the VHH domain. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105. In some embodiments, the substitution, insertion, or deletion occurs in regions other than HVR (i.e., in FR). Optionally, the anti-VHH domain antibody comprises the VH sequence in SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105, including post-translational modifications of that sequence.

[0097] In another aspect, an anti-VHH domain antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence of SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-VHH domain antibody comprising this sequence retains the ability to bind to the VHH domain. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. In some embodiments, the substitution, insertion, or deletion occurs in regions other than HVR (i.e., in FR). Optionally, the anti-VHH domain antibody comprises the VL sequence in SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120, including post-translational modifications of that sequence.

[0098] In another aspect, an anti-VHH domain antibody is provided, wherein the antibody comprises VH as in any of the embodiments provided above and VL as in any of the embodiments provided above.

[0099] In any of the above embodiments, the anti-VHH domain antibody is humanized. In one embodiment, the anti-VHH domain antibody comprises the HVR of any of the above embodiments and further comprises a recipient human frame, such as a human immunoglobulin frame or a human common frame.

[0100] In another aspect, the present invention provides antibodies that bind to the same epitopes as the anti-VHH domain antibodies provided herein. For example, in some embodiments, antibodies that bind to the same epitopes as the anti-VHH domain antibodies provided herein are provided.

[0101] In another aspect of the invention, the anti-VHH domain antibody according to any of the above embodiments is a monoclonal antibody, including chimeric antibodies, humanized antibodies, or human antibodies. In one embodiment, the anti-VHH domain antibody is an antibody fragment, such as Fab, Fab', Fab'-SH, Fv, single-chain variable fragment (scFv), and (Fab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG class antibody or an IgG1 isotype or other antibody class or isotype.

[0102] In another aspect, anti-VHH domain antibodies according to any of the above embodiments may be incorporated, alone or in combination, any of the features described in Sections 1-8 below:

[0103] 1. Antibody affinity

[0104] In some embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10). -8 M or less, such as 10 -8 M to 10 -12 M, for example, 10 -9 M to 10 -12 M, for example, 10 -10 M to 10 -12 M, for example, 10 -11 M to 10 -12 M).

[0105] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab-type antibody of interest and its antigen. For example, by using the minimum concentration in a titration series in the presence of unlabeled antigen. 125 I) Label the antigen to balance Fab, then use an anti-Fab antibody-coated plate to capture the bound antigen to measure the solution-binding affinity of Fab to the antigen (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the conditions for the assay, the VHH domain is used. Multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for 2–5 hours. In non-adsorption plates (Nunc #269620), 100 pM or 26 pM [125I]-antigen was mixed with serially diluted Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, incubation could be prolonged (e.g., approximately 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution was then removed and the plate was coated with 0.1% polysorbate 20 (TWEEN-) in PBS. Wash the plate 8 times. After drying the plate, add 150 μl / well of scintillation buffer (VHH Domain ROSCINT-20). TM Packard), then in Topcount TM The plates were counted for 10 minutes using a gamma counter (Packard). Each Fab was selected to provide a concentration less than or equal to 20% of the maximum binding for the competitive binding assay.

[0106] According to another implementation scheme, Kd is used Measured using surface plasmon resonance assay. For example, at 25°C, using an immobilized antigen CM5 chip at approximately 10 response units (RU). or The assay was performed by (BIAcore, Inc., Piscataway, NJ). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions for use. The antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8 and then injected at a flow rate of 5 μl / min to obtain a conjugated protein of approximately 10 response units (RU). After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, the protein was injected at 25°C at a flow rate of approximately 25 μl / min into a solution containing 0.05% polysorbate 20 (TWEEN-20). TM The surfactant was serially diluted twice in PBS (PBST) to Fab (0.78 nM to 500 nM). A simple one-to-one Langmuir binding model was used. Evaluation Software version 3.2 calculates the binding rate (kon) and dissociation rate (koff) by simultaneously fitting binding and dissociation sensor maps. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the binding rate exceeds 10⁶ M⁻¹ s⁻¹ according to the surface plasmon resonance method described above, then the binding rate can be determined using fluorescence quenching techniques, i.e., using a spectrometer such as an Aviv Instruments spectrophotometer equipped with a flow interruption device or the 8000 series SLM-AMINCO. TM Measurements were taken using a stirred cuvette in a ThermoSpectronic spectrophotometer, in the presence of gradually increasing concentrations of antigen, to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 °C.

[0107] 2. Antibody fragments

[0108] In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); also see WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an extended in vivo half-life, see U.S. Patent No. 5,869,046.

[0109] Biantibodies are antibody fragments with two antigen-binding sites, and can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triantibodies and tetraantibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0110] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1).

[0111] Antibody fragments can be generated using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and the generation of recombinant host cells (such as E. coli or bacteriophages), as described herein.

[0112] 3. Chimeric antibodies and humanized antibodies

[0113] In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a “class-switched” antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragment.

[0114] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains, wherein the HVR, such as the CDR (or a portion thereof), is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, the humanized antibody may also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., an antibody from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0115] Humanized antibodies and their generation methods are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “repaired surface”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach to FR reorganization).

[0116] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using the "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from the common sequence of human antibodies from specific subgroups of the light or heavy chain variable region (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:262). 3 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0117] 4. Human antibodies

[0118] In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Generally, human antibodies are described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0119] Human antibodies can be prepared by administering immunogens to transgenic animals modified to generate complete human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE™ technology; and U.S. Patent No. 5,770,429, which describes… Technology; U.S. Patent No. 7,041,870, which describes KM The technology, and the U.S. Patent Application Publication No. US 2007 / 0061900, which describes (Technology). The human variable region of a complete antibody generated by such animals can be freed, for example, by further modification in combination with different human constant regions.

[0120] Human antibodies can also be generated using hybridoma-based methods. Human myeloma and mouse-human allogeneic myeloma cell lines used for generating human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also documented in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the generation of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0121] Human antibodies can also be generated by isolating variable domain sequences from Fv clones selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. The technique for selecting human antibodies from antibody libraries is described below.

[0122] 5. Library-derived antibodies

[0123] The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (edited by Lo, Human Press, NJ, 2001). Press, Totowa, NJ, 2003); Sidhu et al., J.Mol.Biol.338(2):299-310(2004); Lee et al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132(2004).

[0124] In some phage display methods, complete sets of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library. Antigen-binding phages can then be screened from the phage library, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, natural complete sets can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, an unimmunized library can also be synthesized by cloning the unrearranged V gene segment from stem cells, encoding the highly variable CDR3 region using PCR primers containing random sequences, and then performing rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0125] The antibodies or antibody fragments isolated from the human antibody library are considered to be the human antibodies or human antibody fragments described in this paper.

[0126] 6. Multispecific antibodies

[0127] In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two distinct sites. In some embodiments, one binding specificity targets the VHH domain, while the other targets any other antigen. In some embodiments, a bispecific antibody can bind to two distinct epitopes of the VHH domain. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing the VHH domain. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0128] Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J.10:3655 (1991)), and "segment-entry-cavity" engineering (see, for example, U.S. Patent No. 5,731,168). Engineered electrostatic manipulation effects (WO) can also be used to generate antibody Fc-heterodimeric molecules. 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using “dual antibody” technology for generating bispecific antibody fragments. (See, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the preparation of trispecific antibodies to generate multispecific antibodies using single-chain Fv(sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and as described, for example, Tutt et al., J. Immunol. 147:60 (1991).

[0129] This article also includes engineered antibodies with three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576A1).

[0130] The antibodies or fragments described herein also include “dual-acting FAbs” or “DAFs” that contain an antigen-binding site that binds to both the VHH domain and another different antigen (see, for example, US 2008 / 0069820).

[0131] 7. Antibody variants

[0132] In some embodiments, amino acid sequence variants of the antibodies provided herein are included. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0133] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Substitutional mutagenesis sites of interest include HVR and FR. Conserved substitutions are shown in Table 1 under the heading “Preferred Substitutions.” More substantial variations are provided in Table 1 under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0134] Table 1: Amino Acid Substitutions

[0135] initial residues Exemplary substitution Preferred alternatives Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu(L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0136] Based on their common side-chain characteristics, amino acids can be grouped as follows:

[0137] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0138] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0139] (3) Acidic: Asp, Glu;

[0140] (4) Alkaline: His, Lys, Arg;

[0141] (5) Residues that affect chain orientation: Gly, Pro;

[0142] (6) Aromatics: Trp, Tyr, Phe.

[0143] Non-conservative substitution would require replacing one of these categories with a member of another.

[0144] One class of alternative variants involves one or more hypervariable residues of a substitute parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have certain alterations (e.g., improvements) in biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain some biological properties of the parent antibody. Exemplary alternative variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated, the variant antibody is displayed on a phage, and it is screened for specific biological activities (e.g., binding affinity).

[0145] HVR can be modified (e.g., substituted), for example, to improve antibody affinity. Such modifications can be made to HVR “hotspots,” which are residues encoded by codons that undergo mutations at high frequency during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, where binding affinity is tested for the resulting variant VH or VL. Affinity maturation via the construction and reselection of a secondary library has been documented, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene for maturation selection by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-guided method, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. Alanine scan mutagenesis or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are frequently targeted.

[0146] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) may be made to the HVRs that do not materially reduce binding affinity. For example, such changes may occur outside the antigen-contacting residues in the HVRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0147] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, groups of residues or target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions where the initial substitution indicates functional sensitivity. Alternatively / additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired properties.

[0148] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a peptide that extends the serum half-life of the antibody.

[0149] 8. Antibody derivatives

[0150] In some embodiments, the antibodies provided herein may be further modified to contain additional non-protein property modules known in the art and readily available. Suitable modules for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in production due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions that the antibody is to improve, and whether the antibody derivative will be used for treatment under specified conditions.

[0151] B. Recombination methods and compositions

[0152] Antibodies can be generated using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-VHH domain antibody as described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the antibody VL and / or an amino acid sequence comprising the antibody VH (e.g., the light and / or heavy chains of the antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In yet another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., transformed with the following vectors): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) a first vector and a second vector, the first vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and the second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH. In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method for generating an anti-VHH domain antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody in a culture medium under conditions suitable for antibody expression, as provided above, and optionally, recovering the antibody from the host cell (or host cell culture medium).

[0153] For the recombinant generation of anti-VHH domain antibodies, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0154] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be generated in bacteria such as *Escherichia coli*, particularly where glycosylation and Fc effector function are not required. For the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (also see Charlton, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, HumanaPress, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*). After expression, the antibody can be separated from the bacterial cell clump in a soluble fraction and can be further purified.

[0155] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0156] Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0157] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0158] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, as described in, for example, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK); mouse seltoli cells (TM4 cells, as described in, for example, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT). 060562); TRI cells, such as those described in, for example, Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0159] C. Determination method

[0160] The anti-VHH domain antibodies provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activity using a variety of assays known in the art.

[0161] On the one hand, the antigen-binding activity of the antibodies of the present invention is tested, for example by known methods such as ELISA, Western blotting, etc.

[0162] On the other hand, competitive assays can be used to identify antibodies that compete with any anti-VHH domain antibodies described herein for binding to the VHH domain. In some embodiments, such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) as any anti-VHH domain antibody described herein. Detailed illustrative methods for locating antibody-bound epitopes are given in Morris (1996), “Epitope Mapping Protocols”, Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0163] In an exemplary competitive assay, the VHH domain is immobilized by incubation in a solution containing a first labeled antibody (which binds to the VHH domain, such as any anti-VHH domain antibody described herein) and a second unlabeled antibody (which is to be tested for its ability to compete with the first antibody for binding to the VHH domain). The second antibody may be present in the hybridoma supernatant. As a control, the VHH domain is immobilized by incubation in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the VHH domain, excess unbound antibody is removed, and the amount of label conjugated with the immobilized VHH domain is measured. If the amount of label conjugated with the immobilized VHH domain in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to the VHH domain. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0164] D. Methods and compositions used for detection

[0165] In some implementations, any anti-VHH domain antibody provided herein can be used to detect the presence of the VHH domain in a biological sample. As used herein, the term "detection" encompasses both quantitative and qualitative detection.

[0166] In one embodiment, an anti-VHH domain antibody is provided for use in a detection method. In another aspect, a method is provided for detecting the presence of a VHH domain in a biological sample. In certain embodiments, the method includes contacting the biological sample with the anti-VHH domain antibody, as described herein, under conditions that allow the anti-VHH domain antibody to bind to the VHH domain, and detecting whether a complex is formed between the anti-VHH domain antibody and the VHH domain. Such methods can be in vitro or in vivo.

[0167] In some embodiments, labeled anti-VHH domain antibodies are provided. Labels include, but are not limited to, directly detectable labels or modules (such as fluorescence, chromogenic, electron-dense, chemiluminescence, and radiolabels), and indirectly detectable modules, such as enzymes or ligands, for example via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C, 125 I, 3 H, and 131 I. Fluoresceins such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled with enzymes such as HRP using hydrogen peroxide dye precursors), lactoperoxidase, or microperoxidase, biotin / avidin, spin-labeled, phage-labeled, stable free radicals, etc.

[0168] Sequence description

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] Example

[0185] Example 1: Obtaining hybridoma cell lines of antibodies

[0186] 1) Animal immunization. The antigen used for animal immunization is a recombinant His-tagged camel-derived nanobody, VHH-His (amino acid sequence as follows). Figure 1 (or as shown in SEQ ID NO: 241), this nanobody AS718 is an antigen used for animal immunization, has a C-terminal His tag, contains a total of 121 amino acids, and the CDR domain and His tag are separated by an underscore. Figure 1 As shown in the image.

[0187] Eight New Zealand rabbits were subcutaneously immunized with a 1:1 emulsion containing 200 μg VHH-His and 200 μl Freund's complete adjuvant (Sigma-Aldrich, CAT#:F5881). Booster immunizations were administered every two weeks via subcutaneous injection of a 1:1 emulsion containing 200 μg VHH-His and Freund's incomplete adjuvant (Sigma-Aldrich, CAT#:F5506). Serum antibody titers were measured by ELISA four days before myeloma fusion (e.g., 4 days prior to myeloma fusion). Figure 2 As shown in the figure, the serum titer of the animals was tested. The results showed that the titer of 1:64,000 was greater than 1.0. Two rabbits that met the criteria received intraperitoneal and intravenous booster immunizations of 400 μg VHH-His (without adjuvant).

[0188] Figure 2Using an indirect ELISA method, an ELISA detection plate coated with the immunogen AS718 VHH-His was employed. Animal serum was initially diluted 1:1,000, and then serially diluted 2-fold to 1:512,000 for ELISA detection. Serum titers of the two animals to be fused were determined by OD450 readings. S / N represents the signal-to-noise ratio. The effective serum titer was defined as 2.1 times the background value. Fusion was considered feasible when the serum titer was greater than 1.0 at a dilution of 1:64,000. Results showed that the serum titer of animal #6119 was 1.323 at a dilution of 1:64,000 and 0.488 at a dilution of 1:512,000; the serum titer of animal #563 was 1.637 at a dilution of 1:64,000 and 0.528 at a dilution of 1:512,000. Since the serum values ​​of both animals at a dilution ratio of 1:512,000 were greater than 2.1 times the blank background value, the effective serum titers of both animals were greater than 1:512,000, and the serum values ​​at a dilution ratio of 1:64,000 were greater than 1.0, which met the animal fusion criteria.

[0189] 2) Hybridoma fusion and screening

[0190] Rabbit spleen was extracted and homogenized to produce a single-cell suspension, while a single-cell suspension of myeloma cells was prepared simultaneously. Spleen cells were electrofused with 1.0 × 10⁻⁶ cells. 8 1.2 × 10splenic cells 5 Fusion of myeloma cells was performed; the fused cells were resuspended in 100 ml of DMEM / 10% FBS medium containing hybridoma cell selectants thymidine pyrimidine, hypoxanthine, and aminopterin, and transferred in 100 μl volumes to 150 × 96 well plates; the plates were incubated at 37°C in 6% CO2. After 9 days of incubation, indirect ELISA was used to assess the binding ability of antibodies to VHH-His in the supernatant, indicating the presence of antibodies against VHH-His.

[0191] Coat the ELISA plate overnight at 4°C with 100 μl / well of PBS containing 0.5 μg / ml VHH-His or His protein. Wash the plate with PBS-T (0.05% Tween 20) and block it with 200 μl / well of PBST containing 1% BSA at 37°C for 0.5 h. Discard the blocking solution, add 100 μl of hybridoma cell culture supernatant to each well, and incubate at room temperature for 1 h. After incubation, discard the supernatant, wash the plate three times with PBST, and incubate it with 100 μl / well of goat anti-rabbit IgG (GenScript Goat Anti-Rabbit IgG Antibody (H&L) [HRP], pAb; CAT#: A00098) containing horseradish peroxidase at 37°C for 0.5 h. Wash the plate five times with PBST, then add TMB chromogenic solution and incubate in the dark at room temperature for 15 min. The reaction was terminated by adding 50 μl of 1M HCl stop solution. The plate was read at 450 nm using a microplate reader. Figure 3Using an indirect ELISA method, an ELISA detection plate was coated with the immunogen AS718 VHH-His. The titers of the supernatants of 15 maternal clones were detected by OD450 reading, and their cross-reactivity with His tag proteins was also measured to exclude non-specific binding. Positive serum was used as a positive control for the immunogen AS718 VHH-His, and an anti-His antibody was used as a positive control for the reverse screening of the original His tag protein. A detection value greater than 0.5 was considered positive, and a detection value less than 0.2 was considered negative. The results showed that the binding values ​​of positive serum to VHH-His, positive serum to His-tag protein, anti-His antibody to VHH-His, and anti-His antibody to His-tag protein were 2.953, 0.747, 2.906, and 2.858, respectively, all greater than 0.5, indicating a positive result and valid positive control. The positive serum binding value of 0.747 to His-tag protein indicated that antibodies against the His-tag protein were still produced in the fusion animals. The supernatant of 15 maternal clones was mixed with immunogen AS718. The ELISA values ​​for VHH-His were 2.758, 3.060, 2.908, 2.883, 2.893, 2.556, 2.913, 2.655, 2.561, 2.356, 3.059, 2.130, 2.083, 2.454, and 2.428, all greater than 0.5, indicating that the supernatants of the 15 maternal clones were positive for VHH-His. The original supernatants of the 15 maternal clones were compared with the His standard... The ELISA values ​​for the His-tagged protein were 0.074, 0.130, 0.091, 0.102, 0.082, 0.100, 0.121, 0.083, 0.091, 0.114, 0.123, 0.085, 0.112, 0.082, and 0.079, all less than 0.2. Therefore, the supernatants of the 15 mother clones were determined to be negative for the His-tagged protein, meaning there was no cross-reaction with unrelated His-tagged proteins.

[0192] 3) Hybridoma subcloning: Subcloning was performed using the limiting dilution method.

[0193] Cell counts were determined using a hemocytometer and serially diluted in DMEM / 10% FBS medium containing hybridoma cell selectants thymidine, hypoxanthine, and aminopterin until a cell density of 5-15 cells / ml was achieved. For each hybridoma, 200 μl of cell solution was pipetted into 96 wells at a density of 1-3 cells / well. After incubating the culture at 37°C in 5% CO2 for 1 week, the supernatant was subjected to the above-described ELISA binding assay to assess the presence of antibodies against VHH. ELISA data for the subcloned strain are shown below. Figure 4As shown (ELISA titer assay of subcloning supernatant): An indirect ELISA assay was performed using an AS718 VHH-His immunogen-coated ELISA plate. Positive animal serum diluted 1:1,000 was used as a positive control. The titer of the cell supernatant after subcloning of 15 mother clones was measured by OD450 reading. The first well used the undiluted cell supernatant, and subsequent wells were serially diluted 3-fold to 1:81. A supernatant dilution of 1:9 with a titer greater than 1.0 was considered suitable for antibody production. The results showed that the cell supernatants of the 15 clones had values ​​of 2.460, 1.954, 1.945, 2.143, 2.243, 2.047, 2.064, 2.268, 2.197, 1.528, 2.377, 1.530, 2.485, 1.929, and 1.475, all greater than 1.0, meeting the criteria for hybridoma cell clone identification and allowing for continued antibody production.

[0194] Example 2: Sequencing of the variable region of the antibody and production of recombinant antibodies

[0195] Using TRIzol(Ambion,CAT#:15596-026) from 3×10 6 –5×10 6 Total RNA was extracted from hybridoma cells and analyzed using antibody subtype-specific primers and universal primers (Takara PrimeScript). TM The rabbit immunoglobulin heavy and light chain variable regions were reverse transcribed into cDNA using the 1st Strand cDNA Synthesis Kit (CAT#:6110A). Subsequently, the PCR fragments were amplified by RACE PCR, and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara, CAT#:6011). The inserted fragments were then sequenced using vector-specific primers. Ultimately, the heavy and light chain variable region nucleotide / protein sequences of 15 monoclonal antibodies were obtained, including clone R166.C5, clone R166.G3, clone R166.F2, clone R166.G8, clone R166.H9, clone R166.D1, clone R166.G10, clone R166.H10, clone R166.F9, clone R166.G2, clone R166.E5, clone R166.E3, clone R166.H8, clone R166.E7, and clone R166.A5.

[0196] All antibodies are produced using recombinant expression, taking a selected clone as an example.

[0197] DNA fragments encoding the light chain variable region + constant region (amino acid sequence as shown in SEQ ID NO: 244, nucleotide sequence as shown in SEQ ID NO: 245) and the heavy chain variable region + constant region (amino acid sequence as shown in SEQ ID NO: 242, nucleotide sequence as shown in SEQ ID NO: 243) were synthesized separately and inserted into the pTT5 expression vector to form expression plasmids. The above plasmids were co-transfected into CHO-3E7 cells and cultured in shake flasks at 37°C for 6 days. The supernatant was collected for antibody purification. The tubing and protein A column were depyrogenated with 0.2M NaOH, and then the column was reequilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was diluted 1:1 with 2× the above buffer and filtered for sterilization. The filtered supernatant was incubated with a Protein A column at room temperature for 2 hours. After washing the column with 1× the above buffer, IgG was eluted with sterile 0.1M sodium citrate (pH 3.5). The eluent was collected and neutralized with 1 / 9 volume of sterile 1M Tris-HCl (pH 9.0). Under sterile conditions, the product buffer was replaced with PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibody was quantified using an extinction coefficient of 1.43 (Ec, 0.1%) at OD 280 nm. The purified antibody was analyzed by SDS-PAGE using a 10% precast gel (GenScript) on a BioRad electrophoresis system. The gel was stained with eStain 2.0 (GenScript), and the molecular size and purity were estimated by comparing the staining bands with Protein Ladder (GenScript). The purity identification results of 15 antibody strains are as follows: Figure 5a , 5b As shown, under non-reducing conditions, the two heavy chains and two light chains of the antibody maintain a tetravalent structure through disulfide bonds, resulting in only one target band in the electrophoretic staining results. The displayed molecular weight is slightly lower than the actual molecular weight, comparable to the size of the 116 kDa marker band. Under reducing conditions, the presence of the reducing agent disrupts the disulfide bond structure in the antibody, causing the heavy and light chains to separate and appear as independent electrophoretic bands with sizes of 55 kDa and 25 kDa, respectively. The results show that the purity of all 15 antibody strains is greater than 90%, meeting the standards for antibody production and purification, and can be used for subsequent experimental research.

[0198] Example 3: Binding of the antibody to recombinant camel nanobody

[0199] The binding ability of 15 purified camel-derived nanobodies to 10 different camel-derived nanobodies VHH (amino acid sequences shown in SEQ ID NO: 246-255) was evaluated using an indirect ELISA method.

[0200] ELISA plates were coated overnight at 4°C with 100 μl / well of 10 different VHHs in PBS containing 0.5 μg / ml. The plates were washed with PBS-T (0.05% Tween) and blocked with 200 μl / well of PBST containing 1% BSA at 37°C for 0.5 h. The blocking solution was discarded, and 100 μl of purified antibody (1 μg / ml) was added to the first well. The antibody was then serially diluted 3-fold to obtain 11 test concentration gradients and incubated at room temperature for 1 h. The plates were washed three times with PBST and incubated with 100 μl / well of goat anti-rabbit IgG (GenScript Goat Anti-Rabbit IgG Antibody (H&L) [HRP], pAb; CAT#: A00098) containing horseradish peroxidase at 37°C for 0.5 h. The plates were washed five times with PBST, and then TMB chromogenic solution was added and incubated in the dark at room temperature for 15 min. The reaction was terminated by adding 50 μl of 1M HCl stop solution. The plate was read at 450 nm using a microplate reader; the binding affinity of 15 clones for VHH-His was obtained (e.g., ...). Figure 6a , 6b As shown in the figure, the results showed that the antibodies of the 15 camel-derived nanobodies exhibited varying affinities with different camel-derived nanobodies. On the one hand, antibodies against the same camel-derived nanobodies showed different affinities with different camel-derived nanobodies; on the other hand, antibodies against different camel-derived nanobodies showed different affinities with the same camel-derived nanobodies. However, overall, all 15 purified antibodies could recognize 10 different camel-derived nanobodies. The EC50 calculated based on the ELISA concentration gradient experiment indicated that these antibodies all exhibited considerably high affinity for the antigens.

[0201] The 10 different camel-derived nanobody sequences and the AS718 nanobody sequence used for immunizing animals in Example 1 were analyzed using Kabat to obtain the frames FR1, FR2, FR3, and FR4 sequences. Sequence identity analysis was then performed using BioEdit software. The results showed that the sequence identity of the FR1 region was 53.3%, the FR2 region was 35.7%, the FR3 region was 50%, the FR4 region was 54.5%, and the overall sequence identity of all frame regions was 49.4%. (See [link to relevant documentation]). Figure 7 Moreover, the above 10 camel-derived nanobodies and the AS718 nanobodies showed 74.7% (AS154, AS325), 77.0% (AS587), 79.3% (AS675), 80.5% (AS200, AS519), 81.6% (AS656), 82.8% (AS588, AS988), and 83.9% (AS673) of frame region sequence identity, respectively.

[0202] Similarly, across the full VHH domain, the aforementioned 10 camel-derived nanobodies shared 62.3% (AS154), 62.9% (AS200), 63.6% (AS325), 64.8% (AS656), 66.1% (AS675, AS673), 66.4% (AS587, AS988), 68.0% (AS588), and 69.9% (AS519) sequence identity with the AS718 nanobodies, respectively. The overall identity of the 11 nanobodies was 37.7%.

[0203] Example 4: Determination of affinity between antibodies and multiple camel-derived nanobodies

[0204] In one exemplary implementation, the affinity levels of the antibody clone R166.C5 for multiple camel-derived nanobodies were determined using SPR assays with a Biacore 8K instrument (GE Healthcare).

[0205] CM5 chip pretreatment: After equilibration with HBS 10 μl / min for 5 minutes, the chip was treated with NHS / EDC activator 10 μl / min for 7 minutes, then the conjugated anti-His antibody (GenScript, CAT#:A00186) 10 μl / min was added and reacted for 7 minutes. Finally, the chip was blocked with aminoethanol 10 μl / min. Capture of camel-derived nanobodies: After equilibration with HBS buffer (10 μl / min) for 5 minutes, 10 strains of VHH-His antibody (20 μl / ml) were added and reacted at 30 μl / min for 1 minute. The affinity level of the antibody was determined in the mobile phase at an antibody concentration of 200 nM and a flow rate of 30 μl / min. Immunogen AS718 was used as a positive control for camel-derived nanobodies. The results showed that the affinity of immunogen AS718 for the cloned antibody reached 3.47 pmol. Simultaneously, among the 10 camel-derived nanobodies tested, 6 strains (AS325, AS656, AS673, AS588, AS988, and AS519) all showed affinity for the antibody at the pmol level, with affinities of 4.04 pmol, 1.34 pmol, 4.36 pmol, 7.09 pmol, 7.08 pmol, and 1.66 pmol, respectively. The camel-derived nanobody with the lowest affinity to this cloned antibody was AS587, which still had an affinity of 2.2 nmol. These data indicate that the antibody of clone R166.C5 camel-derived nanobody has high affinity to all 10 different camel-derived nanobodies (e.g., Figure 8 (As shown).

[0206] Example 5: Application of antibodies in the specific isolation of PBMCs from camel-derived heavy chain antibodies

[0207] In one exemplary embodiment, heavy chain antibody-specific PBMCs are separated by flow cytometry using fluorescently labeled camel-derived nanobodies. The PBMCs used in this invention are from non-immune camels.

[0208] Select 5×10 6 One set of PBMC cells was used for analysis. Cells were washed once with 500 μl FACS buffer (PBS, 1% BSA), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. PBMC cells were resuspended in 200 μl FACS buffer and 2 μg of phycoerythrin fluorescein (PE)-labeled camel-derived nanobodies R166.C5, R166.H8, R166.H9, R166.E7, and R166.G8 were added. After incubation on ice for 15 minutes in the dark, the cells were washed once with 500 μl FACS buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were fixed in 2% polymethanol solution for 30 minutes, washed once with 500 μl FACS buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in 200 μl FACS buffer and subjected to flow cytometry sorting for heavy chain antibody-specific PBMC cell separation. FACS results are shown below. Figure 12 As shown, the proportions of PBMC cells with heavy chain antibody specificity obtained by FACS sorting of five PE-labeled different clone antibodies were 22.83%, 23.58%, 24.24%, 23.33%, and 17.24%, respectively, which are consistent with the theoretical value (approximately 20%–30%), indicating that all five antibodies have good heavy chain antibody specificity.

[0209] Example 6: Application of antibodies in the purification of camel-derived heavy chain antibodies

[0210] In one exemplary embodiment, the heavy chain antibody in the serum of a llama or alpaca is purified using the antibody of the present invention, wherein the serum used in the present invention is from a non-immune llama.

[0211] Camel-derived nanobody antibodies (exemplary anti-VHH antibody) were conjugated to NHS-activated magnetic beads at the following mass-to-volume ratios (antibody:magnetic beads = 3:1; 4:1; 5:1; 6:1; 7:1; 8:1) to obtain VHH nanobody purification media. 1 ml of alpaca serum was diluted with 4 ml of PBS and thoroughly mixed. Then, 1 ml of the magnetic bead purification media was added and incubated at 4°C for 1 hour. The magnetic beads were adsorbed using a magnetic rack, and the supernatant was discarded. The beads were washed three times with PBST. Heavy chain antibodies were eluted with sterile 0.1 M sodium citrate (pH 3.5), and the eluent was collected and neutralized with one-ninth volume of sterile 1 M Tris-HCl (pH 9.0). Under sterile conditions, the product buffer was replaced with PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibody was quantified using an extinction coefficient Ec (0.1%) of 1.43 at OD280 nm.

[0212] The purified antibodies (including serum supernatant before magnetic bead purification, eluent after magnetic bead purification, and purified eluted sample) were analyzed by SDS-PAGE using a 10% precast gel (GenScript, CAT#: M00665) on a BioRad electrophoresis system. The gel was then subjected to... L1 Protein Staining System (GenScript) staining and comparison of staining bands with Protein Ladders are used to estimate molecular size and purity. For example... Figure 13 As shown, the molecular weights of the marked Camelidae IgG2 and IgG3 antibodies are 46KD and 43KD, respectively. The obtained magnetic beads can specifically purify the heavy chain antibodies. The overall purity of the purified heavy chain antibodies IgG2 and IgG3 is 77.4%, 78.3%, 71.6%, 80.1%, 81.1%, and 73.8%, respectively.

[0213] Example 7: Application of Antibodies in CAR-T Cell Therapy - Flow Cytometry Identification of CAR-T Cells

[0214] When camel-derived nanobodies are used to construct chimeric receptors in CAR-T cell therapy, the antibodies described in this invention can be used for flow cytometry identification of CAR-T cells.

[0215] CART cells meeting the above criteria were mixed with the initial [cells] at the following ratios (0%, 10%, 20%, 40%, 80%, 95%). T cells were mixed to prepare cell samples for testing; 5 × 10⁶ cells were selected from each sample. 5Cells were washed once with 500 μl FACS buffer (PBS, 1% BSA), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in 400 μl FACS buffer and 4.5 μg of biotin-labeled camel-derived nanobody antibody clone R166.A5 was added. After incubation on ice for 45 minutes, cells were washed once with 500 μl FACS buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in 500 μl FACS buffer and 0.2 μg of PE-labeled streptavidin (Biolegend, CAT#:740452) was added. After incubation at room temperature for 15 minutes, cells were washed once with 500 μl FACS buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were fixed with 2% polymethyl methacrylate solution for 30 minutes, washed once with 500 μl FACS buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 200 μl FACS buffer was used to fix the cells. Cells were resuspended in FACS buffer and analyzed by flow cytometry. The results are as follows: Figure 9 As shown, CAR-T cells were mixed with naïve T cells at different fixed ratios of 0%, 10%, 20%, 40%, 80%, and 95%. Flow cytometry staining was performed using a biotin-labeled antibody against clone R166.A5 and PE-labeled streptavidin to identify the positive CAR-positive cells. The results showed that the proportions of CAR-positive cells were 0.01%, 9.72%, 20.9%, 39.3%, 80.5%, and 93.7%, respectively, with deviations from the premixed ratios less than ±1.5%. These data indicate that the antibody can be well applied to the detection of CAR-T cells constructed using camel-derived nanobodies to create chimeric receptors. This antibody can effectively recognize CAR-positive T cells.

[0216] Example 8: Application of Antibodies in CAR-T Cell Therapy - Magnetic Separation of CAR-T Cells

[0217] When camel-derived nanobodies are used to construct chimeric receptors in CAR-T cell therapy, the antibodies described in this invention can be used for the magnetic separation of CAR-T cells.

[0218] In one exemplary implementation, the number of CART cells to be isolated was 1 x 10-1. 7Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend cells thoroughly in 100 μl PBE buffer (PBS, pH 7.2, 0.5% BSA, 2 mM EDTA). Add 2 μg of biotin-labeled camel-derived nanobody antibody clone R166.A5, mix thoroughly, and incubate at 4°C for 10 minutes. Wash cells once with 2 ml PBE buffer and resuspend cells thoroughly in 100 μl PBE buffer. Add 20 μl of antibiotin-containing magnetic beads (Miltenyi), mix thoroughly, and incubate at 4°C for 10 minutes. Separate cells using a magnetic rack. Perform flow cytometry analysis on the obtained cells to verify the effectiveness of magnetic separation. Figure 10 As shown, naïve T cells were used as a CAR-negative cell control, and a mixture of 10% CAR-positive T cells and naïve T cells was used as the positive sample to be separated. 1x10⁻⁶ cells were taken from each sample. 7 CAR-T cells were magnetically separated using a biotin-labeled antibody against clone R166.A5 and anti-biotin-containing magnetic beads. The effectiveness of the magnetic separation was assessed by flow cytometry. Results showed that the proportion of CAR-positive cells in CAR-negative cells was less than 0.5% both before and after magnetic separation, essentially background noise. However, after magnetic separation, the proportion of CAR-positive cells increased from 10% in the premixed mixture to 88.6% after separation. These data indicate that the antibody is well-suited for the magnetic separation of CAR-T cells. SEQUENCE LISTING <110> Nanjing Genscript Biotechnology Co., Ltd. Qin, Xijian Sun, Liwei Wang, Wanyi Song, Guangwei <120> Anti-VHH domain antibodies and their uses <130> F20W0837PCT <150> CN201910922329.6 <151> 2019-09-27 <160> 255 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.C5 HCDR1 <400> 1 Asn Phe Ala Met Ser 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.H9 HCDR1 <400> 2 Asn Tyr Asn Met Ile 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.F2 HCDR1 <400> 3 Ser His Asp Met Ser 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.H8 HCDR1 <400> 4 Ser Phe Ala Met Ser 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.E7 HCDR1 <400> 5 Ser Phe Ala Met Ser 1 5 <210> 6 <211> 6 <212> PRT <213> Artificial sequence <220> <223> R166.G8 HCDR1 <400> 6 Asn Ile Tyr Trp Ile Cys 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.F9 HCDR1 <400> 7 Ser Asn Ala Met Gly 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.G2 HCDR1 <400> 8 Ser Asp Ala Met Ser 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.D1 HCDR1 <400> 9 Asn Tyr Asp Met Ile 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.G10 HCDR1 <400> 10 Ser Ser Ala Val Ser 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.G3 HCDR1 <400> 11 Arg Tyr Ala Met Gly 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.E5 HCDR1 <400> 12 Gly Tyr Tyr Met Ile 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.E3 HCDR1 <400> 13 Ser Cys Val Leu Ile 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.A5 HCDR1 <400> 14 Asn Tyr Thr Val Ile 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> R166.H10 HCDR1 <400> 15 Ser Tyr Trp Met Ser 1 5 <210> 16 <211> 16 <212> PRT <213> Artificial sequence <220> <223> R166.C5 HCDR2 <400> 16 Ile Ile Tyr Ser Gly Gly Thr Arg Asp Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15 <210> 17 <211> 17 <212> PRT <213> Synthetic sequence <220> <223> R166.H9 HCDR2 <400> 17 Met Ile Gly Asp Gly Asp Asp Ala Ala Trp Tyr Ala Ser Trp Ala Lys 1 5 10 15 [[ID=C]]Gly <210> 18 <211> 17 <212> PRT <213> Synthetic sequence <220> <223> R166.F2 HCDR2 <400> 18 Tyr Ile Tyr Tyr Gly Ser Gly Ser Thr Asp Tyr Ala Ser Trp Ala Glu 1 5 10 15 Gly <210> 19 <211> 17 <212> PRT <213> Synthetic sequence <220> <223> R166.H8 HCDR2 <400> 19 Ile Ile Tyr Ala Thr Gly Gly Thr Arg Asp Tyr Ala Thr Trp Ala Ala 1 5 10 15 Gly <210> 20 <211> 16 <212> PRT <213> Artificial sequence <220> <223> R166.E7 HCDR2 <400> 20 Ile Ile Tyr Thr Gly Gly Thr Arg Asp Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15 <210> twenty one <211> 18 <212> PRT <213> Artificial sequence <220> <223> R166.G8 HCDR2 <400> twenty one Cys Ile Asn Ser Gly Ser Asn Ser Tyr Thr Tyr Tyr Tyr Ala Asn Trp Val 1 5 10 15 Asp Gly <210> twenty two <211> 16 <212> PRT <213> Artificial sequence <220> <223> R166.F9 HCDR2 <400> twenty two Leu Ile Asn Ile Tyr Asp Asn Thr Tyr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> twenty three <211> 16 <212> PRT <213> Artificial sequence <220> <223> R166.G2 HCDR2 <400> twenty three Leu Ile Asn Arg Tyr Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 24 <211> 16 <212> PRT <213> Synthetic Sequence <220> <223> R166.D1 HCDR2 <400> 24 Val Ile Tyr Pro Thr Gly Thr Thr Tyr Tyr Ala Asn Trp Val Lys Gly 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Synthetic Sequence <220> <223> R166.G10 HCDR2 <400> 25 Ile Ile Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Val Asn Gly 1 5 10 15 <210> 26 <211> 16 <212> PRT <213> Synthetic Sequence <220> <223> R166.G3 HCDR2 <400> 26 Ile Ile Gly Tyr Gly Gly Asn Thr Asn Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <210> 27 <211> 16 <212> PRT <213> Synthetic Sequence <220> <223> R166.E5 HCDR2 <400> 27 Ile Val Thr Ser Ser Gly Ser Thr His Tyr Ala Ser Trp Ala Asn Gly 1 5 10 15 <210> 28 <211> 16 <212> PRT <213> Artificial sequence <220> <223> R166.E3 HCDR2 <400> 28 Phe Ile Tyr Gly Ser Gly Asn Ala Tyr Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <210> 29 <211> 16 <212> PRT <213> Artificial sequence <220> <223> R166.A5 HCDR2 <400> 29 Ile Ile Phe Gly Ser Gly Gly Thr Tyr Tyr Tyr Ala Thr Trp Ala Glu Gly 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Artificial sequence <220> <223> R166.H10 HCDR2 <400> 30 Ile Ile Ser Gly Ser Gly Ser Thr Tyr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15 <210> 31 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.C5 HCDR3 <400> 31 Asp Arg Ser Pro Asp Tyr Ser Ala Ala Phe Leu Leu 1 5 10 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> R166.H9 HCDR3 <400> 32 Tyr Leu Ser Phe Thr Arg Leu Asp Leu 1 5 <210> 33 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.F2 HCDR3 <400> 33 Gly Gly Tyr Val Gly Gly Gly Val Asp Ala Phe Asp Pro 1 5 10 <210> 34 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.H8 HCDR3 <400> 34 Asp Arg Ser Pro Asp Tyr Ser Ala Ala Phe Asn Leu 1 5 10 <210> 35 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.E7 HCDR3 <400> 35 Asp Arg Ser Pro Asp Tyr Ser Ala Ala Phe Asn Leu 1 5 10 <210> 36 <211> 14 <212> PRT <213> Artificial sequence <220> <223> R166.G8 HCDR3 <400> 36 Asp Arg Asp Ala Ala Asp Thr Ser Asp Trp Ser Leu Asn Phe 1 5 10 <210> 37 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.F9 HCDR3 <400> 37 Tyr Gly Thr Asp Ser Asp Phe Tyr Tyr Leu Asp Leu 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.G2 HCDR3 <400> 38 Tyr Gly Thr Asp Ser Asp Phe Tyr Phe Leu Asp Leu 1 5 10 <210> 39 <211> 15 <212> PRT <213> Artificial sequence <220> <223> R166.D1 HCDR3 <400> 39 Lys Pro Ile Leu Tyr Val Asp Ser Ser Gly Trp Tyr Ile Asp Leu 1 5 10 15 <210> 40 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.G10 HCDR3 <400> 40 Tyr Gly Gly Asn Ser Gly Gly Tyr Asp Ser Phe Asn Leu 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial sequence <220> <223> R166.G3 HCDR3 <400> 41 Asp Asn Lys Ser Gly Gly Asn Asn Gly Tyr Pro Tyr Tyr Gly Leu Asp 1 5 10 15 Leu <210> 42 <211> 8 <212> PRT <213> Artificial sequence <220> <223> R166.E5 HCDR3 <400> 42 Glu Gly Gly Trp Ala Phe Asp Leu 1 5 <210> 43 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.E3 HCDR3 <400> 43 Ser Gln Glu Asp Asp Ser Phe Gly Tyr Gly Phe Asn Leu 1 5 10 <210> 44 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.A5 HCDR3 <400> 44 Gly Tyr Phe Gly Asn Thr Phe Trp Ala Met Asp Pro 1 5 10 <210> 45 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.H10 HCDR3 <400> 45 Gly Asn Pro His Tyr Ser Phe Gly Phe Asn Ile 1 5 10 <210> 46 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.C5 LCDR1 <400> 46 Gln Ala Ser Glu Ser Val Tyr Ser Asn Asn His Leu Ala 1 5 10 <210> 47 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.H9 LCDR1 <400> 47 Gln Ser Ser Gln Ser Val Tyr His Asn Asn Trp Leu Ala 1 5 10 <210> 48 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.F2 LCDR1 <400> 48 Gln Ala Ser Gln Ser Ile Tyr Thr Tyr Leu Ser 1 5 10 <210> 49 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.H8 LCDR1 <400> 49 Gln Ala Ser Glu Ser Val Tyr Ser Asn Asn His Leu Ala 1 5 10 <210> 50 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.E7 LCDR1 <400> 50 Gln Ala Ser Glu Ser Val Tyr Ser Asn Asn His Leu Ala 1 5 10 <210> 51 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.G8 LCDR1 <400> 51 Gln Ser Ser Asp Ser Val Asn Asn Asp Asn Trp Leu Ala 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.F9 LCDR1 <400> 52 Gln Ala Ser Gln Asn Ile Tyr Thr Tyr Leu Ser 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.G2 LCDR1 <400> 53 Gln Ala Ser Gln Ser Ile Tyr Ser Tyr Leu Ser 1 5 10 <210> 54 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.D1 LCDR1 <400> 54 Gln Ala Ser Glu Asn Ile Asn Asn Tyr Leu Ser 1 5 10 <210> 55 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.G10 LCDR1 <400> 55 Gln Ala Ser Gln Ser Ile Tyr Ser His Leu Ser 1 5 10 <210> 56 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.G3 LCDR1 <400> 56 Gln Ala Ser Gln Ser Ile Ser Ser His Leu Ala 1 5 10 <210> 57 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.E5 LCDR1 <400> 57 Gln Ser Ser Glu Ser Val Ala Asn Ser Asn Trp Leu Ser 1 5 10 <210> 58 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.E3 LCDR1 <400> 58 Gln Ala Ser Gln Ser Ile Gly Thr Tyr Leu Ser 1 5 10 <210> 59 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.A5 LCDR1 <400> 59 Gln Ala Ser Gln Ser Ile Ser Thr Tyr Leu Ser 1 5 10 <210> 60 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.H10 LCDR1 <400> 60 Gln Ala Ser Glu Ser Ile Tyr Ser Trp Leu Ser 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.C5 LCDR2 <400> 61 Ser Ala Ser Thr Leu Glu Ser 1 5 <210> 62 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.H9 LCDR2 <400> 62 Gly Ala Ala Thr Leu Ala Ser 1 5 <210> 63 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.F2 LCDR2 <400> 63 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 64 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.H8 LCDR2 <400> 64 Ser Ala Ser Thr Leu Glu Ser 1 5 <210> 65 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.E7 LCDR2 <400> 65 Ser Ala Ser Thr Leu Glu Ser 1 5 <210> 66 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.G8 LCDR2 <400> 66 Gln Ala Ser Lys Leu Ala Ser 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.F9 LCDR2 <400> 67 Lys Ala Ser Thr Leu Ala Ser 1 5 <210> 68 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.G2 LCDR2 <400> 68 Lys Ala Ser Lys Leu Val Ser 1 5 <210> 69 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.D1 LCDR2 <400> 69 Gln Ala Ser Arg Leu Ala Ser 1 5 <210> 70 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.G10 LCDR2 <400> 70 Gly Ala Ser Thr Leu Ala Ser 1 5 <210> 71 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.G3 LCDR2 <400> 71 Tyr Ala Ser Thr Leu Ala Ser 1 5 <210> 72 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.E5 LCDR2 <400> 72 Trp Ala Ser Lys Leu Ala Ser 1 5 <210> 73 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.E3 LCDR2 <400> 73 Arg Ala Ser Thr Leu Thr Ser 1 5 <210> 74 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.A5 LCDR2 <400> 74 Gln Ala Ser Glu Leu Ala Tyr 1 5 <210> 75 <211> 7 <212> PRT <213> Artificial sequence <220> <223> R166.H10 LCDR2 <400> 75 Ser Ala Ser Tyr Leu Ala Ser 1 5 <210> 76 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.C5 LCDR3 <400> 76 Ala Gly Tyr Lys Ser Ser Asp Thr Asp Gly Thr Ser 1 5 10 <210> 77 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R166.H9 LCDR3 <400> 77 Ala Gly Val Tyr Asn Asp Asp Ser Glu Asn Ala 1 5 10 <210> 78 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.F2 LCDR3 <400> 78 Gln Gln Gly Ala Leu Ser Ser Asn Ile His Asn Thr 1 5 10 <210> 79 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.H8 LCDR3 <400> 79 Ala Gly Tyr Lys Ser Ser Asp Thr Asp Gly Thr Ser 1 5 10 <210> 80 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.E7 LCDR3 <400> 80 Ala Gly Tyr Lys Ser Ser Asp Thr Asp Gly Thr Ser 1 5 10 <210> 81 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.G8 LCDR3 <400> 81 Gln Gly Thr Gly Tyr Ser Ser Thr Trp Tyr Val Ala 1 5 10 <210> 82 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.F9 LCDR3 <400> 82 Gln Ser Asp Trp Leu Ile Ser Ser Asn Gly Asn Thr 1 5 10 <210> 83 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.G2 LCDR3 <400> 83 Gln Ser Asp Trp Leu Ile Ser Ser Asn Gly Asn Thr 1 5 10 <210> 84 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.D1 LCDR3 <400> 84 Gln Gln Gly His Ser Val Ser Asn Asp Val Gly Asn Val 1 5 10 <210> 85 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.G10 LCDR3 <400> 85 Gln Cys Thr Ala Gly Thr Ser Ile Tyr Gly Asn Ala 1 5 10 <210> 86 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.G3 LCDR3 <400> 86 His Gln Ser Tyr Ser Gly Ser Asp Val Asp Asn Thr 1 5 10 <210> 87 <211> 10 <212> PRT <213> Artificial sequence <220> <223> R166.E5 LCDR3 <400> 87 Gln Gly Gly Tyr Thr Ser Asp Arg Arg Ala 1 5 10 <210> 88 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.E3 LCDR3 <400> 88 Gln Glu Gly Tyr Ser Asp Ile Asn Val Asn Asn Ile 1 5 10 <210> 89 <211> 12 <212> PRT <213> Artificial sequence <220> <223> R166.A5 LCDR3 <400> 89 Gln Gln Gly Tyr Ser Asp Ile Asn Val Asp Asn Phe 1 5 10 <210> 90 <211> 13 <212> PRT <213> Artificial sequence <220> <223> R166.H10 LCDR3 <400> 90 Gln Tyr Asn Tyr Asp Ser Gly Asp Gly Ile Thr Asn Gly 1 5 10 <210> 91 <211> 117 <212> PRT <213> Artificial sequence <220> <223> R166.C5 HCVR <400> 91 Gln Ser Val 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 Phe Ser Leu Arg Asn Phe Ala 20 25 三十 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 3 fifty 45 Ile Ile Tyr Ser Gly Gly Thr Arg Asp Tyr Ala Thr Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg 85 90 95 Ser Pro Asp Tyr Ser Ala Ala Phe Leu Leu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 92 <211> 115 <212> PRT <213> Artificial sequence <220> <223> R166.H9 HCVR It should be noted that there may be some inaccuracies in the above translation due to the unclear or incorrect content in the original text (such as "三十" in line 18 which seems to be an incorrect character). Please double-check the original text for a more accurate translation. <400> 92 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 Asn Asn Tyr Asn 20 25 30 Met Ile Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Ile Gly 35 40 45 Met Ile Gly Asp Gly Asp Asp Ala Ala Trp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Glu Val 65 70 75 80 Thr Ser Leu Thr Thr Glu Asp Thr Ala Ile Tyr Phe Cys Ala Arg Tyr 85 90 95 Leu Ser Phe Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val Thr 100 105​​​​​​​​​​​​​​​​​​​​​​ Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Thr Ile Ser Ser His Asp 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Tyr Tyr Gly Ser Gly Ser Thr Asp Tyr Ala Ser Trp Ala Glu 50 55 60 Gly Arg Phe Thr Ile Thr Arg Asn Thr Asn Glu Asn Thr Val Thr Leu 65 70 75 80 Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Gly Gly Tyr Val Gly Gly Gly Val Asp Ala Phe Asp Pro Trp Gly 100 105 110 Pro Gly Thr Val Val Thr Val Ser Ser 115 120 <210> 94 <211> 118 <^212> PRT <213> Artificial Sequence <220> <223> R166.H8 HCVR <400> 94 Gln Ser Val 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 Phe Ser Leu Ser Ser Phe Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Ala Thr Gly Gly Thr Arg Asp Tyr Ala Thr Trp Ala Ala 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Gly Leu Lys Met 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp 85 90 95 Arg Ser Pro Asp Tyr Ser Ala Ala Phe Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 95 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> R166.E7 HCVR <400> 95 Gln Ser Val 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 Phe Ser Leu Ser Ser Phe Ala 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Thr Gly Gly Thr Arg Asp Tyr Ala Thr Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg 85 90 95 Ser Pro Asp Tyr Ser Ala Ala Phe Asn Leu Trp Gly Gln Gly Thr Leu 100 105 110 [[ID=?]]Val Thr Val Ser Ser 115 <210> 96 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> R166.G8 HCVR <400> 96 Gln Leu Leu Glu Gln Ser Gly Gly Gly Ala Glu Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Glu Leu Cys Cys Lys Ala Ser Gly Phe Ser Leu 20 25 30 Ser Asn Ile Tyr Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Thr Gly 35 40 45 Leu Glu Trp Ile Gly Cys Ile Asn Ser Gly Ser Asn Ser Tyr Thr Tyr 50 55 60 It should be noted that there seems to be an error in the original text where "[[ID=?]]" is used instead of a proper ID. I've translated it as best as possible while keeping the error intact for the purpose of following the instructions.Tyr Ala Asn Trp Val Asp Gly Arg Phe Thr Leu Ser Arg Asp Ile Asp 65 70 75 80 Gln Ser Thr Gly Cys Leu Gln Leu Asn Ser Leu Thr Ala Ala Asp Thr 85 90 95 Ala Met Tyr Tyr Cys Ala Arg Asp Arg Asp Ala Ala Asp Thr Ser Asp 100 105 110 Trp Ser Leu Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 97 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> R166.F9 HCVR <400> 97 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Lys Pro Asp Glu Thr 1 5 10 15 Leu Thr Ile Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asn Ala 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Leu Ile Asn Ile Tyr Asp Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Val Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Gly 85 90 95 Thr Asp Ser Asp Phe Tyr Tyr Leu Asp Leu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 98 <211> 117 <212> PRT<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Gly 85 90 95 Thr Asp Ser Asp Phe Tyr Phe Leu Asp Leu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 99 <211> 120 <212> PRT <213> Synthetic Sequence <220> <223> R166.D1 HCVR <400> 99 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Lys Pro Asp Glu Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr Asp 20 25 30 Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile Tyr Pro Thr Gly Thr Thr Tyr Tyr Ala Asn Trp Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Gly Leu Leu Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Lys Pro 85 90 95 Ile Leu Tyr Val Asp Ser Ser Gly Trp Tyr Ile Asp Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 100<00,01644><211> 118 <212> PRT <213> Artificial Sequence <220> <223> R166.G10 HCVR <400> 100 Gln Ser Val 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 Phe Ser Leu Ser Ser Ser Ala 20 25 30 Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Val Asn Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Gly 85 90 95 Gly Asn Ser Gly Gly Tyr Asp Ser Phe Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 101 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> R166.G3 HCVR <400> 101 Gln Ser Val 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 Arg Tyr Ala 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Gly Tyr Gly Gly Asn Thr Asn Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp 85 90 95 Asn Lys Ser Gly Gly Asn Asn Gly Tyr Pro Tyr Tyr Gly Leu Asp Leu 100 105 110 Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 102 <211> 114 <212> PRT <213> Artificial sequence <220> <223> R166.E5 HCVR <400> 102 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 Gly Tyr Tyr 20 25 30 Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Val Thr Ser Ser Gly Ser Thr His Tyr Ala Ser Trp Ala Asn Gly 50 55 60 Arg Phe Ala Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Pro Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu 85 90 95 Gly Gly Trp Ala Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 103 <211> 119 <212> PRT <213> Artificial sequence <220> <223> R166.E3 HCVR <400> 103 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 Ser Cys Val 20 25 30 Leu Ile Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile Gly 35 40 45 Phe Ile Tyr Gly Ser Gly Asn Ala Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ser 85 90 95 Gln Glu Asp Asp Ser Phe Gly Tyr Gly Phe Asn Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 104 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> R166.A5 HCVR <400> 104 Gln Ser Val 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 Phe Ser Leu Asn Asn Tyr Thr 20 25 30 Val Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Phe Gly Ser Gly Gly Thr Tyr Tyr Ala Thr Trp Ala Glu Gly 50 55 60 Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 [[ID=十八]]Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Tyr 85 90 95 Phe Gly Asn Thr Phe Trp Ala Met Asp Pro Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 105 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> R166.H10 HCVR <400> 105 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 Trp 20 25 30 It should be noted that there seems to be an error in the original text where "Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Tyr " was misspelled as "[[ID=十八]]Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Tyr " in the provided text. The above translation is based on the corrected understanding. If this is not what you intended, please clarify. Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Ser Gly Ser Gly Ser Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Asn Pro His Tyr Ser Phe Gly Phe Asn Ile Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Leu 115 <210> 106 <211> 111 <212> PRT <213> Synthetic sequence <220> <223> R166.C5 LCVR <400> 106 Ile Val Met Thr Glu Thr Pro Ser Ser Lys Ser Val Pro Val Gly Asp 1 5 10 15 Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Val Tyr Ser Asn Asn 20 25 30 His Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Lys 50 55 60 Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln 65 70 75 80 Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Ser Ser Asp 85 90 95 Thr Asp Gly Thr Ser Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 107 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> R166.H9 LCVR <400> 107[[ID=*]] * Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly Gly 1 5 10 15 Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr His Asn Asn 20 25 30 Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Ala Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys 50 55 60 It seems there is an error in the provided text as the tag ` ` is repeated as `[[ID=*]] *` in the middle. Please check and correct if necessary for a proper translation. The above translation is based on the correct parts of the text.Gly Ser Gly Ser Gly Thr Gln Phe Thr Phe Thr Ile Thr Asp Val Gln 65 70 75 80 Cys Asp Asp Val Gly Thr Tyr Tyr Cys Ala Gly Val Tyr Asn Asp Asp 85 90 95 Ser Glu Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 108 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> R166.F2 LCVR <400> 108 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 [[ID=N30]]Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Tyr Thr Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Ala Gln Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Leu Ser Ser Asn 85 90 95 Ile His Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 109 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> R166.H8 LCVR <400> 109 Ile Val Met Thr Gln Thr Pro Ser Ser Lys Ser Val Pro Val Gly Asp 1 5 10 15 Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Val Tyr Ser Asn Asn 20 25 30 His Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Lys 50 55 60 Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln 65 70 75 80 Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Ser Ser Asp 85 90 95 Thr Asp Gly Thr Ser Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 110 <211> 111 <212> PRT <213> Artificial sequence <220> <223> R166.E7 LCVR <400> 110 Ile Val Met Thr Gln Thr Pro Ser Ser Lys Ser Val Pro Val Gly Asp 1 5 10 15 Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Val Tyr Ser Asn Asn 20 25 30 His Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Lys 50 55 60 Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln 65 70 75 80 Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Ser Ser Asp 85 90 95 Thr Asp Gly Thr Ser Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> Ⅰ <211> Ⅰ <212> PRT <213> Artificial sequence <220> <223> R166.G8 LCVR <400> 111 Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly Gly 1 5 10 15 Thr Val Thr Ile Ser Cys Gln Ser Ser Asp Ser Val Asn Asn Asp Asn 20 25 30 Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln 65 70 75 80 Cys Asp Asp Ala Ala Asn Tyr Tyr Cys Gln Gly Thr Gly Tyr Ser Ser 85 90 95 Thr Trp Tyr Val Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 112 <211> 116 <212> PRT <213> Artificial Sequence <220> <22​​​​​​​Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Tyr Thr 20 25 30 Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Asp Leu Glu 65 70 75 80 Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Asp Trp Leu Ile Ser 85 90 95 Ser Asn Gly Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Thr Gly 100 105 110 Asp Pro Val Ala 115 <210> 113 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> R166.G2 LCVR <400> 113<00​​​​​​​​Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu 35 40 45 Ile Tyr Lys Ala Ser Lys Leu Val Ser Gly Val Pro Ser Arg Phe Arg 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu 65 70 75 80 Cys Ala Asp Ala Ala Ser Tyr Tyr Cys Gln Ser Asp Trp Leu Ile Ser 85 90 95 See Asn Gly Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Thr 100 105 110 <210> 114 <211> 111 <212> PRT <213> artificial sequence <220> <223> R166.D1 LCVR <400> 114 Ala Tyr Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Ser Ile Lys Cys Gln Ala Ser Glu Asn Ile Asn Asn Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Arg Leu Ala Ser Ala Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Asp Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly His Ser Val Ser Asn 85 90 95 Asp Val Gly Asn Val Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 115 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> R166.G10 LCVR <400> 115 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Tyr Ser His​​​​​​​​​​​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 Cys Thr Ala Gly Thr Ser Ile 85 90 95 Tyr Gly Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Arg 100 105 110 <210> 116 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> R166.G3 LCVR <400> 116 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile Ser Ser His 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Arg Gly Gln Pro Pro Lys Val Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Phe Cys His Gln Ser Tyr Ser Gly Ser Asp 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Arg 100 105 110 <210> 117 <211> 109 <212> PRT <213> Artificial Sequence <220> <​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<212> PRT <213> Artificial Sequence <220> <223> R166.E3 LCVR <400> 118 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile Gly Thr Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Thr Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Glu Gly Tyr Ser Asp Ile Asn 85 90 95 Val Asn Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 119 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> R166.A5 LCVR <400> 119 Ala Asn Ile Val Met Thr Gln Thr Pro Ala Ser Val Ser Gly Ala Val 1 5 10 15 Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Thr 20 25 30 Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Gln Ala Ser Glu Leu Ala Tyr Gly Val Ser Ser Arg Phe Lys 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Val Glu 65 70 75 80 Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Asp Ile [[ID=!]] 85 90 95 Asn Val Asp Asn Phe Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 120 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> R166.H10 LCVR <400> 120 Ala Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val 1 5 10 15 It should be noted that there seems to be a duplicate " " in the original text. I have translated it as is without making any adjustments to this potential error. If this is not intentional, it may need to be corrected in the original source.Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Ile Tyr Ser 20 25 30 Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Ala Ser Tyr Leu Ala Ser Gly Val Pro Ser Gln Phe Arg 50 55 60 Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu 65 70 75 80 Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Tyr Asn Tyr Asp Ser Gly 85 90 95 Asp Gly Ile Thr Asn Gly Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 121 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.C5 HCDR1 <400> 121 aactttgcaa tgagc 15 <210> 122 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.H9 HCDR1 <400> 122 aactacaaca tgatc 15 <210> 123 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.F2 HCDR1 <400> 123 agccacgaca tgagt 15 <210> 124 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.H8 HCDR1 <400> 124 agctttgcaa tgagc 15 <210> 125 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.E7 HCDR1 <400> 125 agctttgcaa tgagc 15 <210> 126 <211> 18 <212> DNA <213> Artificial sequence <220> <223> R166.G8 HCDR1 <400> 126 aatatctact ggatatgt 18 <210> 127 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.F9 HCDR1 <400> 127 agcaatgcaa tgggc 15 <210> 128 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.G2 HCDR1 <400> 128 agcgatgcga tgagc 15 <210> 129 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.D1 HCDR1 <400> 129 aactacgaca tgatc 15 <210> 130 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.G10 HCDR1 <400> 130 agctctgcag tgagc 15 <210> 131 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.G3 HCDR1 <400> 131 agatatgcaa tgggc 15 <210> 132 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.E5 HCDR1 <400> 132 ggctactaca tgatc 15 <210> 133 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.E3 HCDR1 <400> 133 agctgtgtgt tgatc 15 <210> 134 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.A5 HCDR1 <400> 134 aattatactg tcatc 15 <210> 135 <211> 15 <212> DNA <213> Artificial sequence <220> <223> R166.H10 HCDR1 <400> 135 agctactgga tgagc 15 <210> 136 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.C5 HCDR2 <400> 136 atcatttatt ctggtggtac cagggactac gcgacctggg cgaaaggc 48 <210> 137 <211> 51 <212> DNA <213> Artificial sequence <220> <223> R166.H9 HCDR2 <400> 137 atgattggtg atggtgatga tgcagcatgg tacgcgagct gggcgaaagg c 51 <210> 138 <211> 51 <212> DNA <213> The snowstorm <220> <223> R166.F2 HCDR2 <400> 138 tacatttatt atggtagtgg tagcacggac tacgcgagct gggcggaagg c <210> 139 <211> 51 <212> DNA <213> The snowstorm <220> <223> R166.H8 HCDR2 <400> 139 atcatttatg ctactggtgg taccagggac tacgcgacct gggcggcagg c <210> 140 <211> 48 <212> DNA <213> The snowstorm <220> <223> R166.E7 HCDR2 <400> 140 atcatttata ctggtggtac cagggactac gcgacctggg cgaaaggc <210> 141 <211> 54 <212> DNA <213> The snowstorm <220> <223> R166.G8 HCDR2 <400> 141 tgcattaatt ctggtagtaa ttcttatact tactacgcga actgggtgga tggc <210> 142 <211> 48 <212> DNA <213> The snowstorm <220> <223> R166.F9 HCDR2 <400> 142 ctcatcaata tttatgataa cacatactac gcgagctggg cgaaaggc 48 <210> 143 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.G2 HCDR2 <400> 143 ctcatcaatc gttatggtaa cacatactac gcgagctggg cgaaaggc 48 <210> 144 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.D1 HCDR2 <400> 144 gtcatttatc ctactggtac cacatactac gcgaactggg tgaaaggc 48 <210> 145 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.G10 HCDR2 <400> 145 atcattggta gtggtggtag cacatactac gcgagctggg tgaatggt 48 <210> 146 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.G3 HCDR2 <400> 146 atcattggtt atggtggtaa cacaaactac gcgaactggg cgaaaggc 48 <210> 147 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.E5 HCDR2 <400> 147 atcgttacta gtagtggtag cacacactac gcgagctggg cgaatggt 48 <210> 148 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.E3 HCDR2 <400> 148 ttcatttatg gtagtggtaa cgcatactac gcgaactggg cgaaaggc 48 <210> 149 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.A5 HCDR2 <400> 149 atcatttttg gtagtggtgg cacatactac gcgacctggg cggaaggc 48 <210> 150 <211> 48 <212> DNA <213> Artificial sequence <220> <223> R166.H10 HCDR2 <400> 150 atcattagtg gcagtggttc cacatactac gcgacctggg cgaaaggc 48 <210> 151 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.C5 HCDR3 <400> 151 gatcgtagtc ctgattatag tgccgccttt ctcttg 36 <210> 152 <211> 27 <212> DNA <213> Artificial sequence <220> <223> R166.H9 HCDR3 <400> 152 tatcttagtt tcactcggtt ggatctc 27 <210> 153 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.F2 HCDR3 <400> 153 ggtggttatg ttggtggtgg tgttgatgct tttgatccc 39 <210> 154 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.H8 HCDR3 <400> 154 gatcgtagtc ctgattatag tgccgccttt aacttg 36 <210> 155 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.E7 HCDR3 <400> 155 gatcgtagtc ctgattatag tgccgccttt aacttg 36 <210> 156 <211> 42 <212> DNA <213> Artificial sequence <220> <223> R166.G8 HCDR3 <400> 156 gatcgggatg ctgctgatac tagtgattgg tcacttaact tc 42 <210> 157 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.F9 HCDR3 <400> 157 tatggtactg atagtgattt ttattatctc gacttg 36 <210> 158 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.G2 HCDR3 <400> 158 tatggtactg atagtgattt ttattttctc gacttg 36 <210> 159 <211> 45 <212> DNA <213> Artificial sequence <220> <223> R166.D1 HCDR3 <400> 159 aaacccatct tatatgttga tagtagtggt tggtatatcg acttg 45 <210> 160 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.G10 HCDR3 <400> 160 tatggtggta atagtggtgg ttatgattcc tttaacttg 39 <210> 161 <211> 51 <212> DNA <213> Synthetic sequence <220> <223> R166.G3 HCDR3 <400> 161 gataataaaa gtggtggtaa taatggttac ccctactacg gcttggacct c 51 <210> 162 <211> 24 <212> DNA <213> Synthetic sequence <220> <223> R166.E5 HCDR3 <400> 162 gagggtggtt gggcttttga cttg 24 <210> 163 <211> 39 <212> DNA <213> Synthetic sequence <220> <223> R166.E3 HCDR3 <400> 163 tcccaagagg atgatagttt tggttatggc tttaacttg 39 <210> 164 <211> 36 <212> DNA <213> Synthetic sequence <220> <223> R166.A5 HCDR3 <400> 164 ggttattttg gtaatacttt ttgggccatg gacccc 36 <210> 165 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.H10 HCDR3 <400> 165 ggaaatcctc attatagttt tggttttaat atc 33 <210> 166 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.C5 LCDR1 <400> 166 caggccagtg agagtgttta tagtaataac cacttagcc 39 <210> 167 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.H9 LCDR1 <400> 167 cagtccagtc agagtgttta tcataacaac tggttagcc 39 <210> 168 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.F2 LCDR1 <400> 168 caggccagtc agagcattta cacctactta tcc 33 <210> 169 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.H8 LCDR1 <400> 169 caggccagtg agagtgttta tagtaacaac cacttagcc 39 <210> 170 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.E7 LCDR1 <400> 170 caggccagtg agagtgttta tagtaacaac cacttagcc 39 <210> 171 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.G8 LCDR1 <400> 171 cagtccagtg acagcgttaa taatgacaac tggttagcc 39 <210> 172 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.F9 LCDR1 <400> 172 caggccagtc agaacattta cacctactta tcc 33 <210> 173 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.G2 LCDR1 <400> 173 caggccagtc agagcattta cagctactta tcc 33 <210> 174 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.D1 LCDR1 <400> 174 caggccagtg agaacattaa caactactta tcc 33 <210> 175 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.G10 LCDR1 <400> 175 caggccagtc agagcattta cagccacttg tcc 33 <210> 176 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.G3 LCDR1 <400> 176 caggccagtc agagcatcag tagccactta gcc 33 <210> 177 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.E5 LCDR1 <400> 177 cagtccagtg agagtgttgc taatagcaat tggttatcc 39 <210> 178 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.E3 LCDR1 <400> 178 caggccagtc agagcattgg tacctactta tcc 33 <210> 179 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.A5 LCDR1 <400> 179 caggccagtc agagcattag tacttattta tcc 33 <210> 180 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.H10 LCDR1 <400> 180 caggccagtg aaagtattta tagttggtta tcc 33 <210> 181 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.C5 LCDR2 <400> 181 tctgcatcca ctctggaatc t 21 <210> 182 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.H9 LCDR2 <400> 182 ggtgcggcca ctctggcatc t 21 <210> 183 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.F2 LCDR2 <400> 183 agggcgtcca ctctggcatc t 21 <210> 184 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.H8 LCDR2 <400> 184 tctgcgtcca ctctggaatc t 21 <210> 185 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.E7 LCDR2 <400> 185 tctgcatcca ctctggaatc t 21 <210> 186 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.G8 LCDR2 <400> 186 caggcatcca aactggcatc t 21 <210> 187 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.F9 LCDR2 <400> 187 aaggcatcca ctctggcatc t 21 <210> 188 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.G2 LCDR2 <400> 188 aaggcatcca aactggtatc t 21 <210> 189 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.D1 LCDR2 <400> 189 caggcatcca gactggcatc t 21 <210> 190 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.G10 LCDR2 <400> 190 ggtgcatcca ccctggcatc t 21 <210> 191 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.G3 LCDR2 <400> 191 tatgcgtcca ctctggcatc t 21 <210> 192 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.E5 LCDR2 <400> 192 tgggcatcca aattggcatc t 21 <210> 193 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.E3 LCDR2 <400> 193 agggcatcca ctctgacatc t 21 <210> 194 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.A5 LCDR2 <400> 194 caggcatccg aattggcata t 21 <210> 195 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> R166.H10 LCDR2 <400> 195 agtgcatcct atctggcatc t 21 <210> 196 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.C5 LCDR3 <400> 196 gcaggataca aaagtagcga tactgatggt acttct 36 <210> 197 <211> 33 <212> DNA <213> Artificial sequence <220> <223> R166.H9 LCDR3 <400> 197 gcaggcgttt ataatgatga tagtgagaat gct 33 <210> 198 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.F2 LCDR3 <400> 198 caacagggtg ctcttagtag caatattcat aacact 36 <210> 199 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.H8 LCDR3 <400> 199 gcaggataca aaagtagcga tactgatggt acttct 36 <210> 200 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.E7 LCDR3 <400> 200 gcaggataca aaagtagcga tactgatggt acttct 36 <210> 201 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.G8 LCDR3 <400> 201 caaggcactg gttatagtag tacttggtac gttgct 36 <210> 202 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.F9 LCDR3 <400> 202 caaagcgatt ggctttattag tagtaatggg aatact 36 <210> 203 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.G2 LCDR3 <400> 203 caaagcgatt ggctttattag tagtaatggg aatact 36 <210> 204 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.D1 LCDR3 <400> 204 caacagggcc atagtgttag taatgatgtt ggtaatgtt 39 <210> 205 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.G10 LCDR3 <400> 205 caatgtactg ctggtactag tatttatggt aatgct 36 <210> 206 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.G3 LCDR3 <400> 206 caccagtctt atagtggtag tgatgttgat aatact 36 <210> 207 <211> 30 <212> DNA <213> Artificial sequence <220> <223> R166.E5 LCDR3 <400> 207 caaggcggct atactagtga tcgtcgtgct 30 <210> 208 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.E3 LCDR3 <400> 208 caagagggtt atagtgatat taatgttaat aatatt 36 <210> 209 <211> 36 <212> DNA <213> Artificial sequence <220> <223> R166.A5 LCDR3 <400> 209 cagcagggtt atagtgatat taatgtcgat aatttt 36 <210> 210 <211> 39 <212> DNA <213> Artificial sequence <220> <223> R166.H10 LCDR3 <400> 210 caatacaatt atgatagtgg tgatggtatt actaatggt 39 <210> 211 <211> 351 <212> DNA <213> Artificial sequence <220> <223> R166.C5 HCVR <400> 211 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggattctc cctaaggaac tttgcaatga gctgggtccg ccaggctcca 120 gggaagggcc tggaatggat cggaatcatt tattctggtg gtaccaggga ctacgcgacc 180 tgggcgaaag gccgattcac catctccaaa acctcgacca cggtggatct gaaaatgacc 240 agtccgacaa ctgaggacac ggccacctat ttctgtgcca gagatcgtag tcctgattat 300 agtgccgcct ttctcttgtg gggccaaggc accctggtca ccgtctcctc a 351 <210> 212 <211> 345 <212> DNA <213> Artificial sequence <220> <223> R166.H9 HCVR <400> 212 cagtccctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagcct ctggattctc cctcaataac tacaacatga tctgggtccg ccaggctcca 120 ggggaggggc tggaatggat cggaatgatt ggtgatggtg atgatgcagc atggtacgcg 180 agctgggcga aaggccgatt caccatctcc aaaacctcga ccacggtgga tctggaagtg 240 accagtctga caaccgagga cacggccatt tatttttgtg ccagatatct tagtttcact 300 cggttggatc tctggggcca gggcaccctg gtcaccgtct cctca 345 <210> 213 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> R166.F2 HCVR <400> 213 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagcct ctggattcac catcagtagc cacgacatga gttgggtccg ccaggctcca 120 gggaaggggc tggaatggat cggatacatt tattatggta gtggtagcac ggactacgcg 180 agctgggcgg aaggccgatt caccatcacc agaaacacca acgagaacac ggtgactctg 240 aaaatgacca gtctgacaac cgaggacacg gccacctatt tctgtgccag aggtggttat 300 gttggtggtg gtgttgatgc ttttgatccc tggggcccag gcaccgtggt caccgtctcc 360 tca 363 <210> 214 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> R166.H8 HCVR <400> 214 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggattctc cctaagtagc tttgcaatga gctgggtccg ccaggctcca 120 gggaagggcc tggaatggat cggaatcatt tatgctactg gtggtaccag ggactacgcg 180 acctgggcgg caggccgttt caccatctcc aaaacctcga ccacggtggg tctgaaaatg 240 accagtccga caactgagga cacggccacc tatttctgtg ccagagatcg tagtcctgat 300 tatagtgccg cctttaactt gtggggccaa ggcaccctgg tcaccgtctc ctca 354 <210> 215 <211> 351 <212> DNA <213> Artificial sequence <220> <223> R166.E7 HCVR <400> 215 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggattctc cctaagtagc tttgcaatga gctgggtccg ccaggctcca 120 gggaagggcc tggaatggat cggaatcatt tatactggtg gtaccaggga ctacgcgacc 180 tgggcgaaag gccgattcac catctccaaa acctcgacca cggtggatct gaaaatgacc 240 agtccgacaa ctgaggacac ggccacctat ttctgtgcca gagatcgtag tcctgattat 300 agtgccgcct ttaacttgtg gggccaaggc accctggtca ccgtctcctc a 351 <210> 216 <211> 384 <212> DNA <213> The snowstorm <220> <223> R166.G8 HCVR <400> 216 cagctgctgg agcagtccgg aggaggagcc gaaggaggcc tggtcaagcc tgggggatcc ctggaactct gctgcaaagc ctctggattc tcccttagta atatctactg gatatgttgg gtccgccagg ctccagggac ggggctggag tggattggat gcattaattc tggtagtaat 240. tcttatactt actacgcgaa ctgggtggat ggccgattca ctctctccag agacatcgac 300. gttgcctaca actgaacagt ctgacagccg cggacacggc catgtattat tgtgcgagag atcgggatgc tgctgatact agtgattggt cacttaactt ctggggccaa 360 ggcaccctgg tcaccgtctc ctcg 384 <210> 217 <211> 351 <212> DNA <213> The snowstorm <220> <223> R166.F9 HCVR <400> 217 cagtcgctgg aggagtccgg gggtcgcctg gtcaagcctg acgagaccct gacaatcacc 120. tgcacagtct ctggaatcga cctcagtagc aatgcaatgg gctgggtccg ccaggctcca gggaaggggc tggagtggat cggactcatc aatatttatg ataacacata ctacgcgagc 180 tgggcgaaag gccgattcac catctccaaa acctcgacca cggtggattt gaaagtgacc 240 agtctgacaa ccgaggacac ggccacctat ttctgtgcca gatatggtac tgatagtgat 300 ttttattatc tcgacttgtg gggccaaggc accctggtca ccgtctcctc a 351 <210> 218 <211> 351 <212> DNA <213> Artificial sequence <220> <223> R166.G2 HCVR <400> 218 cagtcgctgg aggagtccgg gggtcgcctg gtcaagcctg acgaaaccct gacaatcacc 60 tgcacagtct ctggaatcga cctcagtagc gatgcgatga gctgggtccg ccaggctcca 120 gggaaggggc tggaatggat cggactcatc aatcgttatg gtaacacata ctacgcgagc 180 tgggcgaaag gccgatttac catctccaaa acctcgacca cggtggattt gaaagtgacc 240 agtctgacaa ccgaggacac ggccacctat ttctgtgcca gatatggtac tgatagtgat 300 ttttattttc tcgacttgtg gggccaaggc accctggtca ccgtctcctc a 351 <210> 219 <211> 360 <212> DNA <213> Artificial sequence <220> <223> R166.D1 HCVR <400> 219 cagtcggtgg aggagtccgg cggtcgcctg gtaaagcctg acgaatccct gacactcacc 60 tgcacagtct ctggattctc cctcagcaac tacgacatga tctgggtccg ccaggctcca 120 gggaaggggc tggaatggat cggagtcatt tatcctactg gtaccacata ctacgcgaac 180 tgggtgaaag gccgattcac catctccaaa acctcgacca cggtgggtct gctcatcacc 240 agtccgacga ccgaggacac ggccacctat ttctgtgcca gaaaacccat cttatatgtt 300 gatagtagtg gttggtatat cgacttgtgg ggccaaggca ccctggtcac cgtctcctca 360 <210> 220 <211> 354 <212> DNA <213> Artificial sequence <220> <223> R166.G10 HCVR <400> 220 cagtcggtgg aggagtccgg gggtcgccta gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggattctc cctcagtagc tctgcagtga gctgggtccg ccaggctcca 120 gggaaggggc tggaatacat cggaatcatt ggtagtggtg gtagcacata ctacgcgagc 180 tgggtgaatg gtcgattcac catctccaaa acctcgacca cggtggatct gaaaatgacc 240 agtctgacag ccgcggacac ggccacctat ttctgtgcca gatatggtgg taatagtggt 300 ggttatgatt cctttaactt gtggggccaa ggcaccctgg tcaccgtctc ctca 354 <210> 221 <211> 369 <212> DNA <213> Artificial Sequence <220> [[ID=]18]<223> R166.G3 HCVR <400> 221 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggaatcga cctcagtaga tatgcaatgg gctgggtccg ccaggctcca 120 gggaaggggc tggaatacat cggaatcatt ggttatggtg gtaacacaaa ctacgcgaac 180 tgggcgaaag gccgattcac catctccaaa acctcgtcga ccacggtgga tctgaaaatg 240 accagtccga caaccgagga cacggccacc tatttctgtg ccagagataa taaaagtggt 300 ggtaataatg gttaccccta ctacggcttg gacctctggg gcccagggac cctcgtcacc 360 gtctcttca 369 <210> 222 <211> 342 <212> DNA <213> The snowstorm <220> <223> R166.E5 HCVR <400> 222 cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 120. tgcacagcct ctggattctc cctcagtggc tactacatga tctgggtccg ccaggctcca gggaaggggc tggaatacat cggaatcgtt actagtagtg gtagcacaca ctacgcgagc tgggcgaatg gtcgattcgc catctccaaa acctcgtcga ccacggtgga tctgaaaatg cccagtctga caaccgagga cacggccacc tatttctgtg ccagagaggg tggttgggct tttgacttgt ggggccaagg caccctggtc accgtctcct ca <210> 223 <211> 357 <212> DNA <213> The snowstorm <220> <223> R166.E3 HCVR <400> 223 cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc tgcacagtct ctggaatcga cctcagtagc tgtgtgttga tctgggtccg ccaggctcca gaaaaggggc tggaatggat cggattcatt tatggtagtg gtaacgcata ctacgcgaac 180 tgggcgaaag gccgattcac catctccaaa acctcgtcga ccacggtgga tctgaaaatc 240 accagtccga caaccgagga cacggccacc tatttctgtg ccagatccca agaggatgat 300 agttttggtt atggctttaa cttgtggggc caaggcaccc tggtcaccgt ctcctca 357 <210> 224 <211> 351 <212> DNA <213> Artificial sequence <220> <223> R166.A5 HCVR <400> 224 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagtct ctggattctc cctcaataat tatactgtca tctgggtccg ccaggctcca 120 gggaaggggc tggaatggat cggaatcatt tttggtagtg gtggcacata ctacgcgacc 180 tgggcggaag gccgattcac catctccaga acctcgacca cggtggatct gaaaatgacc 240 agtccgacaa ccgaggacac ggccacctat ttctgtgcca gaggttattt tggtaatact 300 ttttgggcca tggacccctg gggcccaggg accctcgtca ccgtctcttc a 351 <210> 225 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> R166.H10 HCVR <400> 225 cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60 tgcacagcct ctggattctc cctcagtagc tactggatga gctgggtccg ccaggctcca 120 gggaaggggc tggaatatat cggaatcatt agtggcagtg gttccacata ctacgcgacc 180 tgggcgaaag gccgattcac catctccaaa acctcgtcga ccacggtgga tctgaaaatc 240 accagtccga caaccgagga cacggccacc tatttctgtg ccagaggaaa tcctcattat 300 agttttggtt ttaatatctg gggcccaggc accctggtca ccgtctcctt g 351 <210> 226 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> R166.C5 LCVR <400> 226 atcgtgatga cccagactcc atcttccaag tctgtccctg tgggagacac agtcaccatc 60 aattgccagg ccagtgagag tgtttatagt aataaccact tagcctggtt tcaacagaaa 120 ccagggcagc ctcccaagct cctgatctat tctgcatcca ctctggaatc tggggtccca 180 tcgcggttca aaggcagtgg atctgggaca cagttcactc tcaccatcag cggcgtgcag 240 tgtgacgatg ctgccactta ctactgtgca ggatacaaaa gtagcgatac tgatggtact 300 tctttcggcg gagggaccga ggtggtggtc aaa 333 <210> 227 <211> 330 <212> DNA <213> Artificial sequence <220> <223> R166.H9 LCVR <400> 227 gccgtgctga cccagactcc atctcccgtg tctgcagctg tgggaggcac agtcaccatc 60 aattgccagt ccagtcagag tgtttatcat aacaactggt tagcctggta tcagcagaaa 120 ccagggcagc ctcccaagct cctgatctat ggtgcggcca ctctggcatc tggggtccca 180 tcgcggttta aaggcagtgg atctgggaca cagttcactt tcactatcac cgacgtgcag 240 tgtgacgatg ttggcactta ctactgtgca ggcgtttata atgatgatag tgagaatgct 300 ttcggcggag ggaccgaggt ggtggtcaaa 330 <210> 228 <211> 330 <212> DNA <213> Artificial sequence <220> <223> R166.F2 LCVR <400> 228 gcctatgata tgacccagac tccagcctct gtggaggtag ctgtgggagg cacagtcacc 60 atcaagtgcc aggccagtca gagcatttac acctacttat cctggtatca gcagaaacca 120 gggcagcctc ccaagctcct gatctacagg gcgtccactc tggcatctgg ggtctcatcg 180 cggttcaaag gcagtggatc tgggacagat ttcactctca ccatcagcgg cgcgcagtgt 240 gccgatgctg ccacttacta ttgtcaacag ggtgctctta gtagcaatat tcataacact 300 ttcggcggag ggaccgaggt ggtggtcaaa 330 <210> 229 <211> 333 <212> DNA <213> Artificial sequence <220> <223> R166.H8 LCVR <400> 229 atcgtgatga cccagactcc atcttccaag tctgtccctg tgggagacac agtcaccatc 60 aattgccagg ccagtgagag tgtttatagt aacaaccact tagcctggtt tcaacagaaa 120 ccagggcagc ctcccaagct cctgatctat tctgcgtcca ctctggaatc tggggtccca 180 tcgcggttca aaggcagtgg atctgggaca cagttcactc tcaccatcag cggcgtgcag 240 tgtgacgatg ctgccactta ctactgtgca ggatacaaaa gtagcgatac tgatggtact 300 tctttcggcg gagggaccga ggtggtggtc aaa 333 <210> 230 <211> 333 <212> DNA <213> Artificial sequence <220> <223> R166.E7 LCVR <400> 230 atcgtgatga cccagactcc atcttccaag tctgtccctg tgggagacac agtcaccatc 60 aattgccagg ccagtgagag tgtttatagt aacaaccact tagcctggtt tcaacagaaa 120 ccagggcagc ctcccaagct cctgatctat tctgcatcca ctctggaatc tggggtccca 180 tcgcggttca aaggcagtgg atctgggaca cagttcactc tcaccatcag cggcgtgcag 240 tgtgacgatg ctgccactta ctactgtgca ggatacaaaa gtagcgatac tgatggtact 300 tctttcggcg gagggaccga ggtggtggtc aaa 333 <210> 231 <211> 333 <212> DNA <213> Artificial sequence <220> <223> R166.G8 LCVR <400> 231 caagtgctga cccagactcc atcctccgtg tctgcagctg tgggaggcac agtcaccatc 60 caagtgctga cccagactcc atcctccgtg tctgcagctg tgggaggcac agtcaccatc 60 agttgccagt ccagtgacag cgttaataat gacaactggt tagcctggta tcagcagaaa 120 agttgccagt ccagtgacag cgttaataat gacaactggt tagcctggta tcagcagaaa 120 ccagggcagc ctcccaagct cctgatctac caggcatcca aactggcatc tggggtccca 180 ccagggcagc ctcccaagct cctgatctac caggcatcca aactggcatc tggggtccca 180 tcccggttca gcggcagtgg atctgggaca cagttcactc tcaccatcag cggcgtgcag 240 tcccggttca gcggcagtgg atctgggaca cagttcactc tcaccatcag cggcgtgcag 240 tgtgacgatg ctgccaatta ctactgtcaa ggcactggtt atagtagtac ttggtacgtt 300 tgtgacgatg ctgccaatta ctactgtcaa ggcactggtt atagtagtac ttggtacgtt 300 gctttcggcg gagggaccga ggtggtggtc aaa 333 gctttcggcg gagggaccga ggtggtggtc aaa 333 <210> 232<210> 232 <211> 348<211> 348 <212> DNA<212> DNA <213> 人工序列<213> Artificial sequence <220> <220> <223> R166.F9 LCVR <223> R166.F9 LCVR <400> 232 <400> 232 gctgacattg tgatgaccca gactccagcc tccgtgtctg aacctgtggg aggcacagtc 60 gctgacattg tgatgaccca gactccagcc tccgtgtctg aacctgtggg aggcacagtc 60 accatcaagt gccaggccag tcagaacatt tacacctact tatcctggta tcagcagaaa 120 accatcaagt gccaggccag tcagaacatt tacacctact tatcctggta tcagcagaaa 120 ccagggcagc ctcccaagct cctgatctac aaggcatcca ctctggcatc tggggtctca 180 ccagggcagc ctcccaagct cctgatctac aaggcatcca ctctggcatc tggggtctca 180 tcgcggttca aaggcagtgg atctgggaca gagttcactc tcaccatcaa cgacctggag 240 tcgcggttca aaggcagtgg atctgggaca gagttcactc tcaccatcaa cgacctggag 240 tgtgccgatg ctgccactta ctactgtcaa agcgattggc ttattagtag taatgggaat 300 actttcggcg gagggaccga ggtggtggtc acaggtgatc cagttgca 348 <210> 233 <211> 333 <212> DNA <213> Artificial sequence <220> <223> R166.G2 LCVR <400> 233 gctgacattg tgatgaccca gactccagcc tccgtgtctg aacctgtggg aggcacagtc 60 accatcaagt gccaggccag tcagagcatt tacagctact tatcctggta tcagcagaaa 120 ccagggcagc gtcccaaact cctgatctac aaggcatcca aactggtatc tggggtccca 180 tcgcggttca gaggcagtgg atctgggaca gagttcactc tcaccatcag cgacctggag 240 tgtgccgatg ctgcctctta ctactgtcaa agcgattggc ttattagtag taatgggaat 300 actttcggcg gagggaccga ggtggtggtc aca 333 <210> 234 <211> 333 <212> DNA <213> Artificial sequence <220> <223> R166.D1 LCVR <400> 234 gcctatgata tgacccagac tccatcctcc gtgtctgcag ctgtgggagg cacagtcagc 60 atcaagtgcc aggccagtga gaacattaac aactacttat cctggtatca gcagaaacca 120 gggcagcctc ccaagctcct gatctaccag gcatccagac tggcatctgc ggtcccatcg 180 cggttcaaag gcagtggatc tgggacacag ttcactctca ccatcgacga cctggagtgt 240 gccgatgctg ccacttacta ctgtcaacag ggccatagtg ttagtaatga tgttggtaat 300 gttttcggcg gagggaccga ggtggtggtc aaa 333 <210> 235 <211> 330 <212> DNA <213> Artificial sequence <220> <223> R166.G10 LCVR <400> 235 gatgttgtga tgacccagac tccagcctcc gtgtctgaac ctgtgggagg cacagtcacc 60 atcaagtgcc aggccagtca gagcatttac agccacttgt cctggtatca gcagaaacca 120 gggcagcctc ccaagctcct gatccatggt gcatccaccc tggcatctgg ggcctcatcg 180 cggtttaaag ccagtggatc tgggacagag ttcactctca ccatcagcga cctggagtgt 240 gccgatgctg ccacttacta ctgtcaatgt actgctggta ctagtattta tggtaatgct 300 ttcggcggag ggaccgaggt ggtggtcaga 330 <210> 236 <211> 330 <212> DNA <213> Artificial sequence <220> <223> R166.G3 LCVR <400> 236 gcctatgata tgacccagac tccagcctct gtggaggtac ctgtgggagg cacagtcacc 60 atcaattgcc aggccagtca gagcatcagt agccacttag cctggtatca gcagaaacga 120 gggcagcctc ccaaggtcct gatctattat gcgtccactc tggcatctgg ggtctcatcg 180 cggttcaaag gcagtggatc tgggacagag tacactctca ccattagcgg cgtggagtgt 240 gccgatgctg ccacttactt ttgtcaccag tcttatagtg gtagtgatgt tgataatact 300 ttcggcggag ggaccgaggt ggtggtcaga 330 <210> 237 <211> 327 <212> DNA <213> Artificial sequence <220> <223> R166.E5 LCVR <400> 237 caagtgctga cccagactcc attctccgtg tctacagctg tgggaggcac agtcaccatc 60 aattgccagt ccagtgagag tgttgctaat agcaattggt tatcctggta tcagcagaaa 120 ccaggacagc ctcccaagct cctgatctac tgggcatcca aattggcatc tggggtccca 180 tcgcggttca gtggcagtgg atctgggaca cagttcactc tcaccatcag cggcgtgcag 240 tgtgccgatg ctgccactta ctactgtcaa ggcggctata ctagtgatcg tcgtgctttc 300 ggcggaggga ccgaggtggt ggtcaaa 327 <210> 238 <211> 330 <212> DNA <213> Artificial sequence <220> <223> R166.E3 LCVR <400> 238 gcctatgata tgacccagac tccagcctct gtggaggtag ctgtgggagg cacagtcacc 60 atcaattgcc aggccagtca gagcattggt acctacttat cctggtatca acagaaacca 120 gggcagcctc ccaagctcct gatctacagg gcatccactc tgacatctgg ggtctcatcg 180 cggttcaaag gcagtggatc tgggacacag ttcactctca ccatcagcgg cgtggagtgt 240 gccgatgctg ccacttacta ctgtcaagag ggttatagtg atattaatgt taataatatt 300 ttcggcggag ggaccgaggt ggtggtcaaa 330 <210> 239 <211> 333 <212> DNA <213> Artificial sequence <220> <223> R166.A5 LCVR <400> 239 gccaacatcg tgatgaccca gactccagcc tccgtgtctg gagctgtggg aggcacagtc 60 accatcaagt gccaggccag tcagagcatt agtacttatt tatcctggta tcagcagaaa 120 ccagggcagc ctcccaaact cctgatttac caggcatccg aattggcata tggggtctca 180 tcgcggttca aaggcagtgg atctgggaca gagttcactc tcaccatcag cggcgtggag 240 tgtgccgatg ctgccactta ctattgtcag cagggttata gtgatattaa tgtcgataat 300 tttttcggcg gagggaccga ggtggtggtc aaa 333 <210> 240 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> R166.H10 LCVR <400> 240 gctgacattg tgatgaccca gactccagcc tccgtgtctg aacctgtggg aggcacagtc 60 accatcaatt gccaggccag tgaaagtatt tatagttggt tatcctggta tcaacagaaa 120 ccagggcagc ctcccaagct cctgatctat agtgcatcct atctggcatc tggcgtccca 180 tcacaattca gaggcagtgg atctgggaca gagtacactc tcaccatcag cgacctggag 240 tgtgccgatg ctgccactta ttactgtcaa tacaattatg atagtggtga tggtattact 300 aatggtttcg gcggagggac cgaggtggtg gtcaaa 336 <210> 241 <211> 115 <212> PRT <213> Artificial sequence <220> <223> AS718 VHH <400> 241 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Pro Ser Val Asn 20 25 30 Tyr Met Gly Trp Phe Arg Arg Ala Pro Glu Lys Gln Arg Glu Glu Val 35 40 45 Ala Ser Ile Tyr Pro Thr Gly Gly Thr Phe Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Thr Ala Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Gly Lys Trp Gly Thr Asp Tyr Trp Gly Gln Gly Thr Gln Val Ile 100 105 110 Val Ser Ser 115 <210> 242 <211> 323 <212> PRT <213> Synthetic Sequence <220> <223> HCCR <400> 242 Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly 1 5 10 15 Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn 35 40 45 Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys 65 70 75 80 Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala 85 90 95 Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly 100 105 110 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 115 120 125 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 130 135 140 Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val 145 150 155 160 Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile 165 170 175 Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly 180 185 190 Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile 195 200 205 Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val 210 215 220 Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser 225 230 235 240 Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu 245 250 255 Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala 260 265 270 Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val 275 280 285 Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met 290 295 300 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser 305 310 315 320 Pro Gly Lys <210> 243 <211> 969 <212> DNA <213> Artificial sequence <220> <223> HCCR <400> 243 gggcaaccta aggctccatc agtcttccca ctggccccct gctgcgggga cacacccagc 60 tccacggtga ccctgggctg cctggtcaaa ggctacctcc cggagccagt gaccgtgacc 120 tggaactcgg gcaccctcac caatggggta cgcaccttcc cgtccgtccg gcagtcctca 180 ggcctctact cgctgagcag cgtggtgagc gtgacctcaa gcagccagcc cgtcacctgc 240 aacgtggccc acccagccac caacaccaaa gtggacaaga ccgttgcgcc ctcgacatgc 300 agcaagccca cgtgcccacc ccctgaactc ctggggggac cgtctgtctt catcttcccc 360 420. ccaaaaccca aggacaccct catgatctca cgcacccccg aggtcacatg cgtggtggtg gacgtgagcc aggatgaccc cgaggtgcag ttcacatggt acataaacaa cgagcaggtg 540. cgcaccgccc ggccgccgct acggggagcag cagttcaaca gcacgatccg cgtggtcagc accctcccca tcgcgcacca ggactggctg aggggcaagg agttcaagtg caaagtccac 600 660. aacaaggcac tcccggcccc catcgagaa accatctcca aagccagagg gcagcccctg gagccgaagg tctacaccat gggccctccc cgggaggagc tgagcagcag gtcggtcagc 720 ctgacctgca tgatcaacgg cttctaccct tccgacatct cggtggagtg ggagaagaac 780 gggaaggcag aggacaacta caagaccacg ccggccgtgc tggacagcga cggctcctac 840 ttcctctaca gcaagctctc agtgcccacg agtgggcg cgtcttcacc 900 tgctccgtga tgcacgaggc cttgcacaac cactacacgc agaagtccat ctcccgctct ccgggtaaa <210> 244 <211> 104 <212> PRT <213> The snowstorm <220> <223> LCCR <400> 244 Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp 1 5 10 15 Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr 20 25 30 Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr 35 40 45 Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr 50 55 60 Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser 65 70 75 80 His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val 85 90 95 Gln Ser Phe Asn Arg Gly Asp Cys 100 <210> 245 <211> 312 <212> DNA <213> Artificial Sequence <220> <223> LCCR <400> 245 ggtgatccag ttgcacctac tgtcctcatc ttcccaccag ctgctgatca ggtggcaact 60 ggaacagtca ccatcgtgtg tgtggcgaat aaatactttc ccgatgtcac cgtcacctgg 120 gaggtggatg gcaccaccca aacaactggc atcgagaaca gtaaaacacc gcagaattct 180 gcagattgta cctacaacct cagcagcact ctgacactga ccagcacaca gtacaacagc 240 cacaaagagt acacctgcaa ggtgacccag ggcacgacct cagtcgtcca gagcttcaat 300 aggggtgact gt 312 <210> 246 <211> 122 <212> PRT <213> Artificial sequence <220> <223> AS154 VHH <400> 246 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Thr Asp Arg Ala Cys Gly Ser Ser Trp Leu Gly Ala Glu Ser Trp 100 105 110 Ala Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 247 <211> 121 <212> PRT <213> Artificial sequence <220> <223> AS325 VHH <400> 247 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Ile Glu Phe Thr Ile Gly 20 25 30 Val Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Thr Asn Gly Gly Arg Pro Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Leu Asp Arg Leu Phe Lys Ser Pro Asp Gly Leu Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Leu 115 120 <210> 248 <211> 128 <212> PRT <213> artificial sequence <220> <223> AS656 VHH <400> 248 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Val Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Glu Asn Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Ser Trp Ile Pro Arg Thr Ala Tyr Ser Thr Thr Tyr Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Asp Asn Ser Lys 65 70 75 80 Asn Thr Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Ala Ala Gly Gly Ala Thr Gly Pro Leu Ala Leu Asp 100 105 110 Ser His Tyr Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 249 <211> 124 <212> PRT <213> Synthetic sequence <220> <223> AS673 VHH <400> 249 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ile Thr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Ser Gly Ser Met Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Arg Gly Ala Ile Ala Pro Ile Ala Gln Ser Val Tyr Thr 100 105 110 Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 250 <211> 116 <212> PRT <213> Artificial sequence <220> <223> AS587 VHH <400> 250 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Ala 20 25 30 Ala Met Ser Trp Val Arg Gln Val Pro Glu Glu Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Asp Ser Ser Gly Ser Arg Thr Tyr Tyr Ala Gly Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Asp His Met Ser Trp Leu Pro Arg Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115<0003四五四><210> 251 <211> 122 <212> PRT <213> Artificial sequence <220> It should be noted that the tag seems to be misformatted as <0003四五四> in the original. I've translated it as based on the context, but this might need to be double - checked with the correct source. <223> AS588 VHH <400> 251 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Asp Ser Ser Tyr 20 25 30 Cys Gly Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ile Ile Asp Arg Tyr Gly Gly Thr Met Tyr Lys Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ala Lys Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Ala Glu Tyr Arg Gly Ser Ser Cys Asp Ala Glu Ser Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 252 <211> 124 <212> PRT <213> artificial sequence <220> <223> AS200 VHH <400> 252 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Leu Ser Tyr 20 25 30 Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Thr Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Arg Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Arg Thr Ile Ala Thr Val Pro Asn Lys Tyr Glu Tyr Asp 100 105 110 Thr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 253 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> AS988 VHH <400> 253 Gln Val Gln Leu Ala Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Val Ala Ala Ser Gly Tyr 20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val 35 40 45 Ala Ala Ile Ser Ser Asn Asp Leu Val Ala Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Thr Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Gly Gly Tyr Gly Gly Tyr Cys Gly Arg Leu Arg Pro Gly Thr 100 105 110 Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 254 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> AS675 VHH <400> 254 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Leu Thr Tyr 20 25 30 Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Thr Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Arg Trp Ser Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Arg Thr Ile Ala Thr Ile Pro Glu Lys Tyr Glu Tyr Glu 100 105 110 [[ID=2​​​​​​​​​​​​​​​​​​​​​​​20 25 30 Phe Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Val Arg Glu Gly Val 35 40 45 Ala Ala Ile Tyr Thr Gly Thr Ser Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Asp Leu Arg Asp Gly Phe Trp Asp Thr Gly Val Trp Asn Thr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120

Claims

1. An antibody or an antigen-binding fragment thereof, which specifically binds to the VHH domain of a camel-derived antibody, wherein the antibody contains a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 14, HCDR2 shown in SEQ ID NO: 29, and HCDR3 shown in SEQ ID NO: 44, and the light chain variable region comprising LCDR1 shown in SEQ ID NO: 59, LCDR2 shown in SEQ ID NO: 74, and LCDR3 shown in SEQ ID NO:

89.

2. The antibody or its antigen-binding fragment as claimed in claim 1, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 104 or an amino acid sequence having at least 90% identity with the above sequence.

3. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 119 or an amino acid sequence having at least 90% identity with the above sequence.

4. The antibody or antigen-binding fragment thereof as described in claim 1, comprising the heavy chain variable region shown in SEQ ID NO: 104 and the light chain variable region shown in SEQ ID NO:

119.

5. The antibody or its antigen-binding fragment as described in claim 1, wherein it is K D It has a binding affinity of 10 nM to 1 pM and specifically binds to the VHH domain.

6. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the camel-derived antibody is a single-domain antibody or heavy-chain antibody derived from a dromedary camel (Camelus dromedarius), a Bactrian camel (Camelus bactrianus), a llama (Vicugna pacos), or a lama (Lama glama).

7. The antibody or its antigen-binding fragment as described in claim 1, which specifically binds to the VHH domain shown in SEQ ID NO: 241 or the VHH domain having at least 60% amino acid sequence identity with SEQ ID NO:

241.

8. The antibody or antigen-binding fragment thereof as claimed in claim 1, which binds at the frame region of the VHH domain.

9. The antibody or antigen-binding fragment thereof as claimed in claim 1, which binds at a conformational epitope in the VHH domain.

10. The antibody or antigen-binding fragment thereof as claimed in claim 1, which binds at a conformational epitope in the frame region of the VHH domain.

11. The antibody or its antigen-binding fragment as claimed in claim 1, which specifically binds to the frame region of the VHH domain shown in SEQ ID NO: 241 or a frame region having at least 70% amino acid sequence identity with the frame region of the VHH domain shown in SEQ ID NO:

241.

12. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 11, wherein the antibody is selected from Fab, F(ab')2, scFv, chimeric antibodies, and humanized antibodies.

13. One or more polynucleotides encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 12.

14. One or more vectors comprising the polynucleotide as described in claim 13.

15. The vector as described in claim 14, wherein the vector is selected from cloning vectors and expression vectors.

16. A host cell comprising the polynucleotide of claim 13 or the vector of claim 14 or 15.

17. The host cell as described in claim 16, wherein the host cell is selected from prokaryotic cells, yeast cells, insect cells or mammalian cells.

18. A method for generating an antibody or an antigen-binding fragment thereof, comprising culturing a host cell of claim 16 or 17 under conditions suitable for antibody generation to express the antibody or the antigen-binding fragment thereof.

19. The method of claim 18, further comprising recovering the antibody or its antigen-binding fragment.

20. A conjugate comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 12.

21. The conjugate of claim 20, wherein the antibody or its antigen-binding fragment is conjugated with a fluorescein, biotin, enzyme, agarose resin, magnetic beads, or a biochip.

22. A method for detecting the VHH domain of a camel-derived antibody, comprising adding an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 12 or a conjugate as described in claims 20 or 21 to a sample known or suspected of containing the VHH domain of a camel-derived antibody, and detecting a complex formed between the antibody or antigen-binding fragment thereof or the conjugate and the VHH domain.

23. A method for isolating the VHH domain of a camel-derived antibody, comprising adding an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 12 or a conjugate as described in claims 20 or 21 to a sample known or suspected of containing the VHH domain of a camel-derived antibody, and isolating the complex formed between the antibody or antigen-binding fragment thereof or the conjugate and the VHH domain.

24. The method of claim 22 or 23, wherein the VHH domain is in the camel-derived antibody.

25. The method of claim 22 or 23, wherein the VHH domain is in a chimeric antigen receptor.

26. The method of claim 25, wherein the chimeric antigen receptor is on an immune cell.

27. The method of claim 26, wherein the immune cells are selected from PBMCs, T cells, NK cells or macrophages.

28. The method of claim 22 or 23, wherein the method is performed using FACS or MCS.

29. A kit comprising a container containing an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 12 and / or a conjugate as described in claim 20 or 21.

Images