Antibodies and methods of making and using same

Abstract

Antibodies with enhanced biodistribution and / or pharmacokinetics obtained by disrupting one or more surface exposed positively charged amino acid clusters are provided. Methods of enhancing the biodistribution and / or pharmacokinetics of antibodies are also provided. The enhanced antibodies can be used in immunotherapy, radiotherapy, and in vivo imaging.

Description

[0001] Any priority application cited

[0002] Any and all applications that identify foreign or domestic priority claims in the Application Data Sheet filed with the present application are incorporated herein by reference pursuant to 37 CFR 1.57.

[0003] Sequence Listing References

[0004] This application is filed with a sequence listing in electronic format. The sequence listing is provided in the form of a file titled IGNAB063WO_SEQLIST.xml, created on July 2, 2023, and 66,818 bytes in size. The entire contents of the information in the electronic sequence listing are incorporated herein by reference. Technical Field

[0005] The present disclosure generally relates to enhanced biodistribution and / or pharmacokinetics of antibodies (including minibodies and cys-diabodies) and compositions thereof. Background Art

[0006] Description of Related Technology

[0007] The present disclosure generally relates to antibodies, including antigen-binding fragments thereof, such as minibodies and cys-diabodies.Antibodies can be used to bind targets for therapeutic or diagnostic use. Summary of the invention

[0008] Provided herein is a variant antibody comprising at least one disrupted surface-exposed positively charged amino acid cluster, wherein the variant antibody differs from an original antibody comprising the original cluster by replacing at least one surface-exposed positively charged amino acid of the original cluster with a negatively or uncharged amino acid, the original cluster comprising at least two surface-exposed positively charged amino acids within about 30 angstroms of each other.

[0009] Also provided is an antibody comprising a variant polypeptide that is different from an original polypeptide comprising an original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein the original sequence is outside any CDR of the original polypeptide, wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two are surface-exposed positively charged amino acids, which amino acids form a cluster of surface-exposed positively charged amino acids in the original polypeptide, wherein the variant polypeptide differs from the original polypeptide by modifying at least one of the at least two surface-exposed positively charged amino acids of the original sequence, which modification reduces the positive charge attributed to the original sequence (e.g., reduces the positive charge by at least 33%).

[0010] Further provided herein is a variant antibody comprising at least one disrupted cluster of positively charged amino acids, wherein the variant antibody differs from an original antibody comprising the original cluster by replacing at least one positively charged amino acid of the original cluster with a negatively or uncharged amino acid, the original cluster comprising at least two positively charged amino acids within 12 residues of each other, wherein the original cluster is outside any CDR of the original antibody.

[0011] Also provided is a miniantibody or cys-diabody comprising a variant polypeptide, the variant polypeptide and a light chain variable region (V L )FR2, wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two are surface-exposed positively charged amino acids, which form a cluster of surface-exposed positively charged amino acids in the original polypeptide, wherein the variant polypeptide differs from the original polypeptide by modifying at least one of the at least two surface-exposed positively charged amino acids of the original sequence, and the modification reduces at least 33% of the positive charge attributed to the original sequence.

[0012] The present invention provides a light chain variable region (V L ) of a miniantibody or cys-diabody, the light chain variable region comprising a FR2 sequence comprising X1X2X3QAX6X7 (SEQ ID NO: 4), wherein X1 is a surface-exposed positively charged amino acid, wherein X2, X3, and X6 are each independently any negatively charged or uncharged amino acid, and wherein X7 is glutamine or lysine.

[0013] The present invention provides a light chain variable region (V L ) of a minibody or cys-diabody, the light chain variable region comprising (according to IMGT numbering): A49 and either (i) Q51; or (ii) Q48 and K51.

[0014] Also provided is a minibody comprising an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39).

[0015] Provided herein is a composition comprising: any one of the antibodies (eg, variant antibodies), minibodies, or cys-diabodies of the present disclosure; and a pharmaceutically acceptable carrier.

[0016] Also provided is a nucleic acid encoding a variant polypeptide of any of the antibodies herein (eg, variant antibodies), or any of the variant antibodies herein, or any of the minibodies or cys-diabodies of the disclosure.

[0017] Also provided is a genetically engineered host cell comprising any one or a combination of the nucleic acids disclosed herein.

[0018] Provided herein is a method for enhancing the biodistribution and / or pharmacokinetics of an antibody, comprising: identifying (identifying) an original antibody comprising a polypeptide comprising at least one surface-exposed positively charged amino acid cluster, wherein the cluster comprises at least two surface-exposed positively charged amino acids within 30 angstroms of each other; replacing at least one of the at least two surface-exposed positively charged amino acids of at least one cluster with a negatively charged or uncharged amino acid, thereby destroying at least one cluster, thereby generating a variant antibody having enhanced biodistribution and / or pharmacokinetics compared to the original antibody.

[0019] Also provided is a method for enhancing the biodistribution and / or pharmacokinetics of an antibody, comprising: identifying an original antibody comprising a polypeptide comprising at least two surface-exposed clusters of positively charged amino acids within 12 residues of each other; and replacing at least one of the at least two surface-exposed positively charged amino acids of the cluster with a negatively or uncharged amino acid to disrupt the cluster, thereby producing a variant antibody with enhanced biodistribution and / or pharmacokinetics compared to the original antibody.

[0020] The present invention provides a method for preparing a labeled antibody, comprising: selecting a light chain variable region (V L ), wherein the germline sequence does not contain a cluster of at least two positively charged amino acids within 3 residues of each other in framework region 2 (FR2) of the germline sequence; isolating one or more target-specific antibodies in an antibody population, the antibody population comprising V derived from the germline sequence L , and having germline sequence variation within the population, wherein one or more target-specific antibodies are L The FR2 sequence does not contain a cluster of at least two positively charged amino acids within 3 residues of each other; and labeling one or more target-specific antibodies.

[0021] Also provided is an antibody produced by any of the methods of the present disclosure.

[0022] Provided herein is a method of treating a subject, comprising: identifying a subject in need of treatment with any of the antibodies (e.g., variant antibodies), minibodies, or cys-diabodies of the present disclosure; and administering to the subject a therapeutically effective amount of the antibody or minibody, or a composition comprising the same.

[0023] Also provided is a method for treating cancer in a subject, comprising: identifying a subject in need of cancer treatment; and administering to the subject a therapeutically effective amount of any of the antibodies (e.g., variant antibodies), minibodies, or cys-diabodies of the present disclosure, or a composition comprising the same, thereby treating the cancer.

[0024] Provided herein is a method for treating radiation, comprising: identifying a subject in need of radiation therapy; and administering to the subject a therapeutically effective amount of any of the antibodies (e.g., variant antibodies), miniantibodies, or cys-diabodies of the present disclosure, or a composition comprising the same, wherein the antibody, miniantibody, or cys-diabody comprises a radionuclide.

[0025] Also provided is a method of imaging a subject, comprising: administering to the subject a composition comprising an effective amount of any one of the antibodies (e.g., variant antibodies), miniantibodies or cys-diabodies of the present disclosure, or a composition comprising the same, wherein the antibody, miniantibody or cys-diabody is detectably labeled; and imaging the subject to detect the labeled antibody, miniantibody or cys-diabody in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a block diagram showing a non-limiting example of a method for enhancing the biodistribution and / or pharmacokinetics of an antibody.

[0027] Figure 2 is a block diagram showing a non-limiting example of a method for enhancing the biodistribution and / or pharmacokinetics of an antibody.

[0028] Figure 3 is a block diagram showing a non-limiting example of a method for preparing a labeled antibody.

[0029] Figure 4 is a block diagram showing a non-limiting example of a method of treating a subject.

[0030] Figures 5A-5C is a collection of non-limiting examples of amino acid sequences of minibodies.

[0031] Figure 6 is a graph showing a biodistribution comparison between original and variant minibodies according to some non-limiting embodiments of the present disclosure.

[0032] Figures 7A-7D is a collection of non-limiting examples of amino acid sequences of antibody fragments.

[0033] Figure 8 is a graph showing a biodistribution comparison between original and variant minibodies according to some non-limiting embodiments of the present disclosure.

[0034] Fig. 9 is a graph showing a biodistribution comparison between original and variant minibodies according to some non-limiting embodiments of the present disclosure.

[0035] Fig.10is a schematic diagram of some non-limiting structural features of minibodies.

[0036] Fig.11 is a schematic diagram of some non-limiting structural features of cys-diabodies.

[0037] Fig.12 It is C H 3 A collection of non-limiting examples of amino acid sequences. DETAILED DESCRIPTION

[0038] When applied to a subject, the biodistribution and pharmacokinetics of antibodies, miniantibodies and cys-diabodies are important determinants of their safety and effectiveness. Improved biodistribution and pharmacokinetics can affect the dosing and administration schedule of antibody therapeutics, such as during radioimmunotherapy or when used as antibody-drug conjugates. It can also affect the dosing and imaging time points for radiodiagnostic imaging with antibodies, miniantibodies and cys-diabodies. The biodistribution and pharmacokinetics of antibodies may be different according to a variety of factors, including some properties of the electrostatic charge of the surface exposure of the antibody.

[0039] Provided herein are antibodies (including miniantibodies and cys-diabodies) with enhanced biodistribution and / or pharmacokinetics and methods for enhancing the biodistribution and / or pharmacokinetics of antibodies. Compared to the original antibody, antibodies of the present disclosure (e.g., variant antibodies) generally have a destroyed positively charged amino acid cluster, and can have enhanced biodistribution and / or pharmacokinetics compared to the original antibody (the original antibody has a cluster, and the antibody with the destroyed cluster is based on the original antibody).

[0040] Without being bound by theory, it is believed that clusters of positively charged amino acids form patches of positive electrostatic charge, which are described by equipotential surfaces on the antibody, and when administered to a subject, this localized charge concentration may result in accumulation of the antibody in radiosensitive or drug-sensitive tissues such as the kidney. Undesirable antibody accumulation in the kidney may be due to increased capture and retention at the glomerulus or proximal tubules (and / or increased reabsorption and retention in these renal subcompartments) relative to the variant antibodies provided herein.

[0041] As provided herein, destroying positively charged amino acid clusters can reduce the accumulation of antibodies in the kidney. In some embodiments, the destruction of clusters reduces the accumulation of antibodies in the kidney by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, or reduces the percentage within the range defined by any two of the aforementioned values ​​(e.g., 10-80%, 10-50%, 30-80%, 40-70%, etc.). In some embodiments, the destruction of clusters increases the accumulation of antibodies in the liver. In some embodiments, for antibody constructs with a molecular weight (as a dimer) of ~50-80kDa, such as mini-antibodies or cys-double antibodies, it is easy to observe the changes in biodistribution caused by the destruction of clusters. Typically, the original antibody is modified (remodeled) by destroying positively charged amino acid clusters to at least maintain the same binding specificity and binding affinity of the original antibody, while enhancing biodistribution and / or pharmacokinetics. In some embodiments, the molecular weight of the antibody (or its antigen-binding fragment) is 15-160kDa. In some embodiments, the molecular weight of the antibody (or antigen-binding fragment thereof) is 15-110 kDa. In some embodiments, the molecular weight of the antibody (or antigen-binding fragment thereof) is about 50-80 kDa.

[0042] In some embodiments, the biodistribution of the antibody can be controlled by changing the static charge associated with the cluster. As described above, in some embodiments, positively charged amino acid clusters can promote accumulation in the kidney, and reducing the positive charge associated with the cluster or increasing the negative charge associated with the cluster can promote accumulation away from the kidney, such as promoting accumulation in the liver. Therefore, in some embodiments, by increasing the positive charge associated with the cluster, the biodistribution of the cytotoxic agent (e.g., toxin) or radionuclide bound to the antibody can be biased toward the kidney, and by reducing the positive charge associated with the cluster or increasing the negative charge associated with the cluster, the biodistribution of the cytotoxic agent (e.g., toxin) or radionuclide bound to the antibody can be biased toward the liver. In some embodiments, in the case of treating liver disease (e.g., liver cancer) by administering an antibody, the distribution of the antibody to the liver is promoted.

[0043] In the case of radioimmunotherapy, accumulation of antibodies in the kidneys may lead to nephrotoxicity. Therefore, the reduced accumulation of antibodies of the present disclosure in the kidneys can allow the administration of larger doses of radiolabeled antibodies (e.g., greater specific activity, higher frequency of administration, and / or more administration times) while reducing nephrotoxicity.

[0044] the term

[0045] For purposes of this disclosure, all terms have the ordinary customary meanings of those terms as understood by those of ordinary skill in the art.

[0046] As used herein, the term "antibody" includes all kinds of antibodies, including antigen-binding fragments thereof. Further included are constructs comprising 1, 2, 3, 4, 5 and / or 6 CDRs. In some embodiments, a tandem scFv can be provided, which can provide two arms with bivalent binding. In some embodiments, these CDRs can be distributed between their appropriate framework regions in traditional antibodies. In some embodiments, the CDR can be contained in the heavy chain and / or light chain variable region. In some embodiments, the CDR can be in the heavy chain and / or light chain. In some embodiments, the CDR can be in a single peptide chain. Unless otherwise specified herein, the antibodies described herein are bound to the target molecules mentioned. The term "target" or "target molecule" refers to a protein to which an antigen-binding construct specifically binds to a certain degree under physiological conditions.

[0047] The term "antibody" includes, but is not limited to, genetically engineered or otherwise modified immunoglobulin forms such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, single chain variable fragments (scFv), and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, etc.). In addition, the term "antibody" includes camel-derived immunoglobulins such as single heavy chain antibodies and nanobodies. "Antibody fragments" include, but are not limited to, Fab', F(ab')2, Fab, Fv, rIgG (reduced IgG), scFv fragments, scFv-Fc, single domain fragments (e.g., nanobodies), peptibodies, -Fc, mini-antibodies and diabodies. The term "antibody" includes scFv and mini-antibodies. Therefore, unless otherwise expressly stated, each individual embodiment of "antibody" provided herein is also considered to be an scFv and / or mini-antibody embodiment. The term "antibody" includes polypeptides of the immunoglobulin family or polypeptides comprising immunoglobulin fragments, which can bind to the corresponding antigen in a non-covalent, reversible and specific manner. Exemplary antibody structural units include tetramers. In some embodiments, a full-length antibody can be composed of two identical polypeptide chain pairs, each pair having a "light" chain and a "heavy" chain (connected by a disulfide bond). Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, hinge and μ constant region genes, as well as a large number of immunoglobulin variable region genes. For full-length chains, light chains are classified as κ or λ. For full-length chains, heavy chains are classified as γ, μ, α, δ or ε, which define immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. The N-terminus of each chain defines a variable region of up to about 149 or more amino acids primarily responsible for antigen recognition. L ) and variable heavy chain (V H) refer to these regions of the light chain and heavy chain, respectively. As used in this application, "antibody" includes all variants of antibodies and their fragments. Therefore, falling within the scope of this concept are: full-length antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies (scFv), Fab, Fab' and multimeric forms of these fragments (e.g., F(ab')2) with the same binding specificity. In some embodiments, the antibody specifically binds to the desired target.

[0048] "Antibodies" may also include one or more immunoglobulin chains chemically bound to other proteins or expressed as fusion proteins with other proteins. It also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies (hybrid antibodies) with two different heavy / light chain pairs and two different binding sites.

[0049] As used herein, "antibody" may also refer to other antigen-binding fragments or antibody portions of the disclosure, including bispecific scFv antibodies, in which the antibody molecule recognizes two different epitopes, single binding domains (sdAb or ), miniantibodies and cys-diabodies.

[0050] The term "antibody fragment" includes, but is not limited to, one or more antigen-binding fragments of an antibody, alone or in combination with other molecules, including, but not limited to, Fab', F(ab')2, Fab, Fv, rIgG (reduced IgG), scFv fragments, scFv-Fc, single domain fragments peptide body, -Fc, minibodies, diabodies and cys-diabodies. The term "scFv" refers to a single-chain Fv ("fragment variable") antibody in which the variable domains of the heavy and light chains of a traditional diabody are linked to form one chain.

[0051] Minibodies are an antibody form that has a smaller molecular weight than full-length antibodies while maintaining bivalent binding properties to antigens. Due to their small size (~70-90 kDa, preferably ~80 kDa), there is no C-terminal domain for binding to Fc-γ and FcRn receptors. H2 domains, and there is no glycosylation, so the mini-antibody is cleared faster from the blood system and may have enhanced penetration when targeting tumor tissue. With its powerful targeting ability and rapid clearance ability relative to full-size antibodies, mini-antibodies are beneficial for diagnostic and therapeutic applications and the delivery of radioactive payloads, for which extended circulation time may lead to poor patient dosing or dosing. In some embodiments, due to the above-mentioned characteristics, such as tumor penetration and faster clearance, it is also beneficial for the delivery of cytotoxic payloads (e.g., in the case of antibody-drug conjugates, ADCs). As described herein, "mini-antibodies" include homodimers, in which each monomer is connected to a human IgG C by a hinge sequence. H 3-domain single-chain variable fragment (scFv). Fig.10 A non-limiting schematic diagram of a minibody is shown in FIG. In some embodiments, a minibody is a divalent or bispecific covalently bound heterodimer of ∼80 kDa. In some embodiments, each monomer (half molecule) is composed of a variable weight (V H ) domain, which is connected to the corresponding variable light (V L In some embodiments, each single-chain variable fragment (scFv) is connected to a human IgG1, IgG2, IgG3 or IgG4C domain via a hinge sequence. H 3 domains.

[0052] The term "hinge" refers to at least a portion of the hinge region of an antigen-binding construct, such as an antibody or minibody, scFv-Fc or -Fc. The hinge region may include a combination of an upper hinge region, a core (or middle) hinge region, and a lower hinge region. In some embodiments, the hinge is defined according to any one of the definitions of an antibody hinge. Natural IgG1, IgG2, and IgG4 antibodies have a hinge region containing 12-15 amino acids. IgG3 has an extended hinge region with 62 amino acids, including 21 proline and 11 cysteine. It is inferred from crystallographic studies that the functional hinge region of naturally occurring antibodies may extend from amino acid residues 216-237 of the IgG1 H chain (EU numbering) and include the C in the lower hinge. H A short segment at the N-terminus of the 2 domain, the lower hinge is C H The N-terminal end of the 2 domain. The hinge can be divided into three regions; the "upper hinge", the "core" and the "lower hinge".

[0053] The term "upper hinge" refers to the first portion of the hinge, starting from the end of the scFv. The upper hinge includes amino acids from the end of the scFv to (but not including) the first cysteine ​​residue in the core hinge. The term "effective upper hinge" means that sufficient sequence is present to enable the portion to function as an upper hinge; the term includes functional variants and fragments of the specified hinge portion.

[0054] The term "core hinge" refers to the second part of the hinge region, i.e. the C-terminus of the upper hinge. The core hinge may contain interchain disulfide bridges and a high content of proline.

[0055] The term "lower hinge" refers to the third portion of the hinge region, i.e., the C-terminus of the core hinge. In the case of a minibody or antibody fragment, the lower hinge is attached to the C H 3 domain Mb. As described above, the term "effective lower hinge" means that there is enough sequence to enable the portion to function as a lower hinge; the term includes functional variants and fragments of the specified hinge portion. As used herein, the term "lower hinge" can include various amino acid sequences, including naturally occurring IgG lower hinge sequences provided herein and artificial extension sequences or combinations thereof that can replace each other. In some embodiments, various extensions can be considered as lower hinge regions or substitutes in their entirety.

[0056] In some embodiments, the lower hinge can be a native IgG1, 2, 3 or 4 lower hinge, and / or a Gly-Ser sequence (G3)S n or (G4)S n (n can be any number of S; in some embodiments, it is 1 or 2) and / or no lower hinge and / or any combination of amino acids (not necessarily G and S). In some embodiments, the lower hinge can include GGGSSGGGSG (SEQ ID NO: 40).

[0057] The term "diabody" refers to a polypeptide comprising a heavy chain (V H ) domain and light chain variable (V L ) domains. Each heavy chain domain is connected to a light chain domain via a linker to form a monomer. Two monomers are covalently linked via a bridging moiety to form a diabody. "Cys-diabody" refers to a diabody whose monomer chains are covalently linked via disulfide bonds. A non-limiting schematic diagram of a cys-diabody is shown in FIG. Fig.11 As shown. The Cys-diabody can have a molecular weight of ~50 kDa.

[0058] The term "extension sequence" (e.g., in the case of a diabody) refers to the extension of the first V H domain connected to the second V H The domain may be the first V L Connect to the second V LThe extension sequence may be connected to the domains via the C-terminus of each domain. In some embodiments, the extension sequence is connected to the domains via a covalent bond. In some embodiments, the extension sequence includes one or more cysteines, allowing one or more disulfide bonds to form between two such extension sequences. An example of a pair of extension sequences is Fig.11 The schematic diagram on the right is shown as a line with two cysteines connecting two heavy chain domains or two light chain domains. Fig.11 The schematic diagram on the left shows a non-limiting example of a stretch sequence of -(Gly)2-(Cys). In some embodiments, each monomer chain of the stretch sequence includes 1, 2, 3 or more cysteines. Although the stretch sequence will be toward Fig.11 The C-terminus of the construct in the example above is the C-terminus of the construct, but it does not have to be the absolute last amino acid in the variable domain. That is, the extension sequence can be located slightly N-terminal to the C-terminus. For example, the extension sequence can be placed within 10 amino acids of the C-terminus of the monomer. Similarly, additional sequences can be placed between the native C-terminus and the beginning of the extension sequence. The extension sequence can be connected to the V domain by disulfide bonds. H Connect to V H Or V L Connect to V L In some embodiments, the extension sequence comprises GGCPPCPPC (SEQ ID NO: 72).

[0059] In some embodiments, the linker can be any suitable linker of an antibody (e.g., a minibody, a cys-diabody). In some embodiments, the linker sequence can include as (G3)S n or (G4)S n (n can be any integer; in some embodiments, it is 1 or 2). In some embodiments, the linker can include GSTSGGGSGGGSGGGGSS (SEQ ID NO: 41).

[0060] The terms "treating" or "treatment" of a condition can refer to preventing the condition, slowing the onset and / or rate of development of a condition, reducing the risk of developing a condition, preventing and / or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, causing complete or partial regression of the condition, or some combination thereof. The term "preventing" does not require absolute prevention of a condition or disease.

[0061] "Therapeutically effective amount" or "therapeutically effective dose" refers to an amount that produces the desired therapeutic effect in a subject, such as preventing, treating the target condition, delaying the onset of symptoms and / or symptoms, and / or alleviating symptoms associated with the condition. The amount will vary according to various factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the properties of one or more pharmaceutically acceptable carriers in the formulation, and / or the route of administration. In light of the present disclosure, those skilled in the art of clinical and pharmacological medicine will be able to determine the therapeutically effective amount by routine experimentation, such as by monitoring the subject's response to the administration of the compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0062] The terms "complementarity determining domain" or "complementarity determining region ("CDR")" refer interchangeably to a V L and V H CDR is the target molecule binding site of the antibody chain and has specificity for this target molecule. In some embodiments, each V L and / or V H There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in the variable domain, which constitute about 15-20% of the variable domain. The CDRs are structurally complementary to the epitope of the target molecule and are therefore directly responsible for binding specificity. L or V H The remaining stretches, the so-called framework regions (FR), show less variation in amino acid sequence (Kuby, Immunology, 4th edition, Chapter 4, WH Freeman & Co., New York, 2000).

[0063] The positions of CDRs and framework regions can be determined using various definitions known in the art, such as Kabat (Wu, TT, EA Kabat. 1970. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132: 211-250; Kabat, EA, Wu, TT, Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, Bethesda, MD); Chothia (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)); ImMunoGeneTics Database (IMGT) (World Wide Web address imgt.org / ) Giudicelli, V., Duroux, P., Ginestoux, C., Folch, G., Jabado-Michaloud, J., Chaume, D. and Lefranc, M.-P. IMGT / LIGM-DB, the 免疫球蛋白和T细胞受体核苷酸序列的综合数据库,《核酸研究》,34卷,D781 - D784页(2006年),PMID:16381979;勒弗朗,M.-P.,庞米耶,C.,鲁伊斯,M.,朱迪塞利,V.,富尔基耶,E.,特鲁昂,L.,图韦南 - 孔泰,V.和勒弗朗,G.,免疫球蛋白和T细胞受体可变结构域以及Ig超家族V样结构域的IMGT独特编号,《发育与比较免疫学》,27卷,55 - 77页(2003年)。PMID:12477501;布罗谢,X.,勒弗朗,M.-P.和朱迪塞利,V.,IMGT / V - QUEST:用于IG和TR标准化V - J和V - D - J序列分析的高度定制化和集成化系统,《核酸研究》,36卷,W503 - 508页(2008年);AbM(马丁等人,《美国国家科学院院刊》,86:9268 - 9272(1989年));诺斯(诺斯B., Lehmann A., Dunbrack R.L.,抗体CDR环构象的新聚类,《分子生物学杂志》(2011年)406(2):228 - 256);AHo(霍内格A.,普吕克松,免疫球蛋白可变结构域的另一种编号方案:一种自动建模和分析工具,《分子生物学杂志》(2001年)309,657 - 670);接触定义(contact definition)(麦卡勒姆等人,《分子生物学杂志》, 262:732-745 (1996)), and / or automatic modeling and analysis tools Honegger A, Plückthun A. (World Wide Web address bioc dot uzh dot ch / antibody / Numbering / index dot html). In some embodiments, the framework regions are defined by the AHo numbering method as follows: FR1, 1-24; FR2, 41-57; FR3, 78-108, and FR4, 138-149. .

[0064] As used herein, "antibody variable light chain" or "antibody variable heavy chain" refers to a L or V H Endogenous V L Encoded by gene segments V (variable) and J (joining), and endogenous V H Encoded by V, D (diversity) and J. V L or V H Each of the antibody variable light chains and / or antibody variable heavy chains may occasionally be collectively referred to as "antibody chains" in the present application. As will be readily appreciated by those skilled in the art, these terms include sequences that do not disrupt V L or V H In some embodiments, the antibody chains of the basic structure are mutated. In some embodiments, the full-length heavy chain and / or light chain are considered. In some embodiments, only the variable region of the heavy chain and / or light chain is considered.

[0065] Antibodies can exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide bonds in the hinge region to produce F(ab)'2, a dimer of Fab', which itself is linked to V by disulfide bonds. H -C H 1 light chain (V L -C L). F(ab)'2 can be reduced under mild conditions to destroy the disulfide bonds in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is a Fab with a portion of the hinge region. (Paul, WE, "Fundamental Immunology," 3rd edition, New York: Raven Press, 1993). Although various antibody fragments are defined based on the digestion of intact antibodies, it will be appreciated by those skilled in the art that these fragments can be synthesized de novo chemically or using recombinant DNA methods. Therefore, as used herein, the term "antibody" also includes antibody fragments produced by modifying whole antibodies, or antibody fragments (e.g., single-chain Fv) synthesized de novo using recombinant DNA methods, or antibody fragments identified using phage display libraries (e.g., see McCafferty, J. et al., "Phage antibodies: filamentous phage displaying antibody variable domains," Nature, Vol. 348, No. 66301, pp. 552-554, 1990).

[0066] To prepare monoclonal or polyclonal antibodies, any technique known in the art can be used (e.g., see Kohler, G. et al., “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, Vol. 256, No. 5517, pp. 495-497, 1975; Kozbor, D. et al., “The production of monoclonal antibodies from human lymphocytes,” Immunology Today, Vol. 4, No. 3, pp. 72-79, 1983; Cole et al., “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp. 77-96, 1985; Wang, S., “Advances in the production of human monoclonal antibodies,” Antibody Technology Journal, Vol. 1, pp. 1-4, 2011; Sharon, J. et al., “Recombinant polyclonal antibodies for cancer therapy,” J. Cell Biochem., Vol. 96, No. 2, pp. 305-313, 2005; Haurum, JS, "Recombinant polyclonal antibodies: the next generation of antibody therapeutics?," Drug Discov. Today, Vol. 11, No. 13-14, pp. 655-660, 2006). The production technology of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies against the polypeptides of the present invention. In addition, transgenic mice or other organisms, such as other mammals, can be used to express fully human monoclonal antibodies. Alternatively, phage display technology can be used to identify high-affinity binders against selected antigens (e.g., see McCafferty et al., supra; Marks, JD et al., "By-passing immunization: building high affinity human antibodies by chain shuffling," Biotechnology (NY), Vol. 10, No. 7, pp. 779-783, 1992).

[0067] Methods for humanizing or primatizing non-human antibodies are well known in the art. Usually, humanized antibodies have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are commonly referred to as import residues, which are usually taken from the import variable domains. In some embodiments, the terms "donor" and "acceptor" sequences can be used. Humanization can be performed essentially according to the method of Winter and colleagues (e.g., see Jones, PT et al., "Replacing the complementarity-determining regions in a human antibody with those from a mouse," Nature, Vol. 321, No. 6069, pp. 522-525, 1986; Riechmann, L. et al., "Reshaping human antibodies for therapy," Nature, Vol. 332, No. 6162, pp. 323-327, 1988; Verhoeyen, M. et al., "Reshaping human antibodies: grafting anantilysozyme activity," Science, Vol. 239, No. 4847, pp. 1534-1536, 1988; Presta, LG, "Antibody engineering,” Curr. Op. Struct. Biol., Vol. 2, No. 4, pp. 593-596, 1992), by replacing the CDR or CDR sequence of a rodent with the corresponding sequence of a human antibody. Therefore, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less than the entire human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some complementarity determining region (“CDR”) residues and possibly some framework (“FR”) residues are replaced by residues from analogous sites in rodent antibodies.

[0068] The terms "Fc region", "Fc domain" or "Fc" refer to the C-terminal region of an immunoglobulin heavy chain. An "Fc region" can be a native sequence Fc region or a variant Fc region (e.g., a variant having one or more mutations that reduces effector function, such as FcγR binding, and / or binding to Fc neonatal receptor (FcRn), etc.). The Fc region of an immunoglobulin (e.g., IgG) typically comprises two constant domains, C H 2 and C H3. The Fc region may or may not include a hinge region or sequence, as described herein. The Fc region may exist in dimer or monomer form. In some embodiments, the Fc region is a human Fc region or a variant thereof.

[0069] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or to an entirely different molecule (e.g., enzymes, toxins, hormones, growth factors, and drugs) that confers new properties to the chimeric antibody; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity.

[0070] A pharmaceutically acceptable carrier can be a pharmaceutically acceptable substance, composition or vehicle that participates in carrying or transporting a compound of interest from one tissue, organ or part of a body to another tissue, organ or part of a body. For example, a carrier can be a liquid or solid filler, diluent, excipient, solvent or encapsulating material, or a combination thereof. Each component of a carrier is "pharmaceutically acceptable" because it is compatible with the other ingredients of the formulation. It must also be suitable for contact with any tissue, organ or part of the body that may be encountered, which means that it must not carry the risk of toxicity, irritation, allergic reaction, immunogenicity or any other complications that over-exceed its therapeutic benefits. The pharmaceutical compositions described herein can be administered by any suitable route of administration. The route of administration can refer to any route of administration known in the art, including but not limited to aerosols, enteral, intranasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical creams or ointments, patches) or vaginal. "Transdermal" administration can be accomplished using topical creams or ointments or by transdermal patches. "Parenteral" refers to routes of administration typically associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intrauterine, intravenous, subarachnoid, subcapsular, sublingual, subcutaneous, transmucosal, or transtracheal. In some embodiments, the antigen-binding construct can be delivered intraoperatively in a local administration form during intervention or resection.

[0071] When used to describe the interaction between an antigen (e.g., a protein) and an antibody or an antibody-derived binding agent, the phrase "specific (or selective) binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological agents, such as in a biological sample, such as blood, serum, plasma, or a tissue sample. Therefore, under specified immunoassay conditions, in some embodiments, the binding of an antibody or binding agent with a specific binding specificity to a specific antigen is at least twice the background, and substantially does not bind to other antigens present in the sample in significant amounts. Specific binding to an antibody or binding agent under such conditions may require the selection of these antibodies or binding agents for the specificity of a particular protein. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are commonly used to select antibodies that specifically immunoreact with proteins (e.g., see Harlow, E. & Lane D., "Using Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1998, for a description of immunoassay formats and conditions for determining specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal that is at least two times greater than the background signal, more typically at least 10- to 100-fold greater than the background signal.

[0072] The term "equilibrium dissociation constant (K D , M)” refers to the dissociation rate constant (k d ,time -1 ) divided by the association rate constant (k a ,time -1 M -1 The equilibrium dissociation constant can be measured using any method known in the art. The antibodies of the present invention generally have a dissociation constant of less than about 10 -7 or 10 -8 The equilibrium dissociation constant of M is, for example, less than about 10 -9 M or 10 -10 M, in some embodiments, is less than about 10 -11 M, 10 -12 M, or 10 -13 M.

[0073] When applied to nucleic acid or protein, the term "isolated" means that the nucleic acid or protein is substantially free of other cellular components that are combined with it in the native state. In some embodiments, it can be a dry solution (dry solution) or an aqueous solution. Purity and uniformity (homogeneity) can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The main species present in the preparation, i.e., the protein, is substantially purified. In particular, the isolated gene is separated from the open reading frame that is located at the gene flank and encodes the protein other than the gene of interest. The term "purified" means that the nucleic acid or protein basically produces only one band in an electrophoretic gel or size exclusion chromatography analysis. In some embodiments, this can mean that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure molecule present under in vivo conditions.

[0074] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term includes nucleic acids containing known natural nucleotide analogs that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon replacement can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer, MA et al., "Enhanced evolutionary PCR using oligonucleotides with inosine at the 3'-terminus," Nucleic Acid Res., Vol. 19, No. 18, p. 5081, 1991; Ohtsuka, E. et al., "An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions," J. Biol. Chem., Vol. 260, No. 5, pp. 2605-2608, 1985; Rossolini, GM et al., "Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information,” Mol. Cell. Probes, Vol. 8, No. 2, pp. 91-98, 1994).

[0075] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers, and artificial chemical mimetics of non-naturally occurring amino acid polymers.

[0076] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are the amino acids encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, for example, an α-carbon bound to a hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0077] The term "conservatively modified variant" is applicable to both amino acid sequences and nucleic acid sequences. For a specific nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding the same or substantially the same amino acid sequence, or in the case where the nucleic acid does not encode an amino acid sequence, refer to substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. This nucleic acid variation is a "silent variation", which is one of the conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in the nucleic acid (except AUG and TGG, AUG is usually the only codon for methionine, and TGG is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of the nucleic acid encoding a polypeptide is implicit in each described sequence.

[0078] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alters, adds or deletes a single amino acid or a small portion of amino acids in the encoded sequence are "conservatively modified variants," wherein such alterations result in replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs and alleles of the present invention.

[0079] The following eight groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, TE, "Proteins-Structures and Molecular Properties," WH Freeman & Co. Ltd., 1984).

[0080] The term "sequence identity percentage" can be determined by comparing two optimally aligned sequences within a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide of the present invention) that does not contain additions or deletions to achieve optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the percentage of sequence identity.

[0081] In the case of two or more nucleic acid or polypeptide sequences, the term "same" or "identity" percentage refers to two or more sequences or subsequences as identical sequences. When compared and aligned to obtain maximum correspondence within a comparison window or using one of the following sequence comparison algorithms or in a specified region measured by manual alignment and visual inspection, if the two sequences have a specified percentage of identical amino acid residues or nucleotides (e.g., in a specified region, or when not specified, on the entire sequence of a reference sequence, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), then the two sequences are "substantially identical". Some embodiments provided herein provide polypeptides or polynucleotides substantially identical to the polypeptides or polynucleotides exemplified herein, respectively. Optionally, identity is present in a region having a length of at least about 15, 25 or 50 nucleotides, or more preferably in a region having a length of 100 to 500 or 1000 or more nucleotides, or in the full length of a reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region of at least 5, 10, 15, or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally at least about 150, 200, or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, in some embodiments, substantial identity exists when one or two amino acid residues are conservatively substituted according to conservative substitutions as defined herein.

[0082] In some embodiments, the percent identity is between a portion of an antibody described herein (framework region, C H 3 and / or Fc region). In this case, the percent identity of the polypeptide portion can be identified separately from the rest of the protein or nucleic acid sequence. Thus, two portions (e.g., two FR, C H 3 and / or Fc region) can have a specified percentage of identical amino acid residues or nucleotides (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, if not specified, over the entire sequence of a reference sequence), while allowing the remainder of the protein to remain 100% identical to the compared protein, while also allowing the remainder of the protein to also have a variation of the specified identity percentage.

[0083] For sequence comparison, usually one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0084] As used herein, a "comparison window" includes reference to a segment selected from any of a number of consecutive positions of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be, for example, by the local homology algorithm of Smith and Waterman (1970) Adv Appl. Math. 2:482c, by the homology alignment algorithm of Needleman, SB et al., "A general method applicable to the search for similarities in the amino acid sequence of two proteins," J. Mol. Biol., Vol. 48, No. 3, pp. 443-453, 1970, by the similarity search method of Pearson, WR et al., "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci. USA, Vol. 85, No. 8, pp. 2444-2448, 1988, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel, FM et al., Current Protocols in Molecular Biology, Supplement, 1995).

[0085] Two examples of algorithms suitable for determining percentages of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul, SF et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res., Vol. 25, No. 17, pp. 3389-3402, 1977 and Altschul, SF et al., "Basic local alignment search tool," J. Mol. Biol., Vol. 215, No. 3, pp. 403-410, 1990. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy a positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul, SF et al., supra). These initial neighborhood word hits (matches, hits) act as seeds (starts) for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatched residues; always<0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Stop extending word hits in each direction when the following occurs, i.e., the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative scoring residue alignments; or reaches the end of either sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11 by default, an expected value (E) of 10, M=5, N=-4, and two chain comparisons. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff, S. et al., "Amino acid substitution matrices from protein blocks," Proc. Natl. Acad. Sci. USA, Vol. 89, No. 22, pp. 10915-10919, 1992) with an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains.

[0086] The BLAST algorithm also performs statistical analysis on the similarity between two sequences (e.g., see Karlin, S. et al., "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA, Vol. 90, No. 12, pp. 5873-5787, 1993). One similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability of an accidental match between two nucleotide or amino acid sequences. For example, if the minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0087] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, in some embodiments, a polypeptide is generally substantially identical to a second polypeptide, e.g., wherein the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0088] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. The term is intended to include non-radioactive payloads (e.g., ADCs), radioactive isotopes (e.g., 177 Lu, 225 Ac, 67 Cu, 227 Th, 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 213 Bi, 32 P. 149 Tb, 161 Tb, 212 Pb and radioactive isotopes of Lu), chemotherapeutic agents (as defined elsewhere herein). Other cytotoxic agents are described below. Tumoricidal agents destroy tumor cells.

[0089] A "toxin" is any substance that is capable of having a deleterious effect on the growth or proliferation of cells. Non-radioactive payloads include those commonly used in antibody-drug conjugates (ADCs) and fragment-drug conjugates (FDCs), such as toxins belonging to the families of auristains, maytansines, maytansinoids, calicheamicins, duocarymycins, pyrrolobenzodiazepine dimers, and amatoxins. A "therapeutic ion" refers to a charged particle that can be used to treat a condition associated with a target molecule. Examples of therapeutic ions include 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 2. 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir,198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 Ac. These are also therapeutic agents of choice.

[0090] A "chemotherapeutic agent" is a compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and CYTOXAN™ cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, and triethylephosphamide. nephosphoramide), triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOLTM); beta-lapachone; lapachol; colchicine; betulinic acid camptothecins (including the synthetic analogs topotecan (HYCAMTINT™), CPT-11 (irinotecan, CAMPTOSAR™), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; candidins (particularly candidin 1 and candidin 8); dolastatin; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1 and calicheamicin omega 1) (see Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994); anthracyclines (dynemicins), including anthracycline A; esperamicin; and neocarzinostatin chromophore (neocarzinostatin chromophore and related chromophores of the enediyne class of antibiotics), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), acid), nogamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rhodorubicin, streptomycin, streptozotocin, tuberculocidin, ubenimex, zinostatin, zorubicin; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid acid; acetoglucuronide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; belemet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM. Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran;Spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethanes; vindesine (ELDISINETM, FILDESINTM); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxanes such as paclitaxel RTM. Paclitaxel (Bristol-Myers Squibb =Oncology, Princeton, NJ), ABRAXANET™ Cremophor-free, albumin-engineered paclitaxel nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXORE™ docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZART™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBANT™); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVINT™); oxaliplatin; leucovovin; vinorelbine (NAVELBINE™); mitoxantrone; edatrexate; daunomycin; aminopterin; ibandronate (ibandron ate); topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids such as retinoic acid; capecitabine (XELODATM); any of the above pharmaceutically acceptable salts, acids or derivatives; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for the combination therapy of oxaliplatin (ELOXATINTM) with 5-FU and folinic acid. These are also options for therapeutic agents. ;

[0091] The terms "subject," "patient," and "individual" refer interchangeably to an entity being examined and / or treated. This can include, for example, a mammal, such as a human or non-human primate. The mammal can also be a laboratory mammal, such as a mouse, rat, rabbit, hamster. In some embodiments, the mammal can be an agricultural mammal (e.g., horse, sheep, cattle, pig, camelid) or a livestock mammal (e.g., dog, cat).

[0092] Antibody

[0093] As discussed herein, there is provided an antibody with enhanced biodistribution and / or pharmacokinetics obtained by destroying one or more surface-exposed positively charged amino acid clusters. A variant antibody (e.g., miniantibody, cys-double antibody) is provided herein, the antibody includes at least one surface-exposed positively charged amino acid cluster of destruction, wherein the variant antibody is different from the original antibody, and the original antibody includes at least two (e.g., 2,3,4,5,6 or more) surface-exposed, positively charged amino acid original clusters within about 30 angstroms each other. The variant antibody can be different from the original antibody by replacing at least one of the surface-exposed positively charged amino acids of the original cluster with one or more negatively charged or uncharged amino acids. The replacement of the surface-exposed positively charged amino acid of the cluster can destroy the original cluster (e.g., by making the surface-exposed positive charge at the corresponding position on the variant antibody less than the original antibody). In some embodiments, the original or variant antibody is any antibody construct. In some embodiments, this destruction is particularly useful for a construct equal to or less than a miniantibody in size. In some embodiments, the antibody is a cys-double antibody. In some embodiments, the antibody is a scFv-Fc (e.g., a scFv fused to an Fc) or -Fc (e.g., camelid fused to Fc or single domain fragments).

[0094] The distance between the surface exposed positively charged amino acids in the cluster can be the distance between the center of mass of each amino acid residue in a suitable three-dimensional model of the antibody or a portion thereof. The positively charged amino acids of the cluster are surface exposed as determined by van der Waals radii, solvent accessible area, equipotential surface representation of the electrostatic field calculated by an adaptive Poisson-Boltzmann Solver, or a combination thereof.

[0095] Typically, positively charged amino acids are amino acids that are positively charged under physiological conditions (e.g., physiological pH, or pH 5.6-7.5, pH 6.0-7.5, or pH 7.3-7.4). In some embodiments, positively charged amino acids are selected from: arginine, lysine, and histidine. In some embodiments, positively charged amino acids are selected from: arginine and lysine. The amino group at the N-terminus of the polypeptide or protein can also provide a positive charge. Negatively or uncharged amino acids include amino acids that are not positively charged under physiological conditions (e.g., physiological pH, or pH 5.6-7.5, pH 6.0-7.5, or pH 7.3-7.4). In some embodiments, negatively charged amino acids are selected from: aspartic acid and glutamic acid. The carboxyl group at the C-terminus of the polypeptide or protein can also provide a negative charge. In some embodiments, the uncharged amino acid is selected from the group consisting of alanine, glycine, asparagine, glutamine, isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine ​​and methionine. In some embodiments, the negatively charged or uncharged amino acid is glutamine.

[0096] The surface-exposed positively charged amino acids within the cluster can form continuous positively charged patches on the surface (e.g., isoelectric electrostatic surface) of the antibody (e.g., the original antibody with the cluster). In some embodiments, the cluster has at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed positively charged amino acids within about 30 angstroms, 25 angstroms, 20 angstroms, 15 angstroms, 14 angstroms, 13 angstroms, 12 angstroms, 11 angstroms, 10 angstroms or 5 angstroms or less or within a distance defined by any two of the aforementioned values ​​(e.g., 30-5 angstroms, 30-10 angstroms, 20-5 angstroms, 15-10 angstroms, 12-10 angstroms). In some embodiments, at least two surface-exposed positively charged amino acids of the cluster are within about 15 angstroms of each other. In some embodiments, where a cluster includes 3 or more surface-exposed positively charged amino acids, the distance between the two residues that are farthest from each other is within about 30 angstroms, such as 25 angstroms, 20 angstroms, 15 angstroms, 14 angstroms, 13 angstroms, 12 angstroms, 11 angstroms, 10 angstroms, or about 5 angstroms or less, or within a distance in a range defined by any two of the foregoing values ​​(e.g., 30-5 angstroms, 30-10 angstroms, 20-5 angstroms, 15-10 angstroms, 12-10 angstroms). In some embodiments, there are no intervening surface-exposed negatively charged amino acids between at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed positively charged amino acids of a cluster. In some embodiments, there are no surface-exposed negatively charged amino acids between any two of the surface-exposed positively charged amino acids of a cluster.

[0097] A variant antibody is also provided herein, the antibody includes at least one destruction cluster of positively charged amino acids, wherein the variant antibody is different from the original antibody, and the original antibody includes an original cluster of amino acids with at least two (e.g., 2, 3, 4, 5, 6 or more) positively charged (e.g., linearly along the peptide backbone) within 12 residues of each other. The variant antibody can be different from the original antibody by replacing at least one of the positively charged amino acids of the original cluster with a negatively charged or uncharged amino acid, wherein the cluster is outside any CDR of the original antibody. The replacement of positively charged amino acids can destroy the original cluster (e.g., by making the surface exposed positive charge at the corresponding position on the variant antibody less than the original antibody). In some embodiments, the original cluster has at least two positively charged amino acids within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues of each other. As used herein, two amino acids are within 1 residue of each other when they are immediately adjacent to each other in a polypeptide chain, and are within 2 residues of each other when there is one other amino acid residue between them in the polypeptide chain, and so on. In some embodiments, the CDR regions and / or framework regions are based on the Kabat definition. In some embodiments, the CDR regions and / or framework regions are based on the AHo definition.

[0098] In some embodiments, the original cluster is destroyed, wherein the number of positively charged amino acids exposed on the surface in the variant antibody is reduced by at least one (e.g., reduced by one, two, three or more) compared to the original antibody. In some embodiments, the variant antibody differs from the original antibody by up to 3 (e.g., 1, 2 or 3) amino acid substitutions (e.g., negatively charged or uncharged amino acids), thereby destroying the cluster. In some embodiments, the number of positively charged amino acids exposed on the surface in the variant antibody is reduced, reduced by about, or reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 100% compared to the original antibody, or reduced by any two of the aforementioned values. In the case of percentages in the range defined (e.g., reduced by 30-90%, 30-70%, 50-100%, 30-60%, etc.), the cluster is destroyed. In some embodiments, the original cluster is destroyed, wherein the number of positively charged amino acids exposed on the surface in the variant antibody is reduced by about 33% or more compared to the original antibody. In some embodiments, the original cluster is destroyed, wherein the number of positively charged amino acids exposed on the surface in the variant antibody is reduced by about 66% or more compared to the original antibody. In some embodiments, the variant antibody includes a destruction cluster, which has or has about 70%, 60%, 50%, 40%, 30%, 20%, 10% or 0% of the total positive charge attributable to the original cluster in the original antibody due to the destruction of the cluster, or a percentage (e.g., 0-70%, 30-70%, 0-40%, etc.) within the range defined by any two of the aforementioned values. For example, but not limited to, the original antibody can have a cluster of 3 positively charged amino acids (e.g., lysine and / or arginine), and one of the positively charged amino acids can be replaced by an uncharged or negatively charged amino acid (e.g., glutamine) in the variant antibody, thereby destroying the original cluster. In some embodiments, the variant antibody includes a destruction cluster, which has about 66% or less of the total positive charge attributable to the cluster in the original antibody due to the destruction of the cluster. In some embodiments, the variant antibody comprises a disrupted cluster, which has about 33% or less of the total positive charge attributed to the cluster in the original antibody due to the disruption of the cluster. In some embodiments, the total positive charge attributed to the cluster is determined by using an algorithm suitable for solving the Poisson-Boltzmann equation, such as an adaptive Poisson-Boltzmann solver (APBS).

[0099] The relevant characteristics of the amino acid residues of the antibody (e.g., surface exposure, distance between amino acids) can be determined using any suitable options. Suitable options include, but are not limited to, solving crystal structures, homology modeling, molecular dynamics simulations, adaptive Poisson-Boltzmann solvers, van der Waals radius, solvent accessible area, or a combination thereof.

[0100] In some embodiments, at least two of the surface-exposed positively charged amino acids of the cluster in the original antibody are within a contiguous stretch of 15 or less (e.g., 15, 12, 10, 8, 7, 6, 5, 4, 3, or 2) residues in the original antibody polypeptide. In some embodiments, in the polypeptide of the original antibody, at least two of the surface-exposed positively charged amino acids of the cluster in the original antibody are at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200 or more residues apart, or the number of residues within a range defined by any two of the foregoing values ​​(e.g., 20-200, 20-50, 40-75, 30-100, 50-150, 100-200, etc.).

[0101] The replacement of the surface-exposed positively charged amino acids of the original cluster can be in any suitable non-CDR region of the antibody (e.g., the original antibody). In some embodiments, the replacement of at least one of the surface-exposed positively charged amino acids of the original cluster is located in the light chain variable region (V L ) framework region (FR). In some embodiments, the replacement of at least one of the surface exposed positively charged amino acids of the original cluster is located in the V L The surface exposed positively charged amino acid cluster that is disrupted in an antibody (e.g., a variant antibody) can be located in any suitable non-CDR region of the antibody (e.g., the original antibody). In some embodiments, the disruption cluster is located in the light chain variable region (V L ) (for example, in V L In some embodiments, the disruption cluster is located in the V L FR2.

[0102] In some embodiments, the original antibody comprises a polypeptide or original polypeptide having an original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), wherein X1 is a positively charged amino acid, wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid, and wherein X4 and X7 are each independently any amino acid, provided that at least one is a positively charged amino acid, wherein the sequence is outside any CDR of the antibody, and wherein at least one of X4 and X7, which are positively charged amino acids, is surface exposed and is part of the original cluster. In some embodiments, the original sequence is located in the V LIn FR2. In some embodiments, the replacement includes replacing at least one of X4 and X7 with a negatively charged or uncharged amino acid. In some embodiments, X1 is part of the original cluster. In some embodiments, X1 as a positively charged amino acid and at least one of X4 and X7 is part of the original cluster. In some embodiments, all positively charged amino acids of the sequence are part of the original cluster. In some embodiments, X2, X3, X5, and X6 are each independently any uncharged amino acid. Antibodies, such as variant antibodies, can include the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), and can also replace at least one of the surface-exposed positively charged amino acids of the original cluster with a negatively charged or uncharged amino acid, and this replacement destroys the original cluster. In some embodiments, antibodies (such as variant antibodies) include the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), and replace at least one of X4 and X7 with a negatively charged or uncharged amino acid, and this replacement destroys the original cluster. In some embodiments, the antibody (e.g., variant antibody) comprises the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), and replaces X4 and X7 with negatively charged or uncharged amino acids, which destroys the original cluster. In some embodiments, the original antibody comprises an original polypeptide comprising the sequence QQX1X2X3X4X5X6X7 (SEQ ID NO: 3), which comprises the original sequence of X1X2X3X4X5X6X7 (SEQ ID ID: 2), as described herein.

[0103] In some embodiments, in the variant polypeptide, the positive charge attributed to the original sequence (X1X2X3X4X5X6X7 (SEQ ID No: 2)) is reduced, reduced by about, or reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or about 100%, or a percentage within the range defined by any two of the foregoing values ​​(e.g., 30-95%, 30-40%, 30-50%, 30-70%, 60-70%, 60-100%, etc.). In some embodiments, the positive charge attributed to the original sequence (X1X2X3X4X5X6X7 (SEQ ID No: 2)) in the variant polypeptide is reduced by about 33% or more. In some embodiments, the positive charge in the variant polypeptide attributed to the original sequence (X1X2X3X4X5X6X7 (SEQ ID No: 2)) is reduced by about 66% or more. In some embodiments, X7 is a positively charged amino acid. In some embodiments, X4 is a positively charged amino acid. In some embodiments, the positively charged amino acid is lysine, arginine or histidine. In some embodiments, the positively charged amino acid is lysine. In some embodiments, at least one of X2 and X6 is proline. In some embodiments, both X2 and X6 are proline. In some embodiments, X3 is glycine or glutamic acid. In some embodiments, X5 is alanine, valine, serine or proline.

[0104] In some embodiments, the original antibody comprises at least one of the following original sequences: KX2X3KX5X6K (SEQID NO:73), wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid; KX2X3X4X5X6R (SEQID NO:74), wherein X2, X3, X4, X5, and X6 are each independently a negatively charged or uncharged amino acid; or KX2X3X4X5X6K (SEQ ID NO:75), wherein X2, X3, X4, X5, and X6 are each independently a negatively charged or uncharged amino acid. In some embodiments, the original sequence is one of the following: KPGKAPK (SEQ ID NO: 5), KPGQAPR (SEQ ID NO: 6), KPEKAPK (SEQ ID NO: 7), KPGKVPK (SEQ ID NO: 8), KPGQPPR (SEQ ID NO: 9), KPGQSPR (SEQ ID NO: 10), KPGLAPR (SEQ ID No: 11), or KPGQPPK (SEQ ID NO: 12), corresponding to X1X2X3X4X5X6X7 (SEQ ID NO: 2). In some embodiments, the original sequence is KPGKAPK (SEQ ID NO: 5) or KPGQAPR (SEQ ID NO: 6), corresponding to X1X2X3X4X5X6X7 (SEQ ID NO: 2).

[0105] The original sequence can be located in any suitable non-CDR region of the antibody (e.g., the original antibody). In some embodiments, the original sequence is located in the light chain variable region (V L ) (for example, in V L In some embodiments, the original sequence is located in the V region of an antibody (e.g., a variant antibody). L FR2.

[0106] In some embodiments, the negatively or uncharged amino acid replacing at least one of the surface-exposed positively charged amino acids of the cluster is glutamine (eg, a K to Q, or R to Q substitution).

[0107] In some embodiments, the variant antibody comprises at least one of the following (which comprises a substitution of at least one of the positively charged amino acids): KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID No: 16), and QQKPGQSPQ (SEQ ID NO: 15), for example in V LIn some embodiments, the variant antibody comprises at least one of the following (which comprises a substitution of at least one of the positively charged amino acids): KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15), for example in V L In some embodiments, the variant antibody comprises V L FR2, has the amino acid sequence: WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

[0108] Also provided is an antibody comprising a variant polypeptide different from an original polypeptide comprising an original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein the original sequence is outside any CDR of the antibody, wherein at least two of X1, X2, X3, X4, X5, X6, and X7 are each independently a surface-exposed positively charged amino acid and form a cluster of surface-exposed positively charged amino acids in the original polypeptide, wherein the variant polypeptide differs from the original polypeptide by modifying at least one of the at least two surface-exposed positively charged amino acids of the original sequence, and the modification reduces at least about 33% of the positive charge attributed to the original sequence. In some embodiments, at least 33% of the positive charge attributed to the original sequence in the variant polypeptide is reduced compared to the original polypeptide. In some embodiments, about 60% or more of the positive charge attributed to the original sequence in the variant polypeptide is reduced. In some embodiments, the modification in the variant polypeptide reduces the positive charge attributed to the sequence by about or at least about 33%, 40%, 50%, 60%, 70%, 80%, 90% or about 100%, or a percentage within the range defined by any two of the foregoing values ​​(e.g., reduced by 33-90%, 33-50%, 50-100%, 33-60%, etc.) compared to the original polypeptide. In some embodiments, the number of positively charged amino acids in the original sequence is reduced by one or more (e.g., at least 2, 3, 4, or 5 or more) in the variant polypeptide. In some embodiments, in the variant polypeptide, at least one of at least two positively charged amino acids of the original sequence (e.g., at least 2, 3, 4, 5, 6 or more) is replaced by a negatively charged or uncharged amino acid.

[0109] In some embodiments, at least X4 and X7 are each independently positively charged amino acids. In some embodiments, positively charged amino acids are lysine, arginine or histidine. In some embodiments, X2, X3, X5 and X6 are each independently any negatively charged or uncharged amino acids in the original polypeptide. In some embodiments, X2, X3, X5 and X6 are each independently any uncharged amino acids. In some embodiments, at least one of X4 and X7 is replaced by negatively charged or uncharged amino acids to reduce positive charge. In some embodiments, the negatively charged or uncharged amino acids replacing at least one of X4 and X7 are glutamine (e.g., K to Q or R to Q replacement). In some embodiments, the position of variant polypeptide corresponding to X5 in the original sequence of the original polypeptide includes alanine or serine.

[0110] In some embodiments, the variant polypeptide comprises a light chain variable region (V L ). L The variant polypeptide may include 3 CDRs and 4 FRs of the light chain. L and heavy chain variable region (V H ), for example as a scFv fragment. In some embodiments, the variant polypeptide comprises a scFv fragment. L Sequence (e.g., V L FR sequences) can be based on human germline sequences. For example, but not limited to, V L The FRs of are the corresponding FRs of human germline sequences, independently in each FR, there are no substitutions, or up to 1, 2, 3, 4, or 5 or more substitutions to disrupt clusters of surface exposed positive charges. In some embodiments, as described herein, there are up to 1, 2, 3, 4, or 5 substitutions in FR2 to disrupt clusters of surface exposed positive charges, while there are no substitutions in other FRs. In some embodiments, V L (For example, V L FR sequences) are based on human germline belonging to the IGKV1 family. In some embodiments, V L The FRs of are the corresponding FRs of human germline sequences belonging to the IGKV1 family, independently in each FR, there are no substitutions, or up to 1, 2, 3, 4, or 5 substitutions to disrupt the clusters of surface-exposed positive charges. In some embodiments, there are up to 1, 2, 3, 4, or 5 or more substitutions in FR2 to disrupt the clusters of surface-exposed positive charges, as described herein, and no substitutions in the other FRs. In some embodiments, V L (For example, V L FR sequence) is based on the human germline sequence IGKV1-39*01. In some embodiments, V LThe FRs of are the corresponding FRs of the human germline sequence IGKV1-39*01, independently in each FR, there are no substitutions, or up to 1, 2, 3, 4, or 5 or more substitutions to disrupt the clusters of surface-exposed positive charges. In some embodiments, as described herein, there are up to 1, 2, 3, 4, or 5 or more substitutions in FR2 to disrupt the clusters of surface-exposed positive charges, while there are no substitutions in the other FRs. In some embodiments, V L (For example, V L FR sequences) are based on human germline belonging to the IGKV3 family. In some embodiments, V L The FRs of are the corresponding FRs of human germline sequences belonging to the IGKV3 family, independently in each FR, there are no substitutions, or up to 1, 2, 3, 4, or 5 or more substitutions to disrupt the clusters of surface-exposed positive charges. In some embodiments, as described herein, there are up to 1, 2, 3, 4, or 5 or more substitutions in FR2 to disrupt the clusters of surface-exposed positive charges, while there are no substitutions in the other FRs. In some embodiments, V L (For example, V L FR sequence) is based on the human germline sequence IGKV3-15*01. In some embodiments, V L The FRs of are the corresponding FRs of the human germline sequence IGKV3-15*01, independently in each FR, there are no substitutions, or up to 1, 2, 3, 4, or 5 or more substitutions to disrupt the clusters of surface-exposed positive charges. In some embodiments, as described herein, there are up to 1, 2, 3, 4, or 5 or more substitutions in FR2 to disrupt the clusters of surface-exposed positive charges, while there are no substitutions in the other FRs. In some embodiments, V L (For example, V L FR sequences) are based on human germline sequences from subgroup 1 from both the proximal and distal clusters: IGKV1, IGKV1D. In some embodiments, V L (For example, V LFR sequences) are based on human germlines from subgroups 2-7 of both the proximal and distal clusters: IGKV2, IGKV3, IGKV4, IGKV5, IGKV6, IGKV7, IGKV2D, IGKV3D, IGKV6D. As used herein, the variable regions of the antibodies of the present disclosure "based on" a reference sequence (e.g., a human germline sequence) mean that the framework regions (e.g., FR1, FR2, FR3, and FR4) of the antibodies have sequences of corresponding framework regions derived from the reference sequence (e.g., a human germline sequence), independently in each FR, with no substitutions, or with up to 1, 2, 3, 4, or 5 or more substitutions to disrupt clusters of surface-exposed positive charges. In some embodiments, as described herein, there are up to 1, 2, 3, 4, or 5 or more substitutions in FR2 to disrupt clusters of surface-exposed positive charges, while there are no substitutions in other FRs. In some embodiments, the variable regions of an antibody based on a reference sequence (e.g., a human germline sequence) have framework regions that have, have about, or have at least 80%, 85%, 90%, 95%, or about 100% identity to the corresponding framework regions of the reference sequence (e.g., a human germline sequence), or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 85-95%, 90-100%, etc.). In some embodiments, the variable regions of an antibody based on a reference sequence (e.g., a human germline sequence) have framework regions that have the sequence of the corresponding framework region of the reference sequence (e.g., a human germline sequence) with up to 8, 7, 6, 5, 4, 3, 2, 1 substitutions thereto, or no substitutions thereto.

[0111] In some embodiments, the cluster is located in the V L In some embodiments, the cluster is located in the V L Within FR2.

[0112] In some embodiments, the original polypeptide is based on V L The first human germline sequence, the V L having an original FR2 comprising the original sequence, and wherein the variant polypeptide is obtained by replacing the original FR2 with the original FR3 L The second FR2 of the second human germline sequence of the present invention is different from the original polypeptide by replacing at least the original sequence, wherein the second FR2 comprises a second sequence corresponding to the original sequence and the positive charge attributed to it is reduced by at least about 33% compared to the original sequence. For example, the V L Can be based on the original V with FR2 L , FR2 contains a cluster of positively charged amino acids. In a non-limiting example, the original V LIt can be based on germline IGKV1-39*01, with the original sequence KPGKAPK (SEQ ID NO: 5) in FR2. In order to destroy the cluster of positively charged amino acids in the original antibody, the key residues in FR2 are replaced with the corresponding residues in FR2 of germline IGKV2-28*01 with the original sequence KPGQSPQ (SEQ ID NO: 16). In some embodiments, this involves at least three replacements of the original FR2 sequence. In some embodiments, a subsequence of FR2 of at least 3, 4, 5, 6, 7 or more residues is replaced to destroy the cluster of positively charged amino acids. In some embodiments, the entire FR2 is replaced to destroy the cluster of positively charged amino acids.

[0113] The first human germline sequence can be any suitable human germline sequence having a cluster of at least two positively charged amino acids in the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1), such as in V as discussed above. L The second human germline sequence may be in V L Any suitable human germline sequence having a disruption cluster in FR2. Suitable second human germline sequences include, but are not limited to, IGKV2-28*01. In some embodiments, the second human germline sequence includes, but is not limited to, one of IGKV2-29*02, IGKV2D-29*02, IGLV3-1*01, IGKV3-19*01, IGKV2-28*01, IGLV6-57*01, IGKV1-40*01, IGLV3-21*02, and IGLV3-21*03.

[0114] In some embodiments, the antibody (e.g., variant antibody) includes at least one of the following (which includes a substitution of at least one of the positively charged amino acids): KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID No: 16), and QQKPGQSPQ (SEQ ID NO: 15), for example in V L In some embodiments, the antibody (e.g., variant antibody) includes at least one of the following (which includes a substitution of at least one of the positively charged amino acids): KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15), for example in V L In some embodiments, the antibody comprises V LFR2 has the amino acid sequence: WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

[0115] In some embodiments, the cluster is located in the hinge region of the antibody, such as the upper hinge region. In some embodiments, the upper hinge region with the cluster (e.g., in the original antibody without substitution or modification as described herein) includes the original sequence EPKSSDKTHT (SEQ ID NO: 38). In some embodiments, the antibody (e.g., variant antibody) includes the upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39).

[0116] In some embodiments, the molecular weight of the antibody (e.g., original antibody and / or variant antibody) (as a dimer of a polypeptide, e.g., excluding any non-polypeptide features) is about 10 kDa, 20 kDa, 30 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, or about 110 kDa, or a molecular weight within a range defined by any two of the foregoing values ​​(as a dimer of a polypeptide, e.g., excluding any non-polypeptide features). In some embodiments, the molecular weight of the antibody (as a dimer of a polypeptide, e.g., excluding any non-polypeptide features) is about 10-110 kDa, e.g., about 10-90 kDa, about 30-110 kDa, about 40-90 kDa, or about 30-80 kDa. In some embodiments, the molecular weight of the antibody (as a dimer of a polypeptide, e.g., excluding any non-polypeptide features) is about 40-60 kDa, about 70-90 kDa, or about 45-85 kDa. In some embodiments, the molecular weight of the antibody (as a dimer of a polypeptide, e.g., excluding any non-polypeptide features) is about 110 kDa or less, e.g., about 90 kDa or less, or about 80 kDa or less.

[0117] In some embodiments, the variant antibody is substantially identical to the original antibody except that at least one of the surface-exposed positively charged amino acids of the cluster is replaced with a negatively or uncharged amino acid. In some embodiments, the variable region (e.g., V L and V H) is substantially the same as the original antibody. In some embodiments, the pattern (pattern, pattern) of the isoelectric electrostatic surface (e.g., patches of positive and negative charges) is substantially the same as the original antibody, except that the surface-exposed positive charge attributable to the destruction cluster of positively charged amino acids is reduced due to the replacement of at least one of the surface-exposed positively charged amino acids of the cluster with negatively charged or uncharged amino acids. In some embodiments, the variant antibody differs from the original antibody by up to 3 (e.g., 1, 2, or 3) amino acid substitutions. In some embodiments, the amino acid substitutions are outside any CDR of the antibody.

[0118] In some embodiments, the antibody (including, for example, the original antibody, the variant antibody) comprises an antigen binding fragment, such as, but not limited to, a scFv fragment. In some embodiments, the antibody is a miniantibody or a cys-diabody. In some embodiments, the antibody is a miniantibody. In some embodiments, the antibody is a miniantibody and comprises Fig.12 C of any one of the amino acid sequences shown (SEQ ID NOs: 49-69) H 3. In some embodiments, the antibody is a cys-diabody.

[0119] Also provided herein is a minibody or cys-diabody comprising a variant polypeptide and a light chain variable region (V L )FR2, wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two are surface-exposed positively charged amino acids, which form a cluster of surface-exposed positively charged amino acids in the original polypeptide, wherein the variant polypeptide is different from the original polypeptide by modifying at least one of the at least two surface-exposed positively charged amino acids of the original sequence, and the modification reduces at least 33% of the positive charge attributed to the original sequence. Suitable options for the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1) are as discussed herein.

[0120] In some embodiments, the negatively charged or uncharged amino acid replacing at least one of X4 and X7 is glutamine (e.g., a K to Q or R to Q substitution). In some embodiments, the variant polypeptide includes an alanine or serine at the position corresponding to X5 in the sequence of the original polypeptide.

[0121] Also provided is a light chain variable region (V L) of a miniantibody or cys-diabody, the light chain variable region having a FR2 sequence of X1X2X3QAX6X7 (SEQ ID NO: 4), wherein X1 is a surface-exposed positively charged amino acid, wherein X2, X3, and X6 are each independently any negatively charged or uncharged amino acid, and wherein X7 is glutamine or lysine. In some embodiments, X2, X3, and X6 are each independently any uncharged amino acid. In some embodiments, the FR2 sequence comprises KPGQAPK (SEQ ID NO: 13) or KPGQAPQ (SEQ ID ID: 14).

[0122] In some embodiments, X1 is lysine. In some embodiments, X2 and / or X6 are proline. In some embodiments, X2 and X6 are each proline. In some embodiments, X3 is glycine or glutamic acid.

[0123] In some embodiments, at V L In FR2, the minibody or cys-diabody comprises at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15). In some embodiments, in V L In FR2, the miniantibody or cys-diabody comprises at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID ID: 14), KPGQSPQ (SEQ ID NO: 16), and QQKPGQSPQ (SEQ ID NO: 15). In some embodiments, the miniantibody or cys-diabody comprises V L FR2, V L FR2 has an amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

[0124] The present invention also provides an antibody, a minibody or a cys-diabody, comprising a light chain variable region (V L ), the light chain variable region includes (according to IMGT numbering): A49 and either (i) Q51; or (ii) Q48 and K51. In some embodiments, V L Also included is K45 (IMGT number). In some embodiments, V L Also included is G47 (according to IMGT numbering). The present invention also provides an antibody, a mini-antibody or a cys-diabody, including a light chain variable region (V L), the light chain variable region includes (according to IMGT numbering): Q43, A49, and either (i) Q51; or (ii) Q48 and K51. In some embodiments, V L Also included is K45 (IMGT number). In some embodiments, V L Also includes G47 (according to IMGT numbering).

[0125] Also provided herein is a miniantibody having an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39). In some embodiments, the miniantibody comprises a hinge region connected to the C H 3, the hinge region comprises an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39). In some embodiments, the miniantibody comprises a hinge sequence of EPGSSDGTHTCPPCPPC (SEQ ID NO: 71). In some embodiments, the miniantibody comprises a hinge region connected to C H 3, the hinge region comprises EPGSSDGTHTCPPCPPC (SEQ ID NO: 71). In some embodiments, the minibody comprises a V having a disruption cluster of positively charged amino acids. L Any of the FR2 sequences, as described herein.

[0126] In any of the antibodies, minibodies, or cys-diabodies herein, in some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) comprises a light chain variable region (V L ... L ) FR1, FR3 and FR4: IGKV1, IGKV1D. In some embodiments, V L FR1, FR3, and FR4 are each from the corresponding V sequences of human germlines of subgroups 2-7 of both the proximal and distal clusters. L FR1, FR3, and FR4: IGKV2, IGKV3, IGKV4, IGKV5, IGKV6, IGKV7, IGKV2D, IGKV3D, IGKV6D. In some embodiments, V L FR1, FR3, and FR4 are each derived from the corresponding V sequences of human germline IGKV1-39*01 or IGKV3-15*01. L FR1, FR3, and FR4. In some embodiments, V L FR1, FR3, and FR4 are each derived from Figures 5A-5C and Figures 7A-7D The corresponding V of any of the sequences shown in L FR1, FR3, and FR4. In some embodiments, V LFR1, FR3, and FR4 are each Figures 5A-5C and Figures 7A-7D The corresponding V of any of the sequences shown in L FR1, FR3, and FR4 have at least 80%, 85%, 90%, 95%, 97%, or about 100% identity, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.). In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) comprises Figures 5A-5C and Figures 7A-7D The heavy chain variable region (V H ) framework sequences (FR1, FR2, FR3 and FR4). In some embodiments, V H FR1, FR2, FR3, and FR4 are each Figures 5A-5C and Figures 7A-7D The corresponding V of any of the sequences shown in H FR1, FR2, FR3, and FR4 are at least 80%, 85%, 90%, 95%, 97%, or about 100% identical, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.). In some embodiments, the V of an antibody, minibody, or cys-diabody (or variant thereof) is L and V H FR sequence according to Figures 5A-5C and Figures 7A-7D Any of the constructs shown can be paired or combined. In some embodiments, the framework regions are based on the Kabat definition. In some embodiments, the framework regions are based on the AHo definition.

[0127] In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) comprises a light chain variable region (V L )FR1, the light chain variable region includes residues 1-23 of SEQ ID NO:42 (excluding the signal peptide). In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) includes V L FR3, the V L FR3 includes residues 57-88 of SEQ ID NO:42 (excluding the signal peptide). In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) includes V L FR4, the V LFR4 includes residues 98-107 of SEQ ID NO:42 (excluding the signal peptide). In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) includes: a light chain variable region (V L ... L )FR1, V comprising residues 57-88 (excluding the signal peptide) of SEQ ID NO:42 L FR3, and V comprising residues 98-107 of SEQ ID NO:42 (excluding the signal peptide) L In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) comprises a light chain variable region (V L )FR1, the light chain variable region (V L )FR1 includes residues 1-30 of SEQ ID NO: 45. In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) includes V L FR3, the V L FR3 includes residues 59-97 of SEQ ID NO: 45. In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) includes V L FR4, the V L FR4 includes residues 109-119 of SEQ ID NO: 45. In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) includes: a light chain variable region (V L ... L )FR1, comprising V residues 59-97 of SEQ ID NO:45 L FR3, and V comprising residues 109-119 of SEQ ID NO:45 L FR4.

[0128] In any of the minibodies disclosed herein, in some embodiments, the minibody comprises Fig.12 C of any one of the amino acid sequences shown in (SEQ ID NOs: 49-69) H 3. In some embodiments, the miniantibody comprises C H 3. The C H 3 and Fig.12 Any of the amino acid sequences shown in (SEQ ID NOs: 49-69) has at least 80%, 85%, 90%, 95%, 97% or about 100% identity, or a percentage within a range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.).

[0129] Also provided is an antibody comprising at least one disrupted cluster of positively charged amino acids, the cluster having at least three positively charged amino acids in a framework region (FR) of the original antibody, wherein the antibody differs from the original antibody having the cluster in that at least one of the positively charged amino acids of the cluster is replaced with a negatively charged or uncharged amino acid, whereby the cluster is disrupted. In some embodiments, the number of positively charged amino acids is reduced by 1, 2, 3 or more residues, thereby providing a disrupted cluster in the antibody. In some embodiments, the cluster is located in the V region of the original antibody. L In some embodiments, the antibody is a minibody or a cys-diabody.

[0130] In any of the antibodies, miniantibodies or cys-diabodies (or variants thereof) disclosed herein, in some embodiments, the antibody, miniantibody or cys-diabodies specifically binds to any suitable antigen target. In some embodiments, the antibody, miniantibody or cys-diabodies specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA. In some embodiments, the antibody, miniantibody or cys-diabodies (or variants thereof) comprises a heavy chain having Figure 5B , Figure 5C , Figures 7B-7D In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) comprises a light chain variable region and a heavy chain, wherein the light chain variable region has Figure 5B , Figure 5C , Figures 7B-7D , and the heavy chain has Figure 5B , Figure 5C , Figures 7B-7D In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) comprises V H , the V H have Figure 5B , Figure 5C , Figures 7B-7D V in any of the sequences H In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) comprises an amino acid sequence having Figure 5B , Figure 5C , Figures 7B-7D V in any of the sequences L The amino acid sequence of V L , and having Figure 5B , Figure 5C , Figures 7B-7D V in any of the sequences H The amino acid sequence of V H In some embodiments, the antibody, minibody or cys-diabody (or variant thereof) comprises V H , the V H With Figure 5B , Figure 5C , Figures 7B-7D V in any of the sequences H An amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 80-90%, 85-99%, 90-97%, etc.). In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) comprises V L and V H , the V L With Figure 5B , Figure 5C , Figures 7B-7D V in any of the sequences L An amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 80-90%, 85-99%, 90-97%, etc.), and the V H With Figure 5B , Figure 5C , Figures 7B-7D V in any of the sequences H An amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 80-90%, 85-99%, 90-97%, etc.). In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100% identity. Figure 5B , Figure 5C , Figures 7B-7DAn amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 80-90%, 85-99%, 90-97%, etc.), with or without a signal peptide. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 80-90%, 85-99%, 90-97%, etc.), with or without a signal peptide. Figure 5B , Figure 5C , Figures 7B-7D An amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 80-90%, 85-99%, 90-97%, etc.) identity to any of the sequences of, without a signal peptide.

[0131] In any of the antibodies, minibodies or cys-diabodies disclosed herein, in some embodiments, the antibody, minibodies or cys-diabodies do not include SEQ ID NO: 46. In some embodiments, the antibody, minibodies or cys-diabodies do not include a V sequence in SEQ ID NO: 46. L The amino acid sequence of V L In some embodiments, the antibody, minibody, or cys-diabody does not bind to FAP (eg, human FAP).

[0132] The antibody, miniantibody or cys-diabody can have monovalent, divalent or multivalent antigen binding specificity. In some embodiments, the antibody, miniantibody or cys-diabody is monovalent. In some embodiments, the antibody, miniantibody or cys-diabody is divalent. The bispecific antibody, miniantibody or cys-diabody can have two different heavy chain / light chain pairs, and / or it can recognize two different epitopes. In some embodiments, the antibody, miniantibody or cys-diabody is multivalent.

[0133] In some embodiments, the antibody comprises an Fc region (e.g., scFv-Fc, nanobody-Fc). In some embodiments, the antibody comprises an amino acid sequence identical to the amino acid sequence of a native or naturally occurring Fc region (e.g., a human IgG1 Fc region). In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 76, as shown below.

[0134]

[0135] In some embodiments, the Fc region comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to SEQ ID NO: 76, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.). In some embodiments, the Fc region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) that alter (e.g., increase or decrease) effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 76, having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) that alter (e.g., increase or decrease) effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, the Fc region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) that reduce effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, the Fc region includes the amino acid sequence of SEQ ID NO: 76, having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) that reduce effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, one or more mutations that reduce FcRn binding are mutations (e.g., substitutions) of any one or more (e.g., one, two, or all three) of I253, H310, and H435 (EU numbering). In some embodiments, one or more mutations that reduce binding to FcRn are any one or more (e.g., one, two, or all three) of I253A, H310A, and H435A (EU numbering). In some embodiments, the Fc region includes one or more (e.g., one, two, or all three) of I253A, H310A, and H435A (EU numbering). In some embodiments, one or more mutations that reduce binding to the Fc neonatal receptor are mutations (e.g., substitutions) of N297 (EU numbering). In some embodiments, one or more mutations that reduce binding to the Fc neonatal receptor are N297Q, N297A, or N297G (EU numbering). In some embodiments, the Fc region includes N297Q (EU numbering).

[0136] In some embodiments, one or more mutations that reduce Fc effector function are any one or more of the following mutations (e.g., substitutions): L234, L235, G236, G237, P238, H268, K322, L328, P329, A330, P331. In some embodiments, one or more mutations that reduce Fc effector function are any one or more of the following: L234A, L235A, G236R, G237A, P238S, H268A, K322A, L328R, P329G, A330S, P331S. Other non-limiting examples of known mutations that alter Fc effector function can be found in Wilkinson and Hale (2022) "Systematic analysis of the varied designs of 819 therapeutic antibodies and Fc fusion proteins assigned international nonproprietary names." MAbs. 2022 Jan-Dec; 14(1): 2123299. doi: 10.1080 / 19420862.2022.2123299. In some embodiments, the Fc region does not include a C-terminal lysine.

[0137] In some embodiments, the antibodies of the present disclosure are scFv-Fc (e.g., scFv fused to Fc). In some embodiments, the scFv-Fc comprises a variable weight (V H ) domain-linked variable light (V L ) domain, hinge domain and Fc region. In some embodiments, the antibodies of the present disclosure are - Fc (eg, a camelid nanobody (or a single domain fragment) fused to Fc).

[0138] Use as imaging agent

[0139] In any of the antibodies, minibodies or cys-diabodies of the present disclosure, in some embodiments, the antibody, minibody or cys-diabody comprises a detectable label. In some embodiments, the antibody, minibody or cys-diabody comprises a radionuclide or an organic dye. In some embodiments, the radionuclide is one or more of the following: 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe,59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 211 At, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 225 Ac, and 227Th. In some embodiments, exemplary paramagnetic ion substances that can be used as detectable markers include, but are not limited to, transition metals and lanthanide metal ions (e.g., metals with atomic numbers of 6 to 9, 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. In some embodiments, the detectable marker is a radionuclide, such as yttrium-90, lutetium-177, zirconium-89, copper-64, fluorine-18, gallium-68, or actinium-225. Other embodiments of radionuclides include copper-67, astatine-211, lead-212, bismuth-212, bismuth-213, and thorium-227. In some embodiments, treatment of target cells with these radionuclides can result in cell damage and death of target tissues.

[0140] In some embodiments, the detectable label is a bioluminescent or fluorescent compound. Examples include fluorescein, fluorescein isothiocyanate (FITC), OREGON GREEN TM , rhodamine, Texas Red, tetramethylrhodamine isothiocyanate (tetrarhodamine isothiocyanate, TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), self-quenching fluorescent compounds activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, or a combination thereof.

[0141] In some embodiments, the organic dye is indocyanine green (ICG) or one of the dyes that fluoresce in the near-infrared region, such as IR800.

[0142] In some embodiments, any of the detectable labels, radionuclides or organic dyes described herein can be conjugated to an antibody, minibody or cys-diabody. In some embodiments, an antibody, minibody or cys-diabody further comprises a chelating ligand, or is conjugated to a chelating ligand. Examples of chelating ligands that can be used according to embodiments of the present invention include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), dodecanetetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane, 1-(pentanedioic acid)-4,7,10-triacetic acid (DOTAGA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-pentanedioic acid-5,7-triacetic acid (NODAGA), 4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl-acetic acid (4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl-acetic acid ( acid, NETA), deferoxamine (Df, also known as DFO), porphyrin, polyamine, crown ether, bis-thiosemicarbazones, polyoxime and the like.

[0143] Also provided herein is a composition comprising any one of the antibodies, minibodies or cys-diabodies disclosed herein; and a pharmaceutically acceptable carrier.

[0144] Use as a therapeutic agent

[0145] In some embodiments, any of the antibodies, minibodies or cys-diabodies of the present disclosure can be included in a therapeutic composition or pharmaceutical composition, for example, for treating a subject in need of treatment for a disease or condition. In some embodiments, the therapeutic composition is used to treat cancer. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable buffer.

[0146] In some embodiments, antibodies, miniantibodies or cys-diabodies or variants thereof as described herein are combined with therapeutic agents. As used herein, "therapeutic agents" are atoms, molecules or compounds that can be used to treat disorders associated with target molecules. Examples of therapeutic agents include, but are not limited to, drugs, chemotherapeutics, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes (e.g., enzymes that cut prodrugs into cytotoxic agents at antigen binding construct binding sites), nucleases, hormones, immunomodulators, antisense oligonucleotides, chelating agents, boron compounds, photoactive agents and dyes, elastin-like polypeptides such as PLGA and nanoparticles. Examples of disorders include disorders associated with one or more target molecules.

[0147] In some embodiments, antibodies, miniantibodies or cys-diabodies, or variants thereof as described herein are combined with therapeutic agents. While miniantibodies or cys-diabodies, or other antigen-binding fragments may have a shorter circulation half-life than full-length antibodies, in some embodiments, these forms may exhibit improved tumor penetration based on their smaller size and are therapeutically effective when appropriately matched with cytotoxic drugs or radioisotopes. In some embodiments, antibody, miniantibody or cys-diabody, drug conjugate methods may be used. In some embodiments, the treatment method includes radioimmunotherapy by attaching (connecting) appropriate radiolabels (such as iodine-131), beta-emitters or alpha-emitters (such as yttrium-90, lutetium-177, copper-67, terbium-149, terbium-161, astatine-211, lead-212, bismuth-212, actinium-225, bismuth-213 and thorium-227), which can deliver cell damage and death to target tissues. In some embodiments, the radiolabel comprises terbium-149, terbium-161, or lead-212. In some embodiments, treatment with these fragments conjugated to cytotoxic drugs or radionuclides results in less nonspecific toxicity because they are cleared more rapidly from the body.

[0148] In some embodiments, the marker and / or therapeutic agent comprises 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y. 90 Y.89 Sr. 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 2. 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th and 225 Ac, or any combination thereof.

[0149] In some embodiments, antibodies, minibodies and / or cys-diabodies or variants thereof as described herein are combined with a therapeutic agent (e.g., a chemotherapeutic agent). Chemotherapeutic agents generally have cytotoxic or cytostatic properties and may include alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, hormone therapy, targeted therapeutic agents, and immunotherapeutic agents. In some embodiments, according to embodiments of the present disclosure, chemotherapeutic agents that may be used as detectable labels include, but are not limited to, 13-cis-Retinoic acid (13-cis-Retinoic acid Acid), 2-chlorodeoxyadenosine, 5-azacitidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-trans retinoic acid, alpha-interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacytidine, bacillus Calmette-Guérin (BCG) calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide, cytarabine, darbepoetin alfa, dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexamethasone, dexrazoxane, dactinomycin, daunorubicin, dacarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epoetin alfaalfa), erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab, ozogamicin, goserelin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, altretinoin, hydrocortisone, hydroxyurea, ibritumomab, interferon alfa, interleukin-2, interleukin-11, isotretinoin, ixabepilone, idarubicin, imatinib mesylate mesylate), ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, pegylated interferon, pegaspargase, pegfilgrastim, pegylated L-tian PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozotocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolomide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, trimesatide, aclarubicin, vincristine, vinblastine, vindesine, vinorelbine, vorinostat, or zoledronic acid.

[0150] In some embodiments, an antibody, minibody and / or cys-diabody or variants thereof as described herein is conjugated to a cytotoxic agent (eg, a toxin). Cytotoxic agents or toxins that can be used according to embodiments of the present disclosure include, but are not limited to, auristatin E, auristatin F, dolastatin 10, dolastatin 15, combretastatin and its analogs, maytansinoids, calicheamicin, alpha-amanitin, pyrrolobenzodiazepine dimers, epothilones, duocarmycins and their analogs, tubulysin D, basillistatins, ricin, abrin, RNase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and the like. exotoxin) and Pseudomonas endotoxin.

[0151] Any of the antibodies, minibodies or cys-diabodies described herein, or variants thereof described herein, may be further combined with one or more additional therapeutic agents, detectable markers, nanoparticles, carriers or combinations thereof. For example, the antigen binding construct may be radiolabeled with iodine-131 and bound to a lipid carrier such that the anti-target molecule-lipid conjugate forms micelles. The micelles may incorporate one or more therapeutic or detectable markers.

[0152] Medicine or therapeutic composition described herein can be applied by any suitable route of administration.Route of administration can refer to any route of administration known in the art, including but not limited to aerosol, enteral, intranasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch) or vagina. "Transdermal" application can be completed using a topical cream or ointment or by a transdermal patch. "Parenteral" refers to the route of administration usually associated with injection, including suborbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, subarachnoid, subcapsular, sublingual, subcutaneous, transmucosal or transtracheal. In some embodiments, the antigen-binding construct can be delivered in surgery in a topical form during intervention or resection.

[0153] Nucleic acid, expression vector, host cell

[0154] Also provided herein are nucleic acids encoding antibodies of the present disclosure. As used herein with respect to antibodies, miniantibodies or cys-diabodies comprising monomeric chains dimers, "encoding antibodies, miniantibodies or cys-diabodies" refers to encoding at least one monomer in an antibody, miniantibody or cys-diabody, wherein the nucleic acid encoding the monomeric chain can produce a dimeric antibody, miniantibody, or cys-diabody when expressed under appropriate conditions. In some embodiments, the nucleic acid encodes a variant polypeptide of the present disclosure.

[0155] In some embodiments, a nucleic acid is provided that encodes any of the antibodies, minibodies, or cys-diabodies disclosed herein. In some embodiments, a nucleic acid is provided that encodes any of the variant polypeptides disclosed herein. In some embodiments, an expression vector comprising these nucleic acid sequences is provided. In some embodiments, the expression vector comprises pcDNA3.1 for mammalian expression. TM In some embodiments, the expression vector comprises a plasmid. In some embodiments, the vector comprises a viral vector, such as a retrovirus or adenovirus vector. In embodiments, the vector comprises a cosmid, a YAC, or a BAC.

[0156] A host cell is also provided, such as a genetically engineered host cell, which produces any of the antibodies, miniantibodies or cys-diabodies described herein. The host cell can be a mammalian cell, such as HEK293, 293f, Epxi293 cell line, or CHO-K1 cell line. In some embodiments, one or more of a variety of mammalian or non-mammalian expression systems are used to produce antibodies, miniantibodies or cys-diabodies disclosed herein, including but not limited to mammalian expression systems (e.g., CHO-K1 cells), bacterial expression systems (e.g., Escherichia coli (E.coli), Bacillus subtilis (B.subtilis)), yeast expression systems (e.g., Pichia (Pichia), Saccharomyces cerevisiae (S.cerevisiae)) or any other known expression systems. Other systems may include insect cells and / or plant cells.

[0157] method

[0158] Also provided are methods for enhancing the biodistribution and / or pharmacokinetics of antibodies (e.g., minibodies, cys-diabodies) having clusters of surface-exposed positively charged amino acids by disrupting the clusters. Figure 1 , a method for enhancing the biodistribution and / or pharmacokinetics of an antibody (e.g., a variant antibody) is provided. Method 100 may include identifying an original antibody having a polypeptide at box 110, the polypeptide including at least one surface-exposed positively charged amino acid cluster, the cluster including at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed positively charged amino acids within 30 angstroms of each other. The original antibody may be identified using any suitable option. In some embodiments, an antibody having at least one surface-exposed positively charged amino acid cluster is identified using an adaptive Poisson-Boltzmann solver, a van der Waals radius, a solvent accessible area, or a combination thereof. In some embodiments, an antibody having at least one surface-exposed positively charged amino acid cluster is identified by analyzing the isoelectric electrostatic surface of the antibody and calculating the linear distance between the centroids of the charged amino acid residues in a suitable model of the antibody (e.g., modeled using an adaptive Poisson-Boltzmann solver). The method may also include replacing at least one of at least two surface-exposed positively charged amino acids of at least one cluster with a negatively charged or uncharged amino acid at block 120, thereby disrupting at least one cluster. Variant antibodies having disrupted clusters of surface-exposed positively charged amino acids may have enhanced biodistribution and / or pharmacokinetics compared to the original antibody. The original antibody may include any suitable surface-exposed positively charged amino acid clusters disclosed herein.

[0159] refer to Figure 2 , a method for enhancing the biodistribution and / or pharmacokinetics of an antibody is provided. Method 200 may include, at box 210, identifying an original antibody comprising a polypeptide comprising a cluster of at least two surface-exposed positively charged amino acids within 12 residues of each other. The method may also include, at box 220, replacing at least one of at least two surface-exposed positively charged amino acids of at least one cluster with a negatively charged or uncharged amino acid, thereby destroying at least one cluster. Variant antibodies having a destroyed cluster of surface-exposed positively charged amino acids can enhance biodistribution and / or pharmacokinetics compared to the original antibody. In some embodiments, the cluster comprises at least two positively charged amino acids within 6 residues of each other. In some embodiments, the cluster comprises at least two positively charged amino acids within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residues of each other.

[0160] In some embodiments, the polypeptide comprises an amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, wherein at least two (e.g., 2, 3, 4 or more) surface-exposed positively charged amino acids comprise at least two of X1, X2, X3, X4, X5, X6, and X7. In some embodiments, at least two surface-exposed positively charged amino acids comprise at least X1. In some embodiments, at least two surface-exposed positively charged amino acids comprise at least X4 and X7. In some embodiments, X2, X3, X5, and X6 are each independently any negatively charged or uncharged amino acid residue in the original polypeptide. In some embodiments, X2, X3, X5, and X6 are each independently any uncharged amino acid. In some embodiments, at least one of X4 and X7 is replaced by a negatively charged or uncharged amino acid. In some embodiments, each of X4 and X7 is independently replaced by an uncharged amino acid. In some embodiments, the at least two surface exposed positively charged amino acids include arginine, lysine and / or histidine.

[0161] In some embodiments, the negatively charged or uncharged amino acid that replaces at least one of the at least two surface-exposed positively charged amino acids comprises a polar amino acid residue. In some embodiments, the polar amino acid is selected from the group consisting of asparagine, glutamine, serine, threonine, and cysteine. In some embodiments, the polar amino acid is selected from the group consisting of asparagine, glutamine, serine, and threonine. In some embodiments, the negatively charged or uncharged amino acid that replaces at least one of the at least two surface-exposed positively charged amino acids is glutamine. In some embodiments, the positively charged amino acid and the negatively charged or uncharged amino acid that are replaced in the cluster have similar side chain lengths. In some embodiments, the positively charged amino acid and the negatively charged or uncharged amino acid that are replaced in the cluster have side chain lengths that differ by less than one or two carbons.

[0162] In some embodiments, the method further comprises replacing up to 3 (eg, 1, 2 or 3) amino acids of the polypeptide to disrupt at least one cluster. In some embodiments, the amino acid substitution is outside any CDR of the antibody.

[0163] As described herein, clusters of positively charged amino acids are present in the original antibody. In some embodiments, the cluster is located in the V L In some embodiments, the cluster is located in the V region of the original antibody. L In some embodiments, the polypeptide having a cluster is based on V LThe first human germline sequence, the V L having an original FR2 containing clusters, and the method comprising using from V L The second FR2 or a corresponding portion thereof of the second human germline sequence of the present invention replaces the original FR2 or a portion thereof comprising the cluster, wherein the second FR2 comprises at least one less surface exposed positively charged amino acid compared to the cluster. In some embodiments, a stretch of amino acids in the original FR2 (e.g., a stretch of 7 amino acids, such as X1X2X3X4X5X6X7 (SEQ ID NO: 1)) is modified by one or more amino acid substitutions to match the corresponding amino acid stretch in the second human germline sequence. In a non-limiting example, the original V L It can be based on germline IGKV1-39*01, with the sequence KPGKAPK (SEQ ID NO: 5) in FR2. The method can include replacing key residues in the original FR2 with corresponding residues in FR2 of germline IGKV2-28*01 with the sequence KPGQSPQ (SEQ ID NO: 16) to destroy clusters of positively charged amino acids in the original antibody. This involves making at least three replacements of the original FR2 sequence. In some embodiments, a subsequence of FR2 of at least 3, 4, 5, 6 or more residues is replaced to destroy clusters of positively charged amino acids. In some embodiments, the entire FR2 is replaced to destroy clusters of positively charged amino acids.

[0164] In some embodiments, the method further comprises binding a chelating ligand to the variant antibody. Any suitable chelating ligand, such as but not limited to those disclosed herein, can be bound to the variant antibody. In some embodiments, the method comprises labeling the antibody with a radionuclide, such as but not limited to those disclosed herein. In some embodiments, the method comprises labeling the antibody with a detectable marker, such as but not limited to those disclosed herein.

[0165] refer to Figure 3 , provides a non-limiting method for preparing a labeled antibody. Method 300 may include selecting a light chain variable region (V L ) of a germline sequence (e.g., a human germline sequence), wherein the germline sequence does not include a cluster of at least two (e.g., 2 or 3) positively charged amino acids within 3 residues of each other in framework region 2 (FR2) of the germline sequence. Any suitable option can be used to select a germline sequence. In some embodiments, by searching for human V L In some embodiments, the germline sequence does not include a cluster of at least two positively charged amino acids within 3, 4, 5, 6, 7, 8, 9, 10 or more residues of each other in framework region 2 (FR2) of the germline sequence.

[0166] Method 300 may also include isolating one or more target-specific antibodies from a population of antibodies at block 320, the population of antibodies comprising V sequences derived from germline sequences. L , and have germline sequence variation throughout the population, wherein one or more target-specific antibodies are L The FR2 sequence does not contain a cluster of at least two positively charged amino acids within 3 residues of each other. The method may also include, at block 330, labeling one or more target-specific antibodies.

[0167] Any suitable option can be used to separate one or more target-specific antibodies. In some embodiments, separation of one or more target-specific antibodies includes screening an antibody colony derived from a germline sequence. In some embodiments, the antibody colony derived from the germline sequence is produced in a genetically modified host organism expressing antibodies based only on a selected germline sequence (e.g., a genetically modified mouse producing antibody clones based on humanization or human germline sequences). In some embodiments, the antibody colony derived from the germline sequence is a part of an antibody phage library based only on a selected germline sequence. In some embodiments, the germline sequence is a human germline sequence. In some embodiments, one or more target-specific antibodies are humanized.

[0168] In some embodiments, the antibody comprises an antigen binding fragment (e.g., a scFv fragment). The target-specific antibodies produced can be reformatted into a suitable antibody format (e.g., a minibody or cys-diabody format). In some embodiments, the method comprises producing one or more target-specific minibodies or cys-diabodies from one or more target-specific antibodies.

[0169] In some embodiments, the label is a radionuclide, including but not limited to those disclosed herein. In some embodiments, labeling one or more target-specific antibodies (including target-specific miniantibodies or cys-diabodies) comprises binding a chelating ligand to one or more target-specific antibodies (or miniantibodies or cys-diabodies).

[0170] Antibodies (e.g., miniantibodies, cys-diabodies) of the present disclosure can be produced using any suitable options. In some embodiments, any method of enhancing an antibody of the present disclosure includes producing an antibody, such as a variant antibody. In some embodiments, antibodies (e.g., miniantibodies, cys-diabodies) are produced by expressing a nucleic acid encoding an antibody (e.g., a monomeric chain encoding an antibody) in a suitable host cell expression system (e.g., a mammalian cell line, a bacterium, an insect cell line, a yeast, etc.).

[0171] Also provided herein is a method of treating a subject using the antibodies, minibodies or cys-diabodies disclosed herein. Figure 4 , a non-limiting method for treating a subject is provided. Method 400 may include, at box 410, identifying a subject who needs to be treated (e.g., cancer) with an antibody, mini-antibody, or cys-diabody or variant thereof disclosed herein, and at box 420, administering a therapeutically effective amount of the antibody, mini-antibody, or cys-diabody to the subject. In some embodiments, the method includes administering a therapeutically effective amount of a composition, such as a pharmaceutical composition, containing an antibody, mini-antibody, or cys-diabody disclosed herein to the subject. In some embodiments, the method is a radiotherapy method, in which a subject who needs radiotherapy (e.g., a subject with cancer) is identified, and the antibody, mini-antibody, or cys-diabody administered to the subject is labeled with a radionuclide. In some embodiments, the subject suffers from cancer. In some embodiments, the subject suffers from cancer or other conditions associated with an antigen to which the antibody, mini-antibody, or cys-diabody specifically binds. In some embodiments, a larger dose of radiation can be administered using the antibody, mini-antibody, or cys-diabody disclosed herein compared to the original antibody, mini-antibody, or cys-diabody.

[0172] A method of radioimmunotherapy is also provided. The method may include identifying a subject in need of radioimmunotherapy, and administering to the subject a therapeutically effective amount of an antibody, minibody or cys-diabody of the present disclosure, or a variant thereof.

[0173] A method for treating cancer in a subject is also provided. The method may include identifying a subject in need of cancer treatment; and administering to the subject a therapeutically effective amount of any of the antibodies, miniantibodies, or cys-diabodies disclosed herein, or variants thereof, thereby treating the cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), thymic carcinoma, lymphoma, myxoid / round cell liposarcoma, liposarcoma, synovial sarcoma, recurrent adult soft tissue sarcoma, gliosarcoma, astrocytoma, acute myeloid leukemia (AML), malignant solitary fibrous tumor of the pleura (MSFT), penile cancer, diffuse intrinsic pontine glioma (Diffuse Intrinsic Pontine Glioma,DIPG), thyroid cancer, squamous cell carcinoma of the head and neck (SCCHN), lung adenocarcinoma, vulvar cancer (squamous cell carcinoma), bladder cancer, cervical squamous cell carcinoma, germ cell tumor, testicular cancer, pancreatic ductal adenocarcinoma, pancreatic adenocarcinoma, non-melanoma skin cancer, retroperitoneal and peritoneal cancer, melanoma, unresectable or metastatic melanoma, mucosal melanoma of the head and neck, uveal melanoma, non-cutaneous melanoma, cutaneous T-cell lymphoma, occult primary tumor, bile duct cancer, gastrointestinal stromal tumor (GIST), mesothelioma, biphasic mesothelioma, malignant pleural mesothelioma, kidney cancer, myelodysplastic syndrome, hepatocellular carcinoma, esophageal and esophagogastric junction cancer (Esophageal and Esophagogastric Junction Carcinoma), Extrahepatic bile duct adenocarcinoma, Small intestinal malignancies, Gastric adenocarcinoma, Cholangiocarcinoma, Intrahepatic and extrahepatic bile duct carcinoma, Ovarian surface epithelial carcinoma, Non-epithelial and epithelial ovarian cancer, Breast cancer, Triple-negative breast cancer, Endometrial carcinoma, Uterine sarcoma, Bone cancer, Colorectal adenocarcinoma, Prostate adenocarcinoma, Hormone-resistant prostate cancer (PC), Neuroendocrine prostate cancer (NEPC), Neuroendocrine tumors, Solid tumors, Follicular lymphoma, Kaposi's sarcoma, Genitourinary tract cancer, Fallopian tube cancer, Malignant glioma, Waldenstrom's macroglobulinemia, Richter syndrome, Refractory splenic marginal zone lymphoma, Refractory small lymphocytic lymphoma, Refractory nodal marginal zone lymphoma, Refractory lymphoplasmacytic lymphoma, Refractory extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue ZoneLymphoma of the Mucosa-Associated Lymphoid Tissue), Refractory Chronic Lymphocytic Leukemia, Multiple Myeloma, Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, Diffuse Large B-cell Lymphoma, Nasopharyngeal Carcinoma, Gastroesophageal Junction Adenocarcinoma, Renal Cell Carcinoma, Colon Cancer, Transitional Cell Carcinoma (TCC), Urothelial Carcinoma (UCC), Glioblastoma Multiforme (GBM), Gallbladder Cancer, and Merkel Cell Carcinoma.

[0174] In some embodiments, the cancer is: Prostate Cancer, Lung Cancer, Melanoma, Breast Malignancies, Central Nervous System and Brain Malignancies, Skin Malignancies, Occult Primary Tumors, Kidney Cancer, Gastrointestinal Malignancies, Ovarian Tumors, Kidney Cancer, Bile Duct Cancer, Bladder Cancer, Esophageal Cancer, Cervical Cancer, Solid Tumors, Head and Neck Cancer, Genitourinary Tumors, Germ Cell Tumors, Testicular Cancer, Pancreatic Cancer, Glioma, Liver Cancer, Bone Malignancies, Colorectal Cancer, Thyroid Cancer, Chest and Respiratory Tumors, Lymphoma, Male and Female Genitourinary Malignancies, Cholangiocarcinoma, Hematological Malignancies, Multiple Myeloma, Gallbladder Cancer, Endocrine Tumors, Eye Cancer, and Hematopoietic and Lymphoid Tissue Tumors.

[0175] In some embodiments, the method is a method for imaging a subject, wherein the antibody, mini-antibody or cys-diabody is detectably labeled, and the method also includes imaging the subject to detect the antibody, mini-antibody or cys-diabody labeled in the subject. Any suitable option for detecting a detectable label can be used to image the subject. In some embodiments, imaging includes positron emission tomography (PET), computed tomography (CT), single photon emission computed tomography (SPECT), magnetic resonance imaging (NMR) or fluorescence emission detection. In some embodiments, detection can be performed by near infrared (NIR) imaging and / or Cerenkov luminescence imaging (Cerenkov luminesceneceimaging).

[0176] In some embodiments, the antibody, minibody, or cys-diabody specifically binds to an antigenic target, such as, but not limited to, those disclosed herein.

[0177] Other embodiments of the present disclosure are provided in the following numbered arrangements.

[0178] 1. A variant antibody comprising a disrupted cluster of at least one surface-exposed positively charged amino acid, wherein the variant antibody differs from an original antibody comprising the original cluster by replacing at least one surface-exposed positively charged amino acid of the original cluster with a negatively or uncharged amino acid, wherein the original cluster comprises at least two surface-exposed positively charged amino acids within about 30 angstroms of each other.

[0179] 2. The variant antibody according to setting 1, wherein at least two surface-exposed positively charged amino acids of the original cluster are within about 15 angstroms of each other.

[0180] 3. The variant antibody according to setting 1 or 2, wherein due to the destruction of the cluster, the variant antibody has a total positive charge attributable to the cluster that is reduced by at least about 10% compared to the original antibody.

[0181] 4. A variant antibody according to any of the preceding settings, wherein the variant antibody differs from the original antibody by up to 3 amino acid substitutions, thereby disrupting the cluster.

[0182] 5. A variant antibody according to any one of the aforementioned settings, wherein the replacement of at least one of the surface-exposed positively charged amino acids of the original cluster is located in the light chain variable region (V L ) in the framework region (FR).

[0183] 6. The variant antibody according to setting 5, wherein the replacement of at least one of the surface-exposed positively charged amino acids of the original cluster is located at V L FR2.

[0184] 7. A variant antibody according to any of the preceding settings, wherein the original antibody comprises a polypeptide comprising an original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), wherein X1 is a positively charged amino acid, wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid, and wherein X4 and X7 are each independently any amino acid, provided that at least one is a positively charged amino acid, wherein the original sequence is outside any CDR of the antibody, wherein at least one of X4 and X7, which are positively charged amino acids, is surface exposed and is part of the original cluster.

[0185] 8. The variant antibody according to setting 7, wherein the positive charge attributed to the original sequence is reduced by about 33% or more in the variant antibody.

[0186] 9. The variant antibody according to setting 8, wherein the positive charge attributed to the original sequence is reduced by about 60% or more in the variant antibody.

[0187] 10. The variant antibody according to any one of settings 7 to 9, wherein the substitution comprises replacing at least one of X4 and X7 with a negatively charged or uncharged amino acid.

[0188] 11. The variant antibody according to any one of settings 7 to 10, wherein the polypeptide comprises QQX1X2X3X4X5X6X7 (SEQ ID NO: 3), which comprises the original sequence of X1X2X3X4X5X6X7 (SEQ ID ID: 2).

[0189] 12. The variant antibody according to any one of settings 7 to 11, wherein X7 is a positively charged amino acid.

[0190] 13. The variant antibody according to any one of settings 7 to 12, wherein X4 is a positively charged amino acid.

[0191] 14. The variant antibody according to any one of settings 7 to 13, wherein the positively charged amino acid is lysine, arginine or histidine.

[0192] 15. The variant antibody according to setting 14, wherein the positively charged amino acid is lysine or arginine.

[0193] 16. The variant antibody according to any one of settings 7 to 13, wherein X2 and / or X6 is proline.

[0194] 17. The variant antibody according to any one of settings 7 to 14, wherein X3 is glycine or glutamic acid.

[0195] 18. The variant antibody according to any one of settings 7 to 15, wherein X5 is alanine, valine, serine or proline.

[0196] 19. A variant antibody according to any one of settings 7-16, wherein the original sequence is one of the following: KX2X3KX5 X6K (SEQ ID NO: 73), wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid; KX2X3X4X5 X6R (SEQ ID NO: 74), wherein X2, X3, X4, X5, and X6 are each independently a negatively charged or uncharged amino acid; or KX2X3X4X5 X6K (SEQ ID NO: 75), wherein X2, X3, X4, X5, and X6 are each independently a negatively charged or uncharged amino acid.

[0197] 20. A variant antibody according to any one of settings 7-17, wherein the original sequence is one of the following: KPGKAPK (SEQ ID NO: 5), KPGQAPR (SEQ ID NO: 6), KPEKAPK (SEQ ID NO: 7), KPGKVPK (SEQ ID NO: 8), KPGQPPR (SEQ ID NO: 9), KPGQSPR (SEQ ID NO: 10), KPGLAPR (SEQ ID NO: 11), or KPGQPPK (SEQ ID NO: 12).

[0198] 21. The variant antibody according to setting 18, wherein the original sequence is KPGKAPK (SEQ ID NO: 5) or KPGQAPR (SEQ ID ID: 6).

[0199] 22. The variant antibody according to any one of settings 7-19, wherein the negatively charged or uncharged amino acid replacing at least one of the surface-exposed positively charged amino acids of the cluster is glutamine.

[0200] 23. A variant antibody according to any one of settings 7-22, wherein the original sequence is located in the light chain variable region (V L ) in the framework region (FR).

[0201] 24. The variant antibody according to setting 23, wherein the original sequence is located at V L FR2.

[0202] 25. The variant antibody according to any one of the preceding settings, wherein the variant antibody is L FR2 contains at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID NO: 16), and QQKPGQSPQ (SEQ ID NO: 15).

[0203] 26. A variant antibody according to any one of the preceding settings, wherein the variant antibody comprises V L FR2, the V L FR2 has an amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

[0204] 27. A method according to any of the preceding settings, wherein the replacement of at least one of the surface exposed positively charged amino acids of the original cluster is located in the hinge region of the original antibody.

[0205] 28. The method according to setting 27, wherein the replacement of at least one of the surface exposed positively charged amino acids of the original cluster is located in the upper hinge region of the original antibody.

[0206] 29. An antibody comprising a variant polypeptide different from an original polypeptide comprising an original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein the original sequence is outside any CDR of the original polypeptide, wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two are surface-exposed positively charged amino acids, and the amino acids form a cluster of surface-exposed positively charged amino acids in the original polypeptide,

[0207] The variant polypeptide differs from the original polypeptide by modifying at least one of at least two surface-exposed positively charged amino acids of the original sequence, wherein the modification reduces at least 33% of the positive charge attributed to the original sequence.

[0208] 30. The antibody according to setting 29, wherein the positive charge attributed to the original sequence is reduced by about 60% or more in the variant polypeptide.

[0209] 31. The antibody according to setting 29 or 30, wherein the number of positively charged amino acids in the original sequence is reduced by one or more in the variant polypeptide.

[0210] 32. An antibody according to any one of settings 29-31, wherein at least one of the at least two positively charged amino acids of the original sequence is replaced by a negatively charged or uncharged amino acid in the variant polypeptide.

[0211] 33. The antibody according to any one of settings 29-32, wherein at least X4 and X7 are each independently a positively charged amino acid.

[0212] 34. The antibody according to any one of settings 29-33, wherein the positively charged amino acid is lysine, arginine or histidine.

[0213] 35. The antibody according to any one of settings 29-34, wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid.

[0214] 36. The antibody according to any one of settings 29-35, wherein at least one of X4 and X7 is replaced by a negatively charged or uncharged amino acid to reduce the positive charge.

[0215] 37. The antibody according to setting 36, wherein the negatively charged or uncharged amino acid replacing at least one of X4 and X7 is glutamine.

[0216] 38. The antibody according to any one of settings 29-37, wherein the variant polypeptide comprises alanine or serine at the position corresponding to X5 in the sequence of the original polypeptide.

[0217] 39. The antibody according to any one of settings 29-38, wherein the variant polypeptide comprises the light chain variable region (V L ).

[0218] 40. The antibody according to any one of settings 29-39, wherein the original sequence is located at V of the original polypeptide. L within the framework region (FR).

[0219] 41. The antibody according to setting 40, wherein the original sequence is located at V of the original polypeptide L Within FR2.

[0220] 42. The antibody according to setting 40 or 41, wherein the original polypeptide is based on V L The first human germline sequence, the V L The first human germline sequence has an original FR2 comprising the original sequence, and wherein the variant polypeptide is obtained by replacing the V L The invention also provides a method for preparing a polypeptide comprising: replacing at least the original sequence with a corresponding second sequence of the second FR2 of a second human germline sequence and differing from the original polypeptide, wherein the second FR2 comprises the second sequence corresponding to the original sequence and having at least 10% less positive charge attributed to it compared to the original sequence.

[0221] 43. A variant antibody comprising at least one disrupted cluster of positively charged amino acids, wherein the variant antibody differs from an original antibody comprising the original cluster by replacing at least one positively charged amino acid of the original cluster with a negatively charged or uncharged amino acid, wherein the original cluster comprises at least two positively charged amino acids within 12 residues of each other, wherein the original cluster is outside any CDR of the original antibody.

[0222] 44. The variant antibody according to setting 43, wherein the original cluster is located in the light chain variable region (V L )FR.

[0223] 45. The variant antibody according to setting 44, wherein the original cluster is located at V L FR2.

[0224] 46. ​​A variant antibody according to setting 45, wherein the variant antibody comprises at least one of the following (which includes a replacement of at least one of the positively charged amino acids): KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQID No: 14), and QQKPGQSPQ (SEQ ID NO: 15).

[0225] 47. The variant antibody according to setting 45, wherein the variant antibody comprises V L FR2, the V L FR2 comprises the amino acid sequence: WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

[0226] 48. A variant antibody according to setting 43, wherein the original cluster is located in the hinge region of the original antibody.

[0227] 49. The variant antibody according to setting 48, wherein the original cluster is located in the upper hinge region of the original antibody.

[0228] 50. The variant antibody according to setting 48 or 49, wherein the original antibody comprises an upper hinge sequence of EPKSSDKTHT (SEQ ID NO: 38).

[0229] 51. The antibody according to any one of settings 48-50, wherein the variant antibody comprises an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39).

[0230] 52. An antibody according to any of the preceding settings, wherein the antibody comprises an antigen binding fragment.

[0231] 53. The antibody according to any of the preceding settings, wherein the molecular weight of the antibody is in the range of about 15 kDa to about 110 kDa.

[0232] 54. The antibody according to any of the preceding settings, wherein the antibody is a minibody, a cys-diabody, a scFv, a scFv-Fc, or a nanobody-Fc.

[0233] 55. The antibody according to any of the preceding settings, wherein the molecular weight of the antibody is in the range of about 10 kDa to about 20 kDa.

[0234] 56. The antibody according to setting 55, wherein the antibody is a nanobody.

[0235] 57. The antibody according to any one of settings 1-54, wherein the molecular weight of the antibody is greater than 90 kDa.

[0236] 58. The antibody according to setting 57, wherein the antibody is scFv-Fc.

[0237] 59. A miniantibody or cys-diabody comprising a variant polypeptide, wherein the variant polypeptide is a light chain variable region (V L )FR2 is different from the original polypeptide, wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two are surface-exposed positively charged amino acids, and the amino acids form a cluster of surface-exposed positively charged amino acids in the original polypeptide,

[0238] The variant polypeptide differs from the original polypeptide by modifying at least one of at least two surface-exposed positively charged amino acids of the original sequence, wherein the modification reduces at least 33% of the positive charge attributed to the original sequence.

[0239] 60. The miniantibody or cys-diabody according to setting 59, wherein at least X4 and X7 are each independently a positively charged amino acid in the original polypeptide.

[0240] 61. The miniantibody or cys-diabody according to setting 59 or 60, wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid in the original polypeptide.

[0241] 62. The minibody or cys-diabody according to any one of settings 59-61, wherein at least one of X4 and X7 is replaced by a negatively charged or uncharged amino acid to reduce the positive charge.

[0242] 63. A method comprising a light chain variable region (V L ) of a miniantibody or cys-diabody, wherein the light chain variable region includes a FR2 sequence comprising X1X2X3QAX6X7 (SEQ ID NO: 4), wherein X1 is a surface-exposed positively charged amino acid, wherein X2, X3, and X6 are each independently any negatively charged or uncharged amino acid, and wherein X7 is glutamine or lysine.

[0243] 64. The minibody or cys-diabody according to any one of settings 59-63, wherein X1 is lysine.

[0244] 65. The miniantibody or cys-diabody according to any one of settings 59-64, wherein X2 and / or X6 is proline.

[0245] 66. The minibody or cys-diabody according to any one of settings 59-65, wherein X3 is glycine or glutamic acid.

[0246] 67. The miniantibody or cys-diabody according to setting 63, wherein the FR2 sequence comprises KPGQAPK (SEQ ID NO: 13) or KPGQAPQ (SEQ ID ID: 14).

[0247] 68. A method comprising a light chain variable region (V L ) of a miniantibody or cys-diabody, wherein the light chain variable region comprises (according to IMGT numbering):

[0248] A49 and:

[0249] Either (i) Q51;

[0250] or (ii) Q48 and K51.

[0251] 69. The miniantibody or cys-diabody according to setting 61, wherein the V L Also includes K45 (IMGT number).

[0252] 70. A miniantibody comprising an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39).

[0253] 71. An antibody, minibody or cys-diabody according to any of the preceding settings, wherein the antibody, minibody or cys-diabody specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA.

[0254] 72. The antibody, minibody or cys-diabody according to any one of the preceding settings, wherein the antibody, minibody or cys-diabody comprises a light chain variable region and a heavy chain variable region (V H ), the light chain variable region comprises Figure 5B , Figure 5C , Figures 7B-7D , wherein the heavy chain variable region comprises 3 LCDR sequences in any one of the sequences of Figure 5B , Figure 5C , Figures 7B-7D 3 HCDR sequences in any one of the sequences.

[0255] 73. The antibody, minibody or cys-diabody according to any of the preceding settings, further comprising a detectable label.

[0256] 74. The antibody, minibody or cys-diabody according to setting 73, wherein the antibody, minibody or cys-diabody is labeled with a radionuclide or an organic dye.

[0257] 75. The antibody, minibody or cys-diabody according to any one of settings 1-72, further comprising a therapeutic agent.

[0258] 76. The antibody, minibody or cys-diabody of setting 75, wherein the therapeutic agent is a cytotoxic agent.

[0259] 77. The antibody, minibody or cys-diabody of setting 75, wherein the therapeutic agent is a radionuclide.

[0260] 78. The antibody, minibody or cys-diabody according to setting 75, wherein the radionuclide is selected from 212 Pb, 149 Tb and 161 Tb.

[0261] 79. A composition comprising:

[0262] An antibody, minibody or cys-diabody according to any of the preceding settings; and

[0263] A pharmaceutically acceptable carrier.

[0264] 80. A nucleic acid encoding a variant polypeptide of the antibody of any one of settings 29-42 or the minibody or cys-diabody of any one of settings 59-62.

[0265] 81. A nucleic acid encoding a variant antibody according to any one of settings 1-28 and 43-58, or an antibody according to any one of settings 29-42, or a minibody or cys-diabody according to any one of settings 59-72.

[0266] 82. A genetically engineered host cell comprising the nucleic acid of setting 80 or 81.

[0267] 83. A method for enhancing the biodistribution and / or pharmacokinetics of an antibody, comprising:

[0268] recognizing an original antibody comprising a polypeptide comprising at least one surface-exposed positively charged cluster of amino acids, the cluster comprising at least two surface-exposed positively charged amino acids within 30 angstroms of each other;

[0269] replacing at least one of the at least two surface-exposed positively charged amino acids of the at least one cluster with a negatively or uncharged amino acid, thereby destroying the at least one cluster,

[0270] Variant antibodies are thereby generated that have enhanced biodistribution and / or pharmacokinetics compared to the original antibody.

[0271] 84. A method for enhancing the biodistribution and / or pharmacokinetics of an antibody, comprising:

[0272] recognizing an original antibody comprising a polypeptide comprising clusters of at least two surface-exposed positively charged amino acids within 12 residues of each other; and

[0273] At least one of the at least two surface-exposed positively charged amino acids of the cluster is replaced with a negatively or uncharged amino acid to disrupt the cluster, thereby generating a variant antibody having enhanced biodistribution and / or pharmacokinetics compared to the original antibody.

[0274] 85. A method according to setting 84, wherein the cluster comprises at least two positively charged amino acids within 6 residues of each other.

[0275] 86. A method according to setting 84 or 85, wherein the polypeptide comprises an amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, and wherein the at least two surface-exposed positively charged amino acids comprise at least two of X1, X2, X3, X4, X5, X6, and X7.

[0276] 87. A method according to setting 86, wherein the at least two surface-exposed positively charged amino acids include at least X1.

[0277] 88. A method according to setting 86 or 87, wherein the at least two surface-exposed positively charged amino acids include at least X4 and X7.

[0278] 89. The method according to any one of settings 86-88, wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid residue in the original polypeptide.

[0279] 90. The method according to any one of settings 86-89, wherein at least one of X4 and X7 is replaced by a negatively charged or uncharged amino acid.

[0280] 91. The method of any one of settings 83-90, wherein the at least two surface-exposed positively charged amino acids comprise arginine, lysine, or histidine.

[0281] 92. A method according to any one of settings 83-91, wherein the negatively charged or uncharged amino acid replacing at least one of the at least two surface-exposed positively charged amino acids comprises a polar amino acid.

[0282] 93. A method according to setting 92, wherein the negatively charged or uncharged amino acid replacing at least one of the at least two surface-exposed positively charged amino acids is glutamine.

[0283] 94. The method of any one of settings 83-93, wherein the positively charged amino acid and the negatively charged or uncharged amino acid have side chain lengths that differ by less than one or two carbons.

[0284] 95. The method according to any one of settings 83-94, comprising replacing up to 3 amino acids of the polypeptide to disrupt the at least one cluster.

[0285] 96. A method according to any one of settings 83-95, wherein the cluster is outside any complementarity determining region (CDR) of the original antibody.

[0286] 97. The method according to any one of settings 83-96, wherein the cluster is located in the light chain variable region (V L )middle.

[0287] 98. The method according to any one of settings 83-97, wherein the at least two surface-exposed positively charged amino acids are located in the V region of the original antibody. L within the framework region (FR).

[0288] 99. The method according to any one of settings 83-98, wherein the at least two surface-exposed positively charged amino acids are in the V L Within FR2.

[0289] 100. The method according to setting 98 or 99, wherein the polypeptide is based on V L The first human germline sequence, the V L The first human germline sequence has an original FR2, wherein the original FR2 comprises the at least two surface-exposed positively charged amino acids, and wherein the method comprises using L The original FR2 or a portion comprising the cluster is replaced by a second FR2 of a second human germline sequence or a corresponding portion thereof, wherein the second FR1 comprises at least one reduced surface-exposed positively charged amino acid compared to the cluster.

[0290] 101. The method according to setting 99 or 100, wherein the original sequence is one of the following: KX2X3KX5X6K (SEQ ID NO: 73), wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid; KX2X3X4X5X6R (SEQ ID NO: 74), wherein X2, X3, X4, X5, and X6 are each independently a negatively charged or uncharged amino acid; or KX2X3X4X5X6K (SEQ ID NO: 75), wherein X2, X3, X4, X5, and X6 are each independently a negatively charged or uncharged amino acid.

[0291] 102. The method according to setting 99 or 101, wherein the original polypeptide is in V L FR2 includes one of the following: KPGKAPK (SEQ ID NO:5), KPGQAPR (SEQ ID NO:6), KPEKAPK (SEQ ID NO:7), KPGKVPK (SEQ ID NO:8), KPGQPPR (SEQ ID NO:9), KPGQSPR (SEQ ID NO:10), KPGLAPR (SEQ ID No:11), or KPGQPPK (SEQ ID NO:12).

[0292] 103. According to the method according to any one of settings 99-101, wherein the variant polypeptide is in V L FR2 contains at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID No: 16), and QQKPGQSPQ (SEQ ID NO: 15).

[0293] 104. The method according to any one of settings 99-101, wherein the variant polypeptide is in V L FR2 includes at least one of the following: WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

[0294] 105. The method according to any one of settings 83-104, further comprising binding a chelating ligand to the variant antibody.

[0295] 106. The method according to any one of settings 83-105, further comprising labeling the antibody with a radionuclide.

[0296] 107. The method of any one of settings 83-105, further comprising labeling the antibody with a detectable label.

[0297] 108. A method for preparing a labeled antibody, comprising:

[0298] Select the light chain variable region (V L ), wherein the germline sequence does not include a cluster of at least two positively charged amino acids within 3 residues of each other in framework region 2 (FR2) of the germline sequence;

[0299] isolating one or more target-specific antibodies from a population of antibodies comprising V sequences derived from the germline sequences L , and having variation of said germline sequence throughout said population, wherein said one or more target-specific antibodies are in V L The FR2 sequence does not contain a cluster of at least two positively charged amino acids within 3 residues of each other; and

[0300] The one or more target-specific antibodies are labeled.

[0301] 109. The method of setting 108, wherein isolating the one or more target-specific antibodies comprises screening a population of antibodies derived from the germline sequences.

[0302] 110. The method of setting 109, wherein the population of antibodies derived from the germline sequences are produced in a host organism that is genetically modified to express antibodies based solely on the selected germline sequences.

[0303] 111. The method of setting 109, wherein the population of antibodies derived from the germline sequences is included in an antibody phage library based solely on selected germline sequences.

[0304] 112. The method of any one of settings 109-111, wherein the germline sequence is a human germline sequence.

[0305] 113. The method according to any one of settings 108-112, wherein the one or more target-specific antibodies are humanized.

[0306] 114. The method according to any one of settings 108-113, further comprising generating one or more target-specific miniantibodies or cys-diabodies from the one or more target-specific antibodies.

[0307] 115. The method according to any one of settings 108-114, comprising labeling the one or more target-specific antibodies with a radionuclide.

[0308] 116. The method of any one of settings 108-115, wherein labeling the one or more target-specific antibodies comprises binding a chelating ligand to the one or more target-specific antibodies.

[0309] 117. The method of any one of settings 83-116, wherein the antibody comprises an antigen binding fragment.

[0310] 118. The method according to any one of settings 83-117, wherein the antibody is a minibody or a cys-diabody.

[0311] 119. The method of any one of settings 83-118, wherein the antibody specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA.

[0312] 120. The method according to any one of settings 83-118, wherein the antibody or minibody comprises a light chain variable region (V L ) and the heavy chain variable region (V H ), the light chain variable region comprises Figures 5A-5C and Figures 7A-7D , wherein the heavy chain variable region comprises 3 LCDR sequences of any one of the 3 LCDR sequences of Figures 5A-5C and Figures 7A-7D 3 HCDR sequences of any one of the 3 HCDR sequences of .

[0313] 121. An antibody prepared by the method of any one of settings 83-120.

[0314] 122. A method of treating a subject, comprising:

[0315] Identifying a subject in need of treatment with the antibody, minibody or cys-diabody of any of settings 1-78; and

[0316] Administering a therapeutically effective amount of the antibody or minibody or composition of setting 79 to the subject.

[0317] 123. A method of treating cancer in a subject, comprising:

[0318] Identifying subjects in need of cancer treatment; and

[0319] A therapeutically effective amount of the antibody, minibody or cys-diabody of any one of settings 1-78, or the composition of setting 79 is administered to the subject, thereby treating the cancer.

[0320] 124. A radiotherapy method comprising:

[0321] Identify subjects who require radiation therapy; and

[0322] A therapeutically effective amount of the antibody, minibody or cys-diabody of any of settings 1-78, or the composition of setting 79, is administered to the subject, wherein the antibody, minibody or cys-diabody comprises a radionuclide.

[0323] 125. A method of imaging a subject, comprising:

[0324] administering to the subject a composition comprising an effective amount of the antibody, minibody, or cys-diabody of any one of settings 1-78, or the composition of setting 79, wherein the antibody, minibody, or cys-diabody is detectably labeled; and

[0325] The subject is imaged to detect the labeled antibody, minibody or cys-diabody in the subject.

[0326] 126. Use of the antibody, minibody or cys-diabody of any one of settings 1-78 or the composition of setting 79 for treating cancer in a subject in need thereof.

[0327] 127. Use of the antibody, minibody or cys-diabody of any one of setting 1-78 in the preparation of a medicament for treating cancer in a subject in need thereof.

[0328] Example

[0329] Example 1

[0330] This non-limiting example shows that the biodistribution of a minibody is enhanced by mutating the positively charged amino acids in the cluster of positively charged amino acids in FR2 of the light chain variable region.

[0331] The humanized heavy chain variable region (V H ) fused to the humanized light chain variable region (V L ) to form a scFv fragment, and the scFv fragment was fused to the C terminus via the hinge region. H 3 domains, and constructed a miniantibody that binds to DLL3. The amino acid sequence of the DLL3 miniantibody (IAB57M1-3) monomer is as follows Figure 5A As shown. Figures 5A-5C In the example, the signal peptide is shown in italics. 89 Zr) labeling, the DLL3 miniantibody can be used for in vivo imaging of DLL3-expressing tumor cells in patients.

[0332] Humanized V L The germline sequence of IGVK3-15*01 was used. L The protein has a framework region 2 (FR2) sequence (WYQQKPGQAPRLLI (SEQ ID NO: 70) (according to North)) that includes a cluster of two basic amino acids (lysine and arginine) at positions 45 and 51 (IMGT numbering). Molecular modeling of the surface charge using an adaptive Poisson-Boltzmann solver identified localized patches of surface-exposed positive charges formed by the two basic amino acids. Localized positively charged patches may lead to the accumulation of microantibodies in the kidney.

[0333] To test this, Figure 5B As shown, R51 is replaced by glutamine (Q). Therefore, the V L The KPGQAPR (SEQ ID NO: 6) in FR2 was changed to KPGQAPQ (SEQ ID NO: 14) in the first variant. Figure 5C As shown in FIG. 1 , a second variant was prepared by further replacing the alanine (A) at position 49 with serine (S). Thus, the V L KPGQAPR (SEQ ID NO: 6) in FR2 was further changed to KPGQSPQ (SEQ ID NO: 16) in the second variant.

[0334] Combined with Df 89 After Zr labeling, the labeled miniantibodies were administered to mice. The average radioactivity uptake in the liver and kidneys was measured 24 hours after administration and expressed as a percentage of the injected dose per gram (% ID / g). Figure 6 As shown. Compared with the original minibody, the variant minibody showed reduced accumulation in the kidney and increased accumulation in the liver. The reduced accumulation in the kidney is expected to reduce renal radiation toxicity. It may also allow the use of higher radiation doses in the setting of radiotherapy.

[0335] Example 2

[0336] This non-limiting example shows that the biodistribution of a minibody is enhanced by mutating the positively charged amino acids in the cluster of positively charged amino acids in FR2 of the light chain variable region.

[0337] IAB16M2-77 is an anti-human FAP miniantibody designed by humanizing an antibody from a mouse hybridoma clone. This original antibody was humanized based on the following germline: IGHV1-69*01 / IGKV1-39*01. Germline IGKV1-39*01 is a commonly used light chain germline sequence.

[0338] In the light chain variable region (V L ) identified a positively charged plaque on germline IGKV1-39*01 in the framework region (FR) 2, such as Fig. 7A The surface patch maps to the sequence KPGKAPK (SEQ ID NO: 5), which contains three charged residues (Lys, Fig. 7A The mutation K48Q ( Figure 7B 24 hours after application, the 89 Renal uptake of Zr radiolabeled microantibodies ( Figure 8 ). The distribution in the liver and spleen remained relatively unchanged. These results are consistent with the formation of positively charged plaques by clusters of lysine leading to the accumulation of this microantibody in the kidney.

[0339] Example 3

[0340] This non-limiting example shows replacement of a fragment of framework region 2 of a light chain germline sequence with the corresponding fragment from another germline sequence to disrupt clusters of positively charged residues.

[0341] As shown in Table 3.1, several human kappa and lambda light chain germline sequences have a pattern of three positively charged residues in framework region 2 (K or R), including germline IGKV1-39*01. Analysis of the isoelectric electrostatic surface of minibodies with a pattern of positively charged residues at framework region 2 in the light chain variable region using an adaptive Poisson-Boltzmann solver revealed a positively charged patch formed by positively charged residues, whose centers of mass are within a linear distance of approximately 11 angstroms from each other.

[0342] Table 3.1

[0343]

[0344]

[0345] like Figure 7CAs shown, KPGKAPK (SEQ ID NO: 5) in germline IGKV1-39*01 was replaced with KPGQSPQ (SEQ ID ID: 16) from IGKV2-28*01. Germline IGKV1-39*01 was modified by replacing a segment of framework region 2 (FR2) (KPGKAPK (SEQ ID NO: 5)) with an analogous sequence from a germline sequence that does not contain this charge pattern, such as framework region 2 (FR2) from IGKV3-20*01 (KPGQAPR (SEQ ID NO: 6)) or IGKV2-28*01 (KPGQSPQ (SEQ ID NO: 16)).

[0346] Prepare and radiolabel a protein containing variant V L Minibodies with modified FR2 were prepared. Radiolabeled minibodies were administered to mice. Twenty-four hours after administration, the animals were imaged and the distribution of the minibodies in the animals was determined. Compared to the original minibodies, the minibodies with variant V L The microantibodies showed reduced accumulation in the kidney.

[0347] Example 4

[0348] This non-limiting example shows that the biodistribution of a cys-diabody is enhanced by mutating a positively charged amino acid in a cluster of positively charged amino acids in FR2 of the light chain variable region.

[0349] By humanizing the heavy chain variable region (V H ) antibody is fused to the humanized light chain variable region (V L ) to form a scFv fragment, and an extension sequence that mediates dimerization is added to the C-terminus to construct a cys-diabody.

[0350] Humanized V L The germline sequence of IGVK3-15*01 was used. L It has a framework region 2 (FR2) sequence (WYQQKPGQAPRLLI (SEQ ID NO: 20) (according to North)), which includes clusters of two basic amino acids (lysine and arginine) at positions 45 and 51 (IMGT numbering). Molecular modeling of surface charge using an adaptive Poisson-Boltzmann solver determined local patches of surface exposed positive charges caused by clustering of these two basic amino acids. Localized positively charged patches may lead to the accumulation of cys-diabodies in the kidneys.

[0351] R51 was replaced by glutamine (Q). 89After Zr labeling, the labeled cys-diabody was administered to mice. The average radioactive uptake in the liver and kidney was measured 24 hours after injection of the radiolabeled cys-diabody. Compared with the original cys-diabody, the variant cys-diabody showed reduced accumulation in the kidney and increased accumulation in the liver.

[0352] Example 5

[0353] This non-limiting example shows that the biodistribution of a minibody is enhanced by mutating the positively charged amino acids in the cluster of positively charged amino acids in the hinge region.

[0354] The humanized heavy chain variable region (V H ) antibody is fused to the humanized light chain variable region (V L ) to form a scFv fragment, and the scFv fragment was fused to the C terminus via the hinge region. H 3 domains, a miniantibody (IAB16M2-78) was constructed. The hinge region includes the upper hinge sequence EPKSSDKTHT (SEQ ID NO: 38), which includes a cluster of two lysines. The lysine residues in the cluster were replaced by glycine (G) to generate the variant miniantibody IAB16M2-79 ( Fig.7D ), with an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39). Combined with Df and 89 After Zr labeling, the labeled minibodies were administered to mice. The average radioactivity uptake in the liver and kidneys was measured 24 hours after injection of the radiolabeled minibodies. The variant minibodies showed reduced accumulation in the kidneys compared to the original minibodies.

[0355] Example 6

[0356] This non-limiting example demonstrates radioimmunotherapy of a subject using the antibodies of the present disclosure.

[0357] A subject in need of cancer treatment is identified. The target is specifically bound to the tumor and is labeled with a radionuclide in the light chain variable region (V L ) variant miniantibody having the sequence KPGQAPQ (SEQ ID NO: 14) in framework region 2 (FR2). The radiolabeled miniantibody is administered to the subject. The radiation dose delivered to the subject is greater than that using V L The original minibody with the sequence KPGQAPR (SEQ ID NO:6) in FR2 delivered a dose that was comparable to that of the original minibody, while the nephrotoxicity was still comparable.

[0358] Example 7

[0359] This non-limiting example shows the treatment of a subject using an antibody of the disclosure.

[0360] A subject in need of liver cancer treatment is identified. L ) A variant miniantibody having the sequence KPGQAPQ (SEQ ID NO: 4) in framework region 2 (FR2) binds to a cytotoxic agent. The bound miniantibody is administered to a subject.

[0361] As used herein, section headings are used only for text organization purposes and shall not be interpreted as limiting the subject matter in any way. All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated by reference in their entirety for any purpose, including the disclosures specifically cited herein. When the definitions of terms in the references incorporated appear to differ from the definitions provided in this teaching, the definitions in this teaching shall prevail. It should be understood that there is an implicit "about" before the temperature, concentration, time, etc. discussed in this teaching, so slight and non-substantial deviations are within the scope of the teachings herein.

[0362] Although the present disclosure is presented in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the present disclosure and obvious modifications and equivalents thereof. In addition, although several variations of the present disclosure have been shown and described in detail, other modifications within the scope of the present disclosure will be apparent to those skilled in the art based on the present disclosure. It is also conceivable that various combinations or sub-combinations may be made to the specific features and aspects of the embodiments and still fall within the scope of the present disclosure. It should be understood that the various features and aspects of the disclosed embodiments may be combined or replaced with each other to form different modes or embodiments of the present disclosure. Therefore, the scope of the present disclosure presented herein should not be limited to the above-mentioned specific disclosed embodiments.

[0363] However, it should be understood that the detailed description, while indicating preferred embodiments of the present disclosure, is given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art.

[0364] The terminology used in the descriptions presented herein is not intended to be interpreted in any limiting or restrictive manner. Rather, the terminology is used merely in conjunction with the detailed description of embodiments of the systems, methods, and related components. Furthermore, embodiments may include several novel features, no single one of which is responsible for its desirable properties, nor is any single one considered essential to practicing the disclosure described herein.

Claims

1. A variant antibody comprising a disrupted cluster of at least one surface-exposed positively charged amino acid, wherein the variant antibody differs from an original antibody comprising the original cluster by replacing at least one surface-exposed positively charged amino acid of the original cluster with a negatively charged or uncharged amino acid, wherein the original cluster comprises at least two surface-exposed positively charged amino acids within about 30 angstroms of each other.

2. The variant antibody of claim 1, wherein at least two surface-exposed positively charged amino acids of the original cluster are within about 15 angstroms of each other.

3. The variant antibody of claim 1 or 2, wherein due to the disruption of the clusters, the variant antibody has a total positive charge attributable to the clusters that is reduced by at least about 10% compared to the original antibody.

4. A variant antibody according to any one of the preceding claims, wherein the variant antibody differs from the original antibody by up to 3 amino acid substitutions that disrupt the cluster.

5. A variant antibody according to any one of the preceding claims, wherein the replacement of at least one of the surface-exposed positively charged amino acids of the original cluster is located in the light chain variable region (V L ) in the framework region (FR).

6. The variant antibody according to claim 5, wherein the replacement of at least one of the surface-exposed positively charged amino acids of the original cluster is located at V L FR2.

7. A variant antibody according to any of the preceding claims, wherein the original antibody comprises a polypeptide comprising the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), wherein X1 is a positively charged amino acid, wherein X2, X3, X5 and X6 are each independently a negatively charged or uncharged amino acid, and wherein X4 and X7 are each independently any amino acid, provided that at least one is a positively charged amino acid, wherein the original sequence is outside any CDR of the original antibody, wherein at least one of X4 and X7, which are positively charged amino acids, is surface exposed and is part of the original cluster.

8. The variant antibody according to claim 7, wherein the positive charge attributed to the original sequence is reduced by about 33% or more in the variant antibody.

9. The variant antibody according to claim 8, wherein the positive charge attributed to the original sequence is reduced by about 60% or more in the variant antibody.

10. The variant antibody according to any one of claims 7 to 9, wherein the substitution comprises replacing at least one of X4 and X7 with a negatively charged or uncharged amino acid.

11. The variant antibody according to any one of claims 7-10, wherein the polypeptide comprises QQX1X2X3X4X5X6X7 (SEQ ID NO: 3) comprising the original sequence of X1X2X3X4X5X6X7 (SEQ ID ID: 2).

12. The variant antibody according to any one of claims 7 to 11, wherein X7 is a positively charged amino acid.

13. The variant antibody according to any one of claims 7 to 12, wherein X4 is a positively charged amino acid.

14. The variant antibody according to any one of claims 7-13, wherein the positively charged amino acid is lysine, arginine or histidine.

15. The variant antibody of claim 14, wherein the positively charged amino acid is lysine or arginine.

16. The variant antibody according to any one of claims 7 to 15, wherein X2 and / or X6 is proline.

17. The variant antibody according to any one of claims 7 to 16, wherein X3 is glycine or glutamic acid.

18. The variant antibody according to any one of claims 7 to 17, wherein X5 is alanine, valine, serine or proline.

19. A variant antibody according to any one of claims 7-18, wherein the original sequence is one of the following: KX2X3KX5 X6K (SEQ ID NO: 73), wherein X2, X3, X5 and X6 are each independently a negatively charged or uncharged amino acid; KX2X3X4X5 X6R (SEQ ID NO: 74), wherein X2, X3, X4, X5 and X6 are each independently a negatively charged or uncharged amino acid; or KX2X3X4X5 X6K (SEQ ID NO: 75), wherein X2, X3, X4, X5 and X6 are each independently a negatively charged or uncharged amino acid.

20. A variant antibody according to any one of claims 7-19, wherein the original sequence is one of the following: KPGKAPK (SEQ ID NO:5), KPGQAPR (SEQ ID NO:6), KPEKAPK (SEQ ID NO:7), KPGKVPK (SEQ ID NO:8), KPGQPPR (SEQ ID NO:9), KPGQSPR (SEQ ID NO:10), KPGLAPR (SEQ ID NO:11), or KPGQPPK (SEQ ID NO:12).

21. The variant antibody according to claim 20, wherein the original sequence is KPGKAPK (SEQ ID NO: 5) or KPGQAPR (SEQ ID ID: 6).

22. The variant antibody of any one of claims 7-21, wherein the negatively or uncharged amino acid replacing at least one of the surface-exposed positively charged amino acids of the cluster is glutamine.

23. according to the variant antibody described in any one of claims 7-22, wherein said original sequence is located in the light chain variable region (V L ) in the framework region (FR).

24. The variant antibody according to claim 23, wherein the original sequence is located at V L FR2.

25. The variant antibody according to any one of the preceding claims, wherein the variant antibody is L FR2 contains at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID NO: 16) and QQKPGQSPQ (SEQ ID NO: 15).

26. The variant antibody according to any one of the preceding claims, wherein the variant antibody comprises V L FR2, the V L FR2 has an amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

27. The method according to any one of the preceding claims, wherein the replacement of at least one of the surface exposed positively charged amino acids of the original cluster is located in the hinge region of the original antibody.

28. The method of claim 27, wherein the replacement of at least one of the surface exposed positively charged amino acids of the original cluster is located in the upper hinge region of the original antibody.

29. An antibody comprising a variant polypeptide different from an original polypeptide comprising an original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein the original sequence is outside any CDR of the original polypeptide, wherein X1, X2, X3, X4, X5, X6 and X7 are each independently any amino acid, provided that at least two are surface-exposed positively charged amino acids, and the surface-exposed positively charged amino acids form a surface-exposed positively charged amino acid cluster in the original polypeptide, The variant polypeptide differs from the original polypeptide by modifying at least one of at least two surface-exposed positively charged amino acids of the original sequence, wherein the modification reduces at least 33% of the positive charge attributed to the original sequence.

30. The antibody of claim 29, wherein the positive charge attributed to the original sequence is reduced by about 60% or more in the variant polypeptide.

31. The antibody of claim 29 or 30, wherein the number of positively charged amino acids in the original sequence is reduced by one or more in the variant polypeptide.

32. The antibody of any one of claims 29-31, wherein at least one of the at least two positively charged amino acids of the original sequence is replaced by a negatively charged or uncharged amino acid in the variant polypeptide.

33. The antibody of any one of claims 29-32, wherein at least X4 and X7 are each independently a positively charged amino acid.

34. The antibody of any one of claims 29-33, wherein the positively charged amino acid is lysine, arginine or histidine.

35. The antibody of any one of claims 29-34, wherein X2, X3, X5 and X6 are each independently a negatively charged or uncharged amino acid.

36. The antibody according to any one of claims 29-35, wherein at least one of X4 and X7 is replaced by a negatively charged or uncharged amino acid to reduce the positive charge.

37. The antibody of claim 36, wherein the negatively charged or uncharged amino acid replacing at least one of X4 and X7 is glutamine.

38. The antibody according to any one of claims 29-37, wherein the variant polypeptide comprises alanine or serine at the position corresponding to X5 in the sequence of the original polypeptide.

39. The antibody according to any one of claims 29-38, wherein the variant polypeptide comprises the light chain variable region (V L ).

40. according to the antibody described in any one of claim 29-39, wherein said original sequence is located at V of said original polypeptide. L within the framework region (FR).

41. according to the antibody described in claim 40, wherein said original sequence is located at the V of described original polypeptide. L Within FR2.

42. The antibody according to claim 40 or 41, wherein the original polypeptide is based on V L The first human germline sequence, the V L The first human germline sequence has an original FR2 comprising the original sequence, and wherein the variant polypeptide is obtained by replacing the V L The invention also provides a method for producing a polypeptide comprising: producing a polypeptide comprising: a first polypeptide having a second FR2 having a second human germline sequence which replaces at least the original sequence and differs from the original polypeptide, wherein the second FR2 comprises the second sequence corresponding to the original sequence and the second sequence has a positive charge attributed to it which is reduced by at least 10% compared to the original sequence.

43. A variant antibody comprising at least one disrupted positively charged amino acid cluster, wherein the variant antibody differs from an original antibody comprising the original cluster by replacing at least one positively charged amino acid of the original cluster with a negatively charged or uncharged amino acid, wherein the original cluster comprises at least two positively charged amino acids within 12 residues of each other, wherein the original cluster is outside any CDR of the original antibody.

44. The variant antibody of claim 43, wherein the original cluster is located in the light chain variable region (V L )FR.

45. The variant antibody according to claim 44, wherein the original sequence is located at V L FR2.

46. ​​The variant antibody of claim 45, wherein the variant antibody comprises at least one of the following, which comprises a replacement of at least one of the positively charged amino acids: KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14) and QQKPGQSPQ (SEQ ID NO: 15).

47. The variant antibody of claim 45, wherein the variant antibody comprises V L FR2, the V L FR2 comprises the amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

48. The variant antibody of claim 43, wherein the original cluster is located in the hinge region of the original antibody.

49. The variant antibody of claim 48, wherein the original cluster is located in the upper hinge region of the original antibody.

50. The variant antibody of claim 48 or 49, wherein the original antibody comprises an upper hinge sequence of EPKSSDKTHT (SEQ ID NO: 38).

51. The antibody of any one of claims 48-50, wherein the variant antibody comprises an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39).

52. The antibody of any one of the preceding claims, wherein the antibody comprises an antigen binding fragment.

53. The antibody of any of the preceding claims, wherein the antibody has a molecular weight in the range of about 15 kDa to about 110 kDa.

54. The antibody of any of the preceding claims, wherein the antibody is a minibody, a cys-diabody, a scFv, a scFv-Fc or a nanobody-Fc.

55. The antibody of any one of the preceding claims, wherein the antibody has a molecular weight in the range of about 10 kDa to about 20 kDa.

56. The antibody of claim 55, wherein the antibody is a nanobody.

57. The antibody of any one of claims 1-54, wherein the molecular weight of the antibody is greater than 90 kDa.

58. The antibody of claim 57, wherein the antibody is a scFv-Fc.

59. A miniantibody or cys-diabody comprising a variant polypeptide, wherein the variant polypeptide is in combination with a light chain variable region (V L )FR2 is different from the original polypeptide, wherein X1, X2, X3, X4, X5, X6 and X7 are each independently any amino acid, provided that at least two of them are surface-exposed positively charged amino acids that form a surface-exposed positively charged amino acid cluster in the original polypeptide, The variant polypeptide differs from the original polypeptide by modifying at least one of at least two surface-exposed positively charged amino acids of the original sequence, wherein the modification reduces at least 33% of the positive charge attributed to the original sequence.

60. The minibody or cys-diabody of claim 59, wherein at least X4 and X7 are each independently a positively charged amino acid in the original polypeptide.

61. The miniantibody or cys-diabody of claim 59 or 60, wherein X2, X3, X5 and X6 are each independently a negatively charged or uncharged amino acid in the original polypeptide.

62. The minibody or cys-diabody of any one of claims 59-61, wherein at least one of X4 and X7 is replaced by a negatively charged or uncharged amino acid to reduce the positive charge.

63. A method comprising a light chain variable region (V L ) of a miniantibody or cys-diabody, wherein the light chain variable region comprises a FR2 sequence comprising X1X2X3QAX6X7 (SEQ ID NO: 4), wherein X1 is a surface-exposed positively charged amino acid, wherein X2, X3 and X6 are each independently any negatively charged or uncharged amino acid, and wherein X7 is glutamine or lysine.

64. The minibody or cys-diabody according to any one of claims 59-63, wherein X1 is lysine.

65. The miniantibody or cys-diabody according to any one of claims 59-64, wherein X2 and / or X6 is proline.

66. The minibody or cys-diabody of any one of claims 59-65, wherein X3 is glycine or glutamic acid.

67. The minibody or cys-diabody of claim 63, wherein the FR2 sequence comprises KPGQAPK (SEQ ID NO: 13) or KPGQAPQ (SEQ ID ID: 14).

68. A method comprising a light chain variable region (V L ) of a miniantibody or cys-diabody, wherein the light chain variable region comprises (according to IMGT numbering): A49 and either (i) Q51; or (ii) Q48 and K51.

69. The miniantibody or cys-diabody according to claim 68, wherein the V L Also includes K45 (IMGT number).

70. A miniantibody comprising an upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39).

71. The antibody, minibody or cys-diabody of any of the preceding claims, wherein the antibody, minibody or cys-diabody specifically binds DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA.

72. The antibody, minibody or cys-diabody according to any of the preceding claims, wherein the antibody, minibody or cys-diabody comprises a light chain variable region and a heavy chain variable region (V H ), the light chain variable region comprises three LCDR sequences in any one of Figures 5B, 5C, and 7B-7D, and the heavy chain variable region comprises three HCDR sequences in any one of Figures 5B, 5C, and 7B-7D.

73. The antibody, minibody or cys-diabody of any of the preceding claims, further comprising a detectable label.

74. The antibody, minibody or cys-diabody of claim 73, wherein the antibody, minibody or cys-diabody is labeled with a radionuclide or an organic dye.

75. The antibody, minibody or cys-diabody of any one of claims 1-72, further comprising a therapeutic agent.

76. The antibody, minibody or cys-diabody of claim 75, wherein the therapeutic agent is a cytotoxic agent.

77. The antibody, minibody or cys-diabody of claim 75, wherein the therapeutic agent is a radionuclide.

78. The antibody, minibody or cys-diabody of claim 75, wherein the radionuclide is selected from 212 Pb, 149 Tb and 161 Tb.

79. A composition comprising: The antibody, minibody or cys-diabody of any preceding claim; and A pharmaceutically acceptable carrier.

80. A nucleic acid encoding a variant polypeptide of the antibody of any one of claims 29-42 or the minibody or cys-diabody of any one of claims 59-62.

81. A nucleic acid encoding the variant antibody of any one of claims 1-28 and 43-54, the antibody of any one of claims 29-42, or the minibody or cys-diabody of any one of claims 59-72.

82. A genetically engineered host cell comprising the nucleic acid of claim 80 or 81.

83. A method for enhancing the biodistribution and / or pharmacokinetics of an antibody, comprising: recognizing an original antibody comprising a polypeptide comprising at least one surface-exposed positively charged cluster of amino acids, the cluster comprising at least two surface-exposed positively charged amino acids within 30 angstroms of each other; At least one of the at least two surface-exposed positively charged amino acids of the at least one cluster is replaced with a negatively or uncharged amino acid, thereby destroying the at least one cluster, thereby generating a variant antibody with enhanced biodistribution and / or pharmacokinetics compared to the original antibody.

84. A method for enhancing the biodistribution and / or pharmacokinetics of an antibody, comprising: recognizing an original antibody comprising a polypeptide comprising at least two clusters of surface-exposed positively charged amino acids within 12 residues of each other; as well as At least one of the at least two surface-exposed positively charged amino acids of the cluster is replaced with a negatively or uncharged amino acid to disrupt the cluster, thereby generating a variant antibody having enhanced biodistribution and / or pharmacokinetics compared to the original antibody.

85. The method of claim 84, wherein the cluster comprises at least two positively charged amino acids within 6 residues of each other.

86. A method according to claim 84 or 85, wherein the polypeptide comprises an amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein X1, X2, X3, X4, X5, X6 and X7 are each independently any amino acid, and wherein the at least two surface-exposed positively charged amino acids comprise at least two of X1, X2, X3, X4, X5, X6 and X7.

87. The method of claim 86, wherein the at least two surface-exposed positively charged amino acids comprise at least X1.

88. The method of claim 86 or 87, wherein the at least two surface-exposed positively charged amino acids comprise at least X4 and X7.

89. The method of any one of claims 86-88, wherein X2, X3, X5 and X6 are each independently a negatively charged or uncharged amino acid residue in the original polypeptide.

90. The method of any one of claims 86-89, wherein at least one of X4 and X7 is replaced by a negatively charged or uncharged amino acid.

91. The method of any one of claims 83-90, wherein the at least two surface-exposed positively charged amino acids comprise arginine, lysine, or histidine.

92. The method of any one of claims 83-91, wherein the negatively or uncharged amino acid replacing at least one of the at least two surface-exposed positively charged amino acids comprises a polar amino acid.

93. The method of claim 92, wherein the negatively or uncharged amino acid replacing at least one of the at least two surface-exposed positively charged amino acids is glutamine.

94. The method of any one of claims 83-93, wherein the positively charged amino acid and the negatively or uncharged amino acid have side chain lengths that differ by less than one or two carbons.

95. The method of any one of claims 83-94, comprising replacing up to 3 amino acids of the polypeptide to disrupt the at least one cluster.

96. The method of any one of claims 83-95, wherein the cluster is outside any complementarity determining region (CDR) of the original antibody.

97. The method of any one of claims 83-96, wherein the cluster is located in the light chain variable region (V L )middle.

98. according to the method described in any one of claims 83-97, wherein said at least two surface exposed positively charged amino acids are located in the V of the original antibody. L within the framework region (FR).

99. according to the method described in any one of claims 83-98, wherein said at least two surface exposed positively charged amino acids are located in the V of said original antibody. L Within FR2.

100. The method of claim 98 or 99, wherein the polypeptide is based on V L The first human germline sequence, the V L The first human germline sequence of the present invention has an original FR2, wherein the original FR2 comprises the at least two surface-exposed positively charged amino acids, and wherein the method comprises replacing the original FR2 or a portion comprising the cluster with a L A second FR2 of a second human germline sequence of the cluster, or a corresponding portion thereof, wherein the second FR2 comprises at least one reduced surface exposed positively charged amino acid compared to the cluster.

101. The method of claim 99 or 100, wherein the original sequence is one of the following: KX2X3KX5X6K (SEQ ID NO: 73), wherein X2, X3, X5 and X6 are each independently a negatively charged or uncharged amino acid; KX2X3X4X5X6R (SEQ ID NO: 74), wherein X2, X3, X4, X5 and X6 are each independently a negatively charged or uncharged amino acid; or KX2X3X4X5X6K (SEQ ID NO: 75), wherein X2, X3, X4, X5 and X6 are each independently a negatively charged or uncharged amino acid.

102. according to the method described in claim 99 or 101, wherein said original polypeptide is in V L FR2 includes one of the following: KPGKAPK (SEQ ID NO:5), KPGQAPR (SEQ ID NO:6), KPEKAPK (SEQ ID NO:7), KPGKVPK (SEQ ID NO:8), KPGQPPR (SEQ ID NO:9), KPGQSPR (SEQ ID NO:10), KPGLAPR (SEQ ID NO:11) or KPGQPPK (SEQ ID NO:12).

103. according to the method described in any one of claims 99-101, wherein said variant polypeptide is in V L FR2 contains at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID NO: 16) and QQKPGQSPQ (SEQ ID NO: 15).

104. according to the method described in any one of claims 99-101, wherein said variant polypeptide is in V L FR2 includes at least one of the following: WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID No: 18) or WYQQKPGQAPKLLIY (SEQ ID NO: 19).

105. The method of any one of claims 83-104, further comprising binding a chelating ligand to the variant antibody.

106. The method of any one of claims 83-105, further comprising labeling the antibody with a radionuclide.

107. The method of any one of claims 83-105, further comprising labeling the antibody with a detectable label.

108. A method for preparing a labeled antibody, comprising: Select the light chain variable region (V L ), wherein the germline sequence does not comprise a cluster of at least two positively charged amino acids within 3 residues of each other in framework region 2 (FR2) of the germline sequence; isolating one or more target-specific antibodies from a population of antibodies comprising V sequences derived from the germline sequences L , and having variation of said germline sequence throughout said population, wherein said one or more target-specific antibodies are in V L The FR2 sequence does not contain a cluster of at least two positively charged amino acids within 3 residues of each other; and The one or more target-specific antibodies are labeled.

109. The method of claim 108, wherein isolating the one or more target-specific antibodies comprises screening a population of antibodies derived from the germline sequences.

110. The method of claim 109, wherein the population of antibodies derived from the germline sequences are produced in a host organism that has been genetically modified to express antibodies based solely on selected germline sequences.

111. The method of claim 109, wherein the population of antibodies derived from the germline sequences is comprised in an antibody phage library based solely on selected germline sequences.

112. The method of any one of claims 109-111, wherein the germline sequence is a human germline sequence.

113. The method of any one of claims 108-112, wherein the one or more target-specific antibodies are humanized.

114. The method of any one of claims 108-113, further comprising generating one or more target-specific miniantibodies or cys-diabodies from the one or more target-specific antibodies.

115. The method of any one of claims 108-114, comprising labeling the one or more target-specific antibodies with a radionuclide.

116. The method of any one of claims 108-115, wherein labeling the one or more target-specific antibodies comprises binding a chelating ligand to the one or more target-specific antibodies.

117. The method of any one of claims 83-116, wherein the antibody comprises an antigen binding fragment.

118. The method of any one of claims 83-117, wherein the antibody is a minibody or a cys-diabody.

119. The method of any one of claims 83-118, wherein the antibody specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA.

120. The method of any one of claims 83-118, wherein the antibody or minibody comprises a light chain variable region (V L ) and the heavy chain variable region (V H ), the light chain variable region comprises 3 LCDR sequences among the 3 LCDRs of any one sequence in Figures 5A-5C and Figures 7A-7D, and the heavy chain variable region comprises 3 HCDR sequences among the 3 HCDRs of any one sequence in Figures 5A-5C and Figures 7A-7D.

121. An antibody prepared by the method of any one of claims 83-120.

122. A method of treating a subject, comprising: Identifying a subject in need of treatment with the antibody, minibody or cys-diabody of any one of claims 1-78; as well as Administering a therapeutically effective amount of the antibody or minibody or the composition of claim 79 to the subject.

123. A method of treating cancer in a subject, comprising: Identify subjects in need of cancer treatment; as well as Administering a therapeutically effective amount of the antibody, minibody or cys-diabody of any one of claims 1-78 or the composition of claim 79 to the subject, thereby treating the cancer.

124. A radiotherapy method comprising: Identify subjects who require radiation therapy; as well as A therapeutically effective amount of the antibody, minibody or cys-diabody of any one of claims 1-78 or the composition of claim 79 is administered to the subject, wherein the antibody, minibody or cys-diabody comprises a radionuclide.

125. A method of imaging a subject, comprising: administering to a subject a composition comprising an effective amount of the antibody, minibody, or cys-diabody of any one of claims 1-78 or the composition of claim 79, wherein the antibody, minibody, or cys-diabody is detectably labeled; as well as The subject is imaged to detect the labeled antibody, minibody or cys-diabody in the subject.

126. Use of the antibody, minibody or cys-diabody of any one of claims 1-78 or the composition of claim 79 for treating cancer in a subject in need thereof.

127. Use of the antibody, minibody or cys-diabody of any one of claims 1-78 in the preparation of a medicament for treating cancer in a subject in need thereof.

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