BR112016029372B1 - HUMAN OR FULLY HUMANIZED MONOCLONAL ANTIBODY, ISOLATED NUCLEIC ACID MOLECULE, VECTOR, METHODS FOR PRODUCING AN ANTIBODY AND FOR PREPARING A CONJUGATE, CONJUGATE, AND, USE OF A CONJUGATE - Google Patents

Abstract

ISOLATED ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, ISOLATED NUCLEIC ACID MOLECULE, VECTOR, METHODS FOR PRODUCING AN ISOLATED ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, FOR PREPARING A CONJUGATE AND FOR ALLEVIATING A SYMPTOM OF A CANCER, CONJUGATE, AND, USE OF A CONJUGATE. Provision of fully human monoclonal antibodies that recognize HER2, and methods for using such monoclonal antibodies in a variety of therapeutic, diagnostic, and prophylactic indications.

Description

RELATED REQUESTS

[001] This application claims priority to, and the benefit of, U.S. Provisional Application Nos. 62/013,944, filed June 18, 2014; 62/034,489, filed August 7, 2014; 62/147,960, filed April 15, 2015; and 62/149,444, filed April 17, 2015. The contents of each of these applications are hereby incorporated by reference in their entireties.

FIELD OF INVENTION

[002] This invention generally relates to the generation of monoclonal antibodies that recognize the human HER2 receptor, to monoclonal antibodies that recognize specific HER2 epitopes within the extracellular domain of the human HER2 receptor, and to methods of using these monoclonal antibodies as therapeutic and/or diagnostic products.

FUNDAMENTALS OF THE INVENTION

Members of the ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. The receptor family includes four distinct members, including epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185neu), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). Both homo- and heterodimers are formed by the four members of the EGFR family, with HER2 being the preferred and most potent dimerization partner for other ErbB receptors (Graus-Porta et al., Embo 3 1997;16:1647-1655; Tao et al., J Cell Sci 2008;121:3207-3217). HER2 has no known ligand, but can be activated via homodimerization when overexpressed, or by heterodimerization with other, ligand-occupied ErbB receptors.

[004] The HER2 gene (also known as the HER2/neue ErbB2 gene) is amplified in 20 to 30% of early-stage breast cancers, which causes them to overexpress epidermal growth factor (EGF) receptors on the cell membrane (Bange, et al., Nature Medicine 7 (5): 548-552). In addition to breast cancer, HER2 expression has also been associated with other types of human carcinoma, including non-small cell lung cancer, ovarian cancer, gastric cancer, prostate cancer, bladder cancer, colon cancer, esogastric cancer, and squamous cell carcinoma of the head and neck (Garcia de Palazzo et al., Int J Biol Markers 1993; 8: 233-239; Ross et al., Oncologist 2003; 8: 307-325; Osman et al., J Urol 2005; 174: 2174-2177; Kapitanovic et al., Gastroenterology 1997; 112: 1103-1113; Turken et al., Neoplasma 2003; 50: 257-261; and Oshima et al., Int J Biol Markers 2001; 16: 250-254).

[005] Trastuzumab (Herceptin®) is a recombinant, humanized monoclonal antibody directed against domain IV of the HER2 protein, thereby blocking ligand-independent HER2 homodimerization, and to a lesser extent HER2 heterodimerization with other family members in cells highly overexpressing HER2 (Cho et al., Nature 2003; 421: 756-760 and Wehrman et al., Proc Natl Acad Sci USA 2006; 103: 19063-19068). Herceptin® is approved for both first-line and adjuvant treatment of metastatic breast cancer overexpressing HER2, in combination with chemotherapy, or as a single agent following one or more chemotherapy regimens. Trastuzumab has been found to be effective in only 20 to 50% of patients with HER2-overexpressing breast cancer, and many of the initial responders relapse after a few months (Dinh et al., Clin Adv Hematol Oncol 2007;5:707-717).

[006] Pertuzumab (Omnitar/Perjeta® also called 2C4) is another humanized monoclonal antibody directed against domain II of the HER2 protein, resulting in inhibition of ligand-induced heterodimerization (i.e., HER2 dimerizing with another ErbB family member to which a ligand has bound); a mechanism reported not to strictly require high levels of HER2 expression (Franklin et al., Cancer Cell 2004; 5: 317-328.). Pertuzumab is approved for the treatment of HER2-positive metastatic breast cancer in combination with trastuzumab and docetaxel.

[007] A HER2 antibody drug conjugate (ADC), Trastuzumab emtansine (ado-trastuzumab emtansine, Kadcila®) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab (Herceptin) linked to the cytotoxic agent mertansine (DM1). Kadcila as a single agent, is approved for the treatment of patients with HER2-positive (HER2+), metastatic breast cancer (MBC) who previously received trastuzumab and a taxane, separately or in combination.

[008] Although many factors are involved in selecting a suitable antibody for HER2-targeted therapy, it is typically an advantage for an ADC method if the HER2-antibody complex efficiently internalizes upon antibody binding. When compared to EGFR, however, HER2 internalization is impaired.

[009] The complex mechanisms that regulate HER2 function further warrant research into new and optimized therapeutic strategies against this proto-oncogene.

[0010] Consequently, there is a need for therapies that target the biological activities of HER2.

SUMMARY OF THE INVENTION

[0011] The present invention provides monoclonal antibodies that specifically recognize HER2, also known as (ErbB2, p185neu and/or HER2/neu). The antibodies described herein are capable of modulating and useful in modulating, e.g., blocking, inhibiting, reducing, antagonizing, neutralizing or otherwise interfering with the PI3K-Akt pathway that promotes cell survival by reducing levels of phosphorylated AKT. The antibodies described herein are also capable of modulating and useful in modulating, e.g., blocking, inhibiting, reducing, antagonizing, neutralizing or otherwise interfering with the ligand-independent homodimerization and/or heterodimerization of HER2. The antibodies described herein also include antibodies that bind soluble HER2.

[0012] The antibodies of the present invention exhibit HER2-binding characteristics that differ from antibodies described in the art. In particular, the antibodies described herein bind to a different epitope of HER2, in that they cross-block each other, but not trastuzumab, pertuzumab, Fab37 or chA21 from binding to HER2. Furthermore, unlike known antibodies, the antibodies described herein can efficiently internalize into HER2-expressing cells without promoting cell proliferation.

[0013] The antibodies described herein are fully human monoclonal antibodies that bind to novel epitopes and/or have other favorable properties for therapeutic use. Exemplary properties include, but are not limited to, favorable binding characteristics to cancer cells expressing human HER2 at high and low levels, specific binding to recombinant human and cynomolgus monkey HER2, efficient internalization upon binding to HER2, high ability to kill cancer cells expressing high or low levels of HER2 when administered as an antibody drug conjugate (ADC), no substantial agonistic effect on the proliferation of HER2-expressing cancer cells, and provide effective antibody-dependent cellular cytotoxicity (ADCC)-mediated killing of HER2-expressing cells, as well as any combination of the foregoing properties.

[0014] The antibodies described herein also include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor that includes residues 452-531 of the extracellular domain of the human HER2 receptor, e.g., residues 474-553 of SEQ ID NO: 38 or residues 452-531 of SEQ ID NO: 39.

[0015] The antibodies described herein include an isolated antibody or an antigen-binding fragment thereof that binds at least a portion of the N-terminus of domain IV of the human HER2 receptor, but do not cross-compete with an antibody that binds to the 4D5 epitope of the human HER2 receptor. For example, the antibodies or antigen-binding fragments thereof described herein do not cross-compete with trastuzumab for binding to the human HER2 receptor, since trastuzumab is known to bind the 4D5 epitope of the human HER2 receptor. As used herein, the term human HER2 receptor 4D5 epitope refers to amino acid residues 529-627 of the extracellular domain of the human HER2 receptor, for example residues 551-649 of SEQ ID NO: 38 or residues 529-627 of SEQ ID NO: 39. In some embodiments, the isolated antibody or antigen-binding fragment thereof also binds at least one epitope on the cynomolgus monkey HER2 receptor.

[0016] The antibodies described herein also include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor that includes residues 452 to 500 of the extracellular domain of the human HER2 receptor, e.g., residues 474 to 522 of SEQ ID NO: 38 or residues 452 to 500 of SEQ ID NO: 39.

[0017] The antibodies described herein also include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor that includes at least one amino acid residue selected from the group consisting of amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor, e.g., residues 543, 547, and 552 of SEQ ID NO: 38, and residues 521, 525, and 530 of SEQ ID NO: 39. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least two amino acid residues selected from the group consisting of amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. The antibodies described herein also include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor that includes at least amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these isolated antibodies or antigen-binding fragments thereof also bind at least one epitope on the cynomolgus monkey HER2 receptor.

[0018] The antibodies described herein also include an isolated antibody or an antigen-binding fragment thereof that binds to at least a portion of domain III and at least a portion of the N-terminus of domain IV of the human HER2 receptor, but does not cross-compete with the Fab37 monoclonal antibody or an antibody that binds to the 4D5 epitope of the human HER2 receptor. For example, the antibodies or antigen-binding fragments thereof described herein do not cross-compete with the Fab37 monoclonal antibody and/or trastuzumab for binding to the human HER2 receptor. In some embodiments, the isolated antibody or antigen-binding fragment thereof also binds at least one epitope on the cynomolgus monkey HER2 receptor.

[0019] The antibodies described herein also include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor that includes residues 520-531 of the extracellular domain of the human HER2 receptor, e.g., residues 542-553 of SEQ ID NO: 38 or residues 520-531 of SEQ ID NO: 39.

[0020] The antibodies described herein also include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor that includes at least one amino acid residue selected from the group consisting of residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor, e.g., residues 475, 478, 495, 498, 517, 518, 519, and 521 of SEQ ID NO: 38 or residues 453, 456, 473, 476, 495, 496, 497, and 499 of SEQ ID NO: 39. For example, the antibodies described herein ... bind to an epitope of the extracellular domain of the human HER2 receptor that includes at least two amino acid residues selected from the group consisting of amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically bind to an epitope of the extracellular domain of the human HER2 receptor that includes at least three amino acid residues selected from the group consisting of amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least four amino acid residues selected from the group consisting of amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least five amino acid residues selected from the group consisting of amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least six amino acid residues selected from the group consisting of amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these isolated antibodies or antigen-binding fragments thereof also bind at least one epitope on the cynomolgus monkey HER2 receptor.

[0021] The antibodies described herein also include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor that includes at least one amino acid residue selected from the group consisting of residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor, e.g., residues 475, 495, 498, 517, 519, and 521 of SEQ ID NO: 38 or residues 453, 473, 476, 495, 497, and 499 of SEQ ID NO: 39. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least two amino acid residues selected from the group consisting of amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least three amino acid residues selected from the group consisting of amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least four amino acid residues selected from the group consisting of amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, the antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least five amino acid residues selected from the group consisting of amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least six amino acid residues selected from the group consisting of amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. For example, antibodies described herein include an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that includes at least amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these isolated antibodies or antigen-binding fragments thereof also bind at least one epitope on the cynomolgus monkey HER2 receptor.

[0022] These and other aspects of the invention are described in more detail below.

[0023] Exemplary monoclonal antibodies described herein include, for example, the XMT 1517 antibody, the XMT 1518 antibody, the XMT 1519 antibody, and the XMT 1520 antibody described herein. Alternatively, the monoclonal antibody is an antibody that cross-blocks each other but does not bind to the same epitope as trastuzumab, pertuzumab, Fab37, or chA21 (which bind to specific epitopes in domain IV, domain II, domain III, and domain I of HER2, respectively) or biosimilars thereof. These antibodies are respectively referred to herein as “HER2” antibodies. HER2 antibodies include fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies. These antibodies show specificity for human HER2, and they have been shown to modulate, e.g., block, inhibit, reduce, antagonize, neutralize or otherwise interfere with the PI3K-Akt pathways that promote cell survival by reducing the levels of phosphorylated AKT. These antibodies internalize from the cell surface of HER2-expressing cells at a rate that is the same or substantially similar to the rate at which trastuzumab or a biosimilar thereof internalizes. For example, these antibodies and antigen-binding fragments have an internalization rate that is approximately 50% of the total surface bound at time 0 being internalized within 4 hours.

[0024] The antibodies described herein contain a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7 and a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8.

[0025] The antibodies described herein contain a combination of heavy chain and light chain amino acid sequences selected from the group consisting of (i) a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 2; (ii) a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 3 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 4; (iii) a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 6; and (iv) a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 7 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 8.

[0026] In some embodiments, the antibodies described herein contain a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 2.

[0027] In some embodiments, the antibodies described herein contain a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 3 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 4.

[0028] In some embodiments, the antibodies described herein contain a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 5 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 6.

[0029] In some embodiments, the antibodies described herein contain a heavy chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 7 and a light chain amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 8.

[0030] The antibodies described herein contain a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7 and a light chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, respectively.

[0031] The antibodies described herein contain a combination of heavy chain and light chain amino acid sequences selected from the group consisting of (i) the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2; (ii) the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4; (iii) the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6; and (iv) the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8.

[0032] In some embodiments, the antibodies described herein contain the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2.

[0033] In some embodiments, the antibodies described herein contain the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4.

[0034] In some embodiments, the antibodies described herein contain the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6.

[0035] In some embodiments, the antibodies described herein contain the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8.

[0036] The antibodies described herein contain a heavy chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, and 15 and a light chain variable region having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16.

[0037] The antibodies described herein contain a combination of heavy chain variable region and light chain variable region amino acid sequences selected from the group consisting of (i) a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10; (ii) a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 11 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12; (iii) a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 13 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14; and (iv) a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 16.

[0038] In some embodiments, antibodies described herein contain a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10.

[0039] In some embodiments, antibodies described herein contain a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 11 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12.

[0040] In some embodiments, antibodies described herein contain a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 13 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14.

[0041] In some embodiments, antibodies described herein contain a heavy chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 16.

[0042] The antibodies described herein contain a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, and 15 and a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16.

[0043] The antibodies described herein contain a combination of heavy chain variable region and light chain variable region amino acid sequences selected from the group consisting of (i) the heavy chain variable region amino acid sequence of SEQ ID NO: 9 and the light chain variable region amino acid sequence of SEQ ID NO: 10; (ii) the heavy chain variable region amino acid sequence of SEQ ID NO: 11 and the light chain variable region amino acid sequence of SEQ ID NO: 12; (iii) the heavy chain variable region amino acid sequence of SEQ ID NO: 13 and the light chain variable region amino acid sequence of SEQ ID NO: 14; and (iv) the heavy chain variable region amino acid sequence of SEQ ID NO: 15 and the light chain variable region amino acid sequence of SEQ ID NO: 16.

[0044] In some embodiments, the antibodies described herein contain the heavy chain variable region amino acid sequence of SEQ ID NO: 9 and the light chain variable region amino acid sequence of SEQ ID NO: 10.

[0045] In some embodiments, the antibodies described herein contain the heavy chain variable region amino acid sequence of SEQ ID NO: 11 and the light chain variable region amino acid sequence of SEQ ID NO: 12.

[0046] In some embodiments, antibodies described herein contain a heavy chain variable region amino acid sequence of SEQ ID NO: 13, and a light chain variable region having an amino acid sequence of SEQ ID NO: 14.

[0047] In some embodiments, antibodies described herein contain the heavy chain variable region amino acid sequence of SEQ ID NO: 15 and the light chain variable region amino acid sequence of SEQ ID NO: 16.

[0048] The three heavy chain CDRs of the antibodies described herein include a heavy chain complementarity determining region 1 (CDRH1) that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 17, 25, and 30; a heavy chain complementarity determining region 2 (CDRH2) that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 18, 23, 26, and 31; and a heavy chain complementarity determining region 3 (CDRH3) that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 19 and 27.

[0049] The three light chain CDRs of the antibodies described herein include a light chain complementarity determining region 1 (CDRL1) that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 20 and 28; a light chain complementarity determining region 2 (CDRL2) that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 21 and 24; and a light chain complementarity determining region 3 (CDRL3) that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 22 and 29.

[0050] The antibodies include a combination of heavy chain CDR and light chain CDR sequences that include a CDRH1 that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 17, 25, and 30; a CDRH2 that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 18, 23, 26, and 31; a CDRH3 that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 19 and 27; a CDRL1 that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 20 and 28; a CDRL2 that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 21 and 24; and a CDRL3 that includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 22 and 29.

[0051] The three heavy chain CDRs of the antibodies described herein include a CDRH1 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 25, and 30; a CDRH2 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 26, and 31; and a CDRH3 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 27.

[0052] The three light chain CDRs of the antibodies described herein include a CDRL1 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 28; a CDRL2 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 24; and a CDRL3 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 and 29.

[0053] The antibodies described herein include a combination of heavy chain CDR and light chain CDR sequences that include a CDRH1 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 25, and 30; a CDRH2 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 26, and 31; a CDRH3 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 27; a CDRL1 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 28; a CDRL2 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 24; and a CDRL3 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 and 29.

[0054] The antibodies described herein contain a combination of heavy chain complementarity determining region and light chain complementarity determining region amino acid sequences selected from the group consisting of (i) the CDRH1 amino acid sequence of SEQ ID NO: 17, the CDRH2 amino acid sequence of SEQ ID NO: 18, the CDRH3 amino acid sequence of SEQ ID NO: 19, the CDRL1 amino acid sequence of SEQ ID NO: 20, the CDRL2 amino acid sequence of SEQ ID NO: 21, and the CDRL3 amino acid sequence of SEQ ID NO: 22; (ii) the CDRH1 amino acid sequence of SEQ ID NO: 17, the CDRH2 amino acid sequence of SEQ ID NO: 23, the CDRH3 amino acid sequence of SEQ ID NO: 19, the CDRL1 amino acid sequence of SEQ ID NO: 20, the CDRL2 amino acid sequence of SEQ ID NO: 24, and the CDRL3 amino acid sequence of SEQ ID NO: 22; (iii) the CDRH1 amino acid sequence of SEQ ID NO: 25, the CDRH2 amino acid sequence of SEQ ID NO: 26, the CDRH3 amino acid sequence of SEQ ID NO: 27, the CDRL1 amino acid sequence of SEQ ID NO: 28, the CDRL2 amino acid sequence of SEQ ID NO: 21, and the CDRL3 amino acid sequence of SEQ ID NO: 29; and (iv) the CDRH1 amino acid sequence of SEQ ID NO: 30, the CDRH2 amino acid sequence of SEQ ID NO: 31, the CDRH3 amino acid sequence of SEQ ID NO: 27, the CDRL1 amino acid sequence of SEQ ID NO: 28, the CDRL2 amino acid sequence of SEQ ID NO: 21, and the CDRL3 amino acid sequence of SEQ ID NO: 29.

[0055] In some embodiments, antibodies described herein contain the CDRH1 amino acid sequence of SEQ ID NO: 17, the CDRH2 amino acid sequence of SEQ ID NO: 18, the CDRH3 amino acid sequence of SEQ ID NO: 19, the CDRL1 amino acid sequence of SEQ ID NO: 20, the CDRL2 amino acid sequence of SEQ ID NO: 21, and the CDRL3 amino acid sequence of SEQ ID NO: 22.

[0056] In some embodiments, antibodies described herein contain the CDRH1 amino acid sequence of SEQ ID NO: 17, the CDRH2 amino acid sequence of SEQ ID NO: 23, the CDRH3 amino acid sequence of SEQ ID NO: 19, the CDRL1 amino acid sequence of SEQ ID NO: 20, the CDRL2 amino acid sequence of SEQ ID NO: 24, and the CDRL3 amino acid sequence of SEQ ID NO: 22.

[0057] In some embodiments, antibodies described herein contain the CDRH1 amino acid sequence of SEQ ID NO: 25, the CDRH2 amino acid sequence of SEQ ID NO: 26, the CDRH3 amino acid sequence of SEQ ID NO: 27, the CDRL1 amino acid sequence of SEQ ID NO: 28, the CDRL2 amino acid sequence of SEQ ID NO: 21, and the CDRL3 amino acid sequence of SEQ ID NO: 29.

[0058] In some embodiments, antibodies described herein contain the CDRH1 amino acid sequence of SEQ ID NO: 30, the CDRH2 amino acid sequence of SEQ ID NO: 31, the CDRH3 amino acid sequence of SEQ ID NO: 27, the CDRL1 amino acid sequence of SEQ ID NO: 28, the CDRL2 amino acid sequence of SEQ ID NO: 21, and the CDRL3 amino acid sequence of SEQ ID NO: 29.

[0059] In some embodiments, the HER2 antibodies described herein also include an agent conjugated to the antibody. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is (a) an auristatin compound; (b) a calicheamicin compound; (c) a duocarmycin compound; (d) SN38, (e) a pyrrolobenzodiazepine; (f) a vinca compound; (g) a tubulysin compound; (h) an unnatural camptothecin compound; (i) a maytansinoid compound; (j) a DNA binding drug; (k) a kinase inhibitor; (l) a MEK inhibitor; (m) a KSP inhibitor; (n) a topoisomerase inhibitor; and analogs thereof or analogues thereof. In some embodiments, the agent is conjugated to the HER2 antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is any of the toxins described herein.

[0060] In one aspect, the conjugated HER2 antibody described herein includes an isolated HER2 antibody or antigen-binding fragment thereof connected directly or indirectly to one or more therapeutic or diagnostic agents (D). In some embodiments, the conjugated HER2 antibody also includes one or more polymeric scaffolds connected to the antibody or antigen-binding fragment thereof, wherein each of the one or more Ds is independently connected to the antibody or antigen-binding fragment thereof via the one or more polymeric scaffolds.

[0061] In some embodiments, each of the one or more polymeric scaffolds that are connected to the isolated HER2 antibody or antigen-binding fragment thereof, independently, comprises poly(1-hydroxymethylethylene hydroxymethyl-formal) (PHF) having a molecular weight ranging from about 2 kDa to about 40 kDa.

[0062] In some embodiments, each of the one or more polymeric scaffolds independently is of formula (Ic): where:LD1 is a carbonyl-containing moiety;each occurrence of in is independently a first linker that contains a biodegradable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect; and in between LD1 and D indicates direct or indirect linkage* of D to LD1; each occurrence of is independently a second ligand not yet attached to the isolated antibody or antigen-binding fragment thereof, wherein LP2 is a moiety containing a functional group that is yet to form a covalent bond with a functional group of the isolated antibody or antigen-binding fragment thereof, and between LD1 and LP2 indicates direct or indirect binding* of LP2 to LD1, and each occurrence of the second ligand is distinct from each occurrence of the first ligand; each occurrence of is independently a third linker that connects each D-bearing polymeric scaffold to the isolated antibody or antigen-binding fragment thereof, in which the terminus bound to LP2 indicates direct or indirect binding of LP2 to the isolated antibody or antigen-binding fragment thereof in the formation of a covalent bond between a functional group of LP2 and a functional group of the isolated antibody or antigen-binding fragment thereof; and each occurrence of the third ligand is distinct from each occurrence of the first ligand; m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m3 is an integer from 0 to about 18, m4 is an integer from 1 to about 10; the sum of m, m1, m2, m3, and m4 ranges from about 15 to about 300; and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof is 10 or less.

[0063] The conjugate described herein may include one or more of the following traits:

[0064] For example, in formula (Ic), the isolated HER2 antibody or antigen-binding fragment thereof has a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater, 80 kDa or greater, 100 kDa or greater, 120 kDa or greater, 140 kDa or greater, 160 kDa or greater, 180 kDa or greater, or 200 kDa or greater, or from about 40 to 200 kDa, 40 to 180 kDa, 40 to 140 kDa, 60 to 200 kDa, 60 to 180 kDa, 60 to 140 kDa, 80 to 200 kDa, 80 to 180 kDa, 80 to 140 kDa, 100 to 200 kDa, 100 to 18 ... kDa, 100 to 140 kDa, or 140 to 150 kDa). In some embodiments, the isolated HER2 antibody or antigen-binding fragment thereof is any antibody or antigen-binding fragment of the disclosure, including, by way of non-limiting example, the XMT 1517 antibody, the XMT 1518 antibody, the XMT 1519 antibody, and the XMT 1520 antibody described herein.

[0065] For example, in formula (Ic), m1 is an integer from 1 to about 120 (e.g., from about 1 to 90) and/or m3 is an integer from 1 to about 10 (e.g., from about 1 to 8).

[0066] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 45 to about 150), m2 is an integer from 2 to about 20, m3 is an integer from 0 to about 9, m4 is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 75 (e.g., m1 being from 4 to 45).

[0067] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 60 to about 110), m2 is an integer from 2 to about 15, m3 is an integer from 0 to about 7, m4 is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 55 (e.g., m1 being from 4 to 30).

[0068] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 2 kDa to about 20 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 15 to about 150), m2 is an integer from 1 to about 20, m3 is an integer from 0 to about 10 (e.g., m3 ranging from 0 to about 9), m4 is an integer from 1 to about 8, and/or m1 is an integer from 1 to about 70, and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7, or 8).

[0069] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 3 kDa to about 15 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 20 to about 110), m2 is an integer from 2 to about 15, m3 is an integer from 0 to about 8 (e.g., m3 ranging from 0 to about 7), m4 is an integer from 1 to about 8, and/or m1 is an integer from 2 to about 50, and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7, or 8).

[0070] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 5 kDa to about 10 kDa, (i.e. the sum of m, m1, m2, m3, and m4 ranges from about 40 to about 75), m2 is an integer from about 2 to about 10 (e.g., m2 being from 3 to 10), m3 is an integer from 0 to about 5 (e.g., m3 ranging from 0 to about 4), m4 is an integer from 1 to about 8 (e.g., m4 ranging from 1 to about 5), and/or m1 is an integer from about 2 to about 35 (e.g., m1 being from 5 to 35), and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7, or 8).

[0071] For example, each occurrence of D independently is a therapeutic agent having a molecular weight of <5 kDa.

[0072] For example, each occurrence of D independently is an anticancer drug, e.g., selected from vinca alkaloids, auristatins, tubulysins, duocarmycins, unnatural camptothecin compounds, maytansinoids, calicheamicin compounds, topoisomerase inhibitors, DNA binding drugs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogues thereof.

[0073] For example, each when not bound to the isolated antibody or antigen-binding fragment thereof, independently comprises a terminal WP group, wherein each WP independently is: where R1K is a leaving group (e.g., halide or RC(O)O- where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety), R1A is a sulfur protecting group, and ring A is cycloalkyl or heterocycloalkyl, and R1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0074] For example, each R1A independently is where r is 1 or 2 and each of Rs1, Rs2, and Rs3 is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0075] For example, the functional group of LP2 that is further to form a covalent bond with a functional group of the isolated antibody or antigen-binding fragment thereof is selected from –SRp, -SS-LG, or a sulfur protecting group, and one of Xa and Xb is H and the other is a water-soluble maleimide blocking moiety, or Xa and Xb, together with the carbon atoms to which they are attached, form a carbon-carbon double bond. For example, the functional group of LP2 that is yet to form a covalent bond is a functional group that is not reacted with a functional group of the isolated antibody or antigen-binding fragment thereof, e.g., as the functional group of LP2, in which one of Xa and Xb is H and the other is a water-soluble maleimide blocking moiety, or Xa and Xb.

[0076] For example, LD1 comprises —X-(CH2)vC(=O)— with X directly connected to the carbonyl group of , where X is CH2, O, or NH, and v is an integer from 1 to 6.

[0077] For example, each occurrence of isindependently —C(=O)-X-(CH2)vC(=O)-NH-(CH2)u- NH-C(=O)-(CH2)w-(OCH2)x-NHC(=O)-(CH2)y—M, where X is CH2, O, or NH, each of v, u, w, x, and y independently is an integer from 1 to 6, and M is , where one of Xa and Xb is H and the other is a water-soluble maleimide blocking moiety, or Xa and Xb, together with the carbon atoms to which they are attached form a carbon-carbon double bond.

[0078] For example, each of v, u, w, x, and y is 2.

[0079] For example, the ratio of D to the isolated HER2 antibody or antigen-binding fragment thereof is about 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0080] For example, the ratio of D to the isolated HER2 antibody or antigen-binding fragment thereof is about 20:1, 15:1, 10:1, 5:1, 2:1, or 1:1.

[0081] For example, the ratio of D to the isolated HER2 antibody or antigen-binding fragment thereof is about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

[0082] For example, the ratio of D to the isolated HER2 antibody or antigen-binding fragment thereof is about 15:1, 14:1, 13:1, 12:1, or 11:1.

[0083] For example, the ratio of D to the isolated HER2 antibody or antigen-binding fragment thereof is about 15:1, 14:1, 13:1, or 12:1.

[0084] For example, the ratio of D to isolated HER2 antibody or antigen-binding fragment thereof is about 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0085] For example, each of one or more polymeric scaffolds that independently carry D is of the formula (Id): where:m3a is an integer from 0 to about 17,m3b is an integer from 1 to about 8, and the terminal indicates the direct binding* of one or more polymeric scaffolds to the isolated HER2 antibody or antigen-binding fragment thereof having a molecular weight of 40 kDa or greater.

[0086] The formula frame (Id) may include one or more of the following traits:

[0087] The sum of m3a and m3b is between 1 and 18.

[0088] When the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 40 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 300, m1 is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m3a is an integer from 0 to about 17, m3b is an integer from 1 to about 8, the sum of m3a and m3b ranges from 1 to about 18, and the ratio of the PHF to the isolated HER2 antibody or antigen-binding fragment thereof is 10 or less.

[0089] When the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 20 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 150, m1 is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m3a is an integer from 0 to about 9, m3b is an integer from 1 to about 8, the sum of m3a and m3b ranges from 1 to about 10, and the ratio of the PHF to the isolated HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8.

[0090] When the PHF in formula (Id) has a molecular weight ranging from about 3 kDa to about 15 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m3a is an integer from 0 to about 7, m3b is an integer from 1 to about 8, the sum of m3a and m3b ranges from 1 to about 8; and the ratio of the PHF to the isolated HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (e.g., from about 2 to about 6 or from about 2 to about 4).

[0091] When the PHF in formula (Id) has a molecular weight ranging from about 5 kDa to about 10 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 40 to about 75, m1 is an integer from about 2 to about 35, m2 is an integer from about 2 to about 10, m3a is an integer from 0 to about 4, m3b is an integer from 1 to about 5, the sum of m3a and m3b ranges from 1 to about 5; and the ratio of the PHF to the isolated HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (e.g., from about 2 to about 6 or from about 2 to about 4).

[0092] In certain embodiments, the ratio of auristatin F hydroxylpropyl amide (“AF HPA”) to isolated HER2 antibody or antigen-binding fragment thereof can be about 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[0093] In certain embodiments, the ratio of HPA AF to isolated HER2 antibody or antigen-binding fragment thereof can be about 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[0094] In other embodiments, the ratio of HPA AF to isolated HER2 antibody or antigen-binding fragment thereof can be about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[0095] In some embodiments, the ratio of HPA AF to isolated HER2 antibody or antigen-binding fragment thereof can be about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

[0096] In some embodiments, the ratio of HPA AF to isolated HER2 antibody or antigen-binding fragment thereof can be about 15:1, 14:1, 13:1, 12:1, or 11:1.

[0097] In some embodiments, the ratio of HPA AF to isolated HER2 antibody or antigen-binding fragment thereof can be about 15:1, 14:1, 13:1, or 12:1.

[0098] In certain embodiments, the ratio of PHF to isolated HER2 antibody or antigen-binding fragment thereof can be about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0099] In certain embodiments, the ratio of PHF to isolated HER2 antibody or antigen-binding fragment thereof can be about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[00100] In other embodiments, the ratio of PHF to isolated HER2 antibody or antigen-binding fragment thereof can be about 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[00101] In other embodiments, the ratio of PHF to isolated HER2 antibody or antigen-binding fragment thereof can be about 6:1, 5:1, 4:1, 3:1, or 2:1.

[00102] In other embodiments, the ratio of PHF to isolated HER2 antibody or antigen-binding fragment thereof can be about 6:1, 5:1, 4:1, or 3:1.

[00103] In some embodiments, the ratio of PHF to isolated HER2 antibody or antigen-binding fragment thereof can be about 5:1, 4:1, or 3:1.

[00104] In some embodiments, the ratio of PHF to isolated HER2 antibody or antigen-binding fragment thereof can be about 4:1, 3:1, or 2:1.

[00105] The water-soluble maleimide blocking moieties (e.g., Xa or Xb) are moieties that can be covalently attached to one of the two carbon atoms of the olefin in the reaction of the maleimide group with a thiol-containing compound of formula (II):R90-(CH2)d-SH(II)where:R90 is NHR91, OH, COOR93, CH(NHR91)COOR93, or a substituted phenyl group;R93 is hydrogen or C1-4 alkyl;R91 is hydrogen, CH3, or CH3CO, and d is an integer from 1 to 3.

[00106] In one embodiment, the water-soluble maleimide blocking compound of formula (II) can be cysteine, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH2SH), benzyl thiol wherein phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol. The one or more hydrophilic substituents on the phenyl comprise OH, SH, methoxy, ethoxy, COOH, CHO, CO, C1-4 alkyl, NH2, F, cyano, SO3H, PO3H, and the like.

[00107] In another aspect, the water-soluble maleimide blocking group is -S-(CH2)d-R90, wherein, R90 is OH, COOH, or CH(NHR91)COOR93; R93 is hydrogen or CH3; R91 is hydrogen or CH3CO; and d is 1 or 2.

[00108] In another embodiment, the water-soluble maleimide blocking group is -S-CH2-CH(NH2)COOH.

[00109] In certain embodiments, the conjugate described herein comprises one or more D-bearing PHFs, each of which independently is of the formula (Se), wherein the PHF has a molecular weight ranging from about 2 kDa to about 40 kDa: where: m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m3a is an integer from 0 to about 17, m3b is an integer from 1 to about 8; the sum of m3a and m3b ranges from 1 to about 18; the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 300; the terminal indicates the binding* of one or more PHF polymeric scaffolds to the isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor and comprises a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 26); a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence GGHGYFDL (SEQ ID NO: 27); a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSSILA (SEQ ID NO: 28); a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GASSRAT (SEQ ID NO: 21); and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); and the ratio of the PHF to the antibody is 10 or less.

[00110] The formula frame (If) may include one or more of the following traits:

[00111] When the PHF in formula (Se) has a molecular weight ranging from about 2 kDa to about 20 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 150, m1 is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m3a is an integer from 0 to about 9, m3b is an integer from 1 to about 8, the sum of m3a and m3b ranges from 1 to about 10, and the ratio of the PHF to the antibody is an integer from 2 to about 8.

[00112] When the PHF in formula (Se) has a molecular weight ranging from about 3 kDa to about 15 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m3a is an integer from 0 to about 7, m3b is an integer from 1 to about 8, the sum of m3a and m3b ranges from 1 to about 8; and the ratio of the PHF to the antibody is an integer from 2 to about 8 (e.g., from about 2 to about 6 or from about 2 to about 4).

[00113] When the PHF in formula (Se) has a molecular weight ranging from about 5 kDa to about 10 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 40 to about 75, m1 is an integer from about 2 to about 35, m2 is an integer from about 2 to about 10, m3a is an integer from 0 to about 4, m3b is an integer from 1 to about 5, the sum of m3a and m3b ranges from 1 to about 5; and the ratio of the PHF to the antibody is an integer from 2 to about 8 (e.g., from about 2 to about 6 or from about 2 to about 4).

[00114] In certain embodiments, the ratio of auristatin F hydroxylpropyl amide (“AF HPA”) to the antibody can be about 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[00115] In certain embodiments, the ratio of HPA AF to antibody may be about 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[00116] In other embodiments, the ratio of HPA AF to antibody can be about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[00117] In some embodiments, the ratio of HPA AF to antibody may be about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

[00118] In some embodiments, the ratio of AF to antibody may be about 15:1, 14:1, 13:1, 12:1, or 11:1.

[00119] In some embodiments, the ratio of HPA AF to antibody may be about 15:1, 14:1, 13:1, or 12:1.

[00120] In certain embodiments, the ratio of PHF to antibody may be about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[00121] In certain embodiments, the ratio of PHF to antibody may be about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[00122] In other embodiments, the ratio of PHF to antibody may be about 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[00123] In other embodiments, the ratio of PHF to antibody may be about 6:1, 5:1, 4:1, 3:1, or 2:1.

[00124] In other embodiments, the ratio of PHF to antibody may be about 6:1, 5:1, 4:1, or 3:1.

[00125] In some embodiments, the ratio of PHF to antibody may be about 5:1, 4:1, or 3:1.

[00126] In some embodiments, the ratio of PHF to antibody may be about 4:1, 3:1, or 2:1.

[00127] In another aspect, the conjugate described herein is of the formula (Ib): wherein: HER2 ANTIBODY indicates the isolated HER2 antibody or antigen-binding fragment thereof described herein; between LP2 and HER2 ANTIBODY indicates direct or indirect binding* of HER2 ANTIBODY to LP2, each occurrence of HER2 ANTIBODY independently has a molecular weight of less than 200 kDa, m is an integer from 1 to about 2200, m1 is an integer from 1 to about 660, m2 is an integer from 3 to about 300, m3 is an integer from 0 to about 110, m4 is an integer from 1 to about 60; and the sum of m, m1, m2, m3, and m4 ranges from about 150 to about 2200.

[00128] In formula (Ib), m1 is an integer from about 10 to about 660 (e.g., from about 10 to 250).

[00129] When the PHF in formula (Ib) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 370 to about 740), m2 is an integer from 5 to about 100, m3 is an integer from 1 to about 40, m4 is an integer from 1 to about 20, and/or m1 is an integer from 1 to about 220 (e.g., m1 being from about 15 to 80).

[00130] In formula (Ib), each HER2 ANTIBODY independently has a molecular weight of 120 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 20 kDa or less, or 10 kDa or less, or about 4 kDa to 80 kDa (e.g., 4 to 20 kDa, 20 to 30 kDa, or 30 to 70 kDa).

[00131] Another aspect of the invention features a method for preparing a conjugate described herein. The method includes reacting the isolated antibody or antigen-binding fragment thereof with a polymeric scaffold of formula (Ia) such that the conjugate is formed: where:LD1 is a carbonyl-containing moiety;each occurrence of in is independently a first linker that contains a biodegradable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect; and between LD1 and D indicates direct or indirect linkage* of D to LD1; each occurrence of is independently a second ligand not yet attached to the isolated antibody or antigen-binding fragment thereof, wherein LP2 is a moiety containing a functional group that is yet to form a covalent bond with a functional group of the isolated antibody or antigen-binding fragment thereof, and between LD1 and LP2 indicates direct or indirect binding* of LP2 to LD1, and each occurrence of the second ligand is distinct from each occurrence of the first ligand; m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m3 is an integer from 1 to about 18, and the sum of m, m1, m2, and m3 ranges from about 15 to about 300.

[00132] In the formulas for the polymeric scaffolds disclosed herein, the disconnection or gap between the polyacetal units indicates that the units may be connected to each other in any order. In other words, the attachment groups containing, for example, D, LP2, and the isolated antibody or antigen-binding fragment thereof, may be randomly distributed along the polymeric backbone.

[00133] The present invention also provides methods of treating, preventing, delaying the progression of or otherwise ameliorating a symptom of one or more pathologies associated with aberrant HER2 expression, function and/or activation, or alleviating a symptom associated with such pathologies, by administering a monoclonal antibody, fragment thereof, and/or conjugate thereof described herein to a subject in whom such treatment or prevention is desired. The subject to be treated is, for example, a human. The monoclonal antibody, fragment thereof, and/or conjugate thereof are administered in an amount sufficient to treat, prevent or alleviate a symptom associated with the pathology.

[00134] The present invention also provides methods of treating, preventing, delaying the progression of or otherwise ameliorating a symptom of one or more pathologies associated with HER2 expression, function and/or activation, or alleviating a symptom associated with such pathologies, by administering a monoclonal antibody, fragment thereof, and/or conjugate thereof described herein to a subject in whom such treatment or prevention is desired. The subject to be treated is, for example, a human. The monoclonal antibody, fragment thereof, and/or conjugate thereof are administered in an amount sufficient to treat, prevent or ameliorate a symptom associated with the pathology.

[00135] The pathologies treated and/or prevented using the monoclonal antibodies, fragments thereof, and/or conjugates thereof described herein include, for example, cancer. For example, the antibodies, fragments thereof, and/or conjugates thereof described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esogastric cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, throat cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, renal cancer, and gastric cancer.

[00136] In some embodiments, the antibodies or antigen-deleting fragments thereof and/or PBRM-polymer-drug conjugates thereof described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of breast cancer.

[00137] In some embodiments, the antibodies or antigen-deleting fragments thereof and/or PBRM-polymer-drug conjugates thereof described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of gastric cancer.

[00138] In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates thereof described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of non-small cell lung cancer (NSCLC).

[00139] In some embodiments, the antibodies or antigen-deleting fragments thereof and/or PBRM-polymer-drug conjugates thereof described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of ovarian cancer.

[00140] In some embodiments, the HER2 antibody or antigen-binding fragment thereof used in the treatment of, prevention of, delaying the progression of, or otherwise ameliorating a symptom of a cancer (e.g., a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esogastric cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, throat cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, renal cancer, and gastric cancer) competes for binding to the same Her-2 epitope with an antibody comprising (1) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); CDRH2 comprising the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); CDRH3 comprising the amino acid sequence GGHGYFDL (SEQ ID NO: 27); and CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSSILA (SEQ ID NO: 28); CDRL2 comprising the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 comprising the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (2) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSGRSMN (SEQ ID NO: 30); CDRH2 comprising the amino acid sequence YISSDSRTIYYADSVKG (SEQ ID NO: 31); CDRH3 comprising the amino acid sequence GGHGYFDL (SEQ ID NO: 27); CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSSILA (SEQ ID NO: 28); CDRL2 comprising the amino acid sequence GASSRAT (SEQ ID NO: 21); and CDRL3 comprising the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (3) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSSYGMH (SEQ ID NO: 17); CDRH2 comprising the amino acid sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 18); CDRH3 comprising the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19), and CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSDILA (SEQ ID NO: 20); CDRL2 comprising the amino acid sequence GASSRAT (SEQ ID NO: 21); and CDRL3 comprising the amino acid sequence QQYVSYWT (SEQ ID NO: 22); or (4) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSSYGMH (SEQ ID NO: 17); CDRH2 comprising the amino acid sequence GIWWDGSNEKYADSVKG (SEQ ID NO: 23); CDRH3 comprising the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19); and CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSDILA (SEQ ID NO: 20); CDRL2 comprising the amino acid sequence GASRRAT (SEQ ID NO: 24); and CDRL3 comprising the amino acid sequence QQYVSYWT (SEQ ID NO: 22).

[00141] In another embodiment, the HER2 antibody or antigen-binding fragment thereof used in the treatment of, prevention, delaying the progression of, or otherwise ameliorating a symptom of a cancer (e.g., a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esogastric cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, throat cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, renal cancer, and gastric cancer) competes for binding to the same Her-2 epitope with an antibody comprising (1) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); CDRH2 comprising the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); CDRH3 comprising the amino acid sequence GGHGYFDL (SEQ ID NO: 27); and CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSSILA (SEQ ID NO: 28); CDRL2 comprising the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 comprising the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (2) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSGRSMN (SEQ ID NO: 30); CDRH2 comprising the amino acid sequence YISSDSRTIYYADSVKG (SEQ ID NO: 31); CDRH3 comprising the amino acid sequence GGHGYFDL (SEQ ID NO: 27); CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSSILA (SEQ ID NO: 28); CDRL2 comprising the amino acid sequence GASSRAT (SEQ ID NO: 21); and CDRL3 comprising the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (3) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSSYGMH (SEQ ID NO: 17); CDRH2 comprising the amino acid sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 18); CDRH3 comprising the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19), and CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSDILA (SEQ ID NO: 20); CDRL2 comprising the amino acid sequence GASSRAT (SEQ ID NO: 21); and CDRL3 comprising the amino acid sequence QQYVSYWT (SEQ ID NO: 22); or (4) CDRH1 heavy chain variable region comprising the amino acid sequence FTFSSYGMH (SEQ ID NO: 17); CDRH2 comprising the amino acid sequence GIWWDGSNEKYADSVKG (SEQ ID NO: 23); CDRH3 comprising the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19); and CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSDILA (SEQ ID NO: 20); CDRL2 comprising the amino acid sequence GASRRAT (SEQ ID NO: 24); and CDRL3 comprising the amino acid sequence QQYVSYWT (SEQ ID NO: 22), wherein the HER2 antibody or antigen-binding fragment thereof is conjugated directly or indirectly to at least one therapeutic agent, wherein the therapeutic agent is a small molecule having a molecular weight < about 5 kDa, < about 4 kDa, < about 3 kDa, < about 1.5 kDa, or < about 1 kDa. The invention also provides kits and/or methods for identifying or otherwise refining, e.g., stratifying, a patient population suitable for therapeutic administration of a HER2 antibody or antigen-binding fragment thereof and/or PBRM-polymer-drug conjugates thereof described herein by identifying patients having low HER2 expression prior to treatment with a HER2 antibody or antigen-binding fragment thereof, and/or conjugates thereof described herein. Low HER2 expression represents those patients having less than or equal to 100,000, less than or equal to 90,000, or less than or equal to 80,000 HER2 molecules per cell, for example, as measured per cell in a test cell population. The level of HER2 expression, i.e., the number of HER2 molecules per cell, can be measured using any art-recognized method, including but not limited to the use of cell-based viability assays shown in the working examples provided herein (see e.g., Example 18).

[00142] The invention also provides kits and/or methods for identifying or otherwise refining, e.g., stratifying, a patient population suitable for therapeutic administration of a HER2 antibody or antigen-binding fragment thereof, and/or conjugates thereof described herein by identifying the subject's HER2 count prior to treatment with a HER2 antibody or antigen-binding fragment thereof, and/or conjugates thereof described herein. In some embodiments, the subject is identified as having a score of 1+ or 2+ for HER2 expression. In some embodiments, the subject is identified as having a score of 1+ or 2+ for HER2 expression as detected by immunohistochemistry (IHC) analysis performed on a test cell population, and wherein the HER2 gene is not amplified in the test cell population. In some embodiments, the test cell population is derived from fresh, unfrozen tissue from a biopsy sample. In some embodiments, the test cell population is derived from frozen tissue from a biopsy specimen.

[00143] The IHC test measures the amount of HER2 receptor protein on the surface of cells in a cancerous tissue sample, for example, a breast cancer tissue sample or a gastric cancer sample, and designates the detected level of a cell surface HER2 receptor HER2 count of 0, 1+, 2+, or 3+. If the individual's HER2 count is in the range of 0 to 1+, the cancer is judged to be “HER2 negative.” If the count is 2+, the cancer is referred to as “borderline,” and a count of 3+ means the cancer is “HER2 positive.”

[00144] In some embodiments, the individual is identified as having a score of 1+ or 2+ for HER2 expression and is refractory to chemotherapy, including standard, front-line chemotherapeutic agents. As used herein, the term individual includes humans and other mammals. In some embodiments, the individual is identified as having a score of 1+ or 2+ for HER2 expression and is suffering from breast cancer, gastric cancer, non-small cell lung cancer (NSCLC), or ovarian cancer.

[00145] In some embodiments, the antibodies or antigen-deleting fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of breast cancer in patients who have HER2 IHC 1+ or HER2 IHC 2+ without gene amplification, e.g., FISH- (or negative fluorescence in situ hybridization).

[00146] In some embodiments, the antibodies or antigen-deleting fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of breast cancer in patients who have advanced HER2-positive breast cancer and who have received prior treatment with Kadcila.

[00147] In some embodiments, the antibodies or antigen-deleting fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of breast cancer in patients who have advanced HER2-positive breast cancer and who have not received prior treatment with Kadcila.

[00148] In some embodiments, the antibodies or antigen-deleting fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of gastric cancer in patients who have HER2 IHC 1+ or HER2 IHC 2+ without gene amplification, e.g., FISH-.

[00149] In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, delaying the progression of, or otherwise ameliorating a symptom of non-small cell lung cancer (NSCLC) in patients who have HER2 IHC 2+, HER2 IHC 3+, any HER2 gene amplification or mutation status.

[00150] In some embodiments, the individual is refractory to chemotherapy, including standard, front-line chemotherapeutic agents. In some embodiments, the individual is resistant to treatment with Kadcila.

[00151] A HER2 antibody or antigen-binding fragment thereof and/or conjugated HER2 antibody or antigen-binding fragment thereof used in any of the embodiments of the methods and uses provided herein can be administered at any stage of the disease. For example, such a HER2 antibody and/or conjugated HER2 antibody can be administered to a patient suffering from cancer of any stage, from early to metastatic.

[00152] A HER2 antibody and/or HER2 antibody conjugate used in any of the embodiments of these methods and uses can be administered alone or in combination with one or more chemotherapeutic agents or other agents. In some embodiments, the agent is any of the toxins described herein. In some embodiments, the agent is (1) HER2 inhibitors, (2) EGFR inhibitors (e.g., tyrosine kinase inhibitors or targeted anti-EGFR antibodies), (3) BRAF inhibitors, (4) ALK inhibitors, (5) hormone receptor inhibitors, (6) mTOR inhibitors, (7) VEGF inhibitors, or (8) cancer vaccines. In some embodiments, the agent is a standard, first-line chemotherapeutic agent such as, for example, trastuzumab, pertuzumab, adotrastuzumab emtansine (Kadcila), lapatinib, anastrozole, letrozole, exemestane, everolimus, fulvestrant, tamoxifen, toremifene, megestrol acetate, fluoxymesterone, ethinyl estradiol, paclitaxel, capecitabine, gemcitabine, eribulin, vinorelbine, cyclophosphamide, carboplatin, docetaxel, albumin-bound paclitaxel, cisplatin, epirubicin, ixabepilone, doxorubicin, fluorouracil, oxaliplatin, fluoropyrimidine, irinotecan, ramucirumab, mitomycin, leucovorin, cetuximab, bevacizumab, erlotinib, afatinib, crizotinib, permetrexed, ceritinib, etoposide, vinblastine, vincristine, ifosfamid, liposomal doxorubicin, topotecan, altretamine, melphalan, or leuprolide acetate. In some embodiments, the second agent is Kadcila.

[00153] In some embodiments, the agent is at least a second antibody or antigen-binding fragment thereof that specifically binds HER2. In some embodiments, the HER2 antibody and/or HER2 antibody conjugate are administered in combination with a HER2 antibody, a HER2 dimerization-inhibiting antibody, or a combination of a HER2 antibody and a HER2 dimerization-inhibiting antibody, such as, for example, trastuzumab or pertuzumab, or a combination thereof. In some embodiments, the HER2 antibody and/or HER2 antibody conjugate are administered in combination with a trastuzumab biosimilar or a pertuzumab biosimilar, or a combination thereof.

[00154] These combinations of HER2 antibodies and/or HER2 antibody conjugates are useful in the treatment of pathologies such as, for example, cancer. For example, these combinations of HER2 antibodies and/or HER2 antibody conjugates, e.g., a HER2 antibody or antigen-binding fragment thereof and/or HER2 antibody conjugates (e.g., HER2 antibody-polymer-drug conjugates) described herein in combination with trastuzumab, pertuzumab, or both trastuzumab and pertuzumab, or a biosimilar of trastuzumab, a biosimilar of pertuzumab, or both biosimilars, are useful in treating, preventing, slowing the progression of, or otherwise improving a symptom of a cancer (e.g., a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esogastric cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, myeloma, prostate cancer, oral cancer, rectal cancer, throat cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, kidney cancer, and gastric cancer).

[00155] These combinations are also useful for enhancing HER2 degradation when a HER2-expressing cell is contacted with these combinations. The level of HER2 degradation is detected using any art-recognized method for detecting HER2 degradation, including, but not limited to, detecting levels of HER2 degradation in the presence and absence of a HER2 antibody combination (or biosimilars thereof) as shown in the working examples provided herein (see, for example, Example 14). For example, the level of HER2 degradation is determined using western analysis of lysates from HER2-expressing cells that have been treated with a HER2 antibody combination, as compared to the level of HER2 degradation in HER2-expressing cells that have not been treated with a HER2 antibody combination.

[00156] In some embodiments, the HER2 antibody and/or conjugated HER2 antibody and additional agent(s) are formulated into a single therapeutic composition, and the HER2 antibody and/or conjugated HER2 antibody and additional agent are administered simultaneously. Alternatively, the HER2 antibody and/or conjugated HER2 antibody and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the HER2 antibody and/or conjugated HER2 antibody and additional agent are administered simultaneously, or the HER2 antibody and/or conjugated HER2 antibody and additional agent are administered at different times during a treatment regimen. For example, the HER2 antibody and/or HER2 antibody conjugate are administered prior to administration of the additional agent, the HER2 antibody and/or HER2 antibody conjugate are administered subsequent to administration of the additional agent, or the HER2 antibody and/or HER2 antibody conjugate and the additional agent are administered in an alternating manner. As described herein, the HER2 antibody and/or HER2 antibody conjugate and the additional agent are administered in single doses or in multiple doses.

[00157] Pharmaceutical compositions according to the invention may include an antibody, fragment thereof, and/or conjugate thereof described herein and a suitable carrier. These pharmaceutical compositions may be included in kits, such as, for example, diagnostic kits.

[00158] One of skill in the art will appreciate that the antibodies described herein have a variety of uses. For example, the proteins disclosed herein are used as therapeutic agents. The antibodies described herein are also used as reagents in diagnostic kits or as diagnostic tools, or these antibodies may be used in competition assays to generate therapeutic reagents.

[00159] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, the present specification, including the definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[00160] Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

[00161] Figure 1a shows epitope binning for antibodies XMT 1517 and XMT 1518 by Octet Red384 Epitope Binning.

[00162] Figure 1b shows epitope binning for antibodies XMT 1519 and XMT 1520 by Octet Red384 Epitope Binning.

[00163] Figure 2 shows cell surface binding of XMT 1517, XMT 1518, XMT 1519 and XMT 1520 antibodies to JIMT-1 cells.

[00164] Figure 3a shows the binding affinities of antibodies XMT 1517, XMT 1518 to recombinant human HER2 and cynomolgus monkey HER2.

[00165] Figure 3b shows the binding affinities of antibodies XMT 1519, XMT 1520 to recombinant human HER2 and cynomolgus monkey HER2.

[00166] Figure 3c shows the binding affinities of XMT 1519 and Example 16H to human recombinant HER2.

[00167] Figure 3d shows the binding affinities of XMT 1519 and Example 16H to cynomolgus monkey HER2.

[00168] Figure 4 shows that antibodies XMT 1518 and XMT 1519 compete for binding to HER2.

[00169] Figure 5 shows the antibody-dependent cellular cytotoxicity (ADCC) for trastuzumab and antibodies XMT 1518, XMT 1519, and XMT 1520.

[00170] Figure 6 shows the internalization rate for trastuzumab and HT antibodies XMT 1518, XMT 1519, and XMT 1520.

[00171] Figure 7 shows the internalization of HER2 antibodies in SKBR3 cells.

[00172] Figure 8 shows HER2 degradation induced by a combination of anti-HER2 antibodies.

[00173] Figure 9 illustrates the antitumor efficacy of Example 16A, trastuzumab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), and Example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), as measured in an NCI-N87 mouse tumor xenograft model.

[00174] Figure 10 illustrates the antitumor efficacy of Kadcila; Example 16A, trastuzumab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); and Example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a JIMT-1 mouse tumor xenograft model.

[00175] Figure 11 illustrates the antitumor efficacy of Kadcila;pertuzumab; a combination of Kadcila and pertuzumab; and Example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a JIMT-1 mouse tumor xenograft model.

[00176] Figure 12 illustrates the antitumor efficacy of Example 16F, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); a combination of trastuzumab and pertuzumab; or a triple combination of trastuzumab; pertuzumab and Example 16F, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in an NCI-N87 mouse tumor xenograft model.

[00177] Figure 13 illustrates the antitumor efficacy of Kadcila, Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a SNU5 mouse tumor xenograft model.

[00178] Figure 14 illustrates the antitumor efficacy of Kadcila and Example16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a TOV-21G mouse tumor xenograft model.

[00179] Figure 15 illustrates the antitumor efficacy of Kadcila, Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in an H522 mouse tumor xenograft model.

[00180] Figure 16 illustrates the antitumor efficacy of Kadcila, Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a SKOV3 mouse tumor xenograft model.

[00181] Figure 17 illustrates the antitumor efficacy of Example 16F,XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a Calu-3 mouse tumor xenograft model.

[00182] Figure 18 illustrates the antitumor efficacy of Kadcila, Example16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a BRE-0333 HER2 1+ patient-derived xenograft model.

[00183] Figure 19 illustrates the antitumor efficacy of Kadcila, Example16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a MAXF_1162 HER2 3+ patient-derived xenograft model.

[00184] Figure 20 illustrates the antitumor efficacy of Kadcila, Example 16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Example 17C, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a BT474 mouse tumor xenograft model.

DETAILED DESCRIPTION

[00185] The present invention provides monoclonal antibodies that specifically bind human HER2 in soluble, or membrane-bound (i.e., when expressed on a cell surface) form. The invention further provides monoclonal antibodies that specifically bind HER2. HER2. These antibodies are collectively referred to herein as “HER2” antibodies.

[00186] The antibodies of the present invention bind to a HER2 epitope with an equilibrium dissociation constant (Kd or KD) of <1 μM, e.g., <100 nM, preferably <10 nM, and most preferably <1 nM. For example, the HER2 antibodies provided herein exhibit a Kd in the range of approximately <1 nM to about 1 pM.

[00187] The HER2 antibodies described herein serve to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of HER2. HER2. Functional activities of HER2 include, for example, modulating the activity of the PI3K-Akt pathway. For example, HER2 antibodies completely or partially inhibit the functional activity of HER2 by modulating, blocking, inhibiting, reducing, antagonizing, partially or completely neutralizing, or otherwise interfering with the activity of the PI3K-Akt pathway. The activity of the PI3K-Akt pathway is assessed using any art recognized method for detecting PI3K-Akt pathway activity, including, but not limited to, detecting levels of phosphorylated Akt in the presence and absence of an antibody or antigen binding fragment described herein.

[00188] HER2 antibodies are considered to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise completely interfere with the functional activity of HER2 when the level of HER2 functional activity in the presence of the HER2 antibody is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% when compared to the level of HER2 functional activity in the absence of binding with a HER2 antibody described herein. HER2 antibodies are considered to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise partially interfere with the functional activity of HER2 when the level of HER2 activity in the presence of the HER2 antibody is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% when compared to the level of HER2 activity in the absence of binding with a HER2 antibody described herein.

Definitions

[00189] Unless otherwise defined, scientific and technical terms used in connection with the present invention should have the meanings that are customarily understood by those of ordinary skill in the art. Furthermore, unless otherwise required by the context, singular terms should include pluralities and plural terms should include the singular. Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and customarily used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications or as customarily performed in the art or as described herein. The foregoing techniques and procedures are generally performed in accordance with conventional methods well known in the art and as described in numerous general and more specific references that are cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[00190] As used in accordance with this description, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[00191] As used herein, the term “HER2” (also known as ErbB-2, NEU, HER-2, and CD340), when used herein, refers to human epidermal growth factor receptor 2 (SwissProt P04626) and includes any of the HER2 variants, isoforms, and species homologs that are naturally expressed by cells, including tumor cells, or are expressed in cells transfected with the HER2 gene. The HER2 species homolog is from rhesus monkey (Macaca mulatta; Genbank accession No. GI: 109114897). These terms are synonymous and may be used interchangeably.

[00192] As used herein, the term “HER2 antibody” or “anti-HER2 antibody” is an antibody that specifically binds to the HER2 antigen.

[00193] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” “or directed against” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 10-6). Antibodies include, but are not limited to, dAb (domain antibody), polyclonal, monoclonal, chimeric, single chain, Fab, Fab’ and F(ab’)2 fragments, scFvs, and a Fab expression library.

[00194] The basic structural unit of the antibody is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a “light” (about 25 kDa) and a “heavy” (about 50 to 70 kDa) chain. The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans refer to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[00195] The terms “monoclonal antibody” (mAb) or “monoclonal antibody composition,” as used herein, refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a single light chain gene product and a single heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all molecules of the population. mAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[00196] In general, antibody molecules obtained from humans refer to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[00197] The terms “antigen-binding site” or “binding portion” refer to the part of the immunoglobulin molecule that participates in antigen binding. The antigen-binding site is formed by the amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “matrix regions,” or “FRs.” Thus, the term “FR” refers to the amino acid sequences that are naturally found between, and adjacent to, the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity determining regions,” or “CDRs.” The amino acid designation for each domain is in accordance with the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901–917 (1987), Chothia et al. Nature 342:878–883 (1989).

[00198] The terms “fragment,” “antibody fragment,” “antigen-binding fragment,” and “antigen-binding fragment” are used interchangeably herein, unless otherwise specified.

[00199] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof, or a T cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitopic determinants usually consist of chemically active surface clusters of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is < 1 μM; for example, < 100 nM, preferably < 10 nM, and most preferably < 1 nM.

[00200] When used herein in the context of two or more antibodies, the terms “competes with” or “cross-competes with” indicate that the two or more antibodies compete for binding to HER2, e.g., compete for binding to HER2 in the assay described in Examples 5 or 8. An antibody “blocks” or “cross-blocks” one or more other antibodies from binding to HER2 if the antibody competes with the one or more other antibodies 25% or more, with 25% to 74% representing “partial blocking” and 75% to 400% representing “complete blocking”, preferably as determined using the assay of Examples 5 and 8. For some pairs of antibodies, competition or blocking in the assay of Examples 5 or 8 is only observed when one antibody is coated to the plate and the other is used to compete, and not vice versa. Unless otherwise defined or negated by the context, the terms “competes with”, “cross-competes with”, “blocks” or “cross-blocks” when used herein are also intended to encompass such antibody pairs.

[00201] As used herein an antibody that “inhibits HER dimerization” shall mean an antibody that inhibits, or interferes with, the formation of a HER dimer. Preferably, such an antibody binds to HER2 at the heterodimeric binding site thereof. In one embodiment the dimerization-inhibiting antibody herein is pertuzumab or MAb 2C4. Other examples of antibodies that inhibit HER dimerization include antibodies that bind to EGFR and inhibit its dimerization with one or more other HER receptors, such as, for example, EGFR monoclonal antibody 806, MAb 806, which binds to activated or “unabsorbed” EGFR (see Johns et al., J. Biol. Chem. 279(29): 30375-30384 (2004)); antibodies that bind to HER3 and inhibit its dimerization with one or more other HER receptors; and antibodies that bind to HER4 and inhibit its dimerization with one or more other HER receptors.

[00202] The term “HER2 dimerization inhibitor” as used herein shall mean an agent that inhibits the formation of a dimer or heterodimer comprising HER2.

[00203] As used herein, the term “internalization” when used in the context of a HER2 antibody includes any mechanism by which the antibody is internalized into a HER2-expressing cell from the cell surface and/or the surrounding medium, e.g., via endocytosis.

[00204] As used herein, the terms “immunological binding,” and “immunological binding properties” refer to non-covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, where a lower Kd represents a higher affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of formation and dissociation of the antigen/antigen binding site complex, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “association constant” (Kon) and the “dissociation constant” (Koff) can be determined by calculating the concentrations and actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). The Koff/Kon ratio allows cancellation of all non-affinity related parameters, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59: 439-473). An antibody of the present invention is said to specifically bind to HER2 when the equilibrium dissociation constant (Kd or KD) is ≥1 μM, preferably <100 nM, more preferably <10 nM, and most preferably <100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[00205] The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide to which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide that is not linked in nature, or (3) does not occur in nature as part of a larger sequence. The polynucleotides according to the invention include the nucleic acid sequences of SEQ ID NOs: 34 and 36, as well as nucleic acid molecules encoding the heavy chain immunoglobulin molecules shown in SEQ ID NOs: 1, 3, 5, and 7, and the nucleic acid sequences of SEQ ID NOs: 35 and 37, as well as nucleic acid molecules encoding the light chain immunoglobulin molecules shown in SEQ ID NOs: 2, 4, 6, and 8.

[00206] The term “isolated protein” as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins of the same source, (3) is expressed by a cell of a different species, or (4) does not occur in nature.

[00207] The term "polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogues of a polypeptide sequence. Accordingly, native protein fragments, and analogues are species of the genus polypeptide. The polypeptides according to the invention comprise the heavy chain immunoglobulin molecules represented in SEQ ID NOs: 1, 3, 5, and 7, and the light chain immunoglobulin molecules represented in SEQ ID NOs: 2, 4, 6, and 8 as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogues thereof.

[00208] The term “naturally occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory or otherwise is naturally occurring.

[00209] The term "operably linked" as used herein refers to the positions of components so described that are in a relationship that allows them to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a manner that expression of the coding sequence is obtained under conditions compatible with the control sequences.

[00210] The term “control sequence” as used herein refers to those polynucleotide sequences that are necessary to effect the expression and processing of the coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term “polynucleotide” as used herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides, or a modified form of each type of nucleotide. The term includes both single-stranded and double-stranded forms of DNA.

[00211] The term "oligonucleotide" as used herein includes naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotide generally comprising a length of 200 bases or less. Preferably oligonucleotides are 10 to 60 bases in length and more preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes, although oligonucleotides may be double stranded, e.g., for use in constructing a gene mutant. The oligonucleotides described herein are sense or nonsense oligonucleotides.

[00212] The term “naturally occurring nucleotides” referred to herein includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term “oligonucleotide linkages” referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranylated, phosphoronmidate, and the like. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16: 3209 (1988), Zon et al. Anti Cancer Drug Design 6: 539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90: 543 (1990). An oligonucleotide may include a label for detection, if desired.

[00213] The term “selectively hybridizes” as used herein means bind detectably and specifically. Polynucleotides, oligonucleotides, and fragments thereof according to the invention selectively hybridize to nucleic acid strands under hybridization and washing conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions may be used to obtain selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments described herein and a nucleic acid sequence of interest will be at least 80%, and more typically with preferably increasing homologies of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in each of the two sequences being compared) that are allowed in the matching-maximizing gap lengths of 5 or less are preferred with 2 or less being most preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as that term is used herein, if they have an alignment score of more than 5 (in standard deviation units) using the ALIGN program with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. Two sequences or parts thereof are most preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program. The term “corresponds to” is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.

[00214] The following terms are used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: “reference sequence”, “comparison window”, “sequence identity”, “percent sequence identity”, and “substantial identity”. A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or it may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, often at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotide or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotide or amino acid sequences, sequence comparison between two (or more) molecules is typically accomplished by comparing the sequences of the two molecules within a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window,” as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids in which a polynucleotide sequence or amino acid sequence can be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and in which the portion of the polynucleotide sequence in the comparison window can comprise additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less when compared to the reference sequence (which comprises no additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences to align a comparison window can be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970), by the similarity search method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the software packages Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology in the comparison window) generated by the various methods is selected.

[00215] The term “sequence identity” means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) within the comparison window. The term “percent sequence identity” is calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs in both sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the percent sequence identity. The terms “substantial identity” as used herein indicates a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity when compared to a reference sequence over a comparison window of at least 18 nucleotide positions (6 amino acids), often over a window of at least 24 to 48 nucleotide positions (8 to 16 amino acids), wherein the percent sequence identity is calculated by comparing the reference sequence to a sequence that may include deletions or additions that total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[00216] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Ssobland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, o-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used here, the leftward direction is the amino-terminal direction and the rightward direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[00217] Similarly, unless otherwise specified, the left-hand end of single-stranded polynucleotide sequences is the 5' end, and the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The 5' to 3' direction of addition of nascent RNA transcripts is referred to as the transcription direction; regions of sequence on the DNA strand having the same sequence as the RNA and that are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences," regions of sequence on the DNA strand having the same sequence as the RNA and that are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences."

[00218] As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

[00219] Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[00220] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. The preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[00221] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are considered to be encompassed by the present invention, provided that the variations in the amino acid sequence remain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) the acidic amino acids are aspartate, glutamate; (2) the basic amino acids are lysine, arginine, histidine; (3) The nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) the uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide-containing family; (iii) alanine, valine, leucine, and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar substitution of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a scaffold site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. The preferred amino and carboxy termini of fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and/or amino acid sequence data with public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predict protein conformational domains that occur in other proteins of known structure and/or function. Methods for identifying protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains according to the invention.

[00222] Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs may include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain(s) that form intermolecular contacts). A conservative amino acid substitution should not substantially change the structural characteristics of the precursor sequence (e.g., an amino acid substitution should not tend to disrupt a helix that occurs in the precursor sequence, or disrupt other types of secondary structure that characterize the precursor sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354: 105 (1991).

[00223] The term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion at the amino terminus and/or carboxy terminus, but where the remaining amino acid sequence is identical to the corresponding positions in the deduced naturally occurring sequence, for example, from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length, and even more preferably at least 70 amino acids in length. The term “analog” as used herein refers to polypeptides that are comprised of a segment of at least 25 amino acids that have substantial identity to a portion of a deduced amino acid sequence and that have specific binding to HER2 under suitable binding conditions. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally occurring sequence. Analogs are typically at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a naturally occurring full-length polypeptide.

[00224] Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the standard peptide. These types of non-peptide compounds are called “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15: 29 (1986), Veber and Freidinger TINS p, 392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987). Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as a human antibody, but have one or more peptide bonds optionally replaced by a bond selected from the group consisting of: --CH2NH--, --CH2S-, --CH2-CH2--, --CH=CH-- (cis and trans), --COCH2--, CH(OH)CH2--, and -CH2SO--, by methods well known in the art. Systematic replacement of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Furthermore, constricted peptides comprising a substantially identical consensus sequence or a variation of a consensus sequence can be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by the addition of internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[00225] The term “agent” is used herein to indicate a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[00226] As used herein, the terms “label” or “labeled” refer to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or attachment* to a polypeptide of biotinyl moieties that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 14 15 35 90 99 111 125 131 ... In some embodiments, the labels are linked by spacer branches of various lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when appropriately administered to a patient.

[00227] Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[00228] As used herein, “substantially pure” means that a subject species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition in which the subject species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

[00229] Generally, a substantially pure composition will comprise greater than about 80 percent of all macromolecular species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%. Most preferably, the subject species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[00230] The use of the articles “a,” “an,” and “the” in both the description and claims below are to be construed to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. The terms “comprising,” “having,” “being of” as in “being of a chemical formula,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including but not limited to”) unless otherwise stated. For example, a polymer scaffold of a certain formula includes all of the monomer units shown in the formula and may also include additional monomer units not shown in the formula. Additionally, whenever “comprising” or another open-ended term is used in an embodiment, it is to be understood that the same embodiment may be more narrowly claimed using the intermediate term “consisting essentially of” or the closed-ended term “consisting of.”

[00231] The term “about,” “approximately,” or “approximate,” when used in connection with a numeric value, means that a collection or range of values are included. For example, “about X” includes a range of values that are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numeric value. In one embodiment, the term “about” refers to a range of values that are 5% more or less than the specified value. In another embodiment, the term “about” refers to a range of values that are 2% more or less than the specified value. In another embodiment, the term “about” refers to a range of values that are 1% more or less than the specified value.

[00232] The recitation of ranges of values is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if individually recited herein. A range as used herein, unless otherwise specified, includes both ends of the range. For example, the expressions “x being an integer between 1 and 6” and “x being an integer from 1 to 6” both mean “x being 1, 2, 3, 4, 5, or 6”, that is, the terms “between X and Y” and “range from X to Y” are inclusive of X and Y and the integers between them.

[00233] All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illustrate the invention and should not be construed as a limitation on the scope of the claims unless otherwise explicitly claimed. No language in the specification should be construed as indicating that any unclaimed element is essential to what is claimed.

[00234] “Protein-based recognition molecule” or “PBRM” refers to a molecule that recognizes and binds to a cell surface marker or receptor such as a transmembrane protein, surface-immobilized protein, or protoglycan. Examples of PBRMs include but are not limited to the XMT 1517 antibody, the XMT 1518 antibody, the XMT 1519 antibody, and the XMT 1520 antibody described herein, as well as other antibodies (e.g., Trastuzumab, Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab, Labetuzumab, B7-H4, B7-H3, CA125, CD33, CXCR2, EGFR, FGFR1, FGFR2, FGFR3, FGFR4, HER2, NaPi2b, c-Met, MUC-1, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1- and anti-5T4), and antibodies or antigen-binding fragments thereof described herein) or peptides (LHRH receptor targeting peptides, EC-1 peptide), lipocalins, such as, for example, anticalins, proteins such as, for example, interferons, lymphokines, growth factors, colony stimulating factors, and the like, peptides or peptide mimetics, and the like. The protein-based recognition molecule, in addition to targeting the modified polymeric conjugate to a specific cell, tissue or location, may also have certain therapeutic effects such as antiproliferative (cytostatic and/or cytotoxic) activity against a target cell or pathway. The protein-based recognition molecule comprises or may be engineered to comprise at least one chemically reactive group such as, -COOH, primary amine, secondary amine, -NHR, -SH, or a chemically reactive amino acid moiety or side chains such as, for example, tyrosine, histidine, cysteine, or lysine.

[00235] “Biocompatible” as used herein is intended to describe compounds that exert minimal destructive effects or host response while in contact with body fluids or living cells or tissues. Thus a biocompatible group, as used herein, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moiety, which falls within the definition of the term biocompatible, as defined above and herein. The term “Biocompatibility” as used herein, is also taken to mean that the compounds exhibit minimal interactions with recognition proteins, e.g., naturally occurring antibodies, cellular proteins, cells and other components of biological systems, unless such interactions are specifically desirable. Thus, substances and functional groups specifically intended to cause the above minimal interactions, e.g., drugs and prodrugs, are considered to be biocompatible. Preferably (with the exception of compounds intended to be cytotoxic, such as, for example, antineoplastic agents), compounds are “biocompatible” if their addition to normal cells in vitro, at concentrations similar to the intended systemic concentrations in vivo, results in less than or equal to 1% cell death during a time equivalent to the half-life of the compound in vivo (e.g., the period of time required for 50% of the compound administered in vivo to be eliminated/cleared), and their administration in vivo induces minimal and medically acceptable inflammation, foreign body reaction, immunotoxicity, chemical toxicity and/or other such adverse effects. In the above sentence, the term “normal cells” refers to cells that are not intended to be killed or otherwise significantly affected by the compound being tested.

[00236] “Biodegradable”: As used herein, “biodegradable” polymers are polymers that are susceptible to biological processing in vivo. As used herein, “biodegradable” compounds or moieties are those that, when taken up by cells, can be broken down by lysosomes or other chemical mechanisms or by hydrolysis into components that the cells can reuse or dispose of without significant toxic effect on the cells. The term “biocleavable” as used herein has the same meaning as “biodegradable”. The degradation fragments preferably induce little or no organ or cell burden or pathological processes caused by such burden or other adverse effects in vivo. Examples of biodegradation processes include enzymatic and non-enzymatic hydrolysis, oxidation and reduction. Suitable conditions for non-enzymatic hydrolysis of the biodegradable protein-polymer-drug conjugates (or components thereof, e.g., the biodegradable polymeric carrier and the linkers between the carrier and the antibody or drug molecule) described herein, for example, include exposing the biodegradable conjugates to water at a temperature and pH of the intracellular lysosomal compartment. Biodegradation of some protein-polymer-drug conjugates (or components thereof, e.g., the biodegradable polymeric carrier and the linkers between the carrier and the antibody or drug molecule) may also be enhanced extracellularly, for example, in low pH regions of the animal body, e.g., an inflamed area, in the immediate vicinity of activated macrophages or other cells that release factors facilitating degradation. In certain preferred embodiments, the effective size of the polymeric carrier at pH ~7.5 does not change detectably in 1 to 7 days, and remains within 50% of the original size of the polymer for at least several weeks. At pH ~5, on the other hand, the polymeric carrier preferably degrades detectably within 1 to 5 days, and is completely transformed into low molecular weight fragments within a time period of two weeks to several months. The integrity of the polymer in such tests can be measured, for example, by size exclusion HPLC. Although more rapid degradation may in some cases be preferable, in general it may be more desirable for the polymer to degrade in the cells at a rate that does not exceed the rate of metabolism or excretion of the polymer fragments by the cells. In preferred embodiments, the polymers and by-products of polymer biodegradation are biocompatible.

[00237] “Maleimide blocking compound”: as used herein refers to a compound that can react with maleimide to convert it to succinimide and “maleimide blocking moiety” refers to the chemical moiety attached to succinimide in the conversion. In certain embodiments, the maleimide blocking compound is a compound having a terminal thiol group for reacting with maleimide. In one embodiment, the maleimide blocking compound is cysteine, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH2SH), benzyl thiol wherein phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol.

[00238] “Hydrophilic”: The term “hydrophilic” as it refers to substituents, for example, on polymer monomer units or on a maleimide blocking moiety to make it hydrophilic or water-soluble, does not differ essentially from the ordinary meaning of this term in the art, and indicates chemical moieties that contain ionizable, polar, or polarizable atoms, or that can otherwise be solvated by water molecules. Thus a hydrophilic group, as used herein, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moiety, which falls within the definition of the term hydrophilic, as defined above. Examples of particular hydrophilic organic moieties that are suitable include, without limitation, aliphatic or heteroaliphatic groups comprising a chain of atoms in a range of between about one and twelve atoms, hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester, thioester, aldehyde, nitrile, isonitrile, nitroso, hydroxylamine, mercaptoalkyl, heterocycle, carbamates, carboxylic acids and their salts, sulfonic acids and their salts, sulfonic acid esters, phosphoric acids and their salts, phosphate esters, polyglycol ethers, polyamines, polycarboxylates, polyesters, and polythioesters. In certain embodiments, hydrophilic substituents comprise a carboxyl group (COOH), an aldehyde group (CHO), a ketone group (COC1-4 alkyl), a methylol (CH2OH) or a glycol (e.g., CHOH-CH2OH or CH-(CH2OH)2), NH2, F, cyano, SO3H, PO3H, and the like.

[00239] The term “hydrophilic” as it refers to the polymers described herein generally does not differ from the use of this term in the art, and indicates polymers comprising hydrophilic functional groups as defined above. In a preferred embodiment, a hydrophilic polymer is a water-soluble polymer. The hydrophilicity of the polymer can be directly measured by determining the hydration energy, or determined by investigation between two liquid phases, or by chromatography on solid phases with known hydrophobicity, such as, for example, C4 or C18.

[00240] “Polymeric Carrier”: The term polymeric carrier, as used herein, refers to a polymer or a modified polymer, which is suitable for covalently binding to or can be covalently bound to one or more drug molecules with a designated ligand and/or one or more PBRMs with a designated ligand.

[00241] “Physiological Conditions”: The phrase “physiological conditions,” as used herein, refers to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered in the extracellular fluids of living tissues. For most normal tissues, the physiological pH ranges from about 7.0 to 7.4. Circulating blood plasma and normal interstitial fluid represent typical examples of normal physiological conditions.

[00242] “Drug”: As used herein, the term “drug” refers to a compound that is biologically active and provides a desired physiological effect following administration to an individual in need thereof (e.g., a pharmaceutically active ingredient).

[00243] “Cytotoxic”: As used herein, the term “cytotoxic” means toxic to cells or a selected cell population (e.g., cancer cells). The toxic effect may result in the death and/or lysis of the cell. In certain cases, the toxic effect may be a sublethal destructive effect on the cell, e.g., slowing or arresting cell growth. In order to achieve a cytotoxic effect, the drug or prodrug may be selected from a group consisting of a DNA damaging agent, a microtubule disrupting agent, or a cytotoxic protein or polypeptide, among others.

[00244] “Cytostatic”: As used herein the term “cytostatic” refers to a drug or other compound that inhibits or stops cell growth and/or multiplication.

[00245] “Small molecule”: As used herein, the term “small molecule” refers to molecules, whether naturally occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Preferred small molecules are biologically active in that they produce a local or systemic effect in animals, preferably mammals, most preferably humans. In certain preferred embodiments, the small molecule is a drug, and the small molecule is referred to as a “drug molecule” or “drug” or “therapeutic agent.” In certain embodiments, the drug molecule has a MW of less than or equal to about 5 kDa. In other embodiments, the drug molecule has a MW of less than or equal to about 1.5 kDa. In embodiments, the drug molecule is selected from vinca alkaloids, auristatins, duocarmycins, tubulysins, unnatural camptothecin compounds, topoisomerase inhibitors, DNA binding drugs, kinase inhibitors, MEK inhibitors, KSP inhibitors, calicheamicins, SN38, pyrrolobenzodiazepines, and analogs thereof. Preferably, although not necessarily, the drug is one that has already been judged safe and effective for use by an appropriate governmental agency or body, e.g., the FDA. For example, the drugs for human use listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361, and 440 through 460; the drugs for veterinary use listed by the FDA under 21 C.F.R. §§ 500 through 589, incorporated herein by reference, are all considered suitable for use with the present hydrophilic polymers.

[00246] “Drug derivative” or “modified drug” or the like as used herein, refers to a compound comprising the drug molecule intended to be delivered by the conjugate described herein and a functional group capable of linking the drug molecule to the polymeric carrier.

[00247] “Active form” as used herein refers to a form of a compound that exhibits the intended pharmaceutical efficacy in vivo or in vitro. In particular, when a drug molecule intended to be delivered by the conjugate described herein is released from the conjugate, the active form may be the drug itself or its derivatives, which exhibit the intended therapeutic properties. Release of the drug from the conjugate may be achieved by cleavage of a biodegradable linker bond that attaches the drug to the polymeric carrier. The active drug derivatives accordingly may comprise a portion of the linker.

[00248] “PHF” refers to poly(1-hydroxymethylethylene hydroxymethyl-formal).

[00249] As used herein, the terms “polymer unit”, “monomer unit”, “monomer”, “monomeric unit”, “unit” all refer to a repeatable structural unit in a polymer.

[00250] As used herein, “molecular weight” or “MW” of a polymer or polymeric carrier/scaffold or conjugated polymer refers to the weight average molecular weight of the unmodified polymer unless otherwise specified.

[00251] The present invention is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[00252] The present invention is intended to include all isomers of the compound, which refers to and includes, optical isomers, and tautomeric isomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers as well as mixtures of optical isomers including racemic and non-racemic mixtures; where an isomer may be in isolated form or in a mixture with one or more other isomers.

HER2 antibodies

[00253] The monoclonal antibodies described herein have the ability to inhibit HER2-mediated activity of the PI3K-Akt pathway.

[00254] Exemplary antibodies described herein include, for example, the XMT 1517 antibody, the XMT 1518 antibody, the XMT 1519 antibody, and the XMT 1520 antibody. These antibodies show specificity for human HER2 and they have been shown to inhibit the functional activity of HER2 in vitro.

[00255] Each of the monoclonal HER2 antibodies described herein includes a heavy chain (HC), heavy chain variable region (VH), light chain (LC), and a light chain variable region (VL), as shown in the corresponding amino acid and nucleic acid sequences presented below. The heavy chain variable region and light chain variable region for each antibody are shaded in the amino acid sequences below. The complementarity determining regions (CDRs) of the heavy chain and light chain are underlined in the amino acid sequences presented below. The amino acids spanning the complementarity determining regions (CDRs) are as defined by E. A. Kabat et al. (See Kabat, E. A., et al., Sequences of Protein of immunological interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)).>Heavy Chain Amino Acid Sequence XMT 1517 (Heavy chain variable region (SEQ ID NO: 9) + IgG1 heavy chain constant region (SEQ ID NO: 32))QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG LEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLILQMNSLRAE DTAVYYCAKEAPYYAKDYMDVWGKGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTIRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO: 1)CDRH1: FTFSSYGMH (SEQ ID NO: 17)CDRH2: VIWYDGSNKYYADSVKG (SEQ ID NO: 18) CDRH3: EAPYYAKDYMDV (SEQ ID NO: 19)>XMT Heavy Chain Variable Region Nucleic Acid Sequence 1517CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGG AGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTA GCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGG AGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCA AGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACA CGGCGGTGTACTACTGCGCCAAGGAAGCTCCCTACTACGCTAAAG ATTACATGGACGTATGGGGCAAGGGTACAACTGTCACCGTCTCCTC A (SEQ ID NO: 34)>XMT 1517 Light Chain Amino Acid Sequence (Region light chain variable region (SEQ ID NO: 10) + light chain constant region (SEQ ID NO: 33) EIVLTQSPGTLSLSPGERATLSCRASQSVSSDILAWYQQKPGQAPR LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYV SYWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2) CDRL1: RASQSVSSDILA (SEQ ID NO: 20) CDRL2: GASSRAT (SEQ ID NO: 21) CDRL3: QQYVSYWT (SEQ ID NO: 22) > Nucleic Acid Sequence of Light Chain Variable Region Lightweight of CAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGT ACGTCAGTTACTGGACTTTTGGCGGAGGGACCAAGGTTGAGATCA AA (SEQ ID NO: 35)>Heavy Chain Amino Acid Sequence of XMT 1518 (Heavy Chain Variable Region (SEQ ID NO: 11) + IgG1 heavy chain constant (SEQ ID NO: 32))QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG LEWVAGIWWDGSNEKYADSVKGRFTISRDNSKNTLILQMNSLRA EDTAVYYCAKEAPYYAKDYMDVWGKGTTVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTIRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO: 3)CDRH1: FTFSSYGMH (SEQ ID NO: 17)CDRH2: GIWWDGSNEKYADSVKG (SEQ ID NO: 23)CDRH3: EAPYYAKDYMDV (SEQ ID NO: 19)>XMT 1518 Light Chain Amino Acid Sequence (Light chain variable region (SEQ ID NO: 12) + Light chain constant (SEQ ID NO: 33))EIVLTQSPGTLSLSPGERATLSCRASQSVSSDILAWYQQKPGQAPR LLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY VSYWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4)CDRL1: RASQSVSSDILA (SEQ ID NO: 20)CDRL2: GASRRAT (SEQ ID NO: 24)CDRL3: QQYVSYWT (SEQ ID NO: 22)>Heavy Chain Amino Acid Sequence XMT 1519 (Heavy chain variable region (SEQ ID NO: 13) + IgG1 heavy chain constant region (SEQ ID NO: 32))EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKG LEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLILQMNSLRAEDT AVYYCARGGHGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTIRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO: 5)CDRH1: FTFSSYSMN (SEQ ID NO: 25)CDRH2: YISSSSSTIYYADSVKG (SEQ ID NO: 26) CDRH3: GGHGYFDL (SEQ ID NO: 27)>XMT Heavy Chain Variable Region Nucleic Acid Sequence 1519GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGG GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTA GCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTTTCATACATTAGTAGTAGTAGTAGTACCATATACTACGC AGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAA GAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACAC GGCGGTGTACTACTGCGCCAGAGGTGGACACGGATATTTCGACCT ATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 36) >XMT 1519 Light Chain Amino Acid Sequence (Light chain variable region (SEQ ID NO: 14) + Light chain constant region (SEQ ID NO: 33))EIVLTQSPGTLSLSPGERATLSCRASQSVSSSILAWYQQKPGQAPR LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYH HSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6)CDRL1: RASQSVSSSILA (SEQ ID NO: 28)CDRL2: GASSRAT (SEQ ID NO: 21)CDRL3: QQYHHSPLT (SEQ ID NO: 29)>XMT Light Chain Variable Region Nucleic Acid Sequence 1519GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCA GCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCA GGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGA CAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGT ACCACCACAGTCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGA TCAAA (SEQ ID NO: 37) > XMT 1520 Heavy Chain Amino Acid Sequence (Heavy chain variable region (SEQ ID NO: 15) + IgG1 heavy chain constant region (SEQ ID NO: 32))EVQLVESGGGLVQPGGSLRLSCAASGFTFSGRSMNWVRQAPGKG LEWVSYISSDSRTIYYADSVKGRFTISRDNAKNSLILQMNSLRAED TAVYYCARGGHGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTIRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* (SEQ ID NO: 7)CDRH1: FTFSGRSMN (SEQ ID NO: 30)CDRH2: YISSDSRTIYYADSVKG (SEQ ID NO: 31) CDRH3: GGHGYFDL (SEQ ID NO: 27)> XMT 1520 Light Chain Amino Acid Sequence (Light chain variable region (SEQ ID NO: 16) + Light chain constant region (SEQ ID NO: 33))EIVLTQSPGTLSLSPGERATLSCRASQSVSSSILAWYQQKPGQAPR LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYH HSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8)CDRL1: RASQSVSSSILA (SEQ ID NO: 28)CDRL2: GASSRAT (SEQ ID NO: 21)CDRL3: QQYHHSPLT (SEQ ID NO: 29)

[00256] Also included in the invention are antibodies and antigen-binding fragments thereof that bind to the same epitope or cross-compete for binding to the same epitope as the antibodies and antigen-binding fragments thereof described herein. For example, antibodies and antigen-binding fragments described herein specifically bind to HER2, wherein the antibody or fragment binds to an epitope that includes one or more amino acid residues in human HER2 (e.g., GenBank Accession No. P04626.1).

[00257] Antibodies and antigen-binding fragments thereof described herein specifically bind to an epitope on the full-length human HER2 receptor comprising the amino acid sequence: 1 MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY51 QGCQVVQGNL ELTILPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR101 IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK151 GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK 201 GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS251 DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP301 YNILSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL351 REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF401 ETLEEITGIL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI451 SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP501 EDECVGEGLA CHQLCARGHC WGPGPTQCVNCSQFLRGQEC VEECRVLQGL551 PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC601 PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP651 LTSIISAVVG ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPL701 TPSGAMPNQA QMRILKETEL RKVKVLGSGA FGTVYKGIWI PDGENVKIPV751 AIKVLRENTS PKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVQLVTQL801 MPYGCLLDHV RENRGRLGSQ DLLNWCMQIAKGMSILEDVR LVHRDLAARN851 VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT901 HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID951 VYMIMVKCWM IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL1001 DSTFYRSLLE DDDMGDLVDA EEILVPQQGF FCPDPAPGAG GMVHHRHRSS1051 STRSGGGDLT LGLEPVEREA PRSPLAPSEG AGSDVFDGDL GMGAAKGLQS1101 LPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV NQPDVRPQPP1151 SPREGPLPAA RPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEILTPQ1201 GGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEILG1251 LDVPV (SEQ ID NO: 38)

[00258] Antibodies and antigen-binding fragments thereof described herein specifically bind to an epitope in the extracellular domain (ECD) of the human HER2 receptor comprising the amino acid sequence: TQVCTGTDMK LRLPASPET LDMLRHLYQG CQVVQGNLEL TILPTNASLS51 FLQDIQEVQG YVLIAHNQVR QVPLQRLRIV RGTQLFEDNY ALAVLDNGDP101 LNNTTPVTGA SPGGLRELQL RSLTEILKGG VLIQRNPQLC YQDTILWKDI151 FHKNNQLALT LIDTNRSRAC HPCSPMCKGS RCWGESSEDC QSLTRTVCAG201 GCARCKGPLP TDCCHEQCAA GCTGPKHSDC LACLHFNHSG ICELHCPALV251 TYNTDTFESM PNPEGRYTFG ASCVTACPYN ILSTDVGSCT LVCPLHNQEV301 TAEDGTQRCE KCSKPCARVC YGLGMEHLRE VRAVTSANIQ EFAGCKKIFG351 SLAFLPESFD GDPASNTAPL QPEQLQVFET LEEITGILYI SAWPDSLPDL401 SVFQNLQVIR GRILHNGAYS LTLQGLGISW LGLRSLRELG SGLALIHHNT451 HLCFVHTVPW DQLFRNPHQA LLHTANRPEDECVGEGLACH QLCARGHCWG501 PGPTQCVNCS QFLRGQECVE ECRVLQGLPR EYVNARHCLP CHPECQPQNG551 SVTCFGPEAD QCVACAHYKD PPFCVARCPS GVKPDLSYMP IWKFPDEEGA601 CQPCPINCTH SCVDLDDKGC PAEQRASPLT (SEQ ID NO: 39)

[00259] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, whether a monoclonal antibody has the same specificity as a monoclonal antibody described herein (e.g., XMT 1517, XMT 1518, XMT 1519, and XMT 1520) by ascertaining whether the former prevents the latter from binding to a natural binding partner or other molecule known to be associated with HER2. If the monoclonal antibody being tested competes with the monoclonal antibody described herein, as shown by a decrease in binding by the monoclonal antibody described herein, then the two monoclonal antibodies bind to the same, or a closely related, epitope.

[00260] An alternative method for determining whether a monoclonal antibody has the monoclonal antibody specificity disclosed herein is to pre-incubate the monoclonal antibody described herein with soluble HER2 (with which it is normally reactive), and then add the monoclonal antibody being tested to determine whether the monoclonal antibody being tested is inhibited in its ability to bind HER2. If the monoclonal antibody being tested is inhibited, then it is certain to have the same or a functionally equivalent epitopic specificity as the monoclonal antibody described herein.

[00261] Screening of monoclonal antibodies described herein may also be performed, for example, by measuring the HER2-mediated activity of the PI3K-Akt pathway, and determining whether the test monoclonal antibody is capable of modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with the activity of the PI3K-Akt pathway.

[00262] HER2 antibodies are generated, for example, using the methods described in the Examples provided herein. Alternatively or in addition, various procedures known in the art can be used to produce monoclonal antibodies directed against HER2, or against derivatives, fragments, analogs, homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference.) Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and heavy chain, including the CDRs, arise from human genes. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies”. Human monoclonal antibodies are prepared, for example, using the procedures described in the Examples provided below. Human monoclonal antibodies can also be prepared using the trioma technique; the human B cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique for producing human monoclonal antibodies (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used and can be produced using human hybridomas (see Cote, et al., 1983 Proc Natl Acad Sci USA 80: 2026-2030) or by transformation of human B cells with Epstein Barr virus in vitro (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[00263] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provides primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the sought immunoglobulin, or an epitope thereof, can be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[00264] Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with HER2-mediated activity of the PI3K-Akt pathway are generated, for example, by immunizing an animal with membrane-bound and/or soluble HER2, such as, for example, murine, rat or human HER2 or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding HER2 such that HER2 is expressed and associated with the surface of the transfected cells. Alternatively, the antibodies are obtained by screening a library containing antibody or antigen binding domain sequences for binding to HER2. This library is prepared, for example, in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the coding DNA sequences contained within the phage particles (i.e., “phage-displayed library”). Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity to HER2. Additionally, antibodies selected from, and optionally optimized in, yeast antibody display libraries and yeast library display systems as described, for example in: Blaise L, Wehnert A, Steukers MP, van den Beucken T, Hoogenboom HR, Hufton SE. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to an affinity maturation of Fab antibodies fragment. Gene. 2004 Nov 24;342(2):211-8; Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. Jun 1997; 15(6): 553-7; Kuroda K, Ueda M. Cell surface engineering of yeast for applications in white biotechnology. Biotechnol Lett. Jan 2011; 33(1): 1-9. doi: 10.1007/s10529-010-0403-9.Review; Lauer TM, Agrawal NJ, Chennamsetty N, Egodage K, Helk B, Trout BL. Developability index: a rapid in silico tool for the screening of antibody aggregation propensity. J Pharm Sci Jan 2012; 101(1): 102-15; Orcutt K. D. and Wittrup K. D. (2010), 207-233 doi: 10.1007/978-3-642-01144-3_15; Rakestraw JA, Aird D, Aha PM, Baynes BM, Lipovsek D. Secretion-and- capture cell surface display for selection of target-binding proteins. Protein Eng Des Sel. 2011 Jun; 24(6): 525-30; US 8,258,082 , US 6,300,064 , US 6,696,248 , US 6,165,718 , US 6,500,644 , US 6,291,158 , US 6,291,159 , US 6,096,551 , US 6,368,805 , US 6,500,644 . Exemplary yeast library display systems are described, for example, in WO2008118476; WO2009/036379; WO2010105256; and WO2012009568. In certain embodiments, such yeast antibody display libraries or yeast library display systems are designed to mimic or reflect the diversity characteristic of the human preimmune antibody repertoire. In certain embodiments such yeast antibody display library diversity or yeast library display system diversity is generated in silico. In certain embodiments such yeast antibody display libraries or yeast library display systems comprise Saccharomyces yeast cells, such as Saccharomyces cerevisiae cells. In certain embodiments such yeast antibody display libraries or yeast library display systems comprise Pichia cells.

[00265] Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to evoke lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[00266] The immunizing agent will typically include the protein antigen, a fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are used. Hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (“HAT medium”), substances that prevent the growth of HGPRT-deficient cells.

[00267] Preferred immortalized cell lines are those that fuse efficiently, stably support high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. The most preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Mouse human myeloma and human heteromyeloma lines have also been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

[00268] The culture medium in which the hybridoma cells are grown can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody, for example, can be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

[00269] Once the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be cultured in vivo as ascites in a mammal.

[00270] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[00271] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). The hybridoma cells disclosed herein serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that otherwise do not produce immunoglobulin protein, to achieve synthesis of monoclonal antibodies in the recombinant host cells. The DNA may also be modified, for example, by substituting the coding sequence for the human constant heavy and light chain domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide may be substituted in place of the constant domains of an antibody described herein, or may be substituted in place of the variable domains of an antigen-combining site of an antibody described herein to create a chimeric bivalent antibody.

[00272] The monoclonal antibodies described herein include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.

[00273] A humanized or fully human HER2 antibody is generated, for example, using the procedures described in the Examples provided below.

[00274] In other alternative methods, a HER2 antibody is developed, for example, using phage display methods using antibodies containing only human sequences. Such methods are well known in the art, for example, in WO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated by reference. In this method, a combinatorial library of random pairs bearing phage heavy and light chains is screened using natural or recombinant source HER2 or fragments thereof. In another method, a HER2 antibody can be produced by a process in which at least one step of the process includes immunizing a transgenic, non-human animal with human HER2 protein. In this method, some of the endogenous heavy and/or light kappa chain loci of this xenogeneic non-human animal have been disabled and are incapable of the rearrangement required to generate genes encoding immunoglobulins in response to an antigen. Furthermore, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal. Thus, in response to an administered antigen, the human loci rearrange to provide genes encoding human variable regions immunospecific for the antigen. Upon immunization, therefore, the xenomouse produces B cells that secrete fully human immunoglobulins.

[00275] A variety of techniques are well known in the art for producing non-human xenogeneic animals. For example, see U.S. Pats. Nos. 6,075,181 and 6,150,584, which are hereby incorporated by reference in their entirety. This general strategy was demonstrated in connection with the generation of the first XenoMouse® strains as published in 1994. See Green et al. Nature Genetics 7: 13-21 (1994), which is hereby incorporated by reference in its entirety. See also, U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and Japanese Pat. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and European Patent No., EP 0 463 151 B1 and International Patent Application Nos. WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related family members.

[00276] In an alternative method, others have utilized a “mini-locus” method in which an exogenous Ig locus is mimicked by including pieces (individual genes) of the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. See, for example, U.S. Pat. Nos. Nos. 5,545,806, 5,545,807, 5,591,669, 5,612,205,5,625,825, 5,625,126, 5,633,425, 5,643,763, 5,661,016, 5,721,367, 5,770,429, 5,789,215, 5,789,650, 5,814,318, 5,877,397, 5,874,299, 6,023,010, and 6,255,458; and European Patent No. 0 546 073 B1; and International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and related family members.

[00277] The generation of human antibodies from mice into which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced, has also been demonstrated. See European Patent Application Nos. 773 288 and 843 961.

[00278] Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. Although chimeric antibodies have a human constant region and an immune variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multiple-dose uses of the antibody. Thus, it would be desirable to provide fully human antibodies against HER2 in order to weaken or otherwise mitigate HAMA or HACA response problems and/or effects.

[00279] Production of antibodies with reduced immunogenicity is also accomplished via humanization, chimerization, and demonstration techniques using appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See, for example, Winter and Harris Immunol Today 14:43-46 (1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). The antibody of interest can be engineered by recombinant DNA techniques to replace the CH1, CH2, CH3, hinge domains, and/or the matrix domain with the corresponding human sequence (See WO 92102190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of Ig cDNA for the construction of chimeric immunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from a hybridoma or other cell to produce the antibody and used to produce cDNA. The cDNA of interest can be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequences of interest. The DNA sequence encoding the antibody variable region is then fused to the human constant region sequences. The human constant region gene sequences can be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. publication no. 913242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. The preferred isotypes are IgG1, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, can be used. The chimeric, humanized antibody is then expressed by conventional methods.

[00280] Antibody fragments such as Fv, F(ab')2 and Fab may be prepared by cleaving the intact protein, for example by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F(ab')2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translation stop codon to produce the truncated molecule.

[00281] The consensus sequences of the J H and L regions can be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent ligation of segments of the V region to segments of the human C region. The C region cDNA can be modified by site-directed mutagenesis to place a restriction site at the analogous position in the human sequence.

[00282] Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be readily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at splicing regions occurring within human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody can be linked to any strong promoter, including retroviral LTRs, e.g., SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl et al. Cell. 41:885 (1985)). Also, as will be appreciated, native Ig promoters and the like can be used.

[00283] In addition, human antibodies or antibodies from other species can be generated by display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art, and the resulting molecules can be individualized for further maturation, such as affinity maturation, as such techniques are well known in the art. Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes and Plückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol. 17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russell et al. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A (1992), and U.S. Patent No. 5,733,743. If demonstration technologies are used to produce antibodies that are non-human, such antibodies can be humanized as described above.

[00284] Using these techniques, antibodies can be generated to cells expressing HER2, soluble forms of HER2, epitopes or peptides thereof, and expression libraries for these (See e.g., U.S. Patent No. 5,703,057) which can then be screened as described above for the activities described herein.

[00285] The HER2 antibodies described herein can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

[00286] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93/64701, which have targeting moiety (e.g., a ligand for a cell surface receptor), and a nucleic acid binding moiety (e.g., polylysine), viral vector (e.g., a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618) which is a fusion protein containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., a protamine), plasmids, phage, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[00287] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex virus I (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64: 487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90: 7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87: 1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259: 988 (1993); Davidson, et al., Nat. Genet 3: 219 (1993); Yang, et al., J. Virol. 69: 2004 (1995) and Adeno-Associated Viral Vectors (see Kaplitt, M. G. et al., Nat. Genet. 8: 148 (1994).

[00288] Pox viral vectors introduce the gene into the cytoplasm of cells. Avipox virus vectors result in only a short-lived expression of the nucleic acid. Adenovirus vectors, adeno-associated viral vectors and herpes simplex virus (HSV) are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter-lived expression (approximately 2 months) than the adeno-associated virus (approximately 4 months), which in turn is shorter than the HSV vectors. The particular vector chosen will depend on the target cell and the condition being treated. Introduction may be by standard techniques, e.g., infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[00289] The vector can be used to target essentially any desired target cell. For example, stereotaxic injection can be used to direct vectors (e.g., adenovirus, HSV) to a desired site. Additionally, the particles can be delivered by intracerebroventricular (i.c.v.) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A bulk flow-based method, termed convection, has also been shown to be effective in delivering large molecules to extensive areas of the brain and may be useful in delivering the vector to the target cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that may be used include catheters, intravenous, parenteral, intraperitoneal, and subcutaneous injection, and oral or other known routes of administration.

[00290] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of HER2 in a sample. The antibody can also be used to attempt to bind to HER2 and disrupt HER2-related signaling.

[00291] Techniques can be adapted for the production of single chain antibodies specific for an antigenic protein disclosed herein (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huso, et al., 1989 Science 246: 1275-1281) to allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments containing the idiotypes for a protein antigen can be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) a Fab fragment generated by reducing the disulfide bonds of an F(ab')2 fragment; (iii) a Fab fragment generated by treating the antibody molecule with papain and a reducing agent and (iv) Fv fragments.

[00292] The invention also includes anti-HER2 Fv, Fab, Fab' and F(ab')2 fragments or anti-HER2 fragments, single chain anti-HER2 antibodies, multispecific antibodies in which at least one branch binds HER2, and heteroconjugate anti-HER2 antibodies.

[00293] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for HER2. The second binding target is any other antigen, including a cell surface protein or receptor or receptor subunit.

[00294] Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the coexpression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are described in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

[00295] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain and, if desired, immunoglobulin light chain fusions are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, e.g., Suresh et al., Methods in Enzymology, 121: 210 (1986).

[00296] According to another method described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end products such as homodimers.

[00297] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al., Science 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[00298] Additionally, Fab' fragments can be directly recovered from E. coli and chemically linked to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized F(ab')2 bispecific antibody molecule. Each Fab' fragment was separately secreted from E. coli and individualized for direct chemical linkage in vitro to form the bispecific antibody. The bispecific antibody thus formed was capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, as well as eliciting the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[00299] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Consequently, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

[00300] Antibodies with more than two valencies are considered. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

[00301] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates from the protein antigen described herein. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm that binds to a trigger molecule on a leukocyte such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FCYR), such as FCYRI (CD64), FCYRII (CD32), and FCYRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing a particular antigen. Such antibodies have an antigen-binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue fact (TF).

[00302] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two antibodies covalently joined. Such antibodies, for example, have been proposed for targeting cells of the immune system to unwanted cells (see U.S. Patent No. 4,676,980), and for the treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089). It is contemplated that antibodies can be prepared in vitro using methods known in protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those described, for example, in U.S. Patent No. 4,676,980.

[00303] It may be desirable to modify the antibodies described herein with respect to effector function so as to enhance, for example, the efficacy of the antibody in treating diseases and disorders associated with aberrant HER2 expression and/or activity. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing formation of the interchain disulfide bond in this region. The homodimeric antibody thus generated may have improved internalization capabilities and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and may thus have enhanced complement lysis and ADCC capability. (See Stevenson et al., Anti Cancer Drug Design, 3: 219-230 (1989)).

HER2 Antibody Conjugates:

[00304] The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[00305] Toxins and enzymatically active fragments thereof that may be used include diphtheria A chain, non-active binding fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.

[00306] Conjugates of the antibody and cytotoxic agent are manufactured using a variety of bifunctional protein binding agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional imidoesters derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bi-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to antibody. (See WO94/11026).

[00307] Those of ordinary skill in the art will recognize that a wide variety of possible moieties can be attached to the resulting antibodies described herein. (See, e.g., “Conjugated Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruso and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[00308] The linkage may be accomplished by any chemical reaction that will link the two molecules as long as the antibody and the other moiety retain their respective activities. This linkage may include many chemical mechanisms, for example covalent linkage, affinity linkage, intercalation, coordinate linkage, and complexation. The preferred linkage, however, is covalent linkage. Covalent linkage may be accomplished by direct condensation of existing side chains or by incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in molecules that link proteins, such as the antibodies of the present invention, to other molecules. For example, representative linking agents may include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of linking agents known in the art but, rather, is exemplary of the more common linking agents. (See Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987).

[00309] Preferred ligands are described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) which describe the use of MBS (M-maleimidabenzoyl-N-hydroxysuccinimidic ester). See also, U.S. Patent No. 5,030,719, which describes the use of a halogenated acetyl hydrazide derivative linked to an antibody via an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. No. (21558G); (iii) SPDP (succinimidyl-6-(1-methyl-3-(1-dimethylamino-propyl)-carbodiimide) hydrochloride); [3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat.# 21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6-[3-(2-pyridyldithio)-propionamide] hexanoate (Pierce Chem. Co. Cat.# 2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide: Pierce Chem. Co., Cat.# 24510) conjugated to EDC.

[00310] The ligands described above contain components that have different attributes, thus leading to conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing ligands are less soluble than sulfo-NHS esters. In addition, the SMPT ligand contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide bonds, in general, are less stable than other bonds because the disulfide bond is cleaved in vitro, resulting in less available conjugate. Sulfo-NHS, in particular, can enhance the stability of carbodiimide bonds. Carbodimide linkages (such as EDC) when used in conjunction with sulfo-NHS, form esters that are more resistant to hydrolysis than the carbodimide linkage reaction alone.

[00311] The antibodies described herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are described in U.S. Pat. No. 5,013,556.

[00312] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters of defined pore size to produce liposomes of the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982) via a disulfide interchange reaction.

[00313] In one aspect, the conjugate described herein includes an isolated HER2 antibody or antigen-binding fragment thereof connected directly or indirectly to one or more independently D-bearing polymeric scaffolds comprising poly(1-hydroxymethylethylene hydroxymethylformal) (PHF) having a molecular weight ranging from about 2 kDa to about 40 kDa, wherein each of the one or more independently D-bearing polymeric scaffolds is of the formula (Ic): where:each occurrence of D, independently, is a therapeutic or diagnostic agent;LD1 is a carbonyl-containing moiety;each occurrence of in is independently a first ligand that contains a biodegradable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect; and the in between LD1 and D indicates direct or indirect connection* of D to LD1; each occurrence of is independently a second ligand not yet attached to the isolated HER2 antibody or antigen-binding fragment thereof, wherein LP2 is a moiety containing a functional group that is yet to form a covalent bond with a functional group of the isolated antibody or antigen-binding fragment thereof, and the betweenLD1 and LP2 indicates direct or indirect binding* of LP2 to LD1, and each occurrence of the second ligand is distinct from each occurrence of the first ligand; each occurrence of is independently a third linker that connects each D-bearing polymeric scaffold to the isolated antibody or antigen-binding fragment thereof, wherein the terminus bound to LP2 indicates direct or indirect binding* of LP2 to the isolated antibody or antigen-binding fragment thereof in the formation of a covalent bond between a functional group of LP2 and a functional group of the isolated antibody or antigen-binding fragment thereof; and each occurrence of the third ligand is distinct from each occurrence of the first ligand; m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m3 is an integer from 0 to about 18, m4 is an integer from 1 to about 10; the sum of m, m1, m2, m3, and m4 ranges from about 15 to about 300; and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof is 10 or less.

[00314] The conjugate may include one or more of the following traits.

[00315] For example, in formula (Ic), m1 is an integer from 1 to about 120 (e.g., from about 1 to 90) and/or m3 is an integer from 1 to about 10 (e.g., from about 1 to 8).

[00316] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 45 to about 150), m2 is an integer from 2 to about 20, m3 is an integer from 0 to about 9, m4 is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 75 (e.g., m1 being from 4 to 45).

[00317] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 60 to about 110), m2 is an integer from 2 to about 15, m3 is an integer from 0 to about 7, m4 is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 55 (e.g., m1 being from 4 to 30).

[00318] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 2 kDa to about 20 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 15 to about 150), m2 is an integer from 1 to about 20, m3 is an integer from 0 to about 10 (e.g., m3 ranging from 0 to about 9), m4 is an integer from 1 to about 8, and/or m1 is an integer from 1 to about 70, and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7, or 8).

[00319] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 3 kDa to about 15 kDa (i.e., the sum of m, m1, m2, m3, and m4 ranging from about 20 to about 110), m2 is an integer from 2 to about 15, m3 is an integer from 0 to about 8 (e.g., m3 ranging from 0 to about 7), m4 is an integer from 1 to about 8, and/or m1 is an integer from 2 to about 50, and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7, or 8).

[00320] For example, when the PHF in formula (Ic) has a molecular weight ranging from about 5 kDa to about 10 kDa, (i.e. the sum of m, m1, m2, m3, and m4 ranges from about 40 to about 75), m2 is an integer from about 2 to about 10 (e.g., m2 being from 3 to 10), m3 is an integer from 0 to about 5 (e.g., m3 ranging from 0 to about 4), m4 is an integer from 1 to about 8 (e.g., m4 ranging from 1 to about 5), and/or m1 is an integer from about 2 to about 35 (e.g., m1 being from 5 to 35), and the total number of LP2 bound to the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7, or 8).

[00321] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (e.g., about 6 to 20 kDa, or about 8 to 15 kDa, about 2 to 20 kDa, or about 3 to 15 kDa, or about 5 to 10 kDa), the number of drugs per PHF (e.g., m2) is an integer from 1 to about 40 (e.g., from about 1 to 20, or from about 2 to 15, or about 3 to 10, or from about 2 to 10). This scaffold can be used, for example, to conjugate isolated antibody or antigen-binding fragment thereof having a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater; 80 kDa or greater; 100 kDa or greater; 120 kDa or greater; 140 kDa or greater; 160 kDa or greater, 180 kDa or greater, or 200 kDa or greater, or about 40 to 200 kDa, 40 to 180 kDa, 40 to 140 kDa, 60 to 200 kDa, 60 to 180 kDa, 60 to 140 kDa, 80 to 200 kDa, 80 to 180 kDa, 80 to 140 kDa, 100 to 200 kDa, 100 to 180 kDa, 100 to 140 kDa or 140 to 150 kDa). In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to the PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2, between about 1:2 and about 1:4, between about 1:2 and about 1:3, between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

[00322] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (e.g., about 6 to 20 kDa, or about 8 to 15 kDa, about 2 to 20 kDa, or about 3 to 15 kDa, or about 5 to 10 kDa), the number of drugs per PHF (e.g., m2) is an integer from 1 to about 40 (e.g., from about 1 to 20, or from about 2 to 15, or about 3 to 10, or from about 2 to 10). This scaffold can be used, for example, to conjugate isolated antibody or antigen-binding fragment thereof having a molecular weight of 140 kDa to 180 kDa or from 140 kDa to 150 kDa. In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to the PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2, between about 1:2 and about 1:4, between about 1:2 and about 1:3, between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

[00323] Isolated antibody or antigen binding fragment thereof in this molecular weight range include but are not limited to, for example, full-length antibodies such as IgG, IgM.

[00324] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, the number of drugs per PHF (e.g., m2) is an integer from 1 to about 40 (e.g., from about 1 to 20 or from about 2 to 15 or from about 3 to 10 or from about 2 to 10). This scaffold can be used, for example, to conjugate the isolated antibody or antigen-binding fragment thereof having a molecular weight of 60 kDa to 120 kDa. In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to the PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2, between about 1:2 and about 1:4, between about 1:2 and about 1:3, between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

[00325] Isolated antibodies or antigen-binding fragments thereof in this molecular weight range include, but are not limited to, for example, fragments of antibodies such as Fab2 and camelid antibodies.

[00326] In certain embodiments, D is a therapeutic agent. In certain embodiments, the therapeutic agent is a small molecule having a molecular weight < about 5 kDa, < about 4 kDa, < about 3 kDa, < about 1.5 kDa, or < about 1 kDa.

[00327] In certain embodiments, the therapeutic agent has an IC50 of about less than 1 nM.

[00328] In another embodiment, the therapeutic agent has an IC50 of about greater than 1 nM, e.g., the therapeutic agent has an IC50 of about 1 to 50 nM.

[00329] Some therapeutic agents having an IC50 of greater than about 1 nM (e.g., “less potent drugs”) are unsuitable for conjugation to an antibody using art-recognized conjugation techniques. Without wishing to be bound by theory, such therapeutic agents have a potency that is insufficient for use in targeted antibody-drug conjugates using conventional techniques since sufficient copies of the drug (i.e., more than 8) cannot be conjugated using techniques known in the art without resulting in diminished pharmacokinetic and physicochemical properties of the conjugate. However, sufficiently high loadings of these less potent drugs can be obtained using the conjugation strategies described herein, thereby resulting in high loadings of the therapeutic agent while maintaining desirable pharmacokinetic and physicochemical properties. Thus, the invention also relates to an antibody-polymer-drug conjugate that includes the isolated antibody or antigen-binding fragment thereof, PHF and at least eight therapeutic agent moieties, wherein D is auristatin, Dolastatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F, AF HPA, phenylenediamine (AFP).

[00330] For example, duocarmycin or analogues thereof are duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, bizelesin, or carzelesin.

[00331] Other examples of D include those described in, e.g., U.S. Application Publication No. 2013-0101546 and U.S. Patent No. 8,815,226; and co-pending applications thereunder U.S. Serial Nos. 14/512,316 filed October 10, 2014, 61/988,011 filed May 2, 2014, and 62/010,972 filed June 11, 2014; the description of each of which is incorporated herein in its entirety.

[00332] In some embodiments, the number of D-bearing polymeric scaffolds that can be conjugated to an antibody is limited by the number of free cysteine. In some embodiments, the free cysteine residues are introduced into the amino acid sequence of the antibody by the methods described herein. Exemplary conjugates described herein can include antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al (2012) Methods in Enzym. 502: 123-138). In some embodiments, one or more of the free cysteine residues are already present in an antibody, without the use of engineering, in which case the existing free cysteine residues can be used to conjugate the antibody to a D-bearing polymeric scaffold. In some embodiments, an antibody is exposed to reducing conditions prior to conjugation of the antibody so as to generate one or more of the free cysteine residues.

[00333] In certain embodiments, in the conjugate described herein, the polymeric scaffold carrying D of formula (Ic) is of formula (Ie): wherein, PHF has a molecular weight ranging from about 2 kDa to about 40 kDa; each occurrence of D independently is a therapeutic agent having a molecular weight of <5 kDa, and the between D and the carbonyl group indicates direct or indirect bonding* of D to the carbonyl group, X is CH2, O, or NH; one of Xa and Xb is H and the other is a water-soluble maleimido blocking moiety, or Xa and Xb, together with the carbon atoms to which they are attached, form a carbon-carbon double bond, m1 is an integer from 1 to about 140, m7 is an integer from 1 to about 40, and the sum of m1 and m7 is from about 2 to about 180, m is an integer from 1 to about 300, m3a is an integer from 0 to about 17, m3b is an integer from 1 to about 8, and the sum of m3a and m3b is between 1 and 18, and the sum of m, m1, m7, m3a, and m3b ranges from about 15 to about 300.

[00334] In certain embodiments, in the conjugate described herein, the polymeric scaffold carrying D of formula (Ie) is of formula (Id): where: one of Xa and Xb is H and the other is a water-soluble maleimido blocking moiety, or Xa and Xb, together with the carbon atoms to which they are attached, form a carbon-carbon double bond; m3a is an integer from 0 to about 17, m3b is an integer from 1 to about 8, the sum of m3a and m3b is between 1 and 18, and the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 300.

[00335] For example, the ratio of m2 to m3b is greater than 1:1 and less than or equal to 10:1.

[00336] For example, the ratio of m2 to m3b is about 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, or 2:1.

[00337] For example, the ratio of m2 to m3b is between 2:1 and 8:1.

[00338] For example, the ratio of m2 to m3b is about 8:1, 7:1, 6:1.5:1, 4:1, 3:1, or 2:1.

[00339] For example, the ratio of m2 to m3b is between 2:1 and 4:1.

[00340] For example, the ratio of m2 to m3b is about 4:1, 3:1, or 2:1.

[00341] For example, the ratio of m2 to m3b is about 3:1 and 5:1.

[00342] For example, the ratio of m2 to m3b is about 3:1, 4:1, or 5:1.

[00343] For example, when the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 20 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 150, m1 is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m3a is an integer from 0 to about 9, m3b is an integer from 1 to about 8, and the ratio of the PHF to the isolated HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8.

[00344] For example, when the PHF in formula (Id) has a molecular weight ranging from about 3 kDa to about 15 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m3a is an integer from 0 to about 7, m3b is an integer from 1 to about 8, and the ratio of the PHF to the isolated HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (e.g., an integer from 2 to about 6 or an integer from 2 to about 4).

[00345] For example, when the PHF in formula (Id) has a molecular weight ranging from about 5 kDa to about 10 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 40 to about 75, m1 is an integer from about 2 to about 35, m2 is an integer from about 2 to about 10, m3a is an integer from 0 to about 4, m3b is an integer from 1 to about 5, and the ratio of the PHF to the isolated HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (e.g., an integer from 2 to about 6 or an integer from 2 to about 4).

[00346] For example, the water-soluble maleimido blocking moieties are moieties that can be covalently attached to one of two carbon atoms of the olefin in the reaction of the maleimido group with a thiol-containing compound of formula (II):R90-(CH2)d-SH(II)where:R90 is NHR91, OH, COOR93, CH(NHR91)COOR93, or a substituted phenyl group;R93 is hydrogen or C1-4 alkyl;R91 is hydrogen, CH3, or CH3CO, and d is an integer from 1 to 3.

[00347] For example, the water-soluble maleimide blocking compound of formula (II) can be cysteine, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH2SH), benzyl thiol wherein phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol. The one or more hydrophilic substituents on the phenyl comprise OH, SH, methoxy, ethoxy, COOH, CHO, CO, C1-4 alkyl, NH2, F, cyano, SO3H, PO3H, and the like.

[00348] For example, the water-soluble maleimide blocking group is -S-(CH2)d-R90, where, R90 is OH, COOH, or CH(NHR91)COOR93; R93 is hydrogen or CH3; R91 is hydrogen or CH3CO; and d is 1 or 2.

[00349] For example, the water-soluble maleimide blocking group is -S-CH2-CH(NH2)COOH.

[00350] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (e.g., about 2 to 20 kDa, or about 3 to 15 kDa, or about 5 to 10 kDa), the number of drugs per PHF (e.g., m2) is an integer from 1 to about 40 (e.g., about 1 to 20, or about 2 to 15, or about 3 to 10, or about 2 to 10). This scaffold can be used, for example, to conjugate an isolated antibody or antigen-binding fragment thereof having a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater; 80 kDa or greater; or 100 kDa or greater; 120 kDa or greater; 140 kDa or greater; 160 kDa or greater, 180 kDa or greater, or 200 kDa or greater, or from about 40 to 200 kDa, 40 to 180 kDa, 40 to 140 kDa, 60 to 200 kDa, 60 to 180 kDa, 60 to 140 kDa, 80 to 200 kDa, 80 to 180 kDa, 80 to 140 kDa, 100 to 20 ... to 180 kDa, from 100 to 140 kDa or from 140 to 150 kDa). In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to the PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2, between about 1:2 and about 1:8, between about 1:2 and about 1:6, between about 1:2 and about 1:5, between about 1:2 and about 1:4, between about 1:2 and about 1:3, between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

[00351] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (e.g., about 2 to 20 kDa, or about 3 to 15 kDa, or about 5 to 10 kDa), the number of drugs per PHF (e.g., m2) is an integer from 1 to about 40 (e.g., about 1 to 20, or about 2 to 15, or about 3 to 10, or about 2 to 10). This scaffold can be used, for example, to conjugate an isolated antibody or antigen-binding fragment having a molecular weight of 140 kDa to 180 kDa or 140 kDa to 150 kDa. In this embodiment the ratio of the isolated antibody or antigen-binding fragment thereof to the PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2, between about 1:2 and about 1:8, between about 1:2 and about 1:6, between about 1:2 and about 1:5, between about 1:2 and about 1:4, between about 1:2 and about 1:3, between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

[00352] Isolated antibodies or antigen-binding fragments in this molecular weight range include, but are not limited to, for example, full-length antibodies such as IgG, IgM.

[00353] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa (e.g., about 2 to 20 kDa, or about 3 to 15 kDa, or about 5 to 10 kDa), the number of drugs per PHF (e.g., m2) is an integer from 1 to about 40, (e.g., about 1 to 20, or about 2 to 15, or about 3 to 10, or 2 to 10). This scaffold can be used, for example, to conjugate an isolated antibody or antigen-binding fragment having a molecular weight of 60 kDa to 120 kDa. In this embodiment the ratio of the isolated antibody or antigen-binding fragment thereof to the PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2, between about 1:2 and about 1:8, between about 1:2 and about 1:6, between about 1:2 and about 1:5, between about 1:2 and about 1:4, between about 1:2 and about 1:3, between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

[00354] Isolated antibodies or antigen binding fragments in this molecular weight range include but are not limited to, for example, fragments of antibodies such as, for example, Fab2, scFcFv and camelids.

[00355] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (e.g., about 2 to 20 kDa, or about 3 to 15 kDa, or about 5 to 10 kDa), the number of drugs per PHF (e.g., m2) is an integer from 1 to about 40 (e.g., about 1 to 20, or about 2 to 15, or about 3 to 10, or 2 to 10). This scaffold can be used, for example, to conjugate isolated antibody or antigen-binding fragment thereof having a molecular weight of 40 kDa to 80 kDa. In this embodiment the ratio of the isolated antibody or antigen-binding fragment thereof to the PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, between about 1:1 and about 1:2, between about 1:2 and about 1:8, between about 1:2 and about 1:6, between about 1:2 and about 1:5, between about 1:2 and about 1:4, between about 1:2 and about 1:3, between about 1:3 and about 1:4, or between about 1:3 and about 1:5.

[00356] Isolated antibodies or antigen-binding fragments in this molecular weight range, i.e., about 40 kDa to about 80 kDa, include but are not limited to, for example, antibody fragments such as, for example, Fabs.

[00357] In certain embodiments, in the conjugate described herein, the polymeric scaffold bearing D of formula (Ie) is of formula (Se), wherein the polymer is PHF having a molecular weight ranging from about 2 kDa to about 40 kDa: where: m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m3a is an integer from 0 to about 17, m3b is an integer from 1 to about 8; the sum of m3a and m3b ranges from 1 to about 18; the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 300; the terminal indicates the binding* of one or more polymeric scaffolds to the isolated antibody or antigen-binding fragment thereof that specifically bind to an epitope of the human HER2 receptor and comprises a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 26); a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence GGHGYFDL (SEQ ID NO: 27); a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSSILA (SEQ ID NO: 28); a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GASSRAT (SEQ ID NO: 21); and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); the ratio of the PHF to the antibody is 10 or less. The formula frame (Se) may include one or more of the following traits:

[00358] When the PHF in formula (Se) has a molecular weight ranging from about 2 kDa to about 20 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 15 to about 150, m1 is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m3a is an integer from 0 to about 9, m3b is an integer from 1 to about 8, the sum of m3a and m3b ranges from 1 to about 10, and the ratio of the PHF to antibody is an integer from 2 to about 8.

[00359] When the PHF in formula (Se) has a molecular weight ranging from about 3 kDa to about 15 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m3a is an integer from 0 to about 7, m3b is an integer from 1 to about 8, the sum of m3a and m3b ranges from 1 to about 8; and the ratio of the PHF to antibody is an integer from 2 to about 8 (e.g., from about 2 to about 6 or from about 2 to about 4).

[00360] When the PHF in formula (Se) has a molecular weight ranging from about 5 kDa to about 10 kDa, the sum of m, m1, m2, m3a, and m3b ranges from about 40 to about 75, m1 is an integer from about 2 to about 35, m2 is an integer from about 2 to about 10, m3a is an integer from 0 to about 4, m3b is an integer from 1 to about 5, the sum of m3a and m3b ranges from 1 to about 5; and the ratio of the PHF to antibody is an integer from 2 to about 8 (e.g., from about 2 to about 6 or from about 2 to about 4).

[00361] In certain embodiments, the ratio of auristatin F hydroxylpropyl amide (“AF HPA”) to the antibody can be about 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[00362] In certain embodiments, the ratio of HPA AF to antibody can be about 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[00363] In other embodiments, the ratio of HPA AF to antibody may be about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, or 6:1.

[00364] In some embodiments, the ratio of HPA AF to antibody may be about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

[00365] In some embodiments, the ratio of AF to antibody may be about 15:1, 14:1, 13:1, 12:1, or 11:1.

[00366] In some embodiments, the ratio of HPA AF to antibody may be about 15:1, 14:1, 13:1, or 12:1.

[00367] In certain embodiments, the ratio of PHF to antibody may be about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[00368] In certain embodiments, the ratio of PHF to antibody may be about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[00369] In other embodiments, the ratio of PHF to antibody may be about 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[00370] In other embodiments, the ratio of PHF to antibody may be about 6:1, 5:1, 4:1, 3:1, or 2:1.

[00371] In other embodiments, the ratio of PHF to antibody may be about 6:1, 5:1, 4:1, or 3:1.

[00372] In some embodiments, the ratio of PHF to antibody may be about 5:1, 4:1, or 3:1.

[00373] In some embodiments, the ratio of PHF to antibody may be about 4:1, 3:1, or 2:1.

[00374] Isolated antibodies or antigen-binding fragments in this molecular weight range include, but are not limited to, for example, antibody fragments such as Fabs.

[00375] Other embodiments of antibody-polymer-drug conjugates are those described in, e.g., U.S. Patent No. 8,815,226; and U.S. Serial Nos. 14/512,316 filed October 10, 2014, and 61/988,011 filed May 2, 2014; the disclosure of each of which is incorporated herein in its entirety.

[00376] This invention also relates to a so-modified drug derivative that can be directly conjugated to an antibody or antigen-binding fragment thereof absent a polymeric carrier, and drug-antibody conjugates thereof.

[00377] In some embodiments, the antibody-drug conjugates include an antibody or antigen-binding fragment thereof conjugated, i.e. covalently linked, to the drug moiety. In some embodiments, the antibody or antigen-binding fragment thereof is covalently linked to the drug moiety via a linker, e.g., a non-polymeric linker.

[00378] The drug portion (D) of antibody-drug conjugates (ADC) can include any compound, moiety, or group that has a cytotoxic or cytostatic effect as defined herein. In certain embodiments, an antibody-drug conjugate (ADC) comprises an antibody (Ab) that targets a tumor cell, a drug portion (D), and a linker portion (L) that links the Ab to the D. In some embodiments, the antibody is linked to the linker portion (L) via one or more amino acid residues, such as lysine and/or cysteine.

[00379] In certain embodiments the ADC has formula (Ig):Ab-(L-D)p(Ig), where p is 1 to about 20.

[00380] In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine residues. In some embodiments, the free cysteine residues are introduced into the amino acid sequence of the antibody by the methods described herein. Exemplary ADCs of the formula Ig include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al (2012) Methods in Enzym. 502: 123-138). In some embodiments, one or more of the free cysteine residues are already present in an antibody, without the use of engineering, in which case the existing free cysteine residues can be used to conjugate the antibody to a drug. In some embodiments, an antibody is exposed to reducing conditions prior to conjugation of the antibody so as to generate one or more of the free cysteine residues.

[00381] In some embodiments the “Linker” (L) is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties (D) to an antibody (Ab) to form an antibody-drug conjugate (ADC) of the formula Ig. In some embodiments, antibody-drug conjugates (ADC) can be prepared using a linker having reactive functionalities to covalently link the drug and the antibody. For example, in some embodiments, a cysteine thiol of an antibody (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.

[00382] In one aspect, a linker has a functionality that is capable of reacting with a free cysteine present in an antibody to form a covalent bond. Non-limiting exemplary such reactive functionalities include maleimide, haloacetamides, α-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, for example, the conjugation method on page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4): 765-773, and the Examples therein.

[00383] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody moiety. Exemplary non-limiting such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[00384] A linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-succinimidyl-4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“MCC”). Various linker components are known in the art, some of which are described below.

[00385] A linker may be a “cleavable linker,” which facilitates the release of a drug. Exemplary non-limiting cleavable linkers include acid-labile (e.g., hydrazone-comprising) linkers, protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[00386] In certain embodiments, a linker has the following formula (IIg):—Aa—Ww—Yy(IIg)wherein: A is a “stretcher unit,” and A is an integer from 0 to 1; W is an “amino acid unit,” and w is an integer from 0 to 12; Y is a “spacer unit,” and y is an integer 0, 1, or 2. An ADC comprising the linker of formula (IIg) has the formula I(A): Ab—(Aa—Ww—Yy—D)p, wherein Ab, D, and p are defined as above for formula (Ig). Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298, which is incorporated herein by reference in its entirety.

[00387] In some embodiments, a linker component comprises a “stretcher unit” (A) that links an antibody to another linker component or to a drug moiety. Exemplary non-limiting stretcher units are shown below (wherein the wavy line indicates covalent binding sites* to an antibody, drug, or additional linker components):

[00388] In some embodiments, a linker component comprises an “amino acid unit” (W). In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21: 778-784). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit); alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). An amino acid unit may comprise naturally occurring amino acid residues and/or smaller amino acids and/or non-naturally occurring amino acid analogs, such as citrulline. Amino acid units may be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C, and D, or a plasmin protease.

[00389] Typically, peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid-phase synthesis method (e.g., E. Schrider and K. Lubke (1965) “The Peptides”, volume 1, pp 76-136, Academic Press).

[00390] In some embodiments, a linker component comprises a “spacer” unit that links the antibody to a drug moiety, directly or through a stretcher unit and/or an amino acid unit. A spacer unit can be a “self-immolating” or a “non-self-immolating.” A “non-self-immolating” spacer unit is one in which part or all of the spacer unit remains attached to the drug moiety upon cleavage of the ADC. Examples of non-self-immolating spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell associated protease results in the release of a portion of the rest of the ADC. In some such embodiments, the glycine-glycine-drug moiety is individualized for a glycine-glycine-drug hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

[00391] A “self-immolative” spacer unit allows for the release of the drug moiety. In certain embodiments, a spacer unit of a linker comprises a p-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol is linked to an amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103). In some embodiments, the spacer unit comprises p-amino-benzyloxycarbonyl (PAB). In some embodiments, an ADC comprising a self-immolative linker has the structure: where:Q is C1-C8 alkyl, -O-(C1-C8 alkyl), halogen, nitro, or cyano;n6 is an integer from 0 to 4;Xa may be one or more additional spacer units or may be absent; and p is an integer from 1 to about 20.

[00392] In some embodiments, p is an integer from 1 to 10, from 1 to 7, from 1 to 5, or from 1 to 4. Exemplary non-limiting Xa spacer units include: wherein R101 and R102 are independently selected from H and C1-C6 alkyl. In some embodiments, R101 and R102 are each -CH3.

[00393] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzylacetals. In some embodiments, spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2,2,1] and bicyclo[2,2,2] ring systems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amides (Amsberry, et al (1990) J. Org. Chem. 55:5867). Attachment of a drug to the α-carbon of a glycine residue is another example of a self-immolative spacer that can be useful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

[00394] In some embodiments, the linker L may be a dendritic-type linker of more than one drug moiety to an antibody via a branched multifunctional linker moiety (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers may increase the molar ratio of drug to antibody, i.e. the loading that is related to the potency of the ADC. Thus, where an antibody bears only one reactive cysteine thiol group, a large number of drug moieties may be linked via one dendritic linker.

[00395] Exemplary non-limiting ligands are shown below for ADCs of formula (Ig): where R101 and R102 are independently selected from H and C1-C6 alkyl; n5 is an integer from 0 to 12.

[00396] In some embodiments, n is an integer 2 to 10. In some embodiments, n is an integer 4 to 8. In some embodiments, R101 and R102 are each -CH3.

[00397] Other exemplary non-limiting ADCs include the structures: where Xa is: Y is: each R103 is independently H or C1-C6 alkyl; and n7 is an integer from 1 to 12.

[00398] In some embodiments, a ligand is substituted with groups that modulate solubility and/or reactivity. As a non-limiting example, a charged substituent such as a sulfonate

[00399] (-SO3-) or ammonium may increase the water solubility of the linker reagent and facilitate the binding reaction of the linker reagent with the antibody and/or the drug moiety, or facilitate the binding reaction of Ab-L (antibody-linker intermediate) with D, or D-L (drug-linker intermediate) with Ab, depending on the synthetic route used to prepare the ADC. In some embodiments, a linker moiety is linked to the antibody and a linker moiety is linked to the drug, and then Ab-(linker moiety)a is linked to drug-(linker moiety)b to form the ADC of formula Ig.

[00400] The compounds expressly described herein contemplate, but are not limited to, ADCs prepared with the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS), N-(ε-maleimidocaproyloxy) succinimide ester (EMCS), N-[Y-maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), methyl ester Butyric 4-(4-N-maleimidophenyl) (MPBH), Succinimidyl 3-(bromoacetamido)propionate (SBAP), Succinimidyl iodoacetate (SIA), Succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), Succinimidyl N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), Succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC), Succinimidyl 4-(p-maleimido-phenyl)butyrate (SMPB), Succinimidyl 6-[(β-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), and including bis-maleimide reagents: dithiobismaleimidoethane (DTME), 1,4-Bismaleimidobutane (BMB), 1,4-Bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)3 (shown below); bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, bis-maleimide reagents allow for the attachment* of the thiol group of a cysteine on the antibody to a thiol-containing drug moiety, linker, or linker-drug intermediate. Other functional groups that are reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

[00401] Certain useful linker reagents can be obtained from various commercial sources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), Molecular Biosciences Inc. (Boulder, Colo.), or synthesized according to procedures described in the art; for example, in Toki et al (2002) J. Org. Chem. 67: 1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters, 38: 5257-60; Walker, M. A. (1995) J. Org. Chem. 60: 5352-5355; Frisch et al (1996) Bioconjugate Chem. 7: 180-186; U.S. Pat. No. 6,214,345; WO 02/088172; US 2003130189; US.2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.

[00402] Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is exemplary a chelating agent for conjugation of radionucleotide to antibody. See, for example, WO 94/11026.

Methods of Making HER2 Antibody Conjugates:

[00403] In certain embodiments, the conjugates are formed in multiple steps. These steps include (1) modifying a polymer such that it contains a functional group that can react with a functional group of the drug or derivative thereof; (2) reacting the modified polymer with the drug or derivative thereof such that the drug is bound to the polymer; (3) modifying the polymer-drug conjugate such that the polymer contains a functional group that can react with a functional group of the isolated antibody or antigen-binding fragment thereof or derivative thereof; and (4) reacting the modified polymer-drug conjugate with the antibody or antigen-binding fragment thereof to form the conjugate described herein. Step (3) may be omitted if the modified polymer produced by step (1) contains a functional group that can react with a functional group of the antibody or antigen-binding fragment thereof.

[00404] In another embodiment the conjugates are formed in several steps: (1) modifying a polymer so that it contains a functional group that can react with a functional group of a first drug or its derivative; (2) reacting the modified polymer with the first drug or its derivative so that the first drug is bound to the polymer; (3) modifying the polymer-drug conjugate so that it contains a different functional group that can react with a functional group of a second drug or its derivative; (4) reacting the modified polymer-drug conjugate with the second drug or its derivative so that the second drug is bound to the polymer-drug conjugate; (5) modifying the polymer-drug conjugate containing 2 different drugs so that the polymer contains a functional group that can react with a functional group of the antibody or antigen-binding fragment thereof; and (6) reacting the modified polymer-drug conjugate of step (5) with the isolated antibody or antigen-binding fragment thereof or derivative thereof to form the conjugate described herein. Steps (5) and (6) may be repeated if 2 different isolated antibodies or antigen-binding fragments thereof or derivatives thereof are to be conjugated to form a polymer-drug conjugate comprising two different drugs and two different antibodies or antigen-binding fragments thereof.

[00405] In yet another embodiment, the conjugates are formed in multiple steps. These steps include (1) modifying a polymer so that it contains a functional group that can react with a functional group of the drug or its derivative; (2) further modifying the polymer so that it also contains a functional group that can react with a functional group of the antibody or antigen-binding fragment thereof; (3) reacting the modified polymer with the drug or its derivative so that the drug is bound to the polymer; and (4) reacting the modified polymer-drug conjugate with the antibody or antigen-binding fragment thereof to form the conjugate described herein. The sequence of steps (1) and (2) or that of steps (3) and (4) may be reversed. Furthermore, step (1) or (2) may be omitted if the modified polymer contains a functional group that can react with both a functional group of the drug or its derivatives and a functional group of the antibody or antigen-binding fragment thereof.

[00406] In another embodiment the conjugates are formed in several steps: (1) modifying a polymer such that it contains a functional group that can react with a functional group of a first drug or its derivative; (2) further modifying a polymer such that it contains a functional group that can react with a functional group of the antibody or antigen-binding fragment thereof; (3) reacting the modified polymer with the first drug or its derivative such that the first drug is bound to the polymer; (4) modifying the polymer-drug conjugate such that it contains a different functional group that can react with a functional group of a second drug or its derivative; (5) reacting the modified polymer-drug conjugate with the second drug or its derivative such that the second drug is bound to the polymer-drug conjugate; (6) reacting the modified polymer-drug conjugate containing 2 different drugs so that the polymer with the isolated antibody or antigen-binding fragment thereof or derivative thereof forms the conjugate described herein. Step (6) may be repeated if 2 different isolated antibodies or antigen-binding fragments thereof or derivatives thereof are to be conjugated to form a polymer-drug conjugate comprising two different drugs and two different antibodies or antigen-binding fragment thereof. Step (4) may be performed after step (1) so that the modified polymer contains two different functional groups that can react with two different drugs or derivatives thereof. In this embodiment, the modified polymer containing two different functional groups that can react with two different drugs or derivatives thereof may be further modified so that it contains a functional group that can react with a functional group of the antibody or antigen-binding fragment thereof; prior to the reaction of the modified polymer with the two different drugs (step (3) and step (5) or antibody or antigen-binding fragment thereof (step (6).

[00407] In certain exemplary embodiments, the conjugates described herein find use in biomedical applications, such as drug delivery and tissue engineering, and the polymeric carrier is biocompatible and biodegradable. In certain embodiments, the carrier is a soluble polymer, nanoparticle, gel, liposome, micelle, suture, implant, etc. In certain embodiments, the term “soluble polymer” encompasses a biocompatible, biodegradable polymer such as a polyal (e.g., hydrophilic polyacetal or polyetal). In certain other embodiments, the carrier is a fully synthetic, semi-synthetic, or naturally occurring polymer. In certain other embodiments, the carrier is hydrophilic. Examples of polymeric carriers suitable for producing the conjugates described herein are described in U.S. Patent No. 8,815,226, the contents of which are hereby incorporated by reference in their entirety.

[00408] In one embodiment, the polymeric carrier comprises units of formula (IV):

[00409] wherein X' denotes the substituent for the hydroxyl group of the polymer backbone. As shown in formula (IV) and the other formulas described herein, each polyacetal unit has a single hydroxyl group attached to the glycerol portion of the unit and an X' group attached to the glycolaldehyde portion of the unit. This is for convenience only and it should be construed that the polymer having units of formula (IV) and other formulas described herein may contain a random distribution of units having one X group (or another substituent such as a linker comprising a maleimide terminus) attached to the glycolaldehyde portion of the units and those having a single X' group (or another substituent such as a linker comprising a maleimide terminus) attached to the glycerol portion of the units as well as units having two X' groups (or other substituents such as a linker comprising a maleimide terminus) with one attached to the glycolaldehyde portion and the other attached to the glycerol portion of the units.

[00410] In one embodiment, biocompatible biodegradable polyals suitable for practicing the present invention have a molecular weight of between about 0.5 and about 300 kDa. For example, biocompatible biodegradable polyals have a molecular weight of between about 1 and about 300 kDa (e.g., between about 1 and about 200 kDa, between about 2 and about 300 kDa, between about 2 and about 200 kDa, between about 5 and about 100 kDa, between about 10 and about 70 kDa, between about 20 and about 50 kDa, between about 20 and about 300 kDa, between about 40 and about 150 kDa, between about 50 and about 100 kDa, between about 2 and about 40 kDa, between about 6 and about 20 kDa, or between about 8 and about 15 kDa). For example, the biocompatible biodegradable polyal used for the polymeric scaffold or conjugate described herein is PHF having a molecular weight of between about 2 and about 40 kDa (e.g., about 2 to 20 kDa, 3 to 15 kDa, or 5 to 10 kDa.)

[00411] Methods for preparing polymeric carriers (e.g., biocompatible, biodegradable polymeric carriers) suitable for conjugation to modifiers are known in the art. For example, synthetic guidance can be found in U.S. Patent Nos. 5,811,510, 5,863,990, 5,958,398, 7,838,619, 7,790,150, and 8,685,383. The skilled artisan will know how to adapt these methods to manufacture polymeric carriers for use in the practice of the invention.

[00412] In one embodiment, a method for forming the biocompatible biodegradable polyal conjugates of the present invention comprises a process by which a suitable polysaccharide is combined with an efficient amount of a specific glycol oxidizing agent to form an aldehyde intermediate. The aldehyde intermediate, which is itself a polyal, can then be reduced to the corresponding polyol, succinylated, and linked with one or more suitable modifiers to form a biocompatible biodegradable polyal conjugate comprising succinamide-containing linkages.

[00413] In another preferred embodiment, fully synthetic biocompatible biodegradable polyals for use in the present invention can be prepared by reacting a suitable initiator with a suitable precursor compound.

[00414] For example, fully synthetic polyols can be prepared by the condensation of vinyl ethers with protected substituted diols. Other methods, such as loop-opening polymerization, can be used, where the effectiveness of the method may depend on the degree of substitution and size of the protecting groups.

[00415] One of ordinary skill in the art will appreciate that solvent systems, catalysts, and other factors can be optimized to obtain high molecular weight products.

[00416] In certain embodiments, the charger is PHF.

[00417] In embodiments, the polymeric carrier is PHF having a polydispersity index (PDI) of <1.5, e.g., <1.4, <1.3, <1.2, or <1.1.

[00418] For example, to conjugate the isolated antibody or antigen-binding fragment thereof having a molecular weight of 40 kDa to 200 kDa, the polymeric carrier of the scaffold is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., from about 2 to 20 kDa, or from about 3 to 15 kDa, or from about 5 to 10 kDa).

[00419] For example, to conjugate the antibody or antigen-deleting fragment thereof having a molecular weight of 40 kDa to 80 kDa, the polymeric carrier of the scaffold disclosed herein is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., from about 2 to 20 kDa, or from about 3 to 15 kDa, or from about 5 to 10 kDa). For example, the PHF has a molecular weight of about 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

[00420] The antibody or antigen binding fragment thereof in this molecular weight range includes but is not limited to, for example, antibody fragments such as, for example, Fabs.

[00421] For example, to conjugate the antibody or antigen-deleting fragment thereof having a molecular weight of 60 kDa to 120 kDa, the polymeric carrier of the scaffold disclosed herein is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., from about 2 to 20 kDa, or from about 3 to 15 kDa, or from about 5 to 10 kDa). For example, the PHF has a molecular weight of about 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

[00422] Antibody or antigen binding fragment thereof in this molecular weight range include but are not limited to, for example, camelid, Fab2, scFvFc, and the like.

[00423] For example, to conjugate the antibody or antigen-deleting fragment thereof having a molecular weight of 140 kDa to 180 kDa or 140 kDa to 150 kDa, the polymeric carrier of the scaffold described herein is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., from about 2 to 20 kDa, or from about 3 to 15 kDa, or from about 5 to 10 kDa). For example, the PHF has a molecular weight of about 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

[00424] The antibody or antigen binding fragment thereof in this molecular weight range include but are not limited to, for example, full-size antibodies such as IgG, IgM.

[00425] The biocompatible biodegradable conjugates described herein can be prepared to meet desired biodegradability and hydrophilicity requirements. For example, under physiological conditions, a balance between biodegradability and stability can be achieved. For example, it is known that molecules with molecular weights beyond a certain threshold (generally, above 40 to 100 kDa, depending on the physical form of the molecule) are not excreted via the kidneys, whereas small molecules are, and can be cleared from the body only through uptake by cells and degradation in intracellular compartments, most notably lysosomes. This observation exemplifies how functionally stable yet biodegradable materials can be designed by modulating their stability under general physiological conditions (pH = 7.5 ± 0.5) and at lysosomal pH (pH close to 5). For example, the hydrolysis of acetal and ketal groups is known to be acid catalyzed, so polyols will generally be less stable in an acidic lysosomal environment than, for example, in blood plasma. One can design a test to compare the polymer degradation profile, for example, at pH = 5 and pH = 7.5 at 37 °C in aqueous medium, and thus determine the expected equilibrium of polymer stability in a normal physiological environment and in the lysosomal "digestive" compartment after uptake into cells. Polymeric integrity in such tests can be measured, for example, by size exclusion HPLC. One skilled in the art can select other suitable methods to study various fragments of the degraded conjugates described herein.

[00426] In many cases, it will be preferable that at pH = 7.5 the effective size of the polymer will not change detectably in 1 to 7 days, and will remain within 50% of the original for at least several weeks. At pH = 5, on the other hand, the polymer should preferably degrade detectably in 1 to 5 days, and be completely transformed into low molecular weight fragments within a time period of two weeks to several months. Although more rapid degradation may in some cases be preferable, in general it may be more desirable for the polymer to degrade in cells at a rate that does not exceed the rate of metabolism or excretion of the polymer fragments by the cells. Accordingly, in certain embodiments, the conjugates of the present invention are expected to be biodegradable, particularly in uptake by cells, and relatively "inert" with respect to the biological system. The degradation products of the carrier are preferably uncharged and do not significantly change the pH of the environment. It is proposed that the abundance of alcohol groups may provide low rate of polymer recognition by cellular receptors, particularly phagocytes. The polymeric backbones of the present invention generally contain few, if any, antigenic determinants (characteristic, for example, of some polysaccharides and polypeptides) and generally do not comprise rigid structures capable of engaging in “lock and key” interactions in vivo unless the latter is desirable. Thus, the soluble, cross-linked and solid conjugates described herein are predicted to have low toxicity and bioadhesiveness, which make them suitable for various biomedical applications.

[00427] In certain embodiments of the present invention, the biocompatible biodegradable conjugates may form linear or branched structures. For example, the biocompatible biodegradable polyal conjugates of the present invention may be chiral (optically active). Optionally, the biocompatible biodegradable polyal conjugates of the present invention may be scalemic.

[00428] In certain embodiments, the conjugates described herein are water-soluble. In certain embodiments, the conjugates described herein are water-insoluble. In certain embodiments, the inventive conjugate is in a solid form. In certain embodiments, the conjugates described herein are colloids. In certain embodiments, the conjugates described herein are in particulate form. In certain embodiments, the conjugates described herein are in gel form.

[00429] Scheme 1 below shows a synthetic scheme for making a polymeric drug scaffold disclosed herein. In one embodiment, the conjugates are formed in several steps: (1) the polymer, PHF, is modified to contain a COOH moiety (e.g., -C(O)-X-(CH2)2-COOH); (2) the polymer is then further modified such that it contains a maleimide moiety (e.g., EG2-MI) that can react with a functional group of a PBRM; (3) the modified polymer, containing two different functional groups, is reacted with a functional group of a drug or its derivative (e.g., AF-HPA-Ala) to form a polymer-drug conjugate; (4) the disulfide bonds of a PBRM are reduced; (5) the reduced PBRM is then reacted with the polymer-drug conjugate to form the protein-polymer-drug conjugate; and (6) the remaining maleimide moieties are optionally reacted with a maleimide blocking compound (e.g., cysteine).

[00430] In another embodiment the order of steps (2) and (3) may be reversed as depicted in the right-hand path in Scheme 1 below.

[00431] In yet another embodiment, steps (2) and (3) above are performed simultaneously as described in Scheme 2 below.

Use of antibodies against HER2

[00432] It will be appreciated that administration of therapeutic entities according to the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into the formulations to provide transfer, release, tolerability, and the like. A large number of suitable formulations can be found in the form known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly, Chapter 87 by Blaug, Seymour, contained therein. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as Lipofectin®), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbocera emulsions (polyethylene glycols of various molecular weights), semisolid gels, and semisolid blends containing carbocera. Any of the following mixtures may be suitable in the treatments and therapies according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also, Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2): 210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2): 1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug release-some emerging concepts.” J Pharm Sci. 89(8): 967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52: 238-311 (1998) and the citations contained therein for additional information related to formulations, excipients, and carriers well known to pharmaceutical chemists.

[00433] In one embodiment, antibodies, fragments thereof, and/or conjugates thereof described herein can be used as therapeutic agents. Such agents will generally be used for the diagnosis, prognosis, monitoring, treatment, alleviation, prevention, and/or delaying the progression of a disease or disorder associated with, e.g., abnormal HER2 activity and/or expression in a subject. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient, suffering from (or at risk of developing) a disease or disorder associated with abnormal HER2 activity and/or expression, e.g., a cancer, using standard methods. An antibody preparation, preferably one having high specificity and high affinity for the target antigen, is administered to the subject and will generally have an effect due to its binding to the target. Administration of the antibody may abrogate or inhibit or interfere with the signaling function of the target. Administration of the antibody may abrogate or inhibit or interfere with the binding of the target to an endogenous ligand to which it naturally binds. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with HER2 activity and/or expression.

[00434] Diseases or disorders related to abnormal HER2 activity and/or expression include but are not limited to cancer. The target cancer may be anal, astrocytoma, leukemia, lymphoma, head and neck, liver, testicular, cervical, sarcoma, hemangioma, esophageal, ocular, laropharyngeal, oral, mesothelioma, skin, myeloma, oral, rectal, throat, gallbladder, breast, uterine, ovarian, prostate, lung, colon, pancreatic, renal, or gastric cancer.

[00435] In another aspect, the diseases or disorders are cancers selected from the group consisting of breast cancer, gastric cancer, non-small cell lung cancer (NSCLC), and ovarian cancer.

[00436] Generally, alleviating or treating a disease or disorder involves lessening one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, the therapeutically effective amount of the drug may effect one or a combination of the following: reducing the number of cancer cells; reducing the size of the tumor; inhibiting (i.e., decreasing to some degree and/or stopping) the infiltration of cancer cells into peripheral organs; inhibiting tumor metastasis; inhibiting, to some degree, tumor growth; and/or alleviating to some degree one or more of the symptoms associated with the cancer. In some embodiments, a composition disclosed herein may be used to prevent the onset or recurrence of the disease or disorder in a subject.

[00437] A therapeutically effective amount of an antibody, fragment thereof, and/or conjugate thereof described herein generally refers to the amount required to achieve a therapeutic objective. As noted above, this may be an interaction link between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will also be dependent on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume of the other subject to which it is administered. Typical ranges for a therapeutically effective dosage of an antibody or antibody fragment, and/or conjugates thereof described herein may be, by way of non-limiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight, from about 0.1 mg/kg body weight to about 100 mg/kg body weight, or from about 0.1 mg/kg body weight to about 150 mg/kg body weight. Typical dosage frequencies may range, for example, from twice daily to once monthly (e.g., once daily, once weekly; every other week; once every 3 weeks, or monthly). For example, the XMT 1519 conjugates described herein, such as XMT 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) conjugates or XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates can be administered (e.g., as a single weekly dose, every 2 weeks, every 3 weeks, or monthly) at about 0.1 mg/kg to about 20 mg/kg (e.g., 0.2 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, or 13 mg/kg). mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg). For example, XMT 1519 conjugates described herein, such as XMT 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) conjugate or XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugate can be administered (e.g., as a single weekly dose, every 2 weeks to every 3 weeks, or monthly) at about 0.1 mg/kg to about 20 mg/kg (e.g., 0.2 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, or 13 mg/kg). mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg) to treat low-HER2 expressing breast cancer or low-HER2 expressing gastric cancer.

[00438] Effective treatment is determined in association with any method known to diagnose or treat the particular HER2-related disorder. Relief of one or more symptoms of the HER2-related disorders indicates that the antibody confers a clinical benefit.

[00439] Methods for screening antibodies that have the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically mediated techniques known in the art.

[00440] In another embodiment, antibodies directed against HER2 can be used in methods known in the art for localization and/or quantification of HER2 (e.g., for use in measuring HER2 levels within appropriate physiological samples, for use in diagnostic methods, for use in protein visualization, and the like). In a given embodiment, antibodies specific for HER2, or derivatives, fragments, analogs or homologs thereof, which contain the antibody-derived antigen-binding domain, are used as pharmacologically active compounds (hereinafter referred to as “Therapeutics”).

[00441] In another embodiment, an antibody specific for HER2 can be used to isolate a HER2 polypeptide, using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies directed against HER2 protein (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by binding (i.e., physically attaching) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, luminescent biomaterials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamino fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of luminescent biomaterials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S, or 3H.

[00442] In yet another embodiment, an antibody according to the invention can be used as an agent for detecting the presence of HER2 protein (or a fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. The antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. The term “labeled,” with respect to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by binding (i.e., physically attaching) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological specimen” is intended to include tissues, cells, and biological fluids isolated from an individual, as well as tissues, cells, and fluids present within an individual. Included within the use of the term “biological specimen” are therefore blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method described herein can be used to detect an analyte mRNA, protein, or genomic DNA in a biological specimen in vitro as well as in vivo. For example, in vitro techniques for detecting an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting an analyte protein include enzyme-linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detecting an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example, in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo techniques for detecting an analyte protein include introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody may be labeled with a radioactive tracer whose presence and location in a subject can be detected by standard visualization techniques.

Therapeutic Delivery and Formulations of HER2 Antibodies

[00443] The antibodies, derivatives, fragments, analogues and homologues thereof, and/or conjugates thereof described herein (also referred to herein as “active compounds”), may be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in the preparation of such compositions, as well as guidance in the selection of components are provided, for example, in Remington’s Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[00444] Such compositions typically comprise the antibody, fragments thereof, and/or conjugates thereof, and a pharmaceutically acceptable carrier. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be chemically synthesized and/or produced through recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[00445] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous carriers such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.

[00446] Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[00447] A pharmaceutical composition disclosed herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be stored in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

[00448] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extratemporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the degree that easy syringability 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 may be a solvent or dispersion medium, containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[00449] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients enumerated above, as required, followed by sterile filtering. Generally, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any desired additional ingredient from a previously sterile filtered solution thereof.

[00450] Oral compositions generally include an inert diluent or an edible carrier. These may be enclosed in gelatin capsules or compressed into tablets for the purpose of oral therapeutic administration, the active compound may be incorporated with excipient and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared to use a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is orally applied, swished, expectorated, or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, lozenges and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring agent.

[00451] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

[00452] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrating agents appropriate to the barrier to be used are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be effected through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[00453] The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00454] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a sustained/controlled release formulation, including implantable and microencapsulated delivery systems. Biodegradable and biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.

[00455] For example, the active ingredients can be entrapped in microcapsules prepared, for example, through coacervation techniques or through interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions.

[00456] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(polyvinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, nondegradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, some hydrogels release proteins for shorter periods of time.

[00457] The materials may also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Lysosomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) and pharmaceutically acceptable carriers may also be used. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[00458] It is especially advantageous to formulate oral or parenteral compositions in single dosage form for ease of administration and uniformity of dosage. Single dosage form as used herein refers to physically discrete units suitable as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the single dosage forms disclosed herein are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be obtained, and the inherent limitations in the art of compounding such an active compound for the treatment of individuals.

[00459] The pharmaceutical compositions may be included in a container, package, or dispenser together with instructions for administration.

[00460] The formulation may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[00461] In one embodiment, the active compounds are administered in combination therapy, i.e., combined with other agents, e.g., therapeutic agents, that are useful for treating pathological conditions or disorders, such as various forms of cancer, autoimmune disorders, and inflammatory diseases. The term “in combination” in this context means that the agents are given substantially contemporaneously, simultaneously, or sequentially. If given sequentially, at the start of administration of the second compound, the first of the two compounds is preferably still detectable in effective concentrations at the treatment site.

[00462] For example, the combination therapy can include one or more antibodies, fragments thereof, and/or conjugates thereof described herein co-formulated with, and/or co-administered with, one or more additional antibodies, e.g., a HER2 antibody, a HER2 dimerization-inhibiting antibody, or a combination of a HER2 antibody and a HER2 dimerization-inhibiting antibody, such as, e.g., trastuzumab, pertuzumab, or a combination of trastuzumab and pertuzumab, or a biosimilar of trastuzumab and/or pertuzumab or combinations of biosimilars.

[00463] For example, combination therapy may include one or more antibodies, fragments thereof, and/or conjugates thereof described herein co-formulated with, and/or co-administered with, one or more additional therapeutic agents, e.g., a taxane (paclitaxel or docetaxel), an anthracycline (doxorubicin or epirubicin), cyclophosphamide, capecitabine, tamoxifen, letrozole, carboplatin, gemcitabine, cisplatin, erlotinib, irinotecan, fluorouracil, or oxaliplatin. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thereby avoiding potential toxicities or complications associated with the various monotherapies.

[00464] In some embodiments, the additional therapeutic agent(s) used in combination with an antibody, fragment thereof, and/or conjugate thereof described herein are those agents that interfere at different stages in an immune and/or inflammatory response. In one embodiment, one or more antibodies described herein can be co-formulated with, and/or co-administered with, one or more additional agents.

Diagnostic and Prophylactic Formulations

[00465] The HER2 antibody, antigen-binding fragment thereof and/or conjugate thereof described herein are used in diagnostic and prophylactic formulations. In one embodiment, a HER2 antibody, antigen-binding fragment thereof and/or conjugate thereof described herein are administered to patients who are at risk of developing one or more of the aforementioned diseases, such as, for example, without limitation, cancer. A patient or organ predisposition to one or more of the aforementioned indications can be determined using genotypic, serological or biochemical biomarkers.

[00466] In another embodiment of the invention, a HER2 antibody, antigen-binding fragment thereof, and/or conjugate thereof described herein are administered to human subjects diagnosed with a clinical indication associated with one or more of the aforementioned diseases, such as, for example, without limitation, cancer. Upon diagnosis, a HER2 antibody, antigen-binding fragment thereof, and/or conjugate thereof described herein are administered to mitigate or reverse the effects of the clinical indication associated with one or more of the aforementioned diseases.

[00467] In another embodiment of the invention, a method for identifying a patient with breast cancer amenable to treatment with the conjugates described herein comprises measuring the status of certain features in a tumor sample obtained from the patient, and identifying the patient for treatment based on the status of some features in the tumor sample.

[00468] The antibodies described herein are also useful in detecting HER2 in patient samples and are therefore useful as diagnostics. For example, the HER2 antibodies described herein are used in in vitro assays, e.g., ELISA, to detect HER2 levels in a patient sample.

[00469] In one embodiment, a HER2 antibody described herein is immobilized on a solid support (e.g., the wall(s) of a microtiter plate). The immobilized antibody serves as a capture antibody for any HER2 that may be present in a test sample. Prior to communicating the immobilized antibody with a patient sample, the solid support is washed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

[00470] Subsequently, the walls are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is, for example, a serum sample from an individual suspected of having circulating antigen levels considered to be diagnostic of a pathology. After washing the test sample or standard, the solid support is treated with a second antibody that is labeled detectable. The second labeled antibody serves as a detection antibody. The level of detectable label is measured, and the concentration of HER2 antigen in the test sample is determined by comparison with a standard curve developed from the standard samples.

[00471] It will be appreciated that based on the results obtained, using the HER2 antibodies described herein in an in vitro diagnostic assay, it is possible to classify a disease in an individual based on the levels of HER2 antigen expression. For a given disease, blood samples are taken from individuals diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the disease. Using a population of samples that provide the statistically significant results for each stage of progression or therapy, a range of antigen concentrations that can be considered characteristic of each stage is indicated.

[00472] All publications and patent documents cited herein are hereby incorporated by reference as if each publication or document were specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any are pertaining to the prior art, nor does it constitute any admission as to the contents or data thereof. The invention having now been described by way of written description, one skilled in the art will recognize that the invention may be practiced in a variety of embodiments and that the foregoing description and the examples below are for purposes of illustration and not of limitation of the claims which follow.

EXAMPLES

[00473] The following working examples are illustrative of the ligands, drug molecules and PBRMs, and of the methods for preparing these. They are not intended to be limiting and it will be readily understood by one of skill in the art that other reagents or methods may be utilized.

ABBREVIATIONS

[00474] The following abbreviations are used in the reaction schemes and synthetic examples, which follow. This list is not intended to be a comprehensive list of the abbreviations used in the application as additional standard abbreviations, which are readily understood by those skilled in the art of organic synthesis, may also be used in the synthetic schemes and examples. AF-HPA Auristatin F-hydroxypropylamide BSA Bovine serum albumin DMEM Dulbecco's modified Eagle's medium FBS Fetal bovine serum HRP Horseradish peroxidase PBS Buffered saline, 0.9% NaCl TMB 3,3',5,5'-tetramethylbenzidine GENERAL INFORMATION

[00475] Kadcila® (ado-trastuzumab emtansine) for injection manufactured by Genentech was acquired.

[00476] CDRs were identified by the Kabat numbering scheme.

[00477] Tumor growth inhibition (%TGI) was defined as the percent difference in mean tumor volumes (MTVs) between the treated and control groups.

[00478] Treatment efficacy was determined from the incidence and magnitude of tumor size regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of this Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm3 for one or more of these three measurements. In a CR response, the volume was less than 13.5 mm3 for three consecutive measurements during the course of the study. An animal with a CR response at the termination of a study was additionally classified as a tumor-free survivor (TFS). Animals were monitored for regression responses.

[00479] HPLC purification was performed on a Phenomenex Gemini 5 μm 110 Å, 250 x 10 mm, 5 micron, semi-prep column.

[00480] SEC was performed on a Tosoh Biosciences TSKem gel G5000 column (7.8 mm x 30 cm, 10 μm) or Superose 12 column (GE Healthcare).

[00481] WCX was performed on a ProPac WCX-10 column (94 mm x 250 mm) (ThermoFisher).

[00482] If possible, the drug content of the conjugates was determined spectrophotometrically otherwise LC/MS or 1H-NMR was performed for quantitative determination of the drug content.

[00483] The protein content of the protein-polymer-drug conjugates was determined spectrophotometrically at 280 nm or by ELISA.

[00484] The molecular weights of the conjugated polymers (reported as the apparent weight-average molecular weights or molecular peak weights) were determined by SEC with polysaccharide or protein molecular weight standards. More specifically, for the polymer or polymer-drug conjugates, the polysaccharide molecular weight standard was used, and for the protein-drug-polymer conjugates, the protein standards were used. Unless specifically indicated, the molecular weight of the polymeric carrier reported is the weight-average molecular weight of PHF; and the molecular weight of the polymer-drug conjugate and the protein-polymer-drug conjugates is the peak molecular weight. The HER2-polymer-drug conjugates have a peak molecular weight of about 170 kDa to about 230 kDa. The synthesized/measured polymer and polymer conjugates typically have a polydispersity of <1.5.

[00485] Her2-polymer-drug conjugates were separated from residual unreacted polymer-drug conjugates by extensive diafiltration. If necessary, additional purification by size exclusion chromatography and/or WCX chromatography was conducted to remove any aggregated Her2-polymer-drug conjugates. Overall, Her2-polymer-drug conjugates typically contained <5% (w/w, e.g., <2% w/w) aggregated fraction as determined by SEC; <0.5% (w/w, e.g., <0.1% w/w) free (unconjugated) drug as determined by RP-HPLC or LC-MS/MS; <1% (w/w) free polymer-drug conjugate as determined by SEC and/or RP-HPLC and <2% (w/w, e.g., <1% w/w) unconjugated Her2 as determined by HIC-HPLC and/or WCX HPLC. Reduced or partially reduced antibodies were prepared using procedures described in the literature, see, e.g., Francisco et al., Blood 102 (4): 1458-1465 (2003). Total drug concentration (conjugated and unconjugated) was determined by LC-MS/MS.

GENERAL PROCEDURES General Procedure A. Conjugation of polymer with ligand or drug

[00486] In general, the conjugation of the polymer (PHF-BA or PHF-GA) with an amine-containing ligand, such as, for example, EG2-maleimide or an amine-containing drug ligand, such as, for example, AF-HPA-Ala, HPA-Ala, is conducted in an aqueous or 10 to 90% organic/aqueous solvent mixture in the presence of an activating agent, such as, for example, EDC.HCl. Typical organic solvents include, but are not limited to, water-miscible solvents, such as, for example, DMSO, DMF, DMA, NMP and ACN. To accelerate the coupling, a co-activator, such as, for example, NHS, is added. The polymer is first mixed with the amino-containing compound followed by the addition of the co-activator (NHS) and then the addition of the activator (EDC.HCl). The reaction is carried out at 0 to 10 °C, pH 4.5 to 7.5 for 1 h to 24 h at room temperature. The resulting polymer conjugate product is purified by diafiltration or SEC. The product is concentrated to 2 to 50 mg/ml, the pH is adjusted to 4.5 to 6.5 to ensure the stability of the drug-polymer ligand, and the conjugate is stored frozen at -20 to -80 °C until further use.

[00487] Conjugation of the polymer with the amine-containing ligand or drug may proceed sequentially, in any order, or simultaneously.

General Procedure B. Selective partial reduction of protein (HER2 antibody)

[00488] Selective partial reduction of the interchain or unpaired disulfide groups on the target HER2 antibody prior to conjugation to the polymer-drug conjugate is achieved using a reducing agent, such as, for example, TCEP, DTT or β-mercaptoethanol. When reduction is performed with an excess of the reducing agent, the reducing agent is removed prior to conjugation by SEC. The degree of conversion of the HER2 disulfide groups to reactive sulfhydryl groups depends on the stoichiometry of HER2, reducing agent, pH, temperature and/or duration of the reaction. When some but not all of the disulfide groups on the PBRM are reduced, the reduced PBRM is a partially reduced HER2.

[00489] General Procedure C. Conjugation of partially reduced HER2 with polymer-drug conjugate

[00490] Conjugation of the partially reduced PBRM to the polymer-drug conjugate is conducted under neutral or slightly basic conditions (pH 6.5 to 8.5) at PBRM concentrations of 1 to 10 mg/ml and polymer-drug conjugate concentration of 0.5 to 10 mg/ml. The polymer-drug conjugate is typically used in a 1 to 5.0-fold excess relative to the stoichiometry of the desired protein-polymer-drug conjugate. When the PBRM is conjugated to the maleimido group of the polymer-drug conjugate, the conjugation is optionally terminated by the addition of a water-soluble maleimido blocking compound, such as, for example, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH2SH), benzyl thiol, and others.

[00491] The resulting HER2-polymer-drug conjugate is typically purified by diafiltration to remove any impurities from the unconjugated polymer-drug conjugate, unconjugated drug or small molecule. Alternatively or additionally, appropriate chromatographic separation procedures, such as, for example, size exclusion chromatography, hydrophobic interaction chromatography, ion chromatography, such as, for example, WCX chromatography; reversed phase chromatography, hydroxyl apatite chromatography, affinity chromatography or a combination thereof may be used to purify the HER2-polymer-drug conjugate. The resulting purified HER2-polymer-drug conjugate is typically formulated in a buffer of pH 5.0 to 6.5.

Example 1: FACS selection for HER2 antibodies

[00492] Two of the HER2 antibodies described herein, XMT 1517 and XMT 1519, were selected using the procedure described below. Eight naive human-synthetic yeast libraries each of ~109 diversity were propagated as previously described (see, e.g., WO 2009036379; WO2010105256; WO2012/009568; and Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663-70). For the first two rounds of selection, a magnetic bead sorting technique using the Miltenyi MACs system was performed, as described (Siegel et al., J Immunol Methods. 2004 Mar;286(1-2):141-53). Briefly, yeast cells (~1010 cells/library) were incubated with 3 ml of 200 nM biotinylated monomeric HER2 antigen or 10 nM biotinylated HER2-Fc fusion antigen for 15 min at room temperature in FACS PBS wash buffer with 0.1% BSA (biotinylations were done using the EZ-Link Sulfo-NHS-Biotinylation Kit, Thermo Scientific, Cat.# 21425). After washing once with 50 ml of ice-cold wash buffer, the cell pellet was resuspended in 40 ml of wash buffer, and 500 μl of Streptavidin MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany. Cat.# 130-048-101) were added to the yeast and incubated for 15 min at 4 °C. The yeast were then pelleted, resuspended in 5 ml wash buffer, and loaded onto a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany. Cat.# 130-042-401). After the 5 ml were loaded, the column was washed 3 times with 3 ml FACS wash buffer. The column was then removed from the magnetic field, and the yeast were eluted with 5 ml culture medium and then grown overnight. The following three rounds of sorting were performed using flow cytometry. Approximately 1 × 108 yeast were pelleted, washed three times with wash buffer, and incubated with 200 nM, 100, or 10 biotinylated HER2 for 10 min at room temperature, respectively. The yeasts were then washed twice and stained with 1:100 diluted goat anti-human F(ab’)2 kappa-FITC (Southern Biotech, Birmingham, Alabama, Cat.# 2062-02) and 1:500 diluted streptavidin-Alexa Fluor 633 (Life Technologies, Grand Island, NY, Cat.# S21375), or 1:50 diluted Extravidin-phycoerythrin (Sigma-Aldrich, St Louis, Cat.# E4011) secondary reagents for 15 min at 4 °C. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.4 ml wash buffer and transferred to strainer-capped sorting tubes. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sorting gates were set to select only HER2-binding clones for two rounds and the third round was a negative sort to decrease reagent binders. After the final round of sorting, yeast were plated and individual colonies were picked for characterization.

Example 2: Affinity maturation of HER2 antibodies

[00493] The affinity matured HER antibodies described herein, XMT 1518 and XMT 1520, were manufactured using the procedure described below. A binding population from Round 5 was used for a light chain batch diversification (LCBD). Heavy chain plasmids were extracted and transformed into a light chain library with a diversity of 1 x 106. Selections were performed as described above with one round of MACS sorting and two rounds of FACS sorting using 10 nM or 1 nM biotinylated antigen for the respective rounds.

[00494] Further affinity maturation was performed on the best clones from the LCBD. Each of the CDRH3 heavy chains from these clones were individually recombined into a pre-made library with variants of 1 x 108 diversity and selections were performed as described above. Affinity pressures were applied by incubation with the yeast antigen antibody complex with precursor Fab for varying amounts of time to select for the highest affinity antibodies.

[00495] A third round of affinity maturation included error-prone PCR of both the heavy chain and light chain. Selections were performed similar to the previous rounds using FACS sorting for all three rounds and with increased times for Fab pressure.

Example 3: Antibody production and purification

[00496] Yeast clones were grown to saturation and then induced for 48 h at 30 °C with shaking. After induction, yeast cells were pelleted and supernatants were harvested for purification. IgGs were purified using a Protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified on KappaSelect (GE Healthcare LifeSciences, Cat.# 17-5458-01).

[00497] Mammalian expression of IgG was achieved by subcloning antibodies into a new expression vector followed by transient transfection and expression in HEK. Briefly, expression vectors containing the antibody of interest are transfected by complexing with a transfection reagent followed by exposure to HEK cells for one hour followed by dilution of the culture medium to a final density of 4 million cells per ml. The cells are then cultured for 7 days with fresh may fed every 48 hours. After 7 days, the supernatant is collected following centrifugation and purification was performed using protein A and if necessary a CHT column purification added to achieve >95% monomer.

Example 4: HER2 Antibody Affinity Measurements

[00498] Affinity for HER2 antibodies was determined by measuring their KD by ForteBio or MSD-SET. ForteBio affinity measurements were generally performed as previously described (Estep et al., MAbs. 2013 Mar-Apr;5(2):270-8). Briefly, ForteBio affinity measurements were performed by loading IgGs online onto AHQ sensors. Sensors were equilibrated offline in assay buffer for 30 min and then monitored online for 60 s to establish baseline. Sensors with loaded IgGs were exposed to 100 nM antigen for 5 min, after which they were transferred to assay buffer for 5 min for off-rate measurements. Kinetics were analyzed using the 1:1 binding model.

[00499] Equilibrium affinity measurements were performed as previously described (Etapa et al., 2013). Equilibrium solution titrations (SET) were performed in PBS + 0.1% IgG-free BSA (PBSF) with antigen held constant at 50 pM and incubated with 3- to 5-fold serial dilutions of antibody starting at 10 nM. Antibodies (20 nM in PBS) were coated onto standard MSD-ECL binding plates overnight at 4 °C or at room temperature for 30 min. Plates were then blocked for 30 min with shaking at 700 rpm, followed by three washes with wash buffer (PBSF + 0.05% Tween 20). SET samples were spotted and incubated on the plates for 150 s with shaking at 700 rpm followed by one wash. Antigen captured on a plate was detected with 250 ng/ml sulfotag-labeled streptavidin in PBSF by incubating on the plate for 3 min. Plates were washed three times with wash buffer and then read on the MSD Sector Imager 2400 instrument using 1x Reading Buffer T with surfactant. The percentage of free antigen was plotted as a function of the titrated antibody on the Prism and fitted to a quadratic equation to extract the KD. To improve throughput, liquid handling robots were used for all MSD-SET experiments, including SET sample preparation. Table I gives the results for the ForteBio and MSD-SET affinity measurements. 1 Measured on ForteBio instrument. IgG on tip and human HER2 monomer in solution. 2 Measured on Meso Scale Discovery instrument. IgG against biotinylated human HER2 monomer.

Example 5: Epitope Binning in Octet Red384

[00500] Epitope binning of antibodies was performed on a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park, CA) using a standard sandwich binning assay. Control anti-target IgG was loaded onto AHQ sensors and unoccupied Fc binding sites on the sensor were blocked with a non-relevant human IgG1 antibody. The sensors were then exposed to 100 nM target antigen followed by a second anti-target antibody. Data were processed using ForteBio Data Analysis Software 7.0. Additional binding by the second antibody after antigen binding indicates an unoccupied (non-competitor) epitope, while no binding indicates epitope blocking (competitor). This process was repeated for the 4 control antibodies: (i) trastuzumab that binds to Domain IV of HER2 (Cho et al., Nature. 2003 Feb 13;421(6924):756-60); (ii) pertuzumab that binds to Domain II of HER2 (Franklin et al., Cancer Cell. 2004 Apr;5(4):317-28); (iii) Fab37 that binds to Domain III of HER2 (Fisher et al., J Mol Biol. 2010 Sep 10;402(1):217-29); and (iv) chA21 which binds to Domain I of HER2 (Zhou et al., J Biol Chem. 2011 Sep 9;286(36):31676-83.; Cheng et al., Cell Res. 2003 Feb;13(1):35-48). The sequences for the control antibodies are shown below: > Trastuzumab Heavy chain variable region amino acid sequence 1 15 30 45 >Trastuzumab light chain variable region amino acid sequence >Pertuzumab heavy chain variable region amino acid sequence >Pertuzumab light chain variable region amino acid sequence >Fab37 heavy chain variable region amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFSIWWSWIHWVRQAPGKGLEWVASISPSSGWTSYADSVKGRFTISADTSKNTAILQMNSLRAEDTAVYYCARWWSSAMDYWGQGTLVTVSS (SEQ ID NO: 44)>Fab37 light chain variable region amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWWWWPSTFGQGTKVEIK (SEQ ID NO: 45)>chA21 light chain variable region amino acid sequence heavyEVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWIGHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYAMDYWGQGTSVTVSS (SEQ ID NO: 46)>chA21 light chain variable region amino acid sequenceDIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNILAWYQQKPG QSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQ QYSNYPWTFGGGTRLEIK (SEQ ID NO: 47)

[00501] The highest affinity antibodies XMT 1519, XMT 1520, XMT 1517, XMT 1518 that did not compete with the four control antibodies were selected.

[00502] As shown in Figure 1A, antibodies XMT 1517 and XMT 1518 and in Figure 1B, antibodies XMT 1519 and XMT 1520 did not compete with the 4 control antibodies.

Example 6: Antibody binding constant measurements with JIMT-1 cells

[00503] Cell surface binding of HER2 antibodies to JIMT-1 cells was assessed using a Macsquant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). JIMT-1 cells, grown overnight to approximately 90% confluent cultures in DMEM medium (ATCC, Manassas, VA) with 10% FBS (Gibco®, Life Technologies, Grand Island, NY), were released from the plate surface by treatment with Accutase cell detachment solution (Innovative Cell Technologies, San Diego, CA). Detached cells were washed once with ice-cold medium containing 6% goat serum and resuspended in the same medium. 100,000 cells were aliquoted per well of a 96-well V-bottom plate and incubated with a range of HER2 antibody concentrations (0.05 to 100 nM) in 150 μL DMEM with 6% goat serum on ice for 3 hours. Cells were then washed once with ice-cold PBS and resuspended in 100 μL DMEM with 2% goat serum and 6 μg/ml of a fluorescently labeled secondary antibody, Alexa Fluor® 647-labeled goat anti-human IgG (Life Technologies Cat.# A-21445) for 1 hour on ice. Cells were washed once with ice-cold PBS (Gibco®, Life Technologies, Cat.# 10010049) and resuspended in 200 μL ice-cold PBS with 1% paraformaldehyde. The amount of bound fluorescence per cell was determined by running 5000 cells for each treatment on the flow cytometer. The mean fluorescence value for each treatment was plotted, and the dissociation constant, Kd, was calculated for each antibody with Graphpad Prism software by nonlinear regression using the single-site specific binding model. Figure 2 shows the binding of HER2 antibodies to JIMT-1 cells. The calculated Kd values were 3.4 nM for XMT 1517, 0.4 nM for XMT 1518, 1.2 nM for XMT 1519, and 1.1 nM for XMT 1520.

Example 7: Antibody affinity measurements by ELISA

[00504] The affinity of antibodies for recombinant human and cynomolgus monkey HER2 was determined by an ELISA assay. The extracellular domain of purified, recombinant HER2, derived from human (amino acids 23 to 652, Acro Biosystems Cat.# HE2-H5225) or derived from cynomolgus monkey (amino acids 1 to 652, Sino Biological, Cat.# 90295-C08H) was coated onto the surface of 96-well plates by incubating each well with 50 μL of a 1 μg/ml solution in 50 mM bicarbonate buffer, pH 9.0, for 2 hours at room temperature. The wells were washed 4 times with TTBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20). The wells were blocked by incubation with 50 μl of TTBS containing 5% bovine serum albumin for 1 h. A range of dilutions (0.005 to 10 nM, 3-fold serial dilutions) of each test antibody or Example 16H in 50 μl of TTBS with 3% BSA was then added for 2 h at room temperature. Unbound test antibodies were removed by washes with TTBS (4x). An HRP-conjugated anti-human IgG secondary (Bethyl Laboratories, #A80-115P) at a concentration of 0.2 μg/ml in TTBS with 3% BSA was incubated in each well for 1 h. Unbound secondary antibody was removed by 4 washes with TTBS. The HRP substrate, TMB Bethyl Laboratories#E102) was added to each well and incubated until a yellow color was visible. The reaction was stopped by the addition of 50 μL of 0.2 N sulfuric acid. Absorbance at 450 nm was measured in a plate reader (Molecular Devices, Spectramax M5). Values were plotted using GraphPad Prism software. Kd was determined by nonlinear regression using the site-specific binding model.

[00505] Figure 3a gives the results for the binding of anti-HER2 antibodies XMT 1517, XMT 1518 to human HER2 and cynomolgus monkey HER2. Figure 3b gives the results for the binding of anti-HER2 antibodies XMT 1519, XMT 1529 to human HER2 and cynomolgus monkey HER2. The calculated Kd values are given in Table II. Table II

[00506] The binding affinities for the 4 test antibodies to human HER2 and cynomolgus monkey HER2 were similar.

[00507] Figures 3c and 3d show the results for the binding of anti-HER2 antibodies XMT 1519 and Example 16H to human HER2 and cynomolgus monkey HER2 respectively. The calculated Kd values are given in Table IIA below. Table IIA

[00508] The binding affinities for XMT 1519 and Example 16H for Human HER2 and cynomolgus monkey HER2 were similar. Example 8: Antibody Competition Assay with JIMT-1 Cells

[00509] To confirm that XMT 1518 and XMT 1519 antibodies bind to overlapping epitopes, the antibodies were analyzed in a competition binding assay on JIMT-1 cells. JIMT-1 cells were cultured as described in Example 6. 50,000 cells in 50 μL were aliquoted into wells of a 96-well V-bottom plate. Then 30 nM of Alexa Fluor® -647-conjugated XMT 1519 antibody in 25 μL of medium was added alone, or mixed with a second unlabeled antibody (XMT 1518, XMT 1519, or trastuzumab) diluted to a range of concentrations (0.05 to 100 nM), and incubated on ice for 3 hours. Cells were then washed once with ice-cold PBS and resuspended in 100 μl DMEM with 2% goat serum and 6 μg/ml Alexa Fluor® 647-labeled goat anti-human IgG (Life Technologies, Cat.# A-21445) for 1 h on ice. Cells were washed once with ice-cold PBS and resuspended in 200 μl ice-cold PBS with 1% paraformaldehyde. The amount of fluorescence bound per cell was determined by running 5000 cells for each treatment on a MacsQuant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). The mean fluorescence value for each treatment was plotted, and the dissociation constant KD was calculated for each antibody with GraphPad Prism by nonlinear regression using the single-site specific binding model. As shown in Figure 4, the XMT 1518 antibody competed for binding to Alexa Fluor® 647-labeled XMT 1519 to a similar degree as unlabeled XMT 1519 indicating that both antibodies recognize overlapping epitopes on HER2 whereas trastuzumab did not compete for binding to Alexa Fluor® 647-labeled XMT 1519.

Example 9: Antibody-dependent cell-mediated cytotoxicity assay

[00510] Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-HER2 antibodies was quantified in BT474 cells. 5000 cells were plated per well of 96-well plates in DMEM containing 10% FBS and cultured overnight at 37° in 5% CO2. A range of antibody concentrations to XMT 1518, XMT 1519, XMT 1520, or trastuzumab from 0.001 to 100 nM (8 serial dilutions, 5-fold) were added to the cells and ADCC activity was measured with a Promega bioassay kit (Cat.# G7018) that uses effector cells engineered to produce luciferase enzyme as a reporter of ADCC activity. Luciferase activity was measured on a spectrophotometer (Molecular Devices, Spectramax M5). Values (expressed as a ratio of the luciferase activity of each sample of antibody-treated cells to that of a sample of cells that were not treated with antibody) were plotted by nonlinear regression using the four-parameter, variable-slope, log agonist vs response model using GraphPad Prism software.

[00511] Figure 5 is a graph showing ADCC values. XMT1519 and XMT 1520 show maximal activity similar to that of trastuzumab: the ratio of signal with antibody to signal without antibody is 30.1 for XMT 1519 and 29.7 for XMT 1520, while that of trastuzumab is 34.6. XMT 1518 has maximal activity of 11.0. The EC50 (half-maximal binding) values for ADCC activity are 0.16 nM for XMT 1518, 0.18 nM for XMT 1519, 0.54 nM for XMT 1520, and 0.07 nM for trastuzumab, respectively.

Example 10: Ligand-dependent HER-2 signaling in MCF7 cells

[00512] The effects of anti-HER2 antibodies on ligand-dependent HER-2 signaling in MCF7 cells (ATCC, HTB-22) were determined by measuring the amount of phosphorylated AKT after treatment of the cells with the HER3 ligand, neurregulin-β1. 300,000 MCF7 cells were plated in MEM medium with 0.01 mg/ml bovine insulin and 10% FBS in each well of 6-well culture plates and cultured overnight at 37° in a 5% CO2 atmosphere. Media were removed and replaced with fresh media containing 10 μg/ml test antibody and incubated for 1 hour, followed by a 15-minute treatment with 40 pM neurregulin-β1 (R&D Systems, Minneapolis, MN, Cat.# 377-HB-050). Cells were washed once with ice-cold PBS and lysed by the addition of 200 μl of buffer containing 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, Complete Protease Inhibitor Cocktail tablets (Roche, Indianapolis, IN), and PhosSTOP phosphatase inhibitor cocktail tablets (Roche, Indianapolis, IN). Lysates were centrifuged at 15,000 rpm for 15 minutes at 4° to remove insoluble fragments. Phosphorylated AKT levels were determined using Cell Signaling Technology PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit, following the manufacturer's instructions.

[00513] Table III shows the increase in AKT phosphorylation induced by neuregulin treatment of MCF7 cells, expressed as a percentage of that measured in unstimulated cells.Table III

[00514] Neuregulin treatment increased phosphorylated AKT levels fivefold (522% relative to unstimulated cells). XMT 1517, XMT 1518, XMT 1519, and XMT 1520 caused little or no reduction in the neurregulin-induced increase in AKT phosphorylation to 564, 441, 381, and 439% respectively, similar to that caused by trastuzumab (369%). Pertuzumab, which has been shown to inhibit ligand-dependent signaling (Franklin et al., Cancer Cell. 2004 Apr 19;5:317-28), reduced neurregulin stimulation to near that of unstimulated cells (127%). These results suggest that the test antibodies do not act by inhibiting the heterodimerization of HER2 and HER3 that is promoted by neurregulin and is inhibited by pertuzumab.

Example 11: Ligand-independent HER-2 signaling in MCF7, SKBR3, and JIMT-1 cells

[00515] The effects of anti-HER2 antibodies on HER2 signaling in MCF7 (ATCC, HTB-22), SKBR3 (ATCC, HTB-30), JIMT-1 (ATCC, HTB-30) cells were determined by measuring changes in the amount of phosphorylated AKT after a 4-hour antibody treatment. Cells were plated in cell-specific medium with 10% FBS in each well of 6-well culture plates and grown overnight at 37° in a 5% CO2 atmosphere. The media were removed and replaced with fresh medium containing 10 μg/ml test antibody and incubated for 4 hours. Cells were washed once with ice-cold PBS and lysed by the addition of 200 μl of buffer containing 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, Complete Protease Inhibitor Cocktail tablets (Roche, Indianapolis, IN), and PhosSTOP Phosphatase Inhibitor Cocktail tablets (Roche, Indianapolis, IN). Lysates were centrifuged at 15,000 rpm for 15 min at 4° to remove insoluble fragments. Phosphorylated AKT levels were determined using Cell Signaling Technology PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit following the manufacturer's instructions.

[00516] Table IV shows the ligand-independent inhibition of HER2 signaling in MCF7, SKBR3, and JIMT-1 cells as indicated by the amount of AKT phosphorylation. Results are presented relative to the percentage of untreated cells. Table IV

[00517] Anti-HER2 test antibodies caused a strong inhibition of HER2 signaling in SKBR3 cells where XMT 1517, XMT 1518, XMT 1519, and XMT 1520 reduced phosphorylated AKT levels to 43%, 59%, 34%, and 29% of that in untreated cells, compared to a 64% reduction caused by trastuzumab.

[00518] Anti-HER2 antibodies inhibited HER2 signaling in JIMT-1 cells as indicated by a reduction in phosphorylated AKT to 66%, 62%, 84%, and 69% of that occurring in untreated cells for XMT 1517, XMT 1518, XMT 1519, and XMT 1520, respectively, similar to the 56% reduction caused by trastuzumab.

[00519] In MCF7 cells XMT 1518, like trastuzumab, did not inhibit HER2 signaling, however XMT 1517, XMT 1519 and XMT 1520 caused modest reductions in signaling, reducing AKT phosphorylation to 79%, 76%, and 66% of that occurring in untreated cells.

Example 12: Ligand-independent HER-2 signaling in BT474 cells

[00520] The effects of anti-HER2 antibodies on HER2 signaling in BT474 cells (ATCC, HTB-20) were determined by measuring changes in the amount of phosphorylated AKT after a 4-hour antibody treatment. BT474 cells (300,000 cells) were plated in DMEM medium with 10% FBS in each well of 6-well culture plates and grown overnight at 37° in a 5% CO2 atmosphere. The media was removed and replaced with fresh medium containing 10 μg/ml test antibody and incubated for 4 hours. Cells were washed once with ice-cold PBS and lysed by the addition of 200 μl of buffer containing 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, complete protease inhibitor cocktail tablets (Roche, Indianapolis, IN), and PhosSTOP phosphatase inhibitor cocktail tablets (Roche, Indianapolis, IN). Lysates were centrifuged at 15,000 rpm for 15 min at 4° to remove insoluble fragments. Levels of phosphorylated AKT were determined by Western analysis. 20 μL of each extract was mixed with 7 μL of NUPAGE loading dye (Life Technologies, Cat.# NP0007) and 2 μL of 10x reducing agent (Life Technologies Cat.# NP0004) and loaded onto a 4 to 12% Bis-Tris polyacrylamide gel (Life Technologies, Cat.# NP0341) that was run in MOPS running buffer (Life Technologies Cat.# NP000102) for 90 min at 120 volts. The separated proteins were transferred to a nitrocellulose membrane in a semidry electrophoretic transfer system (Bio-Rad, Transblot system) for 30 min at 10 volts in transfer buffer (Life Technologies, Cat.# NP0006) containing 10% methanol. The membranes were incubated for 1 hour in blocking buffer (Li-cor, Cat.# 927-40000) and then with a mouse antibody recognizing AKT protein (Cell Signaling Technology, #2920) diluted 1:1000, a rabbit antibody recognizing AKT phosphorylated at serine 473 (Cell Signaling Technology, #4060) and a rabbit antibody recognizing actin (LiCor, Cat.# 926-42210) diluted 1:5,000 in the same blocking buffer for 1 hour. After incubation, the membrane was washed 3 times with 10 ml of TTBS and then incubated for 1 hour with secondary antibodies: a goat anti-rabbit IgG conjugated to IRdye® 800CW (Li-Cor, Cat.# 926-32211) and a goat anti-mouse IgG conjugated to IRdye® 680RD (Li-Cor, Cat.# 926-68070) both diluted 1:10,000 in blocking buffer. The membrane was again washed 3 times with 10 ml of TTBS and scanned on a Li-Cor Odyssey scanner. The bands corresponding to AKT, phospho-AKT, and actin were quantified using the scanner software. Each phospho-AKT band was normalized to the total AKT band and expressed as a percentage of phospho-AKT protein from cells that were not treated with any of the anti-HER2 antibodies.

[00521] The effects of anti-HER2 antibodies on HER3 phosphorylation were also examined in the same experiment. The nitrocellulose blots described above were also probed with a rabbit antibody that recognizes HER3 phosphorylated at tyrosine 1289 (Cell Signaling Technology, #4791) diluted 1:1000 in blocking buffer for 1 hour. After incubation, the membrane was washed 3 times with 10 ml of TTBS and then incubated for 1 hour with a goat anti-rabbit IgG conjugated to IRdye® 800CW (Li-Cor, Cat.# 926-32211) diluted 1:10,000 in blocking buffer. The membrane was again washed 3 times with 10 ml of TTBS and scanned on a Li-Cor Odyssey scanner. The corresponding bands for phospho-HER3 and actin were quantified using the scanner software. Each phospho-HER3 band was normalized to the total AKT band and expressed as a percentage of phospho-HER3 protein from cells that were not treated with any of the anti-HER2 antibodies.

[00522] Table V gives the inhibition of ligand-independent HER2 signaling in BT474 cells as determined by the amount of AKT phosphorylation or Her3 phosphorylation. Results are presented relative to the percentage of unstimulated cells. Table V

[00523] The anti-HER2 antibodies XMT 1519 reduced the phosphorylation of AKT to approximately 21% and HT19B to approximately 30% of that observed in untreated cells in BT474, compared with trastuzumab which caused a reduction to approximately 31%, and pertuzumab which caused a reduction to approximately 44% in untreated cells. HER3 phosphorylation was modestly affected by all antibodies: 71%, 76%, 80%, and 85% of that observed in untreated cells by XMT 1519, XMT 1520, trastuzumab and pertuzumab, respectively.

Example 13: Measurements on the internalization rate

[00524] The rate at which each antibody, XMT 1518, XMT 1519, XMT 1520, and trastuzumab, is internalized from the cell surface of SKBR3 cells was determined by fluorescence in a 96-cell based assay. SKBR3 cells were plated in 96-well plates and allowed to attach overnight by cultured in DMEM (ATCC, Manassas, VA, Cat.# 30-2002) with 10% fetal bovine serum (FBS) (Gibco®, Life Technologies, Grand Island, NY, Cat.# 16140-071) at 37° in 5% CO2. Cells were incubated in medium containing 10 μg/ml antibody on ice for 1 hour, then washed with ice-cold medium and incubated on ice with 75 μl of medium containing 1 μg/ml of a monovalent goat anti-human IgG Fab fragment (Rockland Immunochemicals, Gilbertsville, PA, Cat.# 8091102) conjugated to Alexa Fluor® -647 for 1 hour. After washing, cells were transferred to a 37° incubator to allow internalization at various time points. Plates were scanned on an Odyssey scanner (Li-Cor Biosciences, Lincoln, NE) using the 700 nm channel. The plates were then acid washed by incubation with 100 mM glycine, 20 mM MgSO4, 50 mM KCl, pH 2.2, on ice to remove cell surface-bound antibodies, washed with ice-cold PBS, and scanned again to determine the amount of fluorescence internalized by the cells. Figure 6 shows the rate of antibody internalization at 4 h. The rate of internalization of all antibodies is nearly identical to that of trastuzumab, with all having approximately 50% of the total surface area bound at time 0 internalized at 4 h (Figure 6).

Example 13A: HER2 internalization in SKBR3 cells

[00525] The effect of a combination of anti-HER2 antibodies on HER2 internalization in SKBR3 cells was investigated by fluorescence microscopy. Trastuzumab was conjugated to Alexa Fluor-647 and was used to visualize the localization of trastuzumab and presumably HER2 within the cells. SKBR3 cells were observed on 4-chamber microscope slides and allowed to attach overnight in DMEM (ATCC, Manassas, VA, Cat.# 30-2002) with 10% fetal bovine serum (FBS) (Gibco®, Life Technologies, Grand Island, NY, Cat.# 16140-071) at 37° in 5% CO2. SKRB3 cells were incubated for 90 min in medium containing (i) a combination of trastuzumab-Alexa-647 and pertuzumab or (ii) a combination of trastuzumab-Alexa-647 and pertuzumab and HER2 antibody XMT 1519. Trastuzumab localization was visualized using fluorescence microscopy. As shown in Figure 7 , while most of trastuzumab-Alexa Fluor-627 in combination with pertuzumab alone colocalized with orange cell mask, suggesting plasma membrane localization, most of trastuzumab-Alexa Fluor-647 in combination with both pertuzumab and XMT 1519 colocalized with Lysotracker, suggesting lysosomal localization. This result suggests that the presence of XMT 1519 in combination with trastuzumab and pertuzumab promotes the internalization and trafficking of HER2 to lysosomes.

Example 13B: Internalization rate measurements with anti-HER2 antibody combination

[00526] The effects of combining multiple anti-HER2 antibodies that recognize different epitopes on the rate of HER2 internalization was determined with a 96-well plate-based assay similar to that described in Example 13. In this experiment, cells were treated with a HER2 test antibody alone, in combination with trastuzumab, or with trastuzumab and pertuzumab, and the amount of anti-HER2 antibody internalized at various times was measured.

[00527] For the assay, 40,000 SKBR3 cells were plated in DMEM medium containing 10% FBS, in each well of three 96-well plates (black colored plates with clear bottom wells) and allowed to attach overnight at 37° in 5% CO2. The next day the cells were treated with antibodies as follows. The medium in each well was replaced with fresh, ice-cold medium containing 5 μg/ml of one of the test HER2 antibodies, or trastuzumab, and incubated on ice for 1 hour. Unbound antibody was removed by washing the cells twice with ice-cold medium. For detection, 5 μg/ml of an Alexa 647-labeled anti-human IgG Fab fragment was added to each well in ice-cold medium and incubated on ice for 1 hour. Unbound secondary antibody was removed by washing each well twice with ice-cold medium. Then, medium containing 5 μg/ml trastuzumab, 5 μg/ml each of trastuzumab and pertuzumab, or no additional antibody was added to each well. Each treatment was performed in triplicate. One set of wells was treated with the monovalent Alexa 647-labeled anti-human IgG Fab fragment alone, and the fluorescence measured in these wells was used to subtract background fluorescence.

[00528] One plate was left on ice to determine time 0 values for each treatment, and the other plates were incubated at 37 °C. After 1.5 hours, one plate at 37 °C, and the time 0 plate, were washed twice with ice-cold PBS and scanned using the 680 nm channel of a Licor Odyssey scanner. The fluorescence measured in each well is used as the total fluorescence for each treatment. The cells were then acid washed twice with 100 mM glycine, 50 mM KCl, and 20 mM MgSO4, pH 2.2, then washed twice with ice-cold PBS to remove surface-bound antibodies. The plates were scanned once more to determine the amount of fluorescence internalized in each well. The 24-hour plates were analyzed in the same manner the following day.

[00529] The percentage of each HER2 antibody (or trastuzumab in control wells) internalized is calculated by subtracting the background, averaging each of the 3 replicates, then dividing the internalized fluorescence by the total fluorescence for each treatment and multiplying by 100.

[00530] As shown in Table VA., 80 to 90% of XMT 1518, XMT 1519, and XMT 1520 are internalized within one and a half hours when combined with either trastuzumab or pertuzumab. XMT 1517 shows 60% internalization, likely due to its very high dissociation, which causes it to diffuse out of the cells during incubations. When combined with trastuzumab, 20 to 40% of each test antibody is internalized within one and a half hours, and about 15% is internalized when each test antibody is present alone. Trastuzumab alone shows about 15% internalization within one and a half hours, and about 30% when combined with pertuzumab.

[00531] Thus, the combination of three HER2 antibodies; any of the test HER2 antibodies, trastuzumab and pertuzumab, increases the rate of antibody internalization, most likely by increasing the rate of HER2 endocytosis.Table VA

Example 14: HER2 degradation

[00532] The effects of anti-HER2 antibodies on HER2 degradation in SKBR3 cells were determined by western analysis after antibody treatment. 300,000 SKBR3 cells were plated in DMEM medium with 10% FBS in each well of 6-well culture plates and grown overnight at 37° in a 5% CO2 atmosphere. The media were removed and replaced with fresh medium containing 10 μg/ml of each antibody and incubated for 4 hours. Cells were washed once with ice-cold PBS and lysed by the addition of 200 μl of buffer containing 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, complete protease inhibitor cocktail tablets (Roche, Indianapolis, IN), and PhosSTOP phosphatase inhibitor cocktail tablets (Roche, Indianapolis, IN). Lysates were centrifuged at 15,000 rpm for 15 min at 4° to remove insoluble fragments. 20 μL of each extract was mixed with 7 μL of NUPAGE loading dye (Life Technologies, Cat.# NP0007) and 2 μL of 10x reducing agent (Life Technologies Cat.# NP0004) and loaded onto a 4 to 12% Bis-Tris polyacrylamide gel (Life Technologies, Cat.# NP0341) that was run in MOPS running buffer (Life Technologies Cat.# NP000102) for 90 min at 120 volts. Separated proteins were transferred to a nitrocellulose membrane in a semidry electrophoretic transfer system (Bio-Rad, Transblot system) for 30 min at 10 volts in transfer buffer (Life Technologies, Cat.# NP0006) containing 10% methanol. The membrane was incubated for 1 hour in blocking buffer (Li-cor, Cat.# 927-40000) and then with a rabbit antibody recognizing HER2 protein (Cell Signaling Technology, Cat.# 2165) diluted 1:1,000, and a rabbit antibody recognizing actin (LiCor, Cat.# 926-42210) diluted 1:5,000 in the same blocking buffer for 1 hour. After incubation, the membrane was washed 3 times with 10 ml of TTBS and then incubated for 1 hour with secondary antibodies: a goat anti-rabbit IgG conjugated to IRdye® 800CW (Li-Cor, Cat.# 926-32211) and a goat anti-mouse IgG conjugated to IRdye® 680RD (Li-Cor, Cat.# 92668070) both diluted 1:10,000 in blocking buffer. The membrane was again washed 3 times with 10 ml of TTBS and scanned on a Li-Cor Odyssey scanner. The bands corresponding to full-length HER2 protein and actin were quantified using the scanner software. Each HER2 band was normalized to the actin band and expressed as a percentage of the HER2 protein of cells that were not treated with either anti-HER2 antibody.

[00533] Figure 8 shows that treatment of SKBR3 cells with the test antibodies alone or in combination with trastuzumab had little or no effect on HER2 levels, with the exception of XMT 1518 and XMT 1520 which caused reductions in full-length HER2 to 78 and 68%, respectively, and caused the appearance of lower molecular weight degradation products. However, triple combinations of trastuzumab, pertuzumab, and one of the HER2 test antibodies caused extensive degradation of HER2, as well as the appearance of HER2 degradation products. The largest reductions are observed when XMT 1518, or XMT 1520, are combined with trastuzumab and pertuzumab, with 20%, or 31% of HER2 remaining. When the antibody XMT 1517 or XMT 1519 is combined with trastuzumab and pertuzumab, the reduction is 40% or 42% respectively of the normal amount of remaining HER2.

Example 15: Epitope Mapping

[00534] Epitope mapping of antibodies XMT 1517 and XMT1519 was performed by Integral Molecular Inc., 3711 Market Street, Suite 900, Philadelphia, Pa., USA, using their Shotgun Mutagenesis Technology. Shotgun Mutagenesis uses a patented high-throughput cellular expression technology that enables the expression and analysis of large libraries of mutated target proteins within eukaryotic cells. Each residue in a protein is individually mutated, usually to multiple other amino acids, in order to assay for changes in function. The proteins are expressed within standard mammalian cell lines, so that difficult proteins that would require eukaryotic translation or post-translational processing can be mapped.

[00535] Shotgun mutagenesis mapping identified six critical amino acids for XMT 1517 binding (C453, H473, N476, R495, H497, and W499) indicating that XMT 1517 binds to the C-terminal regions of Domain III and the N-terminal regions of Domain IV of HER2. Two critical secondary mutations at H456 and G496 would also be involved in XMT 1517 binding to HER2.

[00536] Three critical amino acids for XMT1519 binding (E521, L525, and R530) were identified indicating that XMT 1519 binds a region at the N-terminus of Domain IV of Her2. Example 16: Synthesis of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)))

[00537] HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates were prepared using the procedure described in US Serial No. 14/512,316 filed October 10, 2014. Table VI gives the details of the antibody-polymer-drug conjugates.Table VI

[00538] The HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates had a peak molecular weight of about 170 kDa to about 230 kDa. The synthesized/measured polymer and polymer conjugates typically have a polydispersity of <1.5. The polymer-drug conjugates (i.e., the antibody linked to the drug-carrying polymer chain) contained about 27 mol % to about 33 mol % beta-alanine, about 6.4 mol % to about 9.6 mol % AF-HPA-Ala, and about 1.5 mol % to about 4 mol % EG2-MI.Example 17: Synthesis of Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)))

[00539] Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) (non-binding control) conjugates were prepared using the procedure described in US Serial No. 14/512,316 filed October 10, 2014. Table VII gives the details of the antibody-polymer-drug conjugates.Table VII

[00540] The HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates had a peak molecular weight of about 170 kDa to about 230 kDa. The polymer and polymer conjugates synthesized/measured typically have a polydispersity <1.5. The polymer-drug conjugates (i.e., the polymer chain carrying drug attached to the antibody) contained about 27 mol % to about 33 mol % beta-alanine, about 6.4 mol % to about 9.6 mol % AF-HPA-Ala, and about 1.5 mol % to about 4 mol % EG2-MI.

Example 18: Cytotoxicity assays for HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates

[00541] HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates were evaluated for their antiproliferation properties in tumor cell lines in vitro using Cell Titer-Glo (Promega Corp). JIMT-1 (HER2-expressing cells, DSMZ, Braunschweig, Germany, Cat.# ACC589), MDA-MB-361 (HER2-expressing human breast cancer cells, ATCC, Cat.# HTB-27), MDA-MB-453 cells (triple negative breast cancer cell line, ATCC, Cat.# HTB-131), were cultured in DMEM medium (ATCC, Manassas, VA, Cat.# 30-2002) with 10% FBS (Gibco®, LifeTechnologies, Grand Island, NY, Cat.# 16140-071). NCI-H522 (non-small cell lung carcinoma cell line, non-amplified, ATCC, Cat.# CRL-5810), and NCI-H2170 (non-small cell lung carcinoma cell line, ATCC, Cat.# CRL-5928) cells were cultured in RPMI-1640 medium (ATCC, Cat.# 30-2001). NCI-H1581 (non-small cell lung carcinoma medium-expressing cell line, ATCC, Cat.# CRL-5878) were cultured in DMEM:F12 medium (ATCC, Cat.# 30-2006). SNU5 (gastric carcinoma cell line, non-amplified, ATCC, Cat.# CRL-5973) were cultured in Iscove's Modified Dulbecco's Medium (Invitrogen Life Technologies, Cat.# 12440053) with 20% FBS. OVCAR3 (ovarian adenocarcinoma cell line, non-amplified, ATCC, Cat.# HTB-161) was cultured in RPMI medium with 20% FBS. MDA-MB-175-VII (human breast cancer cell line, non-amplified, ATCC, Cat.# HTB-25), was cultured in Leibovitz's L-15 Medium with 20% FBS. CAMA-1 (human breast cancer cell line, non-amplified, ATCC, Cat.# HTB-21), was cultured in 90% Eagle's Minimum Essential Medium formulated by ATCC. ZR75-1 (human breast cancer cell line, non-amplified, ATCC, Cat.# CRL-1500), was cultured in RPMI-1640 Medium. HCC1187 (human breast cancer cell line, non-amplified, ATCC, Cat.# CRL-2322), was cultured in RPMI-1640 Medium. HCC38 (human breast cancer cell line, non-amplified, ATCC, Cat.# CRL-2314), was cultured in RPMI-1640 Medium. T47D (human breast cancer cell line, non-amplified, ATCC, Cat.# HTB-133), was cultured in RPMI-1640 Medium. HCC70 (human breast cancer cell line, non-amplified, ATCC, Cat.# CRL-2315), was cultured in RPMI-1640 Medium. MDA-MB-231 (human breast cancer cell line, non-amplified, ATCC, Cat.# HTB-26), was cultured in DMEM Medium. CALU3 (human lung adenocarcinoma cell line, ATCC, Cat.# HTB-55) was cultured in Eagle's Minimum Essential Medium formulated by ATCC. A549 (human lung carcinoma cell line, non-amplified, ATCC, Cat.# CCL- 185) was cultured in F12K Medium. NCI-H2122 (human lung adenocarcinoma cell line, non-amplified, ATCC, Cat.# CRL- 5985) was cultured in RPMI-1640 Medium, (ATCC, Cat.# 30-2001). NCI-H460 (human lung carcinoma cell line, non-amplified, ATCC, Cat.# HTB-177) was cultured in RPMI-1640 Medium, (ATCC, Cat.# 30-2001). SHP-77 (human non-small cell lung cancer cell line, non-amplified, ATCC, Cat.# CRL-2195) was cultured in RPMI-1640 Medium, (ATCC, Cat.# 30-2001). KATO III (human gastric carcinoma cell line, non-amplified, ATCC, Cat.# HTB-103) was cultured in Iscove's Modified Dulbecco's Medium with 20% FBS. MKN-45 III (human gastric adenocarcinoma cell line, non-amplified, DSMZ, Braunschweig, Germany, Cat.# ACC409) was cultured in RPMI-1640 Medium, (ATCC, Cat.# 30-2001). SKOV3 (human ovarian adenocarcinoma cell line ATCC, Cat.# HTB-77), was cultured in McCoy's Medium 5a. TOV-21G (human ovarian adenocarcinoma cell line, non-amplified, ATCC, Cat.# CRL-11730) was cultured in 1:1 mixture of MCDB 105 Medium containing a final concentration of 1.5 g/L sodium bicarbonate and Medium 199 containing a final concentration of 2.2 g/L sodium bicarbonate. BT474 (human HER2 high expressing breast cancer cells, ATCC, Cat.# HTB-20) was cultured in DMEM Medium with 10% FBS. NCI-N87 (HER2 high expressing gastric cancer cell line, ATCC, Cat.# CRL-5822), was cultured in RPMI-1640 Medium 10% FBS.

[00542] For the cytotoxicity assay, cells were plated at a density of 3000 cells per well in 96-well plates and allowed to attach by incubating overnight at 37°C in the presence of 5% CO2. Media was then replaced with fresh media containing a strip of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates (100 nM to 0.1 pM), or Kadcil (Genentech) and cells were incubated for 72 hours or 6 days at 37° in the presence of 5% CO2. Cell survival was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI) as described in the kit instructions. Cell viability was normalized to the untreated control and expressed as a percentage. The values were plotted and IC50 values calculated with Graphpad Prism software (San Diego, CA) using a 4-parameter, variable-slope dose-response curve fitting algorithm. Table VIII gives illustrative results for the cytotoxicity of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Kadcila conjugates.Table VIII ND = not determinedb = Example 16C was used in the assay instead of Example 16B c = equivalent potency for the irrelevant antibody control

[00543] As shown in Table VIII, all HER2 antibody-polymer-drug conjugates are more potent than Kadcila in all cell lines tested.

Example 19: Cytotoxicity assays for HER2-(EG2-MI-(10 kDa PHF- BA-(AF-HPA-Ala))) conjugated in a cell line with a mutant HER2.

[00544] HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates were evaluated for their antiproliferation properties on tumor cell lines in vitro using Cell Titer-Glo (Promega Corp). NCI-H1781 (human lung cancer cells expressing mutant HER2, ATCC, Cat.# CRL-5894) were cultured in RPMI-1640 Medium containing 10% FBS.

[00545] For cytotoxicity assays, cells were plated at a density of 3000 cells per well in 96-well plates and allowed to attach by incubating overnight at 37°C in the presence of 5% CO2. The media were then replaced with fresh media containing a range of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates (100 nM to 0.1 pM), Example 17A (rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), or Kadcil (Genentech) and the cells were incubated for 72 h at 37 °C in the presence of 5% CO2. Cell survival was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI) as described in the kit instructions. Cell viability was normalized to the untreated control and expressed as a percentage. Values were plotted and IC50 values calculated with Graphpad Prism software (San Diego, CA) using a 4-parameter, variable-slope dose-response curve fitting algorithm. Table IX gives the results of these tests.Table IX

[00546] As shown in Table IX, all HER2 antibody-polymer-drug conjugates are more potent than rituximab-antibody-polymer-drug conjugate or Kadcila in the cell lines tested.

Example 20. Tumor Growth Response to Administration of PBRM-Polymer-Drug Conjugates

[00547] Female CB-17 SCID mice were inoculated subcutaneously with NCI-N87 cells (n = 10 for each group), JIMT-1 cells (n = 10 for each group), SNU-5 cells (n = 10 for each group), H522 cells (n = 10 for each group), SKOV3 cells (n = 10 for each group), Calu-3 cells (n = 10 for each group), Kadcila-resistant NCI-N87 cells (n = 10 for each group), Kadcila-resistant NCI-N87-MSA cells (n = 10 for each group), or BT474 tumor fragments (n = 10 for each group). Female NCr nu/nu mice were inoculated subcutaneously with TOV-21G cells (n = 10 for each group). Test compounds or carriers were dosed IV as a single dose on day 1 or as indicated. Tumor size was measured at the times indicated in Figures 8 to 18 using digital calipers. Tumor volume was calculated and was used to determine tumor growth retardation. Mice were sacrificed when tumors reached a size of 800 to 1500 mm3. Tumor volumes are reported as the mean ± SEM for each group.

[00548] Figure 9 provides the results for the tumor response in mice subcutaneously inoculated with NCI-N87 cells (n = 10 for each group) after IV administration of carrier; Example 16A, trastuzumab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 0.67 mg/kg; or Example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 0.67 mg/kg as a single dose on day 1. Both carrier and trastuzumab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), showed an increase in tumor volume. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) showed 60% partial regressions and resulted in potential therapeutic activity (TGI 88%) based on tumor growth inhibition analysis at Day 29.

[00549] Figure 10 provides the results for tumor response in mice subcutaneously inoculated with JIMT-1 cells (n = 10 for each group) after IV administration of carrier; Kadcila at 20 mg/kg; Example 16A, trastuzumab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 0.67 mg/kg; or Example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 0.67 mg/kg as a single dose on day 1. Carrier and Kadcila showed an increase in tumor volume. The conjugate of trastuzumab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) showed therapeutic potential and had regressions of 40 % and 70 % respectively. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) had 5 partial regressions and 2 complete regressions of survivors who remained tumor free at the end of the study on Day 69.

[00550] Figure 11 provides the results for tumor response in mice inoculated subcutaneously with JIMT-1 cells (n = 10 for each group) after IV administration of carrier; Kadcila at 10 mg/kg as a single dose on day 1; a combination of Kadcila at 10 mg/kg and pertuzumab at 15 mg/kg dosed weekly for 3 weeks; or Example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 0.67 mg/kg as a single dose on day 1. The carrier, Kadcila, and the combination of Kadcila and pertuzumab all showed an increase in tumor volume. The XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugate showed 60% partial regressions and was the most effective.

[00551] Figure 12 provides the results for the tumor response in mice inoculated subcutaneously with NCI-N87 cells (n = 10 for each group) after IV administration of carrier; Example 16F, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 0.67 mg/kg as a single dose on day 1; a combination of trastuzumab at 15 mg/kg and pertuzumab at 15 mg/kg dosed weekly for 3 weeks (i.e., on day 1, 8, and 15); or a triple combination of trastuzumab at 15 mg/kg and pertuzumab at 15 mg/kg each dosed weekly for 3 weeks (i.e., on days 1, 8, and 15) together with Example 16F, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 0.67 mg/kg as a single dose on day 1. The carrier showed an increase in tumor volume. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) alone or the combinations all showed a reduction in tumor with the trastuzumab triple combination; pertuzumab and Example 16F being the most effective and resulting in 100% partial regressions, whereas Example 16F alone or the combination of trastuzumab and pertuzumab each resulted in one partial response out of ten. The triplet had a numerically higher rate of partial responses, and led to a significantly greater reduction in tumor volume (p<.05, Mann-Whitney test) compared to Example 16F monotherapy or the trastuzumab + pertuzumab doublet. Partial response is defined as regression to less than 50% of baseline tumor volume sustained on at least 3 sequential tumor measurements.

[00552] The overall survival benefit for XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) in combination with pertuzumab and trastuzumab differed significantly versus administration of XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) alone (P < 0.011, logrank test) and was superior to results obtained by the combination of pertuzumab and trastuzumab (P < 0.273, logrank test) or the triple combination of XMT-1519, pertuzumab, and trastuzumab (P < 0.05, logrank test).

[00553] Figure 13 provides the results for the tumor response in mice inoculated subcutaneously with SNU-5 cells (n = 10 for each group) after IV administration of carrier; Kadcil at 10 mg/kg as a single dose; Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg, 2 mg/kg, or 0.67 mg/kg as a single dose; or Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg as a single dose on day 1. By day 18 the carrier showed an increase in tumor volume. The XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugate showed tumor reduction and was more effective than Kadcila or rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and resulted in 100% tumor-free survival at the 5 mg/kg and 2 mg/kg doses and 90% tumor-free survival at the 0.67 mg/kg dose.

[00554] Figure 14 provides the results for tumor responses in mice inoculated subcutaneously with TOV-21G cells (n = 10 for each group) after IV administration of carrier; Kadcila at 10 mg/kg as a single dose; Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg or 2 mg/kg as a single dose on day 1. The XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugate showed tumor retardation and was more efficacious than Kadcila which did not meet the threshold for therapeutic activity.

[00555] Figure 15 provides the results for tumor responses in mice inoculated subcutaneously with H522 cells (n = 10 for each group) after IV administration of carrier; Kadcil at 10 mg/kg as a single dose; Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg as a single dose; or Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg as a single dose on day 1. The XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugate showed tumor retardation and was more efficacious than Kadcila and rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) which did not meet the threshold for therapeutic activity.

[00556] Figure 16 provides the results for the tumor response in mice inoculated subcutaneously with SKOV3 cells (n = 10 for each group) after IV administration of carrier; Kadcil at 10 mg/kg as a single dose; Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg as a single dose; or Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg as a single dose on day 1. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) resulted in 100% regressions consisting of 10 complete responses and was more efficacious than Kadcila or rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)) which did not meet threshold for therapeutic activity.

[00557] Figure 17 provides the results for the tumor response in mice inoculated subcutaneously with Calu-3 cells (n = 10 for each group) after IV administration of carrier; or Example 16F, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg as a single dose on Day 1. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) resulted in 100% regression responses consisting of two partial responses and eight complete responses, six of which remained tumor free at Day 60.

[00558] Figure 18 provides the results for tumor response in a BRE-0333 HER2 1+ patient-derived xenograft model (n = 8 for each group) following IV administration of carrier; Kadcil at 10 mg/kg as a single dose; Example 16H, 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) showed tumor retardation and was more effective than Kadcila or rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))).

[00559] Figure 19 provides the results for tumor response in a MAXF_1162 HER2 3+ patient-derived xenograft model (n = 10 for each group) following IV administration of carrier; Kadcila at 10 mg/kg as a single dose; Example 16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 1 or 3 mg/kg or Example 17B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 3 mg/kg as a single dose on day 1. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at both 1 and 3 mg/kg resulted in 100% regression responses and tumor-free survivals and was more efficacious than Kadcila or rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 3 mg/kg.

[00560] Figure 20 provides the results for the tumor response in mice inoculated subcutaneously with BT474 tumor fragments (n = 10 for each group) after IV administration of carrier; Kadcila at 5 mg/kg as a single dose; Example 16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg or 2 mg/kg as a single dose on day 1; Example 17C, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 mg/kg as a single dose on day 1 or XMT 1519 at 5 mg/kg as a single dose. The XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugate showed complete tumor regressions and was more effective than Kadcila, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) or XMT-1519.

[00561] Single doses of 1 mg/kg or 0.67 mg/kg of XMT1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) conjugate showed complete regression in low HER2 expressing breast and gastric cancer models, where trastuzumab emtansine (Kadcila) was inactive at doses of 10 mg/kg and above. In HER2 driven tumor models, XMT 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) conjugate showed synergistic efficacy in combination with the widely used anti-HER2 therapies trastuzumab and pertuzumab. The XMT 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) conjugate demonstrated an excellent pharmacokinetic profile and was well tolerated in non-human primates at therapeutic doses.

[00562] Preclinical data suggest that the XMT 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) conjugate has the potential to greatly expand the number of patients who can benefit from HER2-targeted therapies. The conjugate provides efficient drug delivery in cancers where there are as few as 10,000 HER2 receptors, where other therapies are inactive.

Other Modalities

[00563] Although the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (27)

1. A human or fully humanized monoclonal antibody that specifically binds to an epitope of the human HER2 receptor, comprising (a) a variable heavy chain complementarity determining region 1 (CDRH1) consisting of the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); a variable heavy chain complementarity determining region 2 (CDRH2) consisting of the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); a variable heavy chain complementarity determining region 3 (CDRH3) consisting of the amino acid sequence GGHGYFDL (SEQ ID NO: 27); and a variable light chain complementarity determining region 1 (CDRL1) consisting of the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); a variable light chain complementarity determining region 2 (CDRL2) consisting of the amino acid sequence GASSRAT (SEQ ID NO: 21); and a variable light chain complementarity determining region 3 (CDRL3) consisting of the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); or (b) a CDRH1 consisting of the amino acid sequence FTFSSYGMH (SEQ ID NO: 17); a CDRH2 consisting of the amino acid sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 18); a CDRH3 consisting of the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19); a CDRL1 consisting of the amino acid sequence RASQSVSSDYLA (SEQ ID NO: 20); a CDRL2 consisting of the amino acid sequence GASSRAT (SEQ ID NO: 21); and a CDRL3 consisting of the amino acid sequence QQYVSYWT (SEQ ID NO: 22). 2. The antibody of claim 1, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 6; a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10; or a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. 3. Antibody according to claim 1 or 2, characterized in that the antibody is a chimeric, humanized or fully human monoclonal antibody. 4. The antibody of any one of claims 1 to 3, wherein the monoclonal antibody exhibits a Kd of less than 100 nM for the human HER2 receptor, as measured by a radioligand binding assay. 5. Antibody according to any one of claims 1 to 4, characterized in that the antibody is a monoclonal antibody, a Fab fragment, an F(ab')2 fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, or a single chain antibody (scAb). 6. Antibody according to any one of claims 1 to 5, characterized in that the antibody is an IgG isotype or an IgG1 isotype. 7. Isolated nucleic acid molecule, characterized by the fact that it encodes the antibody as defined in any one of claims 1 to 6 and that comprises the sequences set forth in (A) SEQ ID NO: 34 and SEQ ID NO: 35, or (B) SEQ ID NO: 36 and SEQ ID NO: 37. 8. Vector, characterized by the fact that it comprises the isolated nucleic acid molecule as defined in claim 7. 9. A method for producing an antibody as defined in any one of claims 1 to 6, characterized in that it is culturing a cell under conditions that lead to the expression of the antibody, wherein the cell comprises the isolated nucleic acid molecule as defined in claim 7 or the vector as defined in claim 8. 10. Conjugate, characterized by the fact that it comprises an antibody as defined in any one of claims 1 to 6, and one or more therapeutic or diagnostic agents (D), each of which is independently connected directly or indirectly to the antibody. 11. The conjugate of claim 10, further comprising one or more polymeric scaffolds both connected to the antibody, wherein each of the one or more Ds is independently connected to the antibody via the one or more polymeric scaffolds. 12. The conjugate of claim 11, wherein each of the one or more polymeric scaffolds independently comprises poly(1-hydroxymethylethylene hydroxymethyl-formal) (PHF) having a molecular weight ranging from 2 kDa to 40 kDa. 13. Conjugate according to claim 12, characterized by the fact that each of the one or more polymeric frames independently is of the formula (Ic): where:LD1 is a carbonyl-containing moiety;each occurrence of in is independently a first ligand that contains a biodegradable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect; and in between LD1 and D indicates direct or indirect linkage of D to LD1; each occurrence of is independently a second ligand not yet attached to the isolated humanized or fully human monoclonal antibody, wherein LP2 is a moiety containing a functional group that is yet to form a covalent bond with a functional group of the isolated humanized or fully human monoclonal antibody, and between LD1 and LP2 indicates direct or indirect binding of LP2 to LD1, and each occurrence of the second ligand is distinct from each occurrence of the first ligand; each occurrence of is is independently a third linker that connects each D-bearing polymeric scaffold to the isolated humanized or fully human monoclonal antibody, in which the terminus linked to LP2 indicates direct or indirect linkage of LP2 to the isolated humanized or fully human monoclonal antibody in the formation of a covalent bond between a functional group of LP2 and a functional group of the isolated humanized or fully human monoclonal antibody; and each occurrence of the third linker is distinct from each occurrence of the first linker; m is an integer from 1 to 300, m1 is an integer from 1 to 140, m2 is an integer from 1 to 40, m3 is an integer from 0 to 18, m4 is an integer from 1 to 10; the sum of m, m1, m2, m3, and m4 ranges from 15 to 300; and the total number of LP2 linked to the isolated antibody is 10 or less. 14. The conjugate of claim 13, wherein the sum of m, mi, m2, m3, and m4 ranges from 15 to 150, m1 is an integer from 1 to 70, m2 is an integer from 1 to 20, m3 is an integer from 0 to 10, and PHF has a molecular weight ranging from 2 kDa to 20 kDa, or wherein the sum of m, m1, m2, m3, and m4 ranges from 20 to 110, m1 is an integer from 2 to 50, m2 is an integer from 2 to 15, m3 is an integer from 0 to 8; and PHF has a molecular weight ranging from 3 kDa to 15 kDa,or wherein the sum of m, m1, m2, m3, and m4 ranges from 40 to 75, m1 is an integer from 2 to 35, m2 is an integer from 2 to 10, m3 is an integer from 0 to 5; and PHF has a molecular weight ranging from 5 kDa to 10 kDa,or wherein the functional group of LP2 is selected from -SRp, -SoS-LG, , and halo, where LG is a leaving group, Rp is H or a sulfur protecting group, and one of Xa and Xb is H and the other is a water-soluble maleimide blocking moiety, or Xa and Xb, together with the carbon atoms to which they are attached form a carbon-carbon double bond, or where LD1 comprises —X-(CH2)vC(=O)— with X directly connected to the carbonyl group of , where X is CH2, O, or NH, and v is an integer from 1 to 6, or where each occurrence of isindependently —C(=O)-X-(CH2)vC(=O)-NH-(CH2)u-NHC(=O)-(CH2)w-(OCH2)x-NHC(=O)-(CH2)y—M, where X is CH2, O, or NH, each of v, u, w, x, and y independently is an integer from 1 to 6, and M is , where one of Xa and Xb is H and the other is a water-soluble maleimide blocking moiety, or Xa and Xb, together with the carbon atoms to which they are attached form a carbon-carbon double bond. 15. The conjugate of claim 14, wherein each of v, u, w, x, and y is 2. 16. The conjugate of any one of claims 10 to 15, wherein each of one or more D is a therapeutic agent having a molecular weight of <5 kDa. 17. Conjugate according to claim 15, characterized by the fact that each of the one or more polymeric frames independently is of the formula (Id): where: m3a is an integer from 0 to 17, m3b is an integer from 1 to 8, and the terminal indicates the direct binding of one or more polymeric scaffolds to the isolated humanized or fully human monoclonal antibody. 18. Conjugate according to any one of claims 10 to 15, characterized in that at least one of the one or more Ds is a diagnostic agent. 19. The conjugate of any one of claims 10 to 18, wherein the isolated humanized or fully human monoclonal antibody has a molecular weight of 40 kDa or greater. 20. The conjugate of claim 10, wherein each of the one or more Ds is independently connected to the antibody via a non-polymeric linker moiety. 21. Conjugate according to claim 12, characterized by the fact that each of the one or more polymeric frames independently is of the formula (If): where: m is an integer from 1 to 300, m1 is an integer from 1 to 140, m2 is an integer from 1 to 40, m3a is an integer from 0 to 17, m3b is an integer from 1 to 8; the sum of m3a and m3b ranges from 1 to 18; and the sum of m, mi, m2, m3a, and m3b ranges from 15 to 300; the terminal indicates the attachment of one or more polymeric scaffolds to the isolated humanized or fully human monoclonal antibody that specifically binds to an epitope of the human HER2 receptor, wherein the isolated humanized or fully human monoclonal antibody is an isolated anti-HER2 antibody that comprises a variable heavy chain complementarity determining region 1 (CDRH1) consisting of the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); a variable heavy chain complementarity determining region 2 (CDRH2) consisting of the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 26); a variable heavy chain complementarity determining region 3 (CDRH3) consisting of the amino acid sequence GGHGYFDL (SEQ ID NO: 27); a variable light chain complementarity determining region 1 (CDRL1) consisting of the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); a variable light chain complementarity determining region 2 (CDRL2) consisting of the amino acid sequence GASSRAT (SEQ ID NO: 21); and a variable light chain complementarity determining region 3 (CDRL3) consisting of the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); and the ratio of the PHF to the antibody is 10 or less. 22. The conjugate of claim 21, wherein the PHF in formula (If) has a molecular weight ranging from 2 kDa to 20 kDa, the sum of m, m1, m2, m3a, and m3b ranges from 15 to 150, m1 is an integer from 1 to 70, m2 is an integer from 1 to 20, m3a is an integer from 0 to 9, m3b is an integer from 1 to 8, the sum of m3a and m3b ranges from 1 to 10, and the ratio of the PHF to the isolated anti-HER2 antibody is an integer from 2 to 8, or wherein the PHF in formula (If) has a molecular weight ranging from 3 kDa to 15 kDa, the sum of m, m1, m2, m3a, and m3b ranges from 20 to 110, m1 is an integer from 2 to 50, m2 is an integer from 2 to 15, m3a is an integer from 0 to 7, m3b is an integer from 1 to 8, the sum of m3a and m3b ranges from 1 to 8, and the ratio of PHF to isolated anti-HER2 antibody is an integer from 2 to 8, or where PHF in formula (If) has a molecular weight ranging from 5 kDa to 10 kDa, the sum of m, m1, m2, m3a, and m3b ranges from 40 to 75, m1 is an integer from 2 to 35, m2 is an integer from 2 to 10, m3a is an integer from 0 to 4, m3b is an integer from 1 to 5, the sum of m3a and m3b ranges from 1 to 5, and the ratio of PHF to isolated antibody is an integer from 2 to 8,or wherein the PHF in formula (If) has a molecular weight ranging from 5 kDa to 10 kDa, the sum of m, m1, m2, m3a, and m3b ranges from 40 to 75, m1 is an integer from 2 to 35, m2 is an integer from 2 to 10, m3a is an integer from 0 to 4, m3b is an integer from 1 to 5, the sum of m3a and m3b ranges from 1 to 5, and the ratio of the PHF to the isolated anti-HER2 antibody is an integer from 2 to 6. 23. A method for preparing a conjugate as defined in any one of claims 10 to 22, comprising reacting the isolated humanized or fully human monoclonal antibody that specifically binds to an epitope of the human HER2 receptor with a polymeric scaffold of formula (Ia) such that the conjugate is formed: where:LD1 is a carbonyl-containing moiety;each occurrence of in is independently a first ligand that contains a biodegradable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect; and in between LD1 and D indicates direct or indirect linkage of D to LD1; each occurrence of is independently a second ligand not yet attached to the isolated humanized or fully human monoclonal antibody, wherein LP2 is a moiety containing a functional group that is yet to form a covalent bond with a functional group of the isolated humanized or fully human monoclonal antibody, and between LD1 and LP2 indicates direct or indirect binding of LP2 to LD1, and each occurrence of the second ligand is distinct from each occurrence of the first ligand; m is an integer from 1 to 300, m1 is an integer from 1 to 140, m2 is an integer from 1 to 40, m3 is an integer from 1 to 18, and the sum of m, m1, m2, and m3 ranges from 15 to 300. 24. Use of a conjugate as defined in any one of claims 12 to 22, characterized in that it is in the manufacture of a medicament for alleviating a symptom of a cancer in an individual in need thereof. 25. Use according to claim 24, characterized in that the individual is human. 26. Use according to claim 24, characterized in that the cancer is selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esogastric cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, throat cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, kidney cancer, and gastric cancer. 27. Use according to claim 24, characterized in that the cancer is selected from the group consisting of breast cancer, gastric cancer, non-small cell lung cancer (NSCLC), and ovarian cancer.