A single variable domain antibody targeting human programmed death ligand 1 (PD-L1) and derivatives thereof

Abstract

The present disclosure provides a single variable domain antibody targeting human programmed death ligand 1 (PD-L1) and derivatives thereof. A phage surface display VHH antibody library is constructed by immunizing a camel with PBMCs of human PD-L1, and an anti-human PD-L1 specific single variable domain antibody 2-2F2 is obtained through screening and identification. On this basis, a chimeric antibody chF2 and a humanized antibody hzF2 mutant are prepared. The affinity of the hzF2 mutant can reach or even exceed that of the initial single variable domain antibody 2-2F2, and the hzF2 mutant can block the binding of PD-1 and PD-L1 in vitro and inhibit the growth of tumors in tumor-bearing mice in vivo.

Description

[0001] This patent application claims priority to Chinese Patent Application No. CN 202010324761.8, filed on April 22, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibody drugs, and more specifically, to monovariable domain antibodies targeting human programmed death-ligand 1 (PD-L1), their derived proteins, and their use in the preparation of drugs, particularly in the treatment and / or prevention, or diagnosis of PD-L1-related diseases such as tumors. Background Technology

[0003] PD-1 and its ligand PD-L1 are important targets in tumor immunity. PD-1 and PD-L1 are a pair of immunosuppressive molecules, crucial components of the immune system in preventing autoimmune overreaction. Activation of their pathways can suppress tumor immune responses and induce tumor-specific T cell apoptosis, thus being closely related to tumor development. PD-1 (CD279) is a type I transmembrane protein belonging to the immunoglobulin superfamily, primarily expressed on activated CD4+ T cells, CD8+ T cells, and B cells. Its ligand PD-L1 (also known as B7-H1, CD274) belongs to the B7 family and is highly expressed in tumor-infiltrating immune cells (TICs) and various malignant tumor cells, such as malignant melanoma, non-small cell lung cancer, and head and neck squamous cell carcinoma. Using monoclonal antibodies to block the PD-1 and PD-L1 pathways to treat tumors has shown good efficacy and safety in clinical practice. Several antibody drugs have been approved for marketing, with indications including melanoma, non-small cell lung cancer, advanced renal cell carcinoma and other malignant tumors. At the same time, many ongoing clinical trials are trying to develop more new indications.

[0004] Although PD-1 / PD-L1 monoclonal antibodies have shown good therapeutic effects in the clinical treatment of various malignant tumors, there are problems such as large dosage and low overall response rate. The main reasons include low PD-L1 expression and T cell depletion in the tumor microenvironment. Therefore, it is still necessary to further develop the PD-1 / PD-L1 target and develop therapeutic drugs with better clinical effects. In terms of drug development for this target, the following approaches may increase the benefits for clinical patients: (1) Further development of antibody molecules with higher affinity and better activity; (2) Bispecific antibodies or analogs based on this target; (3) Preliminary studies have shown that the response rate of PD-L1 positive tumor patients to PD-L1 inhibitors is much higher than that of PD-L1 negative tumor patients. Therefore, effective biomarkers are needed to predict PD-L1 positive tumor patients or screen patients in advance to reduce treatment costs and possible serious adverse reactions; (4) In order to improve the response rate, combination therapy has become a development trend in tumor immunotherapy, such as combination with other tumor immunotherapies, and combination with targeted drugs, chemotherapy or radiotherapy.

[0005] Monovariable domain antibodies (MDVAs) are currently the smallest antibody molecules, with a molecular weight one-tenth that of ordinary antibodies. Originally discovered in camel blood by Belgian scientist R. Hamers, they are a highly anticipated class of engineered antibody products. In addition to the antigenic reactivity of monoclonal antibodies, MDVAs possess unique functional characteristics such as small molecular weight, high stability, good solubility, easy expression, strong targeting, and simple humanization, making them particularly suitable for the development of bispecific / multispecific therapeutic antibodies and CAR-T / M / NK therapies. Currently, the development of MDVAs and / or bispecific / multispecific antibodies based on MDVAs has become a research hotspot. Internationally, Ablynx has made extensive investments in the field of MDVAs. Caplacizumab, developed by Ablynx, was approved by the FDA in February 2019 for the treatment of the rare disease acquired thrombotic thrombocytopenic purpura (aTTP). Acquired thrombotic thrombocytopenic purpura (ITP) is characterized by excessive blood clotting in small vessels. Caplacizumab was the first drug approved for this disease and the first to target von Willebrand factor (vWF), a key protein in the blood clotting cascade. Caplacizumab was also the first single-domain antibody approved by the FDA, marking a significant milestone in the development of single-domain antibodies for treating human diseases. Chinese companies are also actively developing single-domain antibodies, including Shenzhen Guochuang Single-domain Antibody Technology Co., Ltd., Shenzhen Prigen Biopharmaceutical Co., Ltd., Suzhou Boshengji (Anke) Co., Ltd., and Suzhou Kangning Jierui Co., Ltd. Among them, Kangning Jierui was the first in China to enter the field of single-domain antibodies. Its PD-L1 single-domain antibody (KN035, subcutaneous administration) received approval from the CFDA and FDA for clinical trials in 2016 and obtained clinical trial approval from the Japanese Pharmaceuticals and Drug Administration (PMDA) at the end of June 2017. KN035 is the world's first PD-L1 single variable domain antibody.

[0006] Currently, PD-1 / PD-L1 monoclonal antibodies have shown good therapeutic effects in the clinical treatment of various malignant tumors, but there are problems such as large dosage and low overall response rate. The main reasons include low PD-L1 expression and T cell depletion in the tumor microenvironment. Therefore, it is necessary to discover new anti-PD-L1 antibody drugs. In terms of candidate molecule development, the following approaches may be used to obtain antibody molecules with better therapeutic effects: (1) further develop antibody molecules with higher affinity and better activity; (2) bispecific antibodies or analogs based on this target; (3) develop more effective diagnostic antibodies to predict PD-L1 positive tumor patients or screen patients in advance by detecting PD-L1 expression, so as to reduce treatment costs and possible serious adverse reactions. Camel-derived monovariable domain antibodies are expected to be used to solve the above problems well due to their unique properties. Summary of the Invention

[0007] To address the aforementioned issues, this disclosure provides a single-domain antibody targeting human programmed death-ligand 1 (PD-L1) and its derivatives. A phage surface-displaying VHH antibody library was constructed by extracting PBMCs from camels immunized with human PD-L1. Anti-human PD-L1 specific single-domain antibody 2-2F2 was screened and identified. Based on this, a chimeric antibody chF2 and a humanized mutant antibody hzF2 were prepared. The hzF2 mutant exhibits affinity equal to or even exceeding that of the initial single-domain antibody 2-2F2, blocking the binding of PD-1 and PD-L1 in vitro and inhibiting tumor growth in tumor-bearing mice in vivo. Specifically:

[0008] In a first aspect, the present invention provides an anti-PD-L1 single variable domain antibody, characterized in that CDR1-CDR3 in the variable region of the single variable domain antibody are respectively as shown in SEQ ID NO:43-45.

[0009] Furthermore, the anti-PD-L1 monovariable domain antibody of the present invention is characterized in that the monovariable domain antibody does not have a constant region or has 1-3 heavy chain constant regions.

[0010] Furthermore, the anti-PD-L1 single variable domain antibody of the present invention is characterized in that the amino acid sequence of the variable region of the single variable domain antibody is as shown in SEQ ID NO:1.

[0011] In a second aspect, the present invention provides an anti-PD-L1 monovariable domain antibody, characterized in that the monovariable domain antibody is a human-camel chimeric monovariable domain antibody, comprising the variable region and the human heavy chain constant region of the monovariable domain antibody described in the first aspect of the present invention.

[0012] Furthermore, the anti-PD-L1 monovariable domain antibody of the present invention is characterized in that the chimeric monovariable domain antibody has the amino acid sequence shown in SEQ ID NO:3.

[0013] Thirdly, the present invention provides an anti-PD-L1 single variable domain antibody, characterized in that the single variable domain antibody is a humanized single variable domain antibody, the variable region of which is obtained by humanization modification based on the variable region of the single variable domain antibody described in the first aspect of the present invention.

[0014] Furthermore, the anti-PD-L1 monovariable domain antibody of the present invention is characterized in that the variable region of the monovariable domain antibody has the amino acid sequence shown in SEQ ID NO:7.

[0015] Furthermore, the anti-PD-L1 monovariable domain antibody of the present invention is characterized in that the monovariable domain antibody has the amino acid sequence shown in SEQ ID NO:9.

[0016] Fourthly, the present invention provides an anti-PD-L1 single variable domain antibody, characterized in that the single variable domain antibody is a mutated anti-PD-L1 humanized single variable domain antibody, which is generated by mutating one, two, three or four amino acid residues in the variable region CDRs based on the anti-PD-L1 single variable domain antibody described in the third aspect of the present invention; the mutated anti-PD-L1 humanized single variable domain antibody at least partially retains the specific binding ability to PD-L1.

[0017] Furthermore, the anti-PD-L1 single variable domain antibody of the present invention is characterized in that its variable region is selected from the group consisting of SEQ ID NO:11-26.

[0018] Fifthly, the present invention provides a composition comprising one or more anti-PD-L1 antibodies, wherein the one or more anti-PD-L1 antibodies are selected from the group consisting of any of the anti-PD-L1 monovariable domain antibodies described in the first to fourth aspects of the present invention.

[0019] Furthermore, the composition of the present invention is characterized by further comprising a pharmaceutically acceptable carrier and being used as a pharmaceutical composition, preferably the pharmaceutical composition being an aqueous solution, an injection, or a powder injection.

[0020] In a sixth aspect, the present invention provides the use of an antibody or a fragment thereof in the preparation of a medicament for treating abnormal proliferative diseases, characterized in that: the antibody is selected from the group consisting of any of the anti-PD-L1 single variable domain antibodies described in the first to fourth aspects of the present invention.

[0021] Furthermore, in the application described in this invention, the abnormal proliferative disease includes tumors, preferably melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, colon cancer, etc.

[0022] In a seventh aspect, the present invention provides the use of an antibody or a fragment thereof in the preparation of a multispecific antibody or an antibody-targeted drug, characterized in that: the antibody is selected from the group consisting of any of the anti-PD-L1 single variable domain antibodies described in the first to fourth aspects of the present invention.

[0023] Eighthly, the present invention provides a polynucleotide encoding an anti-PD-L1 monovariable domain antibody as described in any of the first to fourth aspects of the present invention.

[0024] In a ninth aspect, the present invention provides a carrier comprising the polynucleotide described in the eighth aspect of the present invention.

[0025] In a tenth aspect, the present invention provides a host cell comprising the polynucleotide described in the seventh aspect of the present invention or the vector described in the eighth aspect of the present invention.

[0026] Eleventhly, the present invention provides a method for preparing an anti-PD-L1 monovariable domain antibody, comprising the following steps:

[0027] (1) The host cells described in the tenth aspect of the present invention are cultured under conditions suitable for expressing recombinant anti-PD-L1 single variable domain antibodies;

[0028] (2) Isolate and purify anti-PD-L1 monovariable domain antibody from cell culture.

[0029] Unless otherwise indicated, the term "immunoglobulin sequence" is used as a general term, whether referring to heavy chain antibodies or conventional 4-chain antibodies herein, to include full-size antibodies, their individual chains, and all their portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments such as VHH domains or VH / VL domains, respectively). Furthermore, the term "sequence" as used herein (e.g., in terms such as "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "VHH sequence," or "protein sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive interpretation.

[0030] Immunoglobulin monovariable domains can be used as “binding units,” “binding domains,” or “building units” (these terms are used interchangeably) for preparing peptides containing one or more additional immunoglobulin monovariable domains that can act as binding units (i.e., the same or different epitopes for the same target and / or for one or more different targets).

[0031] The term "immunoglobulin single variable domain" ("ISVD"), used interchangeably with "single variable domain" ("SVD"), defines a molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes an immunoglobulin ISVD from "conventional" immunoglobulins or fragments thereof, where two immunoglobulin domains, specifically two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL will favor the antigen-binding site, meaning a total of six CDRs will be involved in the formation of the antigen-binding site.

[0032] In contrast, the binding site of an immunoglobulin monovariable domain (CDR) is formed by a single VH or VL domain. Therefore, the antigen-binding site of an immunoglobulin CDR is formed by no more than three CDRs.

[0033] The terms "immunoglobulin single variable domain" and "single variable domain" therefore do not include conventional immunoglobulins or fragments thereof that require at least two variable domains to interact to form an antigen-binding site. However, these terms do include fragments of conventional immunoglobulins in which an antigen-binding site is formed via a single variable domain.

[0034] Typically, a single variable domain will be an amino acid sequence consisting essentially of four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3). Such single variable domains and fragments are most preferred, as they contain immunoglobulin folds or are capable of forming immunoglobulin folds under suitable conditions. Thus, a single variable domain may, for example, contain a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, provided that it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding unit does not need to interact with another variable domain to form a functional antigen-binding unit, as is the case, for example, for variable domains present in conventional antibodies and scFv fragments that require interaction with another variable domain, for example, via VH / VL interaction, to form a functional antigen-binding domain).

[0035] In one embodiment of the invention, the immunoglobulin single variable domain is a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence); more specifically, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0036] For example, a single variable domain or an immunoglobulin single variable domain (or an amino acid suitable for use as an immunoglobulin single variable domain) can be a (single) domain antibody (or an amino acid suitable for use as a (single) domain antibody), a “dAb” or dAb (or an amino acid suitable for use as a dAb) or a nanobody (as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment of any of them.

[0037] For a general description of (single)domain antibodies, reference is also made to the prior art cited herein and to EP0368684. The term “dAb” is used, for example, in Ward et al., 1989 (Nature 341:544-546), Holt et al., 2003 (Trends Biotechnol. 21:484-490); and in reference to, for example, WO04 / 068820, WO 06 / 030220, WO06 / 003388, WO 06 / 059108, WO 07 / 049017, WO 07 / 085815 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of this invention because they are not of mammalian origin, single variable domains can be derived from certain species of sharks (e.g., the so-called “IgNAR domain,” see, for example, WO 05 / 18629).

[0038] In particular, immunoglobulin single variable domains can be (as defined in this document) or its appropriate fragment. [Note:] [This is a registered trademark of Ablynx NV.] A general description of nanobodies is given by reference to the following further description, as well as to prior art referenced herein, such as, for example, that described in WO 08 / 020079 (page 16).

[0039] For further description of VHH and nanobodies, refer to the review article in Muyldermans 2001 (Reviews in Molecular Biotechnology) 74:277-302, and the following patent applications mentioned as general background art: Vrije Universiteit Brussel WO94 / 04678, WO95 / 04079 and WO96 / 34103; Unilever WO94 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP 1134231 and WO02 / 48193; Vlaams Instituutvoor Biotechnologie (VIB) WO97 / 49805, WO99 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP 1134231 and WO02 / 48193. WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531 of Algonomics NV and Ebolings Inc.; WO 01 / 90190 of the National Research Council of Canada; WO 03 / 025020 of the Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO The patent applications 06 / 122825 and Ebolings Inc. are also referenced. Further prior art mentioned in these applications is also referenced, and in particular the list of references mentioned on pages 41-43 of International Application WO 06 / 040153, which, together with the references, is incorporated herein by reference. As described in these references, nanobodies (especially VHH sequences and partially humanized nanobodies) may be characterized, in particular, by the presence of one or more “marker residues” in one or more framework sequences.Further descriptions of nanobodies can be found, for example, in WO08 / 101985 and WO 08 / 142164, including humanization and / or camelification of nanobodies, as well as other modifications, portions or fragments, derivatives or “nanobody fusions,” multivalent constructs (including some non-limiting examples of linker sequences), and various modifications to increase the half-life of nanobodies and their preparation.

[0040] Therefore, in the sense of this invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from non-human, preferably camelid, and more preferably camelid heavy chain antibodies. As previously described, they may be humanized. Furthermore, the term includes polypeptides derived from non-camelid sources such as mice or humans that have been "camelized," as described in Davies and Riechmann 1994 (FEBS 339:285-290), 1995 (Biotechnol. 13:475-479), 1996 (Prot. Eng. 9:531-537) and Riechmann and Muyldermans 1999 (J. Immunol. Methods 231:25-38).

[0041] The term "immunoglobulin single variable domain" encompasses immunoglobulin sequences from various sources, including mouse, rat, rabbit, donkey, human, and cameloid immunoglobulin sequences. It also includes fully human, humanized, or chimeric immunoglobulin sequences. For example, it includes cameloid immunoglobulin sequences and humanized cameloid immunoglobulin sequences, or camel-derived immunoglobulin single variable domains, such as camel-derived dAb (see, for example, WO 94 / 04678 and Davies and Riechmann 1994, 1995, and 1996) and camel-derived VH, as described by Ward et al., 1989 (see, for example, WO 94 / 04678 and Davies and Riechmann 1994, 1995, and 1996).

[0042] Similarly, such immunoglobulin single variable domains can be derived from any suitable source in any suitable manner, and can be, for example, naturally occurring VHH sequences (i.e., from suitable camelid species) or synthetic or semi-synthetic amino acid sequences, including but not limited to partially or fully “humanized” VHHs, “camel-derived” immunoglobulin sequences (and especially camel-derived VHs), and nanobodies and / or VHHs obtained by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences such as VHH sequences), CDR transplantation, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to those skilled in the art for modifying immunoglobulin sequences; or any suitable combination of the foregoing.

[0043] The amino acid sequence and structure of an immunoglobulin single variable domain can be considered—however, not limited to—to consist of four framework regions or “FRs,” referred in the art and herein as “framework region 1” or “FR1”; “framework region 2” or “FR2”; “framework region 3” or “FR3”; and “framework region 4” or “FR4”, respectively; said framework regions are separated by three complementarity-determining regions or “CDRs,” referred in the art as “complementarity-determining region 1” or “CDR1”; “complementarity-determining region 2” or “CDR2”; and “complementarity-determining region 3” or “CDR3”, respectively.

[0044] The total number of amino acid residues in the immunoglobulin single variable domain can be in the range of 110-120, preferably 112-115, and most preferably 113.

[0045] As further described in paragraphs q) on pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of the immunoglobulin monovariable domain are numbered according to the general numbering for the VH domain given by Kabat et al. (“Kabat Numbering”) (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as in Riechmann and Muyldermans 2000 (J. Immunol. Methods 240:185-195; see, for example, that publication). Figure 2 The article applies to the VHH domain of camels, and accordingly, the FR1 of the immunoglobulin monovariable domain contains amino acid residues at positions 1-30, the CDR1 of the immunoglobulin monovariable domain contains amino acid residues at positions 31-35, the FR2 of the immunoglobulin monovariable domain contains amino acid residues at positions 36-49, the CDR2 of the immunoglobulin monovariable domain contains amino acid residues at positions 50-65, the FR3 of the immunoglobulin monovariable domain contains amino acid residues at positions 66-94, the CDR3 of the immunoglobulin monovariable domain contains amino acid residues at positions 95-102, and the FR4 of the immunoglobulin monovariable domain contains amino acid residues at positions 103-113.

[0046] Based on the examples of immunoglobulin monovariable domain (CDR) sequences given herein and in WO 08 / 020079, WO 06 / 040153, and in other references cited therein concerning immunoglobulin monovariable domains, it will be clear that the precise number of amino acid residues will also depend on the length of the specific CDR present in the immunoglobulin monovariable domain. Regarding CDRs, as is well known in the art, there are various definitions and conventions for describing CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the position of the structural loop region). See, for example, http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, although reference may be made to CDRs according to Kabat, it is most preferably to define CDRs based on the Abm definition (which is based on the AbM antibody modeling software of OxfordMolecular), as this is considered the best compromise between the Kabat and Chothia definitions. See also the website http: / / www.bioinf.org.uk / abs / .

[0047] In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVSS, i.e., each of positions 109, 110, 111, 112, and 113. The invention also includes an ISVD terminated at position 109, 110, 111, or 112. In one aspect of the invention, FR4 terminates with the C-terminal amino acid sequence VTVS (positions 109-112), FR4 terminates with the C-terminal amino acid sequence VTV (positions 109-111), FR4 terminates with the C-terminal amino acid sequence VT (positions 109-110), or FR4 terminates with the C-terminal amino acid V (position 109). The C-terminal extension may be present at the C-terminus of the last amino acid residue of FR4 in the last (most C-terminal) ISVD, such as V109, T110, V111, S112, or S113, wherein the cysteine ​​moiety of the invention is preferably present or located at the C-terminus of the C-terminal extension. In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVSS and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTVSSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVS and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTVSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTV and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTVC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VT and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTC). In one embodiment, FR4 comprises the C-terminal amino acid V and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VC).

[0048] In one embodiment, the present invention relates to dimers as described herein, wherein ISVD is a light chain variable domain sequence (VL) and a heavy chain variable domain sequence (VH), derived from conventional tetrachain antibodies or derived from heavy chain antibodies.

[0049] In one embodiment, the present invention relates to dimers as described herein, wherein the ISVD is selected from the group consisting of: single-domain antibodies, domain antibodies, amino acid sequences suitable for use as single-domain antibodies, amino acid sequences suitable for use as domain antibodies, dAbs, amino acid sequences suitable for use as dAbs, nanobodies, VHHs, humanized VHHs, and camel-derived VHs. Preferably, the ISVD comprises between 100 and 140 amino acids, such as between 110 and 130 amino acids.

[0050] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0051] First, the antibody of this invention is a high-affinity humanized monovariable domain antibody against PD-L1. The humanized anti-PD-L1 monovariable domain antibody hzF2 specifically binds to human PD-L1 protein with high affinity (KD) of 1.1 nM, comparable to the control antibody KN035. These high affinity and specificity provide a theoretical basis for the inhibitory effect of hzF2 on the PD-1 / PD-L1 signaling pathway. Furthermore, compared to traditional monoclonal antibodies, monovariable domain antibodies offer greater flexibility in application and are more suitable for the development of bispecific / multispecific therapeutic antibodies.

[0052] Second, the antibody of this invention exhibits good biological activity. hzF2 can effectively bind to recombinantly expressed human PD-L1 on the cell surface, with an EC50 of 1.01 nM when binding to CHO cells (CHO-PD-L1) that recombinantly express human PD-L1; it can effectively block the binding of recombinant human PD-L1 to its receptor PD-1, with an IC50 of 4.3 nM; the blocking effect of hzF2 on the PD-1 / PD-L1 signaling pathway was detected using Jurkat-PD1-NFAT cells and CHO-PD-L1-CD3L cells reporter gene assay, and the EC50 of the activity was 5.45 nM; hzF2 has good in vivo stability and can effectively inhibit tumor growth in the humanized xenograft subcutaneous melanoma A375 model of the immune system.

[0053] Third, this invention provides multiple mutants based on hzF2. Some mutants exhibit superior performance parameters compared to the initial antibody hzF2 in terms of specificity, affinity, and other performance parameters. These different single-variable-domain antibody mutants offer more options for PD-L1-based tumor detection, targeted therapy, and drug delivery, further enriching and expanding the application potential of hzF2. Attached Figure Description

[0054] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0055] Figure 1 ELISA was used to detect the inhibitory effect of a single variable domain antibody on the binding of human PD-L1 to its receptor PD-1.

[0056] Figure 2 FACS analysis of the binding activity of chF2 with cell surface antigens.

[0057] Figure 3 ELISA analysis of the specificity of chF2 binding to recombinant PD-L1.

[0058] Figure 4 : Specificity analysis of chF2 binding to PD-L1 on cell surface.

[0059] Figure 5 Figure: Analysis results of the inhibitory effect of hzF2 on the binding of human PD-L1 to its receptor PD-1.

[0060] Figure 6 : Reporter gene system evaluation of the in vitro blocking activity of the anti-PD-L1VHH antibody Fc fusion protein.

[0061] Figure 7 Drug-time curve of hzF2 after a single dose in Balb / C mice.

[0062] Figure 8 Figure 1: Results of antitumor efficacy experiment of hzF2 on human PD-L1 transgenic mice with subcutaneous allogeneic transplantation of MC38-hPDL1 mouse colon cancer tumor model (tumor volume).

[0063] Figure 9 Figure 1: Results of antitumor efficacy experiment of hzF2 on human PD-L1 transgenic mice with subcutaneous allogeneic transplantation of MC38-hPDL1 mouse colon cancer tumor model (tumor weight). Detailed Implementation

[0064] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0065] Example 1: Construction of a phage-displayed camel single-variable-domain antibody immune library

[0066] Camels were immunized with antigens. Peripheral blood mononuclear cells (PBMCs) were isolated, and total RNA was extracted and reverse transcribed. The reverse transcription product was used as a template to amplify the variable domain of the heavy-chain antibody (VHH), which was then ligated into a phage display vector and electroporated into E. coli TG1 competent cells to construct a camel immune library. Camels were immunized every two weeks for a total of four times. Each immunization included an injection of 0.8 mg of recombinant PD-L1 extracellular protein (autologous expression and purification, gene sequence number: NP_054862.1, 19aa-238aa), adjuvanted with Freund's incomplete adjuvant (Sigma, Cat: F5506-10ml), administered via subcutaneous injection at multiple sites. Two weeks after each immunization, 1 mL of blood was collected to separate serum. Using the immunogen as the assay antigen, the titers of total antibody (IgG) and heavy-chain antibody (HcAb) in the serum were measured by ELISA. Once the serum titer met the requirements for library construction, 100 mL of camel peripheral blood was collected, and PBMCs were isolated using a separation kit (Tianjin Haoyang, Cat: TBD2011CM). Total RNA was extracted from the PBMCs, and cDNA was obtained by reverse inversion, serving as a template for subsequent amplification of the VHH fragment. Based on relevant literature and databases, camel-derived VHH antibody genes were retrieved, and primers for VHH antibody library construction were designed and synthesized. The variable region gene sequence of the antibody was amplified by PCR. Subsequently, the vector and amplified antibody fragment were digested with restriction enzymes. The ligation product was constructed using T4 ligase, and electroporation was used to transform the ligation product into TG1 bacteria. Finally, a 1.8 × 10⁻⁶ cDNA library was constructed. 8 A camel anti-human PD-L1 VHH antibody immunoassay library was used for screening specific anti-human PD-L1 single-variable-domain antibodies. To test the accuracy of the library, 50 clones were randomly selected for colony PCR, and the results showed that the insertion rate reached 100%.

[0067] Example 2: Screening of anti-human PD-L1 specific monovariable domain antibodies

[0068] The constructed camel immune library was screened using a solid-phase screening method to obtain specific phage-displaying single-variable-domain antibodies.

[0069] (1) Original library presentation. The camel immunotherapy library was transferred to 2YT medium containing ampicillin and tetracycline and cultured to the logarithmic growth phase. M13 helper phage was added, followed by kanamycin, and the culture was incubated overnight at a low temperature. The culture supernatant was collected the next day, and the phage was concentrated by PEG precipitation to obtain a high-titer antibody library presentation product for subsequent screening.

[0070] (2) Screening. Specific antibodies were screened using a solid-phase method. Specific antigens were coated onto the surface of immunotubes. After blocking the immunotubes and antibody library separately with a blocking agent, the antibody library was added to the immunotubes and incubated. The mixture was then repeatedly washed, and finally eluted with pH 2.2 acid. The eluent was neutralized and then mixed with XL-Blue in the logarithmic growth phase for infection. Phage display was then performed, and specific phage particles were recovered. Monoclonal identification was performed after 2-3 rounds of screening.

[0071] (3) Identification. XL-Blue plates containing recovered specific phage particles were infected and plated. After colonies grew, single-clone identification was performed. Single clones were picked and cultured to the logarithmic growth phase, then infected with M13 helper phage, followed by kanamycin, and incubated overnight at 30°C. The next day, the supernatant was collected and added to an ELISA plate coated with PD-L1 for ELISA. Phage particles (phage display vectors containing antibody genes) were extracted from positive clones and sequenced to determine the VHH antibody gene sequence. Five phage-displayed single-domain antibodies (VHH) capable of binding recombinant human PD-L1 protein were obtained through screening: 1-4G1, 1-6C4, 2-3D6, 2-5B7, and 2-2F2.

[0072] Example 3: Preliminary identification of anti-human PD-L1 specific monovariable domain antibody

[0073] Five single-domain VHH antibodies were induced to express in *E. coli* TG1 under the following conditions: 1 mM IPTG, 30°C, 150 rpm overnight culture. The induced bacterial samples were sonicated, filtered, purified by affinity chromatography using a nickel column, and then ultrafiltered to obtain the single-domain VHH antibodies. The inhibitory effect of the single domains on the binding of human PD-L1 to its receptor PD-1 was then detected by ELISA. Specifically, a fusion protein of the extracellular domain of human PD-1 and human Fc (PD-1-hFc, PD-1 sequence number: NP_005009.2, 21aa-167aa) at a concentration of 0.5 μg / mL was coated overnight at 4°C and blocked with 5% BSA at 37°C for 60 min. Single-domain VHH antibodies (concentrations of 50, 10, and 2 nM) were co-incubated with 1 μg / mL PD-L1-mFc at 37°C for 60 min. The plate was washed four times with PBST; then HRP-anti-mouse Fc (Jackson ImmunoResearch, Cat: 115-035-071) diluted 1:5000 was added, and the reaction was allowed to proceed for 45 min. TMB (Beijing Taitianhe Biotechnology, Cat: ME142) substrate was added for color development for 15 min, and the reaction was terminated with 2M HCl. The absorbance values ​​(A450nm-630nm) of the wells were read and recorded at a wavelength of 450nm using 630nm as the reference wavelength. The results showed that 2-2F2 effectively blocked the binding of recombinant human PD-L1 to its receptor PD-1. The results also showed that 2-2F2 has good blocking activity. Figure 1 This molecule was selected as the initial molecule for subsequent development and is abbreviated as VHH-F2. The amino acid sequence of the variable region of this single variable domain antibody is shown in SEQ ID NO.1, and the nucleotide sequence of the variable region is shown in SEQ ID NO.2.

[0074] Specific primers were designed, and using positive clone phages as templates, the variable region gene of the camel-derived antibody VHH-F2 was obtained by PCR. The variable region gene was then cloned into a eukaryotic expression vector containing the human Fc (IgG1, hFc) encoding gene via enzyme digestion and ligation. After obtaining the expression plasmid with the correct sequence, it was transfected into 293F cells for transient expression. Purification with Protein A yielded the final human-camel chimeric single-variable-domain antibody fusion protein (VHH-F2-human-Fc chimeric antibody, abbreviated as chF2). The full-length amino acid sequence of the chF2 antibody molecule is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0075] Based on the Envafolimab antibody sequence published by the WHO (WHO Drug Information, Vol. 33, No. 3, 2019, Pages 634-635, Envafolimab), the variable region gene of KN035 was fully synthesized. The amino acid sequence of the KN035 variable region is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6. Using the same strategy described above, an Fc fusion protein of KN035 in the same form as chF2 was constructed, abbreviated as KN035.

[0076] Example 4: Binding activity analysis of anti-human PD-L1 chimeric monovariable antibody

[0077] Method 1. BLI assay for binding activity

[0078] The binding ability of chF2 to recombinant antigens was determined using the Fortebio Octet QKe system instrument and an AHC (Anti-Human Antibody Capture) bioprobe to capture the Fc fragment of the anti-human antibody. During the assay, chF2 was diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (Cat: 18-0015, PALL) for 120 s. Human PD-L1 recombinant protein was used as the mobile phase to react with the antibody captured on the chip surface. The concentration of PD-L1 recombinant protein was 60 nM. The binding time for each antigen was 300 s, and the final dissociation time was 300 s. The results (Table 1) show that under the current experimental conditions, chF2 and PD-L1 recombinant protein have high affinity, comparable to that of the control antibody KN035.

[0079] Table 1. Affinity determination of chF2 and KN035 with human PD-L1 recombinant protein

[0080]

[0081] Method 2. FACS analysis of binding activity

[0082] Cells (CHO-PD-L1-CD3L cells) were collected by centrifugation and divided into 5 × 10⁻⁶ cells. 5Cells / sample / 100μL, add serially diluted monovariable domain antibody, with a final antibody concentration of 66nM (maximum concentration), 3-fold serial dilution 10 times. Incubate on ice for 2 h, wash cells twice with ice-cold PBS (containing 0.05% Tween); add FITC-labeled anti-human Fc secondary antibody (Cat.:F9512, Sigma), incubate on ice for 1 h, wash cells twice with ice-cold PBS (containing 0.05% Tween), resuspend in 200μL flow cytometry buffer, and detect mean fluorescence intensity (MFI) of cells using a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER). Results showed that chF2 and KN035 had comparable binding activity to PD-L1 expressed on the cell surface. Their half-maximal effective binding concentrations (EC50) were 1.04 nM and 1.27 nM, respectively. Figure 2 ).

[0083] Example 5: Specificity analysis of anti-human PD-L1 chimeric monovariable domain antibody

[0084] Method 1. ELISA to identify the specificity of chimeric antibodies and recombinant antigens

[0085] Human recombinant proteins (PD-L1, PD-1, B7H3, B7H4, CTLA4, CD28, ICOS, etc.) were diluted to 1 μg / mL with PBS, and 100 μL / well was used to coat ELISA plates overnight at 4°C. The plates were then blocked with 5% BSA at 37°C for 60 min, and washed three times with PBST. Chlorine F2 diluted to 1 μg / mL was added, and the reaction was carried out at 37°C for 60 min, followed by four washes with PBST. HRP-Anti-human IgG diluted 1:5000 was added, and the reaction was carried out for 45 min, followed by four washes with PBST. Finally, TMB substrate was added for color development, and the reaction was carried out at 37°C for 15 min. The reaction was terminated with 2M HCl. The absorbance (A450nm-630nm) of the plate at 450nm was read and recorded using 630nm as the reference wavelength. The results showed ( Figure 3 chF2 specifically binds to PD-L1, but does not bind to other recombinant proteins.

[0086] Method 2. BLI for identifying species specificity

[0087] Using the Fortebio Octet QKe system, the binding of chF2 to recombinant monkey and mouse PD-L1 was determined by capturing the Fc fragment of anti-human antibodies using an antibody-captured antibody (AHC) bioprobe. During the assay, chF2 was diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (PALL, Cat: 18-0015) for 120 s. Recombinant monkey and mouse PD-L1 proteins were used as the mobile phase to react with the antibodies captured on the chip surface. The concentration of PD-L1 recombinant protein was 60 nM. The binding time for each antigen was 300 s, and the final dissociation time was 300 s. The results showed that both chF2 and KN035 bound to recombinant monkey PD-L1 protein with comparable affinity, but did not bind to recombinant mouse PD-L1 protein. (Table 2)

[0088] Table 2. Binding activity analysis of chF2 and KN035 with monkey and mouse PD-L1 recombinant protein

[0089]

[0090] Method 3. FACS identification of chimeric antibody specificity

[0091] Cells were collected by centrifugation and divided into 3 × 10⁻⁶ portions. 5 Cells / sample / 100μL, add 20μg / ml monovariable domain antibody. Incubate on ice for 2h, wash cells twice with ice-cold PBS (containing 0.05% Tween); add FITC-labeled anti-human Fc secondary antibody (Sigma, F9512), incubate on ice for 1h, wash cells twice with ice-cold PBS (containing 0.05% Tween), resuspend in 200μL flow cytometry buffer, and analyze by flow cytometry. Detection results show ( Figure 4 The responsiveness of chF2 to various tumor cells was exactly the same as that of the control antibody KN035. It specifically bound to cell lines expressing human PD-L1, but not to non-expressing cell lines.

[0092] Example 6: Humanization of chF2

[0093] The human heavy chain variable region framework, which has the highest homology with the variable region of the camel antibody VHH-F2, was selected. Through CDR transplantation, while retaining some supporting structural amino acids, the variable region of VHH-F2 was humanized, and a humanized single-variable-domain antibody fusion protein (VHH-F2-human-Fc humanized antibody, abbreviated as hzF2) was designed. The amino acid sequence of the variable region is shown in SEQ ID NO.7, and the full-length amino acid sequence is shown in SEQ ID NO.9. The nucleotide sequence of the hzF2 variable region was synthesized as shown in SEQ ID NO.8, and a recombinant expression vector for hzF2 was constructed. The full-length nucleotide sequence of the hzF2 variable region is shown in SEQ ID NO.10. Subsequent eukaryotic expression was performed, and affinity was determined using the BLI method, followed by relevant analyses.

[0094] Further mutations were performed on the antibody strain hzF2 to obtain a large number of mutant antibodies. The amino acid sequences of the CDR region of the mutant sequence are shown in Table 3 below, the amino acid sequences of the variable region of the mutant are shown in Table 4 (SEQ ID NO.11~SEQ ID NO.26), the nucleotide sequences are shown in SEQ ID NO.27~SEQ ID NO.42, and the changes in binding and dissociation constants of some mutants are shown in Table 5.

[0095] Table 3. Amino acid sequence and affinity changes in the CDR region of the hzF2 mutant.

[0096]

[0097]

[0098] Table 4. Amino acid sequence of the variable region of the hzF2 mutant

[0099]

[0100]

[0101] Table 5. Affinity assay results between hzF2 mutant and human PD-L1 recombinant protein

[0102]

[0103] Example 7: ELISA detection of the inhibitory effect of hzF2 on the binding of human PD-L1 to its receptor PD-1

[0104] Human PD-1-hFc (PD-1 sequence number: NP_005009.2, 21aa-167aa) was diluted to 0.5 μg / mL with PBS, coated overnight at 4°C, and blocked with 5% BSA in a 37°C incubator for 60 min. Serial dilutions of hzF2 and control antibody KN035, as well as isotype control NC-hIgG1 (starting working concentration of 50 nM, 1.5-fold dilution in 10 concentration gradients), were added to PD-L1-mFc (PD-L1 sequence number: NP_054862.1, 19aa-238aa) at a working concentration of 0.5 μg / mL and co-incubated in a 37°C incubator for 60 min. The plate was washed four times with PBST; then HRP-anti-mouse Fc (Cat:115-035-071, Jackson Immuno Research) diluted 1:5000 was added, and the reaction was allowed to proceed for 45 min. TMB (Cat:ME142, Beijing Taitianhe Biotechnology) substrate was added for color development for 15 min, and the reaction was terminated with 2M HCl. The absorbance values ​​(A450nm-630nm) of the wells were read and recorded at a wavelength of 450 nm using 630 nm as the reference wavelength. The results showed ( Figure 5 hzF2 can effectively block the binding of recombinant human PD-L1 to its receptor PD-1. Its half-maximal inhibitory concentration (IC50) is 4.3 nM.

[0105] Example 8: Evaluation of cell blocking activity using PD-1 and PD-L1 reporter gene assay

[0106] The inhibitory effect of hzF2 on the PD-1 and PD-L1 pathways was detected using the reporter gene assay (RGA) in Jurkat-PD1-NFAT cells and CHO-PD-L1-CD3L cells. Specifically, CHO-PD-L1-CD3L cells in logarithmic growth phase were adjusted to a cell density of 5 × 10⁻⁶ cells. 5 Cells / ml were seeded at 100 μl / well and cultured overnight. Antibody samples were pre-diluted stepwise with culture medium to 20 μg / ml, then serially diluted 2-fold for a total of 10 wells. 50 μl of the diluted sample was added to each well of the overnight cultured cells. Simultaneously, 2 × 10⁶ cells / well were added. 6 Jurkat-PD1-NFAT cells at a concentration of [number] cells / ml, 50 μl / well. Incubate in a cell culture incubator for 6 h. Remove an appropriate amount of Bio-Glo [cells] 1–2 h in advance and aliquot. TM Melt the Luciferase substrate and store at room temperature in the dark. Remove the cell plate from the incubator and allow it to equilibrate to room temperature (approximately 10–15 minutes). Add 100 μl of Bio-Glo to each well of the cell plate. TMLuciferase substrate was used, and the cell plate was placed in a microplate shaker and incubated at 800 rpm in the dark for 20 min. The multi-plate reader was set to Luminescence mode, with an Intensity of 500 (instrument default value), and RLU readings were taken. Data were analyzed using SoftMax software, with sample concentration on the X-axis and the average RLU detection value on the Y-axis, and a four-parameter equation was used to plot a standard curve. The EC50 values ​​were calculated based on the curve fitting results of the reference and test samples. 50 The relative biological activity of the test sample was calculated from the value. The results are as follows: Figure 6 As shown, the blocking activity of hzF2 against PD-L1 and PD-1 is basically equivalent to that of KN035. The EC50 values ​​are hzF2 = 5.45 nM and KN035 = 4.90 nM, respectively.

[0107] Example 9: Determination of the half-life of hzF2 in mice

[0108] Healthy female nude mice, in groups of three, were injected with the antibody via the tail vein at a single dose of 15 mg / kg. Blood samples were collected from the tail vein at 2 h, 4 h, 8 h, 24 h, 48 h, 96 h, 144 h, and 196 h post-administration. Serum was separated by centrifugation and stored at -20 °C. The pharmacokinetic characteristics were then observed. After all blood collection was completed, PD-L1-His (serial number: NP_054862.1, 19aa-238aa) was coated onto a 96-well ELISA plate at 0.5ug / ml, 100ul / well, and incubated overnight at 4°C. After washing the plate three times with PBS, 5% BSA PBS was added, and the plate was blocked at 37°C for 60 min. The plate was then washed three times with PBST. Serum samples to be tested (10000, 20000-fold dilution) were added, and hzF2 standard curve wells were set up (starting concentration 0.05μg / mL, 2-fold serial dilution, 12 gradients). The plate was incubated at 37°C for 60 min, and washed four times with PBST. HRP-goat anti-human IgG (Fcr) diluted 1:5000 (Cat:109-035-098, Jackson Immuno) was added. Research), incubated at 37℃ for 40 min, washed 4 times with PBST; added TMB substrate (Cat: ME142, Beijing Taitianhe Biotechnology Co., Ltd.) for color development, incubated at 37℃ for 10 min, and then terminated the reaction with 2M HCl; using 630 nm as the reference wavelength, the absorbance (A450nm-630nm) of the plate at a wavelength of 450 nm was read and recorded. A time-antibody concentration curve was plotted with the standard antibody concentration as the Y-axis and the OD value as the X-axis, and linear fitting was performed. The results were then calculated according to the formula T... 1 / 2 =|0.693 / k|, calculate the drug half-life T 1 / 2 .

[0109] The final results show (Figure 7 Under the current conditions, the half-life of hzF2 in mice is 83.1 hours, indicating that hzF2 has a good in vivo half-life and stability.

[0110] Example 10: Detection of the antitumor efficacy of hzF2 against a human PD-L1 transgenic mouse model with subcutaneous allogeneic transplantation of MC38-hPDL1 colon cancer tumors.

[0111] MC38-hPDL1 mouse colon cancer cells, which highly express human PDL1, were subcutaneously inoculated into the right anterior flank of female B6-hPDL1 mice (C57-derived human PD-L1 transgenic mice). When the tumor grew to approximately 100 mm³, mice were divided into groups and administered hzF2, KN035, or isotype control IgG at a dose of 10 mg / kg, twice weekly for six weeks. Tumor volume and body weight were measured at each administration, and the relationship between changes in body weight and tumor volume and administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, the tumors were removed, weighed, and photographed. The relative tumor volume ratio (T / C) and tumor growth inhibition rate (1-T / C) between the treatment group and the control group were calculated and statistically analyzed. The results showed that the test drug hzF2 effectively inhibited tumor growth. Figure 8 , Figure 9 ).

[0112] The relevant amino acid and nucleotide sequences of this application are as follows:

[0113] SEQ ID NO.1: Amino acid sequence of the variable region of VHH-F2, a camel-derived single-variable-domain antibody.

[0114] QVQLQESGGGSVQTGGSLRLACAVS RDSDDGASCMG WFRQAPGKGREGVA IIFNAGERTDYGDSVKG RFTISQDNAKNTLFLQMNSLKPEDSAMYYCAT VWCGSWVARS FGQGTQVTVSS

[0115] in:

[0116] Heavy chain CDR1 amino acid (SEQ ID NO.43): RDSDDGASCMG

[0117] Heavy chain CDR2 amino acid (SEQ ID NO.44): IIFNAGERTDYGDSVKG

[0118] Heavy chain CDR3 amino acid (SEQ ID NO.45): VWCGSWVARS

[0119] SEQ ID NO.2: Nucleotide sequence of the variable region of VHH-F2, a camel-derived single-variable-domain antibody.

[0120] CAGGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGACTGGAGGGTCTCTGAGACTCGCCTGTGCAGTCTCT AGAGACAGTGACGACGGTGCCAGCTGTATGGGG TGGTTCCGCCAGGCTCCAGGGAAGGGGCGCGAGGGGGTCGCA ATCATTTTTAATGCTGGTGAACGTACCGACTATGGCGACTCCGTGAAGGGC CGATTCACCATCTCCCAAGACAACGCCAAGAACACGCTGTTTCTACAAATGAACAGCCTGAAACCTGAGGACAGTGCCATGTACTATTGTGCGACA GTT TGGTGTGGTTCTTGGGTCGCGCGTTCT TTCGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0121] in:

[0122] Heavy chain CDR1 nucleotides:

[0123] AGAGACAGTGACGACGGTGCCAGCTGTATGGGG

[0124] Heavy chain CDR2 nucleotides:

[0125] ATCATTTTTAATGCTGGTGAACGTACCGACTATGGCGACTCCGTGAAGGGC Heavy chain CDR3 nucleotides:

[0126] GTTTGGTGTGGTTCTTGGGTCGCGCGTTCT

[0127] SEQ ID NO.3: Full-length amino acid sequence of chimeric monovariable domain antibody chF2

[0128] QVQLQESGGGSVQTGGSLRLACAVS RDSDDGASCMG WFRQAPGKGREGVA IIFNAGERTDYGDSVKG RFTISQDNAKNTLFLQMNSLKPEDSAMYYCAT VWCGSWVARSFGQGTQVTVSSASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0129] in:

[0130] Heavy chain CDR1 amino acid: RDSDDGASCMG

[0131] Heavy chain CDR2 amino acid: IIFNAGERTDYGDSVKG

[0132] Heavy chain CDR3 amino acids: VWCGSWVARS

[0133] SEQ ID NO.4: Full-length nucleotide sequence of chimeric monovariable domain antibody chF2

[0134] CAGGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGACTGGAGGGTCTCTGAGACTCGCCTGTGCAGTCTCT AGAGACAGTGACGACGGTGCCAGCTGTATGGGG TGGTTCCGCCAGGCTCCAGGGAAGGGGCGCGAGGGGGTCGCA ATCATTTTTAATGCTGGTGAACGTACCGACTATGGCGACTCCGTGAAGGGC CGATTCACCATCTCCCAAGACAACGCCAAGAACACGCTGTTTCTACAAATGAACAGCCTGAAACCTGAGGACAGTGCCATGTACTATTGTGCGACA GTT TGGTGTGGTTCTTGGGTCGCGCGTTCTTTCGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCTAGCGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCTCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT

[0135] Wherein:

[0136] Heavy chain CDR1 nucleotide:

[0137] AGAGACAGTGACGACGGTGCCAGCTGTATGGGG

[0138] Heavy chain CDR② nucleotide:

[0139] ATCATTTTTAATGCTGGTGAACGTACCGACTATGGCGACTCCGTGAAGGGC

[0140] It should be noted that there is a misspelling in the original text. "重链CDR2核苷酸" should be "重链CDR2核苷酸" in Chinese, and the translated "Heavy chain CDR② nucleotide" has an incorrect "②". It should be "Heavy chain CDR2 nucleotide".Heavy chain CDR3 nucleotides:

[0141] GTTTGGTGTGGTTCTTGGGTCGCGCGTTCT

[0142] SEQ ID NO.5: KN035 variable region amino acid sequence

[0143] QVQLVESGGGLVQPGGSLRLSCAASGKMSS RRCMA WFRQAPGKERERVA K

[0144] LLTTSGSTYLADSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA DSFE

[0145] DPTCTLVTSSGAFQY WGQGTLVTVSS

[0146] Among them:

[0147] Heavy chain CDR1 amino acid: RRCMA

[0148] Heavy chain CDR2 amino acid: KLLTTSGSTYLADSVKG

[0149] Heavy chain CDR3 amino acids: DSFEDPTCTLVTSSGAFQY

[0150] SEQ ID NO.6: KN035 variable region nucleotide sequence

[0151] CAGGTGCAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCTGGCAAGATGTCCCTCCAGACGGTGC ATGGCC TGGTTCCGACAGGCTCCTGGCAAGGAGCGAGAGCGGGTGGCC AAGCTGCTGACCACCTCCGGCTCCACCTACCTGGCCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGCGAGCTGAGGACACCGCCGTGTACTACTGCGCTGCA GACTCC TTCGAGGACCCCACCTGCACCCTGGTGACCTCCTCTGGAGCCTTCCAGTAC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0152] in:

[0153] Heavy chain CDR1 nucleotides:

[0154] AGACGGTGCATGGCC

[0155] Heavy chain CDR2 nucleotides:

[0156] AAGCTGCTGACCACCTCCGGCTCCACCTACCTGGCCGACTCCGTGAAGGGA

[0157] Heavy chain CDR3 nucleotides:

[0158] GACTCCTTCGAGGACCCCACCTGCACCCTGGTGACCTCCTCTGGAGCCTTCCAGTAC

[0159] SEQ ID NO.7: Amino acid sequence of the variable region of humanized single-variable-domain antibody hzF2

[0160] EVQLVESGGGLVQPGGSLRLSCAAS RDSDDGASCMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0161] in:

[0162] Heavy chain CDR1 amino acid: RDSDDGASCMG

[0163] Heavy chain CDR2 amino acid: IIFNAGERTDYGDSVKG

[0164] Heavy chain CDR3 amino acids: VWCGSWVARS

[0165] SEQ ID NO.8: Nucleotide sequence of the variable region of humanized single-variable-domain antibody hzF2

[0166] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGACGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TGGTGTGGCTCCTGGGTGGCTCGGTCCTGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0167] in:

[0168] Heavy chain CDR1 nucleotides:

[0169] CGGGACTCCGACGACGGAGCCTCCTGCATGGGC

[0170] Heavy chain CDR2 nucleotides:

[0171] ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA

[0172] Heavy chain CDR3 nucleotides:

[0173] GTGTGGTGTGGCTCCTGGGTGGCTCGGTCC

[0174] SEQ ID NO.9: Full-length amino acid sequence of humanized monovariable domain antibody hzF2

[0175] EVQLVESGGGLVQPGGSLRLSCAASRDSDDGASCMGWFRQAPGKGLEGVAIIFNAGERTDYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATVWCGSWVARSWGQGTLVTVSSASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0176] in:

[0177] Heavy chain CDR1 amino acid: RDSDDGASCMG

[0178] Heavy chain CDR2 amino acid: IIFNAGERTDYGDSVKG

[0179] Heavy chain CDR3 amino acids: VWCGSWVARS

[0180] SEQ ID NO.10: Full-length nucleotide sequence of humanized monovariable domain antibody hzF2

[0181] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGACGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TGGTGTGGCTCCTGGGTGGCTCGGTCCTGGGGACAGGGCACCCTGGTGACCGTGTCCTCCGCTAGCGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCTCCATCTCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT

[0182] Among them:

[0183] Heavy chain CDR1 nucleotide:

[0184] CGGGACTCCGACGACGGAGCCTCCTGCATGGGC

[0185] Heavy chain CDR2 nucleotide:

[0186] ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA

[0187] Heavy chain CDR3 nucleotides:

[0188] GTGTGGTGTGGCTCCTGGGTGGCTCGGTCC

[0189] SEQ ID NO.11: Amino acid sequence of the variable region of humanized single-variable-domain antibody hzF2-m1

[0190] EVQLVESGGGLVQPGGSLRLSCAAS RDSDDGASSMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWSGSWVARS WGQGTLVTVSS

[0191] SEQ ID NO.12: Amino acid sequence of the variable region of the humanized monovariable domain antibody hzF2-m2

[0192] EVQLVESGGGLVQPGGSLRLSCAAS GDSDDGASCMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0193] SEQ ID NO.13: Amino acid sequence of the variable region of humanized single-variable-domain antibody hzF2-m3

[0194] EVQLVESGGGLVQPGGSLRLSCAAS RDSSSGASCMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0195] SEQ ID NO.14: Amino acid sequence of the variable region of humanized single-variable-domain antibody hzF2-m4

[0196] EVQLVESGGGLVQPGGSLRLSCAAS RDSNDGASCMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0197] SEQ ID NO.15: Amino acid sequence of the variable region of humanized single-variable-domain antibody hzF2-m5

[0198] EVQLVESGGGLVQPGGSLRLSCAAS RDSDDAASCMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0199] SEQ ID NO.16: Amino acid sequence of the variable region of the humanized monovariable domain antibody hzF2-m6

[0200] EVQLVESGGGLVQPGGSLRLSCAAS RDSDEGASCMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0201] SEQ ID NO.17: Amino acid sequence of the variable region of the humanized single-variable-domain antibody hzF2-m7

[0202] EVQLVESGGGLVQPGGSLRLSCAA SRDSDDSASCMG WFRQAPGKGLEGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0203] SEQ ID NO.18: Amino acid sequence of the variable region of the humanized single-variable-domain antibody hzF2-m8

[0204] EVQLVESGGGLVQPGGSLRLSCAA SRDSDDGASCMG WFRQAPGKEREGVA IIFNVGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0205] SEQ ID NO.19: Amino acid sequence of the variable region of humanized single-variable-domain antibody hzF2-m9

[0206] EVQLVESGGGLVQPGGSLRLSCAA SRDSDEGASCMG WFRQAPGKEREGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0207] SEQ ID NO.20: Amino acid sequence of the variable region of humanized monovariable domain antibody hzF2-m10

[0208] EVQLVESGGGLVQPGGSLRLSCAAS RDSDDGASCMG WFRQAPGKEREGVA IIFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VYCGSWVARS WGQGTLVTVSS

[0209] SEQ ID NO.21: Amino acid sequence of the variable region of humanized monovariable domain antibody hzF2-m11

[0210] EVQLVESGGGLVQPGGSLRLSCAASR DSDDGASCMG WFRQAPGKEREGVAI IFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VYCGSYVARS WGQGTLVTVSS

[0211] SEQ ID NO.22: Amino acid sequence of the variable region of humanized monovariable domain antibody hzF2-m12

[0212] EVQLVESGGGLVQPGGSLRLSCAASR DSDEGASCMG WFRQAPGKEREGVAI IFNVGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VWCGSWVARS WGQGTLVTVSS

[0213] SEQ ID NO.23: Amino acid sequence of the variable region of humanized monovariable domain antibody hzF2-m13

[0214] EVQLVESGGGLVQPGGSLRLSCAASR DSDDGASCMG WFRQAPGKEREGVAI IFNAGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VFCGSFVARS WGQGTLVTVSS

[0215] SEQ ID NO.24: Amino acid sequence of the variable region of humanized monovariable domain antibody hzF2-m14

[0216] EVQLVESGGGLVQPGGSLRLSCAASR DSDEGASCMG WFRQAPGKEREGVAI IFNVGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VYCGSYVARS WGQGTLVTVSS

[0217] SEQ ID NO.25: Amino acid sequence of the variable region of humanized monovariable domain antibody hzF2-m15

[0218] EVQLVESGGGLVQPGGSLRLSCAASR DSDEGASCMG WFRQAPGKEREGVAI IFNVGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VFCGSYVARS WGQGTLVTVSS

[0219] SEQ ID NO.26: Amino acid sequence of the variable region of humanized monovariable domain antibody hzF2-m16

[0220] EVQLVESGGGLVQPGGSLRLSCAASR DSDEGASCMG WFRQAPGKEREGVAI IFNVGERTDYGDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAT VFCGSFVARS WGQGTLVTVSS

[0221] SEQ ID NO.27: Nucleotide sequence of the variable region of humanized single-variable-domain antibody hzF2-m1

[0222] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCTCGGGACTCCGACGACGGAGCCTCCAGCATGGGCTGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCCATCATCTTCAACGCTGGCGAGCGGACCGAC TACGGCGACTCCGTGAAGGGACCGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACCGTGTGGTCCGGCTCCTGGGTGGCTCGGTCCTGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0223] SEQ ID NO.28: Nucleotide sequence of the variable region of the humanized single-variable-domain antibody hzF2-m2

[0224] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCTGGAGACTCCGACGACGGAGCCTCCTGCATGGGCTGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCCATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGACGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACCGTGTGGTGTGGCTCCTGGGTGGCTCGGTCCTGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0225] SEQ ID NO.29: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m3

[0226] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCTCGGGACTCCAGCAGCGGAGCCTCCTGCATGGGCTGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCCATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGACGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACCGTGTGGTGTGGCTCCTGGGTGGCTCGGTCCTGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0227] SEQ ID NO.30: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m4

[0228] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCTCGGGACTCCAACGACGGAGCCTCCTGCATGGGCTGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCCATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGACGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACCGTGTGGTGTGGCTCCTGGGTGGCTCGGTCCTGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0229] SEQ ID NO.31: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m5

[0230] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCTCGGGACTCCGACGACGCTGCCTCCTGCATGGGCTGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCCATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGACGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACCGTGTGGTGTGGCTCCTGGGTGGCTCGGTCCTGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0231] SEQ ID NO.32: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m6

[0232] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGAGGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TGGTGTGGCTCCTGGGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0233] SEQ ID NO.33: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m7

[0234] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGACTCTGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGGCCTGGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TGGTGTGGCTCCTGGGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0235] SEQ ID NO.34: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m8

[0236] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGACGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGTGGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TGGTGTGGCTCCTGGGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0237] SEQ ID NO.35: Nucleotide sequence of the variable region of the humanized single-variable-domain antibody hzF2-m9

[0238] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGAGGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TGGTGTGGCTCCTGGGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0239] SEQ ID NO.36: Nucleotide sequence of the variable region of humanized single-variable-domain antibody hzF2-m10

[0240] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGACGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TACTGTGGCTCCTGGGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0241] SEQ ID NO.37: Nucleotide sequence of the variable region of humanized single-variable-domain antibody hzF2-m11

[0242] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGACGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TACTGTGGCTCCTACGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0243] SEQ ID NO.38: Nucleotide sequence of the variable region of humanized single-variable-domain antibody hzF2-m12

[0244] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGAGGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGTGGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TGGTGTGGCTCCTGGGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0245] SEQ ID NO.39: Nucleotide sequence of the variable region of humanized single-variable-domain antibody hzF2-m13

[0246] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGACGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGCTGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TTCTGTGGCTCCTTCGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0247] SEQ ID NO.40: Nucleotide sequence of the variable region of humanized single-variable-domain antibody hzF2-m14

[0248] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGAGGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGTGGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TACTGTGGCTCCTACGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0249] SEQ ID NO.41: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m15

[0250] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGAGGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGTGGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TTCTGTGGCTCCTACGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0251] SEQ ID NO.42: Nucleotide sequence of the variable region of the humanized single variable domain antibody hzF2-m16

[0252] GAGGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCCTGCGCTGCCTCT CGGGACTCCGACGAGGGAGCCTCCTGCATGGGC TGGTTCAGGCAGGCTCCTGGCAAGGAGAGAGAGGGAGTGGCC ATCATCTTCAACGTGGGCGAGCGGACCGACTACGGCGACTCCGTGAAGGGA CGGTTCACCATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCGCTACC GTG TTCTGTGGCTCCTTCGTGGCTCGGTCC TGGGGACAGGGCACCCTGGTGACCGTGTCCTCC

[0253] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. sequence list <110> Maiwei (Shanghai) Biotechnology Co., Ltd. <120> A monovariable domain antibody targeting human programmed death-ligand 1 (PD-L1) and its derivatives <130> none <160> 45 <170> SIPOSequenceListing 1.0 <210> 1 <211> 120 <212> PRT <213> VHH-F2 <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Val Ser Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Phe Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 2 <211> 360 <212> DNA <213> VHH-F2 <400> 2 caggtgcagc tgcaggagtc tggaggaggc tcggtgcaga ctggagggtc tctgagactc 60 gcctgtgcag tctctagaga cagtgacgac ggtgccagct gtatggggtg gttccgccag 120 gctccaggga aggggcgcga gggggtcgca atcattttta atgctggtga acgtaccgac 180 tatggcgact ccgtgaaggg ccgattcacc atctcccaag acaacgccaa gaacacgctg 240 tttctacaaa tgaacagcct gaaacctgag gacagtgcca tgtactattg tgcgacagtt 300 tggtgtggtt cttgggtcgc gcgttctttc ggccagggga cccaggtcac cgtctcctca 360 <210> 3 <211> 353 <212> PRT <213> chF2‑C region <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Val Ser Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Phe Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ser Glu Pro Lys Ser Ser Asp 115 120 125 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 130 135 140 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 145 150 155 160 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 165 170 175 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 180 185 190 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 195 200 205 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 210 215 220 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 225 230 235 240 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 245 250 255 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 260 265 270 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 275 280 285 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 290 295 300 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 305 310 315 320 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 325 330 335 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 340 345 350 Gly <210> 4 <211> 1059 <212> DNA <213> chF2‐C region <400> 4 caggtgcagc tgcaggagtc tggaggaggc tcggtgcaga ctggagggtc tctgagactc 60 gcctgtgcag tctctagaga cagtgacgac ggtgccagct gtatggggtg gttccgccag 120 gctccaggga aggggcgcga gggggtcgca atcattttta atgctggtga acgtaccgac 180 tatggcgact ccgtgaaggg ccgattcacc atctcccaag acaacgccaa gaacacgctg 240 tttctacaaa tgaacagcct gaaacctgag gacagtgcca tgtactattg tgcgacagtt 300 tggtgtggtt cttgggtcgc gcgttctttc ggccagggga cccaggtcac cgtctcctca 360 gctagcgagc ccaaatctag cgacaaaact cacacatgcc caccgtgccc agcacctgaa 420 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 480 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 540 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 600 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 660 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 720 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgcctcca 780 tctcgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 840 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 900 acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac 960 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1020 aaccactaca cgcagaagag cctctccctg tccccgggt 1059 <210> 5 <211> 128 <212> PRT <213> KN035 <400> 5 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 Lys Met Ser Ser Arg Arg 20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val 35 40 45 Ala Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser 100 105 110 Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 384 <212> DNA <213> KN035 <400> 6 caggtgcagc tggtggagtc tggaggaggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctggcaa gatgtcctcc agacggtgca tggcctggtt ccgacaggct 120 cctggcaagg agcgagagcg ggtggccaag ctgctgacca cctccggctc cacctacctg 180 gccgactccg tgaagggacg gttcaccatc tccagggaca actccaagaa caccgtgtac 240 ctgcagatga actccctgcg agctgaggac accgccgtgt actactgcgc tgcagactcc 300 ttcgaggacc ccacctgcac cctggtgacc tcctctggag ccttccagta ctggggacag 360 ggcaccctgg tgaccgtgtc ctcc 384 <210> 7 <211> 120 <212> PRT <213> hzF2 <400> 7 Glu 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 Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 360 <212> DNA <213> hzF2 <400> 8 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccgacgac ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca aggggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 9 <211> 353 <212> PRT <213> hzF2‑C region <400> 9 Glu 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 Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Glu Pro Lys Ser Ser Asp 115 120 125 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 130 135 140 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 145 150 155 160 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 165 170 175 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 180 185 190 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 195 200 205 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 210 215 220 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 225 230 235 240 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 245 250 255 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 260 265 270 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 275 280 285 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 290 295 300 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 305 310 315 320 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 325 330 335 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 340 345 350 Gly <210> 10 <211> 1059 <212> DNA <213> hzF2‐C region <400> 10 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctcggga ctccgacgac ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca agggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 gctagcgagc ccaaatctag cgacaaaact cacacatgcc caccgtgccc agcacctgaa 420 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 480 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 540 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 600 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 660 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 720 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgcctcca 780 tctcgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 840 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 900 acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac 960 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1020 aaccactaca cgcagaagag cctctccctg tccccgggt 1059 <210> 11 <211> 120 <212> PRT <213> hzF2‑m1 <400> 11 Glu 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 Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Ser Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 120 <212> PRT <213> hzF2‑m2 <400> 12 Glu 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 Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 120 <212> PRT <213> hzF2‑m3 <400> 13 Glu 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 Arg Asp Ser Ser Ser Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 120 <212> PRT <213> hzF2‑m4 <400> 14 Glu 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 Arg Asp Ser Asn Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 120 <212> PRT <213> hzF2‑m5 <400> 15 Glu 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 Arg Asp Ser Asp Asp Ala Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 120 <212> PRT <213> hzF2‑m6 <400> 16 Glu 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 Arg Asp Ser Asp Glu Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 120 <212> PRT <213> hzF2‑m7 <400> 17 Glu 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 Arg Asp Ser Asp Asp Ser Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 120 <212> PRT <213> hzF2‑m8 <400> 18 Glu 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 Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Val Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 120 <212> PRT <213> hzF2‑m9 <400> 19 Glu 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 Arg Asp Ser Asp Glu Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 120 <212> PRT <213> hzF2‑m10 <400> 20 Glu 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 Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Tyr Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 21 <211> 120 <212> PRT <213> hzF2‑m11 <400> 21 Glu 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 Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Tyr Cys Gly Ser Tyr Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 120 <212> PRT <213> hzF2‑m12 <400> 22 Glu 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 Arg Asp Ser Asp Glu Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Val Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Trp Cys Gly Ser Trp Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 120 <212> PRT <213> hzF2‑m13 <400> 23 Glu 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 Arg Asp Ser Asp Asp Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Phe Cys Gly Ser Phe Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 120 <212> PRT <213> hzF2‑m14 <400> 24 Glu 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 Arg Asp Ser Asp Glu Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Val Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Tyr Cys Gly Ser Tyr Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 25 <211> 120 <212> PRT <213> hzF2‑m15 <400> 25 Glu 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 Arg Asp Ser Asp Glu Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Val Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Phe Cys Gly Ser Tyr Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 26 <211> 120 <212> PRT <213> hzF2‑m16 <400> 26 Glu 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 Arg Asp Ser Asp Glu Gly Ala 20 25 30 Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ile Ile Phe Asn Val Gly Glu Arg Thr Asp Tyr Gly Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Thr Val Phe Cys Gly Ser Phe Val Ala Arg Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 360 <212> DNA <213> hzF2-m1 <400> 27 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccgacgac ggagcctcca gcatgggctg gttcaggcag 120 gctcctggca aggggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtccggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 28 <211> 360 <212> DNA <213> hzF2-m2 <400> 28 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctggaga ctccgacgac ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca agggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 29 <211> 360 <212> DNA <213> hzF2‑m3 <400> 29 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcctg cctctcggga ctccagcagc ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca agggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 30 <211> 360 <212> DNA <213> hzF2‑m4 <400> 30 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccaacgac ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca agggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 31 <211> 360 <212> DNA <213> hzF2‑m5 <400> 31 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccgacgac gctgcctcct gcatgggctg gttcaggcag 120 gctcctggca agggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 32 <211> 360 <212> DNA <213> hzF2‑m6 <400> 32 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccgacgag ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca agggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 33 <211> 360 <212> DNA <213> hzF2‐m7 <400> 33 60. gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg tcctgcgctg cctctcggga ctccgacgac tctgcctcct gcatgggctg gttcaggcag gctcctggca agggcctgga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 240. tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 300. tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 34 <211> 360 <212> DNA <213> hzF2‐m8 <400> 34 60. gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg tcctgcgctg cctctcggga ctccgacgac ggagcctcct gcatgggctg gttcaggcag gctcctggca aggagaga gggagtggcc atcatcttca acgtgggcga gcggaccgac 180 240. tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 35 <211> 360 <212> DNA <213> hzF2‑m9 <400> 35 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccgacgag ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca aggagagaga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 36 <211> 360 <212> DNA <213> hzF2‑m10 <400> 36 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccgacgac ggagcctcct gcatgggctg gttcaggcag gctcctggca aggagaga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 240. tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 300. tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg tactgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 37 <211> 360 <212> DNA <213> hzF2‐m1 <400> 37 60. gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg tcctgcgctg cctctcggga ctccgacgac ggagcctcct gcatgggctg gttcaggcag gctcctggca aggagaga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 240. tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 300. tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg tactgtggct cctacgtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 38 <211> 360 <212> DNA <213> hzF2‐m1 <400> 38 60. gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg tcctgcgctg cctctcggga ctccgacgag ggagcctcct gcatgggctg gttcaggcag gctcctggca aggagaga gggagtggcc atcatcttca acgtgggcga gcggaccgac 180 240. tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 300. tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg tggtgtggct cctgggtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 39 <211> 360 <212> DNA <213> hzF2‐m1 <400> 39 60. gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg tcctgcgctg cctctcggga ctccgacgac ggagcctcct gcatgggctg gttcaggcag gctcctggca aggagaga gggagtggcc atcatcttca acgctggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 ttctgtggct ccttcgtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 40 <211> 360 <212> DNA <213> hzF2‑m14 <400> 40 gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg 60 tcctgcgctg cctctcggga ctccgacgag ggagcctcct gcatgggctg gttcaggcag 120 gctcctggca aggagagaga gggagtggcc atcatcttca acgtgggcga gcggaccgac 180 tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 240 tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg 300 tactgtggct cctacgtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 41 <211> 360 <212> DNA <213> hzF2‑m15 <400> 41 60. gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg tcctgcgctg cctctcggga ctccgacgag ggagcctcct gcatgggctg gttcaggcag gctcctggca aggagaga gggagtggcc atcatcttca acgtgggcga gcggaccgac 180 240. tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 300. tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg ttctgtggct cctacgtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 42 <211> 360 <212> DNA <213> hzF2‐m1 <400> 42 60. gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggaggctc cctgaggctg tcctgcgctg cctctcggga ctccgacgag ggagcctcct gcatgggctg gttcaggcag gctcctggca aggagaga gggagtggcc atcatcttca acgtgggcga gcggaccgac 180 240. tacggcgact ccgtgaaggg acggttcacc atctccaggg acaacgccaa gaacaccctg 300. tacctgcaga tgaactccct gagagccgag gacacagccg tgtactactg cgctaccgtg ttctgtggct ccttcgtggc tcggtcctgg ggacagggca ccctggtgac cgtgtcctcc 360 <210> 43 <211> 11 <212> PRT <213> VHH‑F2 CDR1 <400> 43 Arg Asp Ser Asp Asp Gly Ala Ser Cys Met Gly 1 5 10 <210> 44 <211> 17 <212> PRT <213> VHH‑F2 CDR2 <400> 44 Ile Ile Phe Asn Ala Gly Glu Arg Thr Asp Tyr Gly Asp Ser Val Lys 1 5 10 15 Gly <210> 45 <211> 10 <212> PRT <213> VHH‑F2 CDR3 <400> 45 Val Trp Cys Gly Ser Trp Val Ala Arg Ser 1 5 10

Claims

1. An anti-PD-Ll single variable domain antibody, characterized in that The CDR1-CDR3 in the variable region of the single variable domain antibody are shown in SEQ ID NO:43-45, respectively.

2. The anti-PD-Ll single variable domain antibody of claim 1, wherein The single variable domain antibody either has no constant region or has 1-3 heavy chain constant regions.

3. The anti-PD-Ll single variable domain antibody of any one of claims 1-2, wherein The amino acid sequence of the variable region of the single variable domain antibody is shown in SEQ ID NO:

1.

4. An anti-PD-Ll single variable domain antibody, characterized in that The single variable domain antibody is a human-camel chimeric single variable domain antibody, comprising the variable region and the human heavy chain constant region of any of the single variable domain antibodies of claims 1-3.

5. The anti-PD-Ll single variable domain antibody of claim 4, wherein The chimeric monovariable domain antibody has the amino acid sequence shown in SEQ ID NO:

3.

6. An anti-PD-Ll single variable domain antibody, characterized in that The single variable domain antibody is a humanized single variable domain antibody, the variable region of which is obtained by humanization modification based on the variable region of any of the single variable domain antibodies of claims 1-3, and CDR1-CDR3 in the variable region are shown in SEQ ID NO:43-45 respectively.

7. The anti-PD-Ll single variable domain antibody of claim 6, wherein The variable region of the single variable domain antibody has the amino acid sequence shown in SEQ ID NO:

7.

8. The anti-PD-Ll single variable domain antibody of claim 6 or 7, wherein The single variable domain antibody has the amino acid sequence shown in SEQ ID NO:

9.

9. An anti-PD-L1 single-variable-domain antibody, characterized in that... The single-variable-domain antibody is a mutated anti-PD-L1 humanized single-variable-domain antibody, which is generated by mutating one, two, three, or four amino acid residues in the variable region CDRs based on any one of the anti-PD-L1 single-variable-domain antibodies described in claims 6-8; the mutated anti-PD-L1 humanized single-variable-domain antibody at least partially retains the specific binding ability to PD-L1, and the CDR1-CDR3 in the variable region of the mutated anti-PD-L1 humanized single-variable-domain antibody are selected from: 1) The CDR1-CDR3 contained in the variable region of the single variable domain antibody shown in SEQ ID NO: 19; 2) CDR1-CDR3 contained in the variable region of the single variable domain antibody shown in SEQ ID NO: 24; 3) CDR1-CDR3 contained in the variable region of the single variable domain antibody shown in SEQ ID NO: 25; or 4) CDR1-CDR3 contained in the variable region of the single variable domain antibody shown in SEQ ID NO:

26.

10. The anti-PD-L1 monovariable domain antibody as described in claim 9, characterized in that... Its variable region includes: 1) The amino acid sequence as shown in SEQ ID NO: 19; 2) The amino acid sequence as shown in SEQ ID NO: 24; 3) An amino acid sequence as shown in SEQ ID NO: 25; or 4) The amino acid sequence shown in SEQ ID NO:

26.

11. A composition comprising one or more anti-PD-L1 monovariable domain antibodies, wherein the one or more anti-PD-L1 monovariable domain antibodies are selected from the group consisting of any one of the anti-PD-L1 monovariable domain antibodies of claims 1-10.

12. The composition of claim 11, characterized in that... It also contains a pharmaceutically acceptable carrier and is used as a pharmaceutical composition.

13. The composition of claim 12, wherein the pharmaceutical composition is an aqueous solution or an injection.

14. The composition of claim 12, wherein the pharmaceutical composition is a powder for injection.

15. The use of an antibody or its antigen-binding fragment in the preparation of a drug for treating abnormal proliferative diseases, characterized in that: The antibody or its antigen-binding fragment is selected from the group consisting of any one of the anti-PD-L1 single variable domain antibodies according to claims 1-10, and the abnormal proliferative disease is selected from PD-L1 positive melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, and colon cancer.

16. A polynucleotide encoding the anti-PD-L1 monovariable domain antibody of any one of claims 1-10.

17. A vector comprising the polynucleotide of claim 16.

18. A host cell comprising the polynucleotide of claim 16 or the vector of claim 17.

19. A method for preparing an anti-PD-L1 monovariable domain antibody, comprising the following steps: (1) The host cells of claim 18 are cultured under conditions suitable for expressing recombinant anti-PD-L1 monovariable domain antibody; (2) Isolate and purify anti-PD-L1 monovariable domain antibody from cell culture.

Images