Anti-B7-H3 antibody and method of use thereof

Abstract

The present disclosure provides antibodies that specifically bind to B7-H3. The antibodies find use in a variety of therapeutic, diagnostic, and monitoring applications, which are also described. The present disclosure provides antibodies that specifically bind to B7-H3. The subject antibodies specifically bind to B7-H3 from humans and other mammals, such as monkeys and mice.

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Patent Application No. 62 / 870,549, filed on 3 July 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] B7-H3 (also known as CD276) is a major glycoprotein expressed on the surface of antigen-presenting cells (APCs). In humans, this protein is encoded by a gene on chromosome 15. B7-H3 may inhibit T cell infiltration from tumor cells (see, e.g., Vigdorovich et al. Structure 2013 21:707-717 and Chen et al., Exp. Cell Res. 2013 319:96-102), and is therefore considered an immune checkpoint inhibitor. This protein originates from molecules of the B7 family, many of which interact with known checkpoint markers such as CTLA4, PD-1, and CD28. Both B7-H3 and PD-1 are members of the B7 / CD28 superfamily. Since both B7-H3 and PD-1 induce inhibitory effects on T cells and alter their microenvironment to evade the anti-tumor immune response, B7-H3 is considered a target for immunotherapies like PD-1.

[0003] B7-H3 is overexpressed in many cancers, but the characteristics of its receptor are not yet fully understood. Its overexpression is associated with poor prognosis in human patients and the potential for tumor invasiveness and metastasis in in vitro models. Recent evidence suggests that B7-H3 influences cancer progression beyond its immunomodulatory role (see, e.g., Castellanos et al, Am.J.Clin Exp.Immunol.2017 6:66-75). Therefore, B7-H3 inhibition may reduce the proliferation, progression, and metastasis of malignant cells.

[0004] Due to the role of B7-H3 in cancer development and as an immune checkpoint, B7-H3 has become an interesting therapeutic target, particularly for cancers of the lung, breast, brain, kidney, and prostate, as well as tumors that are resistant to or have become resistant to other immune checkpoint inhibitors. B7-H3 antibodies have shown positive results in clinical trials for the treatment of metastatic neuroblastoma (Kramer et al, J Neurooncol. 2010 97:409-418).

[0005] Therefore, there is a need for new therapeutic antibodies that inhibit B7-H3 function.

Summary of the Invention

[0006] The present disclosure provides antibodies that specifically bind to B7-H3. The antibodies are useful for various therapeutic, diagnostic, and monitoring applications, which are also provided.

[0007] In some embodiments, the antibody may comprise (a) a variable domain comprising: i. heavy chain CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Figure 1, and ii. light chain CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of an antibody selected from Figure 2, or (b) a variant of the variable domain of (a) that is otherwise identical to the antibody variable domain except for a maximum of 10 (e.g., maximum 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions of the variable domain of (a).

[0008] In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to the amino acid sequence of the heavy chain variable domain of an antibody selected from Figure 1, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to the amino acid sequence of the light chain variable domain of an antibody selected from Figure 2.

[0009] Antibodies with variable domain arrays shown in Figures 1 and 2 are essentially "human" antibodies made in chickens and can thus bind to epitopes in B7-H3 of various mammals such as humans, mice, and monkeys. These epitopes are not immunogenic in mammals (because they already exist), so it is thought that these antibodies bind to some new epitopes. Furthermore, these antibodies can bind to B7-H3 derived from multiple mammals and their therapeutic potential can be easily tested in mammalian cancer model systems (e.g., mice), so these antibodies can be advantageous.

Brief Description of the Drawings

[0010] [Figure 1-1] The amino acid sequences of 54 anti-B7-H3 antibody heavy chain variable regions are shown, and the complementarity-determining regions (CDRs) defined by the Chothia method are boxed. From top to bottom: SEQ ID NOs: 1-54. [Figure 1-2] The amino acid sequences of 54 anti-B7-H3 antibody heavy chain variable regions are shown, and the complementarity-determining regions (CDRs) defined by the Chothia method are boxed. From top to bottom: SEQ ID NOs: 1-54. [Figure 2-1] The amino acid sequences of 54 anti-B7-H3 antibody light chain variable regions are shown, and the complementarity-determining regions (CDRs) defined by the Chothia method are boxed. From top to bottom: SEQ ID NOs: 55-108. [Figure 2-2] The amino acid sequences of 54 anti-B7-H3 antibody light chain variable regions are shown, and the complementarity-determining regions (CDRs) defined by the Chothia method are boxed. From top to bottom: SEQ ID NOs: 55-108. [Figure 3] A dendrogram showing the phylogenetic relationship of 54 anti-B7-H3 antibodies based on sequence comparison (amino acid substitutions per 100 residues). Analysis of the dendrogram shows that 9 phylogenetic groups are represented. Each phylogenetic group has less than 10 amino acid substitutions per 100 residues. The antibodies in each phylogenetic group may have derived from a common ancestor through affinity maturation. The consensus sequences of the CDRs for each phylogenetic group are provided below.

[0011] definition The terms “antibody” and “immunoglobulin” include, but are not limited to, any isotype of antibody or immunoglobulin, or antibody fragment, that retains specific binding to an antigen, including Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins containing antigen-binding moieties of antibody and non-antibody proteins. Antibodies may be labeled to be detectable, for example, with radioisotopes, enzymes that produce detectable products, or fluorescent proteins. Antibodies can be further complexed with other moieties, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies may also be conjugated to solid supports, including, but not limited to, polystyrene plates or beads. The terms also include Fab', Fv, F(ab')2, and other antibody fragments that retain specific binding to an antigen, and monoclonal antibodies. Antibodies may be monovalent or bivalent.

[0012] An "antibody fragment" is a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and the remaining "Fc" fragment; the names reflect their ability to easily crystallize. Pepsin treatment produces an F(ab')2 fragment with two antigen-binding sites that can crosslink antigens. 。

[0013] "Fv" is the smallest antibody fragment containing a complete antigen recognition site and antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent bonds. The three CDRs of each variable domain interact to form V H -V L This configuration defines the antigen-binding site on the surface of the dimer. In summary, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, although with lower affinity than the entire binding site.

[0014] The "Fab" fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the name used herein for Fab' in which the cysteine ​​residue of the constant domain possesses a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments having a hinge cysteine ​​between them. Other chemical bonds of antibody fragments are also known.

[0015] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0016] "Single-chain Fv" or "sFv" antibody fragments are the V of the antibody. H and VL comprising domains, which are present in a single polypeptide chain. In some embodiments, the Fv polypeptide is a V H domain and a V L domain, and further comprises a polypeptide linker between the V and V domains, thereby enabling the sFv to form a structure desirable for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0017] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, and these fragments are light chain variable domains (V H -V L ) connected to heavy chain variable domains (V L ) within the same polypeptide chain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are fully described, for example, in EP404,097, WO93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0018] As used herein, the term “affinity” refers to the equilibrium constant of the reversible binding of two drugs and is expressed as the dissociation constant (Kd). Affinity is at least 1-fold greater than, at least 2-fold greater than, at least 3-fold greater than, at least 4-fold greater than, at least 5-fold greater than, at least 6-fold greater than, at least 7-fold greater than, at least 8-fold greater than, at least 9-fold greater than, at least 10-fold greater than, at least 20-fold greater than, at least 30-fold greater than, at least 40-fold greater than, at least 50-fold greater than, at least 60-fold greater than, at least 70-fold greater than, at least 80-fold greater than, at least 90-fold greater than, at least 100-fold greater than, or at least 1000-fold greater than or greater than, the affinity of the antibody to the unrelated amino acid sequence. The affinity of the antibody to the target protein may be, for example, about 100 nanomoles (nM) to about 0.1 nM, about 100 nM to about 1 picomole (pM), or about 100 nM to about 1 femtomole (fM) or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more drugs to dissociation after dilution. The terms "immunoreactive" and "preferentially binding" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0019] The term "binding" refers to the direct association between two molecules by covalent, electrostatic, hydrophobic, and ionic, and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Anti-B7-H3 antibodies specifically bind to epitopes within the B7-H3 polypeptide. Nonspecific binding refers to approximately 10 -7 Binding with affinity less than M, for example, 10 -6 M, 10 -5 M, 10 -4 This refers to binding based on affinity, such as M.

[0020] As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean non-adjacent antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977), Kabat et al., USDept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and the definition includes overlaps or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition to refer to CDRs of an antibody or a transplanted antibody or its variants is intended to be within the scope of the terms defined and used herein. The CDRs shown in Figures 1 and 2 are defined by the Chothia method. However, CDRs can also be defined using the Kabat and MacCallum methods, as shown below. [Table 1]

[0021] As used herein, the term “framework,” when used in relation to the antibody variable region, is intended to mean all amino acid residues outside the CDR region within the antibody variable region. The variable region framework is generally a discontinuous amino acid sequence between approximately 100 and 120 amino acids in length, but is intended to refer only to the amino acids outside the CDR. As used herein, the term “framework region” is intended to mean each domain of the framework separated by the CDR.

[0022] "Isolated" antibodies are those identified, separated, and / or recovered from components of their natural environment. These contaminants in the natural environment may be substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody may be purified to (1) a concentration of over 90% by weight, over 95% by weight, or over 98% by weight, e.g., over 99% by weight, when determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Because at least one component of the antibody's natural environment is absent, isolated antibodies contain the antibody in situ within recombinant cells. In some cases, isolated antibodies may be prepared by at least one purification step.

[0023] As used herein, “treatment,” “to treat,” and similar terms mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and may be therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” includes any treatment of a disease in mammals, in particular humans, and includes (a) preventing the onset of the disease in subjects who are predisposed to the disease but have not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., preventing its development, and (c) alleviating the disease, i.e., causing the regression of the disease.

[0024] The terms “individual,” “subject,” “host,” and “patient,” as used interchangeably herein, refer to mammals, including but not limited to rodents (rats, mice), non-human primates, humans, cattle, dogs, cats, and ungulates (e.g., horses, cows, sheep, pigs, goats).

[0025] The "therapeutic effective dose" or "effective dose" refers to the amount of anti-B7-H3 antibody that, when administered to a mammal or other subject to treat the disease, is sufficient to perform such treatment of the disease. The "therapeutic effective dose" varies depending on the anti-B7-H3 antibody, the disease and its severity, as well as the age and weight of the subject being treated.

[0026] The term “biological sample” encompasses various types of samples obtained from an individual and can be used in diagnostic or surveillance assays. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells derived therefrom, as well as their offspring. The definition also includes samples that have been manipulated in any way after acquisition, such as through treatment with reagents, solubilization, or concentration of specific components such as polynucleotides. The term “biological sample” encompasses clinical samples and also includes cells in cultures, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0027] As used herein, the term “collective” in the context of variants of the antibody variable domain that are otherwise identical to the antibody variable domain except for a defined number of amino acid substitutions “in the collective CDR region of the antibody variable domain” indicates that the number of amino acid substitutions is counted using all six CDRs. If, as illustrated by the examples, a variant has five amino acid substitutions relative to the antibody variable domain, then the six CDRs of the variant together have a total of five amino acid substitutions relative to the antibody variable domain. This phrase is not intended to imply that each CDR has a defined number of amino acid substitutions.

[0028] Before further explanation of the present invention, it should be understood that the present invention is not limited to the specific embodiments described and is, of course, subject to change. Since the scope of the present invention is limited solely by the appended claims, it should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0029] Where a range of values ​​is provided, unless explicitly indicated in the context, each intervening value is between the upper and lower limits of that range, up to one-tenth of a unit of the lower limit, and any other listed values ​​or intervening values ​​within that listed range are understood to be included in the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in the invention, subject to any restrictions specifically excluded within the listed range. If a listed range includes one or both of the limits, a range excluding either or both of these included limits is also included in the invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the invention pertains. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials in connection with the citation of such publications.

[0031] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include multiple references unless the context otherwise explicitly indicates otherwise. For example, a reference to “antibody” includes multiple such antibodies, and a reference to “anti-B7-H3 antibody” includes one or more anti-B7-H3 antibodies and their equivalents known to those skilled in the art. It should be further noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as a precedent for the use of exclusive terms such as “exclusively,” “only,” or “negative” restrictions in relation to the enumeration of elements of the claims.

[0032] The publications discussed herein are provided solely for the purpose of disclosing publications prior to the filing date of this application. Nothing in this specification should be construed as an understanding that the present invention has no prior rights to such publications by prior inventions. Furthermore, the publication dates indicated may differ from the actual publication dates and should be confirmed separately. [Modes for carrying out the invention]

[0033] This disclosure provides antibodies specific to human B7-H3. These antibodies are useful for a variety of therapeutic, diagnostic, and surveillance applications, and are also provided.

[0034] antibody The target antibody specifically binds to B7-H3 from humans and other mammals, such as monkeys and mice.

[0035] The target antibody may have high affinity binding to B7-H3 from humans and other mammals, such as monkeys and mice. For example, the target antibody may have at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12M, or 10 -12 The target antibody can bind to human, monkey, and / or mouse B7-H3 with affinity exceeding M. -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, or about 10 -11 M~about 10 -12 M, or 10 -12 It binds to epitopes present in human, monkey, and / or mouse B7-H3 with affinity exceeding M.

[0036] In some embodiments, the target antibody may reduce the binding of B7-H3 to its ligand. For example, in some embodiments, the target antibody reduces the binding of B7-H3 to its ligand or reduces the activity of B7-H3 by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the degree of binding between B7-H3 and its ligand or the activity of B7-H3 in the absence of the antibody.

[0037] The term "antibody" refers to a protein containing one or more (e.g., one or two) heavy chain variable regions (VHs) and / or one or more (e.g., one or two) light chain variable regions (VLs), or subfragments thereof, that can bind to an epitope. The VH and VL regions can be further subdivided into hypervariable regions called "complementarity-determining regions (CDRs)," and are interspersed with more conserved regions called "framework regions (FRs)." The extents of FRs and CDRs are precisely defined (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al. (1987) J.Mol.Biol. 196:901-917). VH can contain three CDRs and four FRs arranged from the N-terminus to the C-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Similarly, VL can contain three CDRs and four FRs arranged from the N-terminus to the C-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0038] The VH or VL chain of an antibody further comprises all or part of the heavy chain or light chain constant region, thereby forming the heavy chain or light chain immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy chains and two light chains, where the heavy and light chains are interconnected, for example, by disulfide bonds. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains include binding regions that interact with the antigen. The constant region of the antibody mediates the binding of the antibody to host tissues and factors, typically including various cells of the immune system and the first component of the complement system. The term “antibody” includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM, and their subtypes. In some embodiments, the antibody of interest is an IgG isotype.

[0039] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, and numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains (about 25 kD or 214 amino acids) are encoded by an N-terminal variable region gene (about 110 amino acids) and a C-terminal kappa or lambda constant region. Full-length immunoglobulin heavy chains (about 50 kD or 446 amino acids) are encoded by an N-terminal variable region gene (about 116 amino acids) and one of the other aforementioned C-terminal constant region genes, for example, gamma (encoding about 330 amino acids). In some embodiments, the target antibody comprises a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0040] In some embodiments, the target antibody does not contain full-length immunoglobulin heavy chains and full-length immunoglobulin light chains, but instead contains antigen-binding fragments of full-length immunoglobulin heavy chains and / or full-length immunoglobulin light chains. In some embodiments, the antigen-binding fragments are contained in a separate polypeptide chain, and in other embodiments, the antigen-binding fragments are contained within a single polypeptide chain. The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that can specifically bind to B7-H3 as described above. Examples of binding fragments include: (i) Fab fragment (a monovalent fragment consisting of VL, VH, CL, and CH1 domains), (ii) F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide crosslink at the hinge region), (iii) Fd fragment (consisting of VH and CH1 domains), (iv) Fv fragment (consisting of VH and VL domains of a single arm of the antibody), (v) dAb fragment (consisting of a VH domain), (vi) isolated CDR, and (vii) single-chain Fv(scFv) (using recombinant means to form a monovalent molecule with VH and VL domain pairs). (viii) a single arm of an antibody conjugated by a synthetic linker (consisting of VH and VL domains), (viii) a diabody (consisting of two scFvs conjugated so that the VH and VL domains of each scFv pair do not form a monovalent molecule, with one VH of each scFv pairing with the VL domain of the other scFv to form a bivalent molecule), and (ix) a bispecific antibody (consisting of at least two antigen-binding regions, each region binding to a different epitope). In some embodiments, the target antibody fragment is a Fab fragment. In some embodiments, the target antibody fragment is a single-chain antibody (scFv).

[0041] In some embodiments, the target antibody is a recombinant or modified antibody, e.g., a chimeric, humanized, deimmunized, or in vitro-produced antibody. As used herein, the terms “recombinant” or “modified” antibody are intended to include all antibodies prepared, expressed, produced, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into host cells, (ii) antibodies isolated from a recombinant combinatorial antibody library, (iii) antibodies isolated from an animal (e.g., mouse) that is transgenic to a human immunoglobulin gene, or (iv) antibodies prepared, expressed, produced, or isolated by other means, including splicing a human immunoglobulin gene sequence with another DNA sequence. Such recombinant antibodies include humanized, CDR-grafted, chimeric, deimmunized, and in vitro-produced antibodies, and may optionally include a constant region derived from a human germline immunoglobulin sequence.

[0042] In some embodiments, the target antibody includes a) a variable domain comprising a heavy chain variable domain including i. a CDR1 region whose amino acid sequence is identical to that of the heavy chain CDR1 region of an antibody selected from the antibodies shown in Figures 1 and 2, ii. a CDR2 region whose amino acid sequence is identical to that of the heavy chain CDR2 region of the selected antibody, and iii. a CDR3 region whose amino acid sequence is identical to that of the heavy chain CDR3 region of the selected antibody; and b) a variable domain comprising a light chain variable domain including i. a CDR1 region whose amino acid sequence is identical to that of the light chain CDR1 region of the selected antibody, ii. a CDR2 region whose amino acid sequence is identical to that of the light chain CDR2 region of the selected antibody sequence, and iii. a CDR3 region whose amino acid sequence is identical to that of the light chain CDR3 region of the selected antibody, wherein the antibody specifically binds to B7-H3 in humans, monkeys, rats, and / or mice.

[0043] In certain embodiments, the antibody comprises (a) a variable domain including i. a CDR1 region having the same amino acid sequence as the heavy chain CDR1 region of an antibody selected from the antibodies shown in Figures 1 and 2, ii. a CDR2 region having the same amino acid sequence as the heavy chain CDR2 region of the selected antibody, and iii. a CDR3 region having the same amino acid sequence as the heavy chain CDR3 region of the selected antibody, as well as a light chain variable domain including i. a CDR1 region having the same amino acid sequence as the light chain CDR1 region of the selected antibody, ii. a CDR2 region having the same amino acid sequence as the light chain CDR2 region of the selected antibody, and iii. a CDR3 region having the same amino acid sequence as the light chain CDR3 region of the selected antibody, or (b) a variant of the variable domain of part (a) that is otherwise identical to the variable domain of part (a) except for up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR region of the variable domain of part (a), and the antibody binds to B7-H3.

[0044] In some embodiments, the antibody may contain only the heavy chain variable domains described herein. In these embodiments, the antibody may be a “heavy chain only” antibody.

[0045] The following consensus sequences were determined using the dendrogram or lineage tree in Figure 3. In any embodiment, the CDR may be contained within any of the consensus sequences in Table 2. For example, an anti-B7-H3 antibody may have a CDR contained within the Group I consensus sequence, Group II consensus sequence, Group III consensus sequence, Group IV consensus sequence, Group V consensus sequence, Group VI consensus sequence, Group VII consensus sequence, Group VIII consensus sequence, or Group IV consensus sequence, as defined in Table 2 below. [Table 2-1] [Table 2-2]

[0046] In some embodiments, the target antibody (e.g., a target antibody that specifically binds to B7-H3) may include a) a light chain region comprising i) one, two, or three complementarity-determining regions (CDRs) from the light chain variable region sequence of the selected anti-B7-H3 antibody, and ii) a light chain framework region, e.g., a framework region from a human immunoglobulin light chain; and b) a heavy chain region comprising i) one, two, or three CDRs from the heavy chain variable region sequence of the selected antibody, and ii) a heavy chain framework region, e.g., a framework region from a human immunoglobulin heavy chain.

[0047] The target antibody may contain a heavy chain variable region that includes an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences shown in Figure 1 and Sequence IDs 1-55. The target antibody may contain a heavy chain variable region that includes one, two, or three heavy chain complementarity determining regions (CDRs) of the selected anti-B7-H3 antibody.

[0048] The target antibody may contain a light chain variable region that includes an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences shown in SEQ ID NOs. The target antibody may contain a light chain variable region that includes one, two, or three light chain CDRs of the selected anti-B7-H3 antibody.

[0049] In some embodiments, the target antibody comprises an anti-B7-H3 antibody heavy chain CDR and an anti-B7-H3 antibody light chain CDR in a single polypeptide chain, for example, in some embodiments, the target antibody is scFv. In some embodiments, the target antibody includes, from the N-terminus to the C-terminus, a first amino acid sequence of approximately 5 to 25 amino acids in length, the light chain CDR1 of the selected anti-B7-H3 antibody, a second amino acid sequence of approximately 5 to 25 amino acids in length, the light chain CDR2 of the selected anti-B7-H3 antibody, a third amino acid sequence of approximately 5 to 25 amino acids in length, the light chain CDR3 of the selected anti-B7-H3 antibody, a fourth amino acid sequence of approximately 5 to 25 amino acids in length, the heavy chain CDR1 of the selected anti-B7-H3 antibody, a fifth amino acid sequence of approximately 5 to 25 amino acids in length, the heavy chain CDR2 of the selected anti-B7-H3 antibody, a sixth amino acid sequence of approximately 5 to 25 amino acids in length, and the heavy chain CDR3 of the selected anti-B7-H3 antibody, a seventh amino acid sequence of approximately 5 to 25 amino acids in length.

[0050] In some embodiments, the target antibody may include, in order from the N-terminus to the C-terminus, the light chain FR1 region, the light chain CDR1 of the selected anti-B7-H3 antibody, the light chain FR2 region, the light chain CDR2 of the selected anti-B7-H3 antibody, the light chain FR3 region, the light chain CDR3 of the selected anti-B7-H3 antibody, optionally the light chain FR4 region, the linker region, optionally the heavy chain FR1 region, the heavy chain CDR1 of the selected anti-B7-H3 antibody, the heavy chain FR2 region, the heavy chain CDR2 of the selected anti-B7-H3 antibody, the heavy chain FR3 region, the heavy chain CDR3 of the selected anti-B7-H3 antibody, and the heavy chain FR4 region. In some of these embodiments, each of the FR regions is a human FR region. The linker region may have a length of approximately 5 to 50 amino acids, for example, approximately 5aa to 10aa, 10aa to 15aa, 15aa to 20aa, 20aa to 25aa, 25aa to 30aa, 30aa to 35aa, 35aa to 40aa, 40aa to 45aa, or 45aa to 50aa.

[0051] Linkers suitable for use with target antibodies include "flexible linkers." If present, the linker molecule is generally long enough to allow some flexible movement between linked regions. Linker molecules are generally about 6 to 50 atoms long. Linker molecules can also be, for example, allylacetylene, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacitates, amino acids, or combinations thereof. Other linker molecules that can be bound to polypeptides may be used in light of this disclosure.

[0052] A suitable linker can be easily selected and may be any of several suitable lengths, including 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0053] An example of a flexible linker is glycine polymer (G) n These include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and therefore can function as neutral tethers between components. Glycine polymers are particularly interesting because glycine has access to far more phi-psi spaces than alanine and is far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that the design of peptides complexed with any of the above elements may include a flexible linker, either entirely or partially, and as a result the linker may include one or more parts that confer a flexible linker as well as a less flexible structure.

[0054] The antibodies shown in Figures 1 and 2 are already human because they are composed primarily of human germline sequences. In some embodiments, the target antibody is “humanized.” The term “humanized antibody” refers to an antibody that contains at least one chain (referred to as acceptor immunoglobulin or antibody) containing variable region framework residues substantially derived from a human body chain and at least one CDR (referred to as donor immunoglobulin or antibody) substantially derived from a mouse antibody. See Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 10033 (1989), U.S. Patents 5,530,101, 5,585,089, 5,693,761, WO90 / 07861, and 5,225,539. The constant region, if present, may be substantially or entirely derived from human immunoglobulin. In some embodiments, the target antibody contains one or more CDRs and one or more FR regions from a human antibody. Methods for producing humanized antibodies are known in the art. See, for example, U.S. Patent No. 7,256,273.

[0055] Replacing mouse CDRs with human variable domain frameworks allows for the preservation of their correct spatial orientation; for example, the human variable domain framework adopts the same or similar three-dimensional structure as the mouse variable framework from which the CDRs are derived. This can be achieved by obtaining human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the mouse variable framework domains from which the CDRs are derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences may be sequences of naturally occurring human antibodies or consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991) and Kolbinger et al., Protein Engineering 6:971 (1993).

[0056] Once the complementarity-determining regions of the mouse donor immunoglobulin and the appropriate human acceptor immunoglobulin have been identified, the next step is to determine which residues of these components, if any, should be substituted to optimize the properties of the resulting humanized antibody. In general, mouse substitutions of human amino acid residues should be kept to a minimum, as the introduction of mouse residues increases the risk that the antibody will induce a human anti-mouse antibody (HAMA) response in humans. Technically accepted methods for determining the immune response can be performed to monitor the HAMA response in a specific patient or during a clinical trial. Patients administered with a humanized antibody can provide an immunogenicity assessment at the start of administration and throughout the administration of the therapy. The HAMA response is measured by detecting antibodies against the humanized therapeutic agent in serum samples from the patient using methods known to those skilled in the art, including, for example, surface plasmon resonance (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the target humanized antibody does not substantially induce a HAMA response in human subjects.

[0057] Specific amino acids from the human variable region framework residues are selected for substitution based on their potential effects on the CDR structure and / or binding to the antigen. Unnatural juxtaposition of the mouse CDR region and the human variable framework region can result in unnatural structural constraints, leading to a loss of binding affinity unless corrected by substitution of specific amino acid residues.

[0058] The selection of amino acid residues for substitution can be determined in part by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are constructed starting from a resolved structure of an immunoglobulin chain or its domain. The chain to be modeled is compared for amino acid sequence similarity with the chain or domain of the resolved three-dimensional structure, and the chain or domain showing the greatest sequence similarity is selected as the starting point for constructing the molecular model. A chain or domain sharing at least 50% sequence identity is selected for modeling, preferably one sharing at least 60%, 70%, 80%, or 90% or more sequence identity. The resolved starting structure is modified to accommodate differences between the actual amino acids of the immunoglobulin chain or modeled domain and the amino acids of the starting structure. The modified structure is then assembled into a complex immunoglobulin. Finally, the model is refined by ensuring energy minimization and that all atoms are at appropriate distances from each other and that bond lengths and angles are within chemically acceptable ranges.

[0059] The CDR and framework region are defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). Alternative structural definitions have been proposed by Chothia et al., J.Mol.Biol.196:901 (1987), Nature 342:878 (1989), and J.Mol.Biol.186:651 (1989) (collectively referred to as "Chothia"). If the framework residues defined by Kabat above constitute the structural loop residues defined by Chothia above, amino acids present in mouse antibodies can be selected for substitution in humanized antibodies. The residues “adjacent to the CDR region” include amino acid residues located directly adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., CDRs as defined by Kabat, or CDRs as defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids interact particularly with amino acids within the CDR and, if selected from the acceptor, can distort the donor CDR and reduce its affinity. Furthermore, adjacent amino acids may directly interact with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to maintain all antigen contacts that provide affinity for the original antibody.

[0060] In some embodiments, the target antibody comprises scFv polymers. For example, in some embodiments, the target antibody is an scFv dimer (e.g., containing two tandem scFv (scFv2)), an scFv trimer (e.g., containing three tandem scFv (scFv3)), an scFv tetramer (e.g., four tandem scFv (scFv4)), or a polymer of more than four scFv (e.g., tandem). The scFv monomers can be linked in tandem via linkers of approximately 2 to 10 amino acids in length, for example, 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, or 10aa. Suitable linkers include, for example, (Gly) x (where x is an integer from 2 to 10) is included. Other preferred linkers are discussed above. In some embodiments, each of the scFv monomers in the scFV polymer of interest is humanized as described above.

[0061] In some embodiments, the target antibody includes a constant region of immunoglobulin (e.g., an Fc region). The Fc region, if present, may be a human Fc region. If a constant region is present, the antibody may contain both a light chain constant region and a heavy chain constant region. Suitable heavy chain constant regions include the CH1, hinge, CH2, CH3, and CH4 regions. Antibodies described herein include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, as well as any isotype, including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is the human isotype IgG1 Fc. The light chain constant region may be lambda or kappa. The target antibody (e.g., a target humanized antibody) may contain sequences from two or more classes or isotypes. Antibodies can be represented as tetramers containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab'F(ab')2, and Fv, or as single-chain antibodies in which the heavy chain variable domain and the light chain variable domain are linked via a spacer.

[0062] In some embodiments, the target antibody contains a free thiol (-SH) group at its carboxyl terminus, which can be used to conjugate the antibody to a second polypeptide (e.g., another antibody containing the target antibody), a scaffold, a carrier, etc.

[0063] In some embodiments, the target antibody contains one or more amino acids that do not exist in nature. In some embodiments, the amino acids that do not exist in nature include carbonyl groups, acetyl groups, aminooxy groups, hydrazine groups, hydrazide groups, semicarbazide groups, azide groups, or alkyne groups. For suitable non-natural amino acids, see, for example, U.S. Patent No. 7,632,924. The inclusion of amino acids that do not exist in nature can provide linkage to polymers, second polypeptides, scaffolds, etc. For example, a target antibody linked to a water-soluble polymer can be prepared by reacting a target antibody containing a non-naturally encoded amino acid, such as one containing an aminooxy, hydrazine, hydrazide, or semicarbazide group, with a water-soluble polymer containing a carbonyl group (e.g., PEG). As another example, a target antibody linked to a water-soluble polymer can be prepared by reacting a target antibody containing an alkyne-containing amino acid with a water-soluble polymer containing an azide moiety (e.g., PEG), in which case, the azide or alkyne group is linked to the PEG molecule via an amide bond. "Non-naturally coded amino acids" refers to amino acids that are not one of the 20 common amino acids or pyrrolicin or selenocysteine. Other terms that may be used synonymously with "non-naturally coded amino acids" include "non-natural amino acid," "unnatural amino acid," "non-naturally-occurring amino acid," and various hyphenated and unhyphenated versions. The term "non-naturally coded amino acids" also includes, but is not limited to, amino acids resulting from modifications of naturally coded amino acids (e.g., post-translational modifications) (including, but not limited to, the 20 common amino acids or pyrrolicin and selenocysteine) that are not naturally incorporated into the growing polypeptide chain by the translation complex.Examples of such non-natural amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0064] In some embodiments, the target antibody is linked (e.g., covalently) to a polymer (e.g., a polymer other than a polypeptide). Suitable polymers include, for example, biocompatible polymers and water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and natural polymers. Suitable polymers include, for example, substituted or unsubstituted linear or branched polyalkylene, polyalkene, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homo or heteropolysaccharides.Suitable polymers include, for example, ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or trade name EVAL), polybutyl methacrylate, poly(hydroxyvaleric acid), poly(L-lactic acid), polycaprolactone, poly(lactide-co-glycolide), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid), poly(D,L-lactic acid), poly(glycolic acid-co-trimethylene carbonate), Polyphosphoesters, polyphosphoester urethanes, poly(amino acids), cyanoacrylates, poly(trimethylene carbonates), poly(iminocarbonates), copolymers (ether-esters) (e.g., poly(ethylene oxide)-poly(lactic acid)(PEO / PLA) copolymers), polyalkylene oxalates, polyphosphazenes; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethanes, silicones, polyesters, polyolefins, polyisobutylenes and ethylene-ammonium compounds. This includes olefin copolymers, acrylic polymers and copolymers; halogenated vinyl polymers and copolymers such as polyvinyl chloride; halogenated polyvinylidenes such as polyvinyl methyl ether, polyvinylidene fluoride, and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatics such as polystyrene; polyvinyl esters such as polyvinyl acetate; copolymers of vinyl monomers and olefins, such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, and ethylene-vinyl acetate copolymer; polyamides such as nylon 66 and polycaprolactam; alkyd resins, polycarbonates, polyoxymethylene, polyimides, polyethers, epoxy resins, polyurethanes, rayons, rayon triacetates, cellulose, cellulose acetate, cellulose butyrate, cellulose acetate-butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, amorphous teflon, poly(ethylene glycol), and carboxymethylcellulose.

[0065] Suitable synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and their derivatives, such as substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol), and their derivatives. Suitable natural polymers include, for example, albumin, amylose, dextran, glycogen, and their derivatives.

[0066] Suitable polymers may have an average molecular weight in the range of 500 Da to 50000 Da, for example, 5000 Da to 40000 Da, or 25000 to 40000 Da. For example, in some embodiments in which the target antibody comprises a poly(ethylene glycol) (PEG) or methoxypoly(ethylene glycol) polymer, the PEG or methoxypoly(ethylene glycol) polymer may have a molecular weight in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa, or 40 kDa to 60 kDa.

[0067] As described above, in some embodiments, the target antibody is covalently bonded to the PEG polymer. In some embodiments, the target scFv polymer is covalently bonded to the PEG polymer. See, for example, Albrecht et al. (2006) J. Immunol. Methods 310:100. Suitable methods and reagents for protein PEGylation are well known in the art and can be found, for example, in U.S. Patent No. 5,849,860. PEG suitable for complexing with proteins is generally water-soluble at room temperature and has the general formula R(O-CH2-CH2) n The formula has OR, where R is a hydrogen atom or a protecting group such as an alkyl group or alkanol group, and n is an integer from 1 to 1000. If R is a protecting group, it generally has 1 to 8 carbon atoms.

[0068] PEG complexed with a target antibody may be linear. PEG complexed with a target protein may also be branched. Examples include branched PEG derivatives, such as those described in U.S. Patent No. 5,643,575, "Star-shaped PEG," and multi-armed PEGs, such as those described in the Shearwater Polymers, Inc. catalog, "Polyethylene Glycol Derivatives 1997-1998." Star-shaped PEG is described, for example, in the art including U.S. Patent No. 6,046,305.

[0069] The target antibody can be glycosylated and may include, for example, a covalently bonded carbohydrate or polysaccharide moiety. Antibody glycosylation is typically either N-linked or O-linked. N-linking refers to the binding of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic binding of a carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0070] The addition of a glycosylation site to an antibody is conveniently achieved by altering the amino acid sequence to include one or more of the tripeptide sequences (in the case of an N-linked glycosylation site) described above. The alteration can also be performed by adding or substituting one or more serine or threonine residues into the original antibody sequence (in the case of an O-linked glycosylation site). Similarly, the removal of a glycosylation site can be achieved by altering the amino acids within the native glycosylation site of the antibody.

[0071] The antibodies in question, in some embodiments, include "radiopaque" labels, such as labels that can be easily visualized using X-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodide salts, bromide salts, or barium salts. Other radiopaque materials are also known, including, but are not limited to, organobismuth derivatives (see, e.g., U.S. Patent No. 5,939,045), radiopaque multiurethanes (see, e.g., U.S. Patent No. 5,346,981), organobismuth composites (see, e.g., U.S. Patent No. 5,256,334), and radiopaque barium polymer complexes (see, e.g., U.S. Patent No. 4,866,132).

[0072] The target antibody can be covalently bonded to a second site (e.g., lipids, polypeptides other than the target antibody, synthetic polymers, carbohydrates, etc.) using, for example, glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. Glutaraldehyde crosslinks polypeptides via the amino moiety. Homobifunctional crosslinkers (e.g., homobifunctional imide esters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. Homobifunctional NHS esters and imide esters crosslink amine-containing polypeptides. At a weakly alkaline pH, imide esters react only with primary amines to form imidoamides, and the overall charge of the crosslinked polypeptide remains unaffected. Homobifunctional sulfhydryl reactive crosslinking agents include bismaleimidehexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propinoamidobutane (DPDPB).

[0073] Heterobifunctional crosslinkers have two or more different reactive moieties (e.g., an amine-reactive moiety and a sulfhydryl-reactive moiety) and crosslink with one polypeptide via the amine or sulfhydryl-reactive moiety, and then react with the other polypeptide via the unreacted moiety. Similar to pyridyl disulfide crosslinkers, multiple heterobifunctional haloacetyl crosslinkers are available. Carbodiimides are a typical example of heterobifunctional crosslinking reagents for coupling carboxyls to amines, resulting in amide bonds.

[0074] The target antibody can be immobilized on a solid support. Suitable supports are well known in the art and include, among others, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and silicon surfaces, nitrocellulose strips, nylon membranes, sheets, Durasite, as well as reaction trays (e.g., multiwell plates), plastic tubes, etc. The solid support may include any of the following materials, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. Suitable methods for immobilizing the target antibody on a solid support are well known and include, but are not limited to, ionic, hydrophobic, and covalent interactions. The solid support may be soluble or insoluble in aqueous solutions, for example. In some embodiments, a suitable solid support is generally insoluble in aqueous solutions.

[0075] The target antibody will, in some embodiments, include a detectable label. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable examples include magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), and radioactive labels (e.g.,3 H, 125 I, 35 S, 14 C, or 32 P) includes, but is not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and others commonly used in enzyme-linked immunosorbent assays (ELISA)), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0076] In some embodiments, the target antibody comprises a contrast agent or radioisotope, the contrast agent or radioisotope being suitable for use in imaging, for example, imaging procedures performed in humans. Non-limiting examples of labeling include: 1231 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium), and 67 It contains radioactive isotopes such as Ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotope ( 153Gd) is also available and is suitable for imaging procedures in non-human mammals. The target antibody can be labeled using standard techniques. For example, the target antibody can be iodized using chloramine T or 1,3,4,6-tetrachloro-3α,6α-dephenylglycouryl. In the case of fluorination, fluorine is added to the target antibody during synthesis by a fluoride ion substitution reaction. For an overview of protein synthesis with such radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999). The target antibody can also be labeled with a contrast agent through standard methods. For example, the target antibody can be labeled with Gd by conjugating it with a low-molecular-weight Gd chelate such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA). See Caravan et al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). The target antibody can also be labeled with Gd by conjugating it with a polylysine-Gd chelate. See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998). Alternatively, the target antibody can be labeled with Gd by incubating paramagnetically polymerized liposomes containing a Gd chelating lipid with avidin and biotinylated antibodies. For example, see Sipkins et al., Nature Med., 4:623-626 (1998).

[0077] Suitable fluorescent proteins that can be linked to the target antibody include, for example, the green fluorescent protein or its variants or derivatives from Aequoria victoria, as described in U.S. Patent No. 6,066,476, e.g., those described in U.S. Patents No. 6,020,192, 5,985,577, 5,976,796, 5,968,750, 5,968,738, 5,958,713, 5,919,445, and 5,874,304; enhanced GFP, e.g., many such GFPs commercially available from Clontech, Inc., e.g., the red fluorescent protein, yellow fluorescent protein, or any of the various fluorescent and colored proteins described in Matz et al., (1999) Nature Biotechnol 17:969-973, e.g., but not limited to these.

[0078] In some embodiments, the target antibody is linked (e.g., covalently or non-covalently) to a fusion partner, such as a ligand, epitope tag, peptide, or protein other than the antibody. Suitable fusion partners include peptides and polypeptides that provide improved in vivo stability (e.g., improved serum half-life) and those that provide ease of purification, such as (His) n These include, for example, those that provide the secretion of fusion proteins from cells, such as 6His; epitope tags such as GST, hemagglutinins, FLAG, and c-myc; detectable signals, such as enzymes that produce detectable products (e.g., β-galactosidase, luciferase); or proteins that are themselves detectable, such as green fluorescent protein, red fluorescent protein, and yellow fluorescent protein; and multimerization, such as those that provide multimerization domains, such as the Fc portion of immunoglobulins.

[0079] The fusion may also include affinity domains containing peptide sequences that can interact with the binding partner, for example, immobilized on a solid support, which is useful for identification or purification. Consecutive single amino acids, such as histidine, when fused to a protein, can be used for one-step purification of the fusion protein by binding with high affinity to resin columns such as nickel Sepharose. Exemplary affinity domains include His5, HisX6, C-myc, Flag, StrepTag, hemagglutinin, glutathione-S-transferase (GST), thioredoxin, cellulose-binding domains, chitin-binding domains, S-peptides, T7 peptides, SH2 domains, C-terminal RNA tags, metal-binding domains such as zinc-binding domains, or calcium-binding domains from calcium-binding proteins such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, bicinin, VILIP, neurocalcin, hypocalcin, frekenin, caltractin, calpain large subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, and calretinin; intein, biotin, streptavidin, MyoD, leucine zipper sequences, and maltose-binding proteins.

[0080] In some embodiments, the target antibody will be fused to a polypeptide that binds to an endogenous blood-brain barrier (BBB) ​​receptor. Linking the target antibody to a polypeptide that binds to an endogenous BBB receptor facilitates its passage across the BBB in targeted therapies (see below), for example, that involve administering the target antibody to an individual requiring it. Suitable polypeptides that bind to the endogenous BBB include antibodies that specifically bind to endogenous BBB receptors, such as monoclonal antibodies or their antigen-binding fragments. Suitable endogenous BBB receptors include, but are not limited to, insulin receptors, transferrin receptors, leptin receptors, lipoprotein receptors, and insulin-like growth factor receptors. See, for example, U.S. Patent Publication No. 2009 / 0156498.

[0081] In some embodiments, the target antibody includes polyamine modification. Polyamine modification of the target antibody increases the permeability of the modified antibody at the blood-brain barrier (BBB). The target antibody can be modified with polyamines that are either natural or synthetic. See, for example, U.S. Patent No. 5,670,477. Useful natural polyamines include putrescine, spermidine, spermine, 1,3-deaminopropane, norspermidine, syn-homospermidine, theremine, thermospermine, cardopentamine, homocardopentamine, and canavalmin. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines include those with empirical formula C X H Y N Z Polyamines can be cyclic or acyclic, branched or unbranched hydrocarbon chains of 3 to 12 carbon atoms, comprising 1 to 6 NR or N(R)2 moieties (wherein R is H, (C1-C4) alkyl, phenyl, or benzyl). Polyamines can be linked to antibodies using any standard crosslinking method.

[0082] In some embodiments, the target antibody is modified to include a carbohydrate moiety, which may be covalently bound to the antibody. In some embodiments, the target antibody is modified to include a lipid moiety, which may be covalently bound to the antibody. Suitable lipid moieties include, for example, N-lipid acyl groups such as N-lauroyl and N-oleoyl, fatty amines such as dodecylamine and oleoylamine, and C3-C16 long-chain aliphatic lipids. (See, for example, U.S. Patent No. 6,638,513). In some embodiments, the target antibody is incorporated into liposomes.

[0083] Method for generating target antibodies The target antibody can be produced by any known method, such as conventional synthesis methods for protein synthesis or recombinant DNA methods.

[0084] If the target antibody is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When polypeptides are chemically synthesized, the synthesis can proceed via liquid or solid phase. Solid-phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is bound to an insoluble support, and the remaining amino acids are sequentially added to the sequence, is an example of a preferred method for chemically synthesizing a target antibody. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing components. Solid-phase synthesis techniques are described in Barany and Merrifield, Solid-Phase Peptide Synthesis, The Peptides: Analysis, Synthesis, Biology, Vol.2: Special Methods in Peptide Synthesis, Part A, pp.3-284, Merrifield, et al., J.Am.Chem.Soc., 85:2149-2156 (1963), Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem.Co., Rockford, Ill. (1984), as well as Ganesan A. 2006 Mini Rev.Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8. Briefly, small, insoluble porous beads can be processed with functional units on which peptide chains are constructed. After repeating the coupling / deprotection cycle, the bound free N-terminal amine in the solid phase is coupled to a single N-protected amino acid unit. This unit is then deprotected to expose a new N-terminal amine, to which further amino acids can be attached. The peptide remains immobilized on the solid phase and undergoes a filtration process before being cleaved.

[0085] Standard recombination methods can be used to produce the target antibody. For example, nucleic acids encoding the variable regions of the light and heavy chains are sometimes ligated to a constant region and inserted into the expression vector. The light and heavy chains can be cloned in the same or different expression vectors. DNA segments encoding immunoglobulin chains are operably ligated to a regulatory sequence in the expression vector that ensures the expression of the immunoglobulin polypeptide. The regulatory sequence includes, but is not limited to, a promoter (e.g., a native-related promoter or a heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. The regulatory sequence may be a eukaryotic cell promoter system in the vector that can transform or transfect eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for antibody collection and purification.

[0086] Due to coding degeneracy, various nucleic acid sequences can encode each immunoglobulin amino acid sequence. Desired nucleic acid sequences can be generated by novel solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of previously prepared variants of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a preferred method for preparing substitution, deletion, and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, the target polypeptide DNA is modified by hybridizing an oligonucleotide encoding the desired mutation into a single-stranded DNA template. After hybridization, DNA polymerase is used to synthesize the entire second-phase complementary strand of the template incorporating the oligonucleotide primers, encoding the selected modification of the target polypeptide DNA.

[0087] Suitable expression vectors are typically replicable within the host organism as episomes or as an integral part of host chromosomal DNA. Generally, expression vectors include selection markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, neomycin resistance, etc.) to enable the detection of these cells transformed with the desired DNA sequence.

[0088] Escherichia coli is an example of a prokaryotic host cell that can be used for cloning polynucleotides encoding target antibodies. Other suitable microbial hosts include bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can also be constructed in these prokaryotic hosts, which will typically contain expression regulatory sequences (e.g., origins of replication) compatible with the host cell. Furthermore, there will be any number of different well-known promoters, such as lactose promoter systems, tryptophan (TRP) promoter systems, β-lactamase promoter systems, or λ-phage-derived promoter systems. Promoters will typically contain ribosome-binding sites, etc., to control expression, sometimes using operator sequences, and to initiate and complete transcription and translation.

[0089] Other microorganisms, such as yeast, are also useful for expression. The genera Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells containing suitable vectors, which may include expression regulatory sequences (e.g., promoters), origins of replication, termination sequences, etc., as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization.

[0090] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and generate the polypeptides of this disclosure (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may contain regulatory sequences such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Examples of suitable regulatory sequences include promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papillomavirus, cytomegalovirus, etc. See Co et al., J.Immunol. 148:1149 (1992).

[0091] Once synthesized (chemically or recombinantly), the entire antibody, its dimers, individual light and heavy chains, or other forms of the target antibody (e.g., scFv) can be purified according to standard procedures in the art, including sulfate precipitation, affinity column, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (see Scopes, Protein Purification (Springer-Verlag, NY, (1982))). The control antibody may be substantially pure, for example, at least about 80%–85% pure, at least about 85%–90% pure, at least about 90%–95% pure, or 98%–99% or more pure, and may not contain contaminants such as cell fragments or macromolecules other than the control antibody.

[0092] composition This disclosure provides a composition comprising a target antibody. In addition to the control antibody, the control antibody compound may contain one or more of the following: salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (e.g., Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonate sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solvents, detergents (e.g., nonionic detergents such as Tween-20), protease inhibitors, glycerol, etc.

[0093] nucleic acid This disclosure provides nucleic acids comprising a nucleotide sequence encoding a target antibody. The nucleotide sequence encoding the target antibody can be operably ligated to one or more regulatory elements, such as promoters and enhancers, thereby enabling the expression of the nucleotide sequence in a target cell of interest (e.g., a genetically modified cell for the synthesis of an encoded antibody).

[0094] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambdaP, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements, the cytomegalovirus early promoter, the herpes simplex virus thymidine kinase promoter, the early and late SV40 promoter, promoters present in the terminal repeat sequences of retroviruses, the mouse metallothionein-I promoter, and various tissue-specific promoters known in the art.

[0095] In some embodiments, suitable promoters for expression in yeast cells, for example, are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, and PYK1 promoter, or regulated promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHO5 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (for example, for use in Pichia). The selection of appropriate vectors and promoters is within the realm of those skilled in the art.

[0096] Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter, trp promoter, lac operon promoter, hybrid promoters such as lac / tac hybrid promoter, tac / trc hybrid promoter, trp / lac promoter, T7 / lac promoter, trc promoter, tac promoter, etc., araBAD promoter, in vivo promoters such as ssaG promoter or related promoters (see, for example, U.S. Patent Publication No. 2004 / 0131637), pagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991:173(1):86-93, Alpuche-Aranda et al., PNAS, 1992;89(21):10079-83), nirB promoter (Harborne et al. (1992) Mol. Micro. 6:2805-2813), etc. (e.g., Dunstan et al.) See al. (1999) Infect.Immun. 67:5133-5141, McKelvie et al. (2004) Vaccine 22:3243-3255, and Chatfield et al. (1992) Biotechnol. 10:888-892), sigma70 promoters, e.g., consensus sigma70 promoters (see GenBank deposit numbers AX798980, AX798961, and AX798183), stationary phase promoters, e.g., dps promoter, spv promoter, etc., promoters derived from pathogenic island SPI-2 (see WO96 / 17951), actA promoters (see Shetron-Rama et al. See al. (2002) Infect. Immun. 70:1087-1096), rpsM promoter (e.g., see [1] Valdivia and Falkow {2] ([3] 1996 {4]) [1] Mol. Microbiol. {2] [1] 22 {2]:[3] 367), tet promoter (e.g., {4] Hillen, W. and Wissmann, A. (1989), Saenger, W. and Heinemann, U.See (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol.10, pp.143-162), SP6 promoter (see, for example, Melton et al. (1984) Nucl. Acids Res. 12:7035), etc. Strong promoters suitable for use in prokaryotes such as E. coli include Trc, Tac, T5, T7, and P. Lambda This includes, but is not limited to, these. 。 Non-exclusive examples of operators for use in bacterial host cells include lactose promoter operators (where the LacI repressor protein changes its conformation upon contact with lactose, thereby preventing the LacI repressor protein from binding to the operator), tryptophan promoter operators (where the TrpR repressor protein has a conformation that binds to the operator when complexed with tryptophan, and a conformation that does not bind to the operator when tryptophan is absent), and tac promoter operators (e.g., deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25).

[0097] The nucleotide sequence encoding the target antibody can be present in the expression vector and / or cloning vector. If the target antibody contains two distinct polypeptides, the nucleotide sequences encoding the two polypeptides can be cloned into the same or distinct vectors. The expression vector may include selectable markers, origins of replication, and other features that provide vector replication and / or maintenance.

[0098] Numerous suitable vectors and promoters are known to those skilled in the art, and many are commercially available for generating the target recombinant constructs. The following vectors are provided as examples: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif, USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).

[0099] Expression vectors generally have a convenient restriction site located near the promoter sequence to provide insertion of nucleic acid sequences encoding heterologous proteins. Selectable markers that can function in the expression host may also exist. Suitable expression vectors include viral vectors (e.g., vaccine viruses, poliovirus; adenoviruses (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655)); and adeno-associated viruses (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998; Flannery et al., PNAS 94:6916 6921,1997, Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863,1997, Jomary et al., Gene Ther 4:683 690,1997, Rolling et al., Hum Gene Ther 10:641 648,1999, Ali et al. al., Hum Mol Genet 5:591 594,1996, Srivastava's WO93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828, Mendelson et al., Virol. (1988) 166:154-165, and Flotte et al. See al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997, Takahashi et al.)This includes, but is not limited to, retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus, and retroviral-derived viruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0100] As described above, the target nucleic acid includes a nucleotide sequence encoding the target antibody. The target nucleic acid may include nucleotide sequences encoding heavy chain and light chain CDRs. In some embodiments, the target nucleic acid includes nucleotide sequences encoding heavy chain and light chain CDRs, and these CDR-encoding sequences are interspersed with nucleotide sequences encoding FRs. In some embodiments, the nucleotide sequences encoding FRs are human FR-encoding nucleotide sequences.

[0101] cell This disclosure provides isolated genetically modified host cells (e.g., in vitro cells) genetically modified with a target nucleic acid. In some embodiments, the isolated genetically modified host cells can produce a target antibody.

[0102] Suitable host cells include eukaryotic host cells such as mammalian cells, insect host cells, and yeast cells, and prokaryotic cells such as bacterial cells. The introduction of the target nucleic acid into host cells can be achieved, for example, by calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

[0103] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian host cells include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., ATCC number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human fetal kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, etc.

[0104] Suitable yeast cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, and Chlamydomonas reinhardtii.

[0105] Suitable prokaryotic cells include, but are not limited to, any of the various laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., etc. See, for example, Carrier et al. (1992) J. Immunol 148:1176-1181, U.S. Patent No. 6,447,784, and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains that can be used in this invention include, but are not limited to, Salmonella typhi and Salmonella typhimurium. Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Laboratory strains are usually non-pathogenic. Other suitable bacteria, though not limited to these, include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and Rhodococcus sp. In some embodiments, the host cell is Escherichia coli.

[0106] Formulations and pharmaceutical compositions This disclosure provides compositions comprising a pharmaceutical composition containing a target antibody. Generally, the formulation contains an effective amount of the target antibody. "Effective amount" means a dose sufficient to produce a desired outcome, such as a reduction in tumor size or improvement in symptoms. Generally, the desired outcome is at least a reduction in cancer symptoms compared to a control. The target antibody can be delivered in a manner that circumvents the blood-brain barrier, as described in more detail below. The target antibody can be formulated and / or modified, if necessary, to allow the antibody to cross the blood-brain barrier. Therapy involves treating a patient already suffering from a disease to produce therapeutically beneficial effects such as improvement of existing symptoms, improvement of the underlying metabolic causes of symptoms, delay or prevention of further onset of the disorder, and / or reduction of the severity of symptoms that will develop or are expected to develop.

[0107] formulation In the method described, the target antibody can be administered to the host using any convenient means capable of producing the desired therapeutic or diagnostic effect. Therefore, the drug can be incorporated into various formulations for therapeutic administration. More specifically, the target antibody can be formulated into pharmaceutical compositions in combination with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0108] In pharmaceutical dosage forms, the target antibodies may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone or in appropriate associations and combinations with other pharmaceutically active compounds. The following methods and excipients are merely examples and are not limiting.

[0109] For oral preparations, conventional additives such as lactose, mannitol, corn starch, or potato starch, binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin, disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose, lubricants such as talc or magnesium stearate, and optionally diluents, buffers, wetting agents, preservatives, and flavoring agents may be used alone or in combination with appropriate additives to produce tablets, powders, granules, or capsules.

[0110] The target antibodies can be formulated into injectable preparations by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and, if desired, using common additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0111] A pharmaceutical composition containing the target antibody can be prepared by mixing the antibody of the desired purity with an optional physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer and / or isotonic agent. Acceptable carriers, excipients and / or stabilizers are non-toxic to the recipient at the dosage and concentration used, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propylparaben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, and isoleucine. This includes amino acids such as leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other sugars; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronic, Triton-X, or polyethylene glycol (PEG).

[0112] Pharmaceutical compositions may be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form, and lyophilized preparations will be reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add a certain amount of pure water (typically equivalent to the volume removed during lyophilization), although a solution containing an antimicrobial agent may be used to produce pharmaceutical compositions for parenteral administration. See also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0113] Exemplary antibody concentrations in the pharmaceutical composition in question may range from approximately 1 mg / mL to approximately 200 mg / mL, or from approximately 50 mg / mL to approximately 200 mg / mL, or from approximately 150 mg / mL to approximately 200 mg / mL.

[0114] Aqueous antibody formulations can be prepared in a pH buffer solution at a pH range of, for example, about 4.0 to about 7.0, or about 5.0 to about 6.0, or at about 5.5. Examples of buffers suitable within this pH range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The concentration of the buffer may be, for example, about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on the desired tonicity of the buffer and formulation.

[0115] To adjust the tonicity of the formulation, isotonic agents may be included in the antibody formulation. Exemplary isotonic agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. While hypertonic or hypotonic solutions may be suitable in some embodiments, aqueous formulations are isotonic. The term "isotonic" means a solution that, when compared, has the same tonicity as some other solution, such as physiological saline or serum. Isotonic agents may be used in amounts of about 5 mM to about 350 mM, for example, in amounts of 100 mM to 350 nM.

[0116] Surfactants may also be added to antibody formulations to reduce aggregation of the formulated antibody and / or to minimize the formation of particulate matter in the formulation and / or to reduce adsorption. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of preferred polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (marketed under the trademark Tween 20®) and polysorbate 80 (marketed under the trademark Tween 80®). Examples of preferred polyethylene-polypropylene copolymers are those marketed under the names Pluronic® F68 or Poloxamer 188®. Examples of preferred polyoxyethylene alkyl ethers are those marketed under the trademark Brij®. Examples of surfactant concentrations can range from approximately 0.001% to approximately 1% w / v.

[0117] To protect the active ingredients (e.g., proteins) from destabilizing conditions during the freeze-drying process, cryoprotectants may also be added. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant may be included in an amount of about 10 mM to 500 nM.

[0118] In some embodiments, the composition of interest comprises the antibody of interest and one or more of the agents defined above (e.g., surfactants, buffers, stabilizers, isotonic agents), and essentially does not contain one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propylparaben, benzalkonium chloride, and combinations thereof. In other embodiments, the preservative is included in the formulation at a concentration ranging, for example, from about 0.001% to about 2% (w / v).

[0119] For example, the target formulation may be a liquid or lyophilized formulation suitable for parenteral administration, and may contain approximately 1 mg / mL to approximately 200 mg / mL of the target antibody, approximately 0.001% to approximately 1% of at least one surfactant, approximately 1 mM to approximately 100 mM of a buffer, optionally approximately 10 mM to approximately 500 mM of a stabilizer, approximately 5 mM to approximately 305 mM of a tonic agent, and have a pH of approximately 4.0 to approximately 7.0.

[0120] As another example, the parenteral formulation in question is a liquid or lyophilized formulation containing approximately 1 mg / mL to 200 mg / mL of the target antibody, 0.04% Tween20 w / v, 20 mM L-histidine, and 250 mM sucrose, with a pH of 5.5.

[0121] As another example, the parenteral formulations in question include the following lyophilized formulations: 1) containing 15 mg / mL of the target antibody, 0.04% Tween 20 w / v, 20 mM L-histidine, and 250 mM sucrose, with a pH of 5.5; or 2) containing 75 mg / mL of the target antibody, 0.04% Tween 20 w / v, 20 mM L-histidine, and 250 mM sucrose, with a pH of 5.5; or 3) containing 75 mg / mL of the target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine, and 250 mM sucrose, with a pH of 5.5; or 4) containing 75 mg / mL of the target antibody, 0.04% Tween 20 The solution contains w / v, 20 mM L-histidine and 250 mM trehalose, with a pH of 5.5, or 6) 75 mg / mL of the target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine and 250 mM trehalose, with a pH of 5.5.

[0122] As another example, the parenteral formulations in question are the following liquid formulations: 1) 7.5 mg / mL of the target antibody, 0.022% Tween 20 w / v, 120 mM L-histidine, and 250 mM sucrose, with a pH of 5.5; or 2) 37.5 mg / mL of the target antibody, 0.02% Tween 20 w / v, 10 mM L-histidine, and 125 mM sucrose, with a pH of 5.5; or 3) 37.5 mg / mL of the target antibody, 0.01% Tween 20 w / v, 10 mM L-histidine, and 125 mM sucrose, with a pH of 5.5; or 4) 37.5 mg / mL of the target antibody, 0.02% Tween 20 5) Contains w / v, 10 mM L-histidine, 125 mM trehalose, and pH 5.5, or 6) Contains 37.5 mg / mL of target antibody, 0.01% Tween 20 w / v, 10 mM L-histidine, and 125 mM trehalose, and pH 5.5, or 7) Contains 5 mg / mL of target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine, and 250 mM trehalose, and pH 5.5, or 8) Contains 75 mg / mL of target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine, and 250 mM mannitol, and pH 5.5, or 8) Contains 75 mg / mL of target antibody, 0.02% Tween 20 1) A solution containing w / v, 20 mM L-histidine and 140 mM sodium chloride, with a pH of 5.5, or 9) 150 mg / mL of target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine and 250 mM trehalose, with a pH of 5.5, or 10) 150 mg / mL of target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine and 250 mM mannitol, with a pH of 5.5, or 11) 150 mg / mL of target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine and 140 mM sodium chloride, with a pH of 5.5, or 12) 10 mg / mL of target antibody, 0.01% Tween 20 It contains w / v, 20 mM L-histidine, and 40 mM sodium chloride, with a pH of 5.5.

[0123] The target antibody can be used in aerosol formulations administered by inhalation. The target antibody can be incorporated into pressurized, acceptable sprays such as dichlorodifluoromethane, propane, and nitrogen.

[0124] Furthermore, the target antibody can be made into a suppository by mixing it with various substrates such as emulsifying substrates and water-soluble substrates. The target antibody can be administered rectally via the suppository. The suppository may contain a vehicle such as cocoa butter, carbowx, or polyethylene glycol that melts at body temperature but solidifies at room temperature.

[0125] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided such that each dose unit, e.g., one teaspoon, one tablespoon, a tablet, or a suppository, contains a predetermined amount of composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the target antibody in the composition as a solution in sterile water, saline solution, or another pharmaceutically acceptable carrier.

[0126] The term "unit dosage form," as used herein, refers to physically distinct units suitable as single doses for human and animal subjects, each unit containing a predetermined amount of the compound of the present invention calculated in an amount sufficient to produce the desired effect in conjunction with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of the target antibody may depend on the specific antibody used and the effect to be achieved, as well as the pharmacodynamics associated with each antibody in the host.

[0127] Other modes of administration may also find use in the present invention. For example, the target antibody can be formulated as a suppository, and possibly as an aerosol and intranasal composition. In the case of a suppository, the vehicle composition includes conventional binders and carriers such as polyalkylene glycol or triglycerides. Such suppositories can be formed from a mixture containing about 0.5% to about 10% (w / w) of the active ingredient, for example, in the range of about 1% to about 2%.

[0128] Intranasal formulations typically contain a vehicle that does not cause irritation to the nasal mucosa and does not significantly disrupt ciliary function. Diluents such as water, saline solution, or other known substances can be used in this invention. Intranasal formulations may also contain, but are not limited to, preservatives such as chlorobutanol and benzalkonium chloride. Surfactants may be present to enhance the absorption of the target protein by the nasal mucosa.

[0129] The target antibody can be administered as an injectable formulation. Typically, the injectable composition is prepared as a liquid solution or suspension, and a solid form suitable for dissolution or suspension in a liquid vehicle before injection may also be prepared. The preparation can also be emulsified, or the antibody can be encapsulated in a liposome vehicle.

[0130] Suitable excipient vehicles include, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. Furthermore, if desired, the vehicle may contain small amounts of auxiliary substances such as wetting agents, emulsifiers, or pH buffers. Practical methods for preparing such dosage forms are known or will become apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The administered composition or formulation will, in any case, contain an appropriate amount of the target antibody to achieve the desired state in the subject being treated.

[0131] Pharmacovigilant excipients such as vehicles, adjuvants, carriers, or diluents are generally readily available. Furthermore, pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, stabilizers, and wetting agents are generally readily available.

[0132] In some embodiments, the target antibody is formulated in a controlled-release formulation. Sustained-release preparations can be prepared using methods well known in the art. Preferred examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing the antibody, the matrix being in the form of a molded article, e.g., a film or microcapsules. Examples of sustained-release matrices include polyesters, L-glutamic acid and ethyl-L-glutamic acid copolymers, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Potential loss of biological activity and changes in immunogenicity of antibodies contained in sustained-release preparations can be prevented by using appropriate additives, controlling water content, and developing specific polymer matrix compositions.

[0133] Within the scope of the present invention, controlled release can be interpreted as meaning any one of several extended-release dosage forms. The following terms may be considered substantially equivalent to controlled release for the purposes of the present invention: continuous release, controlled release, delayed release, depot, stepwise release, long-term release, programmed release, sustained release, proportional release, prolonged release, repository, delayed, sustained release, interval release, maintenance release, time coat, timed release, delayed action, extended action, layered timed release, long-acting, prolonged action, repeated action, delayed action, sustained action, sustained-acting drug, and extended release. Details of these terms can be found in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0134] Various controlled-release technologies cover a very wide range of drug dosage forms. Controlled-release technologies include, but are not limited to, physical and chemical systems.

[0135] Physical systems include, but are not limited to, reservoir systems with rate-controlled membranes such as microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-controlled membranes such as hollow fibers, ultramicroporous cellulose triacetate, porous polymer substrates, and foams; monolithic systems including systems physically dissolved in a non-porous, polymer, or elastomer matrix (e.g., non-corrosive, erosive, permeable to environmental factors, and degradable); and laminated structures including a reservoir layer that is chemically similar to or different from an outer control layer, with materials physically dispersed in a non-porous, polymer, or elastomer matrix (e.g., non-corrosive, erosive, permeable to environmental factors, and degradable); and other physical methods such as osmotic pumps or adsorption to ion exchange resins.

[0136] Chemical systems include, but are not limited to, chemical erosion of the polymer matrix (e.g., heterogeneous or homogeneous erosion) or biological erosion of the polymer matrix (e.g., heterogeneous or homogeneous). Further discussion of the category of controlled release systems can be found in Agis F. Kydonieus, Controlled Release Technologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

[0137] Several controlled-release formulations have been developed for oral administration. These include, but are not limited to, osmotically controlled gastrointestinal delivery systems, hydrodynamically controlled gastrointestinal delivery systems, membrane permeability controlled gastrointestinal delivery systems including microporous membrane permeability controlled gastrointestinal delivery devices, controlled-release gastrointestinal delivery devices targeting gastric juice-resistant intestines, gel diffusion controlled gastrointestinal delivery systems, and ion exchange controlled gastrointestinal delivery systems including cationic and anionic drugs. Additional information on controlled-release drug delivery systems is described in Yie W. Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.). Some of these formulations are discussed herein.

[0138] Dosage The appropriate dosage can be determined by the attending physician or other qualified healthcare professional based on various clinical factors. As is well known in medicine, the dosage for one patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, the patient's sex, time, and route of administration, health, and other medications being administered concurrently. The target antibody can be administered in amounts ranging from 1 ng / kg body weight to 20 mg / kg body weight per dose, for example, 0.1 mg / kg body weight to 10 mg / kg body weight, for example, 0.5 mg / kg body weight to 5 mg / kg body weight, although doses below or above this exemplary range are assumed, especially considering the factors mentioned above. If the regimen is a continuous infusion, it can also range from 1 μg to 10 mg per kilogram of body weight per minute.

[0139] Those skilled in the art will readily understand that dose levels may vary as a function of the specific antibody, the severity of the symptoms, and the subject's sensitivity to side effects. A suitable dose of a given compound can be readily determined by various means by those skilled in the art.

[0140] Route of administration The target antibody is administered to the individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and topical administration routes.

[0141] In some embodiments, the antibody may be administered to the patient every 2 to 4 weeks (e.g., 3) over 10 to 60 minutes (e.g., 30 minutes) as an intravenous infusion (200 mg or 2 mg / kg, up to 200 mg) in a pharmaceutically acceptable carrier, e.g., PBS.

[0142] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, intravenous, intra-arterial, intrarectal, nasal, oral, and other enteral and parenteral routes of administration. The routes of administration can be combined or modified as desired, depending on the antibody and / or desired effect. The target antibody composition can be administered in single or multiple doses. In some embodiments, the target antibody composition is administered orally. In some embodiments, the target antibody composition is administered via inhalation. In some embodiments, the target antibody composition is administered intranasally. In some embodiments, the target antibody composition is administered topically. In some embodiments, the target antibody composition is administered intracranially. In some embodiments, the target antibody composition is administered intravenously.

[0143] The drug can be administered to the host using any available conventional method and route suitable for conventional drug delivery, including systemic or local routes. Generally, the routes of administration intended by the present invention include, but are not limited to, enteral, parenteral, or inhalation routes.

[0144] Other parenteral administration routes besides inhalation include, but are not limited to, local, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than the gastrointestinal tract. Parenteral administration may be performed to result in systemic or local delivery of the target antibody. When systemic delivery is desired, administration usually involves local or mucosal administration of the pharmaceutical preparation to allow invasive or systemic absorption.

[0145] The target antibody can also be delivered to the target via enteral administration. Enteral administration routes include, but are not limited to, oral and rectal (e.g., using suppositories) delivery.

[0146] "Treatment" means at least improvement of the symptoms associated with a disease that is afflicting the host, and improvement is used more broadly to refer to at least reduction of the magnitude of a parameter, for example, cancer and associated pain, of the symptoms associated with the treated disease. Thus, treatment also includes situations in which the disease or at least its associated symptoms are completely inhibited, for example, prevented from occurring or stopped, for example, terminated, so that the host no longer suffers from the disease or at least the symptoms that characterize that disease.

[0147] In some embodiments, the target antibody is administered by injection and / or delivery, for example, to a site in the cerebral artery or directly to brain tissue. The target antibody can also be administered directly to a target site, for example, by delivery using a microparticle gun to the target site.

[0148] Various hosts (wherein the term “host” is used interchangeably with the terms “subject,” “individual,” and “patient” in this specification) can be treated according to the methods of the subject. Generally, such hosts are “mammals” or “mammals,” and these terms are widely used to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodenta (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the host is a human.

[0149] Kits are provided that contain a unit dose of the target antibody, for example, an orally or injectably administered dose. Such kits include, in addition to a container containing the unit dose, a package insert describing the use of the antibody in the treatment of the condition of interest and the associated benefits. Preferred compounds and unit doses are those previously described herein.

[0150] treatment This disclosure provides a method for treating cancer, which generally involves administering an effective amount of a target antibody alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy) to an individual who needs it (e.g., an individual with cancer).

[0151] In some embodiments, an effective dose of the target antibody, when administered in one or more doses, either alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is sufficient to reduce cancer adverse events by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the severity of adverse events without antibody treatment.

[0152] In some embodiments, an effective dose of the target antibody is an amount administered in one or more doses, either alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), that is effective in reducing the tumor size of the individual being treated. For example, an effective dose of the target antibody can reduce the tumor size of an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more, compared to the tumor size without antibody treatment.

[0153] Combination therapy In some embodiments, the treatment method for the target involves administering the target antibody and one or more additional therapeutic agents. Preferred additional therapeutic agents include, but are not limited to, immune checkpoint inhibitors such as anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-VISTA antibodies, anti-LAG-3 antibodies, anti-IDO antibodies, or anti-KIR antibodies (others are also known). In some embodiments, the treatment may also include a co-stimulatory antibody, such as an antibody against CD40, GITR, OX40, CD137, or ICOS. In some embodiments, the antibody may be an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. Examples of such antibodies include, but are not limited to, ipilimumab (CTLA-4), nivolumab (PD-1), pembrolizumab (PD-1), atezolizumab (PD-L1), avelumab (PD-L1), and durvalumab (PD-L1). These therapies can be combined with each other and / or other therapies. In some embodiments, the dose administered may range from 1 mg / kg to 10 mg / kg or 50 mg to 1.5 g every few weeks (e.g., every three weeks), depending on the patient's body weight. In certain embodiments, the patient will be treated with the antibody(s) without knowing the PD1, PD-L1, CTLA-4, TIM-3, VISTA, LAG-3, IDO, or KIR status of the tumor.

[0154] Suitable patients for treatment The treatment method described in this subject is suitable for a variety of subjects. Suitable subjects include, for example, any individual who has cancer, has been diagnosed with cancer, is at risk of developing cancer, has had cancer in the past and is at risk of cancer recurrence, or is recovering from cancer. In some embodiments, the patient may have lung, breast, brain, kidney, and prostate cancer. In these embodiments, the B7-H3 antibody may have Fc-dependent immunoeffector mechanisms such as complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP). In these embodiments, the antibody may be bound to a part that kills the cells it binds to.

[0155] In other embodiments, the antibody is used as an immune checkpoint inhibitor in possible combinations with other immune checkpoint inhibitors to treat, for example, patients with cutaneous melanoma, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck cancer, and Hodgkin lymphoma (all of which can be treated by immune blockade). [Examples]

[0156] The following examples are proposed to provide those skilled in the art with a complete disclosure and explanation of how the present invention may be prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to indicate that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy for the figures used (e.g., quantity, temperature, etc.), but some degree of experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near-atmospheric pressure. Standard abbreviations may be used, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.

[0157] Example 1 Production and characterization of anti-B7-H3 antagonist antibodies The anti-B7-H3 human monoclonal antibody was generated using a transgenic chicken (OMNICHICKEN®) (Ching et al. 2018 MAbs. 2018 10:71-80) that expresses the human antibody gene (the chicken constant region of the heavy chain while also expressing the human light chain (VLCL or VKCK) and human VH).

[0158] Three transgenic chickens were immunized with 200 ug of Fc-tagged human B7-H3 (ACRO Biosystems #CD8-H5253) and given three additional immunizations with 100 ug of antigen every 14 days. Serum immune responses were monitored by ELISA using human and mouse histone-tagged B7-H3 (Acro Biosystems #B73-H52E2 and #B73-M52H4, respectively).

[0159] Splenocytes were isolated and tested using a GEM assay, as described in Mettler Izquierdo et al. 2016 Microscopy(Oxf)2016 65:341-352. Briefly, large beads (Life Technologies #A37306) were coated with His-tagged human B7-H3 (Life Technologies #A37296), and small beads (Life Technologies #A37296) were coated overnight with His-tagged mouse B7-H3. The beads were blocked in 3% milk in DPBS at room temperature for 1 hour, then washed three times with DPBS. The beads were mixed with 1e7 primary splenocytes and Alexa Fluor 594-complexed goat anti-chicken IgY (Thermo Fisher Scientific catalog number A-11042) in a 5:1 ratio (small beads to large beads) for detection. The mixtures were plated, and positive clones were detected under a microscope using a CY3 filter. We selected cells that secrete IgY bound to beads.

[0160] Positive clones were transferred to a 96-well plate containing a PCR mixture consisting of dNTPs, primers, and an enzyme mix.

[0161] The variable regions of the heavy and light chains were cloned and incorporated into a single-chain Fv-Fc (ScFv-Fc) mammalian expression vector. Sanger sequencing was performed using GeneWiz, and the sequences were evaluated using DNASTAR Lasergene 15. Unique scFv-Fcs were transiently expressed in Expi293 cells (Life Technologies, #A14527) grown in Expi293 expression medium (Life Technologies, #A1435101) after transfection using ExpiFectamine transfection reagent and supplement (Life Technologies, #A14524). Cells were incubated in a 5% CO2 humidified incubator at 37°C with shaking for 3–5 days. The supernatant was collected and quantified using a Blitz Instrument (Pall ForteBio), and binding activity was tested by ELISA on plates coated with His-tagged B7-H3 protein from various species (human, mouse, and cynomolgus monkey (ACRO Biosystems cat#CD8-H5224, CD8-M5223, and CD8-C5223, respectively)) or rabbit anti-huFc (Rockland #609-4103, positive control) or unrelated his-tagged proteins (negative control).

[0162] The binding of anti-B7-H3 antibodies to cell lines was evaluated by flow cytometry. Briefly, approximately 1.5 e5 B7-H3 expressing CHO cells (Genescript) or parental CHO cells (ATCC) were resuspended in FACS buffer (PBS / 1% BSA / 0.01% NaN3) containing 10 ug / ml of anti-B7-H3 antibody and incubated on ice for 60 minutes. The cells were washed twice and resuspended on ice for 60 minutes in FACS buffer containing AlexaFluor 647-complexed donkey anti-human Fc (Jackson ImmunoResearch Laboratories #709-605-098). The cells were then washed twice, resuspended in FACS buffer, and read using an Attune Flow Cytometer (Life Technologies). Data are expressed as mean fluorescence intensity (MFI). A total of 54 antibodies showed significant binding to the cells. Two of these did not show significant binding by ELISA, so species cross-reactivity could not be determined. Of the remaining 52, all bound to cyno B7-H3, while only one did not bind to mouse B7-H3.

[0163] The binding affinity of anti-B7-H3 antibodies to B7-H3 was measured by surface plasmon resonance (SPR) on a Carterra LSA instrument. Goat anti-human Fc antibodies were amine-conjugated to HC30M tips. ScFv-Fc supernatant was diluted 30-fold with HBSTE and captured on anti-human Fc-conjugated HC30M tips. A 1:2 dilution series of his-tagged B7-H3, starting at 400 nM, was injected into the tips for binding. Data were analyzed using Carterra® Kinetics software.

[0164] In epitope binning, the supernatant containing scFv-Fc (referred to as ligand scFv-Fc) was diluted 50-fold and coupled to an HC2000M tip via sulfo-NHS / EDC coupling chemistry, followed by blocking with ethanolamine. In each cycle of the binning experiment, 100 nM his-tagged human B7-H3 was injected into the entire array, followed by the injection of individual supernatant scFv-Fc (referred to as analyte scFv-Fc), diluted 40-fold with HBSTE + BSA, into the entire array to sandwich the antigen bound to ligand scFv-Fc. At the end of each cycle, the tip was regenerated with 10 mM glycine pH 2 to remove the bound antigen and analyte scFv-Fc. This procedure was repeated for the supernatant of all analytes. Epitope binning analysis was performed using Carterra epitope software. Five epitope bins were identified in this antibody cohort.

[0165] The ADCC reporter assay was performed using reporter cells expressing human FcγRIIIa V158 (high affinity) (Promega #G7010) as effector cells. Target cells were either A498 cells (ATCC #HTB-44, renal cancer cells) or CHO-K1 cells (GenScript #M00536) stably transfected with B7-H3. Purified anti-B7-H3 antibody (scFv-Fcs above was converted to IgG, expressed, and purified) was added to target cells at a maximum concentration of 10 μg / ml, followed by a 7-point logarithmic dilution series and an untreated sample control. Effector cells were added, and the plate was incubated at 37°C in 5% CO2 for 6 hours. The sample was run three times. After adding Bio-Glo luciferase substrate and incubating for 10 minutes, chemiluminescence was measured using a PerkinElmer 2300 EnSpire plate reader. Data were graphed, and the EC of each antibody was analyzed. 50This was determined by curve fitting using GraphPad Prism. Of the 54 antibodies tested, 37 showed ADCC activity against A498 target cells, and of the 54 antibodies, 52 showed ADCC activity against CHO-K1 / B7-H3 target cells.

[0166] The results of these assays are shown in Tables 3-5 below. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

[0167] While the present invention has been described in relation to its particular embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of this disclosure. Furthermore, many modifications may be made to adapt specific circumstances, materials, compositions of substances, processes, or process steps to the spirit and scope of this disclosure. All such modifications are intended to be within the scope of the appended claims. The present invention includes the following embodiments. <1> An antibody that binds to B7-H3, wherein the antibody is (a) i. The heavy chain CDR1, CDR2, and CDR3 regions of the antibody selected from Figure 1 are identical to the heavy chain CDR1, CDR2, and CDR3 regions, ii. A variable domain comprising light chain CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of the antibody selected from Figure 2, or (b) an antibody comprising a variant of the variable domain of (a) which is otherwise identical to the antibody variable domain except for up to 10 amino acid substitutions in the collective CDR region of the variable domain of (a). <2> The aforementioned antibody A heavy chain variable domain of an antibody selected from Figure 1, comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to the amino acid sequence of the heavy chain variable domain, A light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to the light chain variable domain of the antibody selected from Figure 2, <1> The antibody described above. <3> The heavy chain variable domain and the light chain variable domain are located in separate polypeptides. <1> or <2> The antibody described above. <4> The heavy chain variable domain and the light chain variable domain are present in a single polypeptide. <1> from <3> An antibody listed in any of the following. <5> The antibody, 7 M -1 ~10 12 M -1 It binds to B7-H3 with affinity within this range. <1> from <4> An antibody listed in any of the following. <6> The antibody comprises a non-peptide synthetic polymer to which the antibody is covalently bonded. <1> from <5> An antibody listed in any of the following. <7> The aforementioned synthetic polymer is a poly(ethylene glycol) polymer. <6> The antibody described above. <8> The antibody includes a lipid or fatty acid portion to which it is covalently bound. <1> from <7> An antibody listed in any of the following. <9> The antibody includes a polysaccharide or carbohydrate moiety to which it is covalently bound. <1> from <8> An antibody listed in any of the following. <10> The aforementioned antibody is a single-chain Fv(scFv) antibody. <1> from <9> An antibody listed in any of the following. <11> The aforementioned scFv is polymerized, <10> The antibody described above. <12> a) <1> from <11> An antibody described in any of the following, b) A pharmaceutical composition comprising a pharmaceutically acceptable carrier. <13> The aforementioned antibody is encapsulated in liposomes. <12> The pharmaceutical composition described in [reference]. <14> A method for inhibiting B7-H3, wherein cells containing B7-H3 are... <1> from <11> A method comprising contacting an antibody described in any of the following. <15> A method for inhibiting B7-H3 in a target, wherein an effective amount of <1> from <11> A method comprising administering an antibody described in any of the above to the subject. <16> A method of treating cancer, which involves administering an effective amount to a cancer patient. <1> from <11> A method comprising administering an antibody described in any of the following. <17> The aforementioned cancer is multiple myeloma. <16> Methods used. <18> The aforementioned cancer is melanoma of the skin, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck cancer, or Hodgkin lymphoma. <16> Methods used. <19> The aforementioned patient is currently being treated with an immune checkpoint inhibitor, or has been treated with an immune checkpoint inhibitor in the past. <18> Methods used. <20> The aforementioned immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. <19> Methods used.

Claims

1. An antibody that binds to B7-H3, wherein the antibody is (a) i. The heavy chain CDR1, CDR2, and CDR3 regions are identical to the heavy chain CDR1, CDR2, and CDR3 regions of Sequence ID No. 39, ii. The light chain CDR1, CDR2, and CDR3 regions are identical to the light chain CDR1, CDR2, and CDR3 regions of sequence number 93, Variable domains including Includes, Sequence ID 39 and Sequence ID 93 are the heavy chain and light chain of antibody 40633p1.1.G7, respectively. antibody.

2. The aforementioned antibody A heavy chain variable domain containing an amino acid sequence that is at least 90% identical to sequence number 39, The antibody according to claim 1, comprising a light chain variable domain having an amino acid sequence that is at least 90% identical to SEQ ID NO:

93.

3. The antibody according to claim 1 or 2, wherein the heavy chain variable domain and the light chain variable domain are located in separate polypeptides.

4. The antibody according to claim 1 or 2, wherein the heavy chain variable domain and the light chain variable domain are present in a single polypeptide.

5. The antibody is 10 ７ M －１ ~10 １２ M －１ The antibody according to any one of claims 1 to 4, which binds to B7-H3 with affinity within the range of [specify affinity range].

6. The antibody according to any one of claims 1 to 5, comprising a non-peptide synthetic polymer to which the antibody is covalently bonded.

7. The antibody according to claim 6, wherein the synthetic polymer is a poly(ethylene glycol) polymer.

8. The antibody according to any one of claims 1 to 7, comprising a covalently bound lipid or fatty acid portion of the antibody.

9. The antibody according to any one of claims 1 to 8, comprising a covalently bound polysaccharide or carbohydrate moiety of the antibody.

10. The antibody according to any one of claims 1 to 9, wherein the antibody is a single-chain Fv (scFv) antibody.

11. The antibody according to claim 10, wherein the scFv is polymerized.

12. a) The antibody according to any one of claims 1 to 11, b) A pharmaceutically acceptable carrier, A pharmaceutical composition containing [the specified substance].

13. The pharmaceutical composition according to claim 12, wherein the antibody is encapsulated in liposomes.

14. A pharmaceutical composition for use in methods of treating cancer, The method comprises administering an effective amount of the antibody described in any one of claims 1 to 11 to a cancer patient, A pharmaceutical composition comprising the antibody described in any one of claims 1 to 11.

15. The pharmaceutical composition according to claim 14, wherein the cancer is multiple myeloma.

16. The pharmaceutical composition according to claim 14, wherein the cancer is melanoma of the skin, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck cancer, or Hodgkin lymphoma.

17. The pharmaceutical composition according to claim 16, wherein the patient is also being treated with an immune checkpoint inhibitor or has been treated with an immune checkpoint inhibitor in the past.

18. The pharmaceutical composition according to claim 17, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor.

19. The aforementioned antibody A heavy chain variable domain containing an amino acid sequence that is at least 95% identical to sequence number 39, The antibody according to claim 1, comprising a light chain variable domain having an amino acid sequence that is at least 95% identical to SEQ ID NO: 93.

Images