Anti-HER2 antibodies and their uses

Abstract

The present disclosure generally relates to immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) capable of binding to HER2 protein. The antibodies of the present technology are useful in methods for detecting and treating HER2-associated cancers in subjects in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 229,134, filed August 4, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present technology relates generally to the preparation and use of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to the HER2 protein. In particular, the present technology relates to the preparation of HER2-binding antibodies and their use in the detection and treatment of HER2-associated cancers. [Background technology]

[0003] The following description of the background of the present technology is provided merely as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Monoclonal antibody (mAb)-based therapy for cancer is one of the most successful strategies for treating patients with both hematologic and non-hematologic malignancies.

[0005] One of the major clinical achievements of mAbs was the development of trastuzumab / Herceptin, a humanized monoclonal antibody that binds to the extracellular domain of HER2. Indeed, standard care treatment of HER2-positive breast cancer includes chemotherapy in combination with trastuzumab. Trastuzumab is designed to inhibit cell growth and proliferation, killing HER2-positive tumor cells by antibody-dependent cellular cytotoxicity (ADCC). Both the combination of trastuzumab with conventional chemotherapy in breast and gastric cancer, as well as the use of trastuzumab as a single agent, have been shown to extend progression-free survival in patients with amplified HER2. Importantly, standard HER2-targeted therapy is only offered to patients who score HER2 immunohistochemistry (IHC) 3+ (i.e., strongly overexpressed) or SPoT-Light® HER2 chromogenic IHC (CISH) positive (i.e., ERBB2 gene amplification). However, a significant group of patients does not respond to this targeted therapy, and a large proportion of initially responsive patients acquire resistance in response to trastuzumab treatment. There are multiple mechanisms that contribute to trastuzumab resistance, including activation of HER2 downstream signaling pathways and parallel receptor tyrosine kinase pathways, all of which provide potential targets for combating trastuzumab resistance. The primary mechanism of resistance involves activation of bypass signaling pathways that result in inhibition of HER2 signaling independent of tumor progression. Up to 70% of HER2+ breast cancers become resistant to trastuzumab monotherapy. Despite drug resistance, these tumors generally still overexpress HER2.

[0006] Therefore, there is an urgent need for the development of alternative HER2-targeted therapies that overcome resistance to signaling inhibition and benefit more patients. Summary of the Invention [Means for solving the problem]

[0007] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (VL (a) (i) V H is V of SEQ ID NO:1 H -CDR1 sequence, SEQ ID NO: 2 or SEQ ID NO: 7 H -CDR2 sequence, and V of SEQ ID NO: 8 H or (ii) V H is V of SEQ ID NO:1 H - CDR1 sequence, V of SEQ ID NO: 7 H - CDR2 sequence and V of SEQ ID NO: 3 or SEQ ID NO: 8 H and / or (b)(i) V L is V of SEQ ID NO:9 L -CDR1 sequence, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 L - CDR2 sequence and V of SEQ ID NO: 6 or SEQ ID NO: 12 L or (ii) V L is V of SEQ ID NO: 4 or SEQ ID NO: 9 L -CDR1 sequence, SEQ ID NO: 10 or SEQ ID NO: 11 L - CDR2 sequence and V of SEQ ID NO: 6 or SEQ ID NO: 12 L or (iii) V L is V of SEQ ID NO: 4 or SEQ ID NO: 9 L -CDR1 sequence, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 L - CDR2 sequence, and V of SEQ ID NO: 12 L - Includes the CDR3 sequence.

[0008] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof, comprising: H comprises an amino acid sequence selected from any one of SEQ ID NOs: 13, 15, or 17; and / or (b) V LIn some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from any one of SEQ ID NOs: 14, 16, 18, 19, or 20. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain immunoglobulin variable domain (V) selected from the group consisting of SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 14, SEQ ID NOs: 15 and 18, SEQ ID NOs: 15 and 19, and SEQ ID NOs: 15 and 20, respectively. H ) and light chain immunoglobulin variable domains (V L ) amino acid sequence.

[0009] In another aspect, the disclosure provides an antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal direction, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, and (iii) an amino acid sequence (GGGGS). 3 and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope, and the light chain variable domain and the heavy chain variable domain of the second immunoglobulin have the amino acid sequence (GGGGS) 6to form a single-chain variable fragment; and (b) the second polypeptide chain and the third polypeptide chain each comprise, from N-terminal to C-terminal direction, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a heavy chain constant domain of the first immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 13, 15, or 17, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 14, 16, 18, 19, or 20.

[0010] In another aspect, the present disclosure provides SEQ ID NOs:21 and 22, SEQ ID NOs:21 and 23, SEQ ID NOs:21 and 24, SEQ ID NOs:21 and 25, SEQ ID NOs:21 and 26, SEQ ID NOs:21 and 27, SEQ ID NOs:21 and 28, SEQ ID NOs:21 and 29, SEQ ID NOs:21 and 30, SEQ ID NOs:21 and 31, SEQ ID NOs:21 and 32, SEQ ID NOs:21 and 33, SEQ ID NOs:34 and 33, SEQ ID NOs:21 and 35, SEQ ID NOs:36 and 33, SEQ ID NOs:21 and 37, SEQ ID NOs:21 and 38, SEQ ID NOs:21 and 39, SEQ ID NOs:21 and 40, SEQ ID NOs:21 and 41, SEQ ID NOs:21 and 42, SEQ ID NOs:21 and 43, SEQ ID NOs:21 and 44, SEQ ID NOs:21 and 45, SEQ ID NOs:21 and 46, SEQ ID NOs:21 and 47, SEQ ID NOs:21 and 48, SEQ ID NOs: In one embodiment, the present invention provides an antibody or antigen-binding fragment comprising a heavy chain (HC) and a light chain (LC) selected from the group consisting of SEQ ID NOs:21 and 49, SEQ ID NOs:21 and 50, SEQ ID NOs:21 and 51, SEQ ID NOs:21 and 52, SEQ ID NOs:21 and 53, SEQ ID NOs:21 and 54, SEQ ID NOs:21 and 55, SEQ ID NOs:21 and 56, SEQ ID NOs:21 and 57, SEQ ID NOs:21 and 58, SEQ ID NOs:21 and 59, SEQ ID NOs:21 and 60, SEQ ID NOs:21 and 61, SEQ ID NOs:21 and 62, SEQ ID NOs:21 and 63, SEQ ID NOs:21 and 64, SEQ ID NOs:21 and 65, SEQ ID NOs:21 and 66, SEQ ID NOs:21 and 67, SEQ ID NOs:21 and 68, SEQ ID NOs:21 and 69, SEQ ID NOs:21 and 70, SEQ ID NOs:21 and 71, SEQ ID NOs:21 and 72, and SEQ ID NOs:21 and 85.

[0011] Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In certain embodiments, the antibody or antigen-binding fragment comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, and K322A. In other embodiments, the antibody or antigen-binding fragment comprises an IgG4 constant region comprising an S228P mutation.

[0012] In any and all embodiments of the antigen-binding fragments disclosed herein, the antigen-binding fragment may be any of Fab, F(ab') 2 , Fab', scF v , and F v Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment of the present technology is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody, and / or lacks α-1,6-fucose modifications.

[0013] In any and all embodiments of the antibodies or antigen-binding fragments disclosed herein, the multispecific antibodies or antigen-binding fragments bind to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in some embodiments of the antibodies or antigen-binding fragments disclosed herein, the multispecific antibodies or antigen-binding fragments bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

[0014] Additionally or alternatively, in some embodiments, the multispecific antibodies or antigen-binding fragments of the present technology also bind to T cells and / or CD3. In one aspect, the present disclosure provides T cells that are ex vivo armed with a multispecific antibody or antigen-binding fragment of the present technology that also binds to T cells and / or CD3. In another aspect, the present disclosure provides an ex vivo method of generating therapeutic T cells, comprising ex vivo arming of T cells with a multispecific antibody or antigen-binding fragment of the present technology that can bind to T cells and / or CD3, the T cells being optionally human T cells, and the binding being non-covalent. In another aspect, the present disclosure provides a method for treating cancer in a subject in need of such treatment, comprising administering to the subject an effective amount of T cells that are ex vivo armed with a multispecific antibody or antigen-binding fragment of the present technology that also binds to T cells and / or CD3.

[0015] In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies or antigen-binding fragments described herein. In another aspect, the disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0016] In another aspect, the disclosure provides a pharmaceutical composition comprising any of the antibodies or antigen-binding fragments described herein and a pharma- ceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. In some embodiments, the pharmaceutical composition further comprises an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0017] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the antibodies or antigen-binding fragments described herein, or any of the pharmaceutical compositions disclosed herein, wherein the antibody or antigen-binding fragment specifically binds to HER2. In some embodiments, the cancer is a solid tumor. Examples of cancer include, but are not limited to, breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, and neuroblastoma.

[0018] Additionally or alternatively, in some embodiments of the methods, the antibody or antigen-binding fragment is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent, examples of which include one or more of alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapies, T cells, and immune modulating / stimulatory antibodies (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-TIM3 antibodies, anti-4-1BB antibodies, anti-CD73 antibodies, anti-GITR antibodies, or anti-LAG-3 antibodies).

[0019] In another aspect, the present disclosure provides a method for detecting cancer in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody or antigen-binding fragment of the present technology, the antibody or antigen-binding fragment being configured to localize to cancer cells expressing HER2 and being labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody or antigen-binding fragment that is higher than a reference value. In certain embodiments, the cancer is a solid tumor. In some embodiments, the subject is diagnosed with or suspected of having cancer. Examples of cancer include, but are not limited to, breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, and neuroblastoma. The level of radioactivity emitted by the antibody or antigen-binding fragment can be detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody or antigen-binding fragment of the present technology conjugated to a radionuclide.

[0020] In any and all embodiments of the methods disclosed herein, the subject is a human.

[0021] In yet another aspect, the present disclosure provides a method for detecting HER2 protein expression levels in a biological sample, the method comprising contacting the biological sample with any of the antibodies or antigen-binding fragments disclosed herein and detecting binding to HER2 protein in the biological sample.

[0022] Also disclosed herein is a kit for the detection and / or treatment of HER2-associated cancer, comprising at least one immunoglobulin-associated composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) and instructions for use. In certain embodiments, the immunoglobulin-associated composition is coupled to one or more detectable labels. In one embodiment, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-HER2 immunoglobulin-associated composition described herein. In some embodiments, the secondary antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label. [Brief description of the drawings]

[0023] [Figure 1] 1 shows the amino acid sequences of VH CDR1 (SEQ ID NO: 1), VH CDR2 (SEQ ID NO: 2 or 7), VH CDR3 (SEQ ID NO: 3 or 8), VL CDR1 (SEQ ID NO: 4 or 9), VL CDR2 (SEQ ID NO: 5, 10, or 11), and VL CDR3 (SEQ ID NO: 6 or 12) of the anti-HER2 immunoglobulin-related compositions of the present technology. Mutations in the CDR regions of the trastuzumab variants compared to the CDR regions of the parent trastuzumab antibody (SEQ ID NOs: 1-6) are underlined. [Diagram 2]1 shows the amino acid sequences of the variable heavy immunoglobulin domains (VH) and variable light immunoglobulin domains (VL) of six of the anti-HER2 immunoglobulin-related compositions of the present technology: ABP100s.10.1 HER2 (SEQ ID NOs: 13 and 14, respectively), ABP100s.10.2 HER2 (SEQ ID NOs: 15 and 16, respectively), ABP100s.10.3 HER2 (SEQ ID NOs: 17 and 14, respectively), ABP100s.10.4 HER2 (SEQ ID NOs: 15 and 18, respectively), ABP100s.10.5 HER2 (SEQ ID NOs: 15 and 19, respectively), and ABP100s.10.6 HER2 (SEQ ID NOs: 15 and 20, respectively). The amino acid sequences of VH CDR1-3 and VL CDR1-3 are underlined. Mutations in the CDR regions of the trastuzumab variants compared to the CDR regions of the parent trastuzumab antibody (SEQ ID NOs: 1-6) are double underlined. [Figure 3-1] 1 shows the heavy chain (HC) and light chain (LC) amino acid sequences of 40 exemplary HER2xCD3 bispecific antibodies of the present technology. The amino acid sequences of VH CDR1-3 and VL CDR1-3 are underlined. Linker sequences are in italics. Point mutations and disulfide mutations are in bold and double underlined. Fc substitutions are in bold and underlined. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above. [Figure 3-5] Same as above. [Diagram 3-6] Same as above. [Diagram 3-7] Same as above. [Diagram 3-8] Same as above. [Diagram 3-9] Same as above. [Figure 3-10] Same as above. [Figure 3-11] Same as above. [Figure 3-12] Same as above. [Figure 3-13] Same as above. [Figure 3-14] Same as above. [Figure 3-15] Same as above. [Figure 3-16] Same as above. [Figure 3-17] Same as above. [Figure 3-18] Same as above. [Figure 3-19] Same as above. [Figure 3-20] Same as above. [Figure 4] Two groups of BsAbs are outlined: the first, which targets both HER2 and the T cell co-receptor CD3, and the second, which selectively targets and kills HER2-amplified cancer cells. [Figure 5A] (FIG. 5A) Individual mouse tumor responses in mouse xenograft models using BsAbs with various domain spacing formats administered intravenously (IV) (10 pmol, twice weekly) with 20 million huATC and subcutaneous human IL-2 (1000 U) or (FIG. 5B) ex vivo "armed" T cell (EAT) administration (subcutaneous, twice weekly) of BsAb and human IL-2 (1,000 U). Each line represents one mouse, and the dashed line represents the group average. See Santich et al., Sci. Transl. Med. 12: eaax1315 (2020), which is incorporated herein by reference. [Figure 5B] Same as above. [Figure 6] The process for arming activated T cells with the anti-HER2xCD3 bispecific antibody of the present technology is summarized. [Figure 7A] Shown are CD3+ T cells stained in duplicate wells with either SP34-hIgG1 or the bispecific antibody for 30 min at 4° C., followed by washing and incubation with anti-human IgG Fc specific PE conjugate (Jackson 1:200) for 30 min at 4° C., followed by washing, data collection on a BD FACSCelesta, and data analysis with FlowJo and GraphPad PRISM. Notably, the constructs used in this experiment differed only in their HER2 binding arm and all had the same CD3 reactive arm. [Figure 7B]Figure 7 shows differential killing of HER2 low expressing target cells by reducing the affinity of the HER2 arm. TDCC assays with effector:target (E:T) ratio of 5:1 were performed with a dose range of bispecific antibodies, using CD3+ T cells as effector cells (50k / well) while target cells (10k / well) expressed either high relative amounts of HER-2 (Figure 7B, SKBR-3) or low relative amounts of HER2 (Figure 7C, MCF-7). Cells were incubated with antibodies in RPMI1640 / 10% FBS for 28 hours at 37°C / 5% CO2 and luminescence was quantified on a SpectraMax iD3 plate reader and data analysis in GraphPad PRISM constructs in this experiment differed only in their HER2 binding arm and all had the same CD3 reactive arm. [Figure 7C] Same as above. [Figure 8A] Characterization of activated T cells armed with anti-HER2 x CD3 parent antibody (Trastuzumab x huOKT3). Figure 8A shows qualitative flow cytometry data demonstrating binding of Trastuzumab x huOKT3 to activated T cells after arming at various concentrations. Figure 8B shows Trastuzumab x huOKT3-mediated killing of cell lines expressing different levels of HER2. [Figure 8B] Same as above. [Figure 9A] TDCC assay shows differential killing of HER2-high and HER2-low expressing target cells. Figures 9A and 9B show the effect of the HER2xCD3 bispecific antibodies of the present technology (ABP100s.5, ABP100s.5.1, ABP100s.10.2, ABP100s.10.4, ABP100s.10.5, ABP100s.10.6) on SK-BR-3 cells and MCF-7 cells, respectively. [Figure 9B] Same as above. [Figure 10-1] FIG. 3 shows the affinity of the HER2×CD3 bispecific antibody of the present technology for CD3 and HER2 targets. [Figure 10-2] Same as above. [Figure 11-1]1 shows the heavy chain (HC) and light chain (LC) amino acid sequences of 12 additional exemplary HER2xCD3 bispecific antibodies of the present technology. The amino acid sequences of VH CDR1-3 and VL CDR1-3 are shown in bold. The linker sequences are shown in italics. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 12] FIG. 11 shows the affinity of the HER2×CD3 bispecific antibody of the present technology for CD3 and HER2 targets. [Figure 13A] Results of a CD3 / TCR NFAT T cell activation reporter assay. The Jurkat CD3 / TCR NFAT T cell activation reporter assay (Promega) was used to assess bispecific antibody (0.00004-40 nM) activation of the CD3 / TCR complex after incubation with Her2-high (Figure 13A: SK-BR-3, Figure 13B: HCC1954) and Her2-low (Figure 13C: MCF-7, Figure 13D: HT55) target cell lines or no target cells (Figure 13E). Expression of reporter activity was detected, quantified as relative light units (RLU), and plotted after a 7 hour incubation period. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 14A] Figure 14 shows the results of T cell dependent cytotoxicity (TDCC) in Her2-high and Her2-low target cell lines with human CD3+ T cells. Bispecific antibodies were incubated with CD3+ T cells and target cells (effector:target ratio 5:1) for 40 hours at 37°C. A highly sensitive ATP assay (Cell Titer Glo 2.0) was used to quantify % cytotoxicity compared to [effector + target] only wells for Her2-high target cells (Figure 14A: SKBR-3, Figure 14B: HCC1954) and Her2-low cell lines (Figure 14C: MCF-7, Figure 14DHT55). [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 15A] Figure 1 shows the results of an in vitro multiplex cytokine detection assay on Her2-high and Her2-low target cell lines with human PBMCs. Bispecific antibodies (range: 30, 0.3, 0.003, 0.00003 nM) were incubated with human PBMCs and target cells (effector (100,000 cells): target (10,000 cells), E:T ratio 10:1) for 24 hours at 37°C. Cytokine release in SKBR-3 (Her2-high) target cells (Figures 15A-15D) and MCF-7 (Her2-low) target cells (Figures 15E-15H) was quantified by diluting supernatants 1:4 for use in multiplex bead-based assays for TNF-α (Figures 15A, 15E), IL-6 (Figures 15B, 15F), IL-2 (Figures 15C, 15G), and IFN-γ (Figures 15D, 15H), presented here in picograms / mL quantified by Luminex xMAP software. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 15G] Same as above. [Figure 15H] Same as above. [Figure 16A] Figure 16 shows the results of flow cytometry analysis of bispecific antibody binding to activated T cells and Her-2 expressing target cells. Bispecific antibodies were incubated with Her2-high (Figure 16A: SKBR-3, Figure 16B: SKOV-3) or Her2-low (Figure 16C: MCF-7, Figure 16D: HT55) target cells or activated T cells (Figure 16E). 100,000 cells / well were incubated with the primary bispecific antibody followed by incubation with anti-human IgG PE secondary antibody. Results were quantified as median fluorescence intensity (MFI) of single live cells in FlowJo software. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above. [Figure 17A] 1 shows Biacore (SPR) affinity data for the Fab format (linked to a human IgG1 scaffold) of the present technology's anti-HER2xCD3 BsAb, which cross-reacts with both human and non-human primate (Cyno) HER2 and CD3 antigens. [Figure 17B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] It will be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below at varying levels of detail in order to provide a substantial understanding of the present technology.

[0025] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) capable of specifically binding to a HER2 polypeptide. The immunoglobulin-related compositions of the present technology are useful in methods for detecting or treating HER2-associated cancer in a subject in need thereof. Thus, various aspects of the present methods relate to the preparation, characterization, and manipulation of anti-HER2 antibodies. The immunoglobulin-related compositions of the present technology are useful alone or in combination with additional therapeutic agents to treat cancer. In some embodiments, the immunoglobulin-related compositions are monoclonal antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, or multispecific antibodies.

[0026] In carrying out the subject methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, series Methods in Enzymology (Academic Press, Inc., NY), MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press), MacPherson et al. al. (1995) PCR 2: A Practical Approach, Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition, Gait ed. (1984) Oligonucleotide Synthesis, U.S. Patent No. 4,683,195, Hames and Higgins eds.(1984)Nucleic Acid Hybridization, Anderson (1999) Nucleic Acid Hybridization, Hames and Higgins eds. (1984) Transcription and Translation, Immobilized Cells and Enzymes (IRL Press (1986)), Perbal (1984) A Practical Guide to Molecular Cloning, Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory), Makrides ed.See, (2003) Gene Transfer and Expression in Mammalian Cells, Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London), and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0027] The present disclosure provides HER2 bispecific antibodies with reduced affinity that utilize avidity interactions to selectively bind and kill cells with high Her2 density (such as cells in cancer tissues) and not bind and spare cells with low Her2 density. Without wishing to be bound by theory, it is believed that reducing CD3 affinity in HER2xCD3 bispecific antibodies reduces T cell activation and cytokine production, reducing adverse events such as cytokine release syndrome in the clinic.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this technology belongs. As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly indicates otherwise. For example, a reference to "a cell" includes a combination of two or more cells, and the like. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry, and hybridization described below, are well known and commonly used in the art.

[0029] As used herein, the term "about" in reference to a number is generally interpreted as including numbers that fall within 1%, 5%, or 10% in either direction (greater or smaller) of the number, unless otherwise stated or clear from the context (except where such number is less than 0% or greater than 100% of the possible value).

[0030] As used herein, "administration" of an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be performed by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self-administration and administration by another.

[0031] As used herein, the term "antibody" refers collectively to immunoglobulin or immunoglobulin-like molecules, including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate, e.g., mammals such as humans, goats, rabbits, and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, "antibodies" (including intact immunoglobulins) and "antigen-binding fragments" refer to any molecule of interest (or a group of closely related molecules of interest) that specifically binds to the substantial exclusion of binding to other molecules (e.g., with a binding constant at least 10 times higher than the binding constant for other molecules in a biological sample). 3 M -1 Large, at least 10 4 M -1 Greater than or at least 10 5 M -1 Antibodies and antibody fragments that have a large binding constant for a molecule of interest. The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (bispecific antibodies, etc.). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.), Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.

[0032] More specifically, an antibody refers to a polypeptide ligand that contains at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody is composed of a heavy and a light chain, each of which has a variable heavy (V H ) area and variable light (V L ) region. H Area and V LThe regions are responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to the antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs have been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody are the combined framework regions of the constituent light and heavy chains that predominantly adopt a beta-sheet conformation, with the CDRs forming loops that connect to, and in some cases form part of, the beta-sheet structure. Thus, the framework regions function to form a scaffold that provides for the correct orientation of the CDRs through non-covalent interactions between the chains.

[0033] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are usually designated CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are usually identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to the HER2 protein have a specific V H Area and VL Each CDR has a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although it is the CDRs that differ between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). "Immunoglobulin-related compositions" as used herein refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or an antigen-binding fragment thereof specifically binds to an antigen.

[0034] As used herein, the term "antibody-related polypeptide" refers to antigen-binding antibody fragments, including single-chain antibodies, comprising the variable region alone or in combination with the polypeptide elements of an antibody molecule: hinge region, CH 1 , C.H. 2 , and C.H. 3 The present technology also includes a variable region, a hinge region, a CH domain, or a combination of all or part of the variable region and the hinge region, a CH domain, or a combination ... 1 , C.H. 2 , and C.H. 3 Antibody-related molecules useful in the present methods include, for example, Fab, Fab' and F(ab') domains. 2 , Fd, single chain Fv (scFv), single chain antibody, disulfide-linked Fv (sdFv), and V L Or V H Examples of fragments that include any of the V L , V H , C L , and C.H. 1 (ii) a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, called F(ab') 2 Fragment, (iii)V H and C.H. 1 (iv) an Fd fragment consisting of the V domain of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a VH (vi) isolated complementarity determining regions (CDRs), dAb fragments (Ward et al., Nature 341:544-546, 1989), which consist of the IgG1 domain. Such "antibody fragments" or "antigen-binding fragments" may include a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab') 2 , and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0035] "Bispecific antibody" or "BsAb" as used herein refers to an antibody that can simultaneously bind to two targets having different structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion in a bispecific antibody is a V H and / or V L In some such embodiments, the V H and / or V L The V domains are those found in a particular monoclonal antibody. In some embodiments, a bispecific antibody comprises two antigen-binding portions, each of which is a V domain from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody comprises two antigen-binding moieties, one of which comprises a V domain containing the CDRs from a first monoclonal antibody. H and / or V L The other antigen-binding portion comprises an immunoglobulin molecule having a V domain containing CDRs from a second monoclonal antibody. H and / or V L Antibody fragments having regions (e.g., Fab, F(ab'), F(ab') 2 , Fd, Fv, dAB, scFv, etc.

[0036] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a mechanism of cell-mediated immune defense in which effector cells of the immune system actively lyse target cells, such as tumor cells, whose membrane surface antigens have been bound by an antibody, such as an anti-HER2 antibody.

[0037] As used herein, "antigen" refers to a molecule to which an antibody (or an antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., a HER2 polypeptide). The antigen can also be administered to an animal to generate an immune response in the animal.

[0038] The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains the portion of the polypeptide that is involved in binding to an antigen. Examples of antigen-binding fragments useful in the present technology include scFv, (scFv) 2 , scFvFc, Fab, Fab', and F(ab') 2 Any of the above antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies.

[0039] As used herein, "binding affinity" refers to the strength of the totality of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, whereas high affinity complexes generally contain antibodies that tend to remain bound to the antigen for longer periods of time.

[0040] As used herein, the term "biological sample" refers to sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsils, thymus, blood, hair, cheek, skin, serum, plasma, CSF, semen, prostatic fluid, seminal plasma, urine, feces, sweat, saliva, sputum, mucous membrane, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or cancer. Biological samples can be obtained from subjects for diagnosis or research, or from non-diseased individuals as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0041] As used herein, the term "CDR grafting" means replacing at least one CDR of an "acceptor" antibody with a CDR "graft" from a "donor" antibody having the desired antigen specificity.

[0042] As used herein, the term "chimeric antibody" means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a murine Fc constant region) has been replaced, using recombinant DNA techniques, with the Fc constant region from an antibody of another species (e.g., a human Fc constant region). See generally Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 0125,023; Better et al., Science 240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987; Liu et al., J. Immunol 139:3521-3526, 1987; Sun et al. See, e.g., Nishimura et al., Cancer Res 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559, 1988.

[0043] As used herein, the term "complement-dependent cytotoxicity" or "CDC" generally refers to the effector function of IgG and IgM antibodies that trigger the classical complement pathway upon binding to surface antigens, inducing the formation of the membrane attack complex and lysis of the target cell.

[0044] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic stacking.

[0045] As used herein, the term "consensus FR" refers to a framework (FR) antibody region in the consensus immunoglobulin sequence. FR regions of an antibody do not contact the antigen.

[0046] As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent in treating a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive treatment or receives a placebo) are typically used.

[0047] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragments are composed of a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) (V H V L (The use of a linker that is too short to allow pairing between the two domains on the same chain allows the domains to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al., Proc Natl Acad Sci USA, 90:6444-6448 (1993).

[0048] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein, or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics such as general health, age, sex, weight, and tolerance to drugs. One of skill in the art will be able to determine appropriate dosages depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition can be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0049] As used herein, the term "effector cell" refers to an immune cell involved in the effector stage of an immune response, as opposed to the recognition and activation stage of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils that can induce ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes that express FcαR are involved in the specific killing of target cells and present antigens to other components of the immune system or bind to cells that present antigens.

[0050] As used herein, the term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that the binding of the former, but not the latter, is lost in the presence of denaturing solvents. In some embodiments, the "epitope" of the HER2 protein is the region of the protein to which the anti-HER2 antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope. To screen for anti-HER2 antibodies that bind to an epitope, a routine cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). This assay can be used to determine whether an anti-HER2 antibody binds to the same site or epitope as the anti-HER2 antibody of the present technology. Alternatively, or in addition, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In a different method, peptides corresponding to different regions of the HER2 protein can be used in a competition assay with a test antibody, or with an antibody with a characterized or known epitope.

[0051] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA, splicing and other processing of the precursor mRNA to produce a mature mRNA, mRNA stability, translation of the mature mRNA into a protein (including codon usage and tRNA availability), and glycosylation and / or other modifications of the translation product, as required for proper expression and function.

[0052] As used herein, the term "gene" refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0053] As used herein, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions of each sequence, which can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and the homology or percent sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for the alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard, filter=none, strand=both, cutoff=60, expect=10, Matrix=BLOSUM62, Descriptions=50 sequences, sort by=HIGH SCORE, Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percent homology and encode polypeptides having the same or similar biological activity.Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity, or less than 25% identity, with each other.

[0054] As used herein, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance, such as binding affinity. In general, humanized antibodies contain at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab') 2 The humanized antibody may comprise substantially all of a human immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0055] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V L Around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V H around about 31-35B (H1), 50-65 (H2), and 95-102 (H3) in (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or those residues from the "hypervariable loops" (e.g., L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in H These include 26-32 (H1), 52A-55 (H2), and 96-101 (H3) in Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0056] As used herein, the term "identical" or percent "identity", when used in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison width or designated region as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then said to be "substantially identical". The term also refers to or can be applied to the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as sequences that have substitutions, hi some embodiments, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0057] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or VL). H The heavy chain constant region consists of three domains, CH 1 , C.H. 2 , and C.H. 3 Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region consists of one domain, C L It consists of. V H Area and VL The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L consists of three CDRs and four FRs, in the following order from amino terminus to carboxyl terminus: 1 , CDR 1 , F.R. 2 , CDR 2 , F.R. 3 , CDR 3 , F.R. 4 The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0058] As used herein, the term "linker" refers to a functional group (e.g., a chemical or polypeptide) that covalently bonds two or more polypeptides or nucleic acids such that they are linked together. As used herein, a "peptide linker" refers to a peptide linker that is used to couple two proteins together (e.g., V H and V L A linker refers to one or more amino acids used to couple domains. In certain embodiments, the linker is (GGGGS) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. In certain embodiments, the linker comprises an amino acid sequence having the sequence of GGGGSGGGSGGGGGS (SEQ ID NO: 73) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 74).

[0059] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody derived from a single clone, not the method by which it is produced, including any eukaryotic, prokaryotic, or phage clone. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, but is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including, for example, but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0060] As used herein, the term "nucleic acid" or "polynucleotide" refers to any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions that contain RNA or DNA, or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA that contains one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons.

[0061] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0062] As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two different antibody-producing cell lines. Use of this term includes at least two antibody preparations that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0063] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and longer chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 genetically encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as in a voluminous research literature.

[0064] As used herein, the term "recombinant," e.g., when used in reference to a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0065] As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients by different routes, simultaneously or substantially simultaneously.

[0066] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, with the administration routes being the same or different. More specifically, sequential use refers to the total administration of one active ingredient followed by the other(s). Thus, it is possible to administer one active ingredient over a period of minutes, hours, or days, followed by the administration of the other active ingredient(s). In this example, there is no simultaneous treatment.

[0067] As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route, simultaneously, or substantially simultaneously.

[0068] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to a single chain antibody that is composed of two domains of an Fv fragment, V L and V H A single chain antibody molecule may comprise a polymer having several individual molecules, e.g., a dimer, trimer, or other polymer. v The two domains of the fragment are V L and V H are encoded by separate genes, but they can be synthesized using recombinant methods to L and V H The domains can be linked by a synthetic linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule (single-chain F v (scF v ) Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc Natl Acad Sci 85:5879-5883. Such single chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0069] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind other molecules. The terms "specific binding," "specifically binds to," or "is specific for" a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, refer to, for example, a specific binding activity of about 10% to the molecule to which it binds. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K of M D The term "specifically binds" can refer to binding of a molecule (e.g., an antibody or antigen-binding fragment thereof) to a particular polypeptide (e.g., a HER2 polypeptide), or to an epitope on a particular polypeptide, without substantially binding to any other polypeptides or polypeptide epitopes.

[0070] As used herein, the terms "subject," "patient," or "individual" can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient, or individual is a human.

[0071] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0072] As used herein, "treating" or "treatment" encompasses the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., inhibiting its onset, (ii) relieving the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that a symptom associated with the disease is, for example, alleviated, reduced, cured, or put into remission.

[0073] It is also understood that the various treatment modes for disorders described herein include total treatment, but are intended to mean "substantially" less than total treatment, where some biologically or medically relevant result is achieved. Treatment can be continuous long-term treatment for chronic illnesses, or a single or several doses for treatment of acute conditions.

[0074] Amino acid sequence modifications of the anti-HER2 antibodies described herein are contemplated. Such modifications may be made to improve the binding affinity and / or other biological properties of the antibody, for example, to glycosylate the encoded amino acids, or to destroy the ability of the antibody to bind to C1q, Fc receptors, or to activate the complement system. Amino acid sequence variants of the anti-HER2 antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, by peptide synthesis, or by chemical modification. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to obtain the antibody of interest, so long as the resulting antibody has the desired properties. Modifications also include changes in the glycosylation pattern of the protein. The sites of greatest interest for substitutional mutagenesis include hypervariable regions, but FR changes are also contemplated.

[0075] Conservative amino acid substitutions are those that change a given amino acid for another amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, and size). "Conservative substitutions" are shown in the table below. [Table 1-1] [Table 1-2]

[0076] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitution variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent format from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution by the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein and antibodies with similar or superior properties in one or more relevant assays can be selected for further development.

[0077] HER2 HER2 (GenBank: NP 004439.2 (SEQ ID NO: 84)) is a receptor tyrosine kinase of the epidermal growth factor receptor family. Amplification or overexpression of HER2 has been demonstrated in the development and progression of cancer. Herceptin® (Trastuzumab) is an anti-HER2 monoclonal antibody approved for the treatment of HER2-positive metastatic breast cancer and HER2-positive gastric cancer (Trastuzumab [Highlights of Prescribing Information]. South San Francisco, CA: Genentech, Inc.; 2014). Ertumaxomab is a trispecific HER2-CD3 antibody with intact Fc receptor binding (see, e.g., Kiewe et al. 2006, Clin Cancer Res, 12(10):3085-3091). Ertumaxomab is a rat-mouse antibody, so human anti-mouse and human anti-rat antibody responses are expected upon administration to humans. 2502A, the parent antibody of ertumaxomab, has low affinity and low avidity for HER2 (Diermeier-Daucher et al., MAbs, 2012, 4(5):614-622).

[0078] Immunoglobulin-related compositions of the present technology The present technology describes methods and compositions for the production and use of anti-HER2 immunoglobulin-related compositions (e.g., anti-HER2 antibodies or antigen-binding fragments thereof). The antibodies and antigen-binding fragments of the present technology selectively bind to HER2 polypeptides. The anti-HER2 immunoglobulin-related compositions of the present disclosure may be useful for the diagnosis or treatment of HER2-associated cancers. Anti-HER2 immunoglobulin-related compositions within the scope of the present technology include, but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies, and diabodies that specifically bind to a target polypeptide, homolog, derivative, or fragment thereof. The present disclosure also provides an antigen-binding fragment of any of the anti-HER2 antibodies disclosed herein, the antigen-binding fragment being an Fab, F(ab)'2, Fab', scF, or other fragments thereof. v , and F vThe amino acid sequences of the anti-HER2 immunoglobulin related compositions of the present technology are set forth in Figures 1-3. [Table 2]

[0079] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) (i) V H is V of SEQ ID NO:1 H -CDR1 sequence, SEQ ID NO: 2 or SEQ ID NO: 7 H -CDR2 sequence, and V of SEQ ID NO: 8 H or (ii) V H is V of SEQ ID NO:1 H - CDR1 sequence, V of SEQ ID NO: 7 H - CDR2 sequence and V of SEQ ID NO: 3 or SEQ ID NO: 8 H and / or (b)(i) V L is V of SEQ ID NO:9 L -CDR1 sequence, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 L - CDR2 sequence and V of SEQ ID NO: 6 or SEQ ID NO: 12 L or (ii) V L is V of SEQ ID NO: 4 or SEQ ID NO: 9 L -CDR1 sequence, SEQ ID NO: 10 or SEQ ID NO: 11 L - CDR2 sequence and V of SEQ ID NO: 6 or SEQ ID NO: 12 L or (iii) V L is SEQ ID NO: 4 or SEQ ID NO: 9 L - CDR1 sequence, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 L - CDR2 sequence, and V of SEQ ID NO: 12 L - Includes the CDR3 sequence.

[0080] In one aspect, the present disclosure provides a method for the preparation of a heavy chain immunoglobulin variable domain (VH ) and light chain immunoglobulin variable domains (V L (a) an antibody or antigen-binding fragment thereof, comprising: H comprises an amino acid sequence selected from any one of SEQ ID NOs: 13, 15, or 17; and / or (b) V L In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from any one of SEQ ID NOs: 14, 16, 18, 19, or 20. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 14, SEQ ID NOs: 15 and 18, SEQ ID NOs: 15 and 19, and SEQ ID NOs: 15 and 20, respectively. H and V L Includes.

[0081] In any of the above embodiments, the antibody may be of any isotype, such as, for example, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (IgA 1 and IgA 2 The antibody further comprises an Fc domain, including but not limited to IgD, IgE, or IgM, and IgY. Non-limiting examples of constant region sequences include:

[0082] Human IgD constant region, Uniprot:P01880 (SEQ ID NO:75) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0083] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO: 76) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO:77) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0085] Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO:78) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0086] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 79) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0087] Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO:80) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0088] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO: 81) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0089] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 82) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0090] Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 83) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0091] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NOs: 75-82. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to SEQ ID NO: 83.

[0092] Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment binds to an extracellular region of a HER2 polypeptide. In certain embodiments, the epitope is a conformational epitope or a non-conformational epitope.

[0093] In some embodiments, the heavy (HC) and light (LC) chain immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0094] In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one HER2 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one HER2 polypeptide at about 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Dissociation constant of M (K DIn certain embodiments, the immunoglobulin related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0095] In certain embodiments, the immunoglobulin related composition contains an IgG1 constant region that includes one or more amino acid substitutions selected from the group consisting of N297A, K322A, L234A, and L235A. Additionally, or alternatively, in some embodiments, the immunoglobulin related composition contains an IgG4 constant region that includes an S228P mutation.

[0096] In one aspect, the disclosure provides a multispecific (e.g., bispecific) antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal direction, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a light chain constant domain of the first immunoglobulin, and (iii) an amino acid sequence (GGGGS). 3 and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope, and the light chain variable domain and the heavy chain variable domain of the second immunoglobulin have the amino acid sequence (GGGGS) 6to form a single-chain variable fragment; and (b) the second polypeptide chain and the third polypeptide chain each comprise, from N-terminal to C-terminal direction, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, and (ii) a heavy chain constant domain of the first immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 13, 15, or 17, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 14, 16, 18, 19, or 20.

[0097] In one aspect, the immunoglobulin related compositions of the present technology are, respectively, SEQ ID NO:21 and 22, SEQ ID NO:21 and 23, SEQ ID NO:21 and 24, SEQ ID NO:21 and 25, SEQ ID NO:21 and 26, SEQ ID NO:21 and 27, SEQ ID NO:21 and 28, SEQ ID NO:21 and 29, SEQ ID NO:21 and 30, SEQ ID NO:21 and 31, SEQ ID NO:21 and 32, SEQ ID NO:21 and 33, SEQ ID NO:34 and 33, SEQ ID NO:21 and 35, SEQ ID NO:36 and 33, SEQ ID NO:21 and 37, SEQ ID NO:21 and 38, SEQ ID NO:21 and 39, SEQ ID NO:21 and 40, SEQ ID NO:21 and 41, SEQ ID NO:21 and 42, SEQ ID NO:21 and 43, SEQ ID NO:21 and 44, SEQ ID NO:21 and 45, SEQ ID NO:21 and 46, SEQ ID NO:21 and 47, and 65, SEQ ID NO:21 and 66, SEQ ID NO:21 and 67, SEQ ID NO:21 and 68, SEQ ID NO:21 and 69, SEQ ID NO:21 and 70, SEQ ID NO:21 and 71, SEQ ID NO:21 and 72, and SEQ ID NO:21 and 85.

[0098] In some embodiments, the anti-HER2 immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some embodiments, the anti-HER2 immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some embodiments, Fab fragments are used to promote rapid binding and cellular uptake and / or slow release. In some embodiments, F(ab)' 2 Fragments are used to facilitate rapid binding and cellular uptake and / or slow release.

[0099] In one aspect, the technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein.

[0100] In another aspect, the technology provides host cells expressing any nucleic acid sequence encoding any of the immunoglobulin related compositions described herein.

[0101] The immunoglobulin-related compositions of the present technology (e.g., anti-HER2 antibodies) can be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of one or more HER2 polypeptides and for heterogeneous compositions, such as heterologous polypeptides or solid supports. See, e.g., WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al., J.Immunol.147:60-69(1991), U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648, and 6,106,835, and Kostelny et al., J.Immunol.148:1547-1553(1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized.

[0102] The immunoglobulin-related compositions of the present technology can further be recombinantly fused to heterologous polypeptides at the N-terminus or C-terminus, or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugation).For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules useful as labels for detection assays, and effector molecules such as heterologous polypeptides, drugs, or toxins.See, for example, WO92 / 08495, WO91 / 14438, WO89 / 12624, U.S. Patent No. 5,314,995, and EP0396387.

[0103] In any of the above embodiments of the immunoglobulin-related composition of the present technology, the antibody or antigen-binding fragment may optionally be conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. For chemical or physical binding, a functional group on the immunoglobulin-related composition typically associates with a functional group on the agent. Alternatively, a functional group on the agent associates with a functional group on the immunoglobulin-related composition.

[0104] The functional groups on the drug and the immunoglobulin-related composition can be directly associated. For example, a functional group (e.g., a sulfhydryl group) on the drug can be associated with a functional group (e.g., a sulfhydryl group) on the immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can be associated through a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent can be attached to either the drug or the immunoglobulin-related composition. The number of drugs or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of drugs associated with the conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with the drug depends on the number of functional groups present on the drug.

[0105] In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated with one agent. In one embodiment, the conjugate comprises at least one agent chemically bound (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bound to the immunoglobulin-related composition by any method known to those skilled in the art. For example, a functional group on the agent can be directly bound to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0106] Agents may also be chemically linked to the immunoglobulin-related composition by cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. website can provide assistance. Additional cross-linking agents include platinum cross-linkers described in U.S. Patent Nos. 5,580,990, 5,985,566, and 6,133,038 to Kreatech Biotechnology, BV, Amsterdam, The Netherlands.

[0107] Alternatively, the functional groups on the agent and the immunoglobulin-related composition can be the same. Homobifunctional crosslinkers are typically used to crosslink identical functional groups. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-maleimidohexane). Such homobifunctional crosslinkers are also available from Pierce Biotechnology, Inc.

[0108] In other cases, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The Pierce Biotechnology, Inc. website mentioned above can also provide assistance to the skilled artisan in selecting a suitable crosslinker that can be cleaved, for example, by an enzyme in the cell, so that the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0109] In another embodiment, the conjugate comprises at least one agent physically associated with at least one immunoglobulin-related composition. Any method known to those skilled in the art can be used to physically associate the agent with the immunoglobulin-related composition. For example, the immunoglobulin-related composition and the agent can be mixed together by any method known to those skilled in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent can be placed in a container and agitated, for example by shaking the container, to mix the immunoglobulin-related composition and the agent.

[0110] The immunoglobulin-related compositions can be modified by any method known to those of skill in the art, for example, the immunoglobulin-related compositions can be modified with crosslinking agents or functional groups, as described above.

[0111] A. Methods for Preparing the Anti-HER2 Antibodies of the Present Technology General Overview. First, a target polypeptide is selected against which the antibody of the present technology can be raised. For example, the antibody can be raised against the full-length HER2 protein, or a portion of the extracellular domain of the HER2 protein. Techniques for generating antibodies directed against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, those involving, for example, display libraries, xeno or human mice, hybridomas, etc. Target polypeptides within the scope of the present technology include any polypeptide derived from the HER2 protein that contains an extracellular domain capable of eliciting an immune response.

[0112] It will be appreciated that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, directed against the HER2 protein and fragments thereof are suitable for use in accordance with the present disclosure.

[0113] Anti-HER2 antibodies that can be subjected to the techniques described herein include monoclonal and polyclonal antibodies, as well as Fab, Fab', F(ab') 2 Antibody Fv-containing polypeptides, such as Fab' and F(ab') 2 Methods useful for high-yield production of antibody fragments have been described, see U.S. Patent No. 5,648,237.

[0114] Generally, the antibody is obtained from a starting species. More specifically, the nucleic acid or amino acid sequence of the variable part of the light chain, heavy chain, or both of the antibody of the starting species having specificity for the target polypeptide antigen is obtained. The starting species is any species that has been useful for generating the antibody or antibody library of the present technology (e.g., rat, mouse, rabbit, chicken, monkey, human, etc.).

[0115] Phage or phagemid display technology is a useful technique for inducing the antibody of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of the sequence encoding the antibody of the present technology can be carried out in E. coli.

[0116] Due to the degeneracy of nucleic acid coding sequences, other sequences that code for substantially the same amino acid sequence as that of the naturally occurring protein may be used in the practice of the present technology. These include, but are not limited to, nucleic acid sequences that contain all or part of the above-mentioned polypeptide-encoding nucleic acid sequences, which are modified by substitution of different codons that code for functionally equivalent amino acid residues in the sequence, thus resulting in silent changes. It will be understood that the nucleotide sequence of the immunoglobulin according to the present technology allows for up to 25% sequence homology variation, as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1998, Alan R. Liss, Inc.), so long as such variants form an engineered antibody that recognizes the HER2 protein. For example, one or more amino acid residues in a polypeptide sequence can be replaced by another amino acid of similar polarity that functions as a functional equivalent, resulting in a silent change. Substitutions in amino acids in a sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins, or fragments or derivatives thereof, that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, binding to antibody molecules or other cellular ligands, etc.Additionally, immunoglobulin-encoding nucleic acid sequences can be mutated in vitro or in vivo to create and / or destroy translation, initiation, and / or termination sequences, or to create mutations in the coding region and / or create new restriction endonuclease sites or destroy existing ones to further facilitate in vitro modification. Any technique for mutagenesis known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), and the like.

[0117] Preparation of polyclonal antisera and immunogens. The method of generating antibodies or antibody fragments of the present technology typically includes immunizing a subject (generally a non-human subject such as a mouse or rabbit) with purified HER2 protein or a fragment thereof, or with a cell expressing HER2 protein or a fragment thereof. Suitable immunogenic preparations can include, for example, recombinantly expressed HER2 protein or chemically synthesized HER2 peptides. Anti-HER2 antibodies that bind to HER2 protein or a portion or fragment thereof can be generated using standard techniques for preparing polyclonal and monoclonal antibodies, using the extracellular domain of HER2 protein, or a portion or fragment thereof, as an immunogen.

[0118] In some embodiments, the antigenic HER2 peptide comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on the use, according to methods well known to those of skill in the art. Multimers of a given epitope are sometimes more effective than monomers.

[0119] If necessary, the immunogenicity of the HER2 protein (or its fragment) can be increased by fusion or conjugation with a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. The HER2 protein can also be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant, to increase the subject's immune response to the polypeptide. Various adjuvants used to increase immunological responses include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0120] Alternatively, nanoparticles, such as virus-like particles (VLPs), can be used to present antigens, such as HER2, to host animals. Virus-like particles are multiprotein structures that mimic the organization and conformation of authentic native viruses and do not carry any viral genetic material, and therefore are not infectious (Urakami A, et al, Clin Vaccine Immunol 24:e00090-17(2017)). When introduced into the host immune system, VLPs can elicit an effective immune response, making them attractive carriers of foreign antigens. An important advantage of VLP-based antigen presentation platforms is their ability to present antigens in a dense and repetitive manner. Thus, antigen-bearing VLPs can induce strong B-cell responses by effectively enabling cross-linking of B-cell receptors (BCRs). VLPs can be genetically engineered to refine their properties, such as immunogenicity. These techniques are standard in the art.

[0121] Isolation of a protein or polypeptide sufficiently purified to raise antibodies can be time-consuming and sometimes technically challenging. Additional challenges associated with traditional protein-based immunization include concerns over the safety, stability, scalability, and consistency of protein antigens. Nucleic acid (DNA and RNA)-based immunization has emerged as a promising option. DNA vaccines are usually based on bacterial plasmids that encode the polypeptide sequence of a candidate antigen, e.g., HER2. Using a robust eukaryotic promoter, the encoded antigen is expressed when the host is inoculated with the plasmid to obtain sufficient levels of transgene expression (Galvin TA, et al., Vaccine 2000, 18:2566-2583). The generation of modern DNA vaccines relies on DNA synthesis or one-step cloning into a plasmid vector and subsequent isolation of the plasmid, greatly reducing the time and cost of production. The resulting plasmid DNA is also highly stable at room temperature, avoiding refrigerated transport and resulting in a substantially extended shelf life. These techniques are standard in the art.

[0122] Alternatively, the nucleic acid sequence encoding the antigen of interest, for example, HER2, can be synthetically introduced into an mRNA molecule.Then, the mRNA is delivered to a host animal, whose cells recognize and translate the mRNA sequence into the polypeptide sequence of the candidate antigen, for example, HER2, thus inducing an immune response against the foreign antigen.The attractive feature of mRNA antigen or mRNA vaccine is that mRNA is a non-infectious and non-integrating platform.There is no potential risk of infection or insertional mutagenesis associated with DNA vaccines. In addition, mRNA is degraded by normal cellular processes and has a controllable in vivo half-life through modification of the design and delivery method (Kariko, K., et al., Mol Ther 16:1833-1840(2008); Kauffman, KJ, et al., J Control Release 240, 227-234(2016); Guan, S. & Rosenecker, J., Gene Ther 24, 133-143(2017); Thess, A., et al., Mol Ther 23, 1456-1464(2015)). These techniques are standard in the art.

[0123] In describing the present technology, an immune response can be described as either a "primary" or a "secondary" immune response. A primary immune response, also described as a "protective" immune response, refers to an immune response that occurs in an individual as a result of some initial exposure (e.g., initial "immunization" or "priming") to a particular antigen, e.g., HER2 protein. In some embodiments, immunity can occur as a result of inoculating an individual with a vaccine that contains an antigen. For example, the vaccine can be a HER2 vaccine that includes one or more HER2 protein-derived antigens. A primary immune response can weaken or decrease over time, even disappearing or at least diminishing to the point where it is undetectable. Thus, the present technology also relates to a "secondary" immune response, also described herein as a "memory immune response." The term secondary immune response refers to an immune response that is elicited in an individual in whom a primary immune response has already occurred.

[0124] Thus, a secondary immune response can be elicited, for example, to enhance (e.g., boost) an existing immune response that has weakened or diminished, or to reconstitute a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response results in the stimulation of memory B cells that were generated upon the first presentation of the antigen. A delayed type hypersensitivity (DTH) response is a response that is initiated by the activation of the CD4 + It is a type of cellular secondary or memory immune response mediated by T cells. An initial exposure to an antigen primes the immune system and additional exposures result in DTH.

[0125] After appropriate immunization, anti-HER2 antibodies can be prepared from the serum of the subject. If desired, antibody molecules directed against the HER2 protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain an IgG fraction.

[0126] Monoclonal antibody. In one embodiment of the present technology, the antibody is an anti-HER2 monoclonal antibody. For example, in some embodiments, the anti-HER2 monoclonal antibody can be a human or mouse anti-HER2 monoclonal antibody. For the preparation of monoclonal antibodies directed against HER2 protein or its derivatives, fragments, analogs, or homologs, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975. Nature 256:495-497), the trioma technique, the human B cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol. Today 4:72), and the EBV hybridoma technique which produces human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be utilized in the practice of the present technology and can be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). For example, a population of nucleic acids encoding regions of an antibody can be isolated. Using PCR with primers derived from sequences encoding conserved regions of the antibody, sequences encoding portions of the antibody are amplified from the population, and DNA encoding the antibody or a fragment thereof, such as a variable domain, is then reconstructed from the amplified sequences.Such amplified sequences can also be fused to DNA encoding other proteins, such as bacteriophage coats, or bacterial cell surface proteins, for expression and display of the fusion polypeptide in phages or bacteria. The amplified sequences can be expressed and further selected or isolated, for example, based on the affinity of the expressed antibodies or fragments thereof to antigens or epitopes present in the HER2 protein. Alternatively, hybridomas expressing anti-HER2 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using routine methods. See, for example, Milstein et al., (Galfre and Milstein, Methods Enzymol (1981) 73:3-46). Screening the hybridomas using standard methods produces monoclonal antibodies of various specificities (i.e., against different epitopes) and affinities. A selected monoclonal antibody with the desired properties, e.g., HER2 binding, can be used as expressed by a hybridoma, which can be conjugated to a molecule such as polyethylene glycol (PEG) to modify its properties, or the cDNA encoding it can be isolated, sequenced, and manipulated in a variety of ways. Synthetic dendrimer trees can be added to reactive amino acid side chains, e.g., lysine, to enhance the immunogenic properties of the HER2 protein. Also, CPG-dinucleotide techniques can be used to enhance the immunogenic properties of the HER2 protein. Other manipulations include substitution or deletion of certain aminoacyl residues responsible for the instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of the antibody for the HER2 protein.

[0127] Hybridoma technique. In some embodiments, the antibody of the present technology is an anti-HER2 monoclonal antibody produced by a hybridoma, which comprises a B cell obtained from a transgenic non-human animal, such as a transgenic mouse, whose genome comprises a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma technique is known in the art and is taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Hybridoma and other methods for producing monoclonal antibodies are well known to those skilled in the art.

[0128] Phage display techniques. As mentioned above, the antibodies of the present technology can be produced by the application of recombinant DNA and phage display technology. For example, anti-HER2 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Phages with desired binding properties are typically selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection with antigens bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phages, including fd and M13, with Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. In addition, the methods are adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., Science 246:1275-1281, 1989), allowing the rapid and efficient identification of monoclonal Fab fragments having a desired specificity for a HER2 polypeptide, e.g., a polypeptide, or a derivative, fragment, analog, or homolog thereof.Other examples of phage display methods that can be used to generate antibodies of the present technology include those described in Huston et al., Proc. Natl. Acad. Sci USA, 85:5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87:1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182:41-50, 1995; Ames et al., J. Immunol. Methods 184:177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24:952-958, 1994; Persic et al., Gene 187:9-18, 1997; Burton et al., Advances in Immunology 57:191-280,1994, PCT / GB91 / 01134, WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, WO96 / 06213, WO92 / 01047(Medical Research Council et al. al.), WO97 / 08320 (Morphosys), WO92 / 01047 (CAT / MRC), WO91 / 17271 (Affymax), and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743. A method useful for displaying polypeptides on the surface of bacteriophage particles by linking the polypeptides through disulfide bonds is described by Lohning, US Pat. No. 6,753,136.As described in the above references, after phage selection, the antibody coding region from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., Fab, Fab', and F(ab'). 2 Techniques for recombinantly producing fragments can also be employed using methods known in the art, such as those disclosed in WO92 / 22324, Mullinax et al., BioTechniques 12:864-869, 1992, and Sawai et al., AJRI 34:26-34, 1995, and Better et al., Science 240:1041-1043, 1988.

[0129] Generally, antibodies or antibody fragments are present on the surface of phage or phagemid particles, so that hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against the appropriate antigen to identify variants that maintain superior binding activity. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats can be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or lambda Zap system) for selection and / or screening.

[0130] Expression of recombinant anti-HER2 antibodies. As mentioned above, the antibodies of the present technology can be produced by applying recombinant DNA technology. Recombinant polynucleotide constructs encoding the anti-HER2 antibodies of the present technology typically include expression control sequences operably linked to the coding sequences of the anti-HER2 antibody chains, and include naturally associated or heterologous promoter regions. Thus, another aspect of the present technology includes vectors containing one or more nucleic acid sequences encoding the anti-HER2 antibodies of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, a nucleic acid containing all or a part of the nucleotide sequence encoding the anti-HER2 antibody is inserted into a suitable cloning vector, or expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and detailed below. Methods for generating a diverse population of vectors are described in Lerner et al., U.S. Patent Nos. 6,291,160 and 6,680,192.

[0131] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this disclosure, "plasmid" and "vector" may be used interchangeably, since plasmids are the most commonly used form of vector. However, the present technology is intended to include other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions. Such viral vectors allow for infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector that can transform or transfect a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding the anti-HER2 antibody, and collection and purification of the anti-HER2 antibody, e.g., a cross-reactive anti-HER2 antibody. See generally US2002 / 0199213. These expression vectors are typically replicable in the host organism, either as episomes or as an integrated part of the host chromosomal DNA. Commonly, expression vectors contain a selectable marker, e.g., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences. The vector can also encode a signal peptide, e.g., pectate lyase, useful for directing secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0132] The recombinant expression vector of the present technology comprises a nucleic acid encoding a protein having HER2 binding properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory sequences selected based on the host cell used for expression, operably linked to the nucleic acid sequence to be expressed. In the recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the expression level of the polypeptide desired, and the like. Exemplary regulatory sequences useful as promoters of recombinant polypeptide expression (e.g., anti-HER2 antibodies) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, a polynucleotide encoding an anti-HER2 antibody of the present technology is operably linked to an ara B promoter and can be expressed in a host cell. See U.S. Patent No. 5,028,530.The expression vectors of the present technology can be introduced into a host cell to produce a polypeptide or peptide, including a fusion polypeptide, encoded by a nucleic acid as described herein (such as, for example, an anti-HER2 antibody).

[0133] Another aspect of the technology relates to anti-HER2 antibody expressing host cells that contain nucleic acid encoding one or more anti-HER2 antibodies. The recombinant expression vectors of the technology can be designed for expression of anti-HER2 antibodies in prokaryotic or eukaryotic cells. For example, anti-HER2 antibodies can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells such as yeast, yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods useful for preparing and screening polypeptides with predetermined properties, such as anti-HER2 antibodies, via expression of stochastically generated polynucleotide sequences have been described. See U.S. Patent Nos. 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0134] Expression of polypeptides in prokaryotes is often carried out in E. coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add several amino acids to the polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) increase the expression of the recombinant polypeptide, (ii) increase the solubility of the recombinant polypeptide, and (iii) aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide, allowing separation of the recombinant polypeptide from the fusion moiety following purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc., Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0135] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of different active peptides or protein domains to obtain multifunctional polypeptides via polypeptide fusion are described by Pack et al., U.S. Patent Nos. 6,294,353 and 6,692,935. One strategy to maximize recombinant polypeptide expression in E. coli, for example, anti-HER2 antibodies, is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. For example, see Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that each individual codon for each amino acid is the one that is preferentially utilized in the expression host, for example, E. coli (for example, see Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such modification of the nucleic acid sequence of the present technology can be carried out by standard DNA synthesis techniques.

[0136] In another embodiment, the anti-HER2 antibody expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987.EMBO J.6:229-234), pMFa (Kurjan and Herskowitz, Cell 30:933-943, 1982), pJRY88 (Schultz et al., Gene 54:113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, anti-HER2 antibody can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expressing polypeptides, such as anti-HER2 antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3:2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

[0137] In yet another embodiment, the nucleic acid encoding the anti-HER2 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329:840, 1987) and pMT2PC (Kaufman, et al., EMBO J.6:187-195, 1987). When used in mammalian cells, the control function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells useful for expressing the anti-HER2 antibodies of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0138] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific, Pinkert, et al., Genes Dev. 1:268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43:235-275, 1988), T cell receptor (Winoto and Baltimore, EMBO J. 8:729-733, 1989) and immunoglobulin promoters (Banerji, et al., 1983. Cell 33:729-740; Queen and Baltimore, Cell 33:741-748, 1983.), neuron-specific promoters (e.g., neurofilament promoters, Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), pancreatic-specific promoters (Edlund, et al., Cell 33:5473-5477, 1989), and / or endothelial cell-specific promoters (Edlund, et al., Cell 33:5473-5477, 1989). al., 1985. Science 230:912-916), as well as mammary gland-specific promoters (e.g., the milk whey promoter, U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166). Developmentally-regulated promoters, such as the murine hox promoters (Kessel and Gruss, Science 249:374-379, 1990) and the α-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546, 1989), are also encompassed.

[0139] Another aspect of the method relates to a host cell into which the recombinant expression vector of the present technology is introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in subsequent generations, either by mutation or environmental influence, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term as used herein.

[0140] The host cell can be any prokaryotic or eukaryotic cell. For example, anti-HER2 antibodies can be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese Hamster Ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences such as origins of replication, promoters, enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Queen et al., Immunol. Rev. 89:49, 1986. Exemplary expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J Immunol. 148:1149, 1992. Other suitable host cells are known to those of skill in the art.

[0141] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, gene gun, or virus-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0142] Non-limiting examples of suitable vectors include those designed for propagation and expansion, or for expression, or both. For example, cloning vectors can be selected from the group consisting of pUC series, pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as lambda-GT10, lambda-GT11, lambda-ZapII (Stratagene), lambda-EMBL4, and lambda-NM1149 can also be used. Non-limiting examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Non-limiting examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Calsbad, Calif.) can also be used following the manufacturer's recommendations.

[0143] In certain embodiments, the vector is a mammalian vector. In certain embodiments, the mammalian vector contains at least one promoter element that mediates the signals necessary for initiation of transcription of mRNA, antibody coding sequence, termination of transcription and polyadenylation of the transcript. In certain embodiments, the mammalian vector contains additional elements, such as enhancers flanking donor and acceptor sites for RNA splicing, Kozak sequences, and intervening sequences. In certain embodiments, highly efficient transcription can be achieved using, for example, early and late promoters from SV40, long terminal repeats (LTRS) from retroviruses, such as RSV, HTLVI, HIVI, and early promoters of cytomegalovirus (CMV). Cellular elements can also be used (e.g., human actin promoter). Non-limiting examples of mammalian expression vectors include vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), or pcDNA3.1 / Hygro(+ / -) (Invitrogen, Calsbad, Calif.), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr (ATCC37146), and pBC12MI (ATCC67109). Non-limiting examples of mammalian host cells that can be used in combination with such mammalian vectors include human Hela 293, HEK 293, H9, and Jurkat cells, mouse 3T3, NIH3T3, and C127 cells, Cos 1, Cos 7, and CV 1, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

[0144] In certain embodiments, the vector is a viral vector, such as a retroviral vector, a parvovirus-based vector, such as an adeno-associated virus (AAV)-based vector, an AAV-adenovirus chimeric vector, and an adenovirus-based vector, and a lentiviral vector, such as a herpes simplex (HSV)-based vector. In certain embodiments, the viral vector is engineered to be defective in viral replication. In certain embodiments, the viral vector is engineered to eliminate toxicity to the host. These viral vectors can be prepared using standard recombinant DNA techniques, and are described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[0145] In certain embodiments, the vectors or polynucleotides described herein can be transferred into cells (e.g., ex vivo cells) by conventional techniques, and the resulting cells can be cultured by conventional techniques to produce the anti-HER2 antibodies or antigen-binding fragments described herein. Thus, provided herein is a cell comprising a polynucleotide encoding an anti-HER2 antibody or antigen-binding fragment thereof operably linked to a regulatory expression element (e.g., a promoter) for expression of such sequences in a host cell. In certain embodiments, a vector encoding a heavy chain operably linked to a promoter and a vector encoding a light chain operably linked to a promoter can be co-expressed in a cell for expression of the entire anti-HER2 antibody or antigen-binding fragment. In certain embodiments, the cell comprises a vector comprising a polynucleotide encoding both the heavy and light chains of an anti-HER2 antibody or antigen-binding fragment described herein operably linked to a promoter. In certain embodiments, the cell comprises two different vectors, a first vector comprising a polynucleotide encoding a heavy chain operably linked to a promoter, and a second vector comprising a polynucleotide encoding a light chain operably linked to a promoter. In certain embodiments, the first cell comprises a first vector comprising a polynucleotide encoding a heavy chain of an anti-HER2 antibody or antigen-binding fragment described herein, and the second cell comprises a second vector comprising a polynucleotide encoding a light chain of an anti-HER2 antibody or antigen-binding fragment described herein. In certain embodiments, provided herein is a mixture of cells comprising the first cell and the second cell. Examples of cells include, but are not limited to, human cells, human cell lines, E. coli (e.g., E. coli TB-1, TG-2, DH5a, XL-Blue MRF' (Stratagene), SA2821, and Y1090), B. subtilis, P. aerugenosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9, Ea4), and the like.

[0146] It is known that in stable transfection of mammalian cells, only a small percentage of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cell together with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell in the same vector as that encoding the anti-HER2 antibody, or can be introduced in a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selectable marker gene survive, while other cells die).

[0147] A host cell comprising the anti-HER2 antibody of the present technology, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a recombinant anti-HER2 antibody. In one embodiment, the method includes culturing a host cell (into which a recombinant expression vector encoding the anti-HER2 antibody has been introduced) in a suitable medium such that the anti-HER2 antibody is produced. In another embodiment, the method further includes isolating the anti-HER2 antibody from the medium or the host cell. Once expressed, the anti-HER2 antibody, e.g., the collection of anti-HER2 antibody or anti-HER2 antibody-related polypeptides, is purified from the culture medium and the host cell. The anti-HER2 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-HER2 antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Typically, the anti-HER2 antibody chain is expressed with a signal sequence, and thus released into the culture medium. However, if the anti-HER2 antibody chains are not naturally secreted by the host cells, they can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0148] Polynucleotides encoding anti-HER2 antibodies, e.g., anti-HER2 antibody coding sequences, can be incorporated into transgenes for introduction into the genome of transgenic animals and subsequent expression in the milk of transgenic animals. See, e.g., U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include light and / or heavy chain coding sequences in operable linkage with promoters and enhancers from mammary gland-specific genes, such as casein or β-lactoglobulin. For the generation of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[0149] Single chain antibody. In one embodiment, the anti-HER2 antibody of the present technology is a single chain anti-HER2 antibody. According to the present technology, the technique can be adapted to produce the single chain antibody specific to HER2 protein (see, for example, U.S. Patent No. 4,946,778). Examples of the technique that can be used to produce the single chain Fv and antibody of the present technology include those described in U.S. Patent Nos. 4,946,778 and 5,258,498, Huston et al., Methods in Enzymology, 203:46-88, 1991, Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999, 1993, and Skerra et al., Science 240:1038-1040, 1988.

[0150] Chimeric and humanized antibodies. In one embodiment, the anti-HER2 antibody of the present technology is a chimeric anti-HER2 antibody. In one embodiment, the anti-HER2 antibody of the present technology is a humanized anti-HER2 antibody. In one embodiment of the present technology, the donor antibody and the acceptor antibody are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody.

[0151] Recombinant anti-HER2 antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be produced using standard recombinant DNA techniques and are within the scope of the present technology. In some applications, including the in vivo use of the anti-HER2 antibodies of the present technology in humans and the use of these agents in in vitro detection assays, it is possible to use chimeric or humanized anti-HER2 antibodies. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.Such useful methods include, for example, those described in International Application No. PCT / US86 / 02269, U.S. Pat. No. 5,225,539, European Patent No. 184187, European Patent No. 171496, European Patent No. 173494, PCT International Publication No. 86 / 01533, U.S. Pat. Nos. 4,816,567, 5,225,539, and European Patent No. 125023, Better, et al., 1988. Science 240:1041-1043, Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84:3439-3443, Liu, et al., 1987. J. Immunol. 139:3521-3526, Sun, et al., 1988. J. Immunol. 139:3521-3526, and others. al.,1987.Proc.Natl.Acad.Sci.USA 84:214-218, Nishimura, et al.,1987.Cancer Res.47:999-1005, Wood, et al.,1985.Nature 314:446-449, Shaw, et al.,1988.J.Natl.Cancer Inst.80:1553-1559, Morrison(1985)Science 229:1202-1207, Oi, et al.(1986)BioTechniques 4:214, Jones,et al.,1986.Nature 321:552-525, Verhoeyan,et al.,1988.Science 239:1534,Morrison,Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Patent No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141:4053-4060.For example, antibodies can be humanized using a variety of techniques including CDR grafting (EP 0239400, WO 91 / 09967, U.S. Pat. Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516, EP 460167), veneering or resurfacing (EP 0592106, EP 0519596, Padlan EA, Molecular Immunology, 28:489-498, 1991, Studnicka et al., Protein Engineering 7:805-814, 1994, Roguska et al., PNAS 91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332).In one embodiment, the cDNA encoding the murine anti-HER2 monoclonal antibody is digested with specifically selected restriction enzymes to remove sequences encoding the Fc constant region, and the equivalent portion of the cDNA encoding the human Fc constant region is replaced (Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., (1988) Proc. Natl. Acad. Sci. USA 84:3439-3443; See, for example, U.S. Pat. Nos. 6,180,370, 6,300,064, 6,696,248, 6,706,484, and 6,828,422.

[0152] In one embodiment, the present technology provides for the construction of humanized anti-HER2 antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response, but still have effective antibody effector functions. As used herein, with respect to antibodies, the terms "human" and "humanized" refer to any antibody that is expected to induce a therapeutically tolerable weak immunogenic response in human subjects. In one embodiment, the present technology provides humanized anti-HER2 antibodies, heavy and light chain immunoglobulins.

[0153] CDR antibody. In some embodiments, the anti-HER2 antibody of the present technology is an anti-HER2 CDR antibody. Generally, the donor antibody and the acceptor antibody used to generate the anti-HER2 CDR antibody are monoclonal antibodies from different species, and typically the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody. The graft is a single V of the acceptor antibody. H Or V L or of a single CDR (or even a portion of a single CDR) in V H and V L The CDRs may be of multiple CDRs (or portions thereof) in one or both of the variable domains. In most cases, all three CDRs in all variable domains of the acceptor antibody are replaced with the corresponding donor CDRs, but only as many as are necessary to allow sufficient binding of the resulting CDR-grafted antibody to the HER2 protein. Methods for generating CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Patent No. 5,585,089, U.S. Patent No. 5,693,761, U.S. Patent No. 5,693,762, and Winter U.S. Patent No. 5,225,539, and EP0682040. H and V L Useful methods for preparing polypeptides are taught by Winter et al., U.S. Patent Nos. 4,816,397, 6,291,158, 6,291,159, 6,291,161, 6,545,142, EP0368684, EP0451216, and EP0120694.

[0154] After selecting suitable framework region candidates from the same family and / or same family members, either or both of the heavy and light chain variable regions are generated by grafting the CDRs from the starting species into the hybrid framework regions. With respect to any of the above embodiments, the assembly of hybrid antibodies or hybrid antibody fragments with hybrid variable chain regions can be accomplished using conventional methods known to those of skill in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs from the starting species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding the CDR regions can also be isolated from the starting species antibody using suitable restriction enzymes and ligated to the target species framework by ligation with a suitable ligation enzyme. Alternatively, the framework regions of the variable chains of the starting species antibody can be altered by site-directed mutagenesis.

[0155] Because hybrids are constructed from a selection among multiple candidates corresponding to each framework region, there are many combinations of sequences that are suitable for construction according to the principles described herein. Thus, libraries of hybrids can be assembled having members with different combinations of the individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0156] This process typically does not change the FRs of the acceptor antibody adjacent to the grafted CDRs. However, one skilled in the art can sometimes improve the antigen binding affinity of the resulting anti-HER2 CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable positions for substitution include amino acid residues adjacent to or capable of interacting with the CDR (see, e.g., US5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with a donor FR and modify it to make it more similar to the acceptor FR or human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR matches the human consensus FR at that position or is at least 90% or more identical to such consensus FR, doing so may not significantly increase the antigenicity of the resulting modified anti-HER2 CDR-grafted antibody compared to the same antibody with a fully human FR.

[0157] Bispecific antibodies (BsAbs). Bispecific antibodies are antibodies that can simultaneously bind two targets with different structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. BsAbs can be generated, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, by molecular function, a bispecific binding agent binds one antigen (or epitope) with one of its two binding arms (one VH / VL pair) and binds a different antigen (or epitope) with its second arm (different VH / VL pair). By this definition, a bispecific binding agent has two different antigen binding arms (both in specificity and CDR sequence) and is monovalent for each antigen it binds.

[0158] Multispecific antibodies, such as bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs), have, for example, at least one arm that specifically binds to HER2 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B cell, a T cell, a myeloid cell, a plasma cell, or a mast cell. Additionally or alternatively, in certain embodiments, the second target antigen is selected from the group consisting of CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, and KIR. Exemplary Vs that bind to a second target antigen (e.g., CD3) include: H and V L The sequence is shown in Figure 3 (included in the LC sequence). In certain embodiments, the BsAb is capable of binding to tumor cells expressing the HER2 antigen on the cell surface. In some embodiments, the BsAb is engineered to promote tumor cell killing by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those with a first antigen binding site specific for HER2 and a second antigen binding site specific for a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, other DOTA-chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D A. et al, 1994, Cancer Res. 54(22):5937-5946).

[0159] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed to utilize either the entire immunoglobulin framework (e.g., IgG), single chain variable fragments (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprising an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs composed of two scFv units have been shown to be a clinically successful bispecific antibody format. In some embodiments, the BsAb comprises two single chain variable fragments (scFvs) in tandem, where the scFv that binds to a tumor antigen (e.g., HER2) is designed to be linked to a scFv that binds to T cells (e.g., by binding to CD3). In this way, T cells are recruited to the tumor site so that they can mediate cytotoxic killing of tumor cells. See Dreier et al., J.Immunol.170:4397-4402(2003); Bargou et al., Science 321:974- 977(2008)). In some embodiments, the BsAb of the present technology comprises two single chain variable fragments (scFvs) in tandem, where the scFv that binds to a tumor antigen (e.g., HER2) is designed to be linked to a scFv that binds to a small molecule DOTA hapten.

[0160] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues to cross-link more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins to link two or more different single chain antibody or antibody fragment segments with the required dual specificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0161] Bispecific fusion proteins linking two or more different single chain antibodies or antibody fragments are produced in a similar manner. Various fusion proteins can be produced using recombinant methods. In some specific embodiments, the BsAb of the present technology comprises an immunoglobulin, which comprises a heavy and light chain, and an scFv. In some specific embodiments, the scFv is linked to the C-terminus of the heavy chain of any HER2 immunoglobulin disclosed herein. In some specific embodiments, the scFv is linked to the C-terminus of the light chain of any HER2 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for the in-frame connection of the heavy chain Fd with the scFv is extracted by PCR reaction using the V L and V カッパ The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and the V domain of the HER2 antibody is generated. H The region is ligated into a vector containing a DNA sequence encoding the region, and the resulting vector can be used to transfect a suitable host cell, such as a mammalian cell, for expression of the bispecific fusion protein.

[0162] In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized by not tending to adopt a rigid three-dimensional structure, but rather providing flexibility to the polypeptide (e.g., the first and / or second antigen binding site). In some embodiments, linkers are used in the BsAbs described herein based on the particular properties they confer to the BsAb, such as, for example, increased stability. In some embodiments, the BsAbs of the present technology are characterized by the fact that they tend not to adopt a rigid three-dimensional structure, but rather provide flexibility to the polypeptide (e.g., the first and / or second antigen binding site). 4 In some specific embodiments, the BsAb of the present technology comprises a (G 4 S) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0163] Fc Modifications. In some embodiments, the anti-HER2 antibodies of the present technology comprise a variant Fc region, which comprises at least one amino acid modification relative to a wild-type Fc region (or parent Fc region), such that the molecule has an altered affinity for an Fc receptor (e.g., FcγR), provided that the variant Fc region does not have substitutions at positions that make direct contact with an Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions, such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make direct contact with an Fc receptor, such as FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G loop).

[0164] In some embodiments, the anti-HER2 antibody of the present technology has an altered affinity for activating and / or inhibitory receptors and has a variant Fc region with one or more amino acid modifications, where the one or more amino acid modifications are N297 substitution with alanine or K322 substitution with alanine. Additionally or alternatively, in some embodiments, the Fc region of the HER2 antibody disclosed herein contains two amino acid substitutions Leu234Ala and Leu235Ala (referred to as LALA mutations), eliminating FcγRIIa binding. LALA mutations are commonly used to alleviate cytokine induction from T cells, thus reducing the toxicity of the antibody (Wines BD, et al., J Immunol 164:5313-5318 (2000)).

[0165] Glycosylation modifications. In some embodiments, the anti-HER2 antibodies of the present technology have an Fc region with variant glycosylation compared to the parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose, and in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells.

[0166] In some embodiments, the antibodies of the present technology may have modified glycosylation sites compared to a suitable reference antibody that binds to an antigen of interest (e.g., HER2) without altering the functionality of the antibody, e.g., its binding activity with the antigen. As used herein, "glycosylation site" includes any particular amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more simple sugars linked together) can be specifically and covalently attached.

[0167] Oligosaccharide side chains are typically linked to the backbone of the antibody via either N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine. For example, an Fc-glycoform (hHER2-IgGln) lacking certain oligosaccharides containing fucose and terminal N-acetylglucosamine can be produced in special CHO cells and exhibits enhanced ADCC effector function.

[0168] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of an antibody are well known in the art and are included within the present technology, see, for example, U.S. Pat. No. 6,218,149, EP0359096B1, U.S. Patent Publication No. 2002 / 0028486, WO 03 / 035835, U.S. Patent Publication No. 2003 / 0115614, U.S. Pat. No. 6,218,149, U.S. Pat. No. 6,472,511, which are incorporated herein by reference in their entireties. In some embodiments, the carbohydrate content of an antibody (or a relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some specific embodiments, the present technology includes deleting a glycosylation site in the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0169] Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or decreasing effector function. Engineered glycoforms can be produced by any method known to those of skill in the art, such as by using engineered or variant expression strains, by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase III (GnTIII), by expressing molecules containing Fc regions in different organisms or cell lines from different organisms, or by modifying carbohydrates after the molecules containing Fc regions are expressed. Methods for producing engineered glycoforms are known in the art and are described in Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 277:26733-26740; al., 2003, J. Biol. Chem. 278:3466-3473, U.S. Patent No. 6,602,684, U.S. Patent Application No. 10 / 277,370, U.S. Patent Application No. 10 / 113,929, International Patent Application Publication Nos. WO 00 / 61739A1, WO 01 / 292246A1, WO 02 / 311140A1, WO 02 / 30954A1, POTILLEGENT™ technology (Biowa, Inc. Princeton, NJ), GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication No. 00 / 061739, U.S. Patent Application Publication No. 2003 / 0115614, Okazaki et al., 2004, JMB, 336:1239-49.

[0170] Fusion proteins. In one embodiment, the anti-HER2 antibody of the present technology is a fusion protein. The anti-HER2 antibody of the present technology can be used as an antigen tag when fused to a second protein. Examples of domains that can be fused to a polypeptide include heterologous signal sequences as well as other heterologous functional regions. The fusion does not necessarily have to be direct, but can occur via a linker sequence. Furthermore, the fusion protein of the present technology can also be engineered to improve the characteristics of the anti-HER2 antibody. For example, additional amino acids, particularly a region of charged amino acids, can be added to the N-terminus of the anti-HER2 antibody to improve stability and persistence during purification from the host cell or subsequent handling and storage. Peptide moieties can also be added to the anti-HER2 antibody to facilitate purification. Such regions can be removed before final preparation of the anti-HER2 antibody. The addition of peptide moieties to facilitate handling of a polypeptide is a well-known and routine technique in the art. The anti-HER2 antibody of the present technology can be fused to a marker sequence, such as a peptide, to facilitate purification of the fused polypeptide. In selected embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif), many of which are commercially available, among others. For example, hexa-histidine provides for convenient purification of the fusion protein, as described by Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37:767, 1984.

[0171] Thus, any of these above fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology, and in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo.

[0172] Fusion proteins with disulfide-bonded dimeric structures (as occurs with IgG) can be more efficient in binding and neutralizing other molecules compared to monomeric secreted proteins or protein fragments alone. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.

[0173] Similarly, EP-A-O464533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of an immunoglobulin molecule together with another human protein or fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial for therapy and diagnosis and can thus, for example, result in improved pharmacokinetic properties. See EP-A0232262. Alternatively, it may be desirable to delete or modify the Fc portion after the fusion protein has been expressed, detected and purified. For example, the Fc portion may interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug discovery, for example, human proteins such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8:52-58, 1995, Johanson et al., J. Biol. Chem., 270:9459-9471, 1995.

[0174] Labeled anti-HER2 antibody. In one embodiment, the anti-HER2 antibody of the present technology is coupled with a labeling moiety, i.e., a detectable group. The particular label or detectable group conjugated to the anti-HER2 antibody is not a critical aspect of the present technology, so long as it does not significantly interfere with the specific binding of the anti-HER2 antibody of the present technology to the HER2 protein. The detectable group can be any material that has a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, the label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g., 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O. 89 Zr (for positron emission tomography), 99m T.C., 111In (for single photon emission computed tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference in its entirety and for all purposes. Handbook of Fluorescent Probes and Research Chemicals (6 th Ed., Molecular Probes, Inc., Eugene OR.

[0175] The labels can be directly or indirectly coupled to the desired components of the assay according to methods well known in the art. As mentioned above, a wide variety of labels can be used, with the choice of label depending on factors such as the required sensitivity, ease of conjugation with the compound, stability requirements, available equipment, and disposal regulations.

[0176] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently attached to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule that is inherently detectable or covalently attached to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Many ligands and anti-ligands can be used. If the ligand has a natural anti-ligand, such as biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, such as an anti-HER2 antibody.

[0177] Molecules can also be directly conjugated to signal generating compounds, for example, by conjugation with enzymes or fluorophores. Enzymes of interest as labels are primarily hydrolases, particularly phosphatases, esterases, and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, for example, luciferin, and 2,3-dihydrophthalazinediones, such as luminol. For a review of various labeling or signal generating systems that can be used, see U.S. Pat. No. 4,391,904.

[0178] Means for detecting the label are well known to those skilled in the art. Thus, for example, when the label is a radioactive label, the means for detection include a scintillation counter or photographic film as in autoradiography. When the label is a fluorescent label, it can be detected by exciting the fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be detected visually, by photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers. Similarly, enzyme labels can be detected by providing the enzyme with the appropriate substrate and detecting the resulting reaction product. Finally, simple colorimetric labels can be detected by simply observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0179] Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of target antibodies, such as anti-HER2 antibodies. In this case, antigen-coated particles are agglutinated by the sample containing target antibodies. In this format, none of the components need to be labeled, and the presence of target antibodies is detected by simple visual inspection.

[0180] B. Identification and Characterization of Anti-HER2 Antibodies of the Present Technology Methods for identifying and / or screening anti-HER2 antibodies of the present technology. Methods useful for identifying and screening antibodies against HER2 polypeptides for those with the desired specificity to HER2 protein (e.g., those that bind to the extracellular domain of HER2 protein, such as the polypeptide comprising the amino acid sequence of GenBank: NP 004439.2 (SEQ ID NO: 84)) include any immunologically mediated technique known in the art. Components of the immune response can be detected in vitro by a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigen and antigen-presenting cells and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2:373-80, 1995); (3) antigen-presenting cells can be incubated with total protein antigen and the presentation of that antigen on MHC can be detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with agents that crosslink their Fc-epsilon receptors and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4:432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0181] Similarly, the products of an immune response in either a model organism (e.g., mouse) or a human subject can be detected by a variety of methods well known to those of skill in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., ELISA; (2) the migration of immune cells to a site of inflammation can be detected by scratching the surface of the skin and placing a sterile container to capture the migrating cells across the scratch site (Peters et al., Blood, 72:1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be detected by a variety of methods well known to those of skill in the art. 3 H-thymidine, (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72:1310-5, 1988), and (5) differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs expressing these markers.

[0182] In one embodiment, the anti-HER2 antibody of the present technology is selected by using the display of HER2 peptide on the surface of replicable genetic package.See, for example, U.S. Patent No. 5,514,548, U.S. Patent No. 5,837,500, U.S. Patent No. 5,871,907, U.S. Patent No. 5,885,793, U.S. Patent No. 5,969,108, U.S. Patent No. 6,225,447, U.S. Patent No. 6,291,650, U.S. Patent No. 6,492,160, EP585287, EP605522, EP616640, EP1024191, EP589877, EP774511, EP844306.A method useful for generating / selecting filamentous bacteriophage particles containing phagemid genomes that code for binding molecules with desired specificity is described. See, for example, EP774511, US5871907, US5969108, US6225447, US6291650, US6492160.

[0183] In some embodiments, the anti-HER2 antibody of the present technology is selected using the display of HER2 peptide on the surface of yeast host cells. A method useful for isolating scFv polypeptides by yeast surface display is described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0184] In some embodiments, the anti-HER2 antibody of the present technology is selected using ribosome display. A method useful for identifying ligands in a peptide library using ribosome display is described by Mattheakis et al., Proc.Natl.Acad.Sci.USA 91:9022-26, 1994, and Hanes et al., Proc.Natl.Acad.Sci.USA 94:4937-42, 1997.

[0185] In certain embodiments, the anti-HER2 antibody of the present technology is selected using tRNA display of HER2 peptide. A method useful for in vitro selection of ligands using tRNA display is described by Merryman et al., Chem. Biol., 9:741-46, 2002.

[0186] In one embodiment, the anti-HER2 antibody of the present technology is selected using RNA display.A method useful for selecting peptides and proteins using RNA display library is described by Roberts et al.Proc.Natl.Acad.Sci.USA,94:12297-302,1997 and Nemoto et al.,FEBS Lett.,414:405-8,1997.A method useful for selecting peptides and proteins using non-natural RNA display library is described by Frankel et al.,Curr.Opin.Struct.Biol.,13:506-12,2003.

[0187] In some embodiments, the anti-HER2 antibody of the present technology is expressed in the periplasm of Gram-negative bacteria and mixed with labeled HER2 protein. See WO02 / 34886. In clones expressing recombinant polypeptides with affinity to HER2 protein, the concentration of labeled HER2 protein bound to the anti-HER2 antibody increases, allowing the cells to be isolated from the rest of the library, as described in Harvey et al., Proc.Natl.Acad.Sci.22:9193-98 2004 and US Patent Publication No. 2004 / 0058403.

[0188] After selection of the desired anti-HER2 antibody, it is contemplated that the antibody can be produced in large quantities by any technique known to one of skill in the art, such as expression in prokaryotic or eukaryotic cells. For example, but not limited to, an anti-HER2 antibody, an anti-HER2 hybrid antibody or fragment, can be produced using conventional techniques to construct an expression vector encoding an antibody heavy chain in which the CDRs and, optionally, the minimal portion of the variable region framework required to retain the original species' antibody binding specificity (engineered according to the techniques described herein) are derived from a starting species antibody, and the remainder of the antibody is derived from a target species immunoglobulin that can be engineered as described herein, thereby producing a vector for expression of the hybrid antibody heavy chain.

[0189] Measurement of HER2 binding. In some embodiments, HER2 binding assay refers to an assay format in which HER2 protein and anti-HER2 antibody are mixed under suitable conditions to allow binding between HER2 protein and anti-HER2 antibody and to evaluate the amount of binding between HER2 protein and anti-HER2 antibody. The amount of binding is compared to a suitable control, which can be the amount of binding in the absence of HER2 protein, the amount of binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be evaluated by any suitable method. Binding assay methods include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for direct measurement of HER2 protein binding to anti-HER2 antibody are, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assay, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of the candidate anti-HER2 antibody is at least 1% greater than the binding observed in the absence of the candidate anti-HER2 antibody, the candidate anti-HER2 antibody is useful as an anti-HER2 antibody of the present technology.

[0190] Use of the anti-HER2 antibody of this technology General. The anti-HER2 antibodies of the present technology are useful in methods known in the art related to localizing and / or quantifying HER2 protein (e.g., for use in measuring the level of HER2 protein in an appropriate physiological sample, for use in diagnostic methods, for use in imaging of polypeptides, etc.). The antibodies of the present technology are useful for isolating HER2 protein by standard techniques such as affinity chromatography or immunoprecipitation. The anti-HER2 antibodies of the present technology can facilitate the purification of native immunoreactive HER2 protein from biological samples, e.g., mammalian serum or cells, as well as recombinantly produced immunoreactive HER2 protein expressed in a host system. Additionally, anti-HER2 antibodies can be used to detect immunoreactive HER2 protein (e.g., in plasma, cell lysates, or cell supernatants) and evaluate the amount and pattern of expression of immunoreactive polypeptides. The anti-HER2 antibodies of the present technology can be used diagnostically to monitor immunoreactive HER2 protein levels in tissues as part of a clinical trial procedure, e.g., to determine the effectiveness of a given treatment regimen. As described above, detection can be facilitated by coupling (i.e., physically linking) the anti-HER2 antibodies of the present technology to a detectable substance.

[0191] Detection of HER2 protein. An exemplary method for detecting the presence or absence of immunoreactive HER2 protein in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample with the anti-HER2 antibody of the present technology that can detect immunoreactive HER2 protein, thereby detecting the presence of immunoreactive HER2 protein in the biological sample. Detection can be achieved by means of a detectable label attached to the antibody.

[0192] The term "labeled" with respect to an anti-HER2 antibody is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0193] In some embodiments, the anti-HER2 antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, the anti-HER2 antibodies may be detectably labeled by covalent or non-covalent attachment of a chromogenic, enzymatic, radioisotope, isotope, fluorescent, toxin, chemiluminescent, nuclear magnetic resonance contrast agent, or other label.

[0194] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0195] Examples of suitable radioisotope labels include: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU, 90 Y,67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd and others are included. 111 In is an exemplary isotope for which in vivo imaging is used, as it is involved in the uptake of 125 I or 131 This is to avoid the problem of dehalogenation of I-labeled HER2-binding antibodies. In addition, this isotope has a more favorable gamma emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, 1-(P-isothiocyanatobenzyl)-DPTA coupled to monoclonal antibodies 111 In shows little uptake in non-tumor tissues, especially the liver, enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Contains Fe.

[0196] Examples of suitable fluorescent labels include: 152 Examples of suitable toxin labels include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0197] Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron.

[0198] The detection method of the present technology can be used to detect immunoreactive HER2 protein in biological samples in vitro as well as in vivo. In vitro techniques for detecting immunoreactive HER2 protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay, and immunofluorescence. In addition, in vivo techniques for detecting immunoreactive HER2 protein include introducing a labeled anti-HER2 antibody into a subject. For example, the anti-HER2 antibody can be labeled with a radioactive marker, and its presence and location in a subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains HER2 protein molecules from a test subject.

[0199] Immunoassays and imaging. The anti-HER2 antibodies of the present technology can be used to assay immunoreactive HER2 protein levels in biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be tested with classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, and radioisotopes or other radioactive agents, such as iodine ( 125 I, 121 I, 131 I), Carbon ( 14C), sulfur ( 35 S), tritium ( 3 H), Indium ( 112 In), and technetium ( 99 mTc), and fluorescent labels such as fluorescein, rhodamine, and green fluorescent protein (GFP), and biotin.

[0200] In addition to assaying immunoreactive HER2 protein levels in biological samples, the anti-HER2 antibody of the present technology can be used for in vivo imaging of HER2.Antibodies useful in this method include those that can be detected by X-ray radiography, NMR, or ESR.For X-ray radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not obviously harmful to subjects.For NMR and ESR, suitable markers include those that have detectable characteristic spins, such as deuterium, which can be incorporated into anti-HER2 antibody by nutrient labeling in related scFv clones.

[0201] Radioisotopes (e.g. 131 I, 112 In, 99 An anti-HER2 antibody labeled with a suitable detectable imaging moiety, such as mTc), a radiopaque substance, or a material detectable by nuclear magnetic resonance, is introduced into the subject (e.g., parenterally, subcutaneously, or intraperitoneally). It will be understood in the art that the size of the subject and the imaging system used will determine the amount of 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 usually 99 The range is about 5-20 millicuries of mTc. The labeled anti-HER2 antibody then accumulates at the site of cells that contain the specific target polypeptide. For example, the labeled anti-HER2 antibody of the present technology accumulates in the subject in cells and tissues where the HER2 protein is localized.

[0202] Thus, the present technology provides a method for diagnosing a medical condition, comprising: (a) assaying expression of immunoreactive HER2 protein by measuring binding of an anti-HER2 antibody of the present technology in an individual's cells or bodily fluids; and (b) comparing the amount of immunoreactive HER2 protein present in the sample with a standard reference, wherein an increase or decrease in the level of immunoreactive HER2 protein compared to the standard is indicative of the medical condition.

[0203] Affinity purification. The anti-HER2 antibody of the present technology can be used to purify immunoreactive HER2 protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins, and polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth.Enzym.34 Academic Press, NY (1974)).

[0204] The simplest way to bind antigen to an antibody support matrix is ​​to collect the beads in a column and pass the antigen solution through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended using a slow flow rate. The immobilized antibody captures the antigen as it passes through. Alternatively, the antigen can be contacted with the antibody support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or rocking the slurry, allowing maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed to a column for collection of the beads. The beads are washed using a suitable washing buffer, and then the pure or substantially pure antigen is eluted.

[0205] The antibody or polypeptide of interest can be conjugated to a solid support such as beads. In addition, the first solid support such as beads can also be conjugated to a second solid support, which can be a second bead or other support, if necessary, by any suitable means, including those disclosed herein for conjugating a polypeptide to a support. Thus, any of the conjugation methods and means disclosed herein for conjugating a polypeptide to a solid support can also be applied to conjugating a first support to a second support, and the first and second solid supports can be the same or different.

[0206] Linkers suitable for use in conjugating polypeptides to solid supports include various agents that can be crosslinkers and react with functional groups present on the surface of the support, or with the polypeptide, or both. Reagents useful as crosslinkers include homobifunctional and especially heterobifunctional reagents. Useful bifunctional crosslinkers include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Crosslinkers can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, photolabile crosslinkers such as 3-amino-(2-nitrophenyl)propionic acid can be used as a means to cleave the polypeptide from the solid support. (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and U.S. Pat. No. 5,643,722). Other crosslinking reagents are well known in the art. (See, e.g., Wong (1991), supra, and Hermanson (1996), supra).

[0207] The antibody or polypeptide can be immobilized on a solid support such as a bead through a covalent amide bond formed between a carboxyl functionalized bead and the amino terminus of the polypeptide, or vice versa through a covalent amide bond formed between an amino functionalized bead and the carboxyl terminus of the polypeptide. In addition, a bifunctional trityl linker can be attached to a support, for example, a 4-nitrophenyl active ester on a resin such as a Wang resin, through an amino or carboxyl group on the resin via an amino resin. Using the bifunctional trityl approach, the solid support can require treatment with a volatile acid such as formic acid or trifluoroacetic acid to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide can be deposited as a bead-free patch on the bottom of a well of the solid support or on the flat surface of the solid support. After addition of a matrix solution, the polypeptide can be desorbed by MS.

[0208] Hydrophobic trityl linkers can also be utilized as acid-labile linkers by cleaving the amino-linked trityl group from the polypeptide using a volatile acid or a suitable matrix solution, such as a matrix solution containing 3-HPA. The degree of acid lability can also be varied. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be changed to the appropriate p-substitution or more acid-labile tritylamine derivative of the polypeptide, i.e., trityl ether and tritylamine bonds can be created in the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, for example, by breaking the hydrophobic attraction, or by cleaving the trityl ether or tritylamine bond under acidic conditions, including typical MS conditions, where a matrix such as 3-HPA acts as an acid, if desired.

[0209] Orthogonally cleavable linkers can also be useful for binding a first solid support, e.g., beads, to a second solid support or for binding a polypeptide of interest to a solid support. Such linkers can be used to selectively cleave a first solid support, e.g., beads, from a second solid support without cleaving the polypeptide from the support, and then the polypeptide can be cleaved from the beads at a later time. For example, beads can be attached to a second solid support using a disulfide linker that can be cleaved using a reducing agent such as DTT, and an acid-cleavable bifunctional trityl group can be used to immobilize the polypeptide to the support. If desired, the bond of the polypeptide to the solid support can be cleaved first, e.g., leaving the bond between the first support and the second support intact. Trityl linkers can provide covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl group is easily cleaved in acidic conditions.

[0210] For example, beads can be attached to a second support via a linking group that can be selected to have a length and chemical nature that facilitates high density binding of beads to the solid support or high density binding of polypeptides to the beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing a variety of functional groups per attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, penta-erythrol, and tris-hydroxyaminomethane.

[0211] Non-covalent binding. The antibody or polypeptide can be conjugated to a solid support, or the first solid support can be conjugated to a second solid support by non-covalent interactions. For example, magnetic beads made of ferromagnetic materials can be magnetized and attracted to a magnetic solid support and released from the support by removing the magnetic field. Alternatively, the solid support can be provided with ionic or hydrophobic moieties, respectively, allowing the interaction of the ionic or hydrophobic moieties with a polypeptide, for example a polypeptide containing a bound trityl group, or a second solid support with hydrophobic properties.

[0212] A solid support can also be provided with a member of a specific binding pair and thus conjugated to a polypeptide or a second solid support containing the complementary binding moiety. For example, avidin or streptavidin coated beads can be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or a derivative of biotin such as iminobiotin.

[0213] It should be appreciated that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, biotin, for example, can be incorporated into either the polypeptide or the solid support, and conversely, avidin or other biotin-binding moieties can be incorporated into the support or the polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and the antigens with which they specifically interact, and other such pairs known to those of skill in the art.

[0214] A. Diagnostic Uses of the Anti-HER2 Antibody of the Present Technology General. The anti-HER2 antibodies of the present technology are useful in diagnostic methods. Thus, the present technology provides a method of using the antibodies in diagnosing HER2 activity in a subject. The anti-HER2 antibodies of the present technology can be selected to have any level of epitope binding specificity and very high binding affinity to the HER2 protein. In general, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed in the immunoassay to remove non-specifically bound materials without removing the target polypeptide. Thus, the anti-HER2 antibodies of the present technology useful in diagnostic assays are typically about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , or 10 12 M -1 Additionally, it is desirable for anti-HER2 antibodies used as diagnostic reagents to have sufficient kinetic on-rates to reach equilibrium in at least 12 hours, at least 5 hours, or at least 1 hour under standard conditions.

[0215] Anti-HER2 antibodies can be used to detect immunoreactive HER2 protein in a variety of standard assay formats, including immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988), U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,879,262, 4,034,074, 3,791,932, 3,817,837, 3,839,153, 3,850,752, 3, See, for example, Nos. 850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876. The biological sample can be obtained from any tissue or bodily fluid of the subject. In certain embodiments, the subject is at an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of HER2 protein in the sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsy tissue.

[0216] Immunometric or sandwich assay is one format in the diagnostic method of the present technology. See US Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assay uses one antibody, for example, an anti-HER2 antibody or a population of anti-HER2 antibodies immobilized on a solid phase, and another anti-HER2 antibody or a population of anti-HER2 antibodies in solution. Typically, the solution anti-HER2 antibody or a population of anti-HER2 antibodies is labeled. When an antibody population is used, the population can contain antibodies that bind to different epitope specificities within the target polypeptide. Thus, the same population can be used for both solid phase and solution antibodies. When an anti-HER2 monoclonal antibody is used, a first and a second HER2 monoclonal antibody with different binding specificities are used for the solid phase and the solution phase. The solid phase (also referred to as "capture") and solution (also referred to as "detection") antibodies can be contacted with the target antigen either sequentially or simultaneously. If the solid phase antibody is contacted first, the assay is referred to as a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the HER2 protein with the anti-HER2 antibody, the sample is typically incubated for a period that varies from about 10 minutes to about 24 hours, but is typically about 1 hour. A wash step is then performed to remove components of the sample that are not specifically bound to the anti-HER2 antibody used as a diagnostic reagent. If the solid phase and solution antibodies are bound in separate steps, washing can be performed after either or both binding steps. After washing, binding is typically quantified by detecting a label linked to the solid phase via binding of the labeled solution antibody. Typically, for a given pair of antibodies or population of antibodies, and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of the target antigen. The concentration of immunoreactive HER2 protein in the sample being tested is read by interpolation from the calibration curve (i.e., standard curve).The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of HER2 protein in the sample.

[0217] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, as well as particles such as agarose, dextran-based gels, dipsticks, particles, microparticles, magnetic particles, test tubes, microtiter wells, and SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway NJ). Immobilization can be by absorption or covalent binding. Optionally, the anti-HER2 antibody can be bound to a linker molecule such as biotin for binding to a surface-bound linker such as avidin.

[0218] In some embodiments, the present disclosure provides an anti-HER2 antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may include a radioactive or non-radioactive label, an imaging agent (such as magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label can be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. A diagnostic agent is a molecule that is administered conjugated to an antibody moiety, i.e., an antibody or antibody fragment, or subfragment, and is useful for diagnosing or detecting disease by indicating the location of cells containing the antigen.

[0219] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancement agents, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To load an antibody component with a radiometal or paramagnetic ion, it may be necessary to react it with a reagent that has a long tail to which multiple chelating groups are attached for binding the ion. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains having pendant groups to which chelating groups such as, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose can be attached. Chelates can be coupled to the antibodies of the present technology using standard chemistries. Chelates are usually linked to the antibody by a group that allows for the formation of bonds with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal crosslinking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes for radioimaging. The same chelates, when complexed with non-radioactive metals such as manganese, iron, and gadolinium, are useful for MRI when used with the HER2 antibodies of the present technology. Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N',N''-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are used with a variety of metals and radiometals, such as radionuclides of gallium, yttrium, and copper, respectively.About the pictures of the pictures in the pictures of the pictures. We also want to be able to do the same thing. OTA can be found in (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH。 2 、(ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH 2 、(iii)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH 2 、(iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 、(v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 、(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 、(vii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH 2 、(viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH 2 、(ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 、(x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH 2 、(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 、(xii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 、(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH 2 、(xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 、(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 , (xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH 2 , (xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 , (xviii)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH 2 , and (xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH 2 Includes:

[0220] For RAIT 223 Other ring-type chelates, such as macrocyclic polyethers, which are intended to stably bind nuclides such as Ra, are also contemplated.

[0221] B. Therapeutic Uses of the Anti-HER2 Antibodies of the Present Technology In one aspect, the immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology are useful for treating HER2-associated cancers. Examples of HER2-associated cancers include, but are not limited to, breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or any other neoplastic tissue that expresses the HER2 receptor. In some embodiments, the HER2-associated cancer is a solid tumor. Such treatments can be used in patients identified as having pathologically elevated levels of HER2 (e.g., diagnosed by the methods described herein) or diagnosed with a disease known to be associated with such pathological levels.

[0222] The compositions of the present technology may be used in conjunction with other therapeutic agents useful for treating HER2-associated cancer. For example, the antibody or antigen-binding fragment of the present technology may be administered separately, sequentially, or simultaneously with at least one additional therapeutic agent selected from the group consisting of alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, T cells, bisphosphonate therapeutic agents, and targeted biological therapeutic agents (e.g., therapeutic peptides described in US6306832, WO2012 / 007137, WO2005 / 000889, WO2010 / 096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapy agents include cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolamide, topotecan, vincristine, vinblastine, eribulin, mutagenesis, vasodilator, vasodilator serotonin ... In some embodiments, the therapeutic agent may be cyclosporine, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, or combinations thereof.

[0223] Additionally or alternatively, in some embodiments, the antibodies or antigen-binding fragments of the present technology may be administered separately, sequentially, or simultaneously with at least one additional immune modulating / stimulatory antibody, including, but not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-TIM3 antibodies, anti-4-1BB antibodies, anti-CD73 antibodies, anti-GITR antibodies, and anti-LAG-3 antibodies.

[0224] The compositions of the present technology can optionally be administered to a subject in need thereof as a single bolus. Alternatively, the administration regimen can include multiple administrations administered at various times after the appearance of a tumor.

[0225] Administration can be by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intracranially, intratumorally, intrathecally, or topically. Administration includes self-administration and administration by another. It is also understood that the various modes of treatment of the described medical conditions are intended to mean "substantially" including the total treatment, but less than the total treatment, where some biologically or medically relevant result is achieved.

[0226] In some embodiments, the antibodies of the present technology may comprise a pharmaceutical formulation and be administered to a subject in need thereof in one or more doses. Dosage regimens may be adjusted to provide the desired response (e.g., a therapeutic response).

[0227] Typically, an effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect is in the range of about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Typically, the dosage range is about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For administration of anti-HER2 antibodies, the dosage is in the range of about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg, of subject body weight every week, every two weeks, or every three weeks. For example, the dosage can be in the range of 1 mg / kg body weight, or 10 mg / kg body weight every week, every two weeks, or every three weeks, or 1 to 10 mg / kg every week, every two weeks, or every three weeks. In one embodiment, a single dosage of the antibody is in the range of 0.1 to 10,000 micrograms per kg of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimes involve administration once every two weeks, or once a month, or once every three to six months. The anti-HER2 antibody may be administered on multiple occasions. The interval between single administrations may be hourly, daily, weekly, monthly, or yearly. The interval may also be irregular as indicated by measuring blood levels of the antibody in the subject. In some methods, the dosage is adjusted to achieve a serum antibody concentration in the subject of about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL, or about 150 μg / mL to about 200 μg / mL. Alternatively, the anti-HER2 antibody may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the subject. Dosage and frequency can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. In therapeutic applications, relatively high dosages are sometimes required at relatively short intervals until the progression of the disease is reduced or terminated, or until the subject shows partial or complete improvement of the symptoms of the disease. The patient can then be administered a prophylactic regime.

[0228] In another aspect, the present disclosure provides a method for detecting cancer in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody (or an antigen-binding fragment thereof) of the present technology, the antibody being configured to localize to cancer cells expressing HER2 and labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value may be calculated by measuring the radioactivity level present in non-tumor (normal) tissues and calculating the average radioactivity level present in non-tumor (normal) tissues ± standard deviation. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0229] In some embodiments, the subject is diagnosed with or suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0230] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and67 Examples of alpha particle emitting isotopes include: 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, and 255 Examples of Auger emitters include Fm. 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, and 203 Pb. In some embodiments of the method, non-specific FcR dependent binding in normal tissues is eliminated or reduced (e.g., via N297A mutation in the Fc region resulting in aglycosylation). The therapeutic efficacy of such immunoconjugates can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the immunoconjugate has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0231] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-HER2 antibody described herein provides therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the anti-HER2 antibodies described herein can be assessed by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (the dose lethal to 50% of the population) or LD 100The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is not toxic for use in humans. The dosage of the anti-HER2 antibodies described herein is within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the subject's condition. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch.1 (1975).

[0232] Formulation of pharmaceutical compositions. In accordance with the methods of the present technology, the anti-HER2 antibodies can be incorporated into pharmaceutical compositions suitable for administration. The pharmaceutical composition generally comprises a recombinant or substantially purified antibody in a form suitable for administration to a subject, and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18). th (See, e.g., J. Med. Soc., ed., 1990). Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration. The pharmaceutical composition may further comprise an agent selected from the group consisting of an isotope, a dye, a chromagen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0233] The terms "pharmacologically acceptable" and "physiologically tolerable" and their grammatical variations are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to or in a subject without producing undesirable physiological effects that would prohibit administration of the composition. For example, "pharmaceutically acceptable excipients" generally refer to excipients that are safe, non-toxic, and useful in preparing desirable pharmaceutical compositions, including excipients that are acceptable in veterinary use as well as in human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. "Pharmaceutically acceptable salts and esters" refer to salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-HER2 antibody, e.g., C 1-6Alkyl esters are included. When two acidic groups are present, the pharma- ceutically acceptable or ester can be a monoacid-monosalt or ester, or a di-salt or ester; similarly, when more than two acidic groups are present, some or all of such groups can be salified or esterified. The anti-HER2 antibodies named in the present technology can exist in unsalted or unesterified form, or salified and / or esterified form, and the naming of such anti-HER2 antibodies is intended to include both the original (unsalted and unesterified) compound and its pharma- ceutical acceptable salts and esters. Also, certain embodiments of the present technology can exist in two or more stereoisomeric forms, and the naming of such anti-HER2 antibodies is intended to include all single stereoisomers and all mixtures of such stereoisomers (either racemic or otherwise). Those skilled in the art will have no difficulty in determining the appropriate timing, sequence, and dosages of administration of particular drugs and compositions of the present technology.

[0234] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and compounds for pharmaceutically active substances is well known to those skilled in the art. Except to the extent or extent that any conventional media or compound is incompatible with anti-HER2 antibody, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0235] The pharmaceutical composition of the present technology is formulated to be compatible with its intended route of administration.The anti-HER2 antibody composition of the present technology can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally, or intramuscularly, or as an inhalant.The anti-HER2 antibody can optionally be administered in combination with other agents that are at least partially effective in treating various HER2-related cancers.

[0236] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial compounds such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating compounds such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates, or phosphates, and compounds for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0237] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be desirable to include isotonic compounds in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition a compound which delays absorption, such as aluminum monostearate and gelatin.

[0238] Sterile injectable solution can be prepared by incorporating the anti-HER2 antibody of the present technology in the required amount in a suitable solvent with one or a combination of the components listed above, and optionally followed by sterilization by filtration.Generally, dispersion is prepared by incorporating the anti-HER2 antibody in a sterile vehicle containing a basic dispersion medium and other necessary components listed above.In the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and lyophilization, which produces a powder of active ingredient and any additional desired ingredients from the solution already sterile filtered.The antibody of the present technology can be administered in the form of depot injection or implant preparation, which can be formulated in such a way that it allows the active ingredient to be released continuously or pulsatilely.

[0239] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, anti-HER2 antibodies can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied to the oral cavity, swished, and expectorated or swallowed. Pharmaceutically compatible binding compounds and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a lubricant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring; or ingredients of a similar nature.

[0240] For administration by inhalation, the anti-HER2 antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0241] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, anti-HER2 antibody is formulated into ointments, salves, gels, or creams generally known in the art.

[0242] Anti-HER2 antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0243] In one embodiment, the anti-HER2 antibody is prepared with a carrier that protects the anti-HER2 antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes that target infected cells with monoclonal antibodies against viral antigens, can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0244] T cells bound to the HER2 multispecific binding molecules disclosed herein. Without being bound by any theory, it is believed that when the anti-CD3 multispecific binding molecules provided herein (e.g., HER2xCD3) are bound to T cells by the procedures described herein, the anti-CD3 scFv of the multispecific binding molecule binds to CD3 on the surface of the T cells. Without being bound by any theory, it is believed that the binding of the multispecific binding molecule to the T cells (i.e., the binding of the anti-CD3 scFv to CD3 expressed on the T cells) activates the T cells, thereby circumventing MHC restriction and allowing T cell receptor-based cytotoxicity to be redirected to the desired tumor target.

[0245] Therefore, the present disclosure also provides a T cell that is bound to the multispecific binding molecule of the present technology.In a specific embodiment, the T cell is non-covalently bound to the multispecific binding molecule.In a specific embodiment, the T cell is autologous to the subject to which the T cell is administered.In a specific embodiment, the T cell is allogeneic to the subject to which the T cell is administered.In a specific embodiment, the T cell is a human T cell.

[0246] In a specific embodiment, T cells that bind the multispecific binding molecules of the invention are used in accordance with the therapeutic methods described herein. In a specific embodiment, T cells that are bound to the multispecific binding molecules of the present disclosure are used as part of a combination therapy as described below.

[0247] In a specific embodiment involving a combination therapy with infusion of T cells, provided herein is a pharmaceutical composition comprising (a) a multispecific binding molecule as described herein, (b) T cells, and / or (c) a pharma- ceutical effective carrier. In a specific embodiment, the T cells are autologous to the subject to whom the T cells are administered. In a particular embodiment, the T cells are allogeneic to the subject to whom the T cells are administered. In a specific embodiment, the T cells are either bound to the multispecific binding molecule or are not bound to it. In a specific embodiment, the binding of the T cells to the multispecific binding molecule is non-covalent. In a specific embodiment, the T cells are human T cells. Methods that can be used to bind the multispecific binding molecule to the T cells are known in the art. See, for example, Lum et al., 2013, Biol Blood Marrow Transplant, 19:925-33; Janeway et al., Immunobiology: The Immune System in Health and Disease, 5 th edition, New York: Garland Science, Vaishampayan et al., 2015, Prostate Cancer, 2015:285193, and Stromnes et al., 2014, Immunol Rev. 257(1):145-164.

[0248] In a specific embodiment, administration of a multispecific binding molecule, a polynucleotide, vector, or cell encoding a multispecific binding molecule, or a pharmaceutical composition comprising a multispecific binding molecule as provided herein is performed after the patient has been treated with a T cell infusion. In a specific embodiment, the T cell infusion is performed with T cells that are autologous to the subject to whom the T cells are administered. In a specific embodiment, the T cell infusion is performed with T cells that are allogeneic to the subject to whom the T cells are administered. In a specific embodiment, the T cells are capable of binding to a molecule identical to the multispecific binding molecule described herein. In a specific embodiment, the binding of the T cells to a molecule identical to the multispecific binding molecule is non-covalent. In a specific embodiment, the T cells are human T cells.

[0249] C.Kit The present technology provides a kit for detecting HER2 and / or treating HER2-associated cancer, comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein), or a functional variant thereof (e.g., a substitution variant). Optionally, the above-mentioned components of the kit of the present technology are packaged in a suitable container and labeled for the diagnosis and / or treatment of HER2-associated cancer. The above-mentioned components may be stored in unit or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous, preferably sterile solutions, or as lyophilized, preferably sterile formulations for reconstitution. The kit may further comprise a second container holding a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharma- ceutically acceptable excipients of the pharmaceutical composition and saline. Additionally, the kit may comprise instructions for diluting the pharmaceutical composition, and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle). The kit may further include more containers containing pharma- ceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, media for one or more of the suitable hosts. The kit may optionally include instructions customarily included in commercial packaging of a therapeutic or diagnostic product, including, for example, information regarding the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings concerning the use of such therapeutic or diagnostic product.

[0250] The kit is useful for detecting the presence of immunoreactive HER2 protein in biological samples, such as body fluids, including but not limited to serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites, or blood, biopsy samples from body tissues. For example, the kit can include one or more humanized, chimeric, bispecific, or multispecific anti-HER2 antibodies (or antigen-binding fragments thereof) of the present technology that can bind to HER2 protein in biological samples, a means for determining the amount of HER2 protein in the sample, and a means for comparing the amount of immunoreactive HER2 protein in the sample with a standard. One or more of the anti-HER2 antibodies can be labeled. The kit components (e.g., reagents) can be packaged in a suitable container. The kit can further include instructions for using the kit to detect immunoreactive HER2 protein.

[0251] In an antibody-based kit, the kit can include, for example, 1) a first antibody of the present technology, e.g., a humanized, chimeric, bispecific, or multispecific HER2 antibody (or antigen-binding fragment thereof), bound to a solid support that binds to the HER2 protein, and, optionally, 2) a second, different antibody that binds either the HER2 protein or the first antibody, and is conjugated to a detectable label.

[0252] The kit may also include, for example, a buffer, a preservative, or a protein stabilizing agent. The kit may further include components necessary for detecting a detectable label, for example, an enzyme or a substrate. The kit may also include a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package, along with instructions for interpreting the results of the assay performed using the kit. The kit of the present technology may include written material on or in the kit container. The written material describes how to use the reagents included in the kit, for example, for detecting HER2 protein in vitro or in vivo, or for treating HER2-associated cancer in a subject in need of such treatment. In certain embodiments, the use of the reagents may follow the methods of the present technology. EXAMPLES

[0253] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples set forth the preparation, characterization, and use of exemplary anti-HER2 antibodies of the present technology.

[0254] Example 1: Introduction and Preliminary Experiments Early bispecific T cell engager (TCE) efforts have focused primarily on maximizing cytotoxic activity based on in vitro cell-based assays without predicting the biological consequences of high potency on cytokine release and T cell exhaustion or depletion in patients. These safety concerns were summarized in a recent FDA-sponsored workshop focused on safety evaluation of CD3 TCEs (Kamperschroer et al., J Immunotoxicol. 17(1):67-85 (2020)). Later generations of TCEs include Fc or other similar domains with the goal of extending half-life, but adverse events and clinical findings suggest that extending half-life with high potency TCEs may exacerbate serious adverse events related to neurotoxicity and cytokine release syndrome (CRS) (Vafa et al., Front. Oncol. 10:446 (2020)).

[0255] One potential strategy to overcome resistance to current targeted therapies is to harness the killing activity of T cells to defeat cancer by using BsAbs. These T cell-binding antibodies are designed to simultaneously bind to antigens and T cell activators such as co-receptor CD3 on tumor cells. BsAb binding of T cells activates T cells via CD3 binding, mediates tumor cell killing by forming a cytolytic synapse, and redirects killing activity toward antigen-expressing tumor cells in a major histocompatibility complex (MHC)-independent manner.

[0256] Trastuzumab x huOKT3 (ABP100) is a bispecific antibody developed to treat patients suffering from HER2+ type cancers, including but not limited to breast, gastric, and colon. This molecule binds to HER2+ tumors and brings natural immune T cells to the tumor to reduce it. This BsAb is based on two well-known molecules: trastuzumab, a fully humanized HER2-targeting immunoglobulin G1 (IgG1), and humanized muromonab-CD3 (huOKT3), a CD3-targeting IgG1. The main feature of ABP100 is that it was constructed using a symmetric bivalent BsAb platform IgG-[L]-scFv, in which a single-chain variable fragment (scFv) recognizing human CD3 is fused to the C-terminus of each anti-tumor IgG antibody light chain (Figure 4). The symmetric IgG-[L]-scFv design provides potent in vitro and in vivo antitumor activity against multiple tumor antigens (GD2, CD3, GPA33, and HER2), and recent reports have demonstrated that the IgG-L-scFv platform valence and spatial organization drive substantially more robust antitumor responses than many other BsAb formats (Santich et al., Sci. Transl. Med. 12:eaax1315 (2020), Figures 5A-5B). To reduce the risk of CRS, we silenced the Fc domain function of ABP100 to eliminate potential antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities by introducing two well-characterized mutations: N297A to remove glycosylation, and K322A to reduce complement activation.

[0257] Starting with the fully formatted ABP100 described above as a template, a dual strategy of CD3×HER2 bispecific antibody program was proposed to limit the toxic effects commonly associated with first generation TCE and CAR T cell therapeutics. The two products of this dual strategy are 1) ABP100a, a HER2 affinity-tuned BsAb with selective killing of HER2-high expressing cells designed for ex vivo loading of patient T cells for reinfusion, and 2) ABP102, a precisely redesigned BsAb for intravenous delivery with dual affinity-tuned arms for CD3 and HER2 binding (Figure 4).

[0258] ABP100a was constructed by replacing the huOKT3 portion of ABP100 with a novel humanized huSP34 CD3-binding arm and introducing a novel affinity-tuned HER2-binding arm (Figure 4). The humanized huSP34 CD3-binding arm is cross-reactive with non-human primate (Cyno) CD3, making it a suitable model for evaluating and predicting toxicity in humans. The affinity-tuned HER2-binding arm allows ABP100a to selectively kill HER2-high expressing cells while sparing cells expressing low endogenous levels of HER2. We explore the possibility of directly arming patient-derived activated T cells with ABP100a, a technique that has shown great potential in preclinical models (Figure 6). This approach is explored by using a relatively high affinity CD3-binding arm with a low off-rate, keeping in mind that CRS concerns should be minimal by directly arming pre-activated T cells.

[0259] In parallel, ABP102 was developed by additionally affinity tuning the CD3 T cell binding arm to select for CD3 affinity with potent killing of HER2-amplified cancer cells, which have limited T cell production of cytokines such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα). This approach raised the important understanding that tuning each binding arm of a BsAb individually does not necessarily improve the overall efficacy or safety profile of the molecule as a whole, and therefore the dual affinity-tuned BsAb was evaluated in the context of a complete BsAb with all modifications fully implemented. This dual modification of HER2 and CD3 binding affinity in ABP102 resulted in a novel BsAb for systemic delivery with selective killing of HER2-amplified cancer cells, predicted to exhibit reduced cytokine production for better safety in the clinic.

[0260] Example 2: Antibody humanization, affinity tuning, and characterization Antibody humanization and backmutation by rational design The complementarity determining regions (CDRs), hypervariable loops, and framework regions (FRs) of mouse anti-CD3 antibodies were analyzed within the variable sequences and identified by the KABAT delineation system. The CDRs of the mouse antibodies were identified by dividing the heavy chain variable domains (V H ) and the light chain variable domain (V L ) was used to directly graft the mouse antibody onto the human acceptor framework. Homology modeling was then performed to obtain the modeled structure of the mouse antibody and calculate the solvent accessible surface area of ​​the framework residues to identify the buried framework residues. The V H and V L Critical residues in the sequence were identified and backmutated. Finally, the grafted sequences were inspected for potential propensities for N-glycosylation sites, post-translational modifications, and unpaired cysteine ​​residues that may affect the binding activity of the grafted antibody.

[0261] NNK library design and construction, FASEBA screening, and affinity ranking First, affinity measurements of HER2 and parental BsAbs were determined using a surface plasmon resonance (SPR) biosensor, Biacore 8K (GE Healthcare, Marlborough, MA). Equilibrium dissociation constants (KD) were calculated from the ratio of kd to ka. To determine the contribution of specific residues to antibody affinity and expression, paratope mapping was performed by screening the NNK library. Briefly, the V of the parental antibody was H and V L The CDRs were searched using NCBI Ig-Blast (www.ncbi.nlm.nih.gov / projects / igblast / ), and CDRs were defined by the KABAT delineation system. All residues within the CDRs were defined and mutated by the NNK method. Each individual NNK library was generated residue-by-residue based on the FASEBA platform with a theoretical diversity of 20. More than 48 clones were randomly selected from each NNK library for expression in E. coli in 96-deep-well plates. All clones were sequenced, and unique clones were selected. For evaluation of expression and binding specificity, the crude selected proteins secreted in the medium were analyzed by ELISA against bovine serum albumin (BSA), and human and cynomolgus monkey antigen proteins, respectively. "Beneficial mutants" that reduced antibody affinity without compromising antibody expression were confirmed by GenScript's FAst Screening for Expression level, Biophysical properties, and Affinities (FASEBA) platform through screening and affinity ranking.

[0262] Selection and synthesis of affinity-tuned bispecific antibodies From the mutant library, a series of antibodies with different affinities to human and cynomolgus HER2 were constructed in a bispecific antibody format by subcloning into an expression vector for expression in Expi-CHO-S cells. The bispecific antibodies were purified using a Protein A column.

[0263] The binding between the parental and affinity-tuned bispecific antibodies and human and cynomolgus monkey target proteins (HER2 and CD3) was verified by ELISA, and the kinetics of the interaction between the antigen and the bispecific antibodies was studied using Biacore 8K, see Figures 10, 12, and 17A-17B.

[0264] Example 3: In vitro characterization of anti-HER2 antibodies of the present technology Binding of T cells and target cells by flow cytometry Purified CD3+ T cells and total human PBMCs were bulk stained with live / dead stain and then incubated with various concentrations of bispecific antibodies to assess binding. Controls included the appropriate monoclonal antibody (humanized SP34-hIgG1) and an isotype control (hIgG1). A secondary antibody, anti-human IgG Fc specific PE conjugate, was used for detection.

[0265] The cell lines were grown according to the cell line supplier's recommendations, followed by Accutase treatment to detach the cells. The cells were resuspended in medium, washed, and bulk stained with live / dead stain, followed by staining with the bispecific antibody and a control monoclonal antibody (Trastuzumab hIgG1). A secondary antibody, anti-human IgG Fc specific PE conjugate, was used for detection. Data collection was performed using the BD FACSCelesta HTS platform, and data analysis was performed with FlowJo software. Figure 7A shows that the anti-HER2xCD3 BsAb of the present technology shows reduced binding to T cells compared to the humanized SP34-hIgG1 control.

[0266] T-cell dependent cytotoxicity (TDCC) in vitro assay To assess biological activity, we performed established in-house TDCC assays (Cell Titer Glo 2.0 (Promega) and Caspase 3 / 7 Green Apoptosis Assay (IncuCyte)) using effector human CD3+ T cells to test bispecific HER2xCD3 antibodies with different CD3 affinities against different target cell lines. Anti-HER2xCD3 BsAbs showed selective killing of high HER2 expressing target cells (SKBR-3 cell line) while sparing target cells expressing near endogenous HER2 levels (MCF-7 cell line) (Figures 7B-7C, and Figures 9A-9B). In this experiment, ABP100s.10.0 had WT-trastuzumab-like affinity, and ABP100s.10.5 and ABP100s.10.6 had lower HER2 affinity (-64-fold and -108-fold, respectively), although all bispecific constructs contained the same high affinity CD3 scFv arm. Notably, the ABP100s.10.5 and ABP100s.10.6 constructs with low HER2 affinity enabled differential and selective killing of low HER2 expressing target cells (MCF-7) in contrast to the antibody with WT-like affinity (ABP100s.10.0).

[0267] In contrast, the trastuzumab x huOKT3 parent antibody showed non-selective killing of both SKBR-3 and MCF-7 cell lines (see Figures 8A-8B). It is observed that killing of HER2-high cell lines is achieved at lower concentrations of trastuzumab x huOKT3 than HER2-low cell lines, but killing of the HER2-low cell lines cannot be completely eliminated (Figure 8B).

[0268] Readout of the CTG2.0 assay was performed on a standard luminescence 96-well plate reader, Spectramax iD3, using white well plates. The IncuCyte S3 platform and software for image-based analysis allowed determination of the total green area / image from the caspase 3 / 7 green reagent using black clear-bottom 96-well tissue culture treated plates. The assay provided an estimated relative frequency of apoptotic cells within each well.

[0269] These results demonstrate that reducing CD3 scFv affinity has minimal impact on killing of high HER2 expressing target cells (SKBR-3) while showing no killing of low HER2 expressing cells (MCF-7).

[0270] The effect of the different Her2 and CD3 affinities of each of the HER2xCD3 BsAbs of the present technology on antibody binding, T cell activation, T cell-mediated killing, and cytokine production was examined in the presence or absence of tumor cell lines expressing different levels of Her2. SKBR3 and HCC1954 are selected because they represent high-density Her-2 expressing cell lines with similar Her2 expression levels (Ram et al., MAbs. 2014 6(5):1211-1219), which corresponds to a HercepTest result of 3+ for SKBR3, and MCF-7 and HT55 are selected because they represent low-density Her2 expressing cells (MCF-7 represents a HercepTest score of 0-1+) with similar expression levels to each other and to endogenous non-cancer cells expressing Her2 (Rhodes et al., Am J Clin Pathol 2002 118(3):408-17, Subik et al., Breast Cancer (Auckl). 2010; 4: 35-41, Slaga et al., Sci Transl Med 2018 Oct 17; 10 (463): eaat5775). All p values ​​from the following data for NFAT T cell receptor activation assays, cytotoxicity assays, and cytokine assays were derived from two-way ANOVA with Tukey's multiple comparisons performed on Graphpad Prism Version 9.4.0.

[0271] CD3 / TCR NFAT T cell activation reporter assay: To evaluate T cell activation by bispecific and monoclonal antibodies, the T cell activation bioassay kit (Promega J1621 / J1625) was used with TCR / CD3 Jurkat effector cells (NFAT reporter) and detected using Bio-Glo luciferase assay system (Promega G7941). Briefly, white bottom / chimney (white) TC treated plates (Corning 3917) were used to plate 40,000 target cells (Her2-high: SK-BR-3, HCC1954; Her2-low: MCF-7, HT55) overnight in 100 μL medium and were also used in the condition without target cells. The assay was then performed according to the detailed instructions provided with the assay kit, with a 7 hour incubation followed by luminescence readout.

[0272] In general, higher activation was seen in target cells expressing higher amounts of Her2 (Figures 13A-13B). Notably, 10.5.1 and 10.6.1 were only slightly decreased from the parent construct (10.0) in Her2-high targets (SK-BR-3, HCC1954), with all activation readouts for all constructs being within 300,000-600,000 RLU at the highest dose examined (40 nM) (Figures 13A-13B). In Her2-low targets, the 10.0 construct showed similar, if slightly lower, activation (~300,000 RLU at 40 nM in both MCF-7 and HT-55 cell lines), whereas the 10.5 and 10.6 constructs showed significantly decreased activation (~200,000 RLU) compared to 10.0 in both cell lines (p<0.0001 for both comparisons) (Figures 13C-13D). Furthermore, the 10.5.1 and 10.6.1 constructs demonstrated significantly lower activation (approximately 100,000 RLU) at 40 nM in Her2-low target cells (HT55, MCF-7; Figures 13C-13D) than 10.5 and 10.6 (p<0.0001 for both comparisons), which was not statistically different from the isotype control and similar to the background activation seen in the absence of target cells (Figure 13E). Taken together, these data are consistent with the lack of 10.5.1 and 10.6.1 induced T cell activation in low Her2 expressing cells.

[0273] T-cell dependent cytotoxicity (TDCC): To assess the in vitro functional ability of bispecific antibodies to mediate T-cell mediated killing of Her2 expressing target cells, T-cell dependent cytotoxicity (TDCC) assays were performed with CD3+ T cells. Target cells were plated in white bottom / chimney tissue culture treated plates (Corning 3917) at 10,000 cells / well in 100 μL medium and incubated overnight (Her2-high: SK-BR-3, HCC1954; Her2-low: MCF-7, HT55). Bispecific antibodies were diluted in RPMI1640 / 10% heat inactivated FBS (range: 30, 0.3, 0.003, 0.00003 nM final concentration). Culture medium was removed from the target cells and bispecific antibodies were added at 100 μL, followed by purified human cryopreserved T cells (StemCellTechnologies 70024) at an effector:target ratio of 5 to 1 (T cells:target cells). Detection was performed at 40 hours using CellTiterGlo2.0 (Promega) and luminescence detection on a SpectraMax iD3 plate reader. Results in the figures shown are representative of experiments performed with three different donor CD3+ T cell samples.

[0274] In SK-BR-3 target cells, 10.5.1 and 10.6.1 showed only a small but significant (10.0 vs. 10.5.1: p=0.0042; 10.0 vs. 10.6.1: p=0.0002) decrease in killing compared to the parental construct (10.0) at 40 nM (Figure 14A). In the presence of SKBR3 cells, there was no statistically significant difference between the Her2 mutated, CD3 non-mutated 10.5 and 10.6 and the parental 10.0 (Figure 14A), consistent with the intended targeting of high Her2 expressing cells by 10.5.1 and 10.6.1. In HCC1954 target cells, killing was significantly reduced at 10.5 and 10.6 compared to the parental 10.0 (Figure 14B), but this reduction was modest (approximately 10-20% reduction from 10.0 killing levels in SKBR3 and HCC1954) compared to the reduction seen in Her2-low expressing cells (approximately 67-75% reduction from 10.0 killing levels in MCF-7 and HT55). In Her2-low (HT55, MCF-7) target cells at 40 nM, there was no significant difference between isotype control killing levels and those of 10.5, 10.6, 10.5.1, and 10.6.1 (Figures 14C-14D) (except for isotype vs. 10.5 in MCF-7 cell line, Figure 14C). In contrast, at the same dose in both MCF-7 and HT-55 cell lines, all of these constructs showed significantly reduced killing compared to the 10.0 parent (p<0.0001: 10.0 vs. 10.5, 10.0 vs. 10.6, 10.0 vs. 10.5.1, and 10.0 vs. 10.6.1) (Figures 14C-14D), indicating little or no killing of low Her2 expressing cell lines by the Her2 and CD3 mutant constructs. The significant increase in killing attributable to 10.5 in the MCF-7 cell line was consistently observed across multiple experiments, indicating that this may be due to the slightly stronger affinity of 10.5 (KD is approximately 45 nM) for Her2 than 10.6 (KD is approximately 67 nM).

[0275] Multiplex cytokine detection assay and associated cytotoxicity in Her2-high and Her2-low target cell lines in human PBMCs. To analyze cytokine release mediated by bispecific antibody constructs, human PBMCs were contacted with soluble antibodies in SKBR-3 (Her2-high) and MCF-7 (Her2-low) as well as "no target cells" conditions. Monoclonal anti-CD3 / anti-CD28 antibodies served as controls. Briefly, SKBR-3 (Her2-high) and MCF-7 (Her2-low) target cell lines were plated the night before the assay. Bispecific antibodies were diluted in RPMI1640 / 10% heat-inactivated FBS (range: 30, 0.3, 0.003, 0.00003 nM final concentration). Culture medium was removed from the target cells and bispecific antibodies were added in 100 μL, followed by incubation with human PBMCs (1 donor, Stem Cell Technologies PBMCs approximately 5 × 10) in white-chimney / bottom plates in RPMI / 10% HI FBS. 7 Cells / vial) (10:1 E:T ratio; 100,000 PBMCs:10,000 target cells) were added to the wells. Culture supernatants were harvested at 24 hours and frozen at -80°C for multiplex bead-based cytokine release assays (R&D Systems Human High Sensitivity Cytokine Base Kit B: IFN-γ, IL-2, TNF-α, IL-6, GM-CSF) with signal detection using Magpix (Luminex) and quantification of cytokines in picograms / mL compared to standard wells using Luminex xMAP software. CellTiterGlo2.0 (Promega) was used to develop an assay for TDCC cytotoxicity % evaluation with luminescence detection on a SpectraMax iD3 plate reader. Results in the figures shown represent experiments performed with three different donor PBMC samples. Cytotoxicity and cytokine release results were compiled in Excel and graphed in GraphPAD PRISM.

[0276] Because excessive cytokine production associated with T cell engager administration may result in primary toxicity of T cell engagers, cytokine release syndrome (CRS), we examined the effect of weakened affinity of Her2 and CD3 on cytokine production in the presence of tumor cell lines expressing high (SKBR3) or low (MCF-7) Her2 levels and human PBMCs. The 10.0 parent, the Her2 reduced affinity 10.5 and 10.6 constructs, and the Her2 and CD3 weakened affinity 10.5.1 and 10.6.1 constructs all stimulated cytokine production that was significantly higher than the isotype control (a control representing background cytokine production) in the presence of SKBR3 cells (Figures 15A-15D). However, in the presence of MCF-7 cells, only 10.0 showed a significant increase in cytokine production compared to the isotype control, indicating a reduced ability of the Her2 and / or CD3 weakened constructs to stimulate cytokine production (Figures 15E-15H). At 30 nM, all affinity-weakened constructs showed significantly reduced production of IL-2, IFN-γ, and TNF-α in the presence of MCF-7 cells (p<0.0001: 10.0 vs. 10.5, 10.0 vs. 10.6, 10.0 vs. 10.5.1, and 10.0 vs. 10.6.1). Importantly, production of IL-6, a cytokine that is a key mediator of immune-modulator-associated CRS (Morris et al., Nat Rev Immunol 2022;22(2):85-96), was significantly reduced in the Her2 and CD3 doubly attenuated 10.5.1 and 10.6.1 constructs compared to 10.0 in the presence of MCF-7 cells (p=0.0432 and p=0.029, respectively) (Figure 15F). The lack of significant differences in IL-6 production observed with the Her2 affinity weakened 10.5 and 10.6 constructs compared to the 10.0 parent underscores the importance of weakening both Her2 and CD3 affinity to achieve reduced IL-6 levels. The "no target cells" condition demonstrated that PBMCs bearing bispecific antibodies alone do not promote cytokine release. For SKBR-3 (Her2-high) target cells, cytotoxicity was comparable for 10.5.1 and 10.6.1 compared to the parent construct (10.0), with only minor differences observed.

[0277] These results demonstrate that these agents are useful in reducing the incidence of CRS when treating patients with high Her2-expressing (e.g., 3+ Herceptest score) cancers by selectively killing and producing cytokines in response to high Her2-expressing cells while sparing low Her2-expressing tissues with endogenous, non-cancerous tissue levels of Her2 (0-1+ Herceptest score), thus reducing on-target, normal tissue toxicity (off-tumor) in the clinic.

[0278] Flow cytometric analysis of bispecific antibody binding to activated T cells and Her-2 expressing target cells. To assess flow cytometric binding of bispecific antibodies to activated T cells, human PBMCs (StemCell Technologies 70025.2) were stimulated using an OKT3 / IL-2 stimulation protocol for 12 days. Briefly, PBMCs were activated with 100 IU / mL recombinant human IL-2 (Stemcell Technologies, Cat. No. 78145.1) and 20 ng / mL OKT3 (Biolegend, mouse IgG2a, Cat. No. 317326) in soluble format for 3 days, after which the cells were cultured at 1×10 in RPMI1640 / 10% FBS. 6Activated T cells were expanded / maintained by using only fresh media and IL-2, by normalizing to 10 cells / mL. Activated T cells were cryopreserved and stored in a liquid nitrogen freezer. On the day of the assay, T cells were thawed, washed, and subsequently stained with diluted bispecific antibodies (initial working stock was 240 nM (2x) with a final concentration of 120 nM, serially diluted 1:10 in FACS buffer for a total of 7 serial dilutions). Cells were stained with primary antibodies for 30 min at 4°C in cold PBS / 1% BSA, followed by washing and adding secondary antibody (anti-human IgG-PE, Thermo Fisher Scientific) at 1:250 in cold PBS / 1% BSA for 30 min at 4°C. After a washing step, cells were resuspended in 150 μL PBS / 1% BSA and PE signal was detected on a FACSCelesta HTS system with 96-well V-bottom plates. Prior to the assay, live / dead was assessed using the BV421 channel (Zombie violet, Thermo Fisher Scientific) or Trypan Blue.

[0279] To assess flow cytometric binding of bispecific antibodies to cell lines including Her2-high (SKBR-3, SKOV-3) and Her2-low (MCF-7, HT55), cells were grown to 70-80% confluence in their respective culture media according to ATCC protocols. Cell lines were treated with Accutase to preserve cell surface epitopes for flow cytometry. Cells were resuspended in 5 mL PBS (without BSA) and stained with Live / Dead Zombie dye (Biolegend) at 1:1000 dilution for 20 min at room temperature. Cells were then washed, normalized, and added at 100,000 cells / well onto a 96-well V-bottom plate and stained with primary antibodies for 30 min at 4°C in cold PBS / 1% BSA. This was followed by washing and addition of secondary antibody (anti-human IgG-PE, Thermo Fisher Scientific) at 1:250 in cold PBS / 1% BSA for 30 min at 4°C. After a washing step, cells were resuspended in 150 μL PBS / 1% BSA and PE signals were detected on a FACSCelesta HTS system with 96-well V-bottom plates. Prior to the assay, live / dead was assessed using the BV421 channel (Zombie violet, Thermo Fisher Scientific) or Trypan Blue. Analysis was performed with FlowJo software to obtain median PE (anti-human IgG-PE) values ​​(MFI) and data were then organized and plotted with Microsoft Excel and GraphPAD Prism software from GraphPAD Prism software.

[0280] Results Activated T cell binding was reduced for 10.5.1 and 10.6.1 compared to the parent construct (10.0) (Figure 16E). The reduced T cell binding observed with 10.5.1 and 10.6.1 may be at least partially due to having a CD3 arm that exhibits reduced affinity.

[0281] Target cell line binding to Her2-high (SK-BR-3, SK-OV-3) cell lines was slightly reduced for the 10.5.1 and 10.6.1 constructs compared to the parental construct (10.0) (MFI reduced by about 33% for both 10.5.1 and 10.6.1 compared to 10.0 at a concentration of about 100 nM) (Figures 16A-16B). Target cell line binding to Her2-low (MCF-7, HT55) target cell lines was significantly reduced for the 10.5.1 and 10.6.1 constructs compared to the parental construct (10.0) (MFI reduced by about 84% for both constructs compared to 10.0 at a concentration of about 100 nM) (Figures 16C-16D). Taken together, these results demonstrate that the doubly affinity-weakened HER2xCD3 constructs (10.5.1, 10.6.1) have properties that contribute to selectivity for Her2-high target cell lines and maintain the ability to bind CD3+ T cells to enhance cytotoxicity. Overall, the affinity-weakened constructs 10.5.1 and 10.6.1 show reduced cytotoxicity and cytokine release on Her2-low target cells when compared to the parental constructs.

[0282] In summary, HER2 and CD3 reduced affinity bispecific antibody constructs described in Figure 10 that have comparable affinity to 10.5.1 and 10.6.1 are expected to behave similarly to 10.5.1 and 10.6.1 in at least one of NFAT activation, TDCC, and / or FACS.

[0283] Example 4: In vivo efficacy of the HER2 affinity-tuned bispecific antibodies of the present technology To evaluate the in vivo efficacy of the affinity-tuned bispecific antibodies of the present technology, human tumor and peripheral blood mononuclear cell (PBMC) co-xenografts in mice are performed. Given that low affinity binding to HER2 results in selective killing of high HER2-expressing tumors in vitro, sparing low HER2-expressing tumors (used as a surrogate for endogenous HER2 expression in normal non-cancerous tissues) (Figure 14), we intend to evaluate whether this selective killing is also observed in vivo. NSG mice are subcutaneously implanted with a mixture of 1-5 million HER2-expressing tumors and human PBMCs (1:2 or 1:3 PBMC:tumor (E:T) ratios). High Her2 expressing tumor cell lines, such as HCC1954 cells (representing expression levels in tumors expected in the clinic (e.g., those with a 2+ or 3+ HecepTest score), or low Her-2 expressing tumor cell lines, such as HT55 cells (representing Her2 levels in non-cancerous tissues), are implanted into fully immunocompromised mice, such as Nod-Scid-Gamma (NSG) mice or equivalent. Following implantation of tumor cells and PBMCs, mice are administered different dose levels (ranging from at least 5 mg / kg to 0.005 mg / kg inclusive) of various Her2 and CD3 affinity attenuated constructs. Doses are administered parenterally (e.g., iv or ip) once or more per week for one or more weeks. Tumor volumes are measured over the duration of the study.

[0284] The 10.0 parent is expected to non-selectively inhibit the growth of both Her2 high and Her2 low expressing tumor cells at doses where 10.5.1 and 10.6.1 selectively inhibit the growth of Her2 high tumors, but have little or no growth inhibitory activity against Her2 low expressing tumors. Also, levels of cytokines such as IL-2, IL-6, IFN-γ, and TNF-α are expected to be reduced in animals receiving the 10.5.1 and 10.6.1 clones compared to the 10.0 parent.

[0285] Thus, the immunoglobulin-related compositions of the present technology are useful for treating HER2-associated cancer in a subject in need thereof.

[0286] Equivalent The present technology should not be limited with respect to the specific embodiments described in this application, and the application is intended as one example of each aspect of the present technology. Many modifications and variations of the present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the present technology. It is to be understood that the present technology is not limited to specific methods, reagents, compounds compositions, or biological systems that may, of course, vary. It is also to be understood that the terminology used herein is merely for the purpose of describing specific embodiments, and is not intended to be limiting.

[0287] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0288] As will be understood by those skilled in the art, for any and all purposes, all ranges disclosed herein, particularly in terms of providing a written description, also encompass any and all possible subranges and combinations thereof. Any recited range can be readily recognized as fully indicating and allowing the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Also, as will be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than" refers to a range that includes the recited number and can subsequently be divided into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual number. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5, etc. cells, and so on.

[0289] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, unless they conflict with the explicit teachings of this specification.

Claims

1. Heavy chain immunoglobulin variable domain (V Ｈ ) and a light chain immunoglobulin variable domain (V Ｌ and wherein: (a)(i) the V Ｈ is V of SEQ ID NO: 1 Ｈ - CDR1 sequence, V of SEQ ID NO: 2 or SEQ ID NO: 7 Ｈ - CDR2 sequence and V of SEQ ID NO: 3 or SEQ ID NO: 8 Ｈ - comprises a CDR3 sequence, or (ii) the V Ｈ is V of SEQ ID NO: 1 Ｈ - CDR1 sequence, V of SEQ ID NO: 7 Ｈ - CDR2 sequence and V of SEQ ID NO: 3 or SEQ ID NO: 8 Ｈ - comprises a CDR3 sequence, and / or (b)(i) said V L comprises the V L -CDR1 sequence of SEQ ID NO:4 or SEQ ID NO:9, the V L -CDR2 sequence of SEQ ID NO:11 or SEQ ID NO:10, and the V L -CDR3 sequence of SEQ ID NO:6 or SEQ ID NO:12; or (ii) the V Ｌ V of SEQ ID NO: 9 Ｌ - CDR1 sequence, V of SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 Ｌ - CDR2 sequence and V of SEQ ID NO: 6 or SEQ ID NO: 12 Ｌ - comprises a CDR3 sequence, or (iii) the V Ｌ is V of SEQ ID NO: 4 or SEQ ID NO: 9 Ｌ - CDR1 sequence, V of SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11 Ｌ - CDR2 sequence, and V of SEQ ID NO: 12 Ｌ - an antibody or antigen-binding fragment thereof, comprising a CDR3 sequence.

2. (a) The V Ｈ comprises an amino acid sequence selected from any one of SEQ ID NOs: 15, 13, or 17; and / or (b) said V Ｌ The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 19, 14, 16, 18, or 20.

3. The VH and VL amino acid sequences are each (a) SEQ ID NOs: 15 and 19; (b) SEQ ID NOs: 13 and 14; (c) SEQ ID NOs: 15 and 16; (d) SEQ ID NOs: 17 and 14; (e) SEQ ID NOs: 15 and 18, and (f) The antibody or antigen-binding fragment thereof of claim 1, selected from the group consisting of SEQ ID NOs: 15 and 20.

4. an antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently bonded to each other, the second and third polypeptide chains are covalently bonded to each other, and the third and fourth polypeptide chains are covalently bonded to each other; (a) each of the first polypeptide chain and the fourth polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin; and (iii) (GGGGS) ３ a flexible peptide linker comprising the amino acid sequence (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope, and the light chain variable domain and the heavy chain variable domain of the second immunoglobulin have the amino acid sequence (GGGGS) ６ and a light chain variable domain or a heavy chain variable domain linked together via a flexible peptide linker comprising: (b) each of the second polypeptide chain and the third polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; (ii) a heavy chain constant domain of the first immunoglobulin; An antibody, wherein the heavy chain variable domain of the first immunoglobulin or the heavy chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 15, 13, or 17, and / or the light chain variable domain of the first immunoglobulin or the light chain variable domain of the second immunoglobulin comprises any one of SEQ ID NOs: 19, 14, 16, 18, or 20.

5. An antibody or antigen-binding fragment comprising a heavy chain (HC) and a light chain (LC), each of which: (a) SEQ ID NOs: 21 and 46; (b) SEQ ID NOs: 21 and 22; (c) SEQ ID NOs: 21 and 23; (d) SEQ ID NOs: 21 and 24; (e) SEQ ID NOs: 21 and 25; (f) SEQ ID NOs: 21 and 26; (g) SEQ ID NOs: 21 and 27; (h) SEQ ID NOs: 21 and 28; (i) SEQ ID NOs: 21 and 29; (j) SEQ ID NOs: 21 and 30; (k) SEQ ID NOs: 21 and 31; (l) SEQ ID NOs: 21 and 32; (m) SEQ ID NOs: 21 and 33; (n) SEQ ID NOs: 34 and 33; (o) SEQ ID NOs: 21 and 35; (p) SEQ ID NOs: 36 and 33; (q) SEQ ID NOs: 21 and 37; (r) SEQ ID NOs: 21 and 38; (s) SEQ ID NOs: 21 and 39; (t) SEQ ID NOs: 21 and 40; (u) SEQ ID NOs: 21 and 41; (v) SEQ ID NOs: 21 and 42; (w) SEQ ID NOs: 21 and 43; (x) SEQ ID NOs: 21 and 44; (y) SEQ ID NOs: 21 and 45; (z) SEQ ID NOs: 21 and 47; (aa) SEQ ID NOs: 21 and 48, (bb) SEQ ID NOs: 21 and 49; (cc) SEQ ID NOs: 21 and 50; (dd) SEQ ID NOs: 21 and 51; (ee) SEQ ID NOs: 21 and 52; (ff) SEQ ID NOs: 21 and 53; (gg) SEQ ID NOs: 21 and 54; (hh) SEQ ID NOs: 21 and 55; (ii) SEQ ID NOs: 21 and 56; (jj) SEQ ID NOs: 21 and 57, (kk) SEQ ID NOs: 21 and 58; (ll) SEQ ID NOs: 21 and 59, (mm) SEQ ID NOs: 21 and 60; (nn) SEQ ID NOs: 21 and 61; (oo) SEQ ID NOs: 21 and 62, (pp) SEQ ID NOs: 21 and 63; (qq) SEQ ID NOs: 21 and 64, (rr) SEQ ID NOs: 21 and 65, (ss) SEQ ID NOs: 21 and 66, (tt) SEQ ID NOs: 21 and 67; (uu) SEQ ID NOs: 21 and 68, (vv) SEQ ID NOs: 21 and 69, (ww) SEQ ID NOs: 21 and 70; (xx) SEQ ID NOs: 21 and 71, (yy) SEQ ID NOs: 21 and 72, and (zz) An antibody or antigen-binding fragment selected from the group consisting of SEQ ID NOs: 21 and 85. (a) the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE; or (b) the antigen-binding fragment is selected from the group consisting of Fab, F(ab') 2 , Fab', scF v , and F v ; The antibody or antigen-binding fragment of any one of claims 1 to 5. (a) an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, and K322A; or (b) an IgG4 constant region containing the S228P mutation; The antibody or antigen-binding fragment of claim 6, comprising: (a) the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody; and / or (b) the antibody lacks α-1,6-fucose modifications; The antibody or antigen-binding fragment of any one of claims 1 to 7.

9. 9. The antibody or antigen-binding fragment of claim 8, wherein the multispecific antibody or antigen-binding fragment binds to T cells, B cells, myeloid cells, plasma cells, or mast cells.

10. 10. The antibody or antigen-binding fragment of claim 8 or 9, wherein the multispecific antibody or antigen-binding fragment binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

11. The antibody or antigen-binding fragment of any one of claims 8 to 10, wherein the antibody is a multispecific antibody, and the multispecific antibody or antigen-binding fragment binds to T cells and / or CD3.

12. A recombinant nucleic acid sequence encoding the antibody or antigen-binding fragment of any one of claims 1 to 11.

13. A host cell or vector comprising the recombinant nucleic acid sequence of claim 12.

14. A T cell armed ex vivo with the multispecific antibody or antigen-binding fragment of claim 11.

15. 12. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 11 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

16. The pharmaceutical composition of claim 15, further comprising an agent selected from the group consisting of an isotope, a dye, a chromagen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, and any combination thereof.

17. 17. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 11, the T cell of claim 14, or the pharmaceutical composition of claim 15 or 16, for treating cancer in a subject in need thereof, wherein the antibody or antigen-binding fragment specifically binds to HER2.

18. 18. The composition or pharmaceutical composition of claim 17, wherein the cancer is a solid tumor.

19. 19. The composition or pharmaceutical composition of claim 17 or 18, wherein the cancer is breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, or neuroblastoma.

20. The composition or pharmaceutical composition according to any one of claims 17 to 19, wherein the composition or pharmaceutical composition is administered to the subject in combination with an additional therapeutic agent, either separately, sequentially or simultaneously.

21. 21. The composition or pharmaceutical composition of claim 20, wherein the additional therapeutic agent is one or more of an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, a T-cell, and a bisphosphonate therapy.

22. 22. The composition or pharmaceutical composition of claim 21, wherein the additional therapeutic agent is an immunomodulatory / stimulatory antibody, and the immunomodulatory / stimulatory antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4-1BB antibody, an anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody.

23. 12. A composition for use in a method for in vivo detection of cancer in a subject, said composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, said method comprising: (a) administering to the subject the composition, wherein the antibody or antigen-binding fragment is configured to localize to cancer cells that express HER2 and is labeled with a radioisotope; (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody or antigen-binding fragment that is higher than a reference value.

24. 24. The composition of claim 23, wherein the subject has been diagnosed with or is suspected of having cancer.

25. 25. The composition of claim 23 or 24, wherein the level of radioactivity emitted by the antibody or antigen-binding fragment is detected using positron emission tomography or single photon emission computed tomography.

26. A composition according to any one of claims 23 to 25, characterized in that it is administered in combination with an immunoconjugate comprising an antibody or antigen-binding fragment according to any one of claims 1 to 11 conjugated to a radionuclide.

27. The composition of any one of claims 23 to 27, wherein the cancer is a solid tumor.

28. 28. The composition of any one of claims 23 to 27, wherein the cancer is breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, or neuroblastoma.

29. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 11 and instructions for use.

30. 30. The kit of claim 29, wherein the antibody or antigen-binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

31. 31. The kit of claim 29 or 30, further comprising a secondary antibody that specifically binds to the antibody or antigen-binding fragment of any one of claims 1 to 11.

32. 12. An in vitro or ex vivo method for detecting HER2 protein expression levels in a biological sample, comprising contacting the biological sample with an antibody or antigen-binding fragment of any one of claims 1 to 11, and detecting binding to HER2 protein in the biological sample.

33. 12. An ex vivo method of generating therapeutic T cells, comprising binding the multispecific antibody or antigen-binding fragment of claim 11 to a T cell, wherein the T cell is optionally a human T cell, and wherein the binding is non-covalent.

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