CD98 binding constructs for treatment of brain tumors

Abstract

Provided are novel multispecific antibodies comprising at least one of a first antigen-binding region and a second antigen-binding region each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to CD98.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 395,389, filed Aug. 5, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] Reference Sequence Listing Submitted Electronically

[0004] This application contains a Sequence Listing that has been submitted electronically. The content of the electronic Sequence Listing (JBI6744WOPCT1Sequence Listing.xml; size: 131,642 bytes; and creation date: Jul. 11, 2023) is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0005] The present disclosure relates to novel multispecific antibodies that include a first moiety capable of binding to a first antigen; and a second moiety capable of binding to a second antigen on a particular tissue (such as on the blood-brain barrier). BACKGROUND ART

[0006] Numerous methods have been investigated to improve the delivery of therapeutic monoclonal antibodies (mAbs). For example, while the blood-brain barrier (BBB) prevents harmful substances from entering the brain and is essential for brain homeostasis, it poses a formidable obstacle to the effective delivery of drugs to the brain. Macromolecules such as monoclonal antibodies and other biotherapeutics have great therapeutic / diagnostic potential for treating / detecting pathologies in the central nervous system (CNS). However, their entry into the brain is blocked by the BBB. Numerous methods have been investigated to improve the brain delivery of therapeutic mAbs, including the use of receptor-mediated transcytosis (RMT). RMT utilizes receptors that are abundantly expressed on the luminal side of the BBB to transport across brain endothelial cells. Previous attempts to establish clinically viable platforms for delivering therapeutic mAbs to the brain have focused on antibody engineering to improve the efficiency of transcytosis and have made progress by observing binding avidity, pH-dependence, and affinity (reviewed in Goulatis et al., Curr Opin Struct Biol, 2017, Vol. 45: pp. 109-115). However, translation to non-human primates (NHPs) and the clinic has been limited by rapid peripheral clearance from target-mediated drug disposition (TMDD) and the safety from acute reticulocyte depletion (Gadkar K et al., Eur J Pharm Biopharm., 2016; Vol. 101: pp. 53-61).

[0007] The CD98 heavy chain subunit (CD98hc) is a member of the solute carrier family and heterodimerizes with many CD98 light chain members to form amino acid transporters at the BBB (Zuchero YJ et al., Neuron., 2016, Vol. 89, No. 1: pp. 70-82; citing Boado RJ et al., PNAS, 1999; Vol. 96, No. 21: pp. 12079-12084). The intracellular portion of CD98hc plays a role in mediating integrin signaling, which functions in both cell growth and tumorigenesis (Zuchero YJ et al., Neuron., 2016, Vol. 89, No. 1: pp. 70-82; citing Feral CC et al., J Cell Biol, 2007, Vol. 178: pp. 701-711; Cantor JM and Ginsburg MH. J Cell Sci, 2012; Vol. 125: pp. 1373-1382). CD98hc is highly expressed on human brain microvasculature, and anti-CD98hc bispecific antibodies have been used to deliver therapeutic antibodies to the mouse brain (Zuchero YJ et al., Neuron., 2016; Vol. 89, No. 1: pp. 70-82).

[0008] Accordingly, there is a need for a platform that can be used to shuttle drugs effectively and with improved safety and pharmacokinetics into target tissues. SUMMARY OF THE INVENTION

[0009] In one aspect, provided herein is a multispecific antibody or antigen-binding fragment thereof that comprises at least one of a first antigen-binding region and a second antigen-binding region each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to CD98.

[0010] In certain embodiments, the first antigen-binding region comprises a first heavy-chain variable region (VH1) that includes heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively; and a first light-chain variable region (VL1) that includes light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; the second antigen-binding region comprises a second heavy-chain variable region (VH2) that includes heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; and a second light-chain variable region (VL2) that includes light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; and the third antigen-binding region comprises a first single-chain variable fragment (scFv1) that has: a third heavy-chain variable region (VH3) that includes heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and a third light-chain variable region (VL3) that includes light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have any of the amino acid sequences in Table 2.

[0011] In certain embodiments, VH1 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO: 1; and VL1 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO: 2; VH2 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO: 11; VL2 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO: 12; and VH3 and VL3 comprise the same amino acid sequences as the corresponding VH and VL of the scFv as shown in:

[0012] (i) SEQ ID NO: 19;

[0013] (ii) SEQ ID NO: 26;

[0014] (iii) SEQ ID NO: 33;

[0015] (iv) SEQ ID NO: 34;

[0016] (v) SEQ ID NO: 35;

[0017] (vi) SEQ ID NO: 36;

[0018] (vii) SEQ ID NO: 43;

[0019] (viii) SEQ ID NO: 49;

[0020] (ix) SEQ ID NO: 55;

[0021] (x) SEQ ID NO: 59;

[0022] (xi) SEQ ID NO: 66;

[0023] (xii) SEQ ID NO: 71;

[0024] (xiii) SEQ ID NO: 78;

[0025] (xiv) SEQ ID NO: 85;

[0026] (xv) SEQ ID NO: 87;

[0027] (xvi) SEQ ID NO: 88;

[0028] (xvii) SEQ ID NO: 89;

[0029] (xviii) SEQ ID NO: 90;

[0030] (xix) SEQ ID NO: 92;

[0031] (xx) SEQ ID NO: 93;

[0032] (xxi) SEQ ID NO: 94.

[0033] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, and (b) a first light chain (LC1) comprising VL1 and a light chain constant region.

[0034] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a second heavy chain (HC2) comprising VH1 and a first heavy chain constant region containing a second Fc region (Fc2), and (b) a second light chain (LC2) comprising VL1 and a light chain constant region.

[0035] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0036] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2 and a first heavy chain constant region containing a first Fc region (Fc1), (b) a first light chain (LC1) comprising VL2 and a light chain constant region, and (c) a second heavy chain (HC2) comprising scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0037] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region.

[0038] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, (b) a first light chain (LC1) comprising VL1 and a light chain constant region, and (c) a second heavy chain (HC2) comprising a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein scFv2 comprises VH2 and VL2.

[0039] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, (b) a first light chain (LC1) comprising VL2 and a light chain constant region, and (c) a second heavy chain (HC2) comprising a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein scFv2 comprises VH1 and VL1.

[0040] In certain embodiments, scFv1 and / or scFv2 comprises at least one of the following: (a) a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys; and (b) a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys.

[0041] In certain embodiments, scFv1 and scFv2 each independently comprise a first disulfide bond and a second disulfide bond.

[0042] In certain embodiments, scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4.

[0043] In certain embodiments, scFv1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94.

[0044] In certain embodiments, each of Fc1 and Fc2 comprises one or more heterodimer mutations, or one or more knob and hole mutations.

[0045] In certain embodiments, the heterodimer mutation comprises amino acid modifications at positions T350, L351, F405, and Y407 in one of Fc1 and Fc2, and amino acid modifications at positions T350, T366, K392, and T394 in the other of Fc1 and Fc2, wherein the amino acid modification at position T350 is T350V, T350I, T350L, or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T, or F405S; the amino acid modification at position Y407 is Y407V, Y407A, or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M, the amino acid modification at position K392 is K392F, K392L, or K392M, and the amino acid modification at position T394 is T394W, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0046] In certain embodiments, one of Fc1 and Fc2 comprises the mutations T350V, L351Y, F405A, and Y407V, and the other of Fc1 and Fc2 comprises the mutations T350V, T366L, K392L, and T394W.

[0047] In certain embodiments, each of Fc1 and Fc2 comprises one or more knob and hole mutations.

[0048] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that enhances the binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably the amino acid modification enhances binding at acidic pH, more preferably the Fc domain has an M252Y / S254T / T256E (YTE) mutation, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0049] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that reduces or eliminates effector function, preferably the Fc domain has one or more amino acid modifications at positions L234, L235, D265, D270, N297, E318, K320, K322, P331, and P329, such as one, two, three, or four amino acid modifications selected from L234A, L235A, D265S, and P331S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0050] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having one or more amino acid modifications selected from M252Y, S254T, T256E, L234A, L235A, and D265S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0051] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that does not reduce or eliminate effector function.

[0052] Also provided is a multispecific antibody comprising a first heavy chain, a light chain, and a second heavy chain each having an amino acid sequence that is at least 90% identical to: (a) SEQ ID NO:100, SEQ ID NO:12, and SEQ ID NO:101, respectively; or (b) SEQ ID NO:102, SEQ ID NO:12, and SEQ ID NO:103, respectively;

[0053] wherein the first antigen-binding region is capable of specifically binding to a first epitope of HER2, the second antigen-binding region is capable of specifically binding to a second epitope of HER2, and the third antigen-binding region is capable of specifically binding to CD98.

[0054] In certain embodiments, the first heavy chain, light chain, and second heavy chain each comprise the amino acid sequences of: (a) SEQ ID NO: 100, SEQ ID NO: 12, and SEQ ID NO: 101, respectively; or (b) SEQ ID NO: 213, SEQ ID NO: 12, and SEQ ID NO: 103, respectively.

[0055] Another general aspect of the present application relates to an isolated nucleic acid sequence that encodes a multispecific antibody or an antigen-binding fragment thereof according to the present application. Also provided is a vector comprising the isolated nucleic acid of the present application, and a host cell comprising the isolated nucleic acid or vector of the present application.

[0056] Another general aspect of the present application relates to a method of producing a multispecific antibody or an antigen-binding fragment thereof. The method includes culturing a cell comprising the nucleic acid of the present application under conditions for producing a multispecific antibody or an antigen-binding fragment thereof, and recovering the multispecific antibody or an antigen-binding fragment thereof.

[0057] Also provided is a pharmaceutical composition that comprises the multispecific antibody or an antigen-binding fragment thereof of the present application and a pharmaceutically acceptable carrier.

[0058] Yet another general aspect of the present invention relates to a method of treating or detecting a disorder (preferably cancer) in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or antigen-binding fragment or pharmaceutical composition of the present application.

[0059] Based on the following disclosure, including the detailed description of the present invention and its preferred embodiments and the appended claims, other aspects, features, and advantages of the present invention will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 A diagram showing a biparatopic, multispecific antibody fused to a tissue targeting module is shown.

[0061] Figure 2 The molecular structure of the TEM mAb is shown, in which a single CD98 scFv or CD98 spFv is fused to the C-terminus of one heavy chain of a biparatopic therapeutic mAb targeting HER2 using a short flexible linker.

[0062] Figures 3A - 3EShows the binding of the bispecific antibody to (A) BT474, (B) MBA-MB-361, (C) JIMT-1, (D) U87-MG, and (E) HCC1954 cells. Binding was compared to isotype control (CNTO3930) and trastuzumab silenced and pertuzumab silenced.

[0063] Figures 4A - 4D Shows the curves of spheroid area formed by (A, C) BT474 and (B, D) MDA-MB-361 cells co-cultured with iMG at a 1:1 E:T ratio at increasing doses in the presence of anti-HER2 TEM mAb, trastuzumab, or isotype IgG1.

[0064] Figures 5A - 5C Shows the red fluorescence curves of (A) JIMT-1, (B) MDA-MB-361, and (C) BT474 cells co-cultured with human PBMC at a 10:1 E:T ratio at increasing doses of anti-HER2 TEM mAb, trastuzumab, or isotype IgG1.

[0065] Figure 6 Shows the PK of TEM mAb in non-human primates. TEM and control IgG1 mAb were administered to cynomolgus monkeys at 10 mg / kg by slow rapid IV injection. Blood for PK was collected at 1 h, 6 h, 24 h, 72 h, and 168 h after dosing and processed into serum by the test facility laboratory protocol. After the final blood collection, the animals were euthanized at 72 h and 168 h (n = 2 at each time point). Approximately 200 mg of tissue was isolated from pre-determined brain locations (frontal lobe, hippocampus, and temporal lobe).

[0066] Figures 7A - 7B Shows the anti-tumor activity of BBBB1819 against SC BT474 human HER2 amplified breast cancer xenografts in mice (SC BT474 mouse tumor xenograft model). Figure 7A Shows the mean tumor volume in mice treated with vehicle or different doses of BBBB1819 (CD98xHER2) antibody administered on days 12, 15, 18, 21, 24, 27, 30, and 33 (indicated by the black line below the X-axis). Figure 7BShows the mean tumor volume in mice treated with 20 mg / kg of vehicle, BBB1819, ERBB2077 (an antibody form equivalent to BBBB1819 but lacking the CD98 spFv), or trastuzumab on days 12, 19, 26, and 33 (indicated by the black line below the X-axis). Group tumor volumes were plotted as mean ± SEM for n = 10 animals / group. Data were graphed when at least two-thirds of the number of mice remained in the study.

[0067] Figures 8A - 8D Shows the pharmacokinetic (PK) analysis in the SC BT474 mouse tumor xenograft model. Figure 8A Shows the plasma concentration of different doses of the BBB1819 antibody over time. Figure 8B Shows the plasma concentration of the BBBB1819, ERBB2077, and trastuzumab antibodies over time. Figure 8C Shows the tumor concentration of the BBBB1819, ERBB2077, and trastuzumab antibodies over time. Figure 8D Shows the tumor-to-plasma (%) ratio of the BBBB1819, ERBB2077, and trastuzumab antibodies over time.

[0068] Figures 9A - 9C Shows the in vivo biodistribution of the Zr89-DFO*-HER2xCD98 (HER2xCD98) antibody in C57BL6 (B6) and human CD98 knock-in (CD98) mice. Figure 9A Shows the standardized uptake value (SUV) of the Zr89-DFO*-HER2xCD98 antibody in tissues on day 1. Figure 9B Shows the SUV of the Zr89-DFO*-HER2xCD98 antibody in tissues on day 5. Figure 9C Shows the SUV of the Zr89-DFO*-HER2xCD98 antibody in tissues on day 7.

[0069] Figures 10A - 10D Shows the brain and heart uptake of the HER2xCD98 and Zr89-DFO*-HER2 (HER2) antibodies in C57BL6 (BL6) and human CD98 knock-in (huCD98 KI) mice. Figure 10A Shows the SUV in the brain on day 1. Figure 10B Shows the SUV in the brain on day 5. Figure 10C Shows the brain-to-heart ratio of the SUV on day 1. Figure 10D Shows the brain-to-heart ratio of the SUV on day 5.

[0070] Figures 11A - 11CShows the brain, heart, and whole body uptake of HER2xCD98 antibody in C57BL6 (BL6) and human CD98 knock-in (huCD98 KI) mice. Figure 11A Shows the SUV in the brain after injection. Figure 11B Shows the SUV in the heart after injection. Figure 11C Shows the megabecquerel (MBq) measurements of the whole body of the mice after injection.

[0071] Figures 12A - 12C Shows the pharmacokinetics (PK) of BBB1819, ERBB2077, and trastuzumab (ERBB218) antibodies in human CD98 knock-in (CD98) mice at 24 hours after injection. Figure 12A Shows the concentrations of BBB1819, ERBB2077, and ERBB128 antibodies in the brain. Figure 12B Shows the concentrations of BBB1819, ERBB2077, and ERBB128 antibodies in plasma. Figure 12C Shows the brain-to-plasma (%) ratios of BBB1819, ERBB2077, and ERBB128 antibodies. Detailed Description

[0072] The present invention relates to a multispecific antibody that comprises at least one of a first antigen-binding region and a second antigen-binding region each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to CD98.

[0073] Definitions

[0074] The techniques and procedures described or cited herein include those commonly known to and / or routinely employed by those of ordinary skill in the art, such as, for example, the widely utilized methods described in the following documents: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (edited by Ausubel et al., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009); Monoclonal Antibodies: Methods and Protocols (edited by Albitar, 2010); and Antibody Engineering, Volumes 1 and 2 (edited by Kontermann and Dübel, 2nd ed., 2010). Unless otherwise defined herein, technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following terms will be used in the description, and where appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between any description of the terms set forth herein and any document incorporated herein by reference, the description of the terms set forth below shall prevail.

[0075] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and are used in the broadest sense and specifically encompass, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), and fragments thereof (e.g., domain antibodies), as described hereinbelow. Antibodies can be human, humanized, chimeric, and / or affinity matured, as well as antibodies from other species such as mice, rabbits, llamas, etc. The term "antibody" is intended to include the polypeptide products of B cells within the immunoglobulin polypeptide class that are capable of binding to a specific molecular antigen and are composed of two pairs of identical polypeptide chains, where each pair has one heavy chain (about 50 kDa - 70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain includes a constant region. See, for example, Antibody Engineering (edited by Borrebaeck, 2nd edition, 1995); and Kuby, Immunology (3rd edition, 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, including antibodies from camelid species (e.g., llamas or alpacas) or humanized variants thereof, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the foregoing (e.g., antigen-binding fragments), which refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab’) fragments, F(ab) 2 fragments, F(ab’) 2Fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetra-bodies, and minibodies. Specifically, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen-binding domains or molecules containing antigen-binding sites that bind an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, e.g., in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (ed. Myers, 1995); Huston et al., 1993, Cell Biophysics, Vol. 22: pp. 189-224; Plückthun and Skerra, 1989, Meth. Enzymol., Vol. 178: pp. 497-515; and Day, Advanced Immunochemistry (2nd ed., 1990). The antibodies provided herein can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies can be agonistic antibodies or antagonistic antibodies. The antibodies may be neither agonistic nor antagonistic.

[0076] An "antigen" is a structure to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or in the cell.

[0077] A "full-length" antibody is an antibody that contains an antigen-binding site as well as CL and at least the heavy-chain constant regions CH1, CH2, and CH3. The constant regions can include human constant regions or amino acid sequence variants thereof. In certain embodiments, the full-length antibody has one or more effector functions.

[0078] The term "binding" refers to an interaction between molecules, including, e.g., the formation of a complex. The interaction can be, e.g., a non-covalent interaction, including hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. The complex can also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interaction between a single antigen-binding site on an antibody and a single epitope of a target molecule such as an antigen is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k) of a binding molecule (e.g., an antibody) from a monovalent antigen off) The ratio of the association rate (k on ) to the off dissociation rate (k on ) is the dissociation constant K D , which is inversely proportional to the affinity. The lower the K D value, the higher the affinity of the antibody. The K D value varies with different complexes of the antibody and the antigen and depends on both k on and k off . The dissociation constant K D of the antibodies provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of this multiple interaction between the multivalent antibody and the antigen is called avidity.

[0079] Terms related to the binding molecules described herein, such as "bind", "specifically bind", and similar terms, are also used interchangeably herein and refer to binding molecules that specifically bind to an antigen-binding domain of an antigen such as a polypeptide. A binding molecule or antigen-binding domain that binds to or specifically binds to an antigen can, for example, be detected by immunoassay, or other techniques known to those of skill in the art. In some embodiments, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), a binding molecule or antigen-binding domain binds or specifically binds an antigen when it binds the antigen with a higher affinity than it binds to any cross-reactive antigen. Typically, a specific or selective response will be at least twice the background signal or noise and can be more than 10-fold the background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion of binding specificity. In certain embodiments, the binding molecule or antigen-binding domain binds to a "non-target" protein to an extent less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, e.g., as determined by fluorescence-activated cell sorting (FACS) analysis or RIA. A binding molecule or antigen-binding domain that binds an antigen includes a binding molecule or antigen-binding domain that is capable of binding the antigen with sufficient affinity such that the binding molecule can be used, for example, as a therapeutic and / or diagnostic agent targeting the antigen. In certain embodiments, the binding molecule or antigen-binding domain that binds an antigen has a dissociation constant (K D ) of less than or equal to 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, the binding molecule or antigen-binding domain binds an epitope that is conserved among antigens from different species.

[0080] In certain embodiments, the binding molecule or antigen-binding domain may comprise a "chimeric" sequence, where a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass and fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, Vol. 81: pp. 6851-6855). Chimeric sequences can include humanized sequences.

[0081] In certain embodiments, the binding molecule or antigen-binding domain may comprise portions of "humanized" forms of non-human (e.g., camelid, murine, non-human primate) antibodies, which antibodies include sequences from human immunoglobulins (e.g., acceptor antibodies), wherein the native CDR residues are replaced with residues of the corresponding CDRs from non-human species such as camelids, mice, rats, rabbits, or non-human primates (e.g., donor antibodies) having the desired specificity, affinity, and potency. In some instances, one or more FR region residues of the human immunoglobulin sequence are replaced with the corresponding non-human residues. Additionally, the humanized antibody may comprise residues not present in the acceptor antibody or donor antibody. These modifications are made to further improve antibody performance. The humanized heavy or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. For further details, see Jones et al., Nature, Vol. 321: pp. 522-525, 1986; Riechmann et al., Nature, Vol. 332: pp. 323-329, 1988; Presta, Curr. Op. Struct. Biol., Vol. 2: pp. 593-596, 1992; Carter et al., Proc. Natl. Acad. Sci. USA, Vol. 89: pp. 4285-4289, 1992; U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0082] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "fully human antibody" or "human antibody," where these terms are used interchangeably herein and refer to an antibody that comprises human variable regions and, for example, human constant regions. The binding molecule may comprise an antibody sequence. In specific embodiments, these terms refer to an antibody that comprises variable and constant regions of human origin. In certain embodiments, a "fully human" antibody may also encompass an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described, for example, by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or having an amino acid sequence prepared using any technique for the production of human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. A variety of techniques known in the art can be used to generate human antibodies, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., Vol. 227: p. 381, 1991; Marks et al., J. Mol. Biol., Vol. 222: p. 581, 1991) and yeast display libraries (Chao et al., Nature Protocols, Vol. 1: pp. 755-768, 2006). Methods for the preparation of human monoclonal antibodies are also described in Cole et al., Monoclonal Antibodies and Cancer Therapy, p. 77, 1985; Boerner et al., J. Immunol., Vol. 147, No. 1: pp. 86-95, 1991; and van Dijk and van de Winkel, Curr. Opin. Pharmacol., Vol. 5: pp. 368-374, 2001).Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, such as a mouse (see, e.g., Jakobovits, Curr. Opin. Biotechnol., Vol. 6, No. 5: pp. 561-566, 1995; Brüggemann and Taussing, Curr. Opin. Biotechnol., Vol. 8, No. 4: pp. 455-458, 1997; and U.S. Patent Nos. 6,075,181 and 6,150,584 regarding the XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, Vol. 103: pp. 3557-3562, 2006 regarding human antibodies generated via human B cell hybridoma technology. TM See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding the XENOMOUSE™ technology.

[0083] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "recombinant human antibody," where the phrase includes human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from transgenic and / or transchromosomal animals (e.g., mice or cows) of human immunoglobulin genes (see, e.g., Taylor, L.D. et al., Nucl. Acids Res., Vol. 20: pp. 6287-6295, 1992) or antibodies prepared, expressed, produced, or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are mutagenized in vitro (or, when transgenic animals against human Ig sequences are used, by in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur within the in vivo human antibody germline repertoire.

[0084] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "monoclonal antibody," where the term as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations present in minor amounts or well-known post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation, and each monoclonal antibody typically will recognize a single epitope on an antigen. In a particular embodiment, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for preparing the antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature, Vol. 256: p. 495, 1975, or may be prepared using recombinant DNA methods in bacteria or eukaryotic animal or plant cells (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, Vol. 352: pp. 624-628, 1991, and Marks et al., J. Mol. Biol., Vol. 222: pp. 581-597, 1991. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed therefrom are well known in the art. See, e.g., Short Protocols in Molecular Biology (edited by Ausubel et al., 5th ed., 2002).

[0085] A typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H-chain isotype. Each H and L chain also has regularly spaced intra-chain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in each of the α and γ chains and four CH domains in the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, page 71 (edited by Stites et al., 8th edition, 1994); and Immunobiology (edited by Janeway et al., 5th edition, 2001).

[0086] The term "Fab" or "Fab region" refers to the antibody region that binds antigen. Conventional IgG typically contains two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen-binding ability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab region of conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in a different order, as described in more detail below.

[0087] The terms "variable region", "variable domain", "V region" or "V domain" refer to a part of the light or heavy chain of an antibody, which is generally located at the amino terminus of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is responsible for the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable regions vary widely in sequence among antibodies. The V regions mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid span of the variable region. Instead, the V region consists of stretches of about 15 to 30 amino acids of relatively less variability (e.g., relatively invariant), called framework regions (FRs), which are separated by shorter regions of greater variability (e.g., extreme variability), called "hypervariable regions", each of which is about 9 to 12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs, predominantly adopting a β-sheet conformation, which are connected by three hypervariable regions that form loops connecting the β-sheet structures and in some cases form parts thereof. The hypervariable regions in each chain are held together closely by the FRs and with the hypervariable regions from the other chain, contributing to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as the antibody's participation in antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0088] The term "Kabat variable region residue numbering" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system for the heavy chain variable region or the light chain variable region used in the compilation of antibodies by Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat residue numbering of a given antibody can be determined by aligning the homologous regions of the antibody sequence with the "standard" Kabat numbering sequence. When referring to residues in the variable domain (approximately residues 1 - 107 of the light chain and residues 1 - 113 of the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al., supra). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is typically used (e.g., the EU index reported by Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, e.g., by AbM, Chothia, Contact, IMGT, and AHon.

[0089] When used in reference to an antibody, the term "heavy chain" refers to a polypeptide chain of approximately 50 kDa - 70 kDa, wherein the amino-terminal portion includes a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isotypes), designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu). The different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies, namely IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0090] When used in reference to an antibody, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, wherein the amino-terminal portion includes a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxyl-terminal portion includes a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, designated κ (kappa) or λ (lambda).

[0091] As used herein, the terms "hypervariable region", "HVR", "complementary determining region", and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework regions of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework regions of the VL β-sheet framework of an antibody. CDR1, CDR2, and CDR3 in the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. CDR1, CDR2, and CDR3 in the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, CDRs are variable region sequences that are interspersed within the framework region sequences.

[0092] CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat complementary determining regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra; Nick Deschacht et al., J Immunol, 2010; Vol. 184: pp. 5696-5704). Chothia, in contrast, refers to the positions of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol., Vol. 196: pp. 901-917, 1987). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by the AbM antibody modeling software from Oxford Molecular (see, e.g., Antibody Engineering, Vol. 2 (edited by Kontermann and Dübel, 2nd ed., 2010)). The "contact" hypervariable regions are based on the analysis of available complex crystal structures. Another commonly used numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information (Lafranc et al., Dev. Comp. Immunol., Vol. 27, No. 1: pp. 55-77, 2003). IMGT is an integrated information system specialized in immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. In this article, CDRs are referred to according to both the amino acid sequence and the position within the light or heavy chain. Since the "position" of CDRs within the immunoglobulin variable domain structure is conserved among species and exists in structures called loops, CDRs and framework residues can be readily identified by using a numbering system that aligns variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues of immunoglobulins from one species into a receptor framework, typically from a human antibody. Honegger and Plückthun developed an additional numbering system (AHon), J. Mol. Biol., Vol. 309: pp. 657-670, 2001. The correspondence between numbering systems, including for example the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.). Residues from each of these hypervariable regions or CDRs are exemplified in Table 1 below.

[0093] Table 1. Exemplary CDRs According to Various Numbering Systems

[0094]

[0095]

[0096] The boundaries of a given CDR can vary according to the scheme used for identification. Thus, unless otherwise stated, the terms "CDR" and "complementary determining region" of a given antibody or its region (such as a variable region), and the individual CDRs of an antibody or its region (e.g., CDR-H1, CDR-H2) should be understood to encompass the complementary determining regions as defined by any known scheme as described above. In some cases, the scheme used to identify a particular CDR or CDRs is specified, such as the CDRs defined by the IMGT, Kabat, Chothia, or Contact methods. In other cases, the specific amino acid sequences of the CDRs are given. It should be noted that the CDR regions can also be defined by combinations of various numbering systems, such as a combination of the Kabat and Chothia numbering systems or a combination of the Kabat and IMGT numbering systems. Thus, terms such as "CDR1 as shown in a particular VH" include any CDR1 defined by the above exemplary CDR numbering systems, but are not limited thereto. Once a variable region (e.g., VH or VL) is given, those skilled in the art should understand that the CDRs within that region can be defined by different numbering systems or combinations thereof.

[0097] The hypervariable regions can include the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0098] The term "constant region" or "constant domain" refers to the carboxyl-terminal portions of the light and heavy chains, which do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as interaction with Fc receptors. The term refers to the portion of the immunoglobulin molecule that has a more conserved amino acid sequence relative to the other parts of the immunoglobulin (the variable region containing the antigen-binding site). The constant region can include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0099] The term "framework" or "FR" refers to those variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human domain antibodies, bispecific antibodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0100] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which the K447 residues have not been removed, and a population of antibodies having a mixture of antibodies with and without K447 residues. A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain), and can be evaluated using various assays known to those skilled in the art. A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parental polypeptide, e.g., from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions, in the native sequence Fc region or the Fc region of a parental polypeptide. The variant Fc regions herein can have at least about 80% homology with the native sequence Fc region and / or the Fc region of a parental polypeptide, or at least about 90% homology therewith, e.g., at least about 95% homology therewith.

[0101] As used herein, an "epitope" is a term in the art and refers to a local region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be a continuous amino acid of the polypeptide ("linear" epitope), or the epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide ("conformational", "non-linear", or "discontinuous" epitope). Those skilled in the art will understand that, generally speaking, a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a set of amino acids regardless of whether they are folded in a native three-dimensional protein structure. In other embodiments, the binding molecule requires the amino acid residues that make up the epitope to exhibit a particular conformation (e.g., bend, twist, flip, or fold) in order to recognize and bind the epitope.

[0102] As used herein, the term "bispecific" antigen-binding molecule or "bispecific" polypeptide shall mean a polypeptide comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as defined herein, wherein the two variable domains are capable of binding to two different epitopes of an antigen.

[0103] The "percent amino acid sequence identity (%)" relative to a peptide, polypeptide or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. The alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences to be compared.

[0104] The term "specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding protein. For example, a native antibody is monospecific. As used herein, the term "multispecific" indicates that an antigen-binding protein has two or more antigen-binding sites, wherein at least two of the antigen-binding sites bind different antigens. As used herein, "bispecific" indicates that an antigen-binding protein has two different antigen-binding specificities. As used herein, the term "monospecific" antibody refers to an antigen-binding protein having one or more binding sites, each of which binds the same antigen.

[0105] The term "valence" as used herein denotes the specified number of binding sites present in an antigen-binding protein. For example, a native antibody or a full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" denote the presence of two binding sites, three binding sites, four binding sites, five binding sites and six binding sites, respectively, in an antigen-binding protein.

[0106] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified either naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. This definition also includes polypeptides containing, for example, one or more amino acid analogs (including but not limited to non-natural amino acids) and other modifications known in the art. It should be understood that since the polypeptides of the present disclosure can be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the "polypeptide" can occur as a single chain or two or more related chains.

[0107] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can contain modified nucleotides such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to short, generally single-stranded synthetic polynucleotides, which are usually but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides applies equally and fully to oligonucleotides. The cells that produce the binding molecules of the present disclosure can include parental hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of nascent RNA transcripts is referred to as the transcription direction; the sequence region on a DNA strand that has the same sequence as the RNA transcript and is at the 5' to 5' end of the RNA transcript is referred to as the "upstream sequence"; the sequence region on a DNA strand that has the same sequence as the RNA transcript and is at the 3' to 3' end of the RNA transcript is referred to as the "downstream sequence".

[0108] "Isolated nucleic acid" refers to nucleic acids that are substantially separated from other genomic DNA sequences that are naturally associated with the native sequence, as well as proteins or complexes such as ribosomes and polymerases, such as RNA, DNA, or hybrid nucleic acids. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term encompasses nucleic acid sequences that have been removed from their natural environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. Substantially pure molecules may include isolated forms of the molecule. Specifically, an "isolated" nucleic acid molecule encoding an antibody as described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule, which is typically associated with the contaminating nucleic acid molecule in the environment in which it is produced.

[0109] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The phrase "nucleotide sequence" encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some forms.

[0110] The term "control sequence" refers to DNA sequences that are necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

[0111] As used herein, when referring to nucleic acids or amino acids, the terms "operably linked" and similar phrases (e.g., gene fusion) refer to the operative linking of nucleic acid sequences or amino acid sequences placed in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., the expression of the open reading frame). As another example, operably linked peptides are peptides in which the functional domains are appropriately spaced from each other to confer the expected function of each domain.

[0112] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, which includes, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, for introducing the nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Additionally, a vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included, for example, provide resistance to antibiotics or toxins, complement auxotrophies, or provide key nutrients not present in the medium. Expression control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc., well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., an antibody heavy chain and light chain or both antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector or separate expression vectors. For single vector expression, the encoding nucleic acids may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting of mRNA or polymerase chain reaction (PCR) amplification, immunoblotting for gene product expression, or other suitable analytical methods, to test for the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the nucleic acid molecules are expressed in an amount sufficient to produce the desired product, and further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0113] As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0114] As used herein, the term "host cell" refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such cells. The progeny of such cells may differ from the parental cell transfected with the nucleic acid molecule due to mutations that may occur in the progeny or environmental influences or due to the integration of the nucleic acid molecule into the host cell genome.

[0115] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0116] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeias for use in animals and more specifically in humans.

[0117] "Excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, release agents, sterilizing agents, sweetening agents, solubilizers, wetting agents, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0118] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG), and PLURONICS TM . Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th Edition, 1990).

[0119] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and is suitable for contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th Edition; edited by Rowe et al.; The Pharmaceutical Press and the American Pharmaceutical Association, 2009; Handbook of Pharmaceutical Additives, 3rd Edition; edited by Ash and Ash; Gower Publishing Company, 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; edited by Gibson; CRC Press LLC, Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH-buffered solution.

[0120] In some embodiments, the excipient is a sterile liquid such as water and oils, including those of petroleum, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When administering the composition (e.g., a pharmaceutical composition) intravenously, water is an exemplary excipient. Aqueous solutions of saline and dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol and the like. If desired, the composition can also contain minor amounts of wetting or emulsifying agents or pH buffering agents. The composition can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral compositions, including formulations, can contain standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like.

[0121] For example, a composition comprising a pharmaceutical compound can contain a binding molecule (e.g., an antibody), for example in isolated or purified form together with a suitable amount of excipient.

[0122] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an antibody provided herein, or a therapeutic molecule or pharmaceutical composition comprising an agent and an antibody, sufficient to produce a desired result.

[0123] The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non - primate or a primate (e.g., a human). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal, such as a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, such as a human, at risk of developing a disease or disorder.

[0124] "Administering" refers to the act of injecting or otherwise physically delivering a substance that exists in vitro into the body of a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0125] As used herein, the term "treatment" refers to a reduction or improvement in the progression, severity, and / or duration of a disease or condition caused by the administration of one or more therapies. Treatment can be determined by assessing whether there has been a reduction, alleviation, and / or remission of one or more symptoms associated with the underlying disorder, such that an improvement in the patient is observed, although the patient may still be afflicted with the underlying disorder. The term "treatment" includes managing and improving a disease. The term "managing" refers to the beneficial effects obtained by a subject from a therapy that does not necessarily result in a cure of the disease.

[0126] The term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (e.g., diabetes or cancer).

[0127] As used herein, "delaying" the development of cancer means postponing, hindering, slowing, retarding, stabilizing, and / or deferring the development of the disease. Such delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can actually encompass prevention, since the individual does not develop the disease. A method of "delaying" cancer development is a method that reduces the likelihood of disease development and / or reduces the extent of the disease over a given time period as compared to when the method is not used. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. Cancer development can be detected using standard methods, including but not limited to computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Development can also refer to cancer progression that may initially be undetectable and includes occurrence, recurrence, and onset.

[0128] The term "HER2" (also known as HER2 / neu and ErbB-2) represents "human epidermal growth factor receptor 2". As used herein, it is intended to include variants, isoforms, and species homologs of HER2.

[0129] As used herein, the term "HER2-associated disease or disorder" refers to a disease or disorder that includes cells or tissues in which HER2 is expressed or overexpressed. In some embodiments, the HER2-associated disease or disorder includes cells in which HER2 is abnormally expressed. In other embodiments, the HER2-associated disease or disorder includes cells in which HER2 lacks at least one activity.

[0130] The "blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight junctions within the endothelial membranes of brain capillaries, thereby creating a tight barrier that restricts molecular transport into the brain. The BBB can restrict the transport of even very small molecules such as urea (60 daltons) into the brain. Examples of the BBB include the BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina, all of which are continuous capillary barriers within the CNS. The BBB also encompasses the blood-CSF barrier (choroid plexus), where the barrier is formed by ependymal cells rather than capillary endothelial cells.

[0131] The "blood-brain barrier receptor" (abbreviated herein as "R / BBB") is an extracellular membrane-linked receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the BBB or for the transport of exogenously administered molecules. Examples of R / BBB include, but are not limited to, the large neutral amino acid transporter (LAT) complex, including the CD98 component, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low density lipoprotein receptor (including, but not limited to, low density lipoprotein receptor-related protein 1 (LRP1) and low density lipoprotein receptor-related protein 8 (LRP8)), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). An exemplary R / BBB herein is CD98.

[0132] As used herein, the term "CD98" or "CD98hc" refers to an integral membrane protein composed of the cluster of differentiation 98 heavy chain (CD98hc) that is linked by a disulfide bond to any one of a plurality of light chains. When associated with LAT1 or LAT2, the heterodimeric transporter complex functions as an obligatory amino acid exchanger. CD98hc has a molecular weight of approximately 80 kDa. Preferably, CD98hc is human CD98hc (huCD98hc). hcCD98hc is encoded by the SLC3A2 gene.

[0133] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1% or less of a given value or range.

[0134] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include plural forms unless the context clearly indicates otherwise.

[0135] It should be understood that wherever an embodiment is described herein using the term "comprising", other similar embodiments are also provided that are described in terms of "consisting of" and / or "consisting essentially of". It should also be understood that wherever an embodiment is described herein using the phrase "consisting essentially of", other similar embodiments are also provided that are described in terms of "consisting of".

[0136] The term "between" as used in phrases such as "between A and B" or "A - B" refers to a range that includes both A and B.

[0137] The term "and / or" as used in phrases such as "A and / or B" is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0138] Multispecific Antibodies with Tissue - Targeting Moieties

[0139] In one general aspect, the present application relates to an optimized platform for delivery to a specific tissue. In one aspect, the platform utilizes binding molecules, in particular antibodies or antigen - binding fragments thereof that bind to a target expressed on a specific tissue.

[0140] In one aspect, provided herein is a multispecific antibody that comprises at least one of a first antigen - binding region and a second antigen - binding region each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen - binding region capable of specifically binding to CD98.

[0141] HER2 (also known as ErbB2 or Neu; UniProtKB / Swiss-Prot accession number P04626) consists of 1233 amino acids and is structurally similar to EGFR, having an extracellular domain composed of four subdomains I-IV, a transmembrane domain, a juxtamembrane domain, an intracellular cytoplasmic tyrosine kinase, and a regulatory C-terminal domain (Yamamoto et al., 1986, Nature, vol. 319: pp. 230-234). HER2 is activated by forming heteromeric complexes with other ErbB family members and is thus indirectly regulated by EGFR and HER3 ligands (reviewed in Yarden et al., 2001, Nat Rev Mol Cell Biol., vol. 2: pp. 127-137). HER2 is the preferred heterodimerization partner of three other ErbB receptors (Graus-Porta et al., 1997, EMBO J, vol. 16: pp. 1647-1655; Tzahar et al. 1996, Mol Cell Biol., vol. 16: pp. 5276-5287), enhancing the affinity of other ErbB receptors for their ligands by slowing the rate of ligand-receptor complex dissociation, whereby HER2 enhances and prolongs signaling (Pedersen et al., 2009, Mol Cancer Res., vol. 7: pp. 275-284). Heterodimerization of HER2 and another ligand-binding receptor of the ErbB family induces cross-phosphorylation, resulting in phosphorylation of C-terminal amino acids. These in turn serve as scaffolds for signaling molecules (King et al., 1988, EMBO J, vol. 7: pp. 1647-1651). The most active HER2 heterodimer is the HER2-HER3 complex (Pinkas-Kramarski et al., 1996, EMBO J, vol. 15: pp. 2452-2467), in which HER2 complements kinase-deficient HER3 by providing an active kinase (Guy et al., 1994, Proc Natl Acad Sci USA, vol. 91: pp. 8132-8136). In contrast to EGFR, HER2 is internalization-resistant (Hommelgaard et al., 2004, Mol Biol Cell, vol. 15: pp. 1557-1567), avoiding lysosomal degradation and thus remaining on the plasma membrane.

[0142] In certain embodiments, the antigen-binding regions provided herein comprise one or more CDR sequences. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact.

[0143] In some embodiments, the first antigen-binding region provided herein comprises a first heavy-chain variable region (VH1) and a first light-chain variable region (VL1), the VH1 comprising heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3 as shown in the heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:1, and the VL1 comprising light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3 as shown in the light chain (LC) comprising the amino acid sequence of SEQ ID NO:2. In certain embodiments, the first heavy-chain variable region (VH1) comprises heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NOs:5, 6, and 7, respectively; and the first light-chain variable region (VL1) comprises light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NOs:8, 9, and 10, respectively.

[0144] In certain embodiments, VH1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH of the HC sequence as shown in SEQ ID NO:1; and VL1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VL of the LC sequence as shown in SEQ ID NO:2. In certain embodiments, VH1 comprises an amino acid sequence identical to the VH of the HC sequence as shown in SEQ ID NO:1; and VL1 comprises an amino acid sequence identical to the VL of the LC sequence as shown in SEQ ID NO:2.

[0145] In some embodiments, the second antigen-binding region provided herein comprises a first heavy-chain variable region (VH2) and a second light-chain variable region (VL2), the VH2 comprising heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3 as shown in HC comprising the amino acid sequence of SEQ ID NO:11, and the VL2 comprising light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3 as shown in LC comprising the amino acid sequence of SEQ ID NO:12. In certain embodiments, the second heavy-chain variable region (VH1) comprises heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively; and the first light-chain variable region (VL1) comprises light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively.

[0146] In certain embodiments, VH1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to VH of the HC sequence as shown in SEQ ID NO:1; and VL1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to VL of the LC sequence as shown in SEQ ID NO:2. In certain embodiments, VH1 comprises the same amino acid sequence as VH of the HC sequence as shown in SEQ ID NO:1; and VL1 comprises the same amino acid sequence as VL of the LC sequence as shown in SEQ ID NO:2.

[0147] The determination of the percent identity between two sequences (e.g., amino acid or nucleic acid sequences) can be accomplished using a mathematical algorithm. Non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., Vol. 87: pp. 2264-2268, 1990, as Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., Vol. 90: pp. 5873-5877, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol., Vol. 215: p. 403, 1990. The BLAST nucleotide search can be performed with the NBLAST nucleotide program parameter set, e.g., for score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. The BLAST protein search can be performed with the XBLAST program parameter set, e.g., for score 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain a gapped alignment for comparison purposes, as in Altschul et al., Nucleic Acids Res. Vol. 25: pp. 3389-3402, 1997. Alternatively, an iterative search to detect the distance relationship (Id.) between molecules can be performed using PSI BLAST. When using the BLAST, gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS, Vol. 4: pp. 11-17, 1998. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, allowing or not allowing gaps. When calculating percent identity, generally only exact matches are counted.

[0148] In some embodiments, the antibodies provided herein contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the anti-HER2 antibody containing the sequence retains the ability to bind HER2. In some embodiments, a total of 1 to 10 amino acids in the reference amino acid sequence have been substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HER2 antibodies or antigen-binding regions provided herein include post-translational modifications of the reference sequence.

[0149] In some embodiments, functional epitope mapping can be performed, for example, by alanine scanning to identify the amino acids in the HER2 protein that are necessary for interaction with the anti-HER2 antibodies provided herein. In some embodiments, the conformation and crystal structure of an anti-HER2 antibody that binds HER2 can be used to identify the epitope. In some embodiments, the present disclosure provides an antibody that specifically binds the same epitope as any one of the anti-HER2 antibodies provided herein.

[0150] In certain embodiments, the multispecific antibodies of the invention comprise a third antigen-binding portion thereof that binds to primate CD98 (such as human CD98 or monkey CD98), and the antibody or antigen-binding fragment thereof is optimized for delivering an agent to the brain of a subject in need thereof. The inventors of the present invention surprisingly found that the relationship between affinity and endocytic transport efficiency is more subtle than previously described, and the effects from both the association rate and the dissociation rate have an impact on brain concentration. Specifically, in order to achieve optimal brain PK and PD of an agent (such as an mAb) effectively delivered by an anti-CD98 antibody or its antigen-binding fragment, a moderate dissociation rate that is neither too fast nor too slow is required. Anti-CD98 antibodies and their antigen-binding fragments are described in International Publication No. WO 2021205361, which is incorporated herein by reference in its entirety.

[0151] In certain embodiments, the third antigen-binding region comprises a first single-chain variable fragment (scFv1) having: a third heavy-chain variable region (VH3) comprising HCDR1, HCDR2, and HCDR3, and a third light-chain variable region (VL3) comprising LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as the corresponding CDRs of the scFv shown in Table 2.

[0152] In certain embodiments, the third antigen-binding region comprises a first single-chain variable fragment (scFv1) having a third heavy-chain variable region (VH3) comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and a third light-chain variable region (VL3) comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each have any one of the amino acid sequences shown in Table 2.

[0153] Table 2. Exemplary Anti - CD98 Antibodies or Antigen - Binding Fragments Thereof

[0154]

[0155]

[0156] In certain embodiments, VH3 and VL3 comprise amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the corresponding VH and VL of an scFv as shown in the following sequences: SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ IDNO:59, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94.

[0157] In certain embodiments, VH3 and VL3 comprise the same amino acid sequences as the corresponding VH and VL of the scFv shown in the following sequences: SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94.

[0158] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, and (b) a first light chain (LC1) comprising VL1 and a light chain constant region.

[0159] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a second heavy chain (HC2) comprising VH1 and a first heavy chain constant region containing a second Fc region (Fc2), and (b) a second light chain (LC2) comprising VL1 and a light chain constant region.

[0160] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0161] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2 and a first heavy chain constant region containing a first Fc region (Fc1), (b) a first light chain (LC1) comprising VL2 and a light chain constant region. And (c) a second heavy chain (HC2) comprising scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0162] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region.

[0163] In certain embodiments, a multispecific antibody or an antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH1, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, (b) a first light chain (LC1) comprising VL1 and a light chain constant region. And (c) a second heavy chain (HC2) comprising a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein scFv2 comprises VH2 and VL2.

[0164] In certain embodiments, a multispecific antibody or an antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1) comprising VH2, a first heavy chain constant region containing a first Fc region (Fc1), and scFv1, (b) a first light chain (LC1) comprising VL2 and a light chain constant region. And (c) a second heavy chain (HC2) comprising a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein scFv2 comprises VH1 and VL1.

[0165] In certain embodiments, the scFv of the present invention comprises a heavy chain variable region (H V ) covalently linked via a flexible linker to a light chain variable region (L V ). Although the constant regions are removed and a linker is introduced, the scFv can still retain the specificity of the original immunoglobulin. In the scFv, the order of the domains can be H V -linker-L V or L V -linker-H V . The linker can be de novo designed or derived from known protein structures to provide a compatible length and conformation when bridging the variable domains of the scFv without significant steric interference. The linker can have a length of 10 to about 25 amino acids. Preferably, the linker spans between the carboxyl terminus of the variable domain and the amino terminus of the other domain by about 3.5 nm A peptide linker that does not affect the ability of the domain to fold and form a complete antigen-binding site (Huston et al., Methods in Enzymology, Vol. 203, pp. 46 - 88, 1991, the full text of which is incorporated herein by reference). The linker preferably contains a hydrophilic sequence to avoid the peptide being embedded within or between the variable domains during the folding of the entire protein (Argos, Journal of Molecular Biology, Vol. 211, No. 4, pp. 943 - 958, 1990). For example, the linker may contain Gly and Ser residues and / or be interspersed with charged residues such as Glu, Thr, and Lys to increase solubility. In one embodiment, the linker has the amino acid sequence of SEQ ID NO: 97 (GTEGKSSGSGSESKST). In another embodiment, the linker has the amino acid sequence of SEQ ID NO: 98 (GGSEGKSSGSGSESKSTGGS). According to the present disclosure, any other suitable linker may also be used.

[0166] In certain embodiments, the scFv of the present invention can be a stabilized scFv, referred to herein as a stapled Fv (spFv). "Staple" refers to an scFv linker containing one or two Cys residues capable of forming a disulfide bond with an anchor point Cys. As used herein, "VH cysteine" or "VH Cys" refers to a Cys residue present in the VH framework. "VL cysteine" or "VL Cys" refers to a Cys residue present in the VL framework. "Stable" means that the scFv is considered thermostable when it retains binding comparable to hK2 when compared to an unheated scFv sample. Stapled Fv is described in International Publication No. WO2021 / 030657, the full text of which is incorporated herein by reference.

[0167] In certain embodiments, the isolated single-chain variable fragment (scFv) comprises a heavy-chain variable region (VH), a linker (L), and a light-chain variable region (VL), wherein the scFv comprises a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys; a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys; or a first disulfide bond between a structurally conserved surface-exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys.

[0168] In certain embodiments, scFv1 and scFv2 each independently comprise a first disulfide bond and a second disulfide bond.

[0169] In certain embodiments, the linker has the amino acid sequence of SEQ ID NO:99 (GGGSGGSGGCPPCGGSGG).

[0170] In certain embodiments, the scFv also includes histidine at the N-terminus.

[0171] In certain embodiments, scFv1 includes an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94. Preferably, scFv1 includes an amino acid sequence selected from the group consisting of: SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94.

[0172] In certain embodiments, scFv2 includes an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:3 or 4. Preferably, scFv2 includes the amino acid sequence of SEQ ID NO:3 or 4.

[0173] Antibody Variants

[0174] In some embodiments, amino acid sequence modifications of the HER2-binding antibodies described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the HER2-binding antibodies described herein, variants of the HER2-binding antibodies described herein are contemplated. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art of understanding amino acid changes can alter the post-translational processes of the antibody.

[0175] Chemical Modifications

[0176] In some embodiments, the antibodies provided herein are chemically modified, for example, by covalently attaching any type of molecule to the antibody. Antibody derivatives can include antibodies that have been chemically modified, for example, by glycosylation, acetylation, polyethylene glycolylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cell ligand or other protein, or conjugation to one or more immunoglobulin domains (e.g., Fc or a portion of Fc). Any of a number of chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis with tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0177] In some embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0178] When the antibodies provided herein are fused to an Fc region, the carbohydrates attached thereto can be altered. Native antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally attached via an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH, Vol. 15: pp. 26-32, 1997. The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the binding molecules provided herein can be modified to produce variants with certain improved properties.

[0179] In other embodiments, when the antibodies provided herein are fused to an Fc region, the antibody variants provided herein can have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 and US2004 / 0093621. Exemplary disclosures related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US 2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol., Vol. 336: pp. 1239-1249, 2004; Yamane-Ohnuki et al., Biotech. Bioeng., Vol. 87: p. 614, 2004.Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem., Biophys., Vol. 249: pp. 533-545, 1986; US Patent Application No. US 2003 / 0157108; and WO 2004 / 056312), and knockout cell lines such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng., Vol. 87: p. 614, 2004; Kanda, Y. et al., Biotechnol. Bioeng., Vol. 94, No. 4: pp. 680-688, 2006; and WO2003 / 085107).

[0180] Binding molecules comprising the antibodies provided herein are also provided with aliquots of oligosaccharides, e.g., wherein the biantennary oligosaccharide attached to the Fc region is bisected with GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants are also provided in which at least one galactose residue in the oligosaccharide is attached to the Fc region. Such variants may have improved CDC function. Such variants are described, e.g., in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0181] In molecules comprising the antibodies and Fc regions of the present invention, one or more amino acid modifications can be introduced into the Fc region to generate Fc region variants. The Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0182] In some embodiments, the present application contemplates variants that have some but not all effector functions, making them desirable candidates for applications where the in vivo half-life of the binding molecule is important but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the binding molecule lacks FcγR binding (and thus may lack ADCC activity) but retains the ability to bind FcRn. Non-limiting examples of in vitro assays to evaluate the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA, Vol. 83: pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, Vol. 82: pp. 1499-1502, 1985; 5,821,337 (see Bruggemann, M. et al., J. Exp. Med., Vol. 166: pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods can be employed, see, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Available effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA, Vol. 95: pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods, Vol. 202: p. 163, 1996; Cragg, M. S. et al., Blood, Vol. 101: pp. 1045-1052, 2003; and Cragg, M. S and M. J. Glennie, Blood, Vol. 103: pp. 2738-2743, 2004). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol., Vol. 18 No. 12: pp. 1759-1769, 2006).

[0183] Binding molecules with reduced effector function include binding molecules that substitute one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, which has alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581).

[0184] Certain variants with improved or attenuated FcR binding are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem., Vol. 9 No. 2: pp. 6591-6604, 2001.)

[0185] In some embodiments, the variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In some embodiments, changes are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., Vol. 164: pp. 4178-4184, 2000.

[0186] Binding molecules having increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol., Vol. 117: p. 587, 1976; and Kim et al., J. Immunol., Vol. 24: p. 249, 1994), are described in US2005 / 0014934A1 (Hinton et al.). Those molecules comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following residues in the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., a substitution at residue 434 of the Fc region (U.S. Patent No. 7,371,826). See also Duncan and Winter, Nature, Vol. 322: pp. 738-740, 1988; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which relate to other examples of Fc region variants.

[0187] In some embodiments, it may be desirable to generate cysteine-engineered antibodies in which one or more residues of the antibody are replaced with cysteine residues. In some embodiments, the residues to be replaced are at accessible sites of the antibody. By replacing those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody and can be used to conjugate the antibody to other moieties (such as a drug moiety or a linker-drug moiety) to produce an immunoconjugate, as further described herein.

[0188] Substitutions, Deletions or Insertions

[0189] The mutations can be substitutions, deletions or insertions of one or more codons encoding the antibody or polypeptide, which result in a change in the amino acid sequence as compared to the original antibody or polypeptide. The sites of interest for substitution mutagenesis include the CDRs and FRs.

[0190] Amino acid substitutions can be the result of replacing one amino acid with another having similar structure and / or chemical properties, such as replacing leucine with serine, e.g., conservative amino acid substitutions. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. Insertions or deletions can optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitutions, deletions or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions or fewer than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitutions are conservative amino acid substitutions at one or more predicted non-essential amino acid residues. The permissible variants can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for the activity exhibited by the parental antibody.

[0191] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing multiple residues, as well as insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue.

[0192] Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As noted above, families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence such as by saturation mutagenesis, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative (e.g., within amino acid groups having similar properties and / or side chains) substitutions can be made in order to maintain or not significantly alter properties. Exemplary substitutions are shown in Table 3 below.

[0193] Table 3. Amino Acid Substitutions

[0194]

[0195] Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry, pp. 73-75 (2nd ed., 1975): (1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); and (4) Basic: Lys (K), Arg (R), His (H). Alternatively, the naturally occurring residues can be grouped based on common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe. For example, any cysteine residue that does not participate in maintaining the correct conformation of the antibody can also be replaced, for example, with another amino acid such as alanine or serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Non-conservative substitutions would require the exchange of a member of one of these categories for a member of another category.

[0196] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant selected for further study will have an altered (e.g., improved) (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parental antibody in certain biological properties. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0197] Changes (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. Such changes can be made in CDR “hot spots,” i.e., residues encoded by codons that undergo mutation at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol., Vol. 207: pp. 179-196, 2008) and / or in the SDR (a-CDR), and the binding affinity of the resulting variant antibody or fragment thereof is tested. For example, Hoogenboom et al. have described affinity maturation by constructing secondary libraries and reselecting from the secondary libraries in Methods in Molecular Biology, Vol. 178: pp. 1-37 (edited by O’Brien et al., Human Press, Totowa, NJ, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity includes CDR-directed methods, in which several CDR residues (e.g., 4 to 6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. A more detailed description of affinity maturation is provided below.

[0198] In some embodiments, substitutions, insertions, or deletions can occur within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the CDRs. In some embodiments of the variant antibody sequences provided herein, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0199] A useful method for identifying residues or regions of an antibody that are targetable for mutagenesis is called “alanine-scanning mutagenesis,” as described by Cunningham and Wells, Science, Vol. 244: pp. 1081-1085, 1989. In this method, a residue or a set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the points of contact between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they possess the desired properties.

[0200] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminal or C-terminus of an antibody with an enzyme (e.g., for ADEPT) or a polypeptide, which extends the serum half-life of the antibody.

[0201] Variations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, Biochem J., Vol. 237: pp. 1-7, 1986; and Zoller et al., Nucl. Acids Res., Vol. 10: pp. 6487-6500, 1982), cassette mutagenesis (see, e.g., Wells et al., Gene, Vol. 34: pp. 315-323, 1985), or other known techniques can be performed on cloned DNA to generate antibody variant DNA.

[0202] Fc Mutations

[0203] To facilitate the formation of heterodimers between two heavy chains, for example, one having a fusion of an anti-HER2 antibody or an antigen-binding fragment thereof, and one not; or one having an Fc containing an anti-HER2 arm and one having an Fc containing a tissue-targeting arm, heterodimer mutations are introduced into the Fc of the two heavy chains. Examples of such Fc mutations include, but are not limited to, Zymeworks mutations (see, e.g., US10,457,742) and "knobs-into-holes" mutations (see, e.g., Ridgway et al., Protein Eng., 9(7):617-621, 1996). Other heterodimer mutations can also be used in the present disclosure. In some embodiments, the modified CH3 as described herein is used to facilitate the formation of heterodimers between two heavy chains.

[0204] In a specific embodiment, each heavy chain of the two heavy chains of the antibody comprises one or more heterodimer mutations or one or more knob and hole mutations. In a specific embodiment, the one or more heterodimer mutations are in the CH3 domain.

[0205] In certain embodiments, each of the two heavy chains of a multispecific antibody or an antigen-binding fragment thereof comprises a modified constant heavy chain 3 (CH3) domain as compared to the wild-type CH3 domain to promote heterodimer formation between the two heavy chains. Any mutation that promotes heterodimer formation between the two heavy chains can be used. Preferably, the modified CH3 domain of the first heavy chain comprises amino acid modifications at positions T350, L351, F405, and Y407, and the modified CH3 domain of the second heavy chain comprises amino acid modifications at positions T350, T366, K392, and T394. Preferably, the amino acid modification at position T350 is T350V, T350I, T350L, or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T, or F405S; the amino acid modification at position Y407 is Y407V, Y407A, or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M, the amino acid modification at position K392 is K392F, K392L, or K392M, and the amino acid modification at position T394 is T394W. More preferably, the modified heterodimeric CH3 domain of the first heavy chain comprises the mutations T350V, L351Y, F405A, and Y407V, and the modified heterodimeric CH3 domain of the second heavy chain comprises the mutations T350V, T366L, K392L, and T394W. Unless otherwise explicitly stated, throughout the specification, the numbering of amino acid residues in the antibody is performed according to the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991. In a specific embodiment, the CH3 domain of one heavy chain comprises the mutations T350V, L351Y, F405A, and Y407V, and the CH3 domain of the other heavy chain comprises the mutations T350V, T366L, K392L, and T394W.

[0206] In addition to the heterodimer mutations, other mutations can be introduced. In some embodiments, the Fc region of the antibody further comprises one or more mutations that alter (increase or decrease), preferably eliminate, ADCC / CDC (such as the AAS mutations described herein) and / or one or more mutations that alter (increase or decrease), preferably increase, the binding of the antibody to FcRn (such as the YTE mutations described herein). In some embodiments, one or more cysteine residues in the antibody are replaced with other amino acids (such as serine).

[0207] In certain embodiments, the fragment crystallizable region (Fc region) of a multispecific antibody or an antigen-binding fragment thereof contains substitutions that alter (increase or decrease), preferably eliminate effector functions such as antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Preferably, the Fc region of the multispecific antibody or an antigen-binding fragment thereof contains one or more amino acid modifications that reduce or eliminate the binding of the multispecific antibody or an antigen-binding fragment thereof to Fc gamma receptors (FcγR) and avoid effector function-mediated toxicity. For example, the Fc region of the multispecific antibody or an antigen-binding fragment thereof may contain one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, such as one, two, or three mutations among L234A, L235A, and D265S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0208] In certain embodiments, the Fc region of a multispecific antibody or an antigen-binding fragment thereof contains substitutions that alter (increase or decrease), preferably increase the binding of the multispecific antibody or an antigen-binding fragment thereof to the neonatal Fc receptor (FcRn). Preferably, one or more mutations enhance binding at acidic pH, and more preferably, the Fc has the M252Y / S254T / T256E (YTE) mutation, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0209] In certain embodiments, the Fc region of a multispecific antibody or an antigen-binding fragment thereof contains one or more mutations at positions M252Y, S254T, and T256E, and wherein the amino acid residues are numbered according to the EU index as described in Kabat. In certain embodiments, the Fc region of a multispecific antibody or an antigen-binding fragment thereof contains one or more mutations at positions M252Y, S254T, T256E, L234A, L235A, and D265S, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0210] In certain embodiments, the multispecific antibody or an antigen-binding fragment thereof comprises an Fc domain that does not have amino acid modifications that reduce or eliminate effector functions.

[0211] Also provided are multispecific antibodies comprising a first heavy chain, a light chain, and a second heavy chain, wherein the first heavy chain, light chain, and second heavy chain each have an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following sequences: SEQ ID NO:100, SEQ ID NO:12, and SEQ ID NO:101, respectively; or SEQ ID NO:102, SEQ ID NO:12, and SEQ ID NO:103, respectively; wherein the first antigen-binding region is capable of specifically binding to a first epitope of HER2, the second antigen-binding region is capable of specifically binding to a second epitope of HER2, and the third antigen-binding region is capable of specifically binding to CD98. Preferably, the first heavy chain, light chain, and second heavy chain each comprise the amino acid sequences of the following sequences: SEQ ID NO:100, SEQ ID NO:12, and SEQ ID NO:101, respectively; or SEQ ID NO:102, SEQ ID NO:12, and SEQ ID NO:103, respectively.

[0212] Polynucleotides

[0213] In certain embodiments, the present disclosure provides polynucleotides encoding the antibodies of the invention that bind to HER2 and fusion proteins comprising the antibodies that bind to HER2 described herein. The polynucleotides of the present disclosure can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0214] The present disclosure also relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, a fragment, analogue, and / or derivative of an antibody that binds HER2 of the present disclosure. In certain embodiments, the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identical, and in some embodiments at least about 96%, 97%, 98%, or 99% identical to the polynucleotide encoding the antibody that binds HER2 of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or at any position between those terminal positions, or may be scattered individually among the nucleotides of the reference sequence, or may be scattered in one or more contiguous groups within the reference sequence.

[0215] The polynucleotide variant may contain alterations in the coding region, non-coding region, or both. In some embodiments, the polynucleotide variant contains alterations that result in silent substitutions, additions, or deletions without altering the properties or activities of the encoded polypeptide. In some embodiments, the polynucleotide variant contains silent substitutions that do not result in an alteration in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants may be generated for various reasons, for example, to optimize codon expression for a particular host (i.e., changing the codons in a human mRNA to the preferred codons of a bacterial host such as Escherichia coli (E. coli)). In some embodiments, the polynucleotide variant contains at least one silent mutation in the non-coding region or the coding region of the sequence.

[0216] In some embodiments, polynucleotide variants are generated to regulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant has an increased expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variant has a decreased expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0217] Vectors containing the nucleic acid molecules described herein are also provided. In one embodiment, the nucleic acid molecule can be incorporated into a recombinant expression vector. The present disclosure provides recombinant expression vectors containing any nucleic acid of the present disclosure. As used herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a host cell under conditions sufficient to allow expression of the mRNA, protein, polypeptide, or peptide in the cell, permits the host cell to express the mRNA, protein, polypeptide, or peptide. The vectors described herein are not naturally occurring as a whole; however, portions of these vectors can be naturally occurring. The recombinant expression vector can contain any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and which can contain natural, non-natural, or altered nucleotides. The recombinant expression vector can contain natural or non-natural nucleotide linkages, or both types of linkages. Non-natural or altered nucleotides or nucleotide linkages do not prevent transcription or replication of the vector.

[0218] In one embodiment, the recombinant expression vectors of the present disclosure can be any suitable recombinant expression vectors and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. Vectors can be selected from the group consisting of: pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors can be used, such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene). Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector, such as a γ-retroviral vector.

[0219] In one embodiment, recombinant expression vectors are prepared using standard recombinant DNA techniques such as those described in Sambrook et al. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[0220] The recombinant expression vector can contain regulatory sequences such as transcription and translation start and stop codons that are specific for the type of host (e.g., bacterial, plant, fungal, or animal) into which the vector is to be properly introduced and taking into account whether the vector is DNA-based or RNA-based.

[0221] The recombinant expression vector can contain one or more selectable marker genes that allow for the selection of transformed or transfected hosts. Selectable marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic hosts to provide prototrophy, and the like. Suitable selectable marker genes for the expression vector include, for example, the neomycin / G418 resistance gene, the histidinol D resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0222] The recombinant expression vector can contain a native or standard promoter operably linked to the nucleotide sequences of the present disclosure. The choice of promoter (e.g., strong promoter, weak promoter, tissue-specific promoter, inducible promoter, and development-specific promoter) is within the skill of the ordinary artisan. Similarly, the combination of a nucleotide sequence with a promoter is within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter such as the cytomegalovirus (CMV) promoter, the RSV promoter, the SV40 promoter, or the promoter found in the long terminal repeat of murine stem cell virus.

[0223] The recombinant expression vector can be designed for transient expression, for stable expression, or for both. Moreover, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0224] In addition, the recombinant expression vector can be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of the cell expressing the suicide gene. A suicide gene can be a gene that confers sensitivity of the cell expressing the gene to an agent such as a drug and causes cell death upon contact or exposure of the cell to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0225] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure.

[0226] Also provided are host cells comprising the nucleic acid molecules described herein. A host cell can be any cell that contains heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism that has been selected, modified, transformed, grown, used, or manipulated in any way for the production of a substance by the cell, such as the expression of a gene, DNA or RNA sequence, protein, or enzyme by the cell. Suitable hosts can be determined. For example, a host cell can be selected based on the vector backbone and the desired outcome. By way of example, a plasmid or cosmid can be introduced into a prokaryotic host cell for the replication of several types of vectors. Bacterial cells such as, but not limited to, DH5α, JM109, and KCB, competent cells, and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. Additionally, bacterial cells such as Escherichia coli (E. coli) LE392 can be used as host cells for bacteriophage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells for vector replication and / or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, Saos, PC12, SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other available cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

[0227] Preparation and Preparation Methods of Antibodies

[0228] Methods of preparing antibodies have been described. See, e.g., Els Pardon et al., Nature Protocol, Vol. 9, No. 3: pp. 674, 2014. Antibodies, such as scFv fragments, can be obtained using methods known in the art, such as by immunizing Camelid species (such as camels or llamas) and obtaining hybridomas therefrom, or by cloning antibody libraries using molecular biology techniques known in the art and subsequently selecting by ELISA with individual clones of the unselected library or by using phage display.

[0229] The antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing a nucleic acid encoding the antibody. Polynucleotide sequences encoding the polypeptide components of the antibodies of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells such as hybridoma cells or B cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a host cell. Many vectors available and known in the art can be used for the purposes of the present disclosure. The choice of the appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expressing the antibodies of the present disclosure include prokaryotes such as archaebacteria and eubacteria, including Gram-negative or Gram-positive organisms; eukaryotic microorganisms such as filamentous fungi or yeast; invertebrate cells such as insect or plant cells; and vertebrate cells, such as mammalian host cell lines. The host cells are transformed with the above-described expression vectors and cultured in a conventional nutrient medium that is modified to be suitable for inducing the promoter, selecting transformants, or amplifying the gene encoding the desired sequence. The antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0230] Antibody production methods including vector construction, expression, and purification are further described in Plückthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation, pp. 203-252 (edited by McCafferty et al., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, published in Current Protocols in Protein Science, 2009; Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, published in Antibody Expression and Production (edited by Al-Rubeai, 2011); and Therapeutic Monoclonal Antibodies: From Bench to Clinic (edited by An, 2009).

[0231] Of course, it is contemplated that alternative methods well known in the art can be employed to prepare anti-HER2 antibodies. For example, solid-phase techniques can be used to generate the appropriate amino acid sequence or portions thereof by direct peptide synthesis (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, 1969; and Merrifield, J. Am. Chem. Soc., vol. 85: pp. 2149-2154, 1963). In vitro protein synthesis can be carried out using manual techniques or by automation. The various portions of the anti-HER2 antibody can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-HER2 antibody. Alternatively, the antibody can be purified from the cells or body fluids such as milk of transgenic animals engineered to express the antibody, as disclosed, for example, in U.S. Patents 5,545,807 and 5,827,690.

[0232] Pharmaceutical Compositions

[0233] In one aspect, the present disclosure also provides a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the multispecific antibody or antigen-binding fragment thereof provided herein and a pharmaceutically acceptable excipient.

[0234] A pharmaceutical composition comprising a multispecific antibody or an antigen-binding fragment thereof is prepared by mixing the fusion protein having the desired purity with an optional physiologically acceptable excipient for storage in the form of an aqueous solution or in a lyophilized or other dried form (see, e.g., Remington, Remington’s Pharmaceutical Sciences (18th ed., 1980)).

[0235] The multispecific antibodies or antigen-binding fragments thereof of the present disclosure can be formulated in any suitable form for delivery to target cells / tissues, such as, for example, as microcapsules or coarse emulsions (Remington, supra; Park et al., 2005, Molecules, Vol. 10: pp. 146-161; Malik et al., 2007, Curr. Drug. Deliv., Vol. 4: pp. 141-151), as sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol., Vol. 16: pp. 153-157), or in liposomes (Maclean et al., 1997, Int. J. Oncol., Vol. 11: pp. 325-332; Kontermann, 2006, Curr. Opin. Mol. Ther., Vol. 8: pp. 39-45).

[0236] The antibodies or antigen-binding fragments thereof provided herein can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or entrapped in coarse emulsions. Such techniques are disclosed in, for example, Remington, supra.

[0237] A variety of compositions and delivery systems are known and can be used in conjunction with antibodies or antigen-binding fragments thereof as described herein, including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing multispecific antibodies or antigen-binding fragments thereof, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem., Vol. 262: pp. 4429-4432), construction of nucleic acids as part of retroviral or other vectors, etc. In another embodiment, the composition can be provided as a controlled or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see, e.g., Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng., Vol. 14: pp. 201-240; Buchwald et al., 1980, Surgery, Vol. 88: pp. 507-516; and Saudek et al., 1989, N. Engl. J. Med., Vol. 321: pp. 569-574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof as described herein) or the compositions provided herein (see, e.g., Medical Applications of Controlled Release (edited by Langer and Wise, 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (edited by Smolen and Ball, 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem., Vol. 23: pp. 61-126; Levy et al., 1985, Science, Vol. 228: pp. 190-192; During et al., 1989, Ann. Neurol., Vol. 25: pp. 351-356; Howard et al., 1989, J. Neurosurg., Vol. 71: pp. 105-112; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253).Examples of polymers for sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer for use in a sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.

[0238] In another embodiment, a controlled or sustained release system can be placed near a particular target tissue (e.g., nasal passage or lung), such that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, pp. 115 - 138, 1984). Controlled release systems are discussed, for example, by Langer, Science, Vol. 249: pp. 1527 - 1533, 1990. Any technique known to those of skill in the art can be used to produce a sustained release formulation comprising one or more antibodies or antigen-binding fragments thereof as described herein (see, e.g., U.S. Patent No. 4,526,938, PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., Radiotherapy & Oncology, Vol. 39: pp. 179 - 189, 1996; Song et al., PDA J. of Pharma. Sci. & Tech., Vol. 50: pp. 372 - 397, 1995; Cleek et al., Pro. Int’l. Symp. Control. Rel. Bioact. Mater., Vol. 24: pp. 853 - 854, 1997; and Lam et al., Proc. Int’l. Symp. Control Rel. Bioact. Mater., Vol. 24: pp. 759 - 760, 1997).

[0239] Methods of Using Antibodies

[0240] In one aspect, provided herein is a method of treating or detecting a disorder in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or an antibody fragment thereof provided herein.

[0241] In one aspect, provided herein is a method of delivering a therapeutic or diagnostic agent to a particular tissue of a subject in need thereof, the method comprising administering to the subject a multispecific antibody or an antibody fragment provided herein.

[0242] In one aspect, provided herein is a method of inducing antibody-dependent phagocytosis (ADP) in a subject in need thereof without stimulating the secretion of pro-inflammatory cytokines, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment provided herein.

[0243] In one aspect, provided herein is a method of reducing or eliminating effector function.

[0244] In one aspect, provided herein is a method of attenuating HER2 activity on a cell, the method comprising exposing the cell to an effective amount of a multispecific antibody or antigen-binding fragment provided herein.

[0245] In another aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment provided herein. In one embodiment, the disease or disorder is a HER2-mediated disease or disorder. Also provided herein is a method of treating a disease or disorder, wherein one or more therapeutic agents are administered to the subject in combination with an antibody or antigen-binding fragment provided herein.

[0246] The present disclosure also relates to a method of using an antibody provided herein to inhibit (i.e., antagonize) the function of HER2 in order to inhibit HER2 activation, thereby treating a pathological disorder.

[0247] The pathological disorder can be cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer.

[0248] In certain embodiments, the disease or disorder is brain metastasis.

[0249] In another aspect, provided herein is the use of a multispecific antibody or antigen-binding fragment provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0250] In another aspect, provided herein is the use of a pharmaceutical composition provided herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0251] On the other hand, the present disclosure provides the use of the multispecific antibodies or antigen-binding fragments thereof provided herein in the manufacture of a medicament for a method of detecting the presence of HER2 in a biological sample, the method comprising contacting the biological sample with the antibody under conditions that permit binding of the antibody to the HER2 protein and detecting whether a complex is formed between the antibody and the HER2 protein.

[0252] In other aspects, the antibodies and fragments thereof of the present disclosure can be used to detect the presence of HER2 in a biological sample. As used herein, the term "detect" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises a body fluid, cell, or tissue. Diagnostic assays and methods are described in more detail below.

[0253] Methods of Administration and Dosing

[0254] In one specific embodiment, the present disclosure provides a composition for preventing and / or treating a disease or disorder comprising an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the present disclosure provides a composition for preventing a disease or disorder, wherein the composition comprises an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the present disclosure provides a composition for treating a disease or disorder, wherein the composition comprises an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder is a HER2-mediated disease. In some embodiments, the disease or disorder is associated with HER2. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, administration results in prevention, management, treatment, or amelioration of the disease or disorder.

[0255] In one embodiment, provided herein is a composition for preventing and / or treating symptoms of a disease or disorder, wherein the composition comprises an antibody or an antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for preventing symptoms of a disease or disorder, wherein the composition comprises an antibody or an antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a composition for treating symptoms of a disease or disorder, wherein the composition comprises an antibody or an antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder is HER2-mediated and / or HER2-mediated diseases. In some embodiments, the disease or disorder is associated with HER2. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, administration results in prevention or treatment of symptoms of the disease or disorder.

[0256] In another embodiment, provided herein is a method of preventing and / or treating a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder is a HER2-mediated and / or HER2-associated disease. In some embodiments, the disease or disorder is associated with HER2. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, the administration results in prevention or treatment of the disease or disorder.

[0257] In another embodiment, provided herein is a method of preventing and / or treating the symptoms of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of preventing the symptoms of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, provided herein is a method of treating the symptoms of a disease or disorder in a subject, the method comprising administering an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the disease or disorder is a HER2-mediated and / or HER2-associated disease or disorder. In some embodiments, the disease or disorder is associated with HER2. In some embodiments, the disease or disorder is cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer, pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, and head and neck cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has a disease or disorder. In other embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, the administration results in the prevention or treatment of the symptoms of the disease or disorder.

[0258] Also provided herein are methods of preventing and / or treating a disease or disorder by administering to a subject an effective amount of an antibody or antigen-binding fragment thereof provided herein or a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein. In one aspect, the multispecific antibody or antigen-binding fragment thereof is substantially purified (i.e., substantially free of substances that limit its effect or produce undesirable side effects). The subject to whom the therapy is administered can be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey (such as cynomolgus monkey) or human). In one embodiment, the subject is human. In another embodiment, the subject is a human having a disease or disorder.

[0259] A variety of delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., the antibodies or antigen-binding fragments thereof provided herein), including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing multispecific antibodies or antigen-binding fragments thereof / receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem., Vol. 262: pp. 4429-4432, 1987), construction of nucleic acids as part of retroviruses or other vectors, etc. Methods of administering prophylactic or therapeutic agents (e.g., the antibodies or antigen-binding fragments thereof provided herein) or pharmaceutical compositions include but are not limited to parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, and mucosal administration (e.g., intranasal and oral routes). In a specific embodiment, the prophylactic or therapeutic agent (e.g., the antibody or antigen-binding fragment thereof provided herein) or pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The prophylactic or therapeutic agent or composition can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucosal layers (e.g., oral mucosa, intranasal mucosa, rectal, and intestinal mucosa, etc.) and can be co-administered with other bioactive agents. The administration can be systemic or local. Additionally, pulmonary administration can also be employed, e.g., by using an inhaler or nebulizer and formulating with an aerosol. See, e.g., U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0260] In a specific embodiment, it may be desirable to locally administer the prophylactic or therapeutic agent or the pharmaceutical composition provided herein to the area in need of treatment. This can be achieved, for example, but not limited to, by local infusion, by topical application (e.g., by intranasal spray), by injection, or by means of an implant having a porous, non-porous, or gel-like material, including membranes (such as sialastic membranes) or fibers. In some embodiments, when administering the antibody or antigen-binding fragment thereof provided herein, care must be taken to use materials that do not absorb the antibody or antigen-binding fragment.

[0261] In another embodiment, a prophylactic or therapeutic agent or a composition provided herein can be delivered in vesicles, particularly liposomes (see Langer, Science, Vol. 249: pp. 1527-1533, 1990; Treat et al., published in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365, 1989; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0262] In another embodiment, a prophylactic or therapeutic agent or a composition provided herein can be delivered in a controlled release or sustained release system. In one embodiment, a pump can be used to effect controlled release or continuous release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng., Vol. 14: p. 20; Buchwald et al., 1980, Surgery, Vol. 88: p. 507; Saudek et al., 1989, N. Engl. J. Med., Vol. 321: p. 574). In another embodiment, polymeric materials can be used to effect controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody provided herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem., Vol. 23: p. 61; also see Levy et al., 1985, Science, Vol. 228: p. 190; During et al., 1989, Ann. Neurol., Vol. 25: p. 351; Howard et al., 1989, J. Neurosurg., Vol. 71: p. 105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253. Examples of polymers for use in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable.In another embodiment, a controlled or sustained release system can be placed near the therapeutic target (i.e., the nasal passages or the lung), so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2: pp. 115-138, 1984). Controlled release systems are reviewed by Langer (1990, Science 249:1527-1533). Any technique known to those of skill in the art can be used to produce a sustained release formulation containing one or more antibodies or antigen-binding fragments thereof provided herein. See, e.g., U.S. Patent No. 4,526,938; PCT Publication WO 91 / 05548; PCT Publication WO 96 / 20698; Ning et al., 1996, "Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel", Radiotherapy & Oncology, Vol. 39: pp. 179-189; Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA Journal of Pharmaceutical Science & Technology, Vol. 50: pp. 372-397; Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", Pro. Int'l. Symp. Control. Rel. Bioact. Mater., Vol. 24: pp. 853-854; and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery", Proc. Int'l. Symp. Control Rel. Bioact. Mater., Vol. 24: pp. 759-760, each of which is incorporated herein by reference in its entirety.

[0263] In certain embodiments, where the compositions provided herein are nucleic acids encoding prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof provided herein), the nucleic acids can be administered in vivo to promote expression of the prophylactic or therapeutic agent they encode by, for example, using a retroviral vector (see U.S. Patent No. 4,980,286) or by directly injecting or by using particle bombardment (e.g., gene gun; Biolistic, Dupont) to construct the nucleic acid as part of a suitable nucleic acid expression vector and administering it to make it intracellular, or by coating with lipids or cell surface receptors or transfection agents, or by administering the nucleic acid linked to a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA, Vol. 88: pp. 1864-1868), etc. Alternatively, the nucleic acid can be introduced into cells and incorporated into the host cell DNA by homologous recombination for expression.

[0264] In certain embodiments, the compositions provided herein comprise one, two or more antibodies or antigen-binding fragments thereof provided herein. In another embodiment, the compositions provided herein comprise one, two or more antibodies or antigen-binding fragments thereof provided herein and a prophylactic or therapeutic agent other than the antibodies or antigen-binding fragments thereof provided herein. In one embodiment, the known prophylactic or therapeutic agent can be used for or has been used for or is currently used for preventing, managing, treating and / or ameliorating a disease or disorder. In addition to the prophylactic or therapeutic agent, the compositions provided herein can also comprise excipients.

[0265] The compositions provided herein include bulk pharmaceutical compositions that can be used to prepare unit dosage forms and that can be used to manufacture pharmaceutical compositions (e.g., compositions suitable for administration to a subject or patient). In one embodiment, the compositions provided herein are pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., antibodies or antigen-binding fragments thereof provided herein or other prophylactic or therapeutic agents) and a pharmaceutically acceptable excipient. The pharmaceutical compositions can be formulated for a route suitable for administration to a subject.

[0266] In specific embodiments, the term "excipient" may also refer to a diluent, an adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or a vehicle. A pharmaceutical excipient can be a sterile liquid, such as water and oils, including those derived from petroleum, animals, plants or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When a pharmaceutical composition is administered intravenously, water is an exemplary excipient. Aqueous saline and dextrose solutions, as well as glycerol solutions, can also be used as liquid excipients, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifying agents or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, 1990, Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the multispecific antibody or antigen-binding fragment thereof provided herein, such as in purified form, and a suitable amount of excipient so as to provide a form suitable for proper administration to a patient. The formulation should be suitable for the mode of administration.

[0267] In one embodiment, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Generally, a composition for intravenous administration is a sterile isotonic buffered aqueous solution. If necessary, the composition may also contain solubilizing agents and local anesthetics such as lidocaine to alleviate the pain at the injection site. However, such compositions can be administered by routes other than intravenous administration.

[0268] Generally, the components of the compositions provided herein are provided separately or mixed together in unit dosage forms in airtight containers such as ampoules or sachets indicating the amount of the active agent, e.g., as dry lyophilized powders or anhydrous concentrates. In the case where the composition is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline can be used to dispense the composition. In the case where the composition is administered by injection, ampoules of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0269] The antibodies or antigen-binding fragments thereof provided herein can be packaged in airtight containers such as ampoules or sachets that indicate the amount of the antibody. In one embodiment, the multispecific antibody or antigen-binding fragment thereof is provided as a dry, sterilized, lyophilized powder or anhydrous concentrate in an airtight container and can be reconstituted to an appropriate concentration, for example, with water or saline for administration to a subject. The lyophilized antibody or antigen-binding fragment thereof can be stored between 2°C and 8°C in its original container, and the multispecific antibody or antigen-binding fragment thereof can be administered within 12 hours after reconstitution, such as within 6 hours, within 5 hours, within 3 hours, or within 1 hour. In an alternative embodiment, the antibodies or antigen-binding fragments thereof provided herein are provided in liquid form in an airtight container that indicates the amount and concentration of the antibody.

[0270] The compositions provided herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed from anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and those formed from cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0271] The amount of a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof provided herein) or a composition provided herein that will be effective in preventing and / or treating a disease or disorder can be determined by standard clinical techniques. Additionally, in vitro assays can optionally be employed to assist in determining the optimal dosage range. The exact dosage to be employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the physician and the circumstances of each patient.

[0272] An effective dose can be extrapolated from a dose-response curve derived from in vitro or animal model test systems.

[0273] In certain embodiments, the route of administration of a dose of an antibody or antigen-binding fragment thereof provided herein to a patient is intranasal, intramuscular, intravenous, subcutaneous, or a combination thereof, but other routes described herein are also acceptable. Each dose may or may not be administered by the same route of administration. In some embodiments, an antibody or antigen-binding fragment thereof provided herein can be administered simultaneously or subsequently via multiple routes of administration to other doses of the same or different antibodies or antigen-binding fragments thereof provided herein.

[0274] In certain embodiments, a multispecific antibody or antigen-binding fragment thereof provided herein is administered prophylactically or therapeutically to a subject. A multispecific antibody or antigen-binding fragment thereof provided herein can be administered prophylactically or therapeutically to a subject in order to prevent, mitigate, or improve a disease or its symptoms.

[0275] Diagnostic Assays and Methods

[0276] Labeled antibodies, their derivatives, and analogs that immunospecifically bind to the HER2 antigen can be used for diagnostic purposes to detect, diagnose, or monitor HER2-mediated diseases. Accordingly, provided herein are methods for detecting HER2-mediated diseases, the methods comprising: (a) assaying for the expression of the HER2 antigen in a cellular or tissue sample of a subject using one or more antibodies provided herein that immunospecifically bind to the HER2 antigen; and (b) comparing the level of the HER2 antigen to a control level (e.g., the level in a normal tissue sample (e.g., from a patient not suffering from a HER2-mediated disease, or from the same patient prior to the onset of the disease)), whereby an increase in the measured level of the HER2 antigen compared to the control level of the HER2 antigen indicates a HER2-mediated disease.

[0277] Also provided herein is a diagnostic assay for diagnosing a HER2-mediated disease, the diagnostic assay comprising: (a) assaying for the level of the HER2 antigen in a cellular or tissue sample of an individual using one or more antibodies provided herein that immunospecifically bind to the HER2 antigen; and (b) comparing the level of the HER2 antigen to a control level (e.g., the level in a normal tissue sample), whereby an increase in the measured level of the HER2 antigen compared to the control level of the HER2 antigen indicates a HER2-mediated disease. In certain embodiments, provided herein is a method of treating a HER2-mediated disease in a subject, the method comprising: (a) assaying for the level of the HER2 antigen in a cellular or tissue sample of the subject using one or more antibodies provided herein that immunospecifically bind to the HER2 antigen; and (b) comparing the level of the HER2 antigen to a control level (e.g., the level in a normal tissue sample), whereby an increase in the measured level of the HER2 antigen compared to the control level of the HER2 antigen indicates a HER2-mediated disease. In some embodiments, the method further comprises (c) administering to the subject identified as having a HER2-mediated disease an effective amount of an antibody provided herein. A more definitive diagnosis of a HER2-mediated disease can allow health professionals to adopt preventive measures or aggressive treatment earlier, thereby preventing the development or further progression of the HER2-mediated disease.

[0278] The antibodies provided herein can be used to determine the level of HER2 antigen in a biological sample using classical immunohistological methods as described herein or known to those of skill in the art (see, for example, Jalkanen et al., 1985, J. Cell. Biol., Vol. 101: pp. 976-985; and Jalkanen et al., 1987, J. Cell. Biol., Vol. 105: pp. 3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin.

[0279] One aspect provided herein is the detection and diagnosis of HER2-mediated diseases in humans. In one embodiment, the diagnosis comprises: a) administering (e.g., parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody that immunospecifically binds to the HER2 antigen; b) waiting a certain time interval after administration to allow the labeled antibody to accumulate at sites in the subject where the HER2 antigen is expressed (and to clear unbound labeled molecules to background levels); c) determining the background level; and d) detecting the labeled antibody in the subject such that detection of the labeled antibody above the background level indicates that the subject has a HER2-mediated disease. The background level can be determined by various methods, including comparing the amount of labeled molecules detected to a standard value previously determined for a particular system.

[0280] It will be understood in the art that the body size of the subject and the imaging system used will determine the amount of the imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is typically in the range of about 5 millicuries to 20 millicuries of 99Tc. The labeled antibody will then accumulate at the location of cells containing the particular protein. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 of Tumor Imaging: The Radiochemical Detection of Cancer, edited by S.W. Burchiel and B.A. Rhodes, Masson Publishing Inc. (1982)).

[0281] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration that allows the labeled antibody to concentrate in the subject's body and the unbound labeled antibody to be cleared to background levels is from 6 hours to 48 hours or from 6 hours to 24 hours or from 6 hours to 12 hours. In another embodiment, the time interval after administration is from 5 days to 20 days or from 5 days to 10 days.

[0282] In one embodiment, the monitoring of HER2-mediated disease is carried out by repeating the method used for diagnosing HER2-mediated disease, such as one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0283] The presence of the labeled molecule can be detected in the subject's body using methods known in the art for in vivo scanning. These methods depend on the type of label used. Those skilled in the art will be able to determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods provided herein include, but are not limited to, computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasonography.

[0284] In a specific embodiment, the molecule is labeled with a radioisotope and detected in the patient using a radiation-responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence-responsive scanning instrument. In another embodiment, the molecule is labeled with a positron-emitting metal and detected in the patient using positron emission tomography. In another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

[0285] Kits

[0286] Also provided herein are kits that contain the multispecific antibodies (e.g., anti-HER2 antibodies) or compositions (e.g., pharmaceutical compositions) provided herein packaged in suitable packaging materials. The kit optionally includes a label or package insert that includes a description of the components or instructions for the in vitro, in vivo, or ex vivo use of the components therein.

[0287] The term "packaging material" refers to the physical structure that houses the components of the kit. The packaging material can maintain the components sterilely and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0288] The kit provided herein may include a label or instructions for use. The label or instructions for use include a "printing substance", e.g., paper or cardboard, alone or attached to a component, kit or packaging material (e.g., a box), or attached to, e.g., an ampoule, tube or vial containing a kit component. The label or instructions for use may additionally include a computer-readable medium, such as a disk (e.g., a hard disk, card, storage disk), an optical disk (such as a CD or DVD-ROM / RAM, DVD, MP3, magnetic tape) or an electrical storage medium (such as RAM and ROM) or a mixture thereof (such as a magnetic / optical storage medium, a FLASH medium or a memory card). The label or instructions for use may include information identifying the manufacturer, lot number, manufacturer location and date.

[0289] The kit provided herein may additionally include other components. Each component of the kit may be encapsulated in a separate container, and all the various containers may be within a single package. The kit may also be designed for refrigeration. The kit may also be designed to contain an antibody provided herein, or a cell containing a nucleic acid encoding an antibody provided herein. The cells in the kit may be maintained under suitable storage conditions until ready for use.

[0290] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the appropriate methods and materials are described herein.

[0291] As used herein, numerical values are generally given in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an absolute limitation on the scope of the present invention, unless the context clearly dictates otherwise. Thus, a range used herein expressly includes all possible sub-ranges, all individual numerical values within that range, and all numerical or numerical ranges (including integers and fractions or integers of the numerical values within such ranges), unless the context clearly dictates otherwise. This construction applies in all contexts throughout this patent document, regardless of the width of the range. Thus, for example, a reference to a range of 90% to 100% includes 91% to 99%, 92% to 98%, 93% to 95%, 91% to 98%, 91% to 97%, 91% to 96%, 91% to 95%, 91% to 94%, 91% to 93%, etc. A reference to a range of 90% to 100% also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., and 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., and 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc.

[0292] In addition, the ranges mentioned 1 to 3, 3 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, 190 to 200, 200 to 225, 225 to 250 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In another example, the ranges mentioned 25 to 250, 250 to 500, 500 to 1,000, 1,000 to 2,500, 2,500 to 5,000, 5,000 to 25,000, 25,000 to 50,000 include any value or a range within or encompassing such values, such as 25, 26, 27, 28, 29…250, 251, 252, 253, 254…500, 501, 502, 503, 504… etc.

[0293] A series of ranges used herein are disclosed throughout the document. The use of a series of ranges includes combinations of upper and lower limits to provide another range. Regardless of the width of the range, this construct applies in all contexts throughout this patent document. Thus, for example, the mention of a series of ranges such as 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, 100 to 150 includes ranges such as 5 to 20, 5 to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150 and 10 to 30, 10 to 40, 10 to 50, 10 to 75, 10 to 100, 10 to 150 and 20 to 40, 20 to 50, 20 to 75, 20 to 100, 20 to 150, etc.

[0294] For the sake of brevity, certain abbreviations are used herein. One example is the single-letter abbreviations for amino acid residues. Amino acids and their corresponding three-letter and single-letter abbreviations are as follows:

[0295] Alanine Ala (A)

[0296] Arginine Arg (R)

[0297] Asparagine Asn (N)

[0298] Aspartic acid Asp (D)

[0299] Cysteine Cys (C)

[0300] Glutamic acid Glu (E)

[0301] Glutamine Gln (Q)

[0302] Glycine Gly (G)

[0303] Histidine His (H)

[0304] Isoleucine Ile (I)

[0305] Leucine Leu (L)

[0306] Lysine Lys (K)

[0307] Methionine Met (M)

[0308] Phenylalanine Phe (F)

[0309] Proline Pro (P)

[0310] Serine Ser (S)

[0311] Threonine Thr (T)

[0312] Tryptophan Trp (W)

[0313] Tyrosine Tyr (Y)

[0314] Valine Val (V)

[0315] This application uses affirmative language to describe multiple embodiments and generally discloses the invention. The invention also specifically includes embodiments in which specific subject matter such as substances or materials, method steps and conditions, schemes, procedures, assays or analyses are completely or partially excluded. Thus, even though the invention is not generally expressed in terms of what it does not include, aspects that are not expressly included in the invention are still disclosed herein.

[0316] Multiple embodiments of the invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate rather than limit the scope of the invention described in the claims.

[0317] Embodiments

[0318] The disclosure provided herein also provides the following non-limiting embodiments.

[0319] 1. A multispecific antibody or an antigen-binding fragment thereof, the multispecific antibody or the antigen-binding fragment thereof comprising each capable of specifically binding to human epidermal growth factor receptor 2

[0320] At least one of the first antigen-binding region and the second antigen-binding region of (HER2), and a third antigen-binding region capable of specifically binding to CD98, wherein:

[0321] (1) The first antigen-binding region comprises:

[0322] A first heavy chain variable region (VH1), the VH1 comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NO:5, 6 and 7 respectively; and a first light chain variable region (VL1), the VL1 comprising light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NO:8, 9 and 10 respectively;

[0323] (2) The second antigen-binding region comprises:

[0324] A second heavy chain variable region (VH2), the VH2 comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13, 14 and 15 respectively; and a second light chain variable region (VL2), the VL2 comprising light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NO:16, 17 and 18 respectively; and

[0325] (3) The third antigen-binding region comprises a first single-chain variable fragment (scFv1), the scFv1 having: a third heavy chain variable region (VH3) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, and a third light chain variable region (VL3) comprising light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have any one of the amino acid sequences in Table 2.

[0326] 2. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1, wherein:

[0327] (1) The VH1 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:1; and the VL1 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:2;

[0328] (2) The VH2 comprises the same amino acid sequence as the VH of the HC sequence as shown in SEQ ID NO:11; and the VL2 comprises the same amino acid sequence as the VL of the LC sequence as shown in SEQ ID NO:12; and

[0329] (3) The VH3 and the VL3 comprise the same amino acid sequences as the corresponding VH and VL of the scFv shown in the following sequences:

[0330] (xxii) SEQ ID NO:19;

[0331] (xxiii) SEQ ID NO:26;

[0332] (xxiv) SEQ ID NO:33;

[0333] (xxv) SEQ ID NO:34;

[0334] (xxvi) SEQ ID NO:35;

[0335] (xxvii) SEQ ID NO:36;

[0336] (xxviii) SEQ ID NO:43;

[0337] (xxix) SEQ ID NO:49;

[0338] (xxx) SEQ ID NO:55;

[0339] (xxxi) SEQ ID NO:59;

[0340] (xxxii) SEQ ID NO:66; or

[0341] (xxxiii) SEQ ID NO:71;

[0342] (xxxiv) SEQ ID NO:78;

[0343] (xxxv) SEQ ID NO:85;

[0344] (xxxvi) SEQ ID NO:87;

[0345] (xxxvii) SEQ ID NO:88;

[0346] (xxxviii) SEQ ID NO:89;

[0347] (xxxix) SEQ ID NO:90;

[0348] (xl) SEQ ID NO:92;

[0349] (xli) SEQ ID NO:93;

[0350] (xlii) SEQ ID NO:94.

[0351] 3. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises:

[0352] (a) A first heavy chain (HC1), the HC1 comprising the VH1, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1; and

[0353] (b) A first light chain (LC1), the LC1 comprising the VL1 and a light chain constant region.

[0354] 4. The multispecific antibody or antigen-binding fragment thereof according to embodiment 3, wherein the multispecific antibody or antigen-binding fragment thereof further comprises:

[0355] (a) A second heavy chain (HC2), the HC2 comprising the VH1 and a first heavy chain constant region containing a second Fc region (Fc2), and

[0356] (b) A second light chain (LC2), the LC2 comprising the VL1 and a light chain constant region.

[0357] 5. The multispecific antibody or antigen-binding fragment thereof according to claim 3, wherein the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

[0358] 6. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises:

[0359] (a) A first heavy chain (HC1), the HC1 comprising the VH1 and a first heavy chain constant region containing a first Fc region (Fc1),

[0360] (b) A first light chain (LC1), the LC1 comprising the VL1 and a light chain constant region; and

[0361] (c) A second heavy chain (HC2), the HC2 comprising the scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0362] 7. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises:

[0363] (a) A first heavy chain (HC1), the HC1 comprising the VH2, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1; and

[0364] (b) A first light chain (LC1), said LC1 comprising said VL2 and a light chain constant region.

[0365] 8. The multispecific antibody or antigen-binding fragment thereof according to embodiment 7, said multispecific antibody or antigen-binding fragment thereof further comprising:

[0366] (a) A second heavy chain (HC2), said HC2 comprising said VH2 and a first heavy chain constant region containing a second Fc region (Fc2), and

[0367] (b) A second light chain (LC2), said LC2 comprising said VL2 and a light chain constant region.

[0368] 9. The multispecific antibody or antigen-binding fragment thereof according to embodiment 7, said multispecific antibody or antigen-binding fragment thereof further comprising a second Fc region (Fc2).

[0369] 10. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, said multispecific antibody or antigen-binding fragment thereof comprising:

[0370] (a) A first heavy chain (HC1), said HC1 comprising said VH2 and a first heavy chain constant region containing a first Fc region (Fc1),

[0371] (b) A first light chain (LC1), said LC1 comprising said VL2 and a light chain constant region; and

[0372] (c) A second heavy chain (HC2), said HC2 comprising said scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

[0373] 11. The multispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, said multispecific antibody or antigen-binding fragment thereof comprising said first antigen-binding region, said second antigen-binding region and said third antigen-binding region.

[0374] 12. The multispecific antibody or antigen-binding fragment thereof according to embodiment 11, said multispecific antibody or antigen-binding fragment thereof comprising:

[0375] (a) A first heavy chain (HC1), said HC1 comprising said VH1, a first heavy chain constant region containing a first Fc region (Fc1) and said scFv1,

[0376] (b) A first light chain (LC1), said LC1 comprising said VL1 and a light chain constant region; and

[0377] (c) A second heavy chain (HC2), said HC2 comprising a second single-chain variable fragment (scFv2) and a first heavy-chain constant region containing a second Fc region (Fc2), wherein said scFv2 comprises said VH2 and said VL2.

[0378] 13. The multispecific antibody or antigen-binding fragment thereof according to embodiment 11, said multispecific antibody or antigen-binding fragment thereof comprising:

[0379] (a) A first heavy chain (HC1), said HC1 comprising said VH2, a first heavy-chain constant region containing a first Fc region (Fc1), and said scFv1,

[0380] (b) A first light chain (LC1), said LC1 comprising said VL2 and a light-chain constant region; and

[0381] (c) A second heavy chain (HC2), said HC2 comprising a second single-chain variable fragment (scFv2) and a first heavy-chain constant region containing a second Fc region (Fc2), wherein said scFv2 comprises said VH1 and said VL1.

[0382] 14. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein said scFv1 and / or scFv2 comprises at least one of the following: (a) a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys; and b) a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys.

[0383] 15. The multispecific antibody or antigen-binding fragment thereof according to embodiment 14, wherein said scFv1 and scFv2 each independently comprise said first disulfide bond and said second disulfide bond.

[0384] 16. The multispecific antibody or antigen-binding fragment thereof according to embodiment 13, wherein said scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4.

[0385] 17. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the scFv1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 71, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94.

[0386] 18. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 4 to 6, 8 to 10, and 12 to 17, wherein each of Fc1 and Fc2 comprises one or more heterodimer mutations, or one or more knob and hole mutations.

[0387] 19. The multispecific antibody or antigen-binding fragment thereof according to embodiment 18, wherein the heterodimer mutation comprises amino acid modifications at positions T350, L351, F405, and Y407 in one of Fc1 and Fc2, and amino acid modifications at positions T350, T366, K392, and T394 in the other of Fc1 and Fc2, wherein the amino acid modification at position T350 is T350V, T350I, T350L, or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T, or F405S; the amino acid modification at position Y407 is Y407V, Y407A, or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M, the amino acid modification at position K392 is K392F, K392L, or K392M, and the amino acid modification at position T394 is T394W, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0388] 20. The multispecific antibody or antigen-binding fragment thereof according to embodiment 19, wherein one of Fc1 and Fc2 comprises the mutations T350V, L351Y, F405A, and Y407V, and the other of Fc1 and Fc2 comprises the mutations T350V, T366L, K392L, and T394W.

[0389] 21. The multispecific antibody or antigen-binding fragment thereof according to embodiment 18, wherein each of the Fc1 and the Fc2 comprises one or more knob and hole mutations.

[0390] 22. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, the multispecific antibody or antigen-binding fragment thereof comprising an Fc domain having an amino acid modification that enhances the binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably the amino acid modification enhances the binding at acidic pH, more preferably the Fc domain has an M252Y / S254T / T256E (YTE) mutation, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0391] 23. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, the multispecific antibody or antigen-binding fragment thereof comprising an Fc domain having an amino acid modification that reduces or eliminates effector function, preferably the Fc domain has one or more amino acid modifications at positions L234, L235, D265, D270, N297, E318, K320, K322, P331 and P329, such as one, two, three or four amino acid modifications among L234A, L235A, D265S and P331S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0392] 24. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, the multispecific antibody or antigen-binding fragment thereof comprising an Fc domain having one or more amino acid modifications among M252Y, S254T, T256E, L234A, L235A and D265S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

[0393] 25. The multispecific antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, the multispecific antibody or antigen-binding fragment thereof comprising an Fc domain having an amino acid modification that does not reduce or eliminate effector function.

[0394] 26. A multispecific antibody, the multispecific antibody comprising a first heavy chain, a light chain and a second heavy chain each having an amino acid sequence that is at least 90% identical to the following:

[0395] (1) SEQ ID NO: 100, SEQ ID NO: 12 and SEQ ID NO: 101, respectively; or

[0396] (2) are SEQ ID NO:102, SEQ ID NO:12, and SEQ ID NO:103, respectively;

[0397] wherein the first antigen-binding region is capable of specifically binding to a first epitope of HER2, the second antigen-binding region is capable of specifically binding to a second epitope of HER2, and the third antigen-binding region is capable of specifically binding to CD98.

[0398] 27. The multispecific antibody according to embodiment 26, wherein the first heavy chain, the light chain, and the second heavy chain each comprise the amino acid sequences of:

[0399] (1) SEQ ID NO:100, SEQ ID NO:12, and SEQ ID NO:101, respectively; or

[0400] (2) SEQ ID NO:102, SEQ ID NO:12, and SEQ ID NO:103, respectively.

[0401] 28. An isolated nucleic acid encoding the multispecific antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 27.

[0402] 29. A vector comprising the isolated nucleic acid according to embodiment 28.

[0403] 30. A host cell comprising the isolated nucleic acid according to embodiment 28 or the vector according to embodiment 29.

[0404] 31. A method for producing a multispecific antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to embodiment 30 under conditions for producing the multispecific antibody or an antigen-binding fragment thereof, and recovering the multispecific antibody or an antigen-binding fragment thereof.

[0405] 32. A pharmaceutical composition comprising the multispecific antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 27 and a pharmaceutically acceptable carrier.

[0406] 33. A method for treating or detecting a disorder, preferably cancer, in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 27 or the pharmaceutical composition according to embodiment 32.

[0407] 34. The method according to embodiment 33, wherein the disease or disorder is a HER2-related disease or disorder.

[0408] 35. The method according to embodiment 34, wherein the disease or disorder is a brain metastasis.

[0409] 36. A pharmaceutical composition comprising the isolated nucleic acid according to claim 28, the vector according to claim 29, or the host cell according to claim 30, and a pharmaceutically acceptable carrier.

[0410] 37. A method of treating a disorder, preferably cancer, in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition according to embodiment 36.

[0411] 38. The method according to embodiment 37, wherein the disease or disorder is a HER2-associated disease or disorder.

[0412] 39. The method according to embodiment 37, wherein the disease or disorder is a brain metastasis.

[0413] Examples

[0414] The following is a description of the various methods and materials used in the study and is provided to give a complete disclosure and description to those of ordinary skill in the art of how to make and use the present disclosure and is not intended to limit the scope that the inventors consider their disclosure to be, nor is it intended to represent all of the experiments that are performed below and are capable of being performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily to be performed, but rather these descriptions can be performed to generate data etc. associated with the teachings of the present disclosure. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be taken into account.

[0415] Example 1. Materials and Methods

[0416] CD98 Antibody Generation and Screening

[0417] Transgenic rodents are immunized with CD98 protein according to a multi-site repetitive immunization (RIMMS) protocol. Serum titers are evaluated by ELISA to select rodents for fusion. Lymph nodes are harvested from serum-positive rodents and fused with myeloma cells using standard methods to generate hybridomas. Supernatant hybridomas are screened by MSD for binding to protein, CD98-expressing cells, and human brain endothelial cells. Positive clones are further evaluated for internalization using brain endothelial cells, cross-reactivity with human and cynomolgus monkey proteins, or CD98 / LAT1 transport activity. CD98 clones that bind both human CD98 and cynomolgus monkey CD98, internalize without interfering with CD98 / LAT1 activity, are selected for variable sequence recovery and converted into scFv to generate TEM mAb for further characterization.

[0418] Bispecific TEM Antibody Generation and Characterization

[0419] Anti-HER2 and anti-CD98 are used to generate bispecific antibodies using the knob-in-hole heterodimerization technique (Ridgway et al., 1996, Protein Eng., Vol. 9, pp. 617–621). The anti-HER2 conjugate is prepared as a bivalent Fab, monovalent Fab, or monovalent scFv at the N-terminus of Fc, whereas the anti-CD98 conjugate is prepared as a monovalent scFv to be linked to the C-terminus of one heavy chain Fc via a (G4S)4 linker. Except for the knob and hole mutations in Fc, all antibodies except BBBB1819 contain mutations [M252Y / S254T / T256E (YTE)] that enhance FcRn binding at acidic pH for half-life extension (HLE). The BBBB1613 and BBBB1636 antibodies also contain Fc mutations [L234A / L235A / D265S (AAS)] that eliminate effector functions. According to the manufacturer's recommendations, the constructed TEM mAb is expressed in ExpiCHO-S TM cells by transient transfection with purified plasmid DNA. The harvested cell culture supernatant is purified by protein A and cation exchange chromatography. The homogeneity and purity of the final antibody are confirmed by SDS-PAGE, analytical size exclusion chromatography, and mass spectrometry.

[0420] Table 4. TEM Antibodies

[0421]

[0422]

[0423] Culture and Maintenance of HER2 - Expressing Cancer Cells: BT-474 (HTB-20), BT-474 Clone 5 (CRL-3247), HCC-1954 (CRL-2338), MDA-MB-361 (HTB-27), and U87-MG (HTB-14) were obtained from ATCC. JIMT-1 cells were obtained from CreativeBioarray. U87-MG cells were cultured in EMEM supplemented with 10% low IgG FBS. All other cells were cultured in RPMI supplemented with 10% low IgG FBS and 1% non-essential amino acids (for BT474 and BT-474 Clone 5 only). For imaging studies, cells were transduced with NucLight Red (Essen Biosciences #4476) or rFLuc-T2A-GFP (System Biosciences #LL410VA-1) lentivirus. Stable cell lines were selected and maintained in medium containing 0.5 μg / mL (BT-474, BT-474 Clone 5, MDA-MB-361) or 1 μg / mL puromycin (HCC-1954, JIMT-1).

[0424] Antibodies Binding to Cells : MDA-MB-361, BT474, HCC-1954, JIMT-1, and U87-MG cells were detached with Accutase, washed twice with cold DPBS, and resuspended in cold binding buffer (BD binding buffer with 2 mM EDTA) to a concentration of 2x10 6 cells / mL, then 50 μL / well was added to a 96-well plate. 50 μL / well of the antibody solution in cold binding buffer was added, the plate was covered, and incubated at 4 °C for 1 hour. Subsequently, the wells were washed with 100 μL / well of cold DPBS and centrifuged at 300 x g for 5 minutes to remove the supernatant. Next, the pellet was resuspended in 100 μL / well of LIVE / DEAD Fixable Dead Cell Stain (Invitrogen, 1 / 1,000 dilution) in cold DPBS and incubated on ice for 30 minutes. The cells were washed two more times in the above binding buffer, resuspended in a final volume of 50 μL / well, and read on an iQue flow cytometer (Sartorius).

[0425] Human iPSC - Derived Microglial Cell Cultures: Human microglia were derived from epithelial-derived iPSCs (IPSC0028, male, Sigma) and processed as previously described {Haenseler, 2017#607}. Briefly, iPSCs were plated into Aggrewell 800 plates and allowed to form embryoid bodies (EBs) to recapitulate microglial development in the embryo. Macrophage precursors were generated by culturing the EBs in mTESR1 for 3 days, which contained bone morphogenetic protein 4 (BMP4, 50 ng / mL), vascular endothelial growth factor (VEGF, 50 ng / mL), and stem cell factor (SCF, 25 ng / mL). The EBs were then harvested and transferred to six-well plates and cultured in EX-VIVO15 (Lonza) supplemented with Glutamax, penicillin / streptomycin, β-mercaptoethanol, IL-3 (25 ng / mL), and M-CSF (100 ng / mL) for 8 weeks to promote myeloid differentiation. Secreted macrophage precursors were collected from the supernatant and plated at 20,000 cells / well into 96-well plates, where they were matured for 14 days in advanced DMEM / F12 supplemented with Glutamax, penicillin / streptomycin, β-mercaptoethanol, IL-34 (100 ng / mL), and GM-SCF (10 ng / mL). Microglia were characterized by positive immunostaining with antibodies against the following proteins: Iba1 (019–19741; 1:500; Wako), P2RY12 (HPA014518; 1:100; Sigma), CX3CR1 (2091; 1:200; ProSci Inc.), CD11b (MAB1699; 1:500, RnD Systems), and CB68 (M078, 1:500, Dako).

[0426] pH - rodo Cell Phagocytosis : Target cell lines were labeled with red according to the manufacturer's instructions (Essen Biosciences, 4649). Before co-culturing with iPSC-derived microglia at various E:T ratios, the labeled cells were opsonized with TEM mAb or control mAb at 37 °C for 30 minutes. Microglia co-cultures were monitored every 90 minutes in SX5 imaging, and phagocytosis was measured as the total pHrodoRed area (μm 2 / image) and total pHrodoRed integrated intensity (RCU x μm 2 / image).

[0427] Co - culture Killing Assay: Prior to co - culturing with iPSC - derived microglia, the NucLight Red - labeled target cell line was isolated, washed and incubated with TEM mAb or control mAb at 37 °C for 30 minutes. Co - cultures were observed by imaging in SX5. Target cell loss was determined by quantification of the NucLight Red area in each image.

[0428] Non - Human Primate Cynomolgus Monkey PK Study

[0429] Study Design : TEM and control IgG1 mAb were administered to cynomolgus monkeys at 10 mg / kg by slow - bolus IV injection. Blood for PK was collected at 1 h, 6 h, 24 h, 72 h and 168 h post - dosing and processed into serum by the test facility laboratory protocol. To collect brain tissue, cynomolgus monkeys were placed under deep anesthesia and a final blood draw was performed. After the final blood collection, animals were euthanized at 72 h and 168 h (n = 2 at each time point), and upper body perfusion was performed by perfusing cold saline solution at 250 mL / min for at least 5 minutes according to the test facility standard operating procedure (SOP). Approximately 200 mg of tissue was isolated from predetermined brain locations (frontal lobe, hippocampus and temporal lobe), snap - frozen in liquid nitrogen and stored at - 70 °C until capillary depletion processing and tissue homogenization.

[0430] Brain Tissue Preparation : Each right / left hemisphere was weighed and processed into capillary - depleted brain tissue as previously described with some modifications. 25 Briefly, brain tissue samples were slowly thawed on wet ice, added to modified DPBS buffer (containing protease inhibitor (Pierce; A32955)) at a calculated volume (2.5 μL buffer / 1 mg tissue) and transferred to Lysing Matrix D tubes (MP Biomedicals TM ; 6913 - 100). Total cell suspensions were generated by homogenizing the tissue for 15 seconds at 2.8 m / s with a Bead Ruptor 24 Elite (OmniInternational). The total cell suspension was transferred to a new tube and mixed with an equal volume of dextran buffer (Sigma; 31397) to a final dextran concentration of 13%. The dextran - containing cell suspension was centrifuged at 2,000 g for 20 minutes at 4 °C. The upper layer (capillary - depleted fraction) was carefully separated from the remaining sample and transferred to a new tube containing 10x radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore TM; in (20–188). The capillary-depleted samples were vortexed thoroughly with lysis buffer and centrifuged at 14,000 rpm for 30 minutes at 4 °C, and the supernatants were collected for analysis. The protein concentration of the treated brain tissue lysates was tested using a BCA protein assay kit (Pierce TM ; 23227), and the final sample lysates were normalized to a total protein concentration of 7 mg / mL prior to immunoassay determination.

[0431] PK Assay : The concentrations of TEM and control IgG1 mAb in NHP brain tissue and plasma were determined using MSD immunoassays on streptavidin-coated plates. Fresh standard curves were prepared by serially diluting each mAb in assay diluent containing primary mouse matrix (50% brain tissue lysate or 10% pooled plasma). Frozen quality controls prepared in 100% primary mouse matrix were diluted and tested in each assay. Briefly, plates were blocked with a PBS solution of 1% bovine serum albumin for 30 minutes and washed with a PBS solution of 0.05% Tween-20. A master mix containing capture and detection reagents (biotinylated and ruthenium-labeled anti-human Fc mAb) was combined with reference standards, quality controls, and samples in a 1:1 volume ratio in the assay plates and incubated for 1 hour with shaking. Raw data signals were read on a Meso Sector S 600 imager and analyzed by Watson LIMS software (Thermo Scientific). Data regression was performed using a 1 / Y 2 weighted five-parameter logistic fit. The quantifiable curve range for the brain tissue lysate assay was 1 ng / mL to 512 ng / mL, and the minimum required sample dilution for the treated tissue was 1:2. To calculate the total tissue drug concentration in the brain, the mAb concentration in the brain lysate was multiplied by the total volume used to process to the final 7 mg / mL normalized sample. The total tissue drug concentration (ng) was then divided by the wet weight of the brain tissue to determine the drug:tissue (ng:mg) ratio. The quantifiable curve range for the plasma assay was 2 ng / mL to 512 ng / mL, and the minimum required sample dilution was 1:10. The assay had a sensitivity limit of 2 ng / mL in brain tissue lysates and 10 ng / mL in plasma.

[0432] Example 2. Antibody Binding in HER2 - Positive Cancer Cells

[0433] To determine the ability of TEM mAbs to mediate clearance of cells, TEM constructs were generated using the anti-human epidermal growth factor receptor 2 (HER2)-binding antibody trastuzumab (bivalent BBBB1639 and BBBB1640; and bivalent BBBB1636 that lacks Fc-mediated effector functions) and bispecific molecules that contain both trastuzumab and pertuzumab (bispecifics BBBB1819, BBBB1809, BBBB1615, BBBB1616, BBBB1617, BBBB1826, and BBBB1805; bispecific BBBB1613 that lacks Fc-mediated effector functions). Figure 2 Both trastuzumab and pertuzumab have been extensively characterized in vitro and in vivo and are FDA-approved drugs.

[0434] Binding of the TEM mAbs was first tested in HER2-expressing cell lines BT-474, MDA-MB-361, JIMT-1, and U87-MG ( Figures 3A - 3D ) compared to an isotype control. At very high HER2 expression, binding was mediated by HER2 expression independent of CD98hcscFv ( Figure 3A ). In cell lines expressing HER2 at high to moderate levels, binding of the mAbs was affected by CD98hc, and mAbs containing CD98 spFv showed improved binding relative to CD98 scFv ( Figures 3B - 3C ).

[0435] The TEM mAbs were further tested in the HCC1954 cell line with amplified HER2 expression. Monovalent (BBBB1614), bivalent (BBBB1636 and BBBB1640), and bispecific (BBBB1613, BBBB1615, BBBB1616, BBBB1617) were tested together with silenced trastuzumab, silenced pertuzumab, and an isotype control (CNTO3930). All antibodies showed dose-dependent binding ( Figure 3E ).

[0436] Example 3. TEM Antibodies Promote Antibody - Dependent Cellular Cytotoxicity / Phagocytosis

[0437] To further test anti-HER2 TEM, breast cancer cell lines BT474 and MDA-MB-361 expressing HER2 were selected and engineered to stably express red fluorescent protein (RFP) in the nucleus by lentiviral transduction and used to generate tumor spheroids, which are widely considered a more physiological model of tumor growth compared to 2D cultures. In the presence of anti-HER2 TEM or control, the spheroids were co-cultured with human induced pluripotent stem cell (iPSC)-derived microglia (iMG), and the fluorescence signal was monitored over time for 10 days ( Figures 4A - 4D ). For both cell lines, trastuzumab led to a dose-dependent reduction in the red fluorescence area at day 10, indicating tumor cell killing. Two bispecific mAbs (BBBB1809 and BBBB1819) showed a strong dose-dependent reduction in the fluorescence area, which was not significantly different from trastuzumab. Collectively, these results demonstrate that anti-HER2 TEM has a strong cytotoxic effect on HER2+ tumor cells due to the induction of microglial ADCP.

[0438] Example 4. TEM Antibodies Promote ADCC / ADCP of Live Target Cells via PBMC

[0439] Next, the ability of anti-HER2 TEM molecules to promote the clearance of HER2+ tumor cells by human PBMCs was tested. During a 48-hour co-culture, strong cytotoxicity mediated by bispecific TEM against the JIMT-1, MDA-MB-361, and BT474 cell lines was observed ( Figures 5A - 5C ). Effective dose-dependent inhibition of tumor growth by trastuzumab and bispecific TEM was observed. Compared to trastuzumab, bispecific HER2-TEM molecules showed increased potency against HER2 intermediate cell lines (JIMT-1 and MDA-MB-361) and comparable potency in the HER2 high cell line (BT-474).

[0440] Collectively, these data demonstrate that anti-HER2 TEM molecules are capable of enabling microglia and peripheral immune cells to clear tumor cells in an antigen-dependent manner.

[0441] Example 5. TEM Results in Enhanced Brain Delivery in Cynomolgus Monkeys

[0442] NHPs administered with TEM mAbs (BBBB1615, BBBB1616, and BBBB1617) were compared to a group of NHPs injected with trastuzumab. Brain mAb concentrations were measured at 72 hours and 168 hours after IV administration (10 mg / kg) in eight perfused and capillary-depleted brain regions. The presence of TEM increased the mAb concentration in the frontal lobe, hippocampus, and temporal lobe compared to trastuzumab, with a wide range of fold increases ( Figure 6)。These data demonstrate that anti-HER2 TEM results in increased mAb uptake in the brain compared to trastuzumab.

[0443] Example 6. Antitumor Activity of TEM in Mice

[0444] In a xenograft model of BT474 breast ductal carcinoma cell line growing subcutaneously in female immunocompromised NSG (i.e., non-obese diabetic [NOD] severe combined immunodeficiency disease [scid] γ or NOD.Cg Prkdc scid Il2rg TM1Wjl / Szh) mice, the anti-tumor activity of BBBB1819 as a single agent was evaluated. 1×10 7 cells were implanted into the mice, and intraperitoneal (IP) administration was initiated after the subcutaneous tumor was established to a target randomized volume of 146 mm 3 to 155 mm3.

[0445] In one part of the study, the dose-dependent anti-tumor activity of BBBB1819 was evaluated using a schedule of 8 doses every 3 days (Q3Dx8), where groups of n = 10 animals were dosed with 2 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg of BBBB1819 or with phosphate-buffered saline (PBS) control vehicle ( Figure 7A ). ΔTumor growth inhibition (TGI) of subcutaneous BT474 xenografts was calculated on day 52 when at least two-thirds of the control animals remained in the study. Statistically significant ΔTGI was observed with 2 mg / kg (31%, p < 0.05; Table 5), 5 mg / kg (70%, p < 0.05), 10 mg / kg (114%, p < 0.05), and 20 mg / kg (116%, p < 0.05) of BBBB1819. By the end of the study, dose-dependent complete responses (defined as no measurable tumor remaining) to BBBB1819 were observed in 0 of 10 animals at 2 mg / kg, 0 of 10 animals at 5 mg / kg, 4 of 10 animals at 10 mg / kg, and 9 of 10 animals at 20 mg / kg.

[0446] In another part of the study, the anti-tumor activity of BBBB1819 was compared to that of the reference molecule ERBB2077 (an antibody form equivalent to BBBB1819 but lacking the CD98 spFv) and trastuzumab. BBBB1819, ERBB2077, or trastuzumab was administered intraperitoneally at 20 mg / kg to NSG mice bearing established subcutaneous tumors, randomized to groups of n = 10 animals, using a schedule of 4 doses once a week (QWx4) ( Figure 7B)。The PBS-treated group from the parallel Q3Dx8 cohort was used as the control group. When at least two-thirds of the control animals remained in the study, the ΔTGI of the SCBT474 xenograft was calculated on Day 52. Statistically significant ΔTGI was observed with BBBB1819 (111%, p<0.05), ERBB2077 (115%, p<0.05), and trastuzumab (109%, p<0.05). BBBB1819 had antitumor activity similar to that of trastuzumab and ERBB2077. By the end of the study, complete responses were observed in 4 out of 10 animals dosed with BBBB1819, 9 out of 10 animals dosed with ERBB2077, and 1 out of 10 animals dosed with trastuzumab.

[0447] Table 5. Summary of Tumor Volume and %ΔTGI from BT - 474SC Tumor Study 。

[0448]

[0449] PBS, phosphate-buffered saline; Q3Dx8, every 3 days for 8 doses; QWx4, once a week for 4 doses; SC, subcutaneous; TGI, tumor growth inhibition; TV c , mean tumor volume of the given control group; TV c0 , mean initial tumor volume of the given control group; TV t , mean tumor volume of the treatment group; TV t0 , mean initial tumor volume of the treatment group.

[0450] The p-values for the antitumor activity of all treatment groups were p≤0.05 and were thus statistically significant. %ΔTGI = ([(TV c – TV c0 ) – (TV t – TV t0 )] / [TV c – TV c0 ) × 100.

[0451] a The last day when at least two-thirds of the animals remained in the control group.

[0452] In these studies, at any dose, treatment with BBBB1819, ERBB2077, or trastuzumab did not result in significant weight loss compared to the PBS-treated control group, although none of these antibodies cross-reacted with murine Erbb2 or Slc3a2 (Cd98).

[0453] In addition to efficacy evaluation, blood and tumor PK analyses were performed on satellite groups (n = 4 / dose group), with post-orbital blood sampling at 24 hours after the first dose, before the second dose, at 24 hours after the third dose, before the eighth dose (only Q3Dx8 group), and at 24 hours after the eighth dose (only Q3Dx8 group). Figure 8A and Figure 8B ). Additionally, tumor PK analysis was performed on each QWx4 group, with tumor tissue collection at 24 hours after the first dose. Figure 8C and Figure 8D ).

[0454] The tumor-to-plasma ratio of BBBB1819 was found to be approximately 15%. Based on modeling of mouse tumor regression and PK data, the tumor concentration required for 50% tumor cytotoxicity was estimated to be 1.5 μg / mL.

[0455] Example 7. TEM PET / CT Imaging Study

[0456] The in vivo distribution of the [Zr89]-DFO*-CD98xHER2 (BBBB1616) IgG antibody was evaluated in human CD98 knock-in female mice (Biocytogen 110983) and female C57BL6 mice (Jax). On the day of the imaging experiment, the tracer precursor was labeled with [Zr89]. Shortly thereafter, quality control (QC) of the radiochemical purity and specific activity of the compound was measured.

[0457] Animal body weight was measured using a Mettler Toledo balance. Then the animals were anesthetized via Somni AMD-3 in the anesthesia chamber using isoflurane with oxygen (3.0% - 4.5% for induction and 1.0% - 3.0% for maintenance). After ensuring that the animals maintained a stable breathing pattern, the animals were placed on the operating table for injection, and a heating pad was used to maintain body temperature.

[0458] The mice were divided into three groups (A, B, and C). Animals in the same group were injected with the PET tracer at approximately 2-minute intervals. At the designated time points, the mice were placed on a 4-mouse stage of a Sofie GNEXT PET / CT scanner (Sofie, Culver City, CA, USA), and a heating pad was used to maintain body temperature and respiratory monitoring was performed. The imaging protocol started with a 30-minute static PET scan, followed by a 1-minute standard CT scan acquisition protocol.

[0459] Acquisition and Reconstruction Parameters :

[0460] PET / CT studies were performed on days 1, 5, and 7 after tracer injection. The whole body was centered on the axial FOV of the scanner to maximize sensitivity and resolution. CT scans were used for attenuation correction, anatomical images, and scatter correction of PET images. The PET scan energy window was set between 350 KeV and 650 KeV, and the timing window was 3.438 ns. Emission data were collected in list mode for 30 minutes. PET images were reconstructed into single frames using the iterative 2-dimensional ordered subset expectation maximization (OSEM2D) algorithm (4 OSEM2D – iterations, with Fourier rebinning). The data were reconstructed into images with a 128×128 matrix size.

[0461] Results :

[0462] Figures 9A - 9C Shows the distribution of Zr89-DFO*-HER2xCD98 antibody in CD98 knock-in female mice (Biocytogen 110983) and female C57BL6 mice (Jax) at 1, 5, and 7 days after injection. The Zr89-DFO*-HER2xCD98 antibody showed higher and more uniform brain uptake in CD98 knock-in mice ( Figures 10A - 10D ). Higher Zr89-DFO*-HER2xCD98 antibody uptake in the brain was observed throughout the experiment, while the systemic activity was the same in C57BL6 mice and CD98 knock-in mice ( Figures 11A - 11C ). Zr89-DFO*-HER2 showed higher peripheral organ uptake (liver, kidney, and spleen) compared to the Zr89-DFO*-HER2xCD98 antibody (data not shown).

[0463] Example 8. TEM Pharmacokinetics (PK) Analysis in CD98 Knock - In Mice

[0464] Additional studies were conducted to determine the pharmacokinetics (PK) of TEM antibodies in human CD98 knock-in (huCD98KI) female mice (Biocytogen 110983). Mice were injected intravenously (i.v.) with 13 mg / kg of BBB1819, ERBB2077, or trastuzumab (ERBB128) antibodies. Blood was collected by retro-orbital bleeding 24 hours after injection. Mice were sacrificed and brain tissues were collected.

[0465] Compared to ERBB2077 and trastuzumab, BBBB1819 showed higher concentrations in the brains and lower concentrations in the plasma of huCD98KI mice ( Figures 12A - 12C ). These data demonstrate that BBBB1819 TEM results in increased mAb uptake in the brains of huCD98KI mice compared to antibodies without CD98 spFv.

Claims

1. A multispecific antibody or an antigen-binding fragment thereof, said multispecific antibody or antigen-binding fragment thereof comprising at least one of a first antigen-binding region and a second antigen-binding region each capable of specifically binding to human epidermal growth factor receptor 2 (HER2), and a third antigen-binding region capable of specifically binding to CD98, wherein: (4) said first antigen-binding region comprises: a first heavy-chain variable region (VH1), said VH1 comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:5, 6, and 7, respectively; and a first light-chain variable region (VL1), said VL1 comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:8, 9, and 10, respectively; (5) said second antigen-binding region comprises: a second heavy-chain variable region (VH2), said VH2 comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively; and a second light-chain variable region (VL2), said VL2 comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively; and (6) said third antigen-binding region comprises a first single-chain variable fragment (scFv1), said scFv1 having: a third heavy-chain variable region (VH3) comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and a third light-chain variable region (VL3) comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have any of the amino acid sequences in Table 2.

2. The multispecific antibody or antigen-binding fragment thereof according to claim 2, wherein: (4) said VH1 comprises the same amino acid sequence as the VH of the HC sequence shown in SEQ ID NO:1; and said VL1 comprises the same amino acid sequence as the VL of the LC sequence shown in SEQ ID NO:2; (5) said VH2 comprises the same amino acid sequence as the VH of the HC sequence shown in SEQ ID NO:11; and said VL2 comprises the same amino acid sequence as the VL of the LC sequence shown in SEQ ID NO:12; and (6) said VH3 and said VL3 comprise the same amino acid sequences as the corresponding VH and VL of the scFv shown in the following sequences: (xliii) SEQ ID NO:19; (xliv) SEQ ID NO:26; (xlv) SEQ ID NO:33; (xlvi) SEQ ID NO:34; (xlvii) SEQ ID NO:35; (xlviii) SEQ ID NO:36; (xlix) SEQ ID NO:43; (l) SEQ ID NO:49; (li) SEQ ID NO:55; (lii) SEQ ID NO:59; (liii) SEQ ID NO:66; (liv) SEQ ID NO:71; (lv) SEQ ID NO:78; (lvi) SEQ ID NO:85; (lvii) SEQ ID NO:87; (lviii) SEQ ID NO:88; (lix) SEQ ID NO:89; (lx) SEQ ID NO:90; (lxi) SEQ ID NO:92; (lxii) SEQ ID NO:93; (lxiii) SEQ ID NO:

94.

3. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises: (c) A first heavy chain (HC1), wherein the HC1 comprises the VH1, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1; and (d) A first light chain (LC1), wherein the LC1 comprises the VL1 and a light chain constant region.

4. The multispecific antibody or antigen-binding fragment thereof according to claim 3, wherein the multispecific antibody or antigen-binding fragment thereof further comprises: (c) A second heavy chain (HC2), wherein the HC2 comprises the VH1 and a first heavy chain constant region containing a second Fc region (Fc2), and (d) A second light chain (LC2), wherein the LC2 comprises the VL1 and a light chain constant region.

5. The multispecific antibody or antigen-binding fragment thereof according to claim 3, wherein the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

6. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises: (d) A first heavy chain (HC1), wherein the HC1 comprises the VH1 and a first heavy chain constant region containing a first Fc region (Fc1), (e) A first light chain (LC1), wherein the LC1 comprises the VL1 and a light chain constant region; and (f) A second heavy chain (HC2), wherein the HC2 comprises the scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

7. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises: (c) A first heavy chain (HC1), wherein the HC1 comprises the VH2, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1; and (d) A first light chain (LC1), wherein the LC1 comprises the VL2 and a light chain constant region.

8. The multispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the multispecific antibody or antigen-binding fragment thereof further comprises: (c) A second heavy chain (HC2), wherein the HC2 comprises the VH2 and a first heavy chain constant region containing a second Fc region (Fc2), and (d) A second light chain (LC2), wherein the LC2 comprises the VL2 and a light chain constant region.

9. The multispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the multispecific antibody or antigen-binding fragment thereof further comprises a second Fc region (Fc2).

10. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises: (d) A first heavy chain (HC1), wherein the HC1 comprises the VH2 and a first heavy chain constant region containing a first Fc region (Fc1), (e) A first light chain (LC1), wherein the LC1 comprises the VL2 and a light chain constant region; and (f) A second heavy chain (HC2), wherein the HC2 comprises the scFv1 and a second heavy chain constant region containing a second Fc region (Fc2).

11. The multispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the multispecific antibody or antigen-binding fragment thereof comprises the first antigen-binding region, the second antigen-binding region, and the third antigen-binding region.

12. The multispecific antibody or antigen-binding fragment thereof according to claim 11, wherein the multispecific antibody or antigen-binding fragment thereof comprises: (d) A first heavy chain (HC1), wherein the HC1 comprises the VH1, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1, (e) A first light chain (LC1), wherein the LC1 comprises the VL1 and a light chain constant region; and (f) A second heavy chain (HC2), wherein the HC2 comprises a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein the scFv2 comprises the VH2 and the VL2.

13. The multispecific antibody or antigen-binding fragment thereof according to claim 11, wherein the multispecific antibody or antigen-binding fragment thereof comprises: (d) A first heavy chain (HC1), wherein the HC1 comprises the VH2, a first heavy chain constant region containing a first Fc region (Fc1), and the scFv1, (e) A first light chain (LC1), wherein the LC1 comprises the VL2 and a light chain constant region; and (f) A second heavy chain (HC2), wherein the HC2 comprises a second single-chain variable fragment (scFv2) and a first heavy chain constant region containing a second Fc region (Fc2), wherein the scFv2 comprises the VH1 and the VL1.

14. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the scFv1 and / or scFv2 comprises at least one of the following: (a) a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys; and b) a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys.

15. The multispecific antibody or antigen-binding fragment thereof according to claim 14, wherein each of said scFv1 and scFv2 independently comprises said first disulfide bond and said second disulfide bond.

16. The multispecific antibody or antigen-binding fragment thereof according to claim 13, wherein said scFv2 comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 3 or 4.

17. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said scFv1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 71, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:

94.

18. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 4 to 6, 8 to 10 and 12 to 17, wherein each of said Fc1 and Fc2 comprises one or more heterodimer mutations, or one or more knob and hole mutations.

19. The multispecific antibody or antigen-binding fragment thereof according to claim 18, wherein said heterodimer mutation comprises amino acid modifications at positions T350, L351, F405 and Y407 in one of Fc1 and Fc2, and amino acid modifications at positions T350, T366, K392 and T394 in the other of Fc1 and Fc2, wherein the amino acid modification at position T350 is T350V, T350I, T350L or T350M ； The amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T or F405S; The amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W, and wherein the amino acid residues are numbered according to the EU index as described in Kabat.

20. The multispecific antibody or antigen-binding fragment thereof according to claim 19, wherein one of Fc1 and Fc2 comprises the mutations T350V, L351Y, F405A, and Y407V, and the other of Fc1 and Fc2 comprises the mutations T350V, T366L, K392L, and T394W.

21. The multispecific antibody or antigen-binding fragment thereof according to claim 18, wherein each of said Fc1 and said Fc2 comprises one or more knob and hole mutations.

22. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that enhances the binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably the amino acid modification enhances binding at acidic pH, more preferably the Fc domain has the M252Y / S254T / T256E (YTE) mutation, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

23. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that reduces or eliminates effector function, preferably the Fc domain has one or more amino acid modifications at positions L234, L235, D265, D270, N297, E318, K320, K322, P331, and P329, such as one, two, three, or four amino acid modifications selected from L234A, L235A, D265S, and P331S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

24. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having one or more amino acid modifications selected from M252Y, S254T, T256E, L234A, L235A, and D265S, wherein the amino acid residues are numbered according to the EU index as described in Kabat.

25. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the multispecific antibody or antigen-binding fragment thereof comprises an Fc domain having an amino acid modification that does not reduce or eliminate effector function.

26. A multispecific antibody, the multispecific antibody comprising a first heavy chain, a light chain, and a second heavy chain each having an amino acid sequence that is at least 90% identical to the following: (3) SEQ ID NO: 100, SEQ ID NO: 12, and SEQ ID NO: 101, respectively; or (4) SEQ ID NO: 102, SEQ ID NO: 12, and SEQ ID NO: 103, respectively; Wherein the first antigen-binding region is capable of specifically binding to a first epitope of HER2, the second antigen-binding region is capable of specifically binding to a second epitope of HER2, and the third antigen-binding region is capable of specifically binding to CD98.

27. The multispecific antibody according to claim 26, wherein the first heavy chain, the light chain, and the second heavy chain each comprise the amino acid sequences of the following: (3) SEQ ID NO:100, SEQ ID NO:12, and SEQ ID NO:101, respectively; or (4) SEQ ID NO:102, SEQ ID NO:12, and SEQ ID NO:103, respectively.

28. An isolated nucleic acid sequence encoding the multispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 27.

29. A vector comprising the isolated nucleic acid according to claim 28.

30. A host cell comprising the isolated nucleic acid according to claim 28 or the vector according to claim 29.

31. A method for producing a multispecific antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to claim 30 under conditions for producing the multispecific antibody or an antigen-binding fragment thereof, and recovering the multispecific antibody or an antigen-binding fragment thereof.

32. A pharmaceutical composition comprising the multispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

33. A method for treating or detecting a disorder, preferably cancer, in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 32.

34. The method according to claim 33, wherein the disease or disorder is a HER2-associated disease or disorder.

35. The method according to claim 34, wherein the disease or disorder is brain metastasis.

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