Antibodies and methods for making and using them

Abstract

Antibodies are provided that have enhanced in vivo distribution and / or pharmacokinetics obtained by disruption of one or more clusters of positively charged amino acids exposed on the surface. Also provided is a method of enhancing the in vivo distribution and / or pharmacokinetics of an antibody. The enhanced antibodies find use in immunotherapy, radiotherapy, and in vivo imaging.

Description

Technical Field

[0001] Incorporation by reference to any prior application Any application in which a foreign or domestic priority claim is verified in the application data sheet filed together with the present application is incorporated herein by reference under 37 CFR 1.57 of the United States Patent Rules.

[0002] Reference to the Sequence Listing The present application has been filed together with an electronic form of the Sequence Listing. The Sequence Listing is provided as a file entitled IGNAB063WO_SEQLIST.xml created on July 2, 2023, which is 66,818 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure generally relates to enhancing the biodistribution and / or pharmacokinetics of antibodies (including minibodies and cys-diabodies), and compositions therefor.

Background Art

[0004] Description of Related Art The present disclosure generally relates to antibodies, including antigen-binding fragments thereof such as minibodies and cys-diabodies. Antibodies can be used to bind to targets for therapeutic or diagnostic use.

Prior Art Documents

Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0007] A variant antibody comprising at least one disrupted cluster of positively charged amino acids exposed on the surface, wherein the variant antibody has a substitution of at least one positively charged amino acid exposed on the surface of the original cluster by a negatively charged or uncharged amino acid, such that the original antibody comprising the original cluster containing at least two positively charged amino acids exposed on the surface within about 30 angstroms of each other is different from the variant antibody, is provided herein.

[0008] An antibody comprising a variant polypeptide different from the original polypeptide comprising the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein the original sequence is outside of any CDR of the original polypeptide, and wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two of them are positively charged amino acids exposed on the surface forming a cluster of positively charged amino acids exposed on the surface in the original polypeptide, and the variant polypeptide has at least one modification of at least two positively charged amino acids exposed on the surface of the original sequence that reduces the positive charge resulting from the original sequence (e.g., reduces at least 33% of the positive charge), thereby being different from the original polypeptide, is also provided.

[0009] A variant antibody comprising at least one disrupted cluster of positively charged amino acids, wherein the variant antibody has a substitution of at least one positively charged amino acid of the original cluster by a negatively charged or uncharged amino acid, such that the variant antibody differs from the original antibody that comprises an original cluster containing at least two positively charged amino acids within 12 residues of each other, and the original cluster is outside of any CDR of the original antibody. The variant antibody is further provided herein.

[0010] A minibody or cys-d diabody comprising a variant polypeptide that differs from an original polypeptide comprising an original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1) that includes a variable light chain region (V L ) FR2, wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two of them are positively charged amino acids that form a cluster of positively charged amino acids exposed on the surface in the original polypeptide, and the variant polypeptide differs from the original polypeptide by having a modification of at least one of at least two positively charged amino acids exposed on the surface of the original sequence that reduces at least 33% of the positive charge resulting from the original sequence. A minibody or cys-d diabody is also provided.

[0011] A minibody or cys-d diabody comprising a variable light chain region (V L ) that includes an FR2 sequence comprising X1X2X3QAX6X7 (SEQ ID NO: 4), wherein X1 is a positively charged amino acid exposed on the surface, X2, X3, and X6 are each independently any negatively charged or uncharged amino acid, and X7 is either glutamine or lysine. A minibody or cys-d diabody is provided herein.

[0012] A variable light chain region (V LMinibodies or cys-diabodies that include

[0013] Minibodies that include the upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39) are also provided.

[0014] Compositions are provided herein that include any one of the antibodies (e.g., variant antibodies), minibodies, or cys-diabodies of the disclosure; and a pharmaceutically acceptable carrier.

[0015] Also provided are variant polypeptides of any one of the antibodies (e.g., variant antibodies) herein, or any one of the variant antibodies herein, or nucleic acids encoding any one of the minibodies or cys-diabodies of the disclosure.

[0016] Genetically engineered host cells are also provided that include any one of the nucleic acids of the disclosure, or combinations thereof.

[0017] A method for enhancing the in vivo distribution and / or pharmacokinetics of an antibody is provided herein, the method including the steps of identifying a parent antibody that includes a polypeptide that includes at least one cluster of positively charged amino acids exposed on a surface, the cluster including at least two positively charged amino acids exposed on a surface within 30 angstroms of each other; and substituting at least one of the at least two positively charged amino acids exposed on a surface of at least one cluster with a negatively charged or uncharged amino acid, thereby disrupting at least one cluster, whereby a variant antibody is produced that has enhanced in vivo distribution and / or pharmacokinetics compared to the parent antibody.

[0018] Identifying an original antibody comprising a polypeptide comprising at least two clusters of positively charged amino acids exposed on the surface within 12 residues of each other; and disrupting the cluster by substituting at least one of the at least two positively charged amino acids exposed on the surface of the cluster with a negatively charged or uncharged amino acid, thereby generating a variant antibody having enhanced in vivo distribution and / or pharmacokinetics compared to the original antibody, and a method for enhancing the in vivo distribution and / or pharmacokinetics of an antibody is also provided.

[0019] Selecting a germline sequence for the variable light chain region (V L ) of the antibody, wherein the germline sequence does not contain at least two clusters of positively charged amino acids within 3 residues of each other in the framework region 2 (FR2) of the germline sequence; isolating one or more target-specific antibodies from a population of antibodies comprising V L and having variations in the germline sequence across the population, wherein the one or more target-specific antibodies do not contain at least two clusters of positively charged amino acids within 3 residues of each other in the V L FR2 sequence; and a method for producing a labeled antibody comprising labeling the one or more target-specific antibodies is provided herein.

[0020] An antibody produced by any one of the methods of the present disclosure is also provided.

[0021] Identifying a subject in need of treatment with any one of the antibodies (e.g., variant antibodies), minibodies, or cys-dibodies of the present disclosure; and administering to the subject a therapeutically effective amount of the antibody or minibody, or a composition comprising the same, and a method for treating a subject is provided herein.

[0022] A step of identifying a subject in need of treatment for cancer; and a step of treating the cancer by administering to the subject a therapeutically effective amount of any one of the antibodies (e.g., variant antibodies), minibodies, or cys-diabodies of the present disclosure, or a composition comprising the same, are further provided.

[0023] A step of identifying a subject in need of radiation therapy; and a step of administering to the subject a therapeutically effective amount of any one of the antibodies (e.g., variant antibodies), minibodies, or cys-diabodies of the present disclosure, or a composition comprising the same, wherein the antibody, minibody, or cys-dibody comprises a radionuclide, are provided herein.

[0024] A step of administering to a subject a composition comprising an effective amount of any one of the antibodies (e.g., variant antibodies), minibodies, or cys-diabodies of the present disclosure, or a composition comprising the same, wherein the antibody, minibody, or cys-dibody is detectably labeled; and a step of imaging the subject to detect the labeled antibody, minibody, or cys-dibody in the subject are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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Mode for Carrying Out the Invention

[0026] Detailed Description The biodistribution and pharmacokinetics of antibodies, minibodies, and cys-dibodies are important determinants of their safety and efficacy when administered to a subject. Improvement in 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 diagnostic radiology imaging using antibodies, minibodies, and cys-dibodies. The biodistribution and pharmacokinetics of an antibody can vary depending on several factors, including some characteristics of the electrostatic charges exposed on the surface of the antibody.

[0027] Antibodies (including minibodies and cys-diabodies) having enhanced in vivo distribution and / or pharmacokinetics, and methods for enhancing the in vivo distribution and / or pharmacokinetics of antibodies are provided herein. Antibodies of the present disclosure (e.g., variant antibodies) generally have disrupted clusters of positively charged amino acids compared to the original antibody, and may have enhanced in vivo distribution and / or pharmacokinetics compared to the original antibody based on which the antibody having clusters and disrupted clusters is based.

[0028] Without being bound by theory, clusters of positively charged amino acids form patches of positive electrostatic charge explained by the isoelectric plane on the antibody, and this local charge enrichment may contribute to the accumulation of the antibody in radiosensitive or drug-sensitive tissues such as the kidney when administered to a subject. Undesirable antibody accumulation in the kidney may result from increased capture and retention (and / or increased reabsorption and retention in these renal subcompartments) in the renal glomerulus or proximal renal tubule compared to the variant antibodies provided herein.

[0029] The disruption of clusters of positively charged amino acids provided herein can reduce the accumulation of antibodies in the kidney. In some embodiments, the disruption of the clusters reduces the accumulation of antibodies in the kidney by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, or within a range defined by any two of the preceding values (e.g., 10 - 80%, 10 - 50%, 30 - 80%, 40 - 70%, etc.) percentage decrease. In some embodiments, the disruption of the clusters increases the accumulation of antibodies in the liver. In some embodiments, the change in biodistribution resulting from the disruption of the clusters is readily observable for antibody constructs having a molecular weight of about 50 - 80 kDa (as a dimer), such as minibodies or cys-diabodies. Generally, the modification of the original antibody by disrupting clusters of positively charged amino acids enhances biodistribution and / or pharmacokinetics while at least maintaining the same binding specificity and binding affinity of the original antibody. In some embodiments, the antibody (or antigen-binding fragment thereof) has a molecular weight of 15 - 160 kDa. In some embodiments, the antibody (or antigen-binding fragment thereof) has a molecular weight of 15 - 110 kDa. In some embodiments, the antibody (or antigen-binding fragment thereof) has a molecular weight of about 50 - 80 kDa.

[0030] In some embodiments, the biodistribution of an antibody can be controlled by modifying the electrostatic charge associated with the cluster. As described above, in some embodiments, a cluster of positively charged amino acids can promote accumulation in the kidney, and decreasing the positive charge associated with the cluster or increasing the negative charge can promote accumulation away from the kidney, for example, promoting accumulation in the liver. Thus, in some embodiments, the biodistribution of a cytotoxic agent (e.g., a toxin) or a radionuclide conjugated to an antibody can be biased towards the kidney by increasing the positive charge associated with the cluster, and the biodistribution of a cytotoxic agent (e.g., a toxin) or a radionuclide conjugated to an antibody can be biased towards the liver by decreasing the positive charge associated with the cluster or increasing the negative charge. In some embodiments, when treating liver disorders (e.g., liver cancer) by administering an antibody, the distribution of the antibody to the liver is promoted.

[0031] In the context of radioimmunotherapy, the accumulation of an antibody in the kidney can lead to nephrotoxicity. Thus, the antibodies of the present disclosure having reduced accumulation in the kidney can allow for administration of higher doses of radiolabeled antibodies (e.g., higher specific activity, more frequent and / or multiple administrations) while reducing nephrotoxicity.

[0032] The term All terms have their ordinary and customary meanings as understood by one of ordinary skill in the art in light of the present disclosure.

[0033] As used herein, the term "antibody" includes all types of antibodies, including their antigen-binding fragments. Constructs containing 1, 2, 3, 4, 5, and / or 6 CDRs are further included. In some embodiments, tandem scFvs can be provided that can provide two arms with bivalent binding. In some embodiments, these CDRs can be distributed between their appropriate framework regions in a conventional antibody. In some embodiments, the CDRs can be contained within the heavy and / or light chain variable regions. In some embodiments, the CDRs can be in the heavy and / or light chains. In some embodiments, the CDRs can be in a single polypeptide chain. Unless otherwise indicated herein, the antibodies described herein bind to the target molecule(s) referred to. The term "target" or "target molecule" refers to a protein to which the antigen-binding construct binds with a certain level of specificity under physiological conditions.

[0034] The term "antibody" includes, but is not limited to, forms of immunoglobulins that have been genetically engineered or otherwise modified, 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, tribodies, tetrabodies, etc.). The term "antibody" also includes camelid-derived immunoglobulins such as single-chain antibodies and nanobodies. "Antibody fragments" include, without limitation, Fab', F(ab')2, Fab, Fv, rIgG (reduced IgG), scFv fragments, scFv-Fc, single-domain fragments (e.g., nanobodies), peptibodies, nanobodies (registered trademark), nanobody (registered trademark)-Fc, minibodies, and diabodies. The term "antibody" includes scFv and minibodies. Thus, all embodiments provided herein with respect to "antibody" are also contemplated as scFv and / or minibody embodiments unless otherwise explicitly stated. The term "antibody" includes a polypeptide of the immunoglobulin family or a polypeptide comprising a fragment of an immunoglobulin that can bind to a 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 pairs of polypeptide chains, where each pair has one "light" and one "heavy" chain (connected through disulfide bonds). Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, hinge, and mu constant region genes, as well as numerous immunoglobulin variable region genes. With respect to the full-length chain, the light chain is classified as either kappa or lambda. With respect to the full-length chain, the heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which in turn define the 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 that is primarily involved in antigen recognition. Variable light chain (V L ) and variable heavy chain (V HThe terms " " refer to these regions of the light and heavy chains, respectively. As used in this application, "antibody" encompasses all variations of antibodies and their fragments. Thus, within the scope of this concept are full-length antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies (scFv), Fab, Fab', and multimeric versions of these fragments (e.g., F(ab')2) having the same binding specificity. In some embodiments, the antibody specifically binds to a desired target.

[0035] "Antibody" may also include one or more immunoglobulin chains that are chemically conjugated to other proteins or expressed as a fusion protein with other proteins. It includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites.

[0036] As used herein, "antibody" may also mean other antigen-binding fragments or antibody portions of the present disclosure, including bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (sdAb or nanobodies®), minibodies, and cys-diabodies.

[0037] The term "antibody fragment" includes 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 (nanobodies®), peptibodies, nanobodies®, nanobody®-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 two-chain antibody are joined to form one chain.

[0038] A minibody is an antibody format that has a smaller molecular weight than a full-length antibody while maintaining the bivalent binding property against an antigen. Due to its smaller size (about 70 - 90 kDa, preferably about 80 kDa), the absence of the C H 2 domain, and the absence of glycosylation, the minibody has more rapid clearance from the blood system and potentially enhanced penetrability when targeting tumor tissue. Having a strong targeting ability combined with rapid clearance compared to full-size antibodies, the minibody is advantageous for diagnostic and therapeutic applications where long circulation times can result in harmful patient dosing or dosimetry, as well as for the delivery of radioactive payloads. In some embodiments, it can also be advantageous for the delivery of cytotoxic payloads (such as in the case of antibody-drug conjugates, ADCs) due to the above-described features such as tumor penetrability and more rapid clearance. The "minibody" described herein includes homodimers where each monomer is a single-chain variable fragment (scFv) linked by a hinge sequence to a human IgG C H 3 domain. A non-limiting schematic of the minibody is shown in FIG. 10. In some embodiments, the minibody is a bivalent or bispecific, covalently linked heterodimer of about 80 kDa. In some embodiments, each monomer (half molecule) is composed of a variable heavy chain (V L ) domain linked to the corresponding variable light chain (V H ) domain by a Gly-Ser rich linker sequence of approximately 15 - 18 amino acids. In some embodiments, each single-chain variable fragment (scFv) is linked by a hinge sequence to a human IgG1, IgG2, IgG3, or IgG4 C H 3 domain.

[0039] The term "hinge" refers to at least a portion of the hinge region for an antigen-binding construct, such as an antibody or minibody, scFv-Fc, or nanobody®-Fc. The hinge region can include a combination of an upper hinge, a core (or central) hinge, and a lower hinge region. In some embodiments, the hinge is defined according to any of the antibody hinge definitions. Native IgG1, IgG2, and IgG4 antibodies have hinge regions of 12-15 amino acids. IgG3 has an extended hinge region of 62 amino acids, including 21 prolines and 11 cysteines. The functionally active hinge region of a naturally occurring antibody as deduced from crystallographic studies can extend from amino acid residues 216-237 (EU numbering) of the IgG1 H chain and can include a small segment at the N-terminus of the C H 2-domain that may include the N-terminus, and the lower hinge is C H the N-terminus of the 2-domain. The hinge can be divided into three regions: "upper hinge", "core", and "lower hinge".

[0040] The term "upper hinge" refers to the first part of the hinge that starts from the end of the scFv. The upper hinge includes amino acids from the end of the scFv up to, but not including, the first cysteine residue in the core hinge. The term "effective upper hinge" indicates that there is sufficient sequence to allow this segment to function as an upper hinge; the term encompasses functional variants and fragments of the designated hinge segment.

[0041] The term "core hinge" refers to the second part of the hinge region that is C-terminal to the upper hinge. The core hinge can contain interchain disulfide bridges and a high content of proline.

[0042] The term "lower hinge" refers to the third part of the hinge region that is C-terminal to the core hinge. In the context of a minibody or antibody fragment, the lower hinge is C HConnect to the 3-domain Mb. As noted above, the term "functional lower hinge" indicates that there is a sufficient arrangement to enable the segment to function as a lower hinge; the term includes functional variants and fragments of the designated hinge segment. As used herein, the term "lower hinge" may include various amino acid sequences, including native IgG lower hinge sequences and artificial extension sequences, or combinations thereof provided herein, that are interchangeable with each other. In some embodiments, the various extensions may be considered to be part of the lower hinge region as a whole or as replacements.

[0043] In some embodiments, the lower hinge is 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 without a lower hinge, and / or can be any combination of amino acids (not necessarily G and S). In some embodiments, the lower hinge may include GGGSSGGGSG (SEQ ID NO: 40).

[0044] The term "diabody" refers to a dimer comprising a heavy chain (V H ) domain and a light chain variable (V L ) domain. Each heavy chain domain is connected to a light chain domain through a linker to form a monomer. Two monomers are covalently linked through a cross-linking moiety to form a diabody. "Cys-diabody" refers to a diabody in which the monomer chains are covalently linked by disulfide bonds. A non-limiting schematic of a cys-diabody is shown in FIG. 11. A cys-diabody can have a molecular weight of about 50 kDa.

[0045] The term "extension sequence" (e.g., in the context of a diabody) refers to, for example, in a diabody, the first V H domain to the second V H domain, or the first V L to the second VL Represents a region that connects to a domain. The extension sequence can connect domains through the C-terminus of each domain. In some embodiments, the extension sequence connects domains through a covalent bond. In some embodiments, the extension sequence will include one or more cysteines that allow one or more disulfide bonds to be formed between two such extension sequences. An example of a pair of extension sequences is shown in the schematic on the right side of FIG. 11 as a line having two cysteines that connect either two heavy chain domains or two light chain domains. A non-limiting example of an extension sequence of -(Gly)2-(Cys) is shown in the schematic on the left side of FIG. 11. In some embodiments, the extension sequence includes 1, 2, 3, or more than 3 cysteines per monomer chain. The extension sequence will be towards the C-terminus of the construct in FIG. 11, although 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 where the extension sequence begins. The extension sequence, through a disulfide bond, connects V H to V H or connects V L to V L can be connected. In some embodiments, the extension sequence includes GGCPPCPPC (SEQ ID NO: 72).

[0046] In some embodiments, the linker can be any suitable linker for an antibody (e.g., minibody, cys-diabody). In some embodiments, the linker sequence includes (G3)S n or (G4)S n (n can be any integer; in some embodiments, it is 1 or 2) and can include a motif. In some embodiments, the linker can include GSTSGGGSGGGSGGGGSS (SEQ ID NO: 41).

[0047] The term "treating" or "treatment" of a medical condition can refer to preventing the medical condition, slowing the rate of its development and / or progression, reducing the risk of developing the medical condition, preventing and / or delaying the onset of symptoms associated with the medical condition, reducing and / or terminating the symptoms associated with the medical condition, causing complete or partial regression of the medical condition, or some combination thereof. The term "preventing" does not necessarily require absolute prohibition of a disorder or disease.

[0048] A "therapeutically effective amount" or "therapeutically effective dosage" is an amount that produces a desired therapeutic effect in a subject, such as preventing, treating, delaying the onset of a disorder and / or symptoms, and / or alleviating symptoms associated with the medical condition. This amount will vary depending on a variety of 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 dosage, and type of medication), the nature of one or more pharmaceutically acceptable carriers in the formulation, and / or the route of administration. One of ordinary skill in the clinical and pharmacological arts, in view of the present disclosure, would be able to determine a therapeutically effective amount by routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st ed., Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0049] The term "complementary determining domain" or "complementary determining region" ("CDR") interchangeably refers to the hypervariable regions of V L and V H The CDR is the target molecule binding site of an antibody chain that has specificity for such a target molecule. In some embodiments, each VL and / or V H has three CDRs (CDR1-3 numbered sequentially from the N-terminus), which constitute approximately 15-20% of the variable domain. The CDRs are structurally complementary to the epitope of the target molecule and thus are directly involved in binding specificity. V L or V H The remaining stretches of V or V, the so-called framework regions (FRs), exhibit lower variability in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4, W.H. Freeman & Co., New York, 2000).

[0050] The positions of the CDR and framework regions are defined by various well-known definitions in the art, such as Kabat (Wu, T. T., E. A. 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, E. A., Wu, T. T., Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed., 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)); the ImmunoGeneTics database (IMGT) (available at imgt.org / on the World Wide Web) Giudicelli, V., Duroux, P., Ginestoux, C., Folch, G., Jabado-Michaloud, J., Chaume, D., and Lefranc, M.-P. IMGT / LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences Nucl. Acids Res., 34, D781~D784 (2006), PMID: 16381979; Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V., and Lefranc, G., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains Dev. Comp. Immunol., 27, 55-77 (2003). PMID: 12477501; Brochet, X., Lefranc, M.-P., and Giudicelli, V. IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis Nucl. Acids Res, 36, W503-508 (2008); AbM (Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); North (North B., Lehmann A., Dunbrack R.L., A new clustering of antibody CDR loop conformations, J. Mol. Biol. (2011) 406(2): 228-256); AHo (Honegger A., Pluckthun, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol. Biol. (2001) 309, 657-670); contact definition (MacCallum et al., J. Mol. Biol., 262:732-745 (1996)), and / or the automatic modeling and analysis tool Honegger A, Pluckthun A.(which can be determined using the World Wide Web at bioc dot uzh dot ch / antibody / Numbering / index dot html). In some embodiments, the framework regions are defined by the AHo numbering scheme as follows: FR1, 1-24; FR2, 41-57; FR3, 78-108, and FR4, 138-149.

[0051] As used herein, "antibody variable light chain" or "antibody variable heavy chain" refers to a polypeptide containing V L or V H respectively. Endogenous V L is encoded by the gene segments V (variable) and J (joining), and endogenous V H is encoded by V, D (diversity), and J. Each of V L or V H includes CDRs as well as framework regions. In this application, antibody variable light chains and / or antibody variable heavy chains may sometimes be collectively referred to as "antibody chains". As will be readily recognized by those skilled in the art, these terms include antibody chains containing mutations that do not disrupt the basic structure of V L or V H In some embodiments, full-length heavy and / or light chains are contemplated. In some embodiments, only the variable regions of the heavy and / or light chains are present.

[0052] Antibodies can exist as intact immunoglobulins or as several fragments produced by digestion with various peptidases. Thus, for example, pepsin digests the antibody below the disulfide linkage in the hinge region to produce F(ab)'2, which is a dimer of Fab', which itself is a light chain (V H -C H 1) joined to V L -C L) It is so. F(ab)'2 can be reduced under mild conditions to break the disulfide linkages in the hinge region, thereby converting the F(ab)'2 dimer to Fab' monomers. The Fab' monomers are Fabs having a part of the hinge region. (Paul, W. E., "Fundamental Immunology", Part 3, New York: Raven Press, 1993). Although various antibody fragments are defined from the perspective of digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized de novo by using chemical or recombinant DNA methodologies. Thus, the term "antibody" as used herein includes antibody fragments produced by modification of the whole antibody, or those synthesized de novo using recombinant DNA methodologies (e.g., single-chain Fv), or those 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).

[0053] Regarding the preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, for example, 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, J. S., "Recombinant polyclonal antibodies: the next generation of antibody therapeutics?", Drug Discov. Today, Vol. 11, Nos. 13-14, pp. 655-660, 2006). Techniques for the production of single-chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce antibodies against the polypeptides of the present invention. In addition, other organisms such as transgenic mice or other mammals can be used to express fully human monoclonal antibodies.Alternatively, phage display technology can be used to identify high affinity binding agents for selected antigens (see, for example, McCafferty et al., supra; Marks, J. D. et al., "By-passing immunization: building high affinity human antibodies by chain shuffling", Biotechnology (N.Y.), Vol. 10, No. 7, pp. 779-783, 1992).

[0054] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as import residues, which are typically obtained from the import variable domain. In some embodiments, the terms "donor" and "acceptor" sequences may be employed. Humanization can be essentially carried out according to the method of Winter and colleagues by replacing the CDR sequences of a rodent with the corresponding sequences of a human antibody (see, for example, Jones, P.T. 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 an antilysozyme activity", Science, Vol. 239, No. 4847, pp. 1534-1536, 1988; Presta, L.G., "Antibody engineering", Curr. Op. Struct. Biol., Vol. 2, No. 4, pp. 593-596, 1992). Thus, such humanized antibodies are chimeric antibodies in which substantially fewer human variable domains than intact are replaced by the corresponding sequences from non-human species (U.S. Patent No. 4,816,567). In practice, humanized antibodies are typically human antibodies in which some complementarity determining region ("CDR") residues and optionally some framework ("FR") residues are replaced by residues from similar sites in a rodent antibody.

[0055] The term "Fc region" or "Fc domain" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region (e.g., a variant having one or more mutations that reduce effector functions such as FcγR binding and / or binding to the neonatal Fc receptor (FcRn)). The Fc region of an immunoglobulin (e.g., IgG) generally contains two constant domains, C H 2 and C H 3. The Fc region may or may not include a hinge region or sequence as described herein. The Fc region can exist in a dimeric or monomeric form. In some embodiments, the Fc region is a human Fc region or a variant thereof.

[0056] A "chimeric antibody" is an antibody molecule in which (a) the antigen-binding site (variable region) is altered, replaced, or exchanged such that the constant region or a portion thereof is altered, replaced, or exchanged to a different or modified class, effector function, and / or species of constant region, or is linked to a wholly different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region or a portion thereof is altered, replaced, or exchanged with a variable region having a different or modified antigen specificity.

[0057] A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination of some of them. Each component of the carrier is "pharmaceutically acceptable" in that it is compatible with the other components of the formulation. It must also be suitable for contact with any tissue, organ, or part of the body it may encounter, meaning that it must not carry with it the risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that unduly outweighs its therapeutic benefit. 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 aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. "Transdermal" administration can be accomplished using a topical cream or ointment or by means of a transdermal patch. "Parenteral" refers to routes of administration generally associated with injection, including but not limited to suborbital, injection, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intralung, intraspinal, intrasternal, intrathecal, intracranial, intrauterine, intravenous, subarachnoid, subcapsular, sublingual, subcutaneous, transmucosal, or transtracheal. In some embodiments, the antigen-binding construct can be delivered intraoperatively as a local administration during an intervention or resection.

[0058] The phrase "specifically (or selectively) binds" when used in the context of describing the interaction of an antigen, such as a protein, with an antibody or an antibody-derived binding agent, refers to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biological agents, for example, in a biological sample such as blood, serum, plasma, or tissue sample. Thus, under the specified immunoassay conditions, in some embodiments, an antibody or binding agent having a particular binding specificity binds at least 2-fold to a particular antigen and substantially does not bind in significant amounts to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, a solid-phase ELISA immunoassay is routinely used to select antibodies that are specifically immunoreactive with a protein (see, for example, Harlow, E. & Lane D., "Using Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1998 for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal that is at least 2-fold, more typically at least 10- to 100-fold greater than the background signal.

[0059] The term "equilibrium dissociation constant (K D , M)" refers to the dissociation rate constant (k a , time -1 M -1 ) divided by the association rate constant (k d , time -1 ). The equilibrium dissociation constant can be measured using any known method in the art. The antibodies of the present invention generally have an equilibrium dissociation constant of less than about 10 -7 or 10 -8 M, for example, less than about 10 -9 M or 10 -10 M, and in some embodiments about 10-11 M, 10 -12 M, or 10 -13 would have an equilibrium dissociation constant of less than M.

[0060] The term "isolated" when applied to a nucleic acid or protein denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in its natural state. In some embodiments, it can be in either a dry or aqueous solution state. Purity and homogeneity can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the major species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode proteins other than the gene of interest. The term "purified" denotes that the nucleic acid or protein gives essentially 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 of the molecules present under in vivo conditions.

[0061] The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequence. Specifically, degenerate codon substitutions can be achieved by substituting the third position of one or more selected (or all) codons with a mixture of bases and / or deoxyinosine residues (Batzer, M. A. 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, G. M. 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).

[0062] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0063] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, for example, an alpha-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0064] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant is such a nucleic acid that encodes the same or essentially the same amino acid sequence or, if the nucleic acid does not encode an amino acid sequence, refers to an essentially the same sequence. Because of 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. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations" which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except for AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Thus, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0065] With respect to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" where the change results in substitution of an amino acid with a chemically similar amino acid. Tables of conserved substitutions providing functionally similar amino acids are well known in the art. Such conservatively modified variants are additional to the polymorphic variants, interspecies homologs, and alleles of the invention and are not to be excluded therefrom.

[0066] 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, T. E., Proteins - Structures and Molecular Properties, W. H. Freeman & Co. Ltd., 1984).

[0067] The term "percentage of sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., the polypeptide of the present invention) that does not include additions or deletions for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences, counting the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.

[0068] The terms "identical" or "identity" percent in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same sequence. When compared and aligned for maximum match over a comparison window or specified region, measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (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 length of the reference sequence). Some embodiments provided herein provide polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides exemplified herein, respectively. Optionally, the identity exists over a region that is at least about 15, 25, or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least about 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. For shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, in some embodiments, substantial identity exists if one or two amino acid residues are conservatively substituted according to conservative substitutions as defined herein.

[0069] In some embodiments, the percent identity is for a portion of the antibody referred to herein (framework region, C H3. and / or over the Fc region). In such situations, the percent identity for a portion of the polypeptide can be determined separately from the remainder of the protein or nucleic acid sequence. Thus, two portions (e.g., two FRs, C H 3. and / or the Fc region) can have a percentage of specified amino acid residues or nucleotides that are the same (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the specified region or, if not specified, over the entire sequence of the reference sequence), while allowing the remainder of the protein to remain 100% identical to the comparison protein or allowing the remainder of the protein to also have variations by the specified percentage of identity.

[0070] For sequence comparison, typically, one sequence serves as the reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and subsequence coordinates are specified if necessary, and the 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.

[0071] As used herein, a "comparison window" includes a reference to a segment of any one number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, after two sequences have been optimally aligned, where the sequences can be compared to a reference sequence at the same number of contiguous positions. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman, S. B. 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, W. R. et al., "Improved tools for biological sequence comparison", Proc. Natl. Acad. Sci. U.S.A., Vol. 85, No. 8, pp. 2444-2448, 1988, by computerized execution 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, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Supplement, 1995).

[0072] Two examples of algorithms suitable for determining percent sequence identity and similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul, S. F. 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, 1997, and Altschul, S. F. et al., "Basic local alignment search tool," J. Mol. Biol., Vol. 215, No. 3, pp. 403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul, S. F. et al., supra). These initial neighborhood word hits serve as seeds to begin a search for longer HSPs that contain them. The word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated using parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hits in each direction stops when the cumulative alignment score drops by an amount X from its maximum achieved value; when the cumulative score becomes less than or equal to zero due to the accumulation of one or more negative-score residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses, as initial settings, an 11-word length (W), an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, as initial settings, a 3-word length, an expectation value (E) of 10, and the BLOSUM62 score matrix (see Henikoff, S. et al., "Amino acid substitution matrices from protein blocks", Proc. Natl. Acad. Sci. U.S.A., Vol. 89, No. 22, pp. 10915-10919, 1992), an alignment (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands.

[0073] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin, S. et al., "Applications and statistics for multiple high-scoring segments in molecular sequences", Proc. Natl. Acad. Sci. U.S.A., Vol. 90, No. 12, pp. 5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the minimum total probability in a comparison of a test nucleic acid with a 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 to be similar to the reference sequence.

[0074] 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 an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, in some embodiments, for example, if two peptides differ only by conservative substitutions, the polypeptides are typically substantially identical to the second polypeptide. 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 sequences can be amplified using the same primers.

[0075] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or arrests cell function and / or causes cell death or destruction. The term is intended to include non-radioactive payloads (e.g., ADCs), radioisotopes (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 radioisotopes of Lu), chemotherapeutic agents (as defined elsewhere herein). Other cytotoxic agents are described below. An antitumor agent causes the destruction of tumor cells.

[0076] "Toxin" refers to any substance that can have a harmful 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 family of auristatin, maytansine, maytansinoid, calicheamicin, duocarymycin, pyrrolobenzodiazepine dimer, and amatoxin. "Therapeutic ion" refers to a charged particle that is useful in the treatment of disorders related to target molecules. Examples of therapeutic ions are 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 Er, 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 treatment options.

[0077] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN (trademark) cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (notably bratasin and bratasinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL (trademark)); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogs topotecan (HYCAMTIN (trademark)), CPT-11 (irinotecan, CAMPTOSAR (trademark)), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (notably cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eribulin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, noburemabicin, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Engine antibiotics (for example, calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11 (see, for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin including dynemicin A; esperamicin; and neocarzinostatin chromophore and related pigment protein engine antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cardifilin, chromomycin, daunorubicin, doxorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rhodomycin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin and other antibiotics; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epitioestanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline;Diaziquone; elfornithine, elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.R (trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; lysocine; schizophyllan; spirigermanium; tenuazonic acid; triaziquone; 2,2',2''-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine (ELDISINE(trademark), FILDESIN(trademark)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as TAXOL.R (trademark) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE(trademark) paclitaxel Cremophor-free albumin-engineered nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE(trademark) docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR(trademark)); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN(trademark)); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN(trademark)); oxaliplatin; leucovovin; vinorelbine (NAVELBINE(trademark)); novantrone; edatrexate; daunomycin; aminopterin; ibandronate: topoisomerase inhibitor RFS 2000;Difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA (trademark)); any pharmaceutically acceptable salt, acid, or derivative thereof; and abbreviations of combination therapies such as CHOP, which is an abbreviation of a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, which is an abbreviation of a treatment regimen using oxaliplatin (ELOXATIN (trademark)) in combination with 5-FU and leucovorin, etc., including combinations of two or more of the above. These are also options for therapeutic agents.;

[0078] The terms "subject", "patient", and "individual" are used interchangeably to refer to the entity being tested and / or treated. This can include, for example, mammals such as humans or non-human primate mammals. Mammals can also be laboratory mammals such as mice, rats, rabbits, hamsters. In some embodiments, the mammal can be an agricultural mammal (e.g., equidae, sheep, bovine, swine, camelidae) or a domestic mammal (e.g., canidae, felidae).

[0079] antibody As discussed herein, provided are antibodies having enhanced in vivo distribution and / or pharmacokinetics, obtained by disruption of one or more clusters of positively charged amino acids exposed on the surface. Provided herein are variant antibodies (e.g., minibodies, cys-diabodies) that include at least one disrupted cluster of positively charged amino acids exposed on the surface, wherein the variant antibody differs from the original antibody that includes an original cluster having at least two (e.g., 2, 3, 4, 5, 6 or more) positively charged amino acids exposed on the surface within about 30 angstroms of each other. The variant antibody can differ from the original antibody by having a substitution of at least one positively charged amino acid exposed on the surface of the original cluster with one or more negatively charged or uncharged amino acids. Substitution of the positively charged amino acids exposed on the surface of the cluster can disrupt the original cluster (e.g., by having fewer positively charged amino acids exposed on the surface at the corresponding position on the variant antibody as compared to the original antibody). In some embodiments, the original or variant antibody is any antibody construct. In some embodiments, this disruption is particularly useful for constructs that are the same size as or smaller than a minibody. In some embodiments, the antibody is a cys-dibody. In some embodiments, the antibody is scFv-Fc (e.g., scFv fused to Fc) or nanobody®-Fc (e.g., camelid nanobody® or single domain fragment fused to Fc).

[0080] The distance between the positively charged amino acids exposed on the surface within the cluster can be the distance between the mass centers 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 exposed on the surface as determined by van der Waals radius, solvent accessible surface area, isopotential surface representation of the electrostatic field calculated by an Adaptive Poisson-Boltzmann Solver, or a combination thereof.

[0081] Generally, a positively charged amino acid is an amino acid that is 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, the positively charged amino acid is selected from arginine, lysine, and histidine. In some embodiments, the positively charged amino acid is selected from arginine and lysine. The amino group at the N-terminus of a polypeptide or protein can also provide a positive charge. Negatively charged 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, the negatively charged amino acid is selected from aspartic acid and glutamic acid. The carboxyl group at the C-terminus of a polypeptide or protein can also provide a negative charge. In some embodiments, the uncharged amino acid is selected from 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.

[0082] The positively charged amino acids exposed on the surface within the cluster can form a continuous patch of positive charge on the surface (e.g., the equipotential electrostatic surface) of the antibody (e.g., the original antibody having the cluster). In some embodiments, the cluster has at least two (e.g., 2, 3, 4, 5, 6 or more) positively charged amino acids exposed on the surface within a distance of about 30, 25, 20, 15, 14, 13, 12, 11, 10, or 5 angstroms or less from each other, or within a region defined by any two of the preceding values (e.g., 30 - 5 angstroms, 30 - 10 angstroms, 20 - 5 angstroms, 15 - 10 angstroms, 12 - 10 angstroms) from each other. In some embodiments, at least two positively charged amino acids exposed on the surface of the cluster are within about 15 angstroms of each other. In some embodiments, when the cluster contains three or more positively charged amino acids exposed on the surface, the distance between the two most distant residues from each other is within about 30 angstroms, e.g., 25, 20, 15, 14, 13, 12, 11, 10, or about 5 angstroms or less, or within a region defined by any two of the preceding values (e.g., 30 - 5 angstroms, 30 - 10 angstroms, 20 - 5 angstroms, 15 - 10 angstroms, 12 - 10 angstroms). In some embodiments, there are no negatively charged amino acids exposed on the intervening surface between at least two (e.g., 2, 3, 4, 5, 6 or more) positively charged amino acids exposed on the surface of the cluster. In some embodiments, there are no negatively charged amino acids exposed on the surface between any two positively charged amino acids exposed on the surface of the cluster.

[0083] A variant antibody comprising at least one disrupted cluster of positively charged amino acids, wherein the variant antibody differs from the original antibody that comprises an original cluster having at least two (e.g., 2, 3, 4, 5, 6 or more) positively charged amino acids within 12 residues of each other (e.g., linearly along the peptide backbone). The variant antibody can differ from the original antibody by having a substitution of at least one positively charged amino acid of the original cluster with a negatively charged or uncharged amino acid, and the cluster is outside of any CDR of the original antibody. The substitution of the positively charged amino acid can disrupt the original cluster (e.g., by having fewer surface-exposed positive charges at the corresponding position on the variant antibody compared to 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 residue of each other. As used herein, two amino acids are within 1 residue of each other if they are directly adjacent to each other in the polypeptide chain, within 2 residues of each other if 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.

[0084] In some embodiments, if the number of positively charged amino acids exposed on the surface is reduced by at least one (e.g., 1, 2, 3 or more) in the variant antibody compared to the original antibody, the original cluster is disrupted. In some embodiments, the variant antibody differs from the original antibody by at most three (e.g., 1, 2, 3) amino acid substitutions (e.g., by negatively charged or uncharged amino acids) that disrupt the cluster. In some embodiments, if the number of positively charged amino acids exposed on the surface is reduced by 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or within a range defined by any two of the preceding values (e.g., 30 - 90%, 30 - 70%, 50 - 100%, 30 - 60%, etc.) in the variant antibody compared to the original antibody, the cluster is disrupted. In some embodiments, if the number of positively charged amino acids exposed on the surface is reduced by about 33% or more in the variant antibody compared to the original antibody, the original cluster is disrupted. In some embodiments, if the number of positively charged amino acids exposed on the surface is reduced by about 66% or more in the variant antibody compared to the original antibody, the original cluster is disrupted. In some embodiments, the variant antibody includes disrupted clusters having a percentage of the total positive charge attributable to the original cluster in the original antibody of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0% or about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0%, or within a range defined by any two of the preceding values (e.g., 0 - 70%, 30 - 70%, 0 - 40%, etc.) due to disruption of the cluster. For example, without limitation, the original antibody may have a cluster of three positively charged amino acids (e.g., lysine and / or arginine), and one of the positively charged amino acids may be substituted by an uncharged or negatively charged amino acid (e.g., glutamine) in the variant antibody, thereby disrupting the original cluster.In some embodiments, the variant antibody comprises disrupted clusters having no more than about 66% of the total positive charge attributable to the clusters in the original antibody due to disruption of the clusters. In some embodiments, the variant antibody comprises disrupted clusters having no more than about 33% of the total positive charge attributable to the clusters in the original antibody due to disruption of the clusters. In some embodiments, the total positive charge attributable to the clusters is determined by use of an algorithm suitable for solving the Poisson-Boltzmann equation, such as the Adaptive Poisson-Boltzmann Solver (APBS).

[0085] 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 option. Suitable options include, without limitation, resolved crystal structures, homology modeling, molecular dynamics simulations, Adaptive Poisson-Boltzmann Solver, van der Waals radii, solvent accessible surface area, or combinations thereof.

[0086] In some embodiments, at least two surface-exposed positively charged amino acids of the clusters in the original antibody are within a continuous stretch of no more than 15 (e.g., 15, 12, 10, 8, 7, 6, 5, 4, 3, or 2) residues in the polypeptide of the original antibody. In some embodiments, at least two surface-exposed positively charged amino acids of the clusters 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 residues apart in the polypeptide of the original antibody, or are a number of residues apart within a region defined by any two of the preceding values (e.g., 20 - 200, 20 - 50, 40 - 75, 30 - 100, 50 - 150, 100 - 200, etc.).

[0087] Substitutions of positively charged amino acids exposed on the surface of the original cluster can be within any suitable non-CDR region of the antibody (e.g., the original antibody). In some embodiments, substitutions of positively charged amino acids exposed on at least one surface of the original cluster are within the variable light chain region (V L ) framework region (FR) of the original antibody. In some embodiments, substitutions of positively charged amino acids exposed on at least one surface of the original cluster are within the V L FR2 of the original antibody. A cluster of positively charged amino acids exposed on the disrupted surface in an antibody, e.g., a variant antibody, can be within any suitable non-CDR region of the antibody (e.g., the original antibody). In some embodiments, the disrupted cluster is within the variable light chain region (V L ) of the antibody (e.g., within the framework region of V L ). In some embodiments, the disrupted cluster is within the V L FR2 of the antibody, e.g., a variant antibody.

[0088] In some embodiments, the original antibody comprises a polypeptide having the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), i.e., the original polypeptide, wherein X1 is a positively charged amino acid, X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid, X4 and X7 are each independently any amino acid provided that at least one of X4 and X7 is a positively charged amino acid, the sequence is outside any CDR of the antibody, and at least one of X4 and X7, which is a positively charged amino acid, is surface-exposed and is part of the original cluster. In some embodiments, the original sequence is within the V LIt is within FR2. In some embodiments, the substitution comprises substitution of 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, and at least one of X4 and X7 which are positively charged amino acids, are part of the original cluster. In some embodiments, all of the 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. An antibody, such as a variant antibody, may comprise the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), and may further have a substitution of at least one positively charged amino acid exposed on at least one surface of the original cluster with a negatively charged or uncharged amino acid that disrupts the original cluster. In some embodiments, an antibody, such as a variant antibody, comprises the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), and a substitution of at least one of X4 and X7 with a negatively charged or uncharged amino acid that disrupts the original cluster. In some embodiments, an antibody, such as a variant antibody, comprises the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), and substitutions of X4 and X7 with negatively charged or uncharged amino acids that disrupt the original cluster. In some embodiments, the original antibody comprises an original polypeptide comprising the sequence QQX1X2X3X4X5X6X7 (SEQ ID NO: 3) comprising the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2) as described herein.

[0089] In some embodiments, the positive charge resulting from the original sequence (X1X2X3X4X5X6X7 (SEQ ID NO: 2)) decreases by a percentage within a range defined by 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or about 100%, or about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or about 100%, or at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or about 100%, or any two of the preceding values (e.g., 30 - 95%, 30 - 40%, 30 - 50%, 30 - 70%, 60 - 70%, 60 - 100%, etc.) in the variant polypeptide. In some embodiments, the positive charge resulting from the original sequence (X1X2X3X4X5X6X7 (SEQ ID NO: 2)) decreases by about 33% or more in the variant polypeptide. In some embodiments, the positive charge resulting from the original sequence (X1X2X3X4X5X6X7 (SEQ ID NO: 2)) decreases by about 66% or more in the variant polypeptide. 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.

[0090] In some embodiments, the original antibody comprises at least one of the following original sequences: 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). In some embodiments, the original sequence is one of 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).

[0091] The original sequence can be within any suitable non-CDR region of the antibody (e.g., the original antibody). In some embodiments, the original sequence is within the variable light chain region (V L ) of the antibody (e.g., within the framework region of V L ). In some embodiments, the original sequence is within V L FR2 of the antibody, e.g., a variant antibody.

[0092] In some embodiments, the negatively charged or uncharged amino acid that replaces at least one surface-exposed positively charged amino acid of the cluster is glutamine (e.g., substitution from K to Q, or from R to Q).

[0093] In some embodiments, the variant antibody is, for example, V LFR2 includes at least one of the following, which contain substitutions of at least one positively charged amino acid: KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID NO: 16), and QQKPGQSPQ (SEQ ID NO: 15). In some embodiments, the variant antibody is, for example, V L FR2 includes at least one of the following, which contain substitutions of at least one positively charged amino acid: KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15). In some embodiments, the variant antibody has the amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19) in its V L and includes FR2.

[0094] An antibody comprising a variant polypeptide that is different from the original polypeptide comprising the original array X1X2X3X4X5X6X7 (array number 1), wherein the original array is outside any CDR of the antibody, and wherein at least two of X1, X2, X3, X4, X5, X6, and X7 are each independently a positively charged amino acid exposed on the surface, forming a cluster of positively charged amino acids exposed on the surface in the original polypeptide, and the variant polypeptide is different from the original polypeptide by having a modification of at least one of at least two positively charged amino acids exposed on the surface of the original array that reduces at least about 33% of the positive charge resulting from the original array. In some embodiments, at least 33% of the positive charge resulting from the original array is reduced in the variant polypeptide compared to the original polypeptide. In some embodiments, about 60% or more of the positive charge resulting from the original array is reduced in the variant polypeptide. In some embodiments, the modification results in a percentage reduction in the positive charge due to the sequence in the variant polypeptide of about 33%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or at least about 33%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, or within a range defined by any two of the preceding values (e.g., 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 array is reduced by one or more (e.g., at least 2, 3, 4, or 5 or more) in the variant polypeptide. In some embodiments, at least one (e.g., at least 2, 3, 4, 5, 6 or more) of at least two positively charged amino acids of the original array is replaced with a negatively charged or uncharged amino acid in the variant polypeptide.

[0095] In some embodiments, at least X4 and X7 are each independently a positively charged amino acid. In some embodiments, the positively charged amino acid is lysine, arginine, or histidine. In some embodiments, X2, X3, X5, and X6 are each independently any negatively charged or uncharged amino acid in the original polypeptide. In some embodiments, X2, X3, X5, and X6 are each independently any uncharged amino acid. In some embodiments, to reduce the positive charge, at least one of X4 and X7 is replaced with a negatively charged or uncharged amino acid. In some embodiments, the negatively charged or uncharged amino acid that replaces at least one of X4 and X7 is glutamine (e.g., a substitution from K to Q, or from R to Q). In some embodiments, the variant polypeptide comprises alanine or serine at a position corresponding to X5 in the original sequence of the original polypeptide.

[0096] In some embodiments, the variant polypeptide comprises the variable light chain region (V L ) of an antibody. V L can comprise three CDRs and four FRs of the light chain. In some embodiments, the variant polypeptide can comprise the V L and the variable heavy chain region (V H ) of an antibody, for example, as an scFv fragment. In some embodiments, the variant polypeptide comprises an scFv fragment. The V L sequence in the polypeptide (e.g., the V L FR sequence) can be based on a human germline sequence. For example, without limitation, the FRs of V L are the corresponding FRs of a human germline sequence that have no substitutions or, independently in each FR, up to 1, 2, 3, 4, or more than 5 substitutions that disrupt a cluster of positively charged residues exposed on the surface. In some embodiments, as described herein, FR2 has up to 1, 2, 3, 4, or 5 substitutions that disrupt a cluster of positively charged residues exposed on the surface, and the other FRs have no substitutions. In some embodiments, V L(For example, V L FR sequence) is based on a human germline belonging to the IGKV1 family. In some embodiments, the FR of V L has no substitutions or, independently for each FR, up to 1, 2, 3, 4, or 5 substitutions thereto that disrupt a cluster of positively charged residues exposed on the surface, and is the corresponding FR of a human germline sequence belonging to the IGKV1 family. In some embodiments, as described herein, FR2 has up to 1, 2, 3, 4, or more than 5 substitutions that disrupt a cluster of positively charged residues exposed on the surface, and the other FRs have no substitutions. In some embodiments, V L (For example, V L FR sequence) is based on the human germline sequence IGKV1-39*01. In some embodiments, the FR of V L has no substitutions or, independently for each FR, up to 1, 2, 3, 4, or more than 5 substitutions thereto that disrupt a cluster of positively charged residues exposed on the surface, and is the corresponding FR of the human germline sequence IGKV1-39*01. In some embodiments, as described herein, FR2 has up to 1, 2, 3, 4, or more than 5 substitutions that disrupt a cluster of positively charged residues exposed on the surface, and the other FRs have no substitutions. In some embodiments, V L (For example, V L FR sequence) is based on a human germline belonging to the IGKV3 family. In some embodiments, the FR of V L has no substitutions or, independently for each FR, up to 1, 2, 3, 4, or more than 5 substitutions thereto that disrupt a cluster of positively charged residues exposed on the surface, and is the corresponding FR of a human germline sequence belonging to the IGKV3 family. In some embodiments, as described herein, FR2 has up to 1, 2, 3, 4, or more than 5 substitutions that disrupt a cluster of positively charged residues exposed on the surface, and the other FRs have no substitutions. In some embodiments, V L (For example, V L FR sequence) is based on the human germline sequence IGKV3-15*01. In some embodiments, the FR of V LThe FRs are the corresponding FRs of the human germline sequence IGKV3-15*01 that have no substitutions or, independently for each FR, have up to 1, 2, 3, 4, or 5 or more substitutions thereto that disrupt clusters of positively charged residues exposed on the surface. In some embodiments, as described herein, FR2 has up to 1, 2, 3, 4, or 5 or more substitutions that disrupt clusters of positively charged residues exposed on the surface, and the other FRs have no substitutions. In some embodiments, V L (e.g., V L FR sequences) are based on human germline sequences from subgroup 1: IGKV1, IGKV1D, from both proximal and distal clusters. In some embodiments, V L (e.g., V LThe FR sequences are based on the human germline sequences from subgroups 2-7 from both the proximal and distal clusters: IGKV2, IGKV3, IGKV4, IGKV5, IGKV6, IGKV7, IGKV2D, IGKV3D, IGKV6D. As used herein, a variable region of an antibody that is "based on" a reference sequence (e.g., a human germline sequence) means that the framework regions of the antibody (e.g., FR1, FR2, FR3, and FR4) have no substitutions or, independently in each FR, have up to 1, 2, 3, 4, or 5 or more substitutions thereto that disrupt the cluster of positively charged residues exposed on the surface and are derived from the corresponding framework regions of the reference sequence (e.g., a human germline sequence). In some embodiments, as described herein, FR2 has up to 1, 2, 3, 4, or 5 or more substitutions that disrupt the cluster of positively charged residues exposed on the surface and the other FRs have no substitutions. In some embodiments, the variable region of an antibody based on a reference sequence (e.g., a human germline sequence) has a framework region that has a framework region that is 80%, 85%, 90%, 95%, or about 100%, about 80%, 85%, 90%, 95%, or about 100%, or at least 80%, 85%, 90%, 95%, or about 100%, or within a range defined by any two of the preceding values (e.g., 80-100%, 85-95%, 90-100%, etc.) percentage identical to the corresponding framework region of the reference sequence (e.g., a human germline sequence). In some embodiments, the variable region of an antibody based on a reference sequence (e.g., a human germline sequence) has a framework region that has up to 8, 7, 6, 5, 4, 3, 2, 1 substitutions thereto or has no substitutions and is derived from the sequence of the corresponding framework region of the reference sequence (e.g., a human germline sequence).

[0097] In some embodiments, the cluster is within the V L framework region (FR) of the antibody. In some embodiments, the cluster is within the V L FR2 of the antibody.

[0098] In some embodiments, the original polypeptide has an original FR2 containing the original sequence and is V L Based on the first human germline sequence of, the variant polypeptide is V L Derived from the second human germline sequence of, different from the original polypeptide by having at least a substitution of the original sequence by the corresponding second sequence derived from the second FR2, the second FR2 corresponds to the original sequence and has at least about 33% fewer positive charges than the original sequence. For example, the V of the antibody of the present disclosure L Has an original V with an FR2 containing a cluster of positively charged amino acids L Can be obtained based on. In a non-limiting example, the original V L Can be obtained based on the germline IGKV1-39*01 having the original sequence KPGKAPK (SEQ ID NO: 5) in FR2. To disrupt the cluster of positively charged amino acids in the original antibody, the major residues in FR2 are replaced with the corresponding residues in the FR2 of the germline IGKV2-28*01 having the original sequence KPGQSPQ (SEQ ID NO: 16). In some embodiments, this involves adding at least 3 substitutions to the original FR2 sequence. In some embodiments, to disrupt the cluster of positively charged amino acids, a partial sequence of FR2 of at least 3, 4, 5, 6, 7 residues or more is substituted. In some embodiments, the entire FR2 is substituted to disrupt the cluster of positively charged amino acids.

[0099] The first human germline sequence can be any suitable human germline sequence having at least two clusters of positively charged amino acids in the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1) in, for example, V FR2 as described above. The second human germline sequence is V L L ​It can be any suitable human germline sequence having a cluster disrupted in FR2. Suitable second human germline sequences include, without limitation, IGKV2-28*01. In some embodiments, the second human germline sequence includes, without limitation, 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.

[0100] In some embodiments, the antibody (e.g., variant antibody) is, for example, V L In FR2, it includes at least one of the following having a substitution of a positively charged amino acid: KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), KPGQSPQ (SEQ ID NO: 16), and QQKPGQSPQ (SEQ ID NO: 15). In some embodiments, the antibody (e.g., variant antibody) is, for example, V L In FR2, it includes at least one of the following having a substitution of a positively charged amino acid: KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15). In some embodiments, the antibody has the amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19) in V L including FR2.

[0101] In some embodiments, the cluster is within the hinge region of the antibody, such as the upper hinge region. In some embodiments, the upper hinge region having the cluster (e.g., in the original antibody without the substitutions or modifications 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).

[0102] In some embodiments, the antibody (e.g., the original antibody and / or variant antibody) has a molecular weight of about 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, or about 110 kDa (e.g., as a dimer of the polypeptide, excluding any non-polypeptide features), or a molecular weight within a range defined by any two of the preceding values (e.g., as a dimer of the polypeptide, excluding any non-polypeptide features). In some embodiments, the antibody has a molecular weight of about 10-110 kDa, such as about 10-90 kDa, about 30-110 kDa, about 40-90 kDa, or about 30-80 kDa (e.g., as a dimer of the polypeptide, excluding any non-polypeptide features). In some embodiments, the antibody has a molecular weight of about 40-60 kDa, about 70-90 kDa, or about 45-85 kDa (e.g., as a dimer of the polypeptide, excluding any non-polypeptide features). In some embodiments, the antibody has a molecular weight of about 110 kDa or less, such as about 90 kDa or less, or about 80 kDa or less (e.g., as a dimer of the polypeptide, excluding any non-polypeptide features).

[0103] In some embodiments, the variant antibody is substantially identical to the original antibody except for the substitution of at least one surface-exposed positively charged amino acid in a cluster with a negatively charged or uncharged amino acid. In some embodiments, the variable regions of the variant antibody (e.g., V L and V H) is substantially identical to the original antibody except for the substitution of positively charged amino acids exposed on at least one surface of the cluster by negatively charged or uncharged amino acids. In some embodiments, the pattern of the isopotential electrostatic surface (e.g., patches of positive and negative charges) is substantially identical to the original antibody except for a decrease in the positive charges exposed on the surface due to disrupted clusters of positively charged amino acids caused by the substitution of positively charged amino acids exposed on at least one surface of the cluster by negatively charged or uncharged amino acids. In some embodiments, the variant antibody differs from the original antibody by at most three (e.g., 1, 2, or 3) amino acid substitutions. In some embodiments, the amino acid substitutions are outside of any CDR of the antibody.

[0104] In some embodiments, the antibody (e.g., including the original antibody, variant antibody) includes an antigen-binding fragment such as an scFv fragment, without limitation. In some embodiments, the antibody is a minibody or a cys-diabody. In some embodiments, the antibody is a minibody. In some embodiments, the antibody is a minibody and has any one of the amino acid sequences (SEQ ID NOs: 49-69) shown in FIG. 12 C H including 3. In some embodiments, the antibody is a cys-diabody.

[0105] A light chain variable region (V having the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1) L)A minibody or cys - diabody comprising a variant polypeptide that is different from the original polypeptide comprising FR2, wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two of them are positively charged amino acids exposed on the surface that form a cluster of positively charged amino acids exposed on the surface in the original polypeptide, and the variant polypeptide has at least one modification of at least one of the at least two positively charged amino acids exposed on the surface of the original sequence, which reduces at least 33% of the positive charge resulting from the original sequence, so that it is different from the original polypeptide. A minibody or cys - diabody is also provided herein. Suitable alternatives to the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1) are as discussed herein.

[0106] In some embodiments, the negatively charged or uncharged amino acid that replaces at least one of X4 and X7 is glutamine (e.g., a substitution from K to Q, or from R to Q). In some embodiments, the variant polypeptide comprises alanine or serine at the position corresponding to X5 in the sequence of the original polypeptide.

[0107] A minibody or cys - diabody comprising a light - chain variable region (V L ) having the FR2 sequence of X1X2X3QAX6X7 (SEQ ID NO: 4), wherein X1 is a positively charged amino acid exposed on the surface, X2, X3, and X6 are each independently any negatively charged or uncharged amino acid, and X7 is either glutamine or lysine. A minibody or cys - diabody is also provided. 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 NO: 14).

[0108] In some embodiments, X1 is lysine. In some embodiments, X2 and / or X6 is proline. In some embodiments, X2 and X6 are each proline. In some embodiments, X3 is glycine or glutamic acid.

[0109] In some embodiments, the minibody or cys-diabody is V L FR2 contains at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15). In some embodiments, the minibody or cys-diabody is 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). In some embodiments, the minibody or cys-diabody has the amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19) and contains V L FR2.

[0110] Antibodies, minibodies, or cys-dibodies containing A49 and the light chain variable region (V L ) containing either (i) Q51; or (ii) either Q48 and K51 (by IMGT numbering) are also provided herein. In some embodiments, V L further contains K45 (by IMGT numbering). In some embodiments, V L further contains G47 (by IMGT numbering). Antibodies, minibodies, or cys-dibodies containing Q43, A49, and the light chain variable region (V L ) containing either (i) Q51; or (ii) either Q48 and K51 (by IMGT numbering) are also provided herein. In some embodiments, V L further contains K45 (by IMGT numbering). In some embodiments, V L further contains G47 (by IMGT numbering).

[0111] Also provided herein are minibodies having the upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39). In some embodiments, the minibody is linked to C H 3 via a hinge region comprising the upper hinge sequence of EPGSSDGTHT and comprises a scFv. In some embodiments, the minibody comprises the hinge sequence of EPGSSDGTHTCPPCPPC (SEQ ID NO: 71). In some embodiments, the minibody is linked to C H 3 via a hinge region comprising EPGSSDGTHTCPPCPPC (SEQ ID NO: 71) and comprises a scFv. In some embodiments, the minibody comprises any one of the V L FR2 sequences having a disrupted cluster of positively charged amino acids described herein.

[0112] In any of the antibodies, minibodies, or cys-diabodies herein, in some embodiments, the antibody, minibody, or cys-dibody (or variant thereof) comprises the light chain variable region (V L ) FR1, FR3, and FR4 from subgroup 1 of both the proximal and distal clusters: human germline sequences derived from IGKV1, IGKV1D. In some embodiments, V L FR1, FR3, and FR4 are each from the corresponding V L FR1, FR3, and FR4 of subgroups 2-7 of both the proximal and distal clusters: human germline-derived IGKV2, IGKV3, IGKV4, IGKV5, IGKV6, IGKV7, IGKV2D, IGKV3D, IGKV6D. In some embodiments, V L FR1, FR3, and FR4 are each from the corresponding V L FR1, FR3, and FR4 of human germline IGKV1-39*01 or IGKV3-15*01. In some embodiments, V L FR1, FR3, and FR4 are each from the corresponding V LIt is derived from FR1, FR3, and FR4. In some embodiments, V L FR1, FR3, and FR4 are each one of the corresponding Vs of any one of the sequences shown in FIGS. 5A-5C and 7A-7D L FR1, FR3, and FR4 are at least 80%, 85%, 90%, 95%, 97%, or about 100% identical, or percentage identical within a range defined by any two of the preceding values (e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.). In some embodiments, an antibody, minibody, or cys-diabody (or variant thereof) has a heavy chain variable region (V H ) framework sequence (FR1, FR2, FR3, and FR4) included in any one of the sequences shown in FIGS. 5A-5C and 7A-7D. In some embodiments, V H FR1, FR2, FR3, and FR4 are each one of the corresponding Vs of any one of the sequences shown in FIGS. 5A-5C and 7A-7D H FR1, FR2, FR3, and FR4 are at least 80%, 85%, 90%, 95%, 97%, or about 100% identical, or percentage identical within a range defined by any two of the preceding values (e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.). In some embodiments, the V L and V H FR sequences of an antibody, minibody, or cys-diabody (or variant thereof) are paired or combined according to any one of the constructs shown in FIGS. 5A-5C and 7A-7D. In some embodiments, the framework region is based on the Kabat definition. In some embodiments, the framework region is based on the AHo definition.

[0113] In some embodiments, an antibody, minibody, or cys-diabody (or variant thereof) has a light chain variable region (V L)It includes FR1. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) includes V of residues 57 to 88 of SEQ ID NO: 42 (excluding the signal peptide). L )It includes FR3. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) includes V of residues 98 to 107 of SEQ ID NO: 42 (excluding the signal peptide). L )It includes FR4. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) includes a light chain variable region (V that includes residues 1 to 23 of SEQ ID NO: 42 (excluding the signal peptide). L )FR1, V that includes residues 57 to 88 of SEQ ID NO: 42 (excluding the signal peptide). L )FR3, and V that includes residues 98 to 107 of SEQ ID NO: 42 (excluding the signal peptide). L )It includes FR4. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) includes a light chain variable region (V that includes residues 1 to 30 of SEQ ID NO: 45). L )It includes FR1. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) includes V of residues 59 to 97 of SEQ ID NO: 45. L )It includes FR3. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) includes V of residues 109 to 119 of SEQ ID NO: 45. L )It includes FR4. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) includes a light chain variable region (V that includes residues 1 to 30 of SEQ ID NO: 45). L )FR1, V that includes residues 59 to 97 of SEQ ID NO: 45. L )FR3, and V that includes residues 109 to 119 of SEQ ID NO: 45. L )It includes FR4.

[0114] In any of the minibodies of the present disclosure, in some embodiments, the minibody has C having any one of the amino acid sequences (SEQ ID NOs: 49 to 69) shown in FIG. 12. HIt includes 3. In some embodiments, the minibody has at least 80%, 85%, 90%, 95%, 97%, or about 100% identity with any one of the amino acid sequences (SEQ ID NOs: 49-69) shown in FIG. 12, or within a range defined by any two of the preceding values (e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.) of percent identity for C H It includes 3.

[0115] An antibody comprising at least one disrupted cluster of positively charged amino acids, wherein the cluster has at least three positively charged amino acids in the framework region (FR) of the original antibody, and the antibody has a substitution of at least one positively charged amino acid of the cluster by a negatively charged or uncharged amino acid, whereby the cluster is disrupted, thereby providing an antibody different from the original antibody having the cluster. In some embodiments, the number of positively charged amino acids is reduced by 1, 2, 3 residues or more, thereby providing a disrupted cluster in the antibody. In some embodiments, the cluster is in the V of the original antibody L It is within FR2. In some embodiments, the antibody is a minibody or a cys-diabody.

[0116] In any of the antibodies, minibodies, or cys-diabodies (or variants thereof) of the present disclosure, in some embodiments, the antibody, minibody, or cys-dibody specifically binds to any suitable antigen target. In some embodiments, the antibody, minibody, or cys-dibody specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA. In some embodiments, the antibody, minibody, or cys-dibody (or variant thereof) comprises a heavy chain having three heavy chain variable region CDR (HCDR) sequences (e.g., HCDR1, HCDR2, HCDR3) of three corresponding HCDRs in any one of the sequences in FIGS. 5B, 5C, 7B-7D. In some embodiments, the antibody, minibody, or cys-dibody (or variant thereof) comprises a light chain variable region having three light chain variable region CDR (LCDR) sequences (e.g., LCDR1, LCDR2, LCDR3) of three corresponding LCDRs in any one of the sequences in FIGS. 5B, 5C, 7B-7D, and a heavy chain having three heavy chain variable region CDR (HCDR) sequences (e.g., HCDR1, HCDR2, HCDR3) of three corresponding HCDRs in any one of the sequences in FIGS. 5B, 5C, 7B-7D. In some embodiments, the antibody, minibody, or cys-dibody (or variant thereof) comprises a V H having the amino acid sequence of V H . In some embodiments, the antibody, minibody, or cys-dibody (or variant thereof) comprises a V L having the amino acid sequence of V L , and a V H having the amino acid sequence of V H . In some embodiments, the antibody, minibody, or cys-dibody (or variant thereof) comprises a V Hand V having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or within a range defined by any two of the preceding values (e.g., 80 - 100%, 80 - 90%, 85 - 99%, 90 - 97%, etc.) percentage identical H is included. In some embodiments, the antibody, minibody, or cys - diabody (or variant thereof) has a V in any one of the sequences in FIGS. 5B, 5C, 7B - 7D L and V having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or within a range defined by any two of the preceding values (e.g., 80 - 100%, 80 - 90%, 85 - 99%, 90 - 97%, etc.) percentage identical L , and a V in any one of the sequences in FIGS. 5B, 5C, 7B - 7D H and V having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or within a range defined by any two of the preceding values (e.g., 80 - 100%, 80 - 90%, 85 - 99%, 90 - 97%, etc.) percentage identical HIt includes. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) has, regardless of the presence or absence of a signal peptide, at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or within a range defined by any two of the preceding values (e.g., 80 - 100%, 80 - 90%, 85 - 99%, 90 - 97%, etc.) percentage identity with any one of the sequences in FIGS. 5B, 5C, 7B - 7D. In some embodiments, the antibody, minibody, or cys-diabody (or variant thereof) has, without a signal peptide, at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or about 100%, or within a range defined by any two of the preceding values (e.g., 80 - 100%, 80 - 90%, 85 - 99%, 90 - 97%, etc.) percentage identity with any one of the sequences in FIGS. 5B, 5C, 7B - 7D.

[0117] In any antibody, minibody, or cys-diabody of the present disclosure, in some embodiments, the antibody, minibody, or cys-diabody does not include SEQ ID NO: 46. In some embodiments, the antibody, minibody, or cys-diabody does not include the V L having the amino acid sequence of L In some embodiments, the antibody, minibody, or cys-diabody does not bind to FAP (e.g., human FAP).

[0118] The antibody, minibody, or cys-diabody can have monovalent, divalent, or multivalent antigen-binding specificities. In some embodiments, the antibody, minibody, or cys-diabody is monovalent. In some embodiments, the antibody, minibody, or cys-diabody is divalent. A bispecific antibody, minibody, or cys-diabody can have two different heavy / light chain pairs and / or it can recognize two different epitopes. In some embodiments, the antibody, minibody, or cys-diabody is multivalent.

[0119] 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., human IgG1 Fc region). In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 76 as set forth below.

[0120]

Chemical formula

[0121] In some embodiments, the Fc region comprises an amino acid sequence that is at least 70, 80, 90, 95, 96, 97, 98, 99, or about 100% identical to SEQ ID NO: 76, or within a range defined by any two of the preceding values (e.g., 80 - 100%, 85 - 97%, 85 - 95%, 90 - 100%, etc.) of percentage identity. 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 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 decrease effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, one or more mutations that decrease FcRn binding are one or more (e.g., 1, 2, or all 3) mutations (e.g., substitutions) among I253, H310, and H435 (EU numbering). In some embodiments, one or more mutations that decrease binding to FcRn are one or more (e.g., 1, 2, or all 3) of I253A, H310A, and H435A (EU numbering). In some embodiments, the Fc region comprises one or more (e.g., 1, 2, or all 3) 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) at 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 comprises N297Q (EU numbering).

[0122] In some embodiments, one or more mutations that reduce Fc effector function are mutations (e.g., substitutions) of any one or more of 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 L234A, L235A, G236R, G237A, P238S, H268A, K322A, L328R, P329G, A330S, P331S. Further 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. January–December 2022;14(1):2123299. doi: 10.1080 / 19420862.2022.2123299. In some embodiments, the Fc region does not contain a C-terminal lysine.

[0123] In some embodiments, the antibodies of the present disclosure are scFv-Fc (e.g., scFv fused to Fc). In some embodiments, the scFv-Fc is a variable light chain (V H ) domain linked to a variable heavy chain (V L) It includes a domain, a hinge domain, and an Fc region. In some embodiments, the antibody of the present disclosure is a nanobody(registered trademark)-Fc (for example, a camelid nanobody (or single domain fragment) fused to Fc).

[0124] Use as an imaging agent In any of the antibodies, minibodies, or cys-diabodies of the present disclosure, in some embodiments, the antibody, minibody, or cys-dibody includes a detectable label. In some embodiments, the antibody, minibody, or cys-dibody includes a radionuclide or an organic dye. In some embodiments, the radionuclide is 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 227 Th, or one or more of them. In some embodiments, exemplary paramagnetic ion substances that can be used as detectable markers include ions of transition and lanthanide metals (e.g., metals having atomic numbers of 6 - 9, 21 - 29, 42, 43, 44, or 57 - 71), but are not limited thereto. 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, detectable labels are radionuclides such as yttrium - 90, lutetium - 177, zirconium - 89, copper - 64, fluorine - 18, gallium - 68, or actinium - 225. Additional embodiments of radionuclides include copper - 67, astatine - 211, lead - 212, bismuth - 212, bismuth - 213, and thorium - 227. In some embodiments, the treatment of target cells with these radionuclides can cause cell damage and cell death in the target tissue.

[0125] In some embodiments, detectable labels are bioluminescent or fluorescent compounds. Examples include fluorescein, fluorescein isothiocyanate (FITC), OREGON GREEN™, rhodamine, Texas red, tetrarhodimine isothiocyanate (TRITC), Cy3, Cy5, etc., fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), self - quenching fluorescent compounds activated by tumor - related proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, or combinations thereof.

[0126] In some embodiments, the organic dye is one of dyes that fluoresce in the near-infrared region, such as indocyanine green (ICG) or IR800.

[0127] In some embodiments, any of the detectable labels, radionuclides, or organic dyes described herein may be conjugated to an antibody, minibody, or cys-dibod. In some embodiments, the antibody, minibody, or cys-dibod further comprises a chelating ligand or is conjugated to a chelating ligand. Examples of chelating ligands that may be used according to the embodiments herein include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), dodecane tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane,1-glutaric acid-5,7 acetic acid (NODAGA), 4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazonane-1-yl-acetic acid (NETA), deferoxamine (Df, which may also be referred to as DFO), porphyrin, polyamine, crown ether, bis-thiosemicarbazone, polyoxime, and similar groups.

[0128] Also provided herein are compositions comprising any of the antibodies, minibodies, or cys-dibods of the present disclosure; and a pharmaceutically acceptable carrier.

[0129] Use as a therapeutic agent In some embodiments, any of the antibodies, minibodies, or cys-diabodies of the present disclosure can be included in a therapeutic composition or a pharmaceutical composition for use in treating a subject in need thereof, e.g., treating a disease or medical condition. In some embodiments, the therapeutic composition is for use in treating cancer. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable buffer.

[0130] In some embodiments, an antibody, minibody, or cys-dibody, or a variant thereof, described herein is conjugated to a therapeutic agent. As used herein, a “therapeutic agent” is an atom, molecule, or compound that is useful in treating a disorder associated with a target molecule. Examples of therapeutic agents include, but are not limited to, drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes (e.g., enzymes that cleave prodrugs to cytotoxic agents at the site of antigen-binding construct binding), 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 those associated with one or more target molecules.

[0131] In some embodiments, the antibodies, minibodies, or cys-diabodies, or variants thereof described herein are conjugated to a therapeutic agent. Although minibodies, cys-diabodies, or other antigen-binding fragments may have a shorter circulatory half-life compared to full-length antibodies, in some embodiments, these formats may exhibit improved tumor penetration based on their smaller size and may be therapeutically effective when appropriately equipped with a cytotoxic agent or a radioisotope. In some embodiments, antibody, minibody, or cys-dibody drug-conjugation techniques may be employed. In some embodiments, the therapeutic approach may include radioimmunotherapy by attaching a suitable radiolabel such as iodine-131, a beta emitter or an alpha emitter, e.g., yttrium-90, lutetium-177, copper-67, terbium-149, terbium-161, astatine-211, lead-212, bismuth-212, actinium-225, bismuth-213, and thorium-227, etc., which can cause cell damage and cell death to the target tissue. In some embodiments, the radiolabel includes terbium-149, terbium-161, or lead-212. In some embodiments, treatment with these fragments equipped with a cytotoxic agent or a radionuclide results in less non-specific toxicity as they will be cleared from the body more rapidly.

[0132] In some embodiments, the label and / or therapeutic agent is 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 Er, 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.

[0133] In some embodiments, the antibodies, minibodies, and / or cys-diabodies, or variants thereof described herein are conjugated to a therapeutic agent such as a chemotherapeutic agent. Chemotherapeutic agents are often inherently cytotoxic or cytostatic and can include alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, hormonal therapies, targeted therapeutics, and immunotherapeutics. In some embodiments, chemotherapeutic agents that can be used as detectable markers according to the embodiments of the present disclosure are 13-cis-retinoic acid, 2-chlorodeoxyadenosine, 5-azacitidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin-D, doxorubicin, aldesleukin, alemtuzumab, alitretinoin, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacitidine, bacillus 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 alpha, dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexazone, dexrazoxane, dactinomycin, daunorubicin, dacarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epoetin alpha, 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, hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab,Interferon alpha, interleukin-2, interleukin-11, isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronic acid, pemetrexed, panitumumab, PEG interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, trimethotrexate, alrubicin, vincristine, vinblastine, vindestine, vinorelbine, vorinostat, or zoledronic acid, including, but not limited to.

[0134] In some embodiments, the antibodies, minibodies, and / or cys-diabodies, or variants thereof described herein are conjugated to a cytotoxic agent (e.g., 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, duocarmycin and its analogs, tubulysin D, basillistatin, ricin, abrin, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0135] Any of the antibodies, minibodies, or cys-diabodies, or variants thereof described herein can be further conjugated to one or more additional therapeutic agents, detectable markers, nanoparticles, carriers, or combinations thereof. For example, the antigen-binding construct can be radiolabeled with iodine-131 and conjugated to a lipid carrier, whereby the anti-target molecule-lipid conjugate forms micelles. The micelles can incorporate one or more therapeutic or detectable markers.

[0136] The pharmaceutical or therapeutic 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, aerosol, enteral, nasal, 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 generally associated with injection, including, but not limited to, suborbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intralung, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, subarachnoid, subdural, sublingual, subcutaneous, transmucosal, or transtracheal. In some embodiments, the antigen-binding construct can be delivered intraoperative as a local administration during an intervention or resection.

[0137] Nucleic acids, expression vectors, host cells Nucleic acids encoding the antibodies of the present disclosure are also provided herein. When used herein in connection with antibodies, minibodies, or cys-diabodies that include monomeric chains of dimers, "encoding an antibody, minibody, or cys-dibody" is intended to encode at least one monomer of the antibody, minibody, or cys-dibody, and the nucleic acid encoding the monomeric chain can, when expressed under appropriate conditions, generate a dimeric antibody, minibody, or cys-dibody. In some embodiments, the nucleic acid encodes a variant polypeptide of the present disclosure.

[0138] In some embodiments, provided is a nucleic acid encoding any of the antibodies, minibodies, or cys-diabodies disclosed herein. In some embodiments, provided is a nucleic acid encoding any of the variant polypeptides disclosed herein. In some embodiments, provided is an expression vector comprising these nucleic acid sequences. In some embodiments, the expression vector comprises the pcDNA3.1(trademark) / myc-His(-) version A vector (Invitrogen, Inc.) for mammalian expression or a variant thereof. The pcDNA3.1 expression vector features both a CMV promoter for mammalian expression and both mammalian (neomycin) and bacterial (ampicillin) selection markers. In some embodiments, the expression vector comprises a plasmid. In some embodiments, the vector comprises a viral vector, such as a retroviral or adenoviral vector. In embodiments, the vector comprises a cosmid, YAC, or BAC.

[0139] Also provided are host cells, such as genetically engineered host cells, that produce any of the antibodies, minibodies, or cys-diabodies described herein. The host cells can be mammalian cells, such as HEK293, 293f, Epxi293 cell lines, or CHO-K1 cell lines. In some embodiments, one or more of a variety of mammalian or non-mammalian expression systems, including but not limited to mammalian expression systems (e.g., CHO-K1 cells), bacterial expression systems (e.g., E. coli, B. subtilis), yeast expression systems (e.g., Pichia, S. cerevisiae), or any other known expression systems, are used to produce the antibodies, minibodies, or cys-diabodies disclosed herein. Other systems can include insect cells and / or plant cells.

[0140] Method Methods are also provided for enhancing the biodistribution and / or pharmacokinetics of an antibody (e.g., a minibody, a cys-diabody) having a cluster of positively charged amino acids exposed on the surface by disrupting the cluster. Referring to FIG. 1, methods are provided for enhancing the biodistribution and / or pharmacokinetics of an antibody, such as a variant antibody. Method 100 includes, in block 110, identifying an original antibody having a polypeptide that includes at least one cluster of positively charged amino acids exposed on the surface, wherein the cluster includes at least two (e.g., 2, 3, 4, 5, 6 or more) positively charged amino acids exposed on the surface within 30 angstroms of each other. The original antibody can be identified using any suitable option. In some embodiments, an adaptive Poisson-Boltzmann solver, van der Waals radius, solvent accessible surface area, or a combination thereof is used to identify an antibody having at least one cluster of positively charged amino acids exposed on the surface. In some embodiments, an antibody having at least one cluster of positively charged amino acids exposed on the surface is identified by analyzing the isopotential electrostatic surface of the antibody and calculating the linear distance between the mass centers of the charged amino acid residues in a suitable model of the antibody (e.g., modeled using an adaptive Poisson-Boltzmann solver). The method can further include, in block 120, substituting at least one of the at least two positively charged amino acids exposed on the surface of at least one cluster with a negatively charged or uncharged amino acid, thereby disrupting at least one cluster. A variant antibody having a disrupted cluster of positively charged amino acids exposed on the surface can have enhanced biodistribution and / or pharmacokinetics compared to the original antibody. The original antibody can include any suitable cluster of positively charged amino acids exposed on the surface disclosed herein.

[0141] Referring to Figure 2, a method for enhancing the in vivo distribution and / or pharmacokinetics of an antibody is provided. Method 200 may include, in block 210, identifying an original antibody comprising a polypeptide that includes a cluster of at least two surface-exposed positively charged amino acids within 12 residues of each other. The method may further include, in block 220, substituting 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 disrupting at least one cluster. A variant antibody having a disrupted cluster of surface-exposed positively charged amino acids may enhance in vivo distribution and / or pharmacokinetics as compared to the original antibody. In some embodiments, the cluster includes at least two positively charged amino acids within 6 residues of each other. In some embodiments, the cluster includes at least two positively charged amino acids within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue of each other.

[0142] In some embodiments, the polypeptide comprises the 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 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 substituted with a negatively charged or uncharged amino acid. In some embodiments, each of X4 and X7 is independently substituted with an uncharged amino acid. In some embodiments, at least two surface-exposed positively charged amino acids comprise arginine, lysine, and / or histidine.

[0143] In some embodiments, the negatively charged or uncharged amino acid that replaces at least one of the positively charged amino acids exposed on at least two surfaces includes a polar amino acid residue. In some embodiments, the polar amino acid is selected from asparagine, glutamine, serine, threonine, and cysteine. In some embodiments, the polar amino acid is selected from asparagine, glutamine, serine, and threonine. In some embodiments, the negatively charged or uncharged amino acid that replaces at least one of the positively charged amino acids exposed on at least two surfaces is glutamine. In some embodiments, the substituted positively charged amino acids of the cluster, and the negatively charged or uncharged amino acids have similar side chain lengths. In some embodiments, the substituted positively charged amino acids of the cluster, and the negatively charged or uncharged amino acids that replace the positively charged amino acids of the cluster have side chain lengths that differ by one or less than two carbons.

[0144] In some embodiments, the method further comprises replacing up to three (e.g., 1, 2, or 3) amino acids of the polypeptide to disrupt at least one cluster. In some embodiments, the amino acid substitution is outside of any CDR of the antibody.

[0145] The cluster of positively charged amino acids is in the original antibody described herein. In some embodiments, the cluster is within the variable framework region (FR) of the original antibody. L In some embodiments, the cluster is within FR2 of the original antibody. L In some embodiments, the polypeptide having the cluster has the original FR2 containing the cluster based on the first human germline sequence of V L Based on which, the method comprises replacing the original FR2 containing the cluster or a portion thereof with V LA step of substituting with a second FR2 or a corresponding part thereof derived from a second human germline sequence, wherein the second FR2 contains at least one less positively charged amino acid exposed on the surface as compared to the cluster, the step is included. 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), etc.) is modified by one or more amino acid substitutions to match the corresponding stretch of amino acids in the second human germline sequence. In a non-limiting example, the original V L can be based on the germline IGKV1-39*01 which has the sequence KPGKAPK (SEQ ID NO: 5) in FR2. The method can include a step of substituting the major residues in the original FR2 with the corresponding residues in the FR2 of the germline IGKV2-28*01 which has the sequence KPGQSPQ (SEQ ID NO: 16) to disrupt the cluster of positively charged amino acids in the original antibody. This involves adding at least 3 substitutions to the original FR2 sequence. In some embodiments, to disrupt the cluster of positively charged amino acids, a partial sequence of FR2 of at least 3, 4, 5, 6 residues or more is substituted. In some embodiments, the entire FR2 is substituted to disrupt the cluster of positively charged amino acids.

[0146] In some embodiments, the method further includes a step of conjugating a chelating ligand to the variant antibody. Any suitable chelating ligand such as those disclosed herein but not limited thereto can be conjugated to the variant antibody. In some embodiments, the method includes a step of labeling the antibody with a radionuclide such as those disclosed herein but not limited thereto. In some embodiments, the method includes a step of labeling the antibody with a detectable label such as those disclosed herein but not limited thereto.

[0147] Referring to FIG. 3, a non-limiting method of making a labeled antibody is provided. Method 300 has, in block 310, the variable light chain region (V L), where the germline sequence does not contain a cluster of at least two (e.g., two or three) positively charged amino acids within three residues of each other in framework region 2 (FR2) of the germline sequence. The germline sequence may be selected using any suitable option. In some embodiments, the germline sequence is a human V L Selected by searching a database of germline sequences. In some embodiments, the germline sequence does not contain 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.

[0148] The method 300 includes, at block 320, selecting a V L and having variation in germline sequences across the population, wherein the one or more target specific antibodies comprise V L The method may further include not including a cluster of at least two positively charged amino acids within 3 residues of each other in the FR2 sequence.The method may further include, at block 330, labeling the one or more target-specific antibodies.

[0149] One or more target-specific antibodies can be isolated using any suitable option. In some embodiments, the step of isolating one or more target-specific antibodies includes screening a population of antibodies derived from germline sequences. In some embodiments, the population of antibodies derived from germline sequences is generated in a host organism that has been genetically modified to express antibodies based only on the selected germline sequences (e.g., a mouse that has been genetically modified to generate antibody clones based on humanized or human germline sequences). In some embodiments, the population of antibodies derived from germline sequences is part of a phage library of antibodies based only on the selected germline sequences. In some embodiments, the germline sequence is a human germline sequence. In some embodiments, one or more target-specific antibodies are humanized.

[0150] In some embodiments, the antibody includes an antigen-binding fragment (e.g., an scFv fragment). The generated target-specific antibody can be reformatted into a suitable antibody format (e.g., a minibody or cys-diabody format). In some embodiments, the method includes the step of generating one or more target-specific minibodies or cys-dibodies from one or more target-specific antibodies.

[0151] In some embodiments, the label is a radionuclide including, but not limited to, those disclosed herein. In some embodiments, the step of labeling one or more target-specific antibodies (including target-specific minibodies or cys-dibodies) includes conjugating a chelating ligand to the one or more target-specific antibodies (or minibodies or cys-dibodies).

[0152] The antibodies of the present disclosure (e.g., minibodies, cys-diabodies) can be produced using any suitable option. In some embodiments, any method of enhancing the antibodies of the present disclosure includes the step of producing an antibody, such as a variant antibody. In some embodiments, the antibody (e.g., minibody, cys-dibody) is produced by expressing a nucleic acid encoding the antibody (e.g., encoding the monomeric chain of the antibody) in a suitable host cell expression system (e.g., mammalian cell line, bacteria, insect cell line, yeast, etc.).

[0153] Also provided herein are methods of treating a subject using the antibodies, minibodies, or cys-dibodies of the present disclosure. Referring to Figure 4, non-limiting methods of treating a subject are provided. Method 400 may include, at block 410, identifying a subject in need of treatment, such as for cancer, using an antibody, minibody, or cys-dibody of the present disclosure, or a variant thereof, and, at block 420, administering a therapeutically effective amount of the antibody, minibody, or cys-dibody to the subject. In some embodiments, the method includes administering a therapeutically effective amount of a composition containing an antibody, minibody, or cys-dibody of the present disclosure, such as a pharmaceutical composition, to the subject. In some embodiments, the method is a method of radiotherapy that includes identifying a subject in need of radiotherapy (e.g., a subject having cancer) and labeling the antibody, minibody, or cys-dibody administered to the subject with a radionuclide. In some embodiments, the subject has cancer. In some embodiments, the subject has cancer or other medical conditions associated with an antigen to which the antibody, minibody, or cys-dibody specifically binds. In some embodiments, using the antibodies, minibodies, or cys-dibodies of the present disclosure, a higher dose of radiation can be administered compared to the original antibody, minibody, or cys-dibody.

[0154] Also provided is a method of radioimmunotherapy. The method may include identifying a subject in need of radioimmunotherapy and administering a therapeutically effective amount of an antibody, minibody, or cys-dibody of the present disclosure, or a variant thereof, to the subject.

[0155] Methods for treating a subject with cancer are also provided. The methods can include identifying a subject in need of treatment for cancer; and administering to the subject a therapeutically effective amount of any of the antibodies, minibodies, or cys-diabodies, or variants thereof of the present disclosure, 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 (DIPG), thyroid cancer, squamous cell carcinoma of the head and neck (SCCHN), adenocarcinoma of the lung, vulvar cancer (squamous cell carcinoma), bladder cancer, squamous cell carcinoma of the cervix, 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, biliary tract cancer, gastrointestinal stromal tumor (GIST), mesothelioma, biphasic mesothelioma, malignant pleural mesothelioma, kidney cancer, myelodysplastic syndrome, hepatocellular carcinoma of the liver, esophageal and esophagogastric junction cancer, extrahepatic bile duct adenocarcinoma, small intestinal malignancy, gastric adenocarcinoma, cholangiocarcinoma, intrahepatic and extrahepatic bile duct cancer, ovarian surface epithelial cancer, non-epithelial and epithelial ovarian cancer, breast cancer, triple negative breast cancer, endometrial cancer, uterine sarcoma, bone cancer, colorectal adenocarcinoma, prostate adenocarcinoma, hormone refractory prostate cancer (PC), neuroendocrine prostate cancer (NEPC), neuroendocrine tumor, solid tumor, follicular lymphoma, Kaposi sarcoma, carcinoma of the urogenital tract, fallopian tube cancer, malignant glioma, Waldenström macroglobulinemia, Richter syndrome, refractory splenic marginal zone lymphoma, refractory small lymphocyte lymphoma, refractory nodal marginal zone lymphoma, refractory lymphoplasmacytic lymphoma, refractory extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue, refractory chronic lymphocytic leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B cell lymphoma, nasopharyngeal cancer, gastroesophageal junction adenocarcinoma, renal cell carcinoma, colon cancer, transitional cell carcinoma (TCC), urothelial cancer (UCC), glioblastoma multiforme (GBM), gallbladder cancer, and Merkel cell carcinoma.

[0156] In some embodiments, the cancer is prostate cancer, lung cancer, melanoma, breast malignancy, CNS and brain malignancy, skin malignancy, occult primary tumor, kidney cancer, gastrointestinal malignancy, ovarian neoplasm, renal cancer, biliary cancer, bladder cancer, esophageal neoplasm, cervical cancer, solid tumor, head and neck cancer, urogenital neoplasm, germ cell tumor, testicular cancer, pancreatic cancer, glioma, liver cancer, bone malignancy, colorectal cancer, thyroid cancer, thoracic and respiratory tumor, lymphoma, male and female urogenital malignancy, bile duct cancer, hematological malignancy, multiple myeloma, gallbladder cancer, endocrine tumor, eye cancer, and tumors of hematopoietic and lymphoid tissue.

[0157] In some embodiments, the method is a method of imaging a subject in which an antibody, a minibody, or a cys-diabody is detectably labeled, the method further comprising imaging the subject to detect the labeled antibody, minibody, or cys-diabody in the subject. The subject can be imaged using any suitable option for detecting a detectable label. In some embodiments, the imaging includes positron emission tomography (PET), computed tomography (CT), single photon emission computed tomography (SPECT), magnetic resonance imaging (NMR), or detection of fluorescence emission. In some embodiments, the detection can be via near-infrared (NIR) imaging and / or Cerenkov luminescence imaging.

[0158] In some embodiments, the antibody, minibody, or cys-diabody specifically binds to an antigen target such as, but not limited to, those disclosed herein.

[0159] Additional embodiments of the disclosure are provided in the following numbered configurations. 1. A variant antibody comprising at least one disrupted cluster of positively charged amino acids exposed on the surface, having a substitution of at least one positively charged amino acid exposed on the surface of the original cluster by a negatively charged or uncharged amino acid, and differing from the original antibody comprising the original cluster containing at least two positively charged amino acids exposed on the surface within about 30 angstroms of each other. 2. The variant antibody according to Configuration 1, wherein at least two positively charged amino acids exposed on the surface of the original cluster are within about 15 angstroms of each other. 3. The variant antibody according to Configuration 1 or 2, having at least about 10% less total positive charge due to the cluster compared to the original antibody due to disruption of the cluster. 4. The variant antibody according to any one of Configurations 1 to 3, differing from the original antibody by only up to three amino acid substitutions that disrupt the cluster. 5. The substitution of at least one positively charged amino acid exposed on the surface of the original cluster is within the light chain variable region (V L ) framework region (FR) of the original antibody. The variant antibody according to any one of Configurations 1 to 4. 6. The substitution of at least one positively charged amino acid exposed on the surface of the original cluster is within V L FR2 of the original antibody. The variant antibody according to Configuration 5. 7. The original antibody comprises a polypeptide containing the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2), wherein X1 is a positively charged amino acid, X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid, X4 and X7 are each independently any amino acid provided that at least one of them is a positively charged amino acid, the original sequence is outside any CDR of the antibody, at least one of X4 and X7 which are positively charged amino acids is surface-exposed, and is part of the original cluster. The variant antibody according to any one of Configurations 1 to 6. 8. The variant antibody according to Configuration 7, wherein the positive charge resulting from the original array is reduced by at least about 33% in the variant antibody. 9. The variant antibody according to Configuration 8, wherein the positive charge resulting from the original array is reduced by at least about 60% in the variant antibody. 10. The variant antibody according to any one of Configurations 7 to 9, wherein the substitution includes substitution of at least one of X4 and X7 with a negatively charged or uncharged amino acid. 11. The variant antibody according to any one of Configurations 7 to 10, wherein the polypeptide includes QQX1X2X3X4X5X6X7 (SEQ ID NO: 3) including the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 2). 12. The variant antibody according to any one of Configurations 7 to 11, wherein X7 is a positively charged amino acid. 13. The variant antibody according to any one of Configurations 7 to 12, wherein X4 is a positively charged amino acid. 14. The variant antibody according to any one of Configurations 7 to 13, wherein the positively charged amino acid is lysine, arginine, or histidine. 15. The variant antibody according to Configuration 14, wherein the positively charged amino acid is lysine or arginine. 16. The variant antibody according to any one of Configurations 7 to 13, wherein X2 and / or X6 is proline. 17. The variant antibody according to any one of Configurations 7 to 14, wherein X3 is glycine or glutamic acid. 18. The variant antibody according to any one of Configurations 7 to 15, wherein X5 is alanine, valine, serine, or proline. 19. The original sequence is one of: 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), the variant antibody according to any one of Configurations 7 to 16. 20. The original sequence is one of: 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), the variant antibody according to any one of Configurations 7 to 17. 21. The original sequence is KPGKAPK (SEQ ID NO: 5) or KPGQAPR (SEQ ID NO: 6), the variant antibody according to Configuration 18. 22. The negatively charged or uncharged amino acid that replaces a positively charged amino acid exposed on at least one surface of the cluster is glutamine, the variant antibody according to any one of Configurations 7 to 19. 23. The original sequence is within the light chain variable region (V L ) framework region (FR) of the original antibody, the variant antibody according to any one of Configurations 7 to 22. 24. The original sequence is within the V L FR2 of the original antibody, the variant antibody according to Configuration 23. 25. The 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), the variant antibody according to any one of Configurations 1 to 24. 26. A V having the amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19). L A variant antibody according to any one of Configurations 1 to 25, comprising FR2. 27. A variant antibody according to any one of Configurations 1 to 26, wherein the substitution of positively charged amino acids exposed on at least one surface of the original cluster is within the hinge region of the original antibody. 28. A variant antibody according to Configuration 27, wherein the substitution of positively charged amino acids exposed on at least one surface of the original cluster is within the upper hinge region of the original antibody. 29. An antibody comprising a variant polypeptide different from the original polypeptide comprising the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein the original sequence is outside any CDR of the original polypeptide, and wherein X1, X2, X3, X4, X5, X6, and X7 are each independently any amino acid, provided that at least two of them are positively charged amino acids exposed on the surface that form a cluster of positively charged amino acids exposed on the surface in the original polypeptide. The variant polypeptide has at least one modification of at least two positively charged amino acids exposed on the surface of the original sequence, which reduces at least 33% of the positive charge resulting from the original sequence, and is different from the original polypeptide, an antibody. 30. The antibody according to Configuration 29, wherein the positive charge resulting from the original sequence is reduced by about 60% or more in the variant polypeptide. 31. The antibody according to Configuration 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 according to any one of Configurations 29 to 31, wherein at least one of at least two positively charged amino acids of the original sequence is substituted with a negatively charged or uncharged amino acid in the variant polypeptide. 33. The antibody according to any one of Configurations 29 to 32, wherein at least X4 and X7 are each independently a positively charged amino acid. 34. The antibody according to any one of Configurations 29 to 33, wherein the positively charged amino acid is lysine, arginine, or histidine. 35. The antibody according to any one of Configurations 29 to 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 Configurations 29 to 35, wherein at least one of X4 and X7 is substituted with a negatively charged or uncharged amino acid to reduce the positive charge. 37. The antibody according to Configuration 36, wherein the negatively charged or uncharged amino acid substituting at least one of X4 and X7 is glutamine. 38. The antibody according to any one of Configurations 29 to 37, wherein the variant polypeptide contains alanine or serine at the position corresponding to X5 in the sequence of the original polypeptide. 39. The antibody according to any one of Configurations 29 to 38, wherein the variant polypeptide contains the variable region of the light chain of the antibody (V L ). 40. The antibody according to any one of Configurations 29 to 39, wherein the original sequence is within the V L framework region (FR) of the original polypeptide. 41. The antibody according to Configuration 40, wherein the original sequence is within the V L FR2 of the original polypeptide. 42. The original polypeptide has the original FR2 containing the original sequence in V L based on the first human germline sequence, and the variant polypeptide is different from the original polypeptide by having at least a substitution of the original sequence with a corresponding second sequence from the second FR2 derived from the second human germline sequence of V L . The second FR2 contains a second sequence corresponding to the original sequence and having at least 10% less positive charge resulting from comparison with the original sequence. The antibody according to Configuration 40 or 41. 43. A variant antibody comprising at least one disrupted cluster of positively charged amino acids, wherein the variant antibody has a substitution of at least one positively charged amino acid of the original cluster by a negatively charged or uncharged amino acid, such that, unlike the original antibody comprising the original cluster that contains at least two positively charged amino acids within 12 residues of each other, the original cluster is outside any CDR of the original antibody, the variant antibody. 44. The variant antibody according to composition 43, wherein the original cluster is within the variable region of the light chain (V L )FR of the original antibody. 45. The variant antibody according to composition 44, wherein the original cluster is within V L FR2 of the original antibody. 46. The variant antibody according to composition 45, comprising at least one of the following substitutions of at least one positively charged amino acid: at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15). 47. A V L FR2 comprising the amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19), the variant antibody according to composition 45. 48. The variant antibody according to composition 43, wherein the original cluster is within the hinge region of the original antibody. 49. The variant antibody according to composition 48, wherein the original cluster is within the upper hinge region of the original antibody. 50. The variant antibody according to composition 48 or 49, wherein the original antibody comprises the upper hinge sequence of EPKSSDKTHT (SEQ ID NO: 38). 51. The antibody according to any one of compositions 48 to 50, wherein the variant antibody comprises the upper hinge sequence of EPGSSDGTHT (SEQ ID NO: 39). 52. The antibody according to any one of compositions 1 to 51, comprising an antigen-binding fragment. 53. The antibody according to any one of compositions 1 to 52, wherein the molecular weight of the antibody is in the range of about 15 kDa to about 110 kDa. 54. An antibody according to any one of Configurations 1 to 53, which is a minibody, cys-diabody, scFv, scFv-Fc, or nanobody-Fc. 55. An antibody according to any one of Configurations 1 to 54, wherein the molecular weight of the antibody is in the range of about 10 kDa to about 20 kDa. 56. An antibody according to Configuration 55, which is a nanobody. 57. An antibody according to any one of Configurations 1 to 54, wherein the molecular weight of the antibody is greater than 90 kDa. 58. An antibody according to Configuration 57, which is scFv-Fc. 59. A minibody or cys-diabody comprising a variant polypeptide different from the original polypeptide containing the original 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 of them are positively charged amino acids exposed on the surface forming a cluster of positively charged amino acids exposed on the surface in the original polypeptide. The variant polypeptide is different from the original polypeptide by having at least one modification of at least two positively charged amino acids exposed on the surface of the original sequence, which reduces at least 33% of the positive charge resulting from the original sequence, a minibody or cys-diabody. 60. The minibody or cys-diabody according to Configuration 59, wherein at least X4 and X7 are each independently positively charged amino acids in the original polypeptide. 61. The minibody or cys-diabody according to Configuration 59 or 60, wherein X2, X3, X5, and X6 are each independently negatively charged or uncharged amino acids in the original polypeptide. 62. The minibody or cys-diabody according to any one of Configurations 59 to 61, wherein at least one of X4 and X7 is substituted with a negatively charged or uncharged amino acid to reduce the positive charge. A light chain variable region (V L ) containing an FR2 sequence including 63.X1X2X3QAX6X7 (sequence number 4), wherein X1 is a positively charged amino acid exposed on the surface, X2, X3, and X6 are each independently any negatively charged or uncharged amino acid, and X7 is either glutamine or lysine, a minibody or cys-diabody. 64. The minibody or cys-diabody according to any one of configurations 59 to 63, wherein X1 is lysine. 65. The minibody or cys-diabody according to any one of configurations 59 to 64, wherein X2 and / or X6 is proline. 66. The minibody or cys-diabody according to any one of configurations 59 to 65, wherein X3 is glycine or glutamic acid. 67. The minibody or cys-diabody according to configuration 63, wherein the FR2 sequence includes KPGQAPK (sequence number 13) or KPGQAPQ (sequence number 14). 68. A49, and (i) Q51; or (ii) Q48 and K51 A minibody or cys-diabody containing a light chain variable region (V L ) according to any of the above (by IMGT numbering). 69. V L The minibody or cys-diabody according to configuration 61, further containing K45 (by IMGT numbering). 70. A minibody containing an upper hinge sequence of EPGSSDGTHT (sequence number 39). 71. An antibody, minibody, or cys-diabody according to any one of configurations 1 to 70, specifically binding to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA. 72. A light chain variable region containing three LCDR sequences in any one of the sequences in FIG. 5B, FIG. 5C, FIGS. 7B to 7D, and a heavy chain variable region (VH ) including the antibody, minibody, or cys-diabody according to any one of Configurations 1 to 71. 73. The antibody, minibody, or cys-diabody according to any one of Configurations 1 to 72, further comprising a detectable label. 74. The antibody, minibody, or cys-diabody according to Configuration 73, labeled with a radionuclide or an organic dye. 75. The antibody, minibody, or cys-diabody according to any one of Configurations 1 to 72, further comprising a therapeutic agent. 76. The antibody, minibody, or cys-diabody according to Configuration 75, wherein the therapeutic agent is a cytotoxic agent. 77. The antibody, minibody, or cys-diabody according to Configuration 75, wherein the therapeutic agent is a radionuclide. 78. The radionuclide is 212 Pb, 149 Tb, and 161 selected from among Tb, the antibody, minibody, or cys-diabody according to Configuration 75. 79. The antibody, minibody, or cys-diabody according to any one of Configurations 1 to 78; and a pharmaceutically acceptable carrier comprising a composition. 80. A nucleic acid encoding a variant polypeptide of the antibody according to any one of Configurations 29 to 42, or the minibody or cys-diabody according to any one of Configurations 59 to 62. 81. A nucleic acid encoding a variant antibody according to any one of Configurations 1 to 28 and 43 to 58, the antibody according to any one of Configurations 29 to 42, or the minibody or cys-diabody according to any one of Configurations 59 to 72. 82. A genetically engineered host cell comprising the nucleic acid according to Configuration 80 or 81. 83. A step of identifying the original antibody comprising a polypeptide comprising at least one cluster of positively charged amino acids exposed on the surface, wherein the cluster comprises at least two positively charged amino acids exposed on the surface within 30 angstroms of each other; A step of substituting at least one of the positively charged amino acids exposed on at least two surfaces 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 in vivo distribution and / or pharmacokinetics as compared to the original antibody, A method for enhancing the in vivo distribution and / or pharmacokinetics of an antibody, comprising the above steps. A step of identifying an original antibody comprising a polypeptide containing at least two clusters of positively charged amino acids exposed on at least two surfaces within 12 residues of each other; and A step of substituting at least one of the positively charged amino acids exposed on at least two surfaces of the cluster with a negatively charged or uncharged amino acid to destroy the cluster, thereby generating a variant antibody having enhanced in vivo distribution and / or pharmacokinetics as compared to the original antibody A method for enhancing the in vivo distribution and / or pharmacokinetics of an antibody, comprising the above steps. 85. The method according to configuration 84, wherein the cluster contains at least two positively charged amino acids within 6 residues of each other. 86. The polypeptide contains the 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 the positively charged amino acids exposed on at least two surfaces include at least two of X1, X2, X3, X4, X5, X6, and X7. The method according to configuration 84 or 85. 87. The method according to configuration 86, wherein the positively charged amino acids exposed on at least two surfaces include at least X1. 88. The method according to configuration 86 or 87, wherein the positively charged amino acids exposed on at least two surfaces include at least X4 and X7. 89. The method according to any one of configurations 86 to 88, wherein X2, X3, X5, and X6 are each independently a negatively charged or uncharged amino acid residue in the original polypeptide. The method according to any one of Configurations 86 to 89, wherein at least one of X4 and X7 is replaced with a negatively charged or uncharged amino acid. 91. The method according to any one of Configurations 83 to 90, wherein the at least two positively charged amino acids exposed on the surface include arginine, lysine, or histidine. 92. The method according to any one of Configurations 83 to 91, wherein the negatively charged or uncharged amino acid that replaces at least one of the at least two positively charged amino acids exposed on the surface includes a polar amino acid. 93. The method according to Configuration 92, wherein the negatively charged or uncharged amino acid that replaces at least one of the at least two positively charged amino acids exposed on the surface is glutamine. 94. The method according to any one of Configurations 83 to 93, wherein the positively charged amino acid and the negatively charged or uncharged amino acid have side chain lengths that differ by only one or less than two carbons. 95. The method according to any one of Configurations 83 to 94, comprising the step of replacing the top three amino acids of the polypeptide to disrupt at least one cluster. 96. The method according to any one of Configurations 83 to 95, wherein the cluster is outside any complementarity determining region (CDR) of the original antibody. 97. The cluster is within the variable region of the light chain (V L ) of the original antibody, according to the method of any one of Configurations 83 to 96. 98. The at least two positively charged amino acids exposed on the surface are within the V L framework region (FR) of the original antibody, according to the method of any one of Configurations 83 to 97. 99. The at least two positively charged amino acids exposed on the surface are within the V L FR2 of the original antibody, according to the method of any one of Configurations 83 to 98. 100. The polypeptide has an original FR2 containing at least two positively charged amino acids exposed on the surface and a V LBased on the first human germ cell line sequence, the method involves replacing the original FR2 containing the cluster or a portion thereof with a second FR2 or its corresponding portion derived from the second human germ cell line sequence of V L The method according to Configuration 98 or 99, comprising the step of replacing with a second FR2 or its corresponding portion derived from the second human germ cell line sequence of V, wherein the second FR2 contains at least one less positively charged amino acid exposed on the surface compared to the cluster. 101. The original sequence is one of 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), according to Configuration 99 or 100. 102. The original polypeptide is V L The method according to Configuration 99 or 101, wherein the FR2 of V contains one of 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. The variant polypeptide is V L The method according to any one of Configurations 99 to 101, wherein the FR2 of V 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. The variant polypeptide is V L The method according to any one of Configurations 99 to 101, wherein the FR2 of V contains at least one of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19). The method according to any one of Configurations 83 to 104, further comprising the step of conjugating a chelating ligand to the variant antibody. The method according to any one of Configurations 83 to 105, further comprising the step of labeling the antibody with a radionuclide. The method according to any one of Configurations 83 to 105, further comprising the step of labeling the antibody with a detectable label. 108. A step of selecting a germline sequence for the variable region of the light chain of the antibody (V L ), wherein the germline sequence does not contain a cluster of at least two positively charged amino acids within three residues of each other in the framework region 2 (FR2) of the germline sequence; Isolating one or more target-specific antibodies from a population of antibodies comprising V L derived from the germline sequence and having variations in the germline sequence across the population, wherein the one or more target-specific antibodies do not contain a cluster of at least two positively charged amino acids within three residues of each other in the V L FR2 sequence; and A method for producing a labeled antibody, comprising the step of labeling one or more target-specific antibodies. 109. The method according to Configuration 108, wherein the step of isolating one or more target-specific antibodies comprises screening a population of antibodies derived from the germline sequence. 110. The method according to Configuration 109, wherein the population of antibodies derived from the germline sequence is generated in a host organism genetically modified to express antibodies based only on the selected germline sequence. 111. The method according to Configuration 109, wherein the population of antibodies derived from the germline sequence is included in a phage library of antibodies based only on the selected germline sequence. 112. The method according to any one of Configurations 109 to 111, wherein the germline sequence is a human germline sequence. 113. The method according to any one of Configurations 108 to 112, wherein the one or more target-specific antibodies are humanized. 114. The method according to any one of configurations 108 to 113, further comprising the step of generating one or more target-specific minibodies or cys-diabodies from one or more target-specific antibodies. 115. The method according to any one of configurations 108 to 114, comprising the step of labeling one or more target-specific antibodies with a radionuclide. 116. The method according to any one of configurations 108 to 115, wherein the step of labeling one or more target-specific antibodies comprises the step of conjugating a chelating ligand to the one or more target-specific antibodies. 117. The method according to any one of configurations 83 to 116, wherein the antibody comprises an antigen-binding fragment. 118. The method according to any one of configurations 83 to 117, wherein the antibody is a minibody or a cys-diabody. 119. The method according to any one of configurations 83 to 118, wherein the antibody specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA. 120. The antibody or minibody comprises a light chain variable region (V L ) comprising three LCDR sequences of any one of the sequences in FIGS. 5A-5C and FIGS. 7A-7D, and a heavy chain variable region (V H ) comprising three HCDR sequences of any one of the sequences in FIGS. 5A-5C and FIGS. 7A-7D. The method according to any one of configurations 83 to 118. 121. An antibody produced by the method according to any one of configurations 83 to 120. 122. Identifying a subject in need of treatment with an antibody, minibody, or cys-diabody according to any one of configurations 1 to 78; and Administering to the subject a therapeutically effective amount of the antibody or minibody, or the composition according to configuration 79 A method of treating a subject comprising. 123. Identifying a subject in need of treatment for cancer; and, Administering a therapeutically effective amount of the antibody, minibody, or cys-diabody according to any one of Configurations 1 to 78, or the composition according to Configuration 79, to a subject, thereby treating cancer. A method for treating a subject with cancer, comprising: 124. Identifying a subject in need of radiation therapy; and, Administering a therapeutically effective amount of the antibody, minibody, or cys-diabody according to any one of Configurations 1 to 78, or the composition according to Configuration 79, to the subject, wherein the antibody, minibody, or cys-diabody comprises a radionuclide. A method of radiation therapy, comprising: 125. Administering to a subject a composition comprising an effective amount of the antibody, minibody, or cys-diabody according to any one of Configurations 1 to 78, or the composition according to Configuration 79, wherein the antibody, minibody, or cys-diabody is detectably labeled; and, Imaging the subject to detect the labeled antibody, minibody, or cys-diabody in the subject. A method of imaging a subject, comprising: 126. Use of the antibody, minibody, or cys-diabody according to any one of Configurations 1 to 78, or the composition according to Configuration 79, for the treatment of cancer in a subject in need thereof. 127. Use of the antibody, minibody, or cys-diabody according to any one of Configurations 1 to 78 for the preparation of a medicament for the treatment of cancer in a subject in need thereof.

Example

[0160] (Example 1) This non-limiting example shows enhancing the in vivo distribution of a minibody by mutating a positively charged amino acid within a cluster of positively charged amino acids in FR2 of the light chain variable region.

[0161] The humanized light chain variable region (V of the DLL3 antibody via a linker LThe humanized heavy chain variable region (V H ) of the DLL3 antibody was fused to the C-terminus of ( H ) to form an scFv fragment, and the scFv fragment was fused to the C 89 3 domain via a hinge region to construct a minibody that binds to DLL3. The amino acid sequence of the DLL3 minibody (IAB57M1-3) monomer is shown in FIG. 5A. In FIGS. 5A-5C, the signal peptide is shown in italics. By conjugating an appropriate chelating agent (e.g., Df) and labeling with a radionuclide (e.g.,

[0162] Humanized V L used the germline sequence of IGVK3-15*01. V L has a framework region 2 (FR2) sequence (WYQQKPGQAPRLLI (SEQ ID NO: 70) (according to North)) containing a cluster of two basic amino acids (lysine and arginine) at positions 45 and 51 (IMGT numbering). Molecular modeling of the surface charge using the Adaptive Poisson-Boltzmann Solver identified a local patch of positive charge exposed on the surface formed by the two basic amino acids. The locally positively charged patch may contribute to the accumulation of the minibody in the kidney.

[0163] To test this, as shown in FIG. 5B, R51 was replaced with glutamine (Q). Thus, KPGQAPR (SEQ ID NO: 6) in the V L FR2 of the original minibody was changed to KPGQAPQ (SEQ ID NO: 14) in the first variant. As shown in FIG. 5C, a second variant was created by further replacing alanine (A) at position 49 with serine (S). Thus, KPGQAPR (SEQ ID NO: 6) in the V L FR2 of the original minibody was further changed to KPGQSPQ (SEQ ID NO: 16) in the second variant.

[0164] Conjugation using Df and 89 After labeling with Zr, the labeled minibody was administered to mice. The average radioactivity uptake in the liver and kidneys was measured at 24 hours post - administration as the percentage of the injected dose per gram (%ID / g), and the results are shown in Figure 6. The variant minibody showed a decrease in accumulation in the kidneys and an increase in accumulation in the liver compared to the original minibody. The decrease in accumulation in the kidneys is expected to reduce renal radiation toxicity. This may also enable the use of higher radiation doses in the context of radiation therapy.

[0165] (Example 2) This non - limiting example shows enhancing the in - vivo distribution of a minibody by mutating positively charged amino acids within a cluster of positively charged amino acids in FR2 of the light - chain variable region.

[0166] IAB16M2 - 77 is a humanized anti - human FAP minibody engineered by humanizing an antibody from a mouse hybridoma clone. This original antibody was humanized based on the germline: IGHV1 - 69*01 / IGKV1 - 39*01. The germline IGKV1 - 39*01 is a commonly used light - chain germline sequence.

[0167] As shown in the minibody scFv amino acid sequence in Figure 7A, a positively charged patch on the germline IGKV1 - 39*01 was identified in the framework region (FR)2 of the light - chain variable region (V L ) The surface patch maps to the sequence KPGKAPK (SEQ ID NO: 5) containing three charged residues within the cluster (Lys - bold - emphasized in Figure 7A) (corresponding to K45, K48, and K51 according to IMGT numbering). The mutation K48Q (underlined in Figure 7B) on framework 2 of this germline sequence was at 24 hours post - administration 89The renal uptake of the Zr-radiolabeled minibody was reduced (Figure 8). The distributions in the liver and spleen remained relatively unchanged. These results are consistent with a positively charged patch formed by a cluster of lysines involved in accumulating this minibody in the kidney.

[0168] (Example 3) This non-limiting example shows the replacement of a segment of framework region 2 of one light chain germline sequence with a corresponding segment from another germline sequence to disrupt the cluster of positively charged residues.

[0169] As shown in Table 3.1 (Table 1), several human kappa and lambda light chain germline sequences, including the germline IGKV1-39*01, have a pattern of three positively charged residues (either K or R) in framework region 2. Analysis of the isopotential electrostatic surface of a minibody having a pattern of positively charged residues in framework region 2 in the light chain variable region, using an adaptive Poisson-Boltzmann solver, revealed a patch of positive charge formed by positively charged residues whose centers of mass are within a linear distance of about 11 angstroms of each other.

[0170] [Table 1]

[0171] As shown in Figure 7C, KPGKAPK (SEQ ID NO: 5) in the germline IGKV1-39*01 was replaced with KPGQSPQ (SEQ ID NO: 16) from IGKV2-28*01. The germline IGKV1-39*01 was modified by replacing the segment of framework region 2 (FR2) (KPGKAPK (SEQ ID NO: 5)) with a similar 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)).

[0172] Variant V L Produce a minibody containing (having a modified FR2), and radiolabel it. Administer the radiolabeled minibody to a mouse. Twenty-four hours after administration, image the animal and determine the distribution of the minibody in the animal. Variant V L The minibody having [Variant V] shows a reduced accumulation in the kidney as compared to the original minibody.

[0173] (Example 4) This non-limiting example shows enhancing the in vivo distribution of a cys-diabody by mutating positively charged amino acids within a cluster of positively charged amino acids in FR2 of the light chain variable region.

[0174] The humanized heavy chain variable region (V L ) of the antibody is fused to the C-terminus of the humanized light chain variable region (V H ) of the antibody via a linker to form an scFv fragment, and a cys-diabody is constructed by adding an extension sequence that mediates dimerization to the C-terminus.

[0175] Humanized V L uses the germline sequence of IGVK3-15*01. V L has a framework region 2 (FR2) sequence (WYQQKPGQAPRLLI (SEQ ID NO: 20) (according to North)) containing 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 identifies a local patch of positive charge on the surface due to the clustering of these two basic amino acids. The local positively charged patch may contribute to the accumulation of the cys-diabody in the kidney.

[0176] Substitute R51 with glutamine (Q). Conjugation using Df and 89After labeling with Zr, the labeled cys-diabody is administered to mice. The average radioactivity uptake in the liver and kidneys is measured at 24 hours after injection of the radiolabeled cys-diabody. The variant cys-diabody shows a decrease in accumulation in the kidneys and an increase in accumulation in the liver compared to the original cys-diabody.

[0177] (Example 5) This non-limiting example shows enhancing the in vivo distribution of the minibody by mutating the positively charged amino acids within the cluster of positively charged amino acids in the hinge region.

[0178] The humanized light chain variable region (V L ) of the antibody is fused to the C-terminus of the humanized heavy chain variable region (V H ) of the antibody via a linker to form an scFv fragment, and the scFv fragment is fused to the C H 3 domain to construct a minibody (IAB16M2-78). The hinge region included the upper hinge sequence EPKSSDKTHT (SEQ ID NO: 38) containing a cluster of two lysines. The lysine residues within the cluster were replaced with glycine (G) to generate a variant minibody IAB16M2-79 (Figure 7D) having the upper hinge sequence EPGSSDGTHT (SEQ ID NO: 39). After conjugation using Df and 89 labeling with Zr, the labeled minibody was administered to mice. The average radioactivity uptake in the liver and kidneys was measured at 24 hours after injection of the radiolabeled minibody. The variant minibody showed a decrease in accumulation in the kidneys compared to the original minibody (Figure 9).

[0179] (Example 6) This non-limiting example shows the treatment of radioimmunotherapy of a subject using the antibody of the present disclosure.

[0180] Identify a subject in need of cancer treatment. Specifically bind to a target within a tumor and the light chain variable region (V L)Label a variant minibody having the sequence KPGQAPQ (SEQ ID NO: 14) in the framework region 2 (FR2) with a radionuclide. Administer the radiolabeled minibody to a subject. The radiation dose delivered to the subject is V L Higher than the dose that would have been delivered using the original minibody having the sequence KPGQAPR (SEQ ID NO: 6) in FR2, while the nephrotoxicity remains at the same level.

[0181] (Example 7) This non-limiting example demonstrates the treatment of a subject using the antibodies of the present disclosure.

[0182] Identify a subject in need of liver cancer treatment. Specifically bind to a target within the tumor and have a variable light chain region (V L )Conjugate a variant minibody having the sequence KPGQAPQ (SEQ ID NO: 4) in the framework region 2 (FR2) with a cytotoxic agent. Administer the conjugated minibody to a subject.

[0183] As used herein, section headings are for organizational purposes only and should in no way be construed as limiting the subject matter described. 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 disclosure specifically referenced herein. In the event that a definition of a term in an incorporated reference appears to be different from the definition provided in the present teachings, the definition provided in the present teachings shall control. It will be understood that there is an implicit "about" in front of temperatures, concentrations, times, etc. discussed in the present teachings such that minor and non-substantive deviations are within the scope of the present teachings herein.

[0184] Although the present disclosure is presented in the context of certain embodiments and examples, those skilled in the art will understand that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the present disclosure, as well as their obvious modifications and equivalents. In addition, although some variations of the present disclosure are shown and described in detail, other modifications within the scope of the present disclosure will be readily apparent to those skilled in the art based on the present disclosure. Various combinations or sub - combinations of the specific features and aspects of the embodiments can be made and are still intended to be within the scope of the present disclosure. It is to be understood that the various features and aspects of the disclosed embodiments can be combined with each other or substituted for each other to form various aspects or embodiments of the present disclosure. Therefore, it is intended that the scope of the present disclosure presented herein should not be limited by the specific disclosed embodiments described above.

[0185] However, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art, it is to be understood that this detailed description is given by way of example only and shows the preferred embodiments of the present disclosure.

[0186] The terms used in the description presented herein are not intended to be construed in any limiting or restrictive manner. Rather, the terms are merely being used in conjunction with a detailed description of embodiments of the system, method, and related components. Further, embodiments may include several novel features, none of which alone is involved in its desirable attributes or is considered essential for practicing the present disclosure described herein.

Description of Reference Numerals

[0187] 100 Method 110 Block 120 Block 200 Method 210 Block 220 Block 300 Method 310 block 320 block 330 block 400 method 410 block 420 block

Claims

**Claim 1** A variant antibody comprising at least one disrupted cluster of positively charged amino acids exposed on the surface, having a substitution of at least one positively charged amino acid exposed on the surface of the original cluster by a negatively charged or uncharged amino acid, such that the original antibody comprising the original cluster comprising at least two positively charged amino acids exposed on the surface within about 30 angstroms of each other is different from the variant antibody. **Claim 2** The variant antibody according to claim 1, wherein at least two positively charged amino acids exposed on the surface of the original cluster are within about 15 angstroms of each other. **Claim 3** The variant antibody according to claim 1 or 2, having at least about 10% less total positive charge due to the cluster compared to the original antibody as a result of disruption of the cluster. **Claim 4** The variant antibody according to any one of claims 1 to 3, differing from the original antibody by only up to three amino acid substitutions that disrupt the cluster. **Claim 5** Substitution of a positively charged amino acid exposed on at least one surface of the original cluster is within the light chain variable region (V L ) framework region (FR) of the original antibody, the variant antibody according to any one of claims 1 to 4. **Claim 6** Substitution of a positively charged amino acid exposed on at least one surface of the original cluster is within the V of the original antibody L The variant antibody according to claim 5, which is within FR2 of the original antibody **Claim 7** The original antibody is X 1 X 2 X 3 X 4 X 5 X 6 X 7 comprises a polypeptide comprising the original sequence of (SEQ ID NO: 2), wherein X 1 is a positively charged amino acid, and X 2 , X 3 , X 5 , and X 6 are each independently a negatively charged or uncharged amino acid, and X 4 and X 7 are each independently any amino acid, provided that at least one of them is a positively charged amino acid, the original sequence is outside any CDR of the original antibody, and at least one of the positively charged amino acids X 4 and X 7 is surface-exposed and is part of the original cluster, the variant antibody according to any one of claims 1 to 6. **Claim 8** The variant antibody according to claim 7, wherein the positive charge due to the original sequence is reduced by about 33% or more in the variant antibody. **Claim 9** The variant antibody according to claim 8, wherein the positive charge due to the original sequence is reduced by about 60% or more in the variant antibody. **Claim 10** The substitution is an X by a negatively charged or uncharged amino acid 4 and X 7 The variant antibody according to any one of claims 7 to 9, comprising at least one substitution of **Claim 11** The polypeptide is X 1 X 2 X 3 X 4 X 5 X 6 X 7 QQX comprising the original sequence of (SEQ ID NO: 2) 1 X 2 X 3 X 4 X 5 X 6 X 7 The variant antibody according to any one of claims 7 to 10, comprising (SEQ ID NO: 3). **Claim 12** X 7 is a positively charged amino acid, the variant antibody according to any one of claims 7 to 11. **Claim 13** X 4 is a positively charged amino acid, the variant antibody according to any one of claims 7 to 12. **Claim 14** The variant antibody according to any one of claims 7 to 13, wherein the positively charged amino acid is lysine, arginine, or histidine. **Claim 15** The variant antibody according to claim 14, wherein the positively charged amino acid is lysine or arginine. **Claim 16** X 2 and / or X 6 The variant antibody according to any one of claims 7 to 13, wherein X is proline. **Claim 17** X 3 The variant antibody according to any one of claims 7 to 14, wherein X is glycine or glutamic acid. **Claim 18** X 5 The variant antibody according to any one of claims 7 to 15, wherein X is alanine, valine, serine, or proline. **Claim 19** The original array is KX 2 X 3 KX 5 X 6 K (sequence number 73, wherein X 2 , X 3 , X 5 , and X 6 are each independently a negatively charged or uncharged amino acid); KX 2 X 3 X 4 X 5 X 6 R (sequence number 74, wherein X 2 , X 3 , X 4 , X 5 , and X 6 are each independently a negatively charged or uncharged amino acid); or KX 2 X 3 X 4 X 5 X 6 K (sequence number 75, wherein X 2 , X 3 , X 4 , X 5 , and X 6 are each independently a negatively charged or uncharged amino acid), and is one of the above, the variant antibody according to any one of claims 7 to 16. **Claim 20** The variant antibody according to any one of claims 7 to 17, wherein the original sequence is one of 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). **Claim 21** The variant antibody according to claim 18, wherein the original sequence is KPGKAPK (SEQ ID NO: 5) or KPGQAPR (SEQ ID NO: 6). **Claim 22** The variant antibody according to any one of claims 7 to 19, wherein the negatively charged or uncharged amino acid that replaces the positively charged amino acid exposed on at least one surface of the cluster is glutamine.

23. The original array is within the framework region (FR) of the variable light chain (V L ) of the original antibody, and is a variant antibody according to any one of claims 7 to 22.

24. The original array is the V of the original antibody L The variant antibody according to claim 23, which is within FR2.

25. V L A variant antibody according to any one of claims 1 to 24, wherein FR2 comprises 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. V having the amino acid sequence of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19) L A variant antibody according to any one of claims 1 to 25, comprising FR2.

27. The variant antibody according to any one of claims 1 to 26, wherein the replacement of the positively charged amino acid exposed on at least one surface of the original cluster is within the hinge region of the original antibody.

28. The variant antibody according to claim 27, wherein the replacement of the positively charged amino acid exposed on at least one surface of the original cluster is within the upper hinge region of the original antibody.

29. Original array X 1 X 2 X 3 X 4 X 5 X 6 X 7 An antibody comprising a variant polypeptide different from the original polypeptide comprising (array number 1), wherein the original sequence is outside any CDR of the original polypeptide, and wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 are each independently any amino acid, provided that at least two of them are positively charged amino acids exposed on the surface that form a cluster of positively charged amino acids exposed on the surface in the original polypeptide. The variant polypeptide is different from the original polypeptide by having a modification of at least one of the positively charged amino acids exposed on at least two surfaces of the original sequence, which reduces at least 33% of the positive charge resulting from the original sequence, antibody.

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

31. The antibody according to 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 according to any one of claims 29 to 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. At least X 4 and X 7 The antibody according to any one of claims 29 to 32, wherein each is an amino acid independently and positively charged.

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

35. X 2 , X 3 , X 5 , and X 6 is an antibody according to any one of claims 29 to 34, each independently being a negatively charged or uncharged amino acid.

36. To reduce the positive charge, X 4 and X 7 The antibody according to any one of claims 29 to 35, wherein at least one of is substituted with a negatively charged or uncharged amino acid.

37. X 4 and X 7 The antibody according to claim 36, wherein the negatively charged or uncharged amino acid that replaces at least one of is glutamine.

38. The variant polypeptide comprises alanine or serine at a position corresponding to X in the sequence of the original polypeptide 5 The antibody according to any one of claims 29 to 37, wherein the variant polypeptide comprises alanine or serine at a position corresponding to X in the sequence of the original polypeptide

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

40. The original array is within the V L framework region (FR) of the original polypeptide, and is an antibody according to any one of claims 29 to 39.

41. The original array is the V of the original polypeptide L The antibody according to claim 40, which is within FR2 of

42. The original polypeptide has an original FR2 containing the original sequence and is V L Based on the first human germline sequence of, the variant polypeptide is V L Differing from the original polypeptide by having at least a substitution of the original sequence by a corresponding second sequence from a second FR2 from the second human germline sequence of, the second FR2 corresponds to the original sequence and comprises a second sequence having at least 10% fewer positive charges resulting therefrom as compared to the original sequence. The antibody according to claim 40 or 41

43. A variant antibody comprising at least one disrupted cluster of positively charged amino acids, wherein the variant antibody has a substitution of at least one positively charged amino acid of the original cluster by a negatively charged or uncharged amino acid, thereby differing from the original antibody comprising the original cluster containing at least two positively charged amino acids within 12 residues of each other, and the original cluster is outside any CDR of the original antibody, variant antibody.

44. The original cluster is the variant antibody according to claim 43, which is within the framework region (FR) of the light chain variable region (V L ) of the original antibody.

45. The original cluster is the V of the original antibody L A variant antibody according to claim 44, which is within FR2 of the original antibody.

46. The following, comprising a substitution of at least one positively charged amino acid: at least one of KPGQAPK (SEQ ID NO: 13), KPGQAPQ (SEQ ID NO: 14), and QQKPGQSPQ (SEQ ID NO: 15), the variant antibody according to claim 45.

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

48. The original cluster is within the hinge region of the original antibody, the variant antibody according to claim 43.

49. The original cluster is within the upper hinge region of the original antibody, the variant antibody according to claim 48.

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

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

52. An antibody according to any one of claims 1 to 51, comprising an antigen-binding fragment.

53. The molecular weight of the antibody is in the range of about 15 kDa to about 110 kDa, the antibody according to any one of claims 1 to 52.

54. A mini-body, cys-diabody, scFv, scFv-Fc, or nanobody-Fc, the antibody according to any one of claims 1 to 53.

55. The molecular weight of the antibody is in the range of about 10 kDa to about 20 kDa, the antibody according to any one of claims 1 to 54.

56. A nanobody, the antibody according to claim 55.

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

58. An scFv-Fc, the antibody according to claim 57.

59. Original array X 1 X 2 X 3 X 4 X 5 X 6 X 7 (Variable region of the light chain (V containing array number 1) L ) A minibody or cys - diabody comprising a variant polypeptide different from the original polypeptide comprising FR2, wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 are each independently any amino acid, provided that at least two of them are positively charged amino acids that form a cluster of positively charged amino acids exposed on the surface in the original polypeptide The variant polypeptide differs from the original polypeptide by having a modification of at least one of at least two surface-exposed positively charged amino acids of the original sequence that reduces at least 33% of the positive charge resulting from the original sequence, a mini-body or cys-diabody.

60. At least X 4 and X 7 is the minibody or cys-diabody according to claim 59, which are each independently positively charged amino acids in the original polypeptide.

61. X 2 、 X 3 、 X 5 、 and X 6 is the minibody or cys - diabody according to claim 59 or 60, which are each independently negatively charged or uncharged amino acids in the original polypeptide.

62. To reduce the positive charge, X 4 and X 7 wherein at least one of is substituted with a negatively charged or uncharged amino acid, the minibody or cys-diabody according to any one of claims 59 to 61.

63. X 1 X 2 X 3 QAX 6 X 7 (SEQ ID NO:4)-containing FR2 sequence-containing light chain variable region (V L )-containing minibody or cys-diabody, wherein X 1 is a positively charged amino acid exposed on the surface, and X 2 , X 3 , and X 6 are each independently any negatively charged or uncharged amino acid, and X 7 is either glutamine or lysine, a minibody or cys-diabody.

64. X 1 The minibody or cys-diabody according to any one of claims 59 to 63, wherein 1 is lysine.

65. X 2 and / or X 6 The minibody or cys-diabody according to any one of claims 59 to 64, wherein X is proline.

66. X 3 The minibody or cys-diabody according to any one of claims 59 to 65, wherein X is glycine or glutamic acid.

67. The FR2 sequence comprises KPGQAPK (SEQ ID NO: 13) or KPGQAPQ (SEQ ID NO: 14), the mini-body or cys-diabody according to claim 63.

68. A49, and (i) Q51; or (ii) Q48 and K51 A light chain variable region (V by IMGT numbering) containing any of L A minibody or cys-diabody containing

69. V L The minibody or cys-d diabody according to claim 61, further comprising K45 (IMGT numbering).

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

71. The antibody, minibody, or cys-diabody according to any one of claims 1 to 70, which specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNγ, integrin αVβ6, FOLRα, or PSMA.

72. A light chain variable region comprising three LCDR sequences in any one of the sequences in FIGS. 5B, 5C, 7B-7D, and a heavy chain variable region (V H ) comprising three HCDR sequences in any one of the sequences in FIGS. 5B, 5C, 7B-7D, the antibody, minibody, or cys-diabody according to any one of claims 1 to 71.

73. The antibody, minibody, or cys-diabody according to any one of claims 1 to 72, further comprising a detectable label.

74. The antibody, minibody, or cys-diabody according to claim 73, labeled with a radionuclide or an organic dye.

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

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

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

78. The radionuclide is 212 Pb, 149 Tb, and 161 Tb, and is the antibody, minibody, or cys-diabody according to claim 75, which is selected from among them.

79. The antibody, minibody, or cys-diabody according to any one of claims 1 to 78; and A pharmaceutically acceptable carrier A composition comprising.

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

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

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

83. A process for identifying an original antibody comprising a polypeptide comprising at least one cluster of positively charged amino acids exposed on the surface, the cluster comprising at least two positively charged amino acids exposed on the surface within 30 angstroms of each other; A step of substituting at least one of the at least two positively charged amino acids exposed on the surface of at least one cluster with a negatively charged or uncharged amino acid, thereby disrupting at least one cluster, A process for generating a variant antibody having enhanced in vivo distribution and / or pharmacokinetics as compared to the original antibody. A method for enhancing the in vivo distribution and / or pharmacokinetics of an antibody, comprising the step. **Claim 84** Identifying an original antibody comprising a polypeptide containing at least two clusters of positively charged amino acids exposed on the surface within 12 residues of each other; and Disrupting the cluster by substituting at least one of the at least two positively charged amino acids exposed on the surface of the cluster with a negatively charged or uncharged amino acid, thereby generating a variant antibody having enhanced in vivo distribution and / or pharmacokinetics as compared to the original antibody. A method for enhancing the in vivo distribution and / or pharmacokinetics of an antibody, comprising the step. **Claim 85** The method according to claim 84, wherein the cluster comprises at least two positively charged amino acids within 6 residues of each other. **Claim 86** The polypeptide is X 1 X 2 X 3 X 4 X 5 X 6 X 7 (SEQ ID NO: 1) and contains the amino acid sequence, where X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 are each independently any amino acid, and at least two positively charged amino acids exposed on the surface are X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , and X 7 The method according to claim 84 or 85, comprising at least two of them. **Claim 87** At least two positively charged amino acids exposed on the surface contain at least X 1 The method according to claim 86, comprising **Claim 88** At least two positively charged amino acids exposed on the surface are at least X 4 and X 7 The method according to claim 86 or 87, comprising **Claim 89** X 2 、 X 3 、 X 5 、 and X 6 is the method according to any one of claims 86 to 88, which are independently negatively charged or uncharged amino acid residues in the original polypeptide. **Claim 90** X 4 and X 7 The method according to any one of claims 86 to 89, wherein at least one of 4 and 7 is substituted with a negatively charged or uncharged amino acid. **Claim 91** The method according to any one of claims 83 to 90, wherein the at least two positively charged amino acids exposed on the surface comprise arginine, lysine, or histidine. **Claim 92** The method according to any one of claims 83 to 91, wherein the negatively charged or uncharged amino acid substituting at least one of the at least two positively charged amino acids exposed on the surface comprises a polar amino acid. **Claim 93** The method according to claim 92, wherein the negatively charged or uncharged amino acid substituting at least one of the at least two positively charged amino acids exposed on the surface is glutamine. **Claim 94** The method according to any one of claims 83 to 93, wherein the positively charged amino acid and the negatively charged or uncharged amino acid have side chain lengths that differ by one or less than two carbons. **Claim 95** The method according to any one of claims 83 to 94, comprising the step of substituting the top three amino acids of the polypeptide to disrupt at least one cluster. **Claim 96** The method according to any one of claims 83 to 95, wherein the cluster is outside any complementarity determining region (CDR) of the original antibody. **Claim 97** The cluster is within the light chain variable region (V L ) of the original antibody, and is the method according to any one of claims 83 to 96. **Claim 98** At least two positively charged amino acids exposed on the surface are within the V L framework region (FR) of the original antibody, the method according to any one of claims 83 to 97. **Claim 99** At least two positively charged amino acids exposed on the surface are within the V L FR2 of the original antibody, according to the method of any one of claims 83 to 98. **Claim 100** The polypeptide has an original FR2 that includes at least two positively charged amino acids exposed on the surface, and is V L Based on the first human germline sequence of, the method comprises replacing the original FR2 or a portion thereof that includes the cluster with a second FR2 or a corresponding portion thereof derived from the second human germline sequence of V L The method according to claim 98 or 99, comprising the step of substituting with a second FR2 that includes at least one fewer positively charged amino acid exposed on the surface as compared to the cluster. **Claim 101** The original array is KX 2 X 3 KX 5 X 6 K (array number 73, wherein X 2 X 3 X 5 and X 6 are each independently a negatively charged or uncharged amino acid); KX 2 X 3 X 4 X 5 X 6 R (array number 74, wherein X 2 X 3 X 4 X 5 and X 6 are each independently a negatively charged or uncharged amino acid); or KX 2 X 3 X 4 X 5 X 6 K (array number 75, wherein X 2 X 3 X 4 X 5 and X 6 are each independently a negatively charged or uncharged amino acid), which is one of the following, the method according to claim 99 or 100. **Claim 102** The original polypeptide is V L The method according to claim 99 or 101, wherein FR2 contains one of 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). **Claim 103** The variant polypeptide is V L The method according to any one of claims 99 to 101, wherein FR2 comprises 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). **Claim 104** The variant polypeptide is V L The method according to any one of claims 99 to 101, wherein the FR2 comprises at least one of WYQQKPGQAPQLLIY (SEQ ID NO: 17), WYQQKPGQSPQLLIY (SEQ ID NO: 18), or WYQQKPGQAPKLLIY (SEQ ID NO: 19). **Claim 105** The method according to any one of claims 83 to 104, further comprising the step of conjugating a chelating ligand to the variant antibody.

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

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

108. Selecting a germline sequence for the variable light chain region (V L ) of an antibody, wherein the germline sequence does not contain clusters of at least two positively charged amino acids within three residues of each other in framework region 2 (FR2) of the germline sequence; V derived from a germline sequence L A step of isolating one or more target-specific antibodies from a population of antibodies, which contains V and has variations in the germline sequence across the population, wherein the one or more target-specific antibodies L do not contain a cluster of at least two positively charged amino acids within three residues of each other in the FR2 sequence; and A method for producing a labeled antibody, comprising the step of labeling one or more target-specific antibodies.

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

110. The method according to claim 109, wherein the population of antibodies derived from germline sequences is generated in a host organism genetically modified to express antibodies based on selected germline sequences only.

111. The method according to claim 109, wherein the population of antibodies derived from germline sequences is included in a phage library of antibodies based on selected germline sequences only.

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

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

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

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

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

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

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

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

120. The antibody or minibody comprises a light chain variable region (V L ) containing three LCDR sequences of any one of the sequences in FIGS. 5A-5C and FIGS. 7A-7D, and a heavy chain variable region (V H ) containing three HCDR sequences of any one of the sequences in FIGS. 5A-5C and FIGS. 7A-7D, the method according to any one of claims 83 to 118.

121. An antibody produced by the method according to any one of claims 83 to 120.

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

123. Identifying a subject in need of treatment for cancer; and, Administering to the subject a therapeutically effective amount of an antibody, minibody, or cys-diabody according to any one of claims 1 to 78, or the composition according to claim 79, thereby treating the cancer A method of treating a subject for cancer, comprising.

124. Identifying a subject in need of radiation therapy; and, Administering to the subject a therapeutically effective amount of an antibody, minibody, or cys-diabody according to any one of claims 1 to 78, or the composition according to claim 79, wherein the antibody, minibody, or cys-diabody comprises a radionuclide A method of radiation therapy, comprising.

125. Administering to a subject a composition comprising an effective amount of an antibody, minibody, or cys-diabody according to any one of claims 1 to 78, or the composition according to claim 79, wherein the antibody, minibody, or cys-diabody is detectably labeled; and, Imaging the subject to detect the labeled antibody, minibody, or cys-diabody in the subject A method of imaging a subject, comprising.

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

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

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