EA039374B1 - Monoclonal antibodies against her2 epitope and methods of use thereof - Google Patents

Abstract

The invention relates to an isolated fully human monoclonal antibody or antigen binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, a method of producing such antibody or antigen binding fragment thereof, an isolated nucleic acid molecule encoding the same and an antibody expression vector comprising such nucleic acid, a conjugate for treating, preventing or moderating progression of cancer comprising the isolated antibody or antigen binding fragment thereof, a method of preparing such conjugate, a method of treating cancer using the same and the use of such conjugate in the manufacture of a medicament for treating cancer. As opposed to antibodies known in the art, the antibodies of the invention bind to different epitopes of HER2, wherein they cross-block each other but not trastuzumab, pertuzumab, Fab37 or chA21 from binding to HER2. Further, as opposed to the known antibodies, the antibodies of the invention can internalize efficiently into HER2-expressing cells without promoting cell proliferation.

Description

This application claims priority and advantage over U.S. Provisional Application No. 62/013944, filed June 18, 2014; 62/034489, filed August 7, 2014; 62/147960, filed April 15, 2015, and 62/149444, filed April 17, 2015. The contents of each of these applications are hereby incorporated herein in their entirety by reference.

Field of invention

The invention relates generally to the production 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 for using these monoclonal antibodies as therapeutic and/or diagnostic agents.

State of the art invention

Members of the ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. This family of receptors includes four distinct members including the epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185 ^neu ), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). These four members of the EGFR family form both homo- and heterodimers, with HER2 being the preferred and strongest partner of dimerisapium with other ErbB receptors (Graus-Porta et al., Embo 3 1997; 16:1647-1655; Tao et al., J. Cell Sci. 2008; 121:3207-3217). No ligand is known for HER2, but it may be activated by homodimerization, if overexpressed, or by heterodimerization with other ligand-captured ErbB receptors.

The HER2 gene (also known as the HER2/neu and ErbB2 gene) is amplified in 20-30% of early-stage breast cancers, making it an overexpressed epidermal growth factor (EGF) receptor on the cell membrane (Bange et al., Nature Medicine 7(5):548552). In addition to breast cancer, HER2 expression has also been associated with other types of human carcinomas, including non-small cell lung cancer, ovarian cancer, gastrointestinal cancer, prostate cancer, bladder cancer, colon cancer, esophageal cancer, and head and neck squamous cell carcinoma (Garcia de Palazzo et al., Int. J. Biol. Markers 1993; 8:233-239; Ross et al., Oncologist 2003; 8:307325; 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).

Trastuzumab (Herceptin®) is a recombinant humanized monoclonal antibody directed against domain IV of the HER2 protein, thereby blocking ligand-independent homodimerization and, to a lesser extent, heterodimerization of HER2 with other members of the HER2 family 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 as a first line adjuvant treatment for HER2 overexpressing metastatic breast cancer, either in combination with chemotherapy or as the sole agent followed by one or more chemotherapy regimens. Trastuzumab was found to be effective in only 20-50% of patients with HER2-overexpressing breast tumors, and many of those initially responding to treatment relapsed after a few months (Dinh et al., Clin. Adv. Hematol. Oncol. 2007; 5:707-717).

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

HER2 Antibody Drug Conjugate (ADC), trastuzumab emtansine (adotrastuzumab emtansine, Kadcyla®) is an antibody drug conjugate consisting of the monoclonal antibody trastuzumab (Herceptin) linked to the cytotoxic agent mertansine (DM1). Kadcyla has been approved as a single agent for the treatment of patients with HER2-positive (HER2+) metastatic breast cancer (MBC) who were previously treated with trastuzumab and taxane, alone or in combination.

Since many factors are involved in selecting the appropriate antibody for HER2 targeted therapy, the ADC approach is generally favored if the HER2-antibody complex is effectively internalized upon antibody binding. However, compared to EGFR, HER2 internalization is limited.

The complex mechanisms regulating the functioning of HER2 determine the need for additional research into new and optimized therapeutic strategies against this proto-oncogene.

Accordingly, there is a need for therapies that target HER2 biological activities.

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The essence of the invention

The invention provides monoclonal antibodies that specifically recognize HER2, also known as (ErbB2, p185 ^neu and/or HER2/neu). The antibodies described herein are capable and useful for modulating, eg, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise disrupting the PI3K-Akt mechanism that promotes cell survival by reducing levels of phosphorylated AKT. The antibodies described herein are also capable and useful for modulating, eg, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise disrupting HER2 ligand-independent homodimerization and/or heterodimerization. Antibodies described herein also include antibodies that bind soluble HER2.

The antibodies of the invention exhibit HER2 binding characteristics that differ from those described in the art. In particular, the antibodies described herein bind to different HER2 epitopes and are cross-blocked from binding to HER2 by each other but not by trastuzumab, pertuzumab, Fab37 or chA21. Additionally, in contrast to known antibodies, these antibodies described herein can be efficiently internalized into HER2 expressing cells without stimulating cell proliferation.

The antibodies described herein are fully human monoclonal antibodies that bind to novel epitopes and/or have other therapeutically advantageous properties. Typical properties include, but are not limited to, favorable binding characteristics to cancer cells that express human HER2 at high or low levels, bind specifically to recombinant human and cynomolgus monkey HER2, provide efficient internalization upon binding to HER2, and have a high ability to promote cancer death. cells expressing high or low levels of HER2, when administered as an antibody-drug conjugate (ADC), do not have a significant agonistic effect on the proliferation of HER2-expressing cancer cells and provide effective antibody-dependent cellular cytotoxicity (ADCC) mediating the death of HER2-expressing cells, and as well as any combination of the above properties.

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

The antibodies described herein include an isolated antibody, or antigen-binding fragment thereof, that binds at least a portion of the N-terminus of domain IV of the human HER2 receptor, but does 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, as trastuzumab is known to bind the 4D5 epitope of the human HER2 receptor. As used herein, the term human HER2 receptor epitope 4D5 refers to amino acid residues 529 to 627 of the extracellular domain of the human HER2 receptor, such as residues 551 to 649 of SEQ ID NO: 38 or residues 529 to 627 of SEQ ID NO: 39. B In some embodiments, the isolated antibody, or antigen-binding fragment thereof, also binds at least one epitope on the cynomolgus monkey HER2 receptor.

The antibodies described herein also include an isolated antibody, or an 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, such as residues 474 to 522 of SEQ ID NO: 38 or residues 452 to 500 SEQ ID NO: 39.

Antibodies described herein also include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the human HER2 receptor that contains 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 HER2 receptor human, such as residues 543, 547, and 522 of SEQ ID NO: 38, and residues 521, 525, and 530 of SeQ ID NO: 39. For example, antibodies described herein include an isolated antibody, or antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain a human HER2 receptor that contains 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 include an isolated antibody, or antigen-binding fragment thereof, that specifically binds to an epitope of the human HER2 receptor that contains at least the amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of the isolated antibodies, or antigen-binding fragments thereof, also bind at least one epitope on the cynomolgus monkey HER2 receptor.

Antibodies described herein also include an isolated antibody, or antigen-binding fragment thereof, that binds to at least a portion of Domain III and at least

- 2 039374 part of the N-terminus of domain IV of the human HER2 receptor, but does not cross-compete with a 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 a 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.

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

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 contains 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 SEQ ID NO: 39. For example, antibodies described herein include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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 an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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 an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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 contains 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 an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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, antibodies described herein include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains at least the 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 the isolated antibodies, or antigen-binding fragments thereof, also bind at least one epitope on the cynomolgus monkey HER2 receptor.

The antibodies described herein also include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the human HER2 receptor that contains 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, such as 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, those described herein antibodies include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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 human HER2 receptor domain. For example, antibodies described herein include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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 an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains at least four amino acid residues selected from the group consisting of amino acids

- 3 039374 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 an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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, the antibodies described herein include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains 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, the antibodies described herein include an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to an epitope of the extracellular domain of the human HER2 receptor that contains at least the amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the receptor human HER2. In some embodiments, any or all of the isolated antibodies, or antigen-binding fragments thereof, also bind at least one epitope on the cynomolgus monkey HER2 receptor.

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

Exemplary monoclonal antibodies described herein include, for example, XMT 1517 antibody, XMT 1518 antibody, XMT 1519 antibody, and XMT 1520 antibody described herein. Alternatively, the monoclonal antibody is one that cross-blocks with each other but does not bind to the same epitope as trastuzumab, pertuzumab, Fab37, or chA21 (which bind to specific epitopes on domain IV, domain II, domain III, and domain I HER2 respectively) or their biosimilars. These antibodies are referred to herein as anti-HER2 antibodies, respectively. Anti-HER2 antibodies include fully human monoclonal antibodies as well as humanized monoclonal antibodies and chimeric antibodies. These antibodies exhibit specificity for human HER2 and have been shown to modulate, for example, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise disrupting the PI3K-Akt mechanism that promotes cell survival by reducing levels of phosphorylated AKT. These antibodies are internalized from the cell surface of HER2-expressing cells at a rate that is the same or substantially the same as the rate at which trastuzumab or its biosimilar is internalized. For example, these antibodies and antigen-binding fragments have an internalization rate that is about 50% of total surface binding at time 0, internalizing in 4 hours.

The antibodies described herein comprise a heavy chain having an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical to a sequence selected from the group consisting of SEQ ID NO: 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% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8.

The antibodies described herein comprise a combination of heavy chain and light chain amino acid sequences selected from the group consisting of: (i) a heavy chain amino acid sequence of 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 to the amino acid sequence of the light chain 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) the amino acid sequence of the heavy chain is 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 the amino acid sequence of the light chain is at least 90 , 91, 92, 93, 94, 95, 96, 97 98, 99% or more identical amino acid sequence of SEQ ID NO: 4; (iii) the amino acid sequence of the heavy chain is 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 the amino acid sequence of the light chain is 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) the amino acid sequence of the heavy chain 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 the amino acid sequence of the light chain at least 90, 91, 92, 93, 94, 95, 96, 97 98, 99% or more identical amino acid sequence SEQ ID NO: 8.

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

In some embodiments, the antibodies described herein comprise a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of SEQ ID NO: 3 and amino acid

- 4 039374 light chain 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.

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

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

The antibodies described herein comprise 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 NO: 2 , 4, 6, and 8, respectively.

The antibodies described herein comprise 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 amino acid sequence of the heavy chain of SEQ ID NO: 3 and the amino acid sequence of the light chain 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.

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

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

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

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

Antibodies described herein comprise a heavy chain variable region having an amino acid sequence of 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 NO: 9, 11, 13 and 15, and a light chain variable region having an amino acid sequence of 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 NO: 10, 12, 14 and 16.

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

In some embodiments of the invention, the antibodies described herein contain the amino acid sequence of the heavy chain variable region at least 90,

- 5 039374

91, 92, 93, 94, 95, 96, 97 98, 99% or more identical to the amino acid sequence of SEQ ID NO:

and the amino acid sequence of the light chain variable region 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.

In some embodiments of the invention, the antibodies described in the document contain the amino acid sequence of the variable region of the heavy chain 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 the amino acid sequence of the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the invention, the antibodies described in the document contain the amino acid sequence of the variable region of the heavy chain 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 the amino acid sequence of the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the antibodies described herein comprise the amino acid sequence of the heavy chain variable region at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 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% or more identical to the amino acid sequence of SEQ ID NO: 16.

The antibodies described herein comprise a heavy chain variable region of an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, and 15 and a light chain variable region of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 , 12, 14 and 16.

The antibodies described herein comprise a combination of the 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. : ten; (ii) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 11 and the amino acid sequence of the light chain variable region of SEQ ID NO: 12; (iii) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 13 and the amino acid sequence of the light chain variable region of SEQ ID NO: 14; and (iv) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 15 and the amino acid sequence of the light chain variable region of SEQ ID NO: 16.

In some embodiments, the antibodies described herein comprise 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.

In some embodiments, the antibodies described herein comprise 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.

In some embodiments, an antibody described herein comprises 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.

In some embodiments, the antibodies described herein comprise 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.

The three heavy chain CDRs of the antibodies described herein comprise a heavy chain complementarity determining region 1 (CDRH1) that contains an amino acid sequence of 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 contains an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 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 contains an amino acid sequence of 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.

The three antibody light chain CDRs described herein comprise a light chain complementarity determining region 1 (CDRL1) that contains an amino acid sequence of 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 contains 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 contains an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. a specific sequence selected from the group consisting of SEQ ID NOs: 22 and 29.

The antibodies contain a sequence combination of a heavy chain CDR and a light chain CDR that contains a CDRH1 that contains an amino acid sequence of 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 contains 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 contains an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 19 and 27; CDRL1, which contains an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97 98, 99% or more identical to the sequence selected from the group consisting of SEQ ID NO: 20 and 28; CDRL2 which contains an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical to a sequence selected from the group consisting of SEQ ID NOS: 21 and 24, and CDRL3 , which contains an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97 98, 99% or more identical to the sequence selected from the group consisting of SEQ ID NOS: 22 and 29.

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

The three CDRs of the antibody light chains described herein comprise a CDRL1 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 28; CDRL2, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and 24; CDRL3 which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 and 29.

The antibodies described herein comprise a combination of heavy chain CDR and light chain CDR sequences that comprise a CDRH1 that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 25, and 30; CDRH2 which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 26 and 31; CDRH3, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and 27; CDRL1, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and 28; CDRL2, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and 24; CDRL3 which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 and 29.

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

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

In some embodiments, the antibodies described herein comprise the amino acid sequence CDRH1 SEQ ID NO: 17, the amino acid sequence CDRH2 SEQ ID NO: 23, the amino acid sequence CDRH3 SEQ ID NO: 19, the amino acid sequence CDRL1 SEQ ID NO: 20, the amino acid sequence CDRL2 SEQ ID NO:

- 7 039374 and the amino acid sequence of CDRL3 SEQ ID NO: 22.

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

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

In some embodiments, an anti-HER2 antibody described herein includes an agent conjugated to the antibody. In some embodiments of the invention, the agent is a therapeutic agent. In some embodiments of the invention, the agent is an antineoplastic agent. In some embodiments of the invention, the agent is a toxin or a fragment thereof. In some embodiments, the agent is: (a) an auristatin compound; (b) a calicheamicin compound; (c) a duocarmycin compound; (d) SN38; (e) pyrrolobenzodiazepine; (f vinca compound; (g) tubulisin compound; (h) non-naturally occurring captothecin compound; (i) maytansinoid compound; (j) DNA-binding drug; (k) kinase inhibitor; (l) MEK inhibitor; (m) a KSP inhibitor;(n) a topoisomerase inhibitor and analogs or the like thereof.In some embodiments, the agent is conjugated to the anti-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.

In one aspect of the invention, an anti-HER2 antibody conjugate described herein comprises an isolated anti-HER2 antibody, or antigen-binding fragment thereof, directly or indirectly coupled to one or more therapeutic or diagnostic agents (D). In some embodiments, the HER2 antibody conjugate also comprises one or more polymeric scaffolds linked to the antibody or antigen-binding fragment thereof, each of the one or more Ds independently linked to the antibody or antigen-binding fragment thereof via one or more polymeric scaffolds.

In some embodiments, each of the one or more polymer scaffolds that are coupled to the isolated anti-HER2 antibody or antigen-binding fragment thereof independently comprise poly(1-hydroxymethylethylene hydroxymethylformal) (PHF) having a molecular weight ranging from about 2 to about 40 kDa. .

In some embodiments of the invention, each of the one or more polymer backbones is independently represented by formula (Ic)

(Ic), where L ^D1 represents a carbonyl-containing fragment;

---C(=O)-L ^d1 - | — ---C(=O)-L ^d1 -£- _D each presence of ^ς in ^ς is independently the first linker that contains such a biodegradable bond that when the bond is broken, D is released in an active form to manifest its therapeutic effect; and the presence in -c ₍ ^q_ _D ⁴ between L and D denotes the direct or indirect attachment of D to

- 8 039374 l ^d1 ;

---C(=O)-L ^d1 —£-l ^P2 each presence of ^ζ independently represents a second linker not yet connected to the isolated antibody or its antigen-binding fragment, in which L ^P2 is a fragment containing a functional group that is still will form a covalent bond with the functional group of the selected antibody or antigen-binding fragment, and between L ^d1 and L ^P2 denotes direct or indirect attachment of L ^P2 to L ^d1 and each presence of the second linker is different from each presence of the first linker;

---C(=O)-L ^D1 —|-l ^P2 -£— each presence of ^ς is independently a third linker that connects each D-bearing polymer scaffold to an isolated antibody or its antigen-binding fragment, in which the terminal , attached to L ^P2 , denotes the direct or indirect attachment of L to the selected antibody or antigen-binding fragment in the formation of a covalent bond between the functional group L ^P2 and the functional group of the selected antibody or antigen-binding fragment; and each presence of the third linker is different from each presence of the first linker;

m is an integer from 1 to about 300 m1 is an integer from 1 to about 140 m ₂ is an integer from 1 to about 40 m ₃ is an integer from 0 to about 18 m4 is an integer a number from 1 to about 10, the sum of m, m1, m ₂ , m ₃ and m4 is in the range from about 15 to about 300; and the total amount of L ^P2 bound to the isolated antibody or antigen-binding fragment thereof is 10 or less.

The conjugate described in the document may include one or more of the following features:

For example, in formula (Ic), the isolated anti-HER2 antibody or antigen-binding fragment thereof has a molecular weight of 40 kDa or more (e.g., 60 kDa or more, 80 kDa or more, 100 kDa or more, 120 kDa or more, 140 kDa or more , 160 kDa or more, 180 kDa or more, or 200 kDa or more, or about 40-200, 40-180, 40-140, 60-200, 60-180, 60-140, 80-200, 80-180 , 80-140, 100-200, 100-180, 100-140 or 140-150 kDa). In some embodiments, an isolated HER2 antibody, or antigen-binding fragment thereof, if any antibody or antigen-binding fragment as described herein, includes, but is not limited to, e.g., XMT 1517 antibody, XMT 1518 antibody, XMT 1519 antibody, and XMT 1520 antibody described in this document.

For example, in formula (Ic), m1 is an integer from 1 to about 120 (eg, about 190) and/or m3 is an integer from 1 to about 10 (eg, about 1-8).

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, m ₂ , m3 and m ₄ in the range from about 45 to about 150) , m ₂ is an integer from 2 to about 20, m3 is an integer from 0 to about 9, m ₄ is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 75 (for example , m1 is about 4-45).

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, m ₂ , m3 and m ₄ in the range from about 60 to about 110) , m ₂ is an integer from 2 to about 20, 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 is about 4-45).

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, m ₂ , m3 and m ₄ in the range from about 15 to about 150) , m ₂ is an integer from 1 to about 20, m3 is an integer from 0 to about 10 (for example, m3 in the range from 0 to about 9), m ₄ is an integer from 1 to about 8, and/or m1 is an integer from 1 to about 70, and the total number of L ^P2 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 eight).

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, m ₂ , m3 and m ₄ in the range from about 20 to about 110) , m ₂ is an integer from 2 to about 15, m3 is an integer from 0 to about 8 (for example, m3 in the range 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 amount of L ^P2 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 ).

For example, when the PHF in formula (Ic) has a molecular weight in the range of about 5 kDa to about 10 kDa (i.e., the sum of m, m1, m ₂ , m3 and m ₄ in the range of about 40 to about 75) , m ₂ before

- 9 039374 is an integer from 2 to about 10 (for example, m ₂ is about 3-10), m3 is an integer from 0 to about 5 (for example, m3 in the range from 0 to about 4), m ₄ represents is an integer from 1 to about 8 (for example, m ₄ in the range from 1 to about 5) and/or m1 is an integer from about 2 to about 35 (for example, m1 is about 5-35), and the total number of L ^{The P2} associated with the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (eg, about 2, 3, 4, 5, 6, 7, or 8).

For example, each presence of D is independently a therapeutic agent having a molecular weight of <5 kDa.

For example, each presence of D is independently an anti-cancer drug, e.g., selected from vinca alkaloids, auristatins, tubulisins, duocarmycins, non-naturally occurring camptothecin compounds, maytansinoids, calicheamicin compounds, topoisomerase inhibitors, DNA-binding drugs, kinase inhibitors, MEK inhibitors. , KSP inhibitors and their analogues.

For example, each, when not connected to an isolated antibody or antigen-binding fragment thereof, independently contains a ^WP end group in which each ^WP is independently

- 10 039374

in which R ^1K represents a substitutable group (for example, a halide or RC(O)O-, in which R represents a hydrogen atom, an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl fragment), R ^1A represents a sulfur-containing protective group and ring A represents cycloalkyl or heterocycloalkyl, and R ^1J represents a hydrogen atom, aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl fragment.

For example, each R ^1A is independently o

NR ^s1 0 o

COOR ^s3

OSO ₃ R ^s3 or ps1

AbcooR ^s3

Tr ^s2

R ^s1 XOOR ^s3 ,

in which r represents 1 or 2, and each of R ^s1 , R ^s2 and R ^s3 represents a hydrogen atom, aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl fragment.

For example, the functional group LP ² that will still form a covalent bond with the functional group of the isolated antibody or antigen-binding fragment is selected from -SRp, -SS-LG,

, and a halogen wherein LG is a substitutable group, R ^p is H or a sulfur containing protecting group, one of X _a and X _b is H and the other is a water-soluble maleimido blocking moiety, or X _a and X _b , together with the carbon atoms to which they are attached, form a carbon-carbon double bond. For example, the functional group LP ² that will still form a covalent bond is a functional group that does not react with the functional group of the isolated antibody or its antigen-binding fragment, for example

O Xa\J o as an LP ² functional group wherein one of X _a and X _b is H and the other is a water soluble maleimido blocking moiety, or Xa and X _b .

For example, L ^D1 contains -X-(CH ₂ ) _v -C(=O)- with X directly connected to the carbonyl group---C(=O)-L ^D1 ->— poly ^ζ in which X is CH ₂ , O or NH, av is an integer from 1 to 6.

---C(=O)-L ^d1 — £-L ^p2

For example, each presence of ^ζ is independently -C(=O)-X(CH ₂ ) _v -C(=O)-NH-(CH ₂ ) _u -NH-C(=O)-(CH ₂ ) _w - (OCH ₂ ) _x -NHC(=O)-(CH ₂ ) _y -M, wherein X is CH ₂ , O or NH, v, u, w, x and y are each independently an integer of 1 up to 6, and M preO

Xt^l is ⁰ where one of Xa and X _b is H and the other is a water-soluble maleimido blocking moiety, or X _a and X _b , together with the carbons to which they are attached, form a carbon-carbon double bond.

For example, v, u, w, x, and y each represent 2.

For example, the ratio between D and isolated anti-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.

For example, the ratio between D and isolated anti-HER2 antibody or antigen-binding fragment thereof is about 20:1, 15:1, 10:1, 5:1, 2:1, or 1:1.

For example, the ratio between D and isolated anti-HER2 antibody or antigen-binding fragment thereof is about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

For example, the ratio between D and isolated anti-HER2 antibody or antigen-binding fragment thereof is about 15:1, 14:1, 13:1, 12:1, or 11:1.

For example, the ratio between D and isolated anti-HER2 antibody or antigen-binding fragment thereof is about 15:1, 14:1, 13:1, or 12:1.

For example, the ratio between D and isolated anti-HER2 antibody or antigen-binding fragment thereof is about 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

For example, each of the one or more D-supporting polymer scaffolds is independently represented by the formula (Id)

- 12 039374

(Id), where m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, and terminal denotes the direct attachment of one or more polymer scaffolds to an isolated anti-HER2 antibody or antigen-binding fragment having a molecular weight of about 40 kDa or more.

The formula framework (Id) may include one or more of the following features.

The sum of m _3a and m _3b is between 1 and 18.

When the PHF in formula (Id) has a molecular weight ranging from about 2 to about 40 kDa, then the sum of m, m1, m ₂ , m _3a and m _3b is in the range from about 15 to about 300, m ₁ is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is in the range of 1 to about 18, and the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof is 10 or less.

When the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 20 kDa, then the sum of m, m1, m2, m _3a and m _3b is in the range from about 15 to about 150, m ₁ is an integer from 1 to about 70, m ₂ is an integer from 1 to about 20, m _3a is an integer from 0 to about 9, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b ranges from 1 to about 10, and the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8.

When the PHF in formula (Id) has a molecular weight ranging from about 3 kDa to about 15 kDa, then the sum of m, m _b m ₂ , m _3a and m _3b is in the range from about 20 to about 110, m ₁ represents is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m _3a is an integer from 0 to about 7, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b ranges from 1 to about 8, and the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (e.g., about 2 to about 6 or about 2 to about 4).

When the PHF in formula (Id) has a molecular weight ranging from about 5 kDa to about 10 kDa, then the sum of m, m1, m ₂ , m _3a and m _3b is in the range from about 40 to about 75, m ₁ represents 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 m _3a and m _3b ranges from 1 to about 5, and the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (e.g., about 2 to about 6 or about 2 to about 4).

In some embodiments, the ratio between auri-13 039374 statin F hydroxypropylamide (AF HPA) and isolated anti-HER2 antibody or antigen-binding fragment thereof may 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.

In some embodiments, the ratio between HPA AF and isolated anti-HER2 antibody or antigen-binding fragment thereof 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 .

In other embodiments, the ratio between HPA AF and the isolated anti-HER2 antibody or antigen-binding fragment thereof 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.

In some embodiments, the ratio between HPA AF and isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

In some embodiments, the ratio between HPA AF and isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 15:1, 14:1, 13:1, 12:1, or 11:1.

In some embodiments, the ratio between HPA AF and the isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 15:1, 14:1, 13:1, or 12:1.

In some embodiments, the ratio between PHF and the isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3: 1, 2:1 or 1:1.

In some embodiments, the ratio between PHF and the isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and the isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 6:1.5:1.4:1.3:1.2:1, or 1:1.

In other embodiments, the ratio between PHF and the isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 6:1.5:1.4:1.3:1, or 2:1.

In other embodiments, the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 6:1, 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and the isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof may be about 4:1, 3:1, or 2:1.

Water-soluble maleimide blocking moieties (eg X _a or X _b ) are moieties that can be covalently attached to one of the two carbon atoms of the olefin upon reaction of the maleimide group with a thiol-containing compound of formula (II)

R90-(CH ₂ ) _d -SH (II), where R ₉₀ is NHR91, OH, COOR93, CH(NHR ₉₁ )COOR ₉₃ or a substituted phenyl group;

R ₉₃ represents a hydrogen atom or C _1-4 alkyl;

R ₉₁ represents a hydrogen atom, CH ₃ or CH 3 CO; and d is an integer from 1 to 3.

In one embodiment, the water soluble maleimido blocking compound of formula (II) may be cysteine, N-acetylcysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropionic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e. HOCH2SH) , benzylthiol in which the phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol. One or more hydrophilic substituents on phenyl include OH, SH, methoxy, ethoxy, COOH, CHO, _{COC1_4} alkyl, NH ₂ , F, cyano, SO ₃ H, PO3H, and the like.

In another aspect of the invention, the water-soluble maleimido blocking group is -S-(CH ₂ ) _d -R ₉₀ , wherein

R ₉₀ is OH, COOH or CH(NHR ₉₁ )COOR ₉₃ ;

R ₉₃ represents a hydrogen atom or CH ₃ ;

R ₉₁ represents a hydrogen atom or CH3CO; and d is 1 or 2.

In another embodiment, the water-soluble maleimido blocking group is -S-CH ₂ -CH(NH ₂ )COOH.

In some embodiments, the conjugate described herein contains one or more D-bearing PHFs, each independently of formula (If), 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;

m ₁ is an integer from 1 to about 140;

m ₂ is an integer from 1 to about 40;

m _3a is an integer from 0 to about 17;

m _3b is an integer from 1 to about 8;

the sum of m _3a and m _3b ranges from 1 to about 18;

the sum of m, m _b m ₂ , m _3a and m _3b is in the range from about 15 to about 300;

terminal denotes the attachment of one or more PHF polymer backbones to an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor and contains a heavy chain variable complementarity determining region 1 (CDRH1) containing the amino acid sequence FTFSSYSMN (SEQ ID NO: 25) ; heavy chain variable complementarity determining region 2 (CDRH2) containing the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); heavy chain variable complementarity determining region 3 (CDRH3) containing the amino acid sequence GGHGYFDL (SEQ ID NO: 27); light chain variable complementarity determining region 1 (CDRL1) containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); light chain variable complementarity determining region 2 (CDRL2) containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and light chain variable complementarity determining region 3 (CDRL3) containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); and the ratio between PHF and antibody is 10 or less.

The framework of the formula (If) may include one or more of the following features.

When the PHF in formula (If) has a molecular weight ranging from about 2 kDa to about 20 kDa, then the sum of m, m1, m ₂ , m _3a and m _3b is in the range 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, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is in range from 1 to about 10, and the ratio between PHF and antibody is an integer from 2 to about 8.

When the PHF in formula (If) has a molecular weight ranging from about 3 kDa to about 15 kDa, then the sum of m, m _b m ₂ , m _3a and m _3b is in the range from about 20 to about 110, m ₁ represents is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m _3a is an integer from 0 to about 7, m3b is an integer from 1 to about 8, the sum of m _3a and m _3b is in the range of 1 to about 8, and the ratio between PHF and antibody is an integer from 2 to about 8 (eg, about 2 to about 6 or about 2 to about 4).

When the PHF in formula (If) has a molecular weight ranging from about 5 kDa to about 10 kDa, then the sum of m, m1, m ₂ , m _3a and m _3b is in the range 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, m _3a is an integer from 0 to about 4, m _3b is an integer from 1 to about 5, the sum of m _3a and m _3b ranges from 1 to about 5, and the ratio between PHF and antibody is an integer from 2 to about 8 (eg, about 2 to about 6 or about 2 to about 4).

In some embodiments, the ratio between auristatin F hydroxypropylamide (AF HPA) and antibody may be about 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1 ,

- 15 039374

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.

In some embodiments, the ratio between AF HPA and 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.

In other embodiments, the ratio between AF HPA and 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.

In some embodiments, the ratio between AF HPA and antibody may be about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

In some embodiments, the ratio between AF HPA and antibody may be about 15:1, 14:1, 13:1, 12:1, or 11:1.

In some embodiments, the ratio between AF HPA and antibody may be about 15:1, 14:1, 13:1, or 12:1.

In some embodiments, the ratio between PHF and antibody may be about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1 :one.

In some embodiments, the ratio between PHF and antibody may be about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and antibody may be about 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

In other embodiments, the ratio between PHF and antibody may be about 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and antibody may be about 6:1, 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be about 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be about 4:1, 3:1, or 2:1.

In another aspect of the invention, the conjugate described herein is the formula (Ib)

where the designation Antibody against HER2 refers to the selected antibody against HER2 or its antigennegative fragment described in this document;

between L ^P2 and an anti-HER2 antibody, direct or indirect attachment of an anti-HER2 antibody to L ^P2 is indicated, each anti-HER2 antibody present independently has a molecular weight of less than 200 kDa, m is an integer from 1 to about 2200, m ₁ is an integer from 1 to about 660, m ₂ is an integer from 3 to about 300, m3 is an integer from 0 to about 110, m ₄ is an integer from 1 to about 60, and the sum of m, m1, m1, m ₂ , m ₃ and m ₄ range from about 150 to about 2200.

In formula (Ib), m1 is an integer from about 10 to about 660 (eg, about 10250).

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, m _b m ₂ , m ₃ and m ₄ in the range 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 is about 15-80).

- 16 039374

In formula (Ib), each anti-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 from about 4 to 80 kDa (eg, 4-20, 20-30, or 30-70 kDa).

Another aspect of the invention relates to a method for obtaining the conjugate described in this document. This method involves reacting the isolated antibody or its antigen-binding fragment with a polymer backbone of formula (Ia) so that the conjugate is of the formula

(la), where L ^D1 represents a carbonyl-containing fragment;

---C(=O)-L ^d1 -|_ ---C(=O)-L ^D1 -|_ _D each present ^{in is} independently the first linker that contains such a biodegradable bond that when the bond is broken, D is released in an active form to manifest its therapeutic effect; and the presence in ( * ^D between L ^D1 and D denotes the direct or indirect attachment of D to l ^d1 ;

---C'))-L ^D1 --|-L ^P2 each presence of ^ζ is independently a second linker not yet connected to the isolated antibody or its antigen-binding fragment, in which L ^P2 is a fragment containing a functional group that will yet form a covalent bond with the functional group of the selected antibody or antigen-binding fragment, and between L ^D1 and L ^P2 denotes direct or indirect attachment of L ^P2 to L ^D1 , and each presence of the second linker is different from each presence of the first linker;

m is an integer from 1 to about 300, m ₁ is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m ₃ is an integer from 1 to about 18, and the sum of m, m1, m ₂ , m _3a and m _3b range from about 15 to about 300.

In the polymer backbone formulas described herein, separation or spacing between polyacetal units indicates that these units can be connected to each other in any order. In other words, the introduced groups, which contain, for example, D, L ^P2 and an isolated antibody or antigen-binding fragment thereof, can be randomly distributed along the polymer backbone.

The present invention also provides methods for treating, preventing, slowing progression, or otherwise alleviating a symptom of one or more pathologies associated with abnormal expression, function and/or activation of HER2, or alleviating a symptom associated with such pathologies, by administering a monoclonal antibody, a fragment thereof, and /or its conjugate, described in this document, the subject for whom such treatment or prevention is desirable. The subject to be treated is, for example, a human. The monoclonal antibody, its fragment and/or its conjugate is administered in an amount sufficient to treat, prevent or alleviate the symptom associated with this pathology.

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

- 17 039374 symptoms associated with this pathology.

Pathologies treatable and/or preventable using these monoclonal antibodies, their fragments and/or their conjugates described herein include, for example, cancer. For example, the antibodies, fragments thereof, and/or conjugates thereof described herein are useful in treating, preventing, slowing the progression, or otherwise alleviating a symptom of a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head cancer, and neck, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, larynx cancer, mouth 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 gastrointestinal cancer.

In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates thereof described herein are useful in treating, preventing, slowing the progression, or otherwise alleviating a symptom of breast cancer.

In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates thereof described herein are useful in treating, preventing, slowing the progression, or otherwise alleviating a symptom of gastrointestinal cancer.

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

In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates thereof described herein are useful in treating, preventing, slowing the progression, or otherwise alleviating a symptom of ovarian cancer.

In some embodiments, an anti-Her2 antibody, or antigen-binding fragment thereof, used to treat, prevent, slow the progression of, or otherwise alleviate a symptom of a cancer (e.g., a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head cancer and neck, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, mouth 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 gastrointestinal cancer), competes for binding to the same Her epitope -2 with an antibody containing (1) a CDRH1 heavy chain variable region containing the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); CDRH2 containing the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); CDRH3 containing the amino acid sequence GGHGYFDL (SEQ ID NO: 27) and the CDRL1 light chain variable region containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); CDRL2 containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (2) a CDRH1 heavy chain variable region containing the amino acid sequence FTFSGRSMN (SEQ ID NO: 30); CDRH2 containing the amino acid sequence YISSDSRTIYYADSVKG (SEQ ID NO: 31); CDRH3 containing the amino acid sequence GGHGYFDL (SEQ ID NO: 27) and the CDRL1 light chain variable region containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); CDRL2 containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (3) a CDRH1 heavy chain variable region containing the amino acid sequence FTFSSYGMH (SEQ ID NO: 17); CDRH2 containing the amino acid sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 18); CDRH3 containing the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19) and the CDRL1 light chain variable region containing the amino acid sequence RASQSVSSDYLA (SEQ ID NO: 20); CDRL2 containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 containing the amino acid sequence QQYVSYWT (SEQ ID NO: 22) or (4) CDRH1 heavy chain variable region containing the amino acid sequence FTFSSYGMH (SEQ ID NO: 17) ; CDRH2 containing the amino acid sequence GIWWDGSNEKYADSVKG (SEQ ID NO: 23); CDRH3 containing the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19) and the CDRL1 light chain variable region containing the amino acid sequence RASQSVSSDYLA (SEQ ID NO: 20); CDRL2 containing the amino acid sequence GASRRAT (SEQ ID NO: 24) and CDRL3 containing the amino acid sequence QQYVSYWT (SEQ ID NO: 22).

- 18 039374

In another embodiment, an anti-Her2 antibody, or antigen-binding fragment thereof, used to treat, prevent, slow the progression of, or otherwise alleviate a symptom of a cancer (e.g., a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head cancer and neck, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, mouth 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 gastrointestinal cancer), competes for binding to the same Her epitope -2 with an antibody containing (1) a CDRH1 heavy chain variable region containing the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); CDRH2 containing the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); CDRH3 containing the amino acid sequence GGHGYFDL (SEQ ID NO: 27) and the CDRL1 light chain variable region containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); CDRL2 containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (2) a CDRH1 heavy chain variable region containing the amino acid sequence FTFSGRSMN (SEQ ID NO: 30); CDRH2 containing the amino acid sequence YISSDSRTIYYADSVKG (SEQ ID NO: 31); CDRH3 containing the amino acid sequence GGHGYFDL (SEQ ID NO: 27); a CDRL1 light chain variable region comprising the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); CDRL2 containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29); (3) a CDRH1 heavy chain variable region containing the amino acid sequence FTFSSYGMH (SEQ ID NO: 17); CDRH2 containing the amino acid sequence VIWYDGSNKYYADSVKG (SEQ ID NO: 18); CDRH3 containing the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19) and the CDRL1 light chain variable region containing the amino acid sequence RASQSVSSDYLA (SEQ ID NO: 20); CDRL2 containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and CDRL3 containing the amino acid sequence QQYVSYWT (SEQ ID NO: 22) or (4) CDRH1 heavy chain variable region containing the amino acid sequence FTFSSYGMH (SEQ ID NO: 17) ; CDRH2 containing the amino acid sequence GIWWDGSNEKYADSVKG (SEQ ID NO: 23); CDRH3 containing the amino acid sequence EAPYYAKDYMDV (SEQ ID NO: 19) and the CDRL1 light chain variable region containing the amino acid sequence RASQSVSSDYLA (SEQ ID NO: 20); CDRL2 containing the amino acid sequence GASRRAT (SEQ ID NO: 24) and CDRL3 containing 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 of < 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 detecting or otherwise separating, for example stratifying, a patient population suitable for therapeutic administration of an anti-HER2 antibody or antigen-binding fragment thereof and/or its PBRM-polymer-drug conjugates described herein by detecting a patient having low HER2 expression prior to treatment with an anti-HER2 antibody or antigen-binding fragment thereof and/or its conjugates described herein. Low expression of HER2 is present in those patients who have an amount of 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 the cell population under study. HER2 expression level, i.e. the number of HER2 molecules per cell can be measured using any method recognized in the art, including, but not limited to, the use of cell viability based assays, assays are shown in the working examples provided herein (see, for example, example eighteen).

The invention also provides kits and/or methods for detecting or otherwise separating, e.g. stratifying, a patient population suitable for therapeutic administration of an anti-HER2 antibody or antigen-binding fragment thereof and/or its conjugates described herein by detecting a HER2 score in a patient, up to treatment with an anti-HER2 antibody or antigen-binding fragment and/or its conjugates as described herein. In some embodiments, the subject is identified as having an HER2 expression score of 1+ or 2+. In some embodiments, the subject is identified as having a HER2 expression score of 1+ or 2+ as determined by immunohistochemistry (IHC) analysis performed on the cell population under study, and the HER2 gene is not amplified in the cell population under study. In some embodiments of the invention, the cell population under study is obtained from fresh, thawed tissue from a biopsy sample. In some embodiments of the invention, the cell population under study is obtained from frozen tissue from a biopsy sample.

- 19 039374

An IHC study measures the amount of HER2 receptor protein on the cell surface of a cancer tissue sample, such as a breast cancer tissue sample or a gastrointestinal cancer sample, and assigns the detected cell surface HER2 receptor level a HER2 score of 0, 1+, 2+, or 3+ . If the subject has a HER2 score between 0 and 1+, then the cancer is considered HER2 negative. A score of 2+ means the cancer is borderline, and a score of 3+ means the cancer is HER2 positive.

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

In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, slowing progression, or otherwise alleviating a symptom of breast cancer in patients who have a HER2 IHC result of 1 + or HER2 2+ IHC without gene amplification, such as FISH- (or negative fluorescence in situ hybridization).

In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, slowing progression, or otherwise alleviating a symptom of breast cancer in patients who have advanced HER2. -positive breast cancer and who were previously treated with Kadcyla.

In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, slowing progression, or otherwise alleviating a symptom of breast cancer in patients who have advanced HER2. -positive breast cancer and who have not previously been treated with Kadcyla.

In some embodiments, the antibodies or antigen-binding fragments thereof and/or PBRM-polymer-drug conjugates described herein are useful in treating, preventing, slowing progression, or otherwise alleviating a symptom of gastrointestinal cancer in patients who have an IHC result. HER21+ or HER22+ IHC without gene amplification, eg FISH-.

In some embodiments, the antibodies or antigen-binding fragments thereof and/or the PBRM-polymer-drug conjugates described herein are useful in treating, preventing, slowing the progression, or otherwise alleviating a symptom of non-small cell lung cancer (NSCLC) in patients who are HER22+ IHC or HER23+ IHC, any HER2 gene amplification or mutation status.

In some embodiments of the invention, the subject is refractory to chemotherapy, including standard first-line chemotherapeutic agents. In some embodiments, the subject is resistant to treatment with Kadcyla.

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

The anti-HER2 antibody and/or conjugated anti-HER2 antibody used in any of the embodiments of these methods and uses may 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 chemotherapy agent such as, for example, trastuzumab, pertuzumab, ado-trastuzumab emtansine (Kadcyla), lapatinib, anastrozole, letrozole, exemestane, everolimus, fulvestrant, tamoxifen, toremifene, megestrol acetate, fluoxymestron, ethinyl estradiol, paclitaxel, capecitabine, gemcitabine, eribulin, vinorelbine, cyclophosphamide, carboplatin, docetaxel, albumin-bound paclitaxel, cisplatin, epirubicin, ixabepilone, doxorubicin, fluorouracil, oxaliplatin, fluoropyrimidine, irinotecan, ramucirumab, leucoworimitomycin, tuto bevacizumab, erlotinib, afatinib, crizotinib, permetrexed, ceritinib, etoposide, vinblastine, vincristine, ifosfamide, liposomal doxorubicin, topotecan, altretamine, melphalan, or leuprolide acetate. In some embodiments, the second agent is Kadcyla.

In some embodiments, the agent is at least a second antibody, or antigen-binding fragment thereof, that specifically binds HER2. In some embodiments, the anti-HER2 antibody and/or conjugated anti-HER2 antibody is administered in combination with an anti-HER2 antibody, a HER2 dimerization inhibitor antibody, or a combination of an anti-HER2 antibody and a HER2 dimerization inhibitor antibody, such as, for example, trastuzumab or pertuzumab, or their combination. In some embodiments, the anti-HER2 antibody and/or conjugated anti-HER2 antibody is administered in combination with a trastuzumab biosimilar or a pertuzumab biosimilar, or a combination thereof.

These combinations of anti-HER2 antibodies and/or conjugated anti-HER2 antibodies are useful in the treatment of pathologies such as, for example, cancer. For example, these combinations of anti-HER2 antibodies and/or conjugated anti-HER2 antibodies, such as an anti-HER2 antibody or antigen-binding fragment thereof, and/or conjugated anti-HER2 antibodies (e.g., anti-HER2 antibody-polymer-drug conjugates) described herein, in combinations with trastuzumab and pertuzumab, or both trastuzumab and pertuzumab, or a trastuzumab biosimilar, pertuzumab biosimilar, or both biosimilars, are useful in treating, preventing, slowing the progression, or otherwise alleviating 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, esophageal cancer, eye cancer, larynx cancer, mouth cancer, mesothelioma, skin cancer, myeloma, oral cancer, cancer rectal cancer, throat cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, germ lung cancer (NSCLC), colon cancer, pancreatic cancer, kidney cancer, and gastrointestinal cancer).

These combinations are also useful in increasing the destruction of HER2 when an HER2 expressing cell is brought into contact with these combinations. The level of HER2 destruction is detected using any method recognized in the art for detecting HER2 destruction, including, but not limited to, detection of levels of HER2 destruction in the presence and absence of a combination of anti-HER2 antibodies (or biosimilars thereof) as shown in the working examples provided in this document (see, for example, example 14). For example, the level of HER2 destruction is determined using Western analysis of lysates of HER2 expressing cells that have been treated with a combination of anti-HER2 antibodies compared to the level of HER2 destruction in HER2 expressing cells that have not been treated with a combination of anti-HER2 antibodies.

In some embodiments, the anti-HER2 antibody and/or conjugated anti-HER2 antibody and additional agent(s) are formulated as a single therapeutic composition, and the anti-HER2 antibody and/or conjugated anti-HER2 antibody and additional agent are administered simultaneously. Alternatively, the anti-HER2 antibody and/or the conjugated anti-HER2 antibody and the additional agent are separated from each other, e.g., each is formulated as a separate therapeutic composition, and the anti-HER2 antibody and/or the conjugated anti-HER2 antibody and the additional agent are administered simultaneously, or the anti-HER2 antibody HER2 and/or an anti-HER2 conjugated antibody and an additional agent are administered during the treatment regimen at various time points. For example, anti-HER2 antibody and/or conjugated anti-HER2 antibody is administered prior to administration of the additional agent, anti-HER2 antibody and/or conjugated anti-HER2 antibody is administered after administration of the additional agent, or anti-HER2 antibody and/or conjugated anti-HER2 antibody is administered in an alternating manner. As described herein, the anti-HER2 antibody and/or the conjugated anti-HER2 antibody and the additional agent are administered in single doses or multiple doses.

Pharmaceutical compositions in accordance with this invention may include an antibody, its fragment and/or its conjugate described in this document, and a suitable carrier. These pharmaceutical compositions may be included in kits such as, for example, diagnostic kits.

One of skill in the art will appreciate that the antibodies described herein have many uses. For example, the proteins described herein are used as therapeutic agents. The antibodies described herein are also used as reagents in diagnostic kits or diagnostics, or these antibodies can be used in competitive assays to create therapeutic reagents.

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as generally understood by a person skilled in the art to which this invention belongs. In this 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 verification of the present invention, suitable methods and materials are described below. In the event of a conflict, the present disclosure, including definitions, shall govern. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of this invention will become apparent from the following detailed description and claims.

Brief description of graphic materials

In FIG. 1a shows an epitope binding by XMT 1517 and XMT 1518 antibodies by octet binding of Red384 epitopes;

in fig. 1b - epitope binding by XMT 1519 and XMT 1520 antibodies by octet binding of Red384 epitopes;

in fig. 2 - binding to the cell surface of antibodies XMT 1517, XMT 1518, XMT 1519 and XMT 1520 with JIMT-1 cells;

in fig. 3a - binding affinities of antibodies XMT 1517, XMT 1518 with recombinant human HER2 and cynomolgus monkey HER2;

in fig. 3b - binding affinities of antibodies XMT 1519, XMT 1520 with recombinant human HER2 and cynomolgus monkey HER2;

in fig. 3c - binding affinities of antibodies XMT 1519 and example 16H with recombinant human HER2;

in fig. 3d - binding affinities of antibodies XMT 1519 and example 16H with HER2 cynomolgus monkey;

in fig. 4 shows that XMT 1518 and XMT 1519 antibodies compete for binding to HER2;

in fig. 5 shows antibody-dependent cellular cytotoxicity (ADCC) of trastuzumab and antibodies XMT 1518, XMT 1519 and XMT 1520;

in fig. 6 - the rate of internalization of trastuzumab and antibodies HTXMT 1518, XMT 1519 and XMT 1520;

in fig. 7 - internalization of HER2 antibodies into SKBR3 cells;

in fig. 8 - destruction of HER2 induced by a combination of anti-HER2 antibodies;

in fig. 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 the NCI-N87 mouse tumor xenograft model;

in fig. 10 - antitumor efficacy of Kadsila; example 16A, trastuzumab-(EG2-M1-(10 kDa PHF-BA-(AF-HPA-Ala))), and example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA -Ala))) as measured in the JIMT-1 tumor xenograft mouse model;

in fig. 11 - antitumor efficacy of Kadcyla; pertuzumab; combinations of Kadcyla and pertuzumab and Example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in the JIMT-1 mouse tumor xenograft model;

in fig. 12 - 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 the NCI-N87 mouse tumor xenograft model;

in fig. 13 - Antitumor efficacy of Kadcyla, Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), and Example 17B, rituximab-(EG2-M1-(10 kDa PHF- BA-(AF-HPA-Ala))) as measured in the SNU5 mouse tumor xenograft model;

in fig. 14 Antitumor efficacy of Kadcyla and Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) as measured in a TOV-21G mouse tumor xenograft model;

in fig. 15 Antitumor efficacy of Kadcyla, Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), and Example 17B, rituximab-(EG2-M1-(10 kDa PHF- BA-(AF-HPA-Ala))) as measured in the H522 mouse tumor xenograft model;

in fig. 16 - Antitumor efficacy of Kadcyla, Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), and Example 17B, rituximab-(EG2-M1-(10 kDa PHF- BA-(AF-HPA-Ala))) as measured in a mouse SKOV3 tumor xenograft model;

in fig. 17 Antitumor efficacy of Example 16F, XMT 1519-(EG2-MI-(10 kDa PHF-BA(AF-HPA-Ala))) as measured in the Calu-3 mouse tumor xenograft model;

in fig. 18 Antitumor efficacy of Kadcyla, Example 16H, 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 patient xenograft model BRE-0333 HER2 1+;

in fig. 19 Antitumor efficacy of Kadcyla, Example 16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), and Example 17B, rituximab-(EG2-M1-(10 kDa PHF- BA-(AF-HPA-Ala))) as measured in patient xenograft model MAXF_1162 HER23+;

in fig. 20 Antitumor efficacy of Kadcyla, Example 16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), and Example 17C, rituximab-(EG2-M1-(10 kDa PHF- BA-(AF-HPA-Ala))) as measured in the BT474 mouse tumor xenograft model.

Detailed description of the invention

The invention provides monoclonal antibodies that specifically bind human HER2 in soluble or membrane-bound form (ie, when expressed on the cell surface). AT

- 22 039374 This invention further provides monoclonal antibodies that specifically bind HER2. These antibodies are collectively referred to herein as anti-HER2 antibodies. The antibodies of the present invention bind to an HER2 epitope with an equilibrium dissociation constant (Kd or K _D ) < 1 μM, eg < 100 nM, preferably < 10 nM, and more preferably < 1 nM. For example, anti-HER2 antibodies provided herein exhibit a Kd ranging between about < 1 nM to about 1 pM.

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

An anti-HER2 antibody is considered to fully modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise impair HER2 functional activity if the level of HER2 functional activity in the presence of an anti-HER2 antibody is reduced by at least 95%, e.g., 96, 97 , 98, 99, or 100% compared to the level of functional activity of HER2 in the absence of binding to the anti-HER2 antibody described in this document. An anti-HER2 antibody is said to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with HER2 functional activity if the level of HER2 activity in the presence of the anti-HER2 antibody is reduced by less than 95%, e.g., 10, 20, 25, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% compared to the level of HER2 activity in the absence of binding to the anti-HER2 antibody described herein.

Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present invention will have the meanings that are commonly understood by those skilled in the art. Additionally, unless the context otherwise requires, singular forms include the plural, and plural terms will include the singular. In general, the nomenclatural names used in connection with, as well as techniques, cell and tissue culture, molecular biology and chemistry of proteins and oligo- or polynucleotides, and hybridization described in this document, are well known and commonly used in this field. technology. Standard techniques are used for recombinant DNA production, oligonucleotide synthesis, tissue culture, and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification procedures are performed as described by the manufacturers, or as commonly performed in the art, or as described herein. The techniques and procedures set forth above are generally generally performed in accordance with conventional methods well known in the art and as described in various general and more specific sources cited and discussed throughout the text of this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatural names used in connection with, as well as laboratory procedures and techniques, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical formulation, dosage form and delivery, and patient care.

As used in accordance with the present description, the following terms, unless otherwise indicated, should be understood as having the following meanings.

As used herein, the term HER2 (also known as ErbB2, NEU, HER-2, and CD340), as used herein, refers to human epidermal growth factor receptor 2 (SwissProt P04626) and includes any variants, isoforms, and species homologues. HER2, which are naturally expressed by cells, including tumor cells, or expressed on cells transfected with the HER2 gene. Species homologues include HER2 of the rhesus monkey (tacaca mulatto, no. GI: 109114897 in the Genbank database). These terms are synonymous and can be used interchangeably.

As used herein, the term anti-HER2 antibody or anti-HER2 antibody is an antibody that specifically binds to a HER2 antigen.

As used herein, the term antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e. molecules that contain

- 23 039374 an antigen-binding site that specifically binds an antigen (reacts immunologically with it). The term specifically bind or immunologically reacts with or is directed against means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds them with much lower affinity (Kd > 10 ^-6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, F _ab , F _ab and F( _ab ')2 fragments, scFv, and F _ab expression library.

It is known that the basic structural unit of an antibody contains a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair has one light (about 25 kDa) and one heavy chain (about 50-70 kDa). 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, human-derived antibody molecules belong to any of the IgG, IgM, IgA, IgE, and IgD classes, which differ from one another in the nature of the heavy chain present in the molecule. Some classes also contain subclasses such as IgG1, IgG ₂ and others. Also, in humans, the heavy chain can be a k chain or a λ chain.

The term monoclonal antibody (mAb) or monoclonal antibody composition as used herein refers to a population of antibody molecules that contains only one molecular species of the antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical across all molecules in the population. The mAb contains an antigen-binding site capable of immunologically reacting with a particular antigen epitope that has a unique binding affinity for it.

In general, human-derived antibody molecules belong to any of the IgG, IgM, IgA, IgE, and IgD classes, which differ from one another in the nature of the heavy chain present in the molecule. Some classes also contain subclasses such as IgG1, IgG ₂ and others. Also, in humans, the heavy chain can be a k chain or a λ chain.

The term antigen-binding site or binding portion refers to the part of the immunoglobulin molecule that is involved in the binding of antigens. 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 gaps within the V regions of the heavy and light chains, called hypervariable regions, are located between more conserved flanking gaps, known as framework regions or FRs. Thus, the term FR refers to amino acid sequences that are naturally found in immunoglobulins between and adjacent to hypervariable regions. In an antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are located relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional space of the bound antigen, and these three hypervariable regions of each of the heavy and light chains are called complementarity-determining regions, or CDRs. The amino acid assignment of each domain is as defined by 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).

The terms fragment, antibody fragment, antigen-binding fragment, and antigen-binding fragment are used interchangeably herein, unless otherwise indicated.

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

When used herein in the context of two or more antibodies, the term competes with or cross-competes with indicates that 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 when binding to HER2 if the antibody competes with one or more other antibodies by 25% or more, 25-74% represents partial block and 75-400% represents complete block, as preferably determined by using the assay in Examples 5 and 8. For some pairs of antibodies, competition or blocking in the assay in Examples 5 and 8 was observed only when one antibody was plated and

- 24 039374 the other is used for competition, and not vice versa. Unless otherwise defined or denied in context, the terms competes with, cross-competes with, blocks, or cross-blocks as used herein are also intended to encompass such antibody pairs.

As used herein, an antibody that inhibits HER dimerization should mean an antibody that inhibits or interferes with HER dimer formation. Preferably, such an antibody binds to HER2 at its heterodimeric binding site. In one embodiment, the dimerization inhibitory 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, the anti-EGFR 806 monoclonal antibody, MAb 806, which binds to activated or unbound EGFR ( see Johns et al., J Biol Chem 279(29):30375-30384 (2004)); an antibody that binds to HER3 and inhibits its dimerization with one or more other HER receptors; and an antibody that binds to HER4 and inhibits its dimerization with one or more other HER receptors.

The term HER2 dimerization inhibitor, as used herein, should mean an agent that inhibits the formation of a dimer or heterodimer containing HER2.

As used herein, the term internalization, when used in the context of an anti-HER2 antibody, includes any mechanism by which an antibody is internalized into an HER2-expressing cell from the cell surface and/or from the environment, such as by endocytosis.

As used herein, the terms immunological binding and immunological binding properties refer to a non-covalent interaction of the type that occurs 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, with lower Kd values representing greater affinity. The immunological binding properties of individual polypeptides can be quantified using methods well known in the art. One such method involves the measurement of the rates of formation and dissociation of the antigen-binding site/antigen complex, and these rates depend on the concentrations of the partners of the complex, the affinity of the interaction and geometric parameters, which equally affect the rate in both directions. Thus, both the association rate constant (K _on ) and the dissociation rate constant (K _off ) can be determined by the concentration calculation and the actual association and dissociation rates. (See Nature 361:186-87 (1993)). The ratio K _off /K _on makes it possible to compensate for all parameters not related to affinity, and it is equal to the dissociation constant K _d . (See, in general, Davies et al. (1990) Annual Rev. Biochem. 59:439-473). As stated, an antibody of this invention specifically binds to HER2 when the equilibrium dissociation constant (Kd or K _D ) is <1 μM, preferably < 100 nM, more preferably < 10 nM, and most preferably < 100 pM to about 1 pM, as measured assays such as radioligand binding assays or similar assays known to those skilled in the art.

The term isolated polynucleotide as used herein shall mean a polynucleotide of genome, cDNA or synthetic origin, or some combination thereof, for which, due to its origin, the isolated polynucleotide (1) does not associate with all or part of the polynucleotide in which the isolated polynucleotide is found naturally, (2) is operably linked to a polynucleotide to which it is not naturally linked, or (3) does not occur naturally as part of a larger sequence. The polynucleotides of the present invention include the nucleic acid sequences of SEQ ID NOs: 34 and 36, as well as the nucleic acid molecules encoding immunoglobulin heavy chain 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 immunoglobulin light chain molecules shown in SEQ ID NOs: 2, 4, 6 and 8.

The term isolated protein as used herein means a cDNA, recombinant RNA or synthetically derived protein, or a combination thereof, for which, due to its origin or source of origin, the isolated protein (1) is not associated with proteins found in nature, (2) is free from other proteins from the same source, (3) is expressed by a cell derived from a different species, or (4) does not occur naturally.

The term polypeptide is used herein in relation to a native protein, fragments or analogues of a polypeptide sequence. Therefore, native protein fragments and analogs are species of this genus of polypeptide. The polypeptides of the invention comprise immunoglobulin heavy chain molecules as set forth in SEQ ID NOs: 1, 3, 5, and 7 and immunoglobulin light chain molecules as set forth in SEQ ID NOs: 2, 4, 6, and 8, as well as antibody molecules. formed by combinations containing molecules of heavy chains of immunoglobulins with molecules of light chains of immunoglobulins, such as molecules of light chains of immunoglobulins, and vice versa, as well as their fragments and analogues.

The term naturally occurring, as used herein, when applied to an object, refers to the fact that the 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 humans in a laboratory or otherwise, is naturally occurring.

The term operably linked, as used herein, refers to the positions of components described in this manner in relation to their ability to function in the way they are intended. The regulatory sequence is operably linked to the coding sequence, linked in such a way that expression of the coding sequence is achieved under conditions comparable to those of the regulatory sequences.

The term regulatory sequence, as used herein, refers to polynucleotide sequences that are necessary to influence the expression and processing of the coding sequences to which they are associated. The nature of such regulatory sequences differs depending on the host organism in prokaryotes, such control sequences generally include a promoter, a ribosome binding site and a transcription termination sequence in eukaryotes, in general, such regulatory sequences include promoters and a transcription termination sequence. The term regulatory sequences is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is preferred, such as leader sequences and fusion partner sequences. The term polynucleotide as used herein means a polymeric array of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms of DNA.

The term oligonucleotide as used herein refers to naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide bonds. Oligonucleotides are a subset of polynucleotides, generally containing 200 bases or less in length. Preferably the oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 bases in length. Oligonucleotides are typically single stranded, eg for probes, although oligonucleotides may be double stranded, eg for use in gene mutant construction. The oligonucleotides described herein are either sense or antisense oligonucleotides.

The term naturally occurring oligonucleotides 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, phosphoselenoate, phosphorodiselenoate, phosphoranylothioate, phosphoranilladate, phosphoramidate, 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 Drag 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. US patent No. 5151510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). The oligonucleotide, if necessary, may include a label for detection.

The term selectively hybridize, referred to in this document, means the ability to detect and selectively bind. The polynucleotides, oligonucleotides and fragments thereof according to the invention are selectively hybridized to nucleic acid strands under hybridization and washing conditions, which minimizes the observed amounts of detectable binding to non-specific nucleic acids. To achieve selective hybridization conditions, as is known in the art and discussed herein, particularly stringent conditions can be used. In general, nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments described herein and the nucleic acid sequence of interest will be at least 80% or more, and generally with a preferred increase in homology to 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 if two sequences align to the maximum match. For gaps (in either of the two matching sequences), a preferred maximum gap length match of 5 or less is allowed, and more preferably 2 or less. Alternatively, and preferably, two protein sequences (or polypeptide sequences derived from them,

- 26 039374 with at least 30 amino acids in length) are homologous, as the term is used herein, if they have an alignment score greater than 5 (in standard deviation units) using the ALIGN program with a mutation data matrix and gap penalty equal to 6 or more. See Dayhoff M.O., in Atlas of Protein Sequence and Structure, pp. 101-110 (vol. 5, National Biomedical Research Foundation (1972)) and Supplement 2 to that volume, p. 1-10. Two sequences or parts thereof are preferably homologous if their amino acids are more than or equal to 50% identical when they are optimally aligned using the ALIGN program. The term matches is used herein to denote that a polynucleotide sequence is homologous (i.e., identical, not strictly evolutionarily related) to all or part of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term complementary is used herein to mean that a complementary sequence is homologous to all or part of a reference polynucleotide sequence. To illustrate, the TATAS nucleotide sequence corresponds to the TATAS reference sequence and is complementary to the GTATA reference sequence.

The following terms are used to describe sequence relationships between two or more polynucleotide or amino acid sequences: reference sequence, comparison window, sequence identity, percent sequence identity, and significant identity. A reference sequence is a specific sequence used as a basis for comparing sequences, a reference sequence may be a subset of a larger sequence, such as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may contain the entire cDNA or gene sequence. In general, the reference sequence is at least 18 nucleotides or 6 amino acids long, often at least 24 nucleotides or 8 amino acids long, and often at least 48 nucleotides or 16 amino acids long. Because two polynucleotide or amino acid sequences can each (1) contain a sequence (i.e., a portion of an entire polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) can further contain a sequence that is divergent between the two polynucleotide or amino acid sequences , then sequence comparisons between two (or more) molecules are typically performed by comparing the sequences of the two molecules across 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 consecutive nucleotide positions or 6 amino acid sequences in which a polynucleotide sequence or amino acid sequence can be compared to a reference sequence of at least 18 consecutive nucleotide positions or 6 amino acid sequences and In this case, the portion of the polynucleotide sequence in the comparison window may contain additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less compared to the reference sequence (which contains no addition or deletion) to optimally align the two sequences. Optimal sequence alignment for comparison window alignment can be performed by the local homology search algorithm from Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm from Needleman and Wunsch J. Mol. Biol. 48:443 (1970), similarity search method from Pearson and Lipman Proc. Natl. Acad. sci. (U.S.A.) 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, WI), Geneworks or MacVector software) or by browsing, and selecting the best alignment (i.e., one that obtains the highest percentage of homology across the comparison window) achieved by various methods.

The term sequence identity means that two polynucleotide or amino acid sequences are identical (ie, based on a nucleotide-nucleotide or residue-residue comparison) across a window of comparison. The percent sequence identity term is calculated by comparing two optimally aligned sequences over a comparison window by determining the number of positions at which an identical nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs in both sequences to obtain the number of positions that matched. , dividing the number of matched positions by the total number of positions in the comparison window (ie, window size) and multiplying the result by 100 to obtain the percent sequence identity. The terms significant identity as used herein means a characteristic of a polynucleotide or amino acid sequence in which the polynucleotide or amino acid contains a sequence that has at least 85% sequence identity, preferably at least 90 to 95% sequence identity, rather at least at least 99% sequence identity compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, often over a window of

- 27 039374 at least 24-48 nucleotide (8-16 amino acids) positions, and the percentage of sequence identity is calculated by comparing the reference sequence with a sequence that may include deletions or additions that are only 20 percent or less of the reference sequence in the comparison window. The reference sequence may be a subset of a larger sequence.

As used herein, the twenty basic amino acids and their abbreviations correspond to conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland? Mass. (1991)). Stereoisomers (eg, D-amino acids) of the twenty basic amino acids, non-naturally occurring amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-traditional amino acids may also be useful components for the polypeptides of this invention. Examples of non-traditional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Νtrimethyllysine, ε-Ν-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-Ν- methylarginine and other related amino acids and imino acids (eg 4hydroxyproline). In the designation of polypeptides used herein, in accordance with standard usage and convention, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction.

Similarly, unless otherwise indicated, the left-hand side of single-stranded polynucleotide sequences is the 5' end, the left-hand side of double-stranded polynucleotide sequences is the 5'-direction. The direction from 5' to 3', the addition of the resulting RNA transcripts, refers to the direction of transcription of sections of sequences on a DNA strand that has the same sequence as RNA, and which are 5' in relation to the 5' end of the RNA transcript are ascending sequences, portions of sequences on a DNA strand that have the same sequence as RNA and are 3' to the 3' end of the RNA transcript are downstream sequences.

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

Preferably, positions of residues that are not identical differ in conservative amino acid substitutions.

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

As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are intended to be covered by the present invention, provided that the variations in the amino acid sequence conserve at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. %. In particular, conservative amino acid substitutions are contemplated. Conservative amino acids are those that come from a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into two families: (1) acidic amino acids - aspartate, glutamate; (2) basic amino acids - lysine, arginine, histidine; (3) non-polar amino acids - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. 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 an aliphatic hydroxyl 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 leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or a similar amino acid substitution with a structurally related amino acid will not significantly affect the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a scaffold site. . Whether a change in amino acid will affect a functional peptide can easily be determined by analyzing the specific activity of the polypeptide derivative. The analyzes are described in detail in this document. Fragments or analogs of antibodies or immunoglobulin molecules can easily be obtained by one of ordinary skill in the art. The preferred amino and carboxy termini of fragments or analogs occur near functional domain boundaries. Structural and functional domains can be identified by comparing nucleotide and/or amino acid sequence data with publicly available or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or expected protein conformational domains that occur in other proteins of known structure and/or function. Methods are known for identifying protein sequences that fold into a known three-dimensional structure. Bowie et al. Science 253:164 (1991). Thus, the above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural or functional domains in accordance with the present invention.

Preferred amino acid substitutions are those that: (1) desensitize proteolysis, (2) desensitize oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinities, and (4) confer different or modify other physicochemical or functional properties of such analogues. The analogs may include different 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 a naturally occurring sequence (preferably in the portion of the polypeptide outside the junction-forming domain(s). A conservative amino acid substitution should not substantially alter the structural characteristics of the original sequence (eg, an amino acid substitution should not promote helix breakage that occurs in the original sequence or destroy other types of secondary structure that characterizes the original sequence). Examples of secondary polypeptides and tertiary structures recognized in the art 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 at. Nature 354:105 (1991).

The term polypeptide fragment, as used herein, refers to a polypeptide that has an amino-terminal and/or carboxy-terminal division, but in which the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence, as determined, for example, from the full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids long, preferably at least 14 amino acids long, more preferably at least 20 amino acids long, typically at least 50 amino acids long, and even more preferably at least 70 amino acids in length. The term analog, as used herein, refers to polypeptides that contain a segment of at least 25 amino acids that has significant identity with a portion of the established amino acid sequence and that has specific binding to HER2 under suitable binding conditions. As a rule, polypeptide analogs contain a conservative amino acid substitution (or addition or deletion) in relation to the naturally occurring sequence. Analogues are typically at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be the same length as a full-length naturally occurring polypeptide.

Peptide analogues are commonly used in the pharmaceutical industry as non-protein drug substances with properties similar to those of the matrix peptide. These types of non-peptide compounds are called peptide mimetics or peptidomimetics. Fauchere, J. Adv. Drag 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 designed using computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to obtain an equivalent therapeutic or prophylactic effect. In general, peptidomimetics are structurally similar to a reference polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as a human antibody, but has 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)CH ₂ -- 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-li

- 29 039374 zine instead of L-lysine) can be used to create a more stable peptide. In addition, a sterically hindered peptide containing a consensus sequence or a substantially identical variation of the consensus sequence can be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

The term agent is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from a biological material.

As used herein, the term labeled or labeled refers to the insertion of a detectable marker, for example by inserting a radioisotopically labeled amino acid or attaching to the polypeptide a biotin moiety that can be detected labeled™ by avidin (for example, streptavidin containing a fluorescent marker or an 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 well 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 H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ⁿ¹ In, ¹²⁵ I, ¹³¹ I), fluorescent labels ( e.g., FITC, rhodamine, lanthanide complex phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent biotinylated groups, predefined polypeptide epitopes recognized by a secondary reporter (e.g., pairs of leucine zipper sequences zippers, secondary antibody binding sites, metal binding domains, epitope markers). In some embodiments, the tags are attached using spacer arms of various lengths to reduce potential steric hindrances. The term pharmaceutical agent or drug substance, as used herein, refers to a chemical compound or composition capable of inducing the desired therapeutic effect when properly administered to a patient.

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

As used herein, substantially pure means that the target species is the predominant species (i.e., on a molar basis, it is more abundant than any other single species in the composition), and preferably the substantially pure fraction is the composition, with the target species containing at least about 50% (on a molar basis) of all macromolecular species present.

In general, a substantially pure composition will contain greater than about 80% of all macromolecular species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%. Most preferably, the target species is purified to substantial homogeneity (the contaminant species cannot be detected in the composition by conventional detection methods), the composition consisting essentially of a single macromolecular species.

The use of the singular in both the following description and the claims is to be interpreted as embracing singular and plural forms unless otherwise indicated herein or clearly conflicted from context. The terms constituent, having, represented by, represented by a chemical formula, including, and containing are interpreted as non-limiting terms (i.e., meaning including, but not limited to), unless otherwise noted. For example, a polymer backbone of a particular formula includes all of the monomer units shown in the formula and may also include additional monomer units shown in the formula. In addition, whenever a constituent or other non-limiting term is used in an embodiment of the invention, it should be understood that the same embodiment may be claimed more narrowly using the intermediate term consisting essentially of or the limiting term consisting of.

The term about, approximately, or approximately, when used in connection with a numerical value, means that a set or range of values is 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 of the invention, the term about refers to a range of values that are 5% greater than or less than the specified value. In another embodiment of the invention, the term about refers to a range of values that are 2% more or less than the specified value. In another embodiment of the invention, the term about refers to a range of values that are 1% greater than or less than the specified value.

The listing of ranges of values is solely intended to perform the function of a shorthand way of self-reference to each individual value falling within the range, unless otherwise noted in this document, and each individual value is included in

- 30 039374 description, as if it were independently indicated in this document. Unless otherwise noted, the range used in this document includes two range limits. For example, both expressions x is an integer between 1 and 6 and x is an integer between 1 and 6 mean x is 1, 2, 3,

4, 5 or 6, i.e. a term between X and Y, and a range from X to Y, are inclusive of X and Y, and integers in between.

All of the methods described herein may be performed in any suitable order, unless otherwise indicated herein or where there is a clear contradiction from the context. The use of any or all of the examples, or exemplary wording (such as) provided herein, is solely intended to better illustrate the present invention and should not be construed as limiting the scope of the claims, unless expressly stated otherwise. No wording in the specification should be construed as indicating that an element not claimed is essential to what is being claimed.

A 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, an immobilized surface protein, or a protoglycan. Examples of PBRM include, but are not limited to, XMT 1517 antibody, XMT 1518 antibody, XMT 1519 antibody, and XMT 1520 antibody described herein, as were all other antibodies (e.g., trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, veltuzumab, labetuzumab, B7-H4, B7-H3, CA125, CD33, CXCR,2 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 mimics and the like. A protein-based recognition molecule, in addition to targeting a polymer-modified conjugate conjugated to a specific cell, tissue, or site, may also have a specific therapeutic effect, such as anti-proliferative (cytostatic and/or cytotoxic) activity against the target cell or mechanism. The protein-based recognition molecule contains or may be designed to contain at least one chemically reactive group such as -COOH, a primary amine, a secondary amine, -NHR, -SH or a chemically reactive amino acid moiety or side chains such as eg tyrosine, histidine, cysteine or lysine.

Biocompatible, as used herein, is intended to describe compounds that exhibit minimal destructive or other effects in the form of an organism's response when brought into 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 that falls within the definition of biocompatible as defined above and herein. The term biocompatibility, as used herein, also implies that compounds exhibit minimal interactions with recognition proteins, such as naturally occurring antibodies, cellular proteins, cells, and other components of biological systems, unless such interactions are particularly desired. Thus, it is believed that substances and functional groups intended solely to provide the above minimum interactions, such as drugs and prodrugs, should be biocompatible. Preferably (with the exception of compounds suspected of being cytotoxic, such as, for example, anticancer agents), the compounds are biocompatible if their addition to normal cells in vitro at concentrations similar to the expected systemic concentrations in vivo results in less than or equal to 1% degree of cell death over the period of time required for 50% of the amount of the compound administered in vivo, taking into account isolation/elimination), and their administration in vivo induces minimal and medically acceptable inflammation, reaction to the introduction of a foreign body, immunotoxicity, chemical toxicity and/or other similar adverse effects. In the above sentence, the term normal cells refers to cells that are not designed to be destroyed or otherwise significantly affected by the compound to be investigated.

Biodegradable. As used herein, biodegradable polymers are polymers that are susceptible to in vivo biological processing. As used herein, biodegradable compounds or fragments are those that are taken up by cells, can be broken down by a lysosomal or other chemical mechanism, or hydrolyzed into components that cells can either reuse or release without significant toxic effects on cells. The term biodegradable as used herein has the same meaning as biodegradable. The degradation fragments preferably induce little or no overload of organs or cells, or the pathological processes caused by such overload, or other adverse effects in vivo. Examples of biodegradation processes include enzymatic and non-enzymatic hydrolysis, oxidation and reduction. Suitable conditions for the non-enzymatic hydrolysis of biodegradable protein-polymer-drug conjugates (or components thereof, e.g., a biodegradable polymeric carrier and a linker between the carrier and the antibody or drug molecule) described herein, for example, include exposing the biodegradable water conjugates to temperature and pH of the lysosomal intracellular compartment. The biodegradation of some protein-polymer-drug conjugates (or components thereof, such as a biodegradable polymer carrier and a linker between the carrier and the antibody or drug molecule) can also be enhanced by extracellular exposure, such as in low pH areas in animals, such as in an inflamed area, in close proximity to activated macrophages or other cells releasing factors that promote destruction. In some preferred embodiments of the invention, the effective polymer carrier size at pH~7.5 does not change at a detectable level within 1 to 7 days and remains within 50% of the original polymer size for at least several weeks. On the other hand, at pH~5, the polymer carrier preferably degrades detectably within 1 to 5 days and is completely converted to low molecular weight fragments within a time frame of two weeks to several months. Polymer integrity can be measured in tests such as size exclusion HPLC. Although faster degradation may be preferred in some cases, in general it may be more necessary for the polymer to be degraded in cells at a rate that does not exceed the rate of metabolic conversion or excretion of polymer fragments by the cells. In preferred embodiments of the invention, the polymers and biodegradation by-products are biocompatible.

Maleimido blocking compound. As used herein, refers to a compound that can react with maleimide to convert it to succinimide, and maleimide blocking compound refers to a chemical moiety attached to succinimide when it is converted. In some embodiments, the maleimidoblocking compound is a compound having a thiol terminal group for reaction with maleimide. In one embodiment, the maleimido blocking compound is cysteine, N-acetylcysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropionic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH2SH), benzylthiol, wherein phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol.

Hydrophilic. The term hydrophilic, when referring to substituents, such as on polymeric monomer units or on a maleimide blocking moiety to render them hydrophilic or water soluble, is essentially the same as the usual meaning of the term in the art and denotes a chemical moiety that contains ionizable, polar or polarizable atoms, or which 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 that falls within the definition of hydrophilic as defined above. Examples of specific hydrophilic organic moieties that are suitable include, without limitation, aliphatic or heteroaliphatic groups containing a chain of atoms ranging between about one and twelve atoms, hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxyl ether, thioether, 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 esters, polyamines, polycarboxylates, polyesters and polythioethers. In some embodiments of the invention, hydrophilic substituents contain a carboxyl group (COOH), an aldehyde group (CHO), a ketone group (COC1_ ₄ alkyl), methylol (CH ₂ OH), or glycol (for example, CHOH-CH2OH or CH-(CH ₂ OH) ₂ ), NH2, F, cyano, SO ₃ H, PO ₃ H and the like.

The term hydrophilic, when referring to the polymer described herein, is generally the same as the use of the term in the art and refers to polymers containing hydrophilic functional groups as defined above. In a preferred embodiment of the invention, the hydrophilic polymer is a water-soluble polymer. The hydrophilicity of a polymer can be directly measured by determining the energy of hydration, or by determination by examination between two liquid phases, or by chromatography on solid phases of known hydrophobicity, such as, for example, C4 or C18.

polymer carrier. The term polymeric carrier, as used herein, refers to a polymer or modified polymer that is suitable for covalent attachment to, or can be covalently attached to, one or more targeted linker drug molecules and/or one or more targeted linker PBRMs.

Physiological conditions. The phrase physiological conditions, as used herein, refers to the range of chemical (eg, pH, ionic strength) and biochemical (eg, enzyme concentrations) conditions likely to occur in the extracellular fluids of living organisms.

- 32 039374 fabrics. For most normal tissues, the physiological pH is in the range of about 7.0 to 7.4.

Circulating blood plasma and normal interstitial fluid are typical examples of normal physiological conditions.

medicinal substance. As used herein, the term drug refers to a compound that is biologically active and exerts a desired physiological effect upon administration to a subject in need (eg, an active pharmaceutical ingredient).

Cytotoxic. As used herein, the term cytotoxic means toxic to cells or a selected population of cells (eg, cancer cells). The toxic effect can lead to cell death and/or lysis. In some cases, the toxic effect may have a sub-lethal destructive effect on the cell, such as slowing down or stopping cell growth. In order to achieve a cytotoxic effect, the drug or prodrug may be selected, inter alia, from the group consisting of a DNA damaging agent or a cytotoxic protein or polypeptide.

Cytostatic. As used herein, the term cytostatic refers to a drug or other compound that inhibits or stops the growth and/or reproduction of cells.

small molecule. As used herein, the term small molecule refers to molecules, either naturally occurring or artificially created (eg, through chemical synthesis), that have a relatively low molecular weight. Preferred small molecules are biologically active due to the fact that they have a local or systemic effect on the body of animals, preferably mammals, preferably humans. In some preferred embodiments, the small molecule is a drug and the small molecule is referred to as a drug molecule or a drug or therapeutic agent. In some embodiments, the drug molecule has an MW less than or equal to about 5 kDa. In other embodiments, the drug molecule has an MM less than or equal to about 1.5 kDa. In embodiments, the drug molecule is selected from vinca alkaloids, auristatins, duocarmycins, tubulisins, duocarmycins, non-naturally occurring camptothecin compounds, topoisomerase inhibitors, DNA-binding drugs, kinase inhibitors, MEK inhibitors, KSP inhibitors, calicheamicins, SN38, pyrrolobenzodiazepines, and their counterparts. Preferably, though not necessarily, the drug substance is one that has already been recognized as safe and effective for use by an appropriate government agency or authority, such as the FDA. For example, medicinal substances for medical use are listed by the FDA under 21 C.F.R. §§ 330.5, paragraphs 331 to 361 and 440 to 460; medicinal substances for veterinary use are listed by the FDA at 21 C.F.R. §§ 500 to 589, incorporated herein by reference, are all considered suitable for use with the present hydrophilic polymers.

A drug derivative or modified drug or the like as used herein refers to a compound that contains a drug molecule intended to be delivered by the conjugate described herein and a functional group capable of attaching the drug molecule to a polymeric carrier.

Active form, as used herein, refers to the form of a compound that exhibits intended pharmaceutical efficacy in vivo or in vitro. In particular, if the drug molecule intended to be delivered by the conjugate described herein is released from the conjugate, then the active form may be the drug itself or a derivative thereof that exhibits the intended therapeutic properties. Release of the drug from the conjugate can be achieved by cleavage of the biodegradable bond of the linker that attaches the drug to the polymeric carrier. Derivatives of the active drug substance, respectively, may contain part of the linker.

PHF refers to poly(1-hydroxymethylethylene hydroxymethyl formal).

As used herein, the terms polymer unit, monomer unit, monomer, monomer unit, unit all refer to a repeat of a structural unit in a polymer.

As used herein, the molecular weight or MW of a polymer or polymer carrier/framework or polymer conjugates refers to the weight of the average molecular weight of the unmodified polymer, unless otherwise indicated.

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. As a general example, and without limitation, isotopes of the hydrogen atom include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

The present invention is intended to include all isomers of the compound which is

- 33 039374 to and includes optical isomers and tautomeric isomers, and 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; wherein the isomer may be in isolated form or in admixture with one or more other isomers.

Antibodies against HER2.

The monoclonal antibodies described herein have the ability to inhibit the HER2-mediated activity of the PI3K-Akt mechanism.

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

Each of the anti-HER2 monoclonal antibodies described herein includes a heavy chain (HC), a heavy chain variable region (VH), a light chain (LC), and a light chain variable region (VL), as shown in the amino acid sequences and corresponding nucleic acid sequences. acids below. The heavy chain variable region and the light chain variable region for each antibody are indicated by background in the amino acid sequences below. Complementarity determining regions (CDRs) of the heavy chain and light chain are underlined in the amino acid sequences below. Amino acids spanning complementarity determining regions (CDRs) are defined by E.A. Kabat et al. (See Kabat E.A., et al. Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

>XMT 1517, heavy chain amino acid sequence (heavy chain variable region (SEQ ID NO: 9) + IgG1, heavy chain constant region (SEQ ID NO: 32))

OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYGMHWVROAPGKGLEWV AVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

KEAPYYAKDYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG * (SEQ ID NO: 1)

CDRH1: FTFSSYGMH (SEQ ID NO: 17)

CDRH2: VIWYDGSNKYYADSVKG (SEQ ID NO: 18)

CDRH3: EAPYYAKDYMDV (SEQ ID NO: 19) >XMT 1517, heavy chain variable region nucleic acid sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC

CCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTA TATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC AGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGGAAGCTCC CTACTACGCTAAAGATTACATGGACGTATGGGGCAAGGGTACAACTGTCA CCGTCTCCTCA (SEQ ID NO: 34)> XMT 1517 amino acid sequence of the light chain (light chain variable region (SEQ ID NO: 10) + light chain constant region (SEQ ID NO: 33))

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EIVLTOSPGTLSLSPGERATLSCRASOSVSSDYLAWYOOKPGOAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOQYVSYWTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYQEKHKVYACEV SFTHQPVSSK2

CDRL1: RASQSVSSDYLA (SEQ ID NO: 20)

CDRL2: GASSRAT (SEQ ID NO: 21)

CDRL3: QQYVSYWT (SEQ ID NO: 22) >XMT 1517, light chain variable region nucleic acid sequence GAAATTGTGTTGACGCAGTCTCAGGCACCCTGTCTTTGTCTCCAGGGGAA

AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCGACTACTTA GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT GCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTC TGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC AGTGTATTACTGTCAGCAGTACGTCAGTTACTGGACTTTTGGCGGAGGGAC CAAGGTTGAGATCAAA (SEQ ID NO: 35)> XMT 1518 amino acid sequence of the heavy chain (heavy chain variable region (SEQ ID NO: 11) + IgG1, constant heavy chain (SEQ ID NO: 32))

OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYGMHWVROAPGKGLEWV AGIWWDGSNEKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KEAPYYAKDYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG * (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))

EIVLTOSPGTLSLSPGERATLSCRASQSVSSDYLAWYOOKPGOAPRLLIYG ASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOQYVSYWTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYQEKHKVECSEV SFTK4

CDRL1: RASQSVSSDYLA (SEQ ID NO: 20)

CDRL2: GASRRAT (SEQ ID NO: 24)

CDRL3: QQYVSYWT (SEQ ID NO: 22) >XMT 1519, heavy chain amino acid sequence (heavy chain variable region (SEQ ID NO: 13) + IgG1, heavy chain constant region (SEQ ID NO: 32))

- 35 039374

EVOLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWVS YISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGG HGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG * (SEQ ID NO: 5)

CDRH1: FTFSSYSMN (SEQ ID NO: 25)

CDRH2: YISSSSTIYYADSVKG (SEQ ID NO: 26)

CDRH3: GGHGYFDL (SEQ ID NO: 27) >XMT 1519, heavy chain variable region nucleic acid sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC

CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCAT GAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACA TTAGTAGTAGTAGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGAT TCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAAC AGCCTGAGAGCTGAGGACACGGCGGTGTACTACTGCGCCAGAGGTGGACA CGGATATTTCGACCTATGGGGGAGAGGTACCTTGGTCACCGTCTCCTCA (SEQ ID NO: 36)> XMT 1519 amino acid sequence of the light chain (light chain variable region (SEQ ID NO: 14) + light chain constant region (SEQ ID NO: 33))

EIVLTOSPGTLSLSPGERATLSCRASOSVSSSYLAWYOOKPGOAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOQYHHSPLTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSSPTYSLSSTLTLSKADYQEKHKVYACEVTHQSEN

CDRL1: RASQSVSSSYLA (SEQ ID NO: 28)

CDRL2: GASSRAT (SEQ ID NO: 21)

CDRL3: QQYHHSPLT (SEQ ID NO: 29) >XMT 1519, Light chain variable region nucleic acid sequence GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA

AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTA GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT GCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTC TGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC AGTGTATTACTGTCAGCAGTACCACCACAGTCCTCTCACTTTTGGCGGAGG GACCAAGGTTGAGATCAAA (SEQ ID NO: 37)> XMT 1520, the amino acid sequence of the heavy chain (heavy chain variable region (SEQ ID NO: 15) + IgG1, heavy chain constant region (SEQ ID NO: 32))

- 36 039374

EVQLVESGGGLVQPGGSLRLSCAASGFTFSGRSMNWVRQAPGKGLEWVS YISSDSRTIYYADSVKGRFTISRDNAKNSLYLOMNSLRAEDTAVYYCARGG

HGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG * (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))

EIVLTOSPGTLSLSPGERATLSCRASQSVSSSYLAWYOOKPGOAPRLLIYG ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOQYHHSPLTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSSPTYSLSSTLTLSKADYEKHKVYACEVQN

CDRL1: RASQSVSSSYLA (SEQ ID NO: 28)

CDRL2: GASSRAT (SEQ ID NO: 21)

CDRL3: QQYHHSPLT (SEQ ID NO: 29)

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, the 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 on human HER2 (eg, GenBank # P04626.1).

The antibodies and antigen-binding fragments thereof described herein specifically bind to an epitope on the full length human HER2 receptor containing the amino acid sequence

- 37 039374

MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY

QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ

VRQVPLQRLR

101 IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL

QLRSLTEILK

151 GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR

ACHPCSPMCK

201 GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC

AAGCTGPKHS

251 DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT

FGASCVTACP

301 YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL

351 REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA

PLQPEQLQVF

401 ETLEEITGYL YISAWPDSLP DLSVFQNLQVIRGRILHNGA YSLTLQGLGI

451 SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP

- 38 039374

501 EDECVGEGL VEECRVLQGL CHQLCARGHC WGPGPTQCVN CSQFLRGQEC 551 PREYVNARHC KDPPFCVARC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY 601 PSGVKPDLSY GCPAEQRASP MPIWKFPDEE GACQPCPINC THSCVDLDDK

651 LTSIISAVVGILLVVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPL

701 TPSGAMPNQA PDGENVKIPV QMRILKETEL RKVKVLGSGA FGTVYKGIWI 751 AIKVLRENTS TSTVQLVTQL PKANKEILDE AYVMAGVGSP YVSRLLGICL 801 MPYGCLLDHV LVHRDLAARN RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR 851 VLVKSPNHVK LESILRRRFT ITDFGLARLL DIDETEYHAD GGKVPIKWMA 901 HQSDVWSYGV LPQPPICTID TVWELMTFGA KPYDGIPARE IPDLLEKGER 951 VYMIMVKCWM NEDLGPASPL IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ 1001 dstfyrsle GMVHHRHRSS DDDMGDLVDA EEYLVPQQGF FCPDPAPGAG 1051 STRSGGGDLT GMGAAKGLQS LGLEPSEEEA PRSPLAPSEG AGSDVFDGDL 1101 LPTHDPSPLQ NQPDVRPQPP RYSEDPTVPL PSETDGYVAP LTCSPQPEYV 1151 spree VENPEYLTPQ RPAGATLERP KTLSPGKNGV VKDVFAFGGA 1201 GGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT

PTAENPEYLG

1251 LDVPV (SEQ ID NO: 38)

The antibodies and antigen-binding fragments thereof described herein specifically bind to an epitope on the extracellular domain (ECD) of the human HER2 receptor containing the amino acid sequence

- 39 039374

TQVCTGTDMK LRLPASPETH LDMLRHLYQG CQVVQGNLEL

TYLPTNASLS

FLQDIQEVQG YVLIAHNQVR QVPLQRLRIV RGTQLFEDNY

ALAVLDNGDP

101 LNNTPVTGA SPGGLRELQL RSLTEILKGG VLIQRNPQLC YQDTILWKDI

151 FHKNNQLALT LIDTNRSRAC HPCSPMCKGS RCWGESSEDC

QSLTRTVCAG

201 GCARCKGPLP TDCCHEQCAA GCTGPKHSDC LACLHFNHSG

ICELHCPALV

251 TYNTDTFESM PNPEGRYTFG ASCVTACPYN YLSTDVGSCT

LVCPLHNQEV

301 TAEDGTQRCE KCSKPCARVC YGLGMEHLRE VRAVTSANIQ

EFAGCKKIFG

351 SLAFLPESFD GDPASNTAPL QPEQLQVFET LEEITGYLYI SAWPDSLPDL

401 SVFQNLQVIR GRILHNGAYS LTLQGLGISW LGLRSLRELG SGLALIHHNT

451 HLCFVHTVPW DQLFRNPHQA LLHTANRPED ECVGEGLACH QLCARGHCWG

501 PGPTQCVNCS QFLRGQECVE ECRVLQGLPR EYVNARHCLP

CHPECQPQNG

551 SVTCFGPEAD QCVACAHYKD PPFCVARCPS GVKPDLSYMP

IWKFPDEEGA

601 CQPCPINCTH SCVDLDDKGC PAEQRASPLT (SEQ ID NO: 39)

One skilled in the art will recognize that without undue experimentation, it is possible to determine whether a monoclonal antibody has the same specificity as the monoclonal antibody described herein (e.g., XMT 1517, XMT 1518, XMT 1519, and XMT 1520 ) by determining whether the first antibody prevents the latter from binding to a natural binding partner or another molecule known to be associated with HER2. If the monoclonal antibody under investigation competes with the monoclonal antibody described herein, as shown by reduced binding by the monoclonal antibody described herein, then both monoclonal antibodies bind to the same or a closely related epitope.

An alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody described 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 assayed in with respect to determining whether the test monoclonal antibody is inhibited in relation to its ability to bind HER2. If the monoclonal antibody under investigation is inhibited, then it is likely to have the same or functionally equivalent epitope specificity as the monoclonal antibody described herein.

Screening for the monoclonal antibodies described herein can also be performed, for example, by measuring the HER2-mediated mechanism of PI3K-Akt activity and determining whether the monoclonal antibody is capable of modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise disrupting the activity of the PI3K mechanism. -Act.

Anti-HER2 antibodies are generated, for example, using the methods described in the examples provided herein. Alternatively, or in addition to this, to obtain

- 40 039374 monoclonal antibodies directed against HER2 or against derivatives, fragments, analogues of their homologs or orthologs, can be used in various procedures known within the art. (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 and heavy chains, including the CDRs, are derived 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 techniques described in the examples provided herein. Human monoclonal antibodies can also be obtained using the trioma technique; human B-cell hybridoma technique (see Kozbor et al., 1983 Immunol. Today 4: 72) and EBV hybridoma technique to generate 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 obtained by using human hybridomas (see Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming 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).

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

Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise disrupt the activity of the HER2-mediated PI3KAkt mechanism are prepared, 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, antibodies are generated by screening a library that contains antibody or antigen-binding domain sequences for binding to HER2. This library is obtained, for example, on a bacteriophage as fusion proteins or peptides with a bacteriophage coat protein that is expressed on the surface of the assembled phage particles encoding the DNA sequences contained within the phage particles (i.e. phage display library). Hybridomas resulting from myeloma/B cell fusions are then screened for HER2 reactivity. In addition, antibodies are selected, and optionally optimized, in yeast antibody display libraries and yeast library presentation systems as described, for example, in Blaise L., Wehnert A., Steukers M.P., van den Beucken T., Hoogenboom H.R., Hufton S.E. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments. Gene. 2004 Nov 24;342(2):211-8; Boder E.T., Wittrup K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 1997 Jun; 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 T.M., Agrawal N.J., Chennamsetty N., Egodage K., Helk B., Trout B.L. 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: 10.1007/978-3-642-01144315; Rakestraw J.A., Aird D., Aha P.M., Baynes B.M., Lipovsek D. Secretion-and-capture cell-surface display for selection of target-binding proteins. ProteinEng. Des. Sel. Jun 2011; 24(6):525-30; US patents No. 8258082; 6300064; 6696248; 6165718; 6500644; 6291158; 6291159; 6096551; 6368805; No. 6,500,644. Exemplary yeast library presentation systems are described, for example, in WO 2008118476; WO2009/036379; WO 2010105256 and WO 2012009568. In some embodiments, such yeast library displays or yeast library presentation systems aim to mimic or display a characteristic of the diversity of the pre-immune human antibody repertoire. In some embodiments, such a variety of yeast display library or variety of yeast library presentation system is created in silico. In some embodiments, such yeast library displays or yeast library presentation systems comprise yeast Saccharomyces cells, such as Saccharomyces cerevisiae cells. In some embodiments, such yeast library displays or yeast library presentation systems comprise yeast Saccharomyces cells, such as Saccharomyces cerevisiae cells.

Monoclonal antibodies are prepared, for example, using hybridoma techniques such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, the mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent.

- 41 039374 agent for detecting lymphocytes that produce or are capable of producing antibodies that will specifically bind to this immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent will typically include a protein antigen, a fragment thereof, or a fusion protein thereof. In general, any peripheral blood lymphocytes are used if cells of human origin are required, or spleen cells or lymph node cells are used if non-human mammalian sources are required. The lymphocytes are then fused to an immortalized cell line using suitable fusion agents such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed into mammalian cells, in particular myeloma cells derived from a rodent, bovine or human body. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-fused immortalized cells. For example, if the original cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), then the culture medium will include hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, maintain a stable high level of antibody expression by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More 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. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies (see Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc. ., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells were cultured can then be analyzed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or 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 a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and high binding affinity for the target antigen.

Once the desired hybridoma cells have been identified, clones can be subcloned by limiting dilution and growth procedures by standard techniques (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 grown in vivo as ascitic cells in a mammalian body.

Monoclonal antibodies secreted by subclones can be isolated or purified from the culture medium or ascitic fluid by conventional immunoglobulin purification procedures such as, for example, protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be produced by recombinant DNA techniques such as those described in US Pat. with genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells described herein are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to allow the synthesis of monoclonal antibodies in recombinant cells. -owners. DNA can also be modified, for example, by replacing the coding sequence with human heavy and light chain constant domains instead of homologous mouse sequences (see US Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or a portion containing a non-immunoglobulin polypeptide coding sequence. In such a non-immunoglobulin polypeptide, there may be substitutions for the constant domains of the antibody described herein, or there may be substitutions for the variable domains of a single antigen-combining site of the antibody described herein to create a chimeric bivalent antibody.

Monoclonal antibodies described herein include fully human antibodies or humanized antibodies.

These antibodies are suitable for administration to humans without causing an immune response in the human body against the administered immunoglobulin.

A humanized or fully human anti-HER2 antibody is prepared, for example, using the procedures described in the examples provided herein.

In other alternative methods, an anti-HER2 antibody is developed, for example, using phage display techniques using antibodies containing only human sequences. Such approaches are well known in the art, for example in WO 92/01047 and US Pat. No. 6,521,404, which are hereby incorporated by reference. In this approach, a combinatorial phage library carrying random light and heavy chain pairs is screened using a natural or recombinant source of HER2 or fragments thereof. In another approach, an anti-HER2 antibody may be produced by a process wherein at least one step of the process comprises immunizing a transgenic non-human animal with human HER2 protein. In this approach, some of the endogenous heavy and/or light chain loci to 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. In addition, 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 introduced antigen, human loci undergo a rearrangement to produce genes encoding human variable regions immunospecific for the antigen. Therefore, when immunized, the xenogeneic mouse produces B cells that secrete fully human immunoglobulin.

A variety of techniques are well known in the art for producing xenogenic non-human animals. For example, see US Pat. 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 development 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. Patent Nos. 6162963, 6150584, 6114598, 6075181 and 5939598 and Japanese Patent Nos. WO 98/24893, WO 00/76310 and related documents of the family of patents-analogues.

In an alternative approach, other authors have used a minilocus approach in which the exogenous Ig locus is mimicked by incorporating portions (individual genes) from the Ig locus. Thus, one or more VH genes, one or more D _H genes, one or more J _H genes, a mu constant region, and a second constant region (preferably a γ constant region) are formulated into a construct for insertion into an animal body. See, for example, US Pat. Nos. 5,545,806; 5545807; 5591669; 5612205; 5625825; 5625126; 5633425; 5643763; 5661016; 5721367; 5770429; 5789215; 5789650; 5814318; 5877397; 5874299; 6023010 and 6255458; european patent no. 0546073 B1 and international patent applications no. , WO 97/13852 and WO 98/24884 and related patent documents.

It has also been demonstrated that human antibodies are formed from mice in which large portions of chromosomes or entire chromosomes have been introduced by microcellular fusion. See European Patent Applications Nos. 773288 and 843961.

Responses in the form of human anti-mouse antibodies (HAMA) have led to the creation of an industrial approach to obtain chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and an immune variable region, it is expected that certain human antichimeric antibody (HACA) responses will be observed, particularly with long-term or multi-dose applications of this antibody. Thus, it may be necessary to provide fully human anti-HER2 antibodies in order to disrupt or otherwise mitigate problems or impacts associated with a HAMA or NASA response.

Obtaining antibodies with reduced immunogenicity is also accomplished through humanization, chimerization, and display techniques using appropriate libraries. It should be appreciated that murine or other species antibodies 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). Antibodies of interest can be constructed using recombinant DNA techniques to replace the CH1, CH2, CH3, hinge and/or framework domains with the appropriate human sequence (see WO 92102190 and US Pat. Nos. 5,530,101; 5,585,089; 5,693,761; Also known in the art is the use of Ig cDNA for the construction of chimeric immunoglobulin genes (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). The mRNA is isolated from a hybridoma or other antibody-producing cell and used to obtain cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (US Pat.

- 43 039374

US No. 4683195 and 4683202). Alternatively, a library is created and screened to isolate a sequence of interest. The DNA sequence encoding the antibody variable region is then fused to human constant region sequences. Human constant region gene sequences can be found in Kabat et al. (1991) Sequences of Proteins of immunological Interest, N.I.H. No. 91-3242. Human C-site genes are readily available from known clones. The choice of isotype will be driven by the desired effector functions, such as complement fixation or antibody-dependent cellular nitotoxicity activity. Preferred isotypes are IgG1, IgG3 and IgG4. Any of the human light chain constant regions, k or λ, can be used. The chimeric humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab') ₂ and Fab, can be obtained by cleavage of the intact protein, for example, proteases or chemical cleavage. Alternatively, a truncated gene is constructed. For example, a chimeric gene encoding part of an F(ab')2 fragment may include DNA sequences encoding a CH1 domain and an H chain hinge, followed by a translational stop codon to produce a truncated molecule.

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

Expression vectors include plasmid, retroviruses, YACs derived from the EBV episome, and the like. A convenient vector is a vector that encodes a functionally complete human immunoglobulin CH or CL sequence with appropriate restriction sites designed such that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing typically occurs between the donor splice site in the inserted J region and the acceptor splice site preceding the human C region, as well as in splice regions that occur within human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions in the direction of reading. The resulting chimeric antibody can be coupled to any strong promoter that includes retroviral LTRs, such as the 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, it should be noted that native Ig promoters and the like can be used.

Additionally, human or other species antibodies can be generated by display-type technologies, including, but not limited to, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art, and the resulting molecules can be subjected to additional 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 Pluckthun 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), Russel et al. Nucl. Acids Research 21:10811085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:808A (1992) and US Pat. No. 5,733,743. If display technologies are used to produce antibodies that are not human, such antibodies can be humanized as described above.

Using these techniques, antibodies can be generated against HER2 expressing cells, soluble forms of HER2, their epitopes and peptides, and their expression libraries (see, for example, US Pat. No. 5,703,057), which can then be screened as described above. to the activities described in this document.

The anti-HER2 antibodies described herein can be expressed by a vector containing a DNA segment encoding a single chain antibody as described above.

These techniques may include the use of vectors, liposomes, naked DNA followed by a DNA adjuvant, gene gun, catheters, and the like. Vectors include chemical conjugates such as those described in WO 93/64701 which have a targeting moiety (e.g. a ligand to a cell surface receptor) and a nucleic acid binding moiety (e.g. polylysine), a viral vector (e.g. a DNA or RNA viral vector), fusion proteins, such as those described in PCT/US 95/02140 (WO 95/22618), which are a fusion protein containing a target fragment (eg, antibody specific for the target cell) and a nucleic acid binding fragment (eg, protamine), plasmid, phage, and the like. The vectors may be chromosomal, non-chromosomal, or synthetic.

Preferred vectors include the viral vector, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. Viral DNA vectors are preferred. These vectors include poxvirus vectors such as vectors

- 44 039374 orthopox and avipox, herpes virus vectors, such as the herpes simplex virus type 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).

Viral pox vectors introduce the gene into the cytoplasm of cells. Viral avipox vectors result in only transient expression of the nucleic acid. Adenovirus vectors, adeno-associated viral vectors, and the herpes simplex virus (HSV) vector are preferred for introducing nucleic acid into nerve cells. The adenovirus vector results in only transient expression (about 2 months), followed by adeno-associated virus (about 4 months), which in turn is less active than HSV vectors. The particular vector chosen will depend on the target cell and the pathological condition requiring treatment. The introduction can be performed by standard techniques, such as infection, transfection, transduction or transformation. Examples of gene transfer methods include the use of, for example, naked DNA, CaPO ₄ precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

The vector can be used to target essentially any desired target cell. For example, stereotaxic injection can be used to introduce vectors (eg, adenovirus, HSV) at the desired site. In addition, particles can be delivered by intracerebroventricular (ICV) infusion using a mini-pump infusion system such as the SynchroMed Infusion System. A mass flow-based method called convection has also proven effective in delivering large molecules to distant regions of the brain and may be useful for delivering a vector to a 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.

These vectors can be used to express large amounts of antibodies which 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 and disrupt HER2-associated signaling.

Methods can be adapted to obtain single-chain antibodies specific for the antigenic protein described in this document (see, for example, US patent No. 4946778). In addition, methods can be adapted to construct F _ab expression libraries (see, for example, Huse et al., 1989 Science 246: 1275-1281) to enable rapid and efficient identification of F _ab monoclonal fragments with the desired protein specificity. or its derivatives, fragments, analogues or homologues. Antibody fragments that contain idiotypes for a protein antigen can be prepared by techniques known in the art, including but not limited to: (i) an F( _ab ) ₂ fragment formed by pepsin cleavage of an antibody molecule; (i) fragment F _ab formed by reduction of disulfide bridges fragment F( _ab ') ₂ ; (iii) an F _ab fragment formed by treating the antibody molecule with papain and a reducing agent, and (iv) Fv fragments.

The invention also includes anti-HER2 fragments Fv, F _ab , F _ab ' and F( _ab ') ₂ or anti-HER2 fragments, single chain anti-HER2 antibodies, multispecific antibodies in which at least one arm binds HER2, and anti-HER2 heteroconjugate antibodies.

Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. Here, one of the binding specificities is for HER2. The second binding target is any other antigen that includes a cell surface protein or a receptor or receptor subunit.

Methods for creating bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain/light chain pairs in which the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random distribution of heavy and light chains of immunoglobulins, these hybridomas (quadromas) form potential mixtures of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually carried out using affinity chromatography steps. Similar procedures are described in WO 93/08829, published May 13, 1993, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably carried out with an immunoglobulin heavy chain constant domain containing at least a portion of the hinge, CH2 and CH3 regions. It is preferable to have a first heavy chain constant domain (CH1) containing the site required for binding the light chain to

- 45 039374 at least one of the mergers. DNA encoding immunoglobulin heavy chain and, if desired, immunoglobulin light chain fusions are inserted into a separate expression vector and co-transfected into a suitable host organism. For additional details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210(1986).

According to another approach, as described in WO 96/27011, the coupling between a pair of antibody molecules can be designed to maximize the percentage of heterodimers that are isolated from the recombinant cell culture. The preferred conjugation contains at least a portion of the CH3 region of the constant domain of the antibody. In this method, one or more small amino acid side chains from the conjugation of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). Compensating cavities of identical or similar size to the large side chain(s) are created at the conjugation of the second antibody molecule by replacing larger amino acid side chains with smaller ones (eg, alanine or threonine). This action provides a mechanism to increase the yield of the heterodimer over other undesirable end products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be made using chemical linkage. Brennan et al., Science 229:81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to form F(ab') ₂ fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize the vicinal dithiols and prevent the formation of intermolecular disulfides. The generated Fab' fragments are then converted into trinitrobenzoate (TNB) derivatives. Then one of the Fab'-TNB derivatives is converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The obtained bispecific antibodies can be used as agents for the selective immobilization of enzymes.

In addition, Fab' fragments can be directly isolated from E. coli and chemically linked to form bispecific antibodies. In Shalaby et al., J. Exp. Med. 175:217-225 (1992) describes the preparation of a fully humanized F(ab') ₂ bispecific antibody molecule. Each Fab' fragment was individually secreted by E. coli and subjected to targeted chemical binding in vitro to form a bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as to trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques have also been described for generating and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies were generated using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jim proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge to form monomers and then re-oxidized to form antibody heteromers. This method can also be used to produce antibody homodimers. The diabody technology described by Hollinger et al., Proc. Natl. Acad. sci. USA 90:6444-6448 (1993) provides an alternative mechanism for generating bispecific antibody fragments. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (V _L ) by a linker that is too short to allow pairing between two domains on the same chain. Accordingly, the V _H and V _L domains from one fragment are forced to pair with the complementary V _H and V _L domains of another fragment, thereby forming two antigen-binding sites. Another strategy has also been reported to generate bispecific antibody fragments by using single chain Fv dimers (sFv). See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valences are meant. For example, trispecific antibodies can be generated. Tutt et al., J. Immunol. 147:60(1991).

Typical bispecific antibodies can bind to two different epitopes, at least one of which is derived from the protein antigen described herein. Alternatively, the anti-antigen arm of the immunoglobulin molecule can be combined with an arm that binds to a trigger molecule on the leukocyte, such as a T cell receptor molecule (e.g. CD2, CD3, CD28 or B7) or Fc receptors for IgG (Fc γ-R) , such as Fc γ-RI (CD64), Fc γ-RII (CD32) and Fc γ-RIII (CD16) to focus cellular defense mechanisms against cell expression of a particular antigen. Bispecific antibodies can also be used to target cytotoxic agents to cells that express a particular antigen. These antibodies have an antigen binding arm that binds a cytotoxic agent or radionuclide chelating agent such as EOTUBE, DPTA, DOTA or TETA. Other items of interest

- 46 039374 bispecific antibody binds the protein antigen described in this document, and additionally binds tissue factor (TF).

Heteroconjugate antibodies are also within the scope of this invention. Antiteletaheteroconjugates consist of two covalently linked antibodies. Such antibodies have, for example, been proposed for targeting cells of the immune system to unwanted cells (see US Pat. No. 4,676,980), and for the treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089). It is contemplated that these antibodies may be produced in vitro using known methods of synthetic protein chemistry, including methods involving the use of cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate and those described in, for example, US Pat. No. 4,676,980.

It may be necessary to modify the antibody described herein in relation to effector function in order to enhance, for example, the effectiveness of the antibody in the treatment of diseases and disorders associated with abnormal expression and/or activity of HER2.

For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing the formation of an interchain disulfide bond at that region. An antibody thus generated may have improved internalization capacity and/or improved complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176: 11911195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, the antibody can be engineered to have a dual Fc region and thereby have improved complement lysis and ADCC properties (see Stevenson et al., Anti-Cancer Drag Design, 3: 219-230 (1989 )).

Anti-HER2 antibody conjugates.

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

Enzymatically active toxins or fragments thereof that can be used include diphtheria toxin A chain, non-binding diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modexin A chain, α-sarcin, Aleurites proteins fordii, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin, or tricothecenes. Various radionuclides are available for making radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Antibody-cytotoxic agent conjugates are made using various bifunctional protein-binding agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester 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), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene- 2,6 diisocyanate) and compounds with bis-active fluorines (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminopentaacetic acid (MXDTPA) is a typical chelating agent for conjugating a radionucleotide to an antibody (see WO 94/11026).

Those of ordinary skill in the art will appreciate that a wide variety of possible fragments can bind to the resulting antibodies described herein (see, e.g., Conjugate Vaccines, Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds) , Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

Coupling can be carried out by any chemical reaction that will bind two molecules, provided that the antibody and other fragment retain their respective activities. This bonding may involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. The preferred bonding, however, is covalent bonding. Covalent bonding can be achieved either by direct condensation of existing side chains or by the introduction of external bridging molecules. Many bivalent or multivalent binders are useful for binding protein molecules, such as the antibodies of this invention, to other molecules. For example, representative binding agents may include organic compounds such as thioethers, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamines. This list does not purport to be exhaustive for the various classes of coupling agents known in the art, but rather is representative of most

- 47 039374 conventional coupling 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).

Preferred linkers are described in the literature (see, for example, Ramakrishnan S. et al., Cancer Res. 44:201-208 (1984) describing the use of MBS (M-maleimidobenzoyl-Nhydroxysuccinimide ester). See also US Pat. No. 5,030,719 describing the use of a halogenated acetylhydrazide derivative linked to an antibody via an oligopeptide linker Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (ii) SMPT (4-sucpinimidyl-a -methyl-a-(2-pyridyldithio)toluene (Pierce Chem. Co., cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co. cat. no. 2165-G) and (v) sulfo-NHS (N -hydroxysulfosuccinimide: Pierce Chem. Co. Cat No. 24510) conjugated with EDC.

The linkers described above contain components that have different attributes, respectively, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS esters are less soluble than sulfo-NHS esters. Additionally, the SMPT linker contains a sterically hindered disulfide bond and can form conjugates with increased stability. Disulfide bonds are generally less stable than other bonds because the disulfide bond is cleaved in vitro resulting in less conjugate availability. In particular, sulfo-NHS can enhance the stability of carbodiimide bonds. Carbodiimide coupling (such as EDC), when used with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodiimide coupling reaction itself.

The antibodies described herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those 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 US Pat. US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are described in US Pat. No. 5,013,556.

In particular, useful liposomes can be obtained by counter-phase evaporation from a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are forced through filters with a certain pore size to obtain liposomes with the desired diameter. Fab' fragments of an antibody of this invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide exchange reaction.

In one aspect of the invention, the conjugate described herein comprises an isolated anti-HER2 antibody, or an antigen-binding fragment thereof, directly or indirectly linked to one or more D-bearing polymer backbones independently containing poly(1-hydroxymethylethylene hydroxymethyl-formal) (PHF), having a molecular weight ranging from about 2 kDa to about 40 kDa, wherein each of the one or more D-supporting polymer backbones independently has the formula (Ic)

(Ic), where each presence of D, independently represents a therapeutic or diagnostic agent;

L ^D1 is a carbonyl-containing moiety;

---CPO)-L ^D1 -|_ ---CPO)-L ^d1 ->- _D each presence in is independently the first linker that contains such a biodegradable bond that when the bond is broken D is released in active form for manifestations of its therapeutic action; and presence in

- 48 039374 - C(=O)-L°4- _d ⁴ between L to L ^d1 ;

and D stands for direct or indirect attachment of D

---C(=O)-L ^d1 —£-L ^p2 each presence of ^ζ is independently a second linker not yet connected to an isolated anti-HER2 antibody or antigen-binding fragment thereof, in which LP ² is a fragment containing a functional group, which will still form a covalent bond with the functional group of the isolated antibody or its antigen-binding fragment, and between L ^d1 and LP ² denotes a direct or indirect attachment

LP ² to L ^D1 and each presence of the second linker is different from each presence of the first linker;

---C(=O)-L ^D1 —^-L ^p2 —^— each presence is independently a third linker that connects each D-bearing polymer scaffold to the isolated antibody or its antigen-binding fragment, at the terminal attached to LP ² , denotes the direct or indirect attachment of LP ² to the selected antibody or antigennegative fragment when forming a covalent bond between the functional group of LP ² and the functional group of the selected antibody or antigennegative fragment; and each presence of the third linker is different from each presence of the first linker;

m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m ₂ is an integer from 1 to about 40, m ₃ is an integer from 0 to about 18, m ₄ is an integer from 1 to about 10, the sum of m, m1, m ₂ , m ₃ and m ₄ ranges from about 15 to about 300, and the total number of LP ² attached to the isolated antibody or antigen-binding fragment thereof is 10 or less.

The conjugate described in the document may include one or more of the following features.

For example, in formula (Ic), m1 is an integer from 1 to about 120 (eg, about 190) and/or m ₃ is an integer from 1 to about 10 (eg, about 1-8).

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, m ₂ , m ₃ and m ₄ in the range from about 45 to about 150 ), m2 is an integer from 2 to about 20, m ₃ is an integer from 0 to about 9, m ₄ is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 75 ( for example, m1 is about 4-45).

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, m ₂ , m ₃ and m ₄ in the range from about 60 to about 110 ), m2 is an integer from 2 to about 15, m ₃ 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 is about 4-30).

For example, when the PHF in formula (Ic) has a molecular weight in the range of about 2 kDa to about 20 kDa (i.e., the sum of m, m1, m ₂ , m ₃ and m4 in the range of about 15 to about 150) , m2 is an integer from 1 to about 20, m ₃ is an integer from 0 to about 10 (for example, m ₃ in the range 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 LP ² bound to the isolated antibody or antigen-binding fragment thereof ranges from about 2 to about 8 (eg, about 2, 3, 4, 5, 6, 7, or eight).

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, mi, m ₂ , m ₃ and m ₄ in the range from about 20 to about 110 ), m2 is an integer from 2 to about 15, m ₃ is an integer from 0 to about 8 (for example, m ₃ in the range 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 L ^P2 bound to the isolated antibody or antigen-binding fragment thereof is in the range from about 2 to about 8 (e.g., about 2, 3, 4, 5, 6, 7 or 8).

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, m ₂ , m ₃ and m ₄ in the range from about 40 to about 75 ), m2 is an integer from 2 to about 10 (for example, m2 is about 3-10), m ₃ is an integer from 0 to about 5 (for example, m3 in the range from 0 to about 4), m ₄ represents is an integer from 1 to about 8 (for example, m ₄ in the range from 1 to about 5) and/or m1 is an integer from about 2 to about 35 (for example, m1 is about 5-35), and the total number of LP ² connected to

- 49 039374 isolated antibody or antigen-binding fragment, is in the range from about 2 to about 8 (for example, about 2, 3, 4, 5, 6, 7 or 8).

For example, when PHF has a molecular weight in the range of 2 kDa to 40 kDa, (e.g., about 6-20 kDa, or about 8-15 kDa, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), the number of drug molecules per PHF (eg, m ₂ ) is an integer from 1 to about 40 (eg, about 1-20, or about 2-15, or about 3-10, or about 2-10). 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 more (e.g., 60 kDa or more; 80 kDa or more; 100 kDa or more; 120 kDa or more; 140 kDa or more ; 160 kDa or more; 180 kDa or more, or 200 kDa or more, or about 40-200, 40-180, 40-140, 60-200, 60-180, 60-140, 80-200, 80-180 , 80140, 100-200, 100-180, 100-140 or 140-150 kDa). In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to 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.

For example, when the PHF has a molecular weight in the range of 2 to 40 kDa, (e.g., about 6-20 kDa, or about 8-15 kDa, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), the number of drug molecules per PHF (eg, m ₂ ) is an integer from 1 to about 40 (eg, about 1-20, or about 2-15, or about 3-10, or about 2-10). This framework can be used, for example, to conjugate an isolated antibody or antigen-binding fragment having a molecular weight of 140 to 180 kDa or 140 to 150 kDa. In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to 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.

An isolated antibody or antigen-binding fragment thereof in this molecular weight range includes, but is not limited to, for example, full length antibodies such as IgG, IgM.

For example, when the PHF has a molecular weight in the range of 2 to 40 kDa, then the number of drug molecules per PHF (eg, m ₂ ) is an integer from 1 to about 40 (eg, about 1-20, or about 2-15 , or about 3-10 or about 2-10). This framework can be used, for example, to conjugate an isolated antibody or antigen-binding fragment having a molecular weight of 60 to 120 kDa. In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to 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.

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

In one embodiment, D is a therapeutic agent. In some embodiments, the therapeutic agent is a small molecule having a molecular weight of < about 5 kDa, < about 4 kDa, < about 3 kDa, < about 1.5 kDa, or < about 1 kDa.

In some embodiments, the therapeutic agent has an IC _{50 of} less than about 1 nM.

In another embodiment, the therapeutic agent has an IC _{50 of} greater than about 1 nM, eg, the therapeutic agent has an IC ₅₀ of about 1 to 50 nM.

Some therapeutic agents having an IC50 greater than about 1 nM (eg, less active drugs) are not suitable for conjugation to an antibody using conjugation techniques recognized in the art. Without wishing to be bound by any theory, such therapeutic agents have an activity that is not sufficient for use in targeted antibody-drug conjugates using conventional techniques, since enough copies of the drug substance (i.e., more than 8) cannot be conjugated with using techniques recognized in the art without degrading the pharmacokinetic and physicochemical properties of the conjugate. However, significantly higher loadings of such less active drugs can be achieved using the conjugation strategies described herein, thereby resulting in high loadings of the therapeutic agent while maintaining the desired pharmacokinetic and physicochemical properties. Thus,

- 50 039374 this invention also relates to an antibody-polymer-drug conjugate, which includes an isolated antibody or its antigen-binding fragment, PHF and at least eight fragments of therapeutic agents, where D represents auristatin, dolastatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F, AF HPA, phenylenediamine (AFP).

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

Other examples of D include those described in, for example, U.S. Application Publication No. 20130101546 and U.S. Patent No. 8,815,226 and co-pending U.S. Applications Serial No. 14/512,316 filed October 10, 2014, 61/988011, filed May 2, 2014 and 62/010972 filed June 11, 2014; a description of each of which is included in this document in its entirety.

In some embodiments, the number of D-bearing polymer backbones that can be conjugated to an antibody is limited by the number of free cysteine residues. In some embodiments of the invention, free cysteine residues are introduced into the amino acid sequence of an antibody by the methods described herein. Exemplary conjugates described herein may 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 free cysteine residues are already present in the antibody without design, in which case existing free cysteine residues can be used to conjugate the antibody to a D-bearing polymer backbone. In some embodiments, the antibody is subjected to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues.

In some embodiments of the invention in the conjugate described herein, the D-supporting polymer backbone of formula (Ic) is formula (Ie)

where PHF has a molecular weight in the range from about 2 kDa to about 40 kDa;

each presence of D independently represents a therapeutic agent having a molecular weight < 5 kDa, and between D and the carbonyl group denotes the direct or indirect addition of D to the carbonyl group,

X represents CH ₂ , 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, m ₇ is an integer from 1 to about 40, and the sum of m1 and m ₇ is from about 2 to about 180, m is an integer from 1 to about 300, m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8 and the sum of m _3a and m _3b is between 1 and 18, and the sum of m, m _b m ₇ , m _3a and m _3b is in the range from about 15 to about 300.

In some embodiments of the invention in the conjugate described herein, the D-supporting polymer backbone of formula (Ie) is formula (Id)

- 51 039374

(I) wherein one of Xa and _Xb is H and the other is a water-soluble maleimido blocking moiety, or Xa _{and Xb} , together with the carbons to which they are attached, form a carbon-carbon double bond;

m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, and the sum of m _3a and m _3b is between 1 and 18, and the sum of m, m _b m2, m _3a , and m _3b is ranging from about 15 to about 300.

For example, the ratio between m2 and m _3b is greater than 1:1 and less than or equal to 10:1.

For example, the ratio between m2 and m _3b is about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

For example, the ratio between m2 and m _3b is between 2:1 and 8:1.

For example, the ratio between m2 and m _3b is about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

For example, the ratio between m2 and m _3b is between 2:1 and 4:1.

For example, the ratio between m2 and m _3b is about 4:1, 3:1 or 2:1.

For example, the ratio between m2 and m _3b is about 3:1 and 5:1.

For example, the ratio between m2 and m _3b is about 3:1, 4:1 or 5:1.

For example, when the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 20 kDa, then the sum of m, m _b m ₂ , m _3a and m _3b is in the range 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 between PHF and isolated antibody against HER2 or an antigen-binding fragment thereof is an integer from 2 to about 8.

For example, when the PHF in formula (Id) has a molecular weight ranging from about 3 kDa to about 15 kDa, then the sum of m, m _b m ₂ , m _3a and m _3b is in the range from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m _3a is an integer from 0 to about 7, m _3b is an integer from 1 to about 8, and the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (eg, an integer from 2 to about 6 or an integer from 2 to about 4).

For example, when the PHF in formula (Id) has a molecular weight ranging from about 5 kDa to about 10 kDa, then the sum of m, m _b m ₂ , m _3a and m _3b is in the range 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, m _3a is an integer from 0 to about 4, m _3b is an integer from 1 to about 5, and the ratio between The PHF and the isolated anti-HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8 (eg, an integer from 2 to about 6, or an integer from 2 to about 4).

For example, water-soluble maleimide blocking moieties are moieties that can be covalently attached to one of the two carbon atoms of an olefin upon reaction of a maleimide group with a thiol-containing compound of formula (II)

R ₉ o-(CH ₂ ) _d -SH (P), where R ₉₀ represents NHR ₉₁ OH, COOR ₉₃ , CH(NHR ₉₁ )COOR ₉₃ or a substituted phenyl group;

R ₉₃ represents a hydrogen atom or C _1-4 alkyl;

R91 represents a hydrogen atom, CH3 or CH3CO; a

- 52 039374 d is an integer from 1 to 3.

For example, the water soluble maleimido blocking compound of formula (II) may be cysteine, N-acetylcysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropionic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e. HOCH ₂ SH), benzylthiol in which the phenyl is substituted with one or more hydrophilic substituents, or 3aminopropane-1-thiol. One or more hydrophilic substituents on phenyl include OH, SH, methoxy, ethoxy, COOH, CHO, COC _1-4 alkyl, NH2, F, cyano, SO3H, PO3H and the like.

For example, a water-soluble maleimido blocking group is -S-(CH ₂ ) _d -R ₉₀ where R ₉₀ is OH, COOH, or CH(NHR ₉₁ )COOR ₉₃ ; R ₉₃ represents a hydrogen atom or CH ₃ ; R ₉₁ represents a hydrogen atom or CH3CO; and d is 1 or 2.

For example, a water-soluble maleimido blocking group is -S-CH2CH(NH2)COOH.

For example, when the PHF has a molecular weight in the range of 2 to 40 kDa, (e.g., about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), then the number of drug molecules per PHF (e.g., m2) is an integer from 1 to about 40 (eg, about 1-20, or about 2-15, or about 3-10, or about 2-10). This framework can be used, for example, to conjugate an isolated antibody or antigen-binding fragment thereof having a molecular weight of 40 kDa or more (e.g., 60 kDa or more; 80 kDa or more; or 100 kDa or more; 120 kDa or more; 140 kDa or more; 160 kDa or more, 180 kDa or more, or 200 kDa or more, or about 40-200, 40-180, 40-140, 60-200, 60-180, 60-140, 80-200, 180, 80-140, 100-200, 100-180, 100-140 or 140150 kDa). In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to 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.

For example, when the PHF has a molecular weight in the range of 2 to 40 kDa, (e.g., about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), then the number of drug molecules per PHF (e.g., m ₂ ) is an integer from 1 to about 40 (eg, about 1-20, or about 2-15, or about 3-10, or about 2-10). This framework can be used, for example, to conjugate an isolated antibody or antigen-binding fragment having a molecular weight of 140 to 180 kDa or 140 to 150 kDa. In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to 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.

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.

For example, when the PHF has a molecular weight in the range of 2 to 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), then the number of drug molecules per PHF (eg, m2) is is an integer from 1 to about 40 (eg, about 1-20, or about 2-15, or about 3-10 or 2-10). This scaffold can be used, for example, to conjugate an isolated antibody or antigen-binding fragment having a molecular weight of 60 to 120 kDa. In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to 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.

Isolated antibodies or antigen-binding fragments in this molecular weight range include, but are not limited to, eg antibody fragments such as, for example, Fab2, scFcFv, and camelid antibody fragments.

For example, when the PHF has a molecular weight in the range of 2 to 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), then the number of drug molecules per PHF (eg, m2) is is an integer from 1 to about 40 (eg, about 1-20, or about 2-15, or about 3-10 or 2-10). This scaffold can be used, for example, to conjugate an isolated antibody or antigen-binding fragment thereof having a molecular weight of 40 to 80 kDa. In this embodiment, the ratio of the isolated antibody or antigen-binding fragment thereof to 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,

- 53 039374 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.

Isolated antibodies or antigen-binding fragments in this molecular weight range, ie. from about 40 to about 80 kDa, include, but are not limited to, for example, fragments of antibodies, such as, for example, Fab.

In some embodiments, in the conjugate described herein, the D-supporting polymer backbone of formula (Ie) is formula (If), wherein the polymer is PHF, which has a molecular weight in the range of about 2 kDa to about 40 kDa

(if) where m is an integer from 1 to about 300, mi is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8;

the sum of m _3a and m _3b ranges from 1 to about 18, the sum of m, m1, m ₂ , m _3a and m _3b ranges from about 15 to about 300, terminal denotes the attachment of one or more polymer backbones to the isolated antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor and contains a variable heavy chain complementarity determining region 1 (CDRH1) containing the amino acid sequence FTFSSYSMN (SEQ ID NO: 25); heavy chain variable complementarity determining region 2 (CDRH2) containing the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); heavy chain variable complementarity determining region 3 (CDRH3) containing the amino acid sequence GGHGYFDL (SEQ ID NO: 27); light chain variable complementarity determining region 1 (CDRL1) containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO:28); light chain variable complementarity determining region 2 (CDRL2) containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and light chain variable complementarity determining region 3 (CDRL3) containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29), and the ratio between PHF and antibody is 10 or less.

The framework of the formula (If) may include one or more of the following features.

When the PHF in formula (If) has a molecular weight ranging from about 2 kDa to about 20 kDa, then the sum of m, m1, m ₂ , m _3a and m _3b is in the range from about 15 to about 150, m1 is an integer from 1 to about 70, m ₂ is an integer from 1 to about 20, m _3a is an integer from 0 to about 9, m _3b is an integer from 1 to about 8, the sum of m3a and m3b is in range from 1 to about 10, and the ratio between PHF and antibody is an integer from 2 to about 8.

When the PHF in formula (If) has a molecular weight ranging from about 3 kDa to about 15 kDa, then the sum of m, m1, m ₂ , m _3a and m _3b is in the range 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, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is in range from 1 to about 8, and the ratio between PHF and antibody is an integer from 2 to about 8 (eg, about 2 to about 6 or about 2 to about 4).

When the PHF in formula (If) has a molecular weight ranging from about 5 kDa to about 10 kDa, then the sum of m, m1, m ₂ , m _3a and m _3b is in the range from about 40 to about 75, m1 is 54 039374 is an integer from about 2 to about 35, m ₂ is an integer from about 2 to about

10, m _3a is an integer from 0 to about 4, m _3b is an integer from 1 to about

5, the sum of m _3a and m _3b ranges from 1 to about 5, and the ratio between PHF and antibody is an integer from 2 to about 8 (e.g., about 2 to about 6 or about 2 to about 4).

In some embodiments, the ratio between auristatin F hydroxypropylamide (AF HPA) and antibody may 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.

In some embodiments, the ratio between HPA AF and 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.

In other embodiments, the ratio between AF HPA and 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.

In some embodiments, the ratio between AF HPA and antibody may be about 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

In some embodiments, the ratio between AF HPA and antibody may be about 15:1, 14:1, 13:1, 12:1, or 11:1.

In some embodiments, the ratio between AF HPA and antibody may be about 15:1, 14:1, 13:1, or 12:1.

In some embodiments, the ratio between PHF and antibody may be about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1 :one.

In some embodiments, the ratio between PHF and antibody may be about 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and antibody may be about 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

In other embodiments, the ratio between PHF and antibody may be about 6:1.5:1.4:1.3:1 or 2:1.

In other embodiments, the ratio between PHF and antibody may be about 6:1, 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be about 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be about 4:1, 3:1, or 2:1.

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

Other embodiments of antibody-polymer drug conjugates are those described in, for example, U.S. Patent No. 8,815,226 and U.S. Applications Serial Nos. G.; a description of each of which is included in this document in its entirety.

The present invention also relates to a drug derivative modified so that it can be directly conjugated to an antibody or antigen-binding fragment thereof in the absence of a polymeric carrier and form drug-antibody conjugates thereof.

In some embodiments, antibody-drug conjugates include an antibody, or antigen-binding fragment thereof, conjugated, i. covalently attached to the drug moiety. In some embodiments, the antibody, or antigen-binding fragment thereof, is covalently attached to the drug moiety via a linker, such as a non-polymeric linker.

The drug moiety (D) of antibody drug conjugates (ADCs) may include any compound, moiety or group that has a cytotoxic or cytostatic effect as defined herein. In some embodiments, an antibody drug conjugate (ADC) comprises an antibody (Ab) that targets a tumor cell, a drug moiety (D), and a linker moiety (1) that attaches Ab to D. In some embodiments, the antibody is attached to the linker fragment (1) through one or more amino acid residues such as lysine and/or cysteine.

In some embodiments of the invention, the ADC has the formula (Ig)

Ab-(LD) _p (Ig), where p is an integer from 1 to about 20.

In some embodiments, the number of drug fragments that can be conjugated to an antibody is limited by the number of free cysteine wasps.

- 55 039374 tatkov. In some embodiments of the invention, free cysteine residues are introduced into the amino acid sequence of an antibody by the methods described herein. Exemplary Ig ADCs 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 free cysteine residues are already present in the antibody without design, in which case existing free cysteine residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is subjected to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues.

In some embodiments, the linker (1) 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 Ig antibody drug conjugate (ADC). In some embodiments, antibody-drug conjugates (ADCs) can be prepared using a linker having reactive functional groups for covalent attachment to the drug and to the antibody. For example, in some embodiments, the cysteine thiol of an antibody (Ab) can form a bond with a linker reactive functional group or drug-linker intermediate to create an ADC.

In one aspect of the invention, the linker has a functional group that is capable of reacting with free cysteine present on the antibody to form a covalent bond. Non-limiting examples of such reactive functional groups include maleimide, haloacetamides, α-haloacetyl, activated esters such as 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 examples herein.

In some embodiments, the linker has a functional group that is capable of reacting with an electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the linker's reactive functional group may react with an electrophilic group on an antibody to form a covalent bond with the antibody unit. Non-limiting examples of such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

The linker may contain one or more linker components. Typical linker components include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzoyloxycarbonyl (PAB), N-succinimidyl 4-(2pyridylthio ) pentanoate (SPP) and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (MCC). Various linker components are known in the art, some of which are described below.

The linker may be a cleavable linker, facilitating drug release. Non-limiting exemplary cleavable linkers include acid-labile linkers (eg, containing hydrazone), protease-sensitive (eg, peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US Pat. No. 5208020).

In some embodiments of the invention, the linker has the following formula (IIg) -A _a -W _w -Y _y (IIg), where A is a stretching 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 the integer 0, 1, or 2. An ADC containing a linker of formula (IIg) has the formula I(A): Ab-(Aa-Ww-Yy-D)p where Ab, D and p are defined as above for formula (Ig). Exemplary implementations of such linkers are described in US Pat. US No. 7498298, which is incorporated herein in its entirety by reference.

In some embodiments, the linker component contains a stretching unit (A) that binds the antibody to another linker component or drug moiety. Non-limiting examples of stretch units are shown below (where wavy lines indicate covalent attachment sites to antibody, drug, or additional linker components)

- 56 039374 tPEG or

MC MP

In some embodiments of the invention, the linker component contains an amino acid unit (W). In some such embodiments, the amino acid unit allows the protease to cleave the linker, thereby facilitating drug release from the immunoconjugate when exposed 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 homolysin (phe-homolys) and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (glyval-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit may contain naturally occurring amino acid residues and/or minor amino acids and/or non-naturally occurring amino acid analogs such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, such as tumor-associated protease, cathepsin B, C and D, or plasmin protease.

Typically, peptide-type linkers can be made by forming a peptide bond between two or more amino acid and/or peptide moieties. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (eg, E. Schrider and K. Lubke (1965) The Peptides, volume 1, pp 76-136, Academic Press).

In some embodiments, the linker component comprises a spacer unit that binds the antibody to the drug moiety, either directly or through a stretch unit and/or amino acid unit. The spacer unit may be self-fissile or non-self-fissile. A non-self-cleaving spacer unit is one in which some or all of the spacer units remain bound to the drug moiety upon cleavage of the ADC. Examples of non-self-cleaving spacer units include, but are not limited to, glycine spacer unit and glycine-glycine spacer unit. In some embodiments of the invention, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease results in the release of the glycine-glycine-drug moiety from the rest of the ADC. In some such embodiments, the glycine-glycine-drug moiety undergoes a hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

The self-cleaving spacer unit releases the drug moiety. In some embodiments, the linker spacer unit contains a paminobenzyl unit. In some such embodiments, the p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond and a carbamate, methyl carbamate, or carbonate is created between the benzine alcohol and the drug substance (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103). In some embodiments of the invention, the spacer unit contains p-aminobenzyloxycarbonyl (PAB). In some embodiments of the invention, an ADC containing a self-cleaving linker has the structure

- 57 039374

where Q is -C1-C8 alkyl, -O-(C1- _C8 alkyl), halogen, nitro or cyano;

n ₆ is an integer from 0 to 4;

X _a may be one or more additional spacer units or may be absent; and p is an integer from 1 to about 20.

In some embodiments, p is an integer from 1 to 10, 1 to 7, 1 to 5, _or 1 to 4. Non-limiting exemplary Xa spacer units include

where R ₁₀₁ and R ₁₀₂ are independently selected from H and C ₁ -C ₆ alkyl. In some embodiments, R _W1 and R ₁₀₂ are each -CH3.

Other examples of self-cleaving spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (US Pat. No. 7375078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), and ortho- or para-aminobenzylacetals. In some embodiments, a spacer may be used such that it undergoes cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology 2:223), respectively, substituted with 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). The attachment of a drug to the α-carbon of a glycine moiety is another example of a self-cleaving spacer that can be used in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker for covalently attaching 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 11:17611768). Dendritic linkers can increase the molar ratio of drug to antibody, ie. load that correlates with ADC activity. Thus, if an antibody carries only one reactive cysteine thiol group, then multiple drug moieties can be attached via the dendritic linker.

Non-limiting exemplary linkers are shown below for ADC formula (Ig)

- 58 039374

Phe-Lys-PAB-Ab;

where R ₁₀₁ and R102 are independently selected from H and C1-C6 alkyl;

n ₅ is an integer from 0 to 12.

In some embodiments, n is an integer from 2 to 10. In some embodiments, n is an integer from 4 to 8.

In some embodiments, R ₁₀₁ and R ₁₀₂ are each -CH3.

Additional non-limiting examples of ADCs include structures

- 59 039374

where Ha represents

—(CH ₂ ) _n7 -CN-(CH ₂ ) _n7 ^R 103 or Y is

each R103 is independently H or C1-C6 alkyl; and n7 is an integer from 1 to 12.

In some embodiments of the invention, the linker is replaced with groups that modulate solubility and/or reactivity. As a non-limiting example, a charged substituent, such as a sulfonate (-SO3 ^- ) or ammonium, can increase the water solubility of the linker reagent and facilitate the binding reaction of the linker reagent to the antibody and/or drug moiety, or facilitate the binding reaction of Ab-L (antibody intermediate -linker) with D, or DL (drug-linker intermediate) with Ab, depending on the synthetic route used to prepare the ADC. In some embodiments, the linker portion binds to the antibody and the linker portion binds to the drug, and then Ab-(linker portion) ^a binds to drug-(linker portion) ^b to form an Ig ADC.

The compounds described herein are expressly understood to include, but are not limited to, ADCs prepared using the following linker reagents: bis-maleimidotrioxyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyloxy)succinimide ester (EMCS), N-[γ-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), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate ( SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-3-(2-pyridylthio) propionate (SPDP), N-succinimidyl-4

- 60 039374 (2-pyridylthio)pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-[(v- 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-dihydrooxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM (PEG) ₂ (shown below) and WM(PEG) ₃ (shown below); difunctional imidoester derivatives (such as dimethyladipimidate 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 fluoro compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, bis-maleimide reagents provide for the attachment of a cysteine thiol group in an antibody with 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.

VM(PEG) ₃

Some useful linker reagents can be obtained from various commercial sources such as Pierce Biotechnology, Inc. (Rockford, Illinois), Molecular Biosciences Inc. (Boulder, Colorado) 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. 7:180186; Pat. US No. 6214345; WO 02/088172; US2003130189; US2003096743; WO 03/026577; WO 03/043583 and WO 04/032828.

Carbon-14 labeled 1-isothiocyanatebenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugation of a radionucleotide to an antibody. See, for example, WO 94/11026.

Methods for creating anti-HER2 antibody conjugates.

In some embodiments of the invention, the conjugates are obtained in several stages. These steps include: (1) modifying the polymer so that it contains a functional group that reacts with the functional group of the drug or its derivative; (2) reacting the modified polymer with the drug or derivative thereof so that the drug is bound to the polymer; (3) modifying the polymer-drug conjugate so that the polymer contains a functional group that can react with the functional group of the isolated antibody or its antigen-binding fragment, or its derivative, and (4) reacting the modified polymer-drug conjugate with the antibody or its antigennegative fragment with the formation of the conjugate described in this document. Step (3) can be omitted if the modified polymer obtained in step (1) contains a functional group that can react with the functional group of the antibody or antigen-binding fragment thereof.

In another embodiment of the invention, the conjugates are obtained in several stages: (1) modifying the polymer so that it contains a functional group that reacts with the functional group of the drug or its derivative; (2) carrying out the reaction of the modified polymer with the first drug substance or its derivative so that the drug substance

- 61 039374 associated with the polymer; (3) modifying the polymer-drug conjugate so that it contains a different functional group that can react with the functional group of the second drug or its derivative; (4) reacting the modified polymer-drug conjugate with a second drug or a derivative thereof such 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 the functional group of the antibody or its antigen-binding fragment, and (6) reacting the modified polymer-drug conjugate from step ( 5) with an isolated antibody or antigen-binding fragment 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 or derivatives thereof are to be conjugated to form a polymer-drug conjugate containing two different drugs and two different antibodies or antigen-binding fragments thereof.

In another embodiment of the invention, the conjugates are obtained in several stages. These steps include: (1) modifying the polymer so that it contains a functional group that reacts with the functional group of the drug or its derivative; (2) further modification of the polymer so that it also contains a functional group that can react with the functional group of the antibody or its antigen-binding fragment; (3) reacting the modified polymer with the drug or derivative thereof so that the drug binds 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 the sequence of steps (3) and (4) may be reversed. Additionally, any step (1) or (2) can be skipped if the modified polymer contains a functional group that can react with both the functional group of the drug or its derivatives and the functional group of the antibody or its antigen-binding fragment.

In another embodiment of the invention, the conjugates are obtained in several stages: (1) modifying the polymer so that it contains a functional group that reacts with the functional group of the drug or its derivative; (2) additional modification of the polymer so that it contains a functional group that can react with the functional group of the antibody or its antigen-binding fragment; (3) reacting the modified polymer with the first drug or derivative thereof so that the drug is bound to the polymer; (4) modifying the polymer-drug conjugate so that it contains a different functional group that can react with the functional group of the second drug or its derivative; (5) reacting the modified polymer-drug conjugate with a second drug or derivative thereof such that the second drug is bound to the polymer-drug conjugate; (6) reacting a modified polymer-drug conjugate containing 2 different drugs such that the polymer with the isolated antibody or antigen-binding fragment 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 containing two different drugs and two different antibodies or antigen-binding fragments thereof. Step (4) can be performed after step (1) so that the modified polymer contains two different functional groups that can react with two different drugs or their derivatives. In this embodiment, a modified polymer containing two different functional groups that can react with two different drugs or derivatives thereof can be further modified to contain a functional group that can react with a functional group of an antibody or antigen-binding fragment thereof; before reacting the modified polymer with either of the two drugs (step (3) and step (5) or the antibody or antigen-binding fragment thereof (step (6)).

In some exemplary embodiments of the invention, the conjugates described herein find use in biomedical applications such as drug delivery and tissue engineering, and the polymer carrier is biocompatible and biodegradable. In some embodiments, the carrier is a soluble polymer, nanoparticle, gel, liposome, micelle, thread, implant, etc. In some embodiments, the term soluble polymer encompasses a biodegradable, biocompatible polymer, such as a polyal (eg, a hydrophilic polyacetal or polyketal). In some other embodiments of the invention, the carrier is a fully synthetic, semi-synthetic, or naturally occurring polymer. In some other embodiments of the invention, the carrier is

- 62 039374 hydrophilic. Examples of a suitable polymeric carrier for preparing the conjugates described herein are described in US Pat. No. 8,815,226, the contents of which are hereby incorporated by reference in their entirety.

In one embodiment of the invention, the polymer carrier contains units of formula (IV)

OH X'

- J p' (IV), where X' indicates a substituent for the hydroxyl group of the polymer skeleton. As shown in formula (IV) and other formulas described herein, each polyacetal unit has a single hydroxyl group attached to the glycerol moiety of the unit, and the X' group is attached to the glycoaldehyde moiety of the unit. This step is for convenience only and should be construed as meaning that a polymer having formula (IV) and other formulas described herein may contain a random distribution of units having an X' group (or other substituent such as a linker containing a maleimide end) attached to the glycoaldehyde moiety of units, and units having a single X' group (or other substituent, such as a linker containing a maleimide end) attached to the glycerol moiety of units, as well as units having two X' groups (or other substituents , such as a linker containing a maleimide end), with one attached glycoaldehyde moiety and another attached glycerol moiety of units.

In one embodiment, the biodegradable, biocompatible polyales suitable for the practice of the present invention have a molecular weight between about 0.5 and about 300 kDa. For example, biodegradable biocompatible polyals have a molecular weight 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, a biodegradable, biocompatible polyal used for the polymer scaffold or conjugate described herein is PHF having a molecular weight between about 2 and about 40 kDa (e.g., about 2-20 kDa, 3-15 kDa, or 5-10 kDa .)

Methods for preparing polymeric carriers (eg, biocompatible, biodegradable polymeric carriers) suitable for conjugation with modifiers are known in the art. For example, synthesis guidance can be found in US Pat. Nos. 5,811,510; 5863990; 5958398; 7838619; 7,790,150 and 8,685,383. The skilled practitioner should know how to adapt these methods to obtain polymeric carriers for use in the practice of this invention.

In one embodiment of the invention, a method for creating a biodegradable biocompatible polyal conjugate of this invention comprises a process by which a suitable polysaccharide is combined with an effective amount of a glycol-specific oxidizing agent to form an aldehyde intermediate. The aldehyde intermediate, which is itself a polyalm, can then be reduced to the corresponding polyol, succinylated and associated with one or more suitable modifiers to form a biodegradable biocompatible polyal conjugate containing succinamide-containing linkages.

In another preferred embodiment of the invention, fully synthetic biodegradable biocompatible polyales for use in the present invention can be obtained by reacting a suitable initiator with a suitable precursor compound.

For example, fully synthetic polyalis can be prepared by the condensation of vinyl esters with protected substituted diols. Other methods may be used, such as ring-opening polymerization, where the effectiveness of the method may depend on the degree of substitution and the bulkiness of the protecting groups.

One of ordinary skill in the art will recognize that solvent systems, catalysts, and other factors can be optimized to produce high molecular weight products.

In some embodiments of the invention, the carrier is PHF.

In embodiments of the invention, the polymeric carrier is a PHF having a polydispersity index (PDI) of <1.5; for example, <1.4; <1.3; <1.2 or <1.1.

For example, to conjugate an isolated antibody or antigen-binding fragment thereof having a molecular weight of 40 to 200 kDa, the backbone polymeric carrier is a polyacetal, such as PHF, having a molecular weight (e.g., unmodified PHF MW) in the range of about 2 kDa to about 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 510 kDa).

For example, for conjugation of an antibody or antigen-binding fragment thereof having a molecular weight of 40 to 80 kDa, the backbone polymeric carrier described herein is a polyacetal, e.g., PHF, having a molecular weight (e.g., unmodified PHF MW) in the range of about 2 kDa to about 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa). For example, PHF has a molecular weight of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 kDa.

An 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, Fab.

For example, for conjugation of an antibody or antigen-binding fragment thereof having a molecular weight of 60 to 120 kDa, the backbone polymeric carrier described herein is a polyacetal, e.g., PHF, having a molecular weight (e.g., unmodified PHF MW) in the range of about 2 kDa to about 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa). For example, PHF has a molecular weight of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 kDa.

An 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, camelid antibody fragments, Fab2, scFvFc, and the like.

For example, for conjugation of an antibody or antigen-binding fragment thereof having a molecular weight of 140 to 180 kDa or 140 to 150 kDa, the backbone polymer carrier described herein is a polyacetal, such as PHF, having a molecular weight (e.g., MW of unmodified PHF) in the range of about 2 kDa to about 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa). For example, PHF has a molecular weight of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 kDa.

An antibody or antigen-binding fragment thereof in this range of molecular weights includes, but is not limited to, for example, full length antibodies such as IgG, IgM.

Biodegradable biocompatible conjugates described in this document can be obtained in compliance with the required requirements for biodegradability and hydrophilicity. For example, under physiological conditions, a balance between biodegradability and stability can be achieved. For example, molecules with molecular weights above a certain threshold (typically greater than 40-100 kDa, depending on the physical form of the molecule) are known not to be excreted by the kidneys as small molecules and can only be excreted from the body through cellular uptake and degradation in intracellular compartments. primarily lysosomes. This observation provides an example of how functionally stable still biodegradable materials can be engineered by modulating their stability under general physiological conditions (pH 7.5±0.5) and lysosomal pH (pH about 5). For example, the hydrolysis of acetal and ketal groups is known to be catalyzed by acids, so polyalls will generally be less stable in an acidic lysosomal environment than, for example, in blood plasma. One can develop a test to compare the degradation profile of a polymer, for example, at pH 5 and pH 7.5 at 37°C in an aqueous environment and thus to determine the expected balance of polymer stability in the normal physiological environment and in the degrading lysosomal compartment after cellular uptake. Polymer integrity can be measured in tests such as size exclusion HPLC. One of ordinary skill in the art may choose another suitable method to study the various fragments of the disrupted conjugates described herein.

In many cases it will be preferred that at pH~7.5 the effective polymer size does not change at a detectable level within 1 to 7 days and remains within 50% of the original polymer size for at least several weeks. On the other hand, at pH 5, the polymer should preferably degrade detectably within 1 to 5 days and be completely converted to low molecular weight fragments within a time frame of two weeks to several months. Although faster degradation may be preferred in some cases, in general it may be more desirable for the polymer to be degraded in cells at a rate that does not exceed the rate of metabolic conversion or excretion of polymer fragments by the cells. Accordingly, in some embodiments of the invention, it is expected that the conjugate of this invention is biodegradable, in particular when taken up by cells, and relatively inert with respect to biological systems. The carrier degradation products are preferably uncharged and do not significantly shift the pH of the environment. It is assumed that the saturation of alcohol

- 64 039374 groups can provide a low rate of polymer recognition by cell receptors, in particular phagocytes. The polymer backbones of this invention are generally almost free of antigenic determinants (characteristic, for example, of certain polysaccharides and polypeptides) and generally do not include rigid structures capable of being captured in key-lock interactions in vivo, although the latter are desirable. Thus, the soluble cross-linked solid conjugates described herein are predictably low in toxicity and bioadhesiveness, making them suitable for a number of biomedical applications.

In some embodiments of the invention according to this invention biodegradable biocompatible conjugates may form a linear or branched structure. For example, the biodegradable biocompatible polyal conjugates of this invention may be chiral (optically active). Optionally, the biodegradable biocompatible polyal conjugates of this invention may be scalemic.

In some embodiments of the invention, the conjugates described herein are water soluble. In some embodiments of the invention, the conjugates described herein are water insoluble. In some embodiments, the novel conjugate is in solid form. In some embodiments of the invention, the conjugates described herein are colloids. In some embodiments of the invention, the conjugates described herein are in particulate form. In some embodiments of the invention, the conjugates described herein are in the form of a gel.

Scheme 1 below shows the synthesis scheme for obtaining the polymer backbone of the drug substance described in this document. In one embodiment, the conjugates are prepared in several steps: (1) the polymer, PHF, is modified to contain a COOH moiety (eg, -C(O)-X-(CH2)2-COOH); (2) the polymer is then further modified to contain a maleimide moiety (eg, EG2-MI) that can react with the PBRM functionality; (3) a modified polymer containing two different functional groups is reacted with a functional group of the drug or its derivative (eg, AF-HPA-Ala) to form a polymer-drug conjugate; (4) PBRM disulfide bonds are reduced; (5) the reduced PBRM is then reacted with the polymer drug conjugate to form a protein-polymer-drug conjugate; and (6) the remaining maleimide moiety is optionally reacted with a maleimido blocking compound (eg, cysteine).

In another embodiment of the invention, the order of steps (2) and (3) can be reversed, as depicted on the right side of the synthesis route in Scheme 1 below.

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Scheme 1

PBRM

AF-HPA-Ala

T 2. Cysteine

In yet another embodiment of the invention, steps (2) and (3) above are performed simultaneously, as depicted in Scheme 2 below.

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Scheme 2 .o

PBRM

Cysteine

The use of antibodies against HER2

It should be appreciated that the administration of therapeutic agents in accordance with the invention will be carried out with suitable carriers, excipients and other substances that are included in dosage forms to ensure transfer, delivery, tolerability and the like. A variety of appropriate dosage forms can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), in particular chapter 87 by Blaug, Seymour, herein. These dosage forms include, for example, powders, pastes, ointments, gels, waxes, oils, lipids containing lipid (cationic or anionic) vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil, carbovax emulsions (polyethylene glycols of various molecular weights), soft gels and soft blends containing carbovax. Any prior mixtures may be suitable for the methods of treatment and therapy in accordance with the present invention, provided that the active ingredient in the dosage form is not inactivated by the dosage form, and the dosage form is physiologically compatible and tolerable for a given 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 W.N. Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts J. Pharm. sci. 89(8):967-78 (2000), Powell et al. Compendium of excipients

- 67 039374 for parenteral formulations PDA J. Pharm. sci. Technol. 52:238-311 (1998) and references therein for additional information relating to dosage forms, excipients and carriers well known to pharmaceutical chemists.

In one embodiment of the invention, the antibodies, their fragments and/or their conjugates described herein can be used as therapeutic agents. Such agents will generally be used to diagnose, predict, control, treat, alleviate, prevent and/or slow the progression of a disease or pathology associated with, for example, abnormal HER2 activity and/or expression in a subject. The treatment regimen is performed by identifying a subject, eg a human patient, suffering from (or at risk of developing) a disease or disorder associated with abnormal HER2 activity and/or expression, eg a cancer, using standard methods. An antibody preparation, preferably an antibody having high specificity and high affinity for its target antigen, is administered to a subject and, by virtue of its binding to the target, will generally be effective. Administration of an antibody may interrupt or inhibit or impair the target's signaling function. Administration of an antibody may terminate or inhibit or disrupt the binding of the target to the endogenous ligand to which it naturally binds. For example, an antibody binds to a target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise disrupts HER2 activity or expression.

Diseases or disorders associated with abnormal activity and/or expression of HER2 include, but are not limited to, cancer. The target cancer can be anal cancer, astrocytoma, lymphoma, head and neck cancer, liver, testicles, cervix, sarcoma, hemangioma, esophageal cancer, eye, larynx, mouth, mesothelioma, skin cancer, myeloma, cancer of the mouth, throat , bladder, breast, uterus, ovary, prostate, lung, colon, pancreas, kidney, or gastrointestinal cancer.

In another aspect of the invention, the disease or disorder is a cancer selected from the group consisting of breast cancer, gastrointestinal cancer, non-small cell lung cancer (NSCLC), and ovarian cancer.

In general, alleviating or treating a disease or disorder involves alleviating one or more of the symptoms or medical problems of the disease or disorder. For example, in the case of cancer, one or a combination of the following can be achieved by a therapeutically effective amount of the drug: reduction in the number of cancer cells; reduction in tumor size; inhibiting (ie, reducing to some extent and/or stopping) the infiltration of cancer cells into peripheral organs; inhibition of tumor metastases; inhibiting tumor growth to some extent and/or alleviating one or more symptoms associated with the cancer to some extent. In some embodiments, the composition described herein may be used to prevent the onset or recurrence of a disease or disorder in a subject.

A therapeutically effective amount of an antibody, fragment thereof, and/or conjugate thereof described herein generally refers to the amount necessary to achieve the goal of treatment. As noted above, this may be a binding interaction between the antibody and its target antigen, which in some cases disrupts the function of the target. The amount to be administered will also depend on the binding affinity of the antibody to its specific antigen and will also depend on the rate at which the administered antibody is depleted in the headspace of the other subject to which it is administered. Typical therapeutically effective dosage ranges for an antibody or antibody fragment and/or conjugate thereof described herein can 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 dosing frequencies may range, for example, from twice a day to once a month (eg, once a day, once a week; once every second week; once every 3 weeks or once a month). For example, XMT 1519 conjugates described herein, such as XMT 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) or XMT 1519-(EG2-MI-(10 kDa PHF -BA-(AF-HPA-Ala))) may be administered (eg, as a single dose weekly, every 2 weeks, every 3 weeks, or monthly) at a dose of about 0.1 mg/kg to about 20 mg/kg (eg , 0.2, 0.5, 0.67, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg). For example, the XMT 1519 conjugates described herein, such as the XMT 1519 conjugate(EG2-MI-(PHF-BA-(AF-HPA-Ala))) or the XMT 1519-(EG2-MI-(10 kDa PHF- BA-(AF-HPA-Ala))) may be administered (eg, as a single dose weekly, every 2 weeks, every 3 weeks, or monthly) at a dose of about 0.1 mg/kg to about 20 mg/kg (eg, 0.2, 0.5, 0.67, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg) for the treatment of breast cancer with low expression of HER2 or cancer of the gastrointestinal tract with low expression of HER2.

Treatment efficacy is determined in conjunction with any known method for diagnosing or treating a particular HER2-associated disorder. Relief of one or more symptoms of a HER2-associated disorder indicates that the antibody is of clinical benefit.

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Screening methods for 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.

In another embodiment, an antibody directed against HER2 may be used in methods known in the art relating to the localization and/or quantification of HER2 (e.g., for use in measuring HER2 levels in appropriate physiological samples, for use in diagnostic methods , for use in protein imaging and the like). In this embodiment, an antibody specific for HER2 or a derivative, fragment, analog or homologue thereof, which contains an antigen-binding domain derived from the antibody, is used as pharmacologically active compounds (referred to herein as therapeutic agents).

In another embodiment, an antibody specific for HER2 is used to isolate the HER2 polypeptide using standard techniques, such as using immunoaffinity, chromatography, or immunoprecipitation. Antibodies directed against the HER2 protein (or fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical research procedure, for example to determine the effectiveness of a given treatment regimen. Detection can be facilitated by binding (ie physical binding) of the antibody to the substance to be detected. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. 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 substances include umbelliferone, fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent substance includes luminol; examples of bioluminescent substances include luciferase, luciferin and aequorin, and examples of suitable radioactive substances include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

In yet another embodiment, an antibody of the invention may be used as a means of detecting the presence of a HER2 protein (or fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. The antibodies are polyclonal or, preferably, monoclonal. An intact antibody or fragment thereof is used (eg F _ab , scFv or F( _ab ) ₂ ). The term labeled, in relation to a probe or antibody, is intended to cover the direct introduction of a label into a probe or antibody by binding (i.e. physical binding) of the substance to be detected to the probe or antibody, as well as indirect introduction into the probe or antibody by reacting with another reagent, which is labeled directly. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and a biotin-tagged DNA probe so that it can be detected by fluorescently labeled streptavidin. The term biological sample is intended to include tissues, cells, and body fluids isolated from a subject, as well as tissues, cells, and fluids present in the subject's body. Therefore, within the scope of the term biological sample, blood and a fraction or component of blood, including blood serum, blood plasma or lymph, are included. In other words, the detection method described herein can be used to detect an analyzed mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detecting analyte mRNA include northern hybridization and in situ hybridization. In vitro techniques for detecting a protein of interest include enzyme-linked immunosorbent assays (ELISA), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detecting analyzed genomic DNA include Southern hybridization. Procedures for conducting immunoassays are described, for example, in ELISA: Theory and Practice: Methods in Molecular Biology, Vol. 42, JR 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. However, in vivo methods for detecting analyzed protein include introducing into the subject's body a labeled antibody against the analyzed protein. For example, the antibody may be a labeled radioactive marker, the presence and location of which in the subject's body can be detected by standard imaging techniques.

Therapeutic administration and dosage forms of antibodies against HER2.

The antibodies, derivatives, fragments, analogs and homologues and/or conjugates thereof described herein (also referred to herein as active compounds) may be included in pharmaceutical compositions suitable for administration. The principles and considerations involved in the preparation of such compositions, as well as guidance for selecting ingredients, 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.

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Such compositions typically contain the antibody, fragments and/or conjugates thereof, and a pharmaceutically acceptable carrier. When 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 sequences of the variable regions of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be chemically synthesized and/or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).

As used herein, the term pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents 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, standard source text in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin solution. Liposomes and non-aqueous 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 when any conventional media or agents contemplated for use in these compositions are incompatible with the active compound.

Dosage forms intended for in vivo administration must be sterile. This condition is easily achieved by filtration through sterile filtration membranes.

The pharmaceutical composition described herein is prepared in the form of a dosage form, which must be compatible with the intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (eg, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous administration may include the following components: sterile solvent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, polyethylene glycol and other synthetic solvents, antibacterial agents such as benzene alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffering agents such as acetates, citrates or phosphates; and agents for correcting tonicity such as sodium chloride or dextrose. The pH value can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be placed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsipiny, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability is possible. It must be stable under the conditions of manufacture and storage and must be protected from 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 (eg glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents, eg sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of injectable compositions may be due to the inclusion in the composition of an agent that slows absorption, such as aluminum monostearate or gelatin.

Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent, optionally with one or a combination of the ingredients listed above, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the necessary other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze drying, which yield a powder of the active ingredient plus any desired ingredient from its pre-sterilized filtered solution.

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Oral compositions typically include an inert solvent or edible carrier. They can be placed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be formulated with excipients and administered as tablets, lozenges or capsules. Oral compositions may also be formulated using a liquid carrier for use as a mouthwash in which the compound in the liquid carrier is administered orally, rinsed in the mouth and spit or swallowed. As part of the composition, pharmaceutically compatible binding agents and/or adjuvants may be included. 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; an excipient such as starch or lactose; a leavening agent such as alginic acid, Primogel or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as mint flavor, methyl salicate or orange flavor.

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

Systemic administration can also be carried out by transmucosal or transdermal routes. For transmucosal or transdermal administration in dosage form, appropriate penetrants are used to penetrate the barrier. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be performed through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels or creams, as is generally known in the art.

The compounds may also be formulated in the form of suppositories (eg, with a conventional suppository base such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are formulated with a carrier that will protect the compound from rapid excretion from the body to form dosage forms such as sustained/controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such dosage forms will be apparent to those skilled in the art.

For example, active ingredients can be captured into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, into hydroxymethyl cellulose microcapsules or gelatin capsules and poly(methyl methacrylate) microcapsules, respectively, into colloidal delivery systems (for example, liposomes, microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

Sustained release formulations may be prepared. Suitable sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and y-ethyl-b-glutamate, non-degradable ethylene vinyl acetate , degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of a copolymer of lactic acid-glycolic acid and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid copolymer are able to release molecules for more than 100 days, some hydrogels release proteins in shorter periods of time.

These materials are also available commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

A significant advantage in the preparation of oral or parenteral compositions in unit dosage form is ease of administration and uniformity of dosing. Unit dosage form, as used herein, refers to physically separated units adapted for unit dosages to the subject to be treated; each

- 71 039374 unit contains a predetermined amount of the active compound, calculated to obtain the desired therapeutic effect, in combination with the desired carrier. The description of the unit dosage forms described herein is predetermined by and directly dependent on the individual properties of the active compound and the particular therapeutic effect achieved, and the limitations inherent in the art for the formulation of such an active compound for the treatment of individuals.

Pharmaceutical compositions may be enclosed in a container, pack or dispenser along with instructions for administration.

The dosage form may also contain more than one active compound, as appropriate for the particular indication to be treated, preferably compounds with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purposes.

In one embodiment of the invention, the active compounds are administered in combination therapy, i. in combination with other agents, for example therapeutic agents, which are useful in the treatment of 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 funds are given almost at the same time, either simultaneously or sequentially. If given sequentially, when the result of administration of the second compound is observed, the first of the two compounds is preferably still present at an effective concentration at the site of treatment.

For example, combination therapy may include one or more of the antibodies, fragments thereof, and/or conjugates thereof described herein, co-formulated and/or co-administered with one or more additional antibodies, e.g., an anti-HER2 antibody, a dimerization inhibitor antibody HER2 or a combination of an anti-HER2 antibody and an antibody that inhibits HER2 dimerization, such as, for example, trastuzumab, pertuzumab, or combinations of trastuzumab and pertuzumab, or a biosimilar of trastuzumab and/or pertuzumab, or a combination of biosimilars.

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

In some embodiments of the invention, the additional therapeutic agent(s) used in combination with the antibody, fragment thereof and/or its conjugate described herein are agents that interfere with various steps in immune and/or inflammatory response. In one embodiment, one or more of the antibodies described herein may be co-formulated and/or co-administered with one or more additional agents.

Diagnostic and prophylactic formulations.

An anti-HER2 antibody, an antigen-binding fragment thereof, and/or a conjugate thereof as described herein are used in diagnostic and prophylactic formulations. In one embodiment, an anti-HER2 antibody, antigen-binding fragment thereof, and/or a conjugate thereof as described herein is administered to patients who are at risk of developing one or more of the aforementioned diseases, such as, for example, without limitation, cancer. The predisposition of the patient's organs to one or more of the above indications can be determined using genotypic, serological or biochemical markers.

In another embodiment, an anti-HER2 antibody, antigen-binding fragment thereof, and/or conjugate thereof described herein is administered to individuals who have been diagnosed with a clinical indication associated with one or more of the aforementioned diseases, such as, for example, without limitation, cancer. In diagnosis, an anti-HER2 antibody, antigen-binding fragment thereof, and/or a conjugate thereof as described herein is administered to alleviate or reverse the effects of a clinical indication associated with one or more of the aforementioned diseases.

In another embodiment of the invention, a method for identifying a patient with breast cancer treatable with the conjugates described herein comprises measuring the status of certain characteristics of a tumor sample obtained from the patient and identifying the patient as suitable for treatment based on the status of certain characteristics of the tumor sample. .

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The antibodies described herein are also useful for detecting HER2 in patient samples and are therefore useful as diagnostic tools. For example, antibodies against

The HER2s described herein are used in in vitro assays, such as ELISA, to detect HER2 levels in patient samples.

In one embodiment, an anti-HER2 antibody described herein is immobilized on a solid support (eg, well(s) of a microtiter plate). The immobilized antibody serves as a capture antibody for any HER2 that may be present in the test sample. Before contacting 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 non-specific adsorption of the analyte.

Next, the wells are treated with a test sample that is expected to contain antigen, or with a solution containing a standard amount of antigen. Such a sample is, for example, a serum sample taken from a subject that is expected to carry levels of circulating antigen considered diagnostic for the presence of pathology. After washing off the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as the detectable antibody. The level of the detected label is measured and the concentration of the HER2 antigen in the test sample is determined by comparison with the calibration curve obtained using standard samples.

It should be appreciated that based on the results obtained using the anti-HER2 antibodies described herein in an in vitro diagnostic assay, it is possible to determine a subject's disease stage based on expression levels of the HER2 antigen. For a given disease, blood samples taken from subjects are diagnosed as being at different stages of disease progression, and/or at different stages of therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of antigen concentrations can be assigned that can be considered representative of each designated stage.

All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document were specifically and independently identified as being incorporated herein by reference. The citation of publications and patent documents does not imply an acknowledgment that any prior art statement is an integral part of any acknowledgment as to its content or date. The present invention, heretofore described by way of description in writing, will be recognized by those skilled in the art as an invention that can be practiced in a variety of embodiments, and that the foregoing description and examples below are for illustrative purposes and do not limit the following claims. .

Examples

The following working examples are illustrative of linkers, drug molecules, and PBRMs, and methods for their preparation. They are not intended to be limiting, and those skilled in the art will appreciate that other reagents and methods may be used.

Abbreviations.

In the following reaction schemes and in the synthetic examples, the following abbreviations are used. This list is not meant to correspond to an all-encompassing list of abbreviations used in this application, but is provided as additional standard abbreviations that are easily understood by those skilled in the art of organic synthesis, which can also be used in synthesis schemes and in examples.

AF-HPA Auristatin F-hydroxypropylamide

BSA Bovine Serum Albumin

DMEM Medium Needle in Dulbecco's modification

FBS Fetal bovine serum

HRP horseradish peroxidase

FSB Phosphate buffered saline, 0.9% NaCl

TMB 3,3',5,5'-tetramethylbenzidine

General information.

Purchased Genentech Kadcyla® (ado-trastuzumab emtansine) injectable.

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The CDRs were identified by the Kabat numbering scheme.

Tumor growth inhibition (%TGI) was defined as the percentage difference in median tumor volumes (MTV) between treated and control groups.

Treatment efficacy was determined by the incidence and magnitude of regressive responses to tumor size observed during treatment. Treatment can cause partial regression (PR) or complete regression (CR) of the tumor in the animal. At PR response, the tumor volume was 50% or less than the corresponding volume on day 1 for three consecutive measurements during the course of the study and was equal to or greater than 13.5 mm for one or more of these three measurements. With a CR response, the tumor volume was less than 13.5 mm ³ for three consecutive measurements during the course of the study. An animal with a CR response at the end of the study was further classified as tumor-free survivor (TFS). Animals were monitored for regressive responses.

HPLC purification was performed on a Phenomenex Gemini semi-preparative synthesis column 5 µm 110 A, 250 x 10 mm, 5 µm.

Size exclusion chromatography (SEC) was performed on a Tosoh Biosciences TSK gel G5000 column (7.8 mm χ 30 cm, 10 μm) or a Superose 12 column (GE Healthcare).

Weak cation exchange (WCX) chromatography was performed on a ProPac WCX-10 (94 mm x 250 mm) column (ThermoFisher).

In all possible cases, the drug content was determined spectrophotometrically, otherwise LC/MS or 1H-NMR was performed to quantify the drug content.

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

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

The Her2-polymer-drug conjugates were separated from the residual unreacted drug-polymer conjugates by wide sweep diafiltration. Additional purification was performed, if necessary, by size exclusion chromatography and WCX chromatography to remove any aggregated Her2-polymer-drug conjugates. In general, Her2-polymer-drug conjugates typically contained <5% (w/w, eg <2% w/w) of the aggregated fraction as determined by SEC; <0.5% (w/w, e.g. <0.1% w/w) of free (non-conjugated) drug as determined by RP HPLC or LCMS/MS; <1% (w/w) free polymer-drug conjugate determined by SEC and/or RP HPLC, and <2% (w/w, e.g. <1% w/w) unconjugated Her2, determined HPLC-CHC (hydrophobic interaction chromatography) and/or HPLC WCX. Reconstituted or partially reconstituted antibodies were prepared using techniques described in the literature, see, for example, Francisco et al., Blood 102 (4): 1458-1465 (2003). The total drug concentration (conjugated and unconjugated) was determined by LC-MS/MS.

General methods.

General Procedure A. Conjugation of a polymer with a linker or drug.

In general, the conjugation of a polymer (PHF-BA or PHF-GA) with an amine-containing linker, such as, for example, EG2-maleimide, or an amine-containing drug linker, such as, for example, AF-HPA-Ala, HPA-Ala, was carried out in an aqueous solvent or a mixture of 10-90% organic/aqueous solvents in the presence of an activating agent such as, for example, ED-CHCl. Typical organic solvents include, but are not limited to, water-miscible solvents such as, for example, DMSO, DMF, DMA, NMP, and ACN. An activator such as, for example, NHS is added to speed up the binding. First, the polymer is mixed with an amine-containing compound, followed by the addition of a co-activator (NHS) and then the addition of an activator (EDC-HCl). The reaction is carried out at 0-10° C., pH 4.5 to 7.5 for 1 to 24 hours at ambient temperature. The resulting conjugated polymer product is purified by diafiltration or SEC. The product is concentrated to 2-50 mg/ml, the pH is adjusted from 4.5 to 6.5, to ensure drug stability, the polymeric linker and conjugate are stored frozen at -20 to

- 74 039374

-80°C.

Conjugation of the polymer with the amine-containing linker or drug can be carried out sequentially, in any order, or simultaneously.

General procedure B. Partial selective protein reduction (anti-HER2 antibodies).

Partial selective reduction of interstrand disulfide groups or the unpaired disulfide in the respective anti-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. If the reduction is performed with an excess of the reducing agent, then the reducing agent is removed by SEC before conjugation. The extent to which HER2 disulfide groups are converted to reactive sulfhydryl groups depends on the HER2 stoichiometry, reducing agent, pH, temperature, and/or reaction time. When some but not all of the disulfide groups are reduced in PBRM, then the reduced PBRM is partially reduced HER2.

General Procedure C. Conjugation of partially reduced HER2 to a polymer-drug conjugate.

The conjugation of the partially reduced PBRM with the polymer-drug conjugate is carried out under neutral or slightly alkaline conditions (pH 6.5-8.5) at a PBRM concentration of 1-10 mg/ml and a polymer-drug conjugate concentration of 0.5-10 mg/ml. ml. The polymer-drug conjugate is typically used in a 1-5.0-fold excess relative to the required stoichiometry of the protein-polymer-drug conjugate. When PBRM is conjugated to the maleimide group of the polymer-drug conjugate, the conjugation is optionally terminated by the addition of a water-soluble maleimide blocking compound such as, for example, N-acetylcysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropionic acid, 2-mercaptoacetic acid, mercaptomethanol (ie HOCH ₂ SH), benzylthiol and the like.

The resulting HER2-polymer-drug conjugate is typically purified by diafiltration to remove any unconjugated polymer-drug conjugate, unconjugated drug, and small molecular weight impurities. Alternatively or additionally, appropriate separation chromatography techniques such as, for example, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, such as, for example, WCX chromatography, can be used to purify the HER2-polymer-drug conjugate; reverse phase chromatography, hydroxyapatite chromatography, affinity chromatography, or a combination thereof. The resulting purified HER2-polymer drug conjugate is typically formulated in a buffer solution at pH 5.0-6.5.

Example 1 FACS screening for anti-HER2 antibodies.

Using the procedures described below, two anti-HER2 antibodies described herein, XMT 1517 and ^XMT 1519, were selected. 2009036379; WO 2010105256; WO 2012/009568 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 procedure was performed using the Miltenyi MAC system as described (Siegel et al., J. Immunol. Methods. 2004 Mar; 286(1-2): 141-53). Briefly, yeast cells (~10 ¹⁰ cells/library) were incubated with 3 ml of 200 nM biotinylated HER2 antigen monomer or 10 nM biotinylated HER2Fc fusion antigen for 15 min at room temperature in PBS FACS wash buffer with 0.1% BSA ( biotinylation was performed 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. no. 130-048-101) were added to the yeast and incubated for 15 min at 4°C. Next, the yeast was precipitated, resuspended in 5 ml wash buffer and applied to a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany, cat. no. 130-042-401). After loading with 5 ml, the column was washed 3 times with 3 ml of FACS wash buffer. The column was then removed from the magnetic field and the yeast was eluted with 5 ml growth medium and grown overnight. The next three sorting cycles were performed using flow cytometry. Approximately 1x108 yeast was precipitated, washed three times with wash buffer and incubated with 200, 100, or 10 nM biotinylated HER2, respectively, for 10 minutes at room temperature. The yeast was then washed twice and stained with goat anti-human F(ab') ₂ -FITC diluted 1:100 (Southern Biotech, Birmingham, AL, cat. no. 2062-02) and either streptavidin-Alexa Fluor 633 (Life Technologies, Grand Island, cat. no. S21375) diluted 1:500, or extravidin-phycoerythrin (Sigma-Aldrich, St. Louis, cat. no. E4011) diluted 1:50, secondary reagents for 15 min at 4°C. After washing twice with ice-cold wash buffer, the cell pellets were resuspended.

- 75 039374 in 0.4 ml wash buffer and transferred to tubes for sorting colored capped products. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sort gates were adjusted to select only HER2 binding clones in two cycles, with the third cycle being a negative sort to reduce the content of binding reagents. After the last sorting cycle, the yeasts were plated and individual colonies were collected for characterization.

Example 2 Affinity maturation of anti-HER2 antibodies.

Using the procedures described below, the affinity matured anti-HER2 antibodies described herein, XMT 1518 and XMT 1520, were generated. Cycle 5 binding of the population was used to diversify the light chain series (LCBD). Heavy chain plasmids were isolated and transformed into a light chain library with 1x10 ⁶ diversity. 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 cycles.

Additionally, affinity maturation was performed on the best clones after LCBD. Each of the CDRH3 heavy chains from these clones was separately recombined in a pre-built variant library with 1x10 ⁸ diversity and made selections as described above. Affinity pressure was applied by incubating the yeast antibody-antigen complex with the parent Fab for varying lengths of time to select antibodies with the highest affinity.

The third round of affinity maturation included error-prone heavy chain and light chain PCR. Selections were performed in a similar fashion to previous cycles using FACS sorting for all three cycles and with extended time periods for Fab pressure.

Example 3 Preparation and Purification of Antibodies.

Yeast clones were grown to saturation and then induced for 48 hours at 30° C. with shaking. After induction, yeast cells were pelleted and supernatants were collected for purification. IgG was purified using a protein A column and eluted with acetic acid, pH 2.0. Fab fragments were prepared by digestion with papain and purified on KappaSelect (GE Healthcare LifeSciences cat. no. 17-5458-01).

Mammalian IgG expression was performed by subcloning antibodies in a new expression vector followed by transient transfection and expression in HEK. Briefly, expression vectors containing antibodies of interest were transfected by complexation 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 were then cultured for 7 days, fed with fresh medium every 48 hours. After 7 days, the supernatant was collected, followed by centrifugation and purification using protein A, and, if necessary, purification on a CNT column was added to achieve a content of > 95% monomer.

Example 4 Anti-HER2 Antibody Affinity Measurements.

The affinity of HER2 antibodies was determined by measuring their K _D 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 applying IgG to AHQ sensors online. The sensors were equilibrated in assay buffer offline for 30 min and then monitored online for 60 s to establish a baseline. The IgG-coated sensors were exposed to 100 nM antigen for 5 minutes, after which they were transferred to the assay buffer solution for 5 minutes to measure the dissociation rate. The kinetics were analyzed using a 1:1 binding model.

Equilibrium affinity measurements were performed as previously described (Estep 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 on standard MSD-ECL binding plates overnight at 4° C. or at room temperature for 30 minutes. The plates were then blocked for 30 minutes with shaking at 700 rpm followed by three washes with wash buffer (PBSF+ 0.05% Tween 20). SET samples were applied and incubated on the plates for 150 seconds with shaking at 700 rpm followed by a single wash. The antigen captured on the plate was detected with 250 ng/mL sulfo-labeled streptavidin in PBSF by incubation on the plate for 3 min. The plates were washed three times with wash buffer and then read on an MSD Sector Imager 2400 using 1x T reading buffer with surfactant. The percentage of free antigen was plotted as a function of titrated antibody in the Prism program and a quadratic equation was fitted to find K _D . To improve performance, all MSD-SET experiments utilized fluid manipulation robots, including sample preparation for SET. In table. I give the results of affinity measurements using the ForteBio and MSD-SET methods.

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Table I

Test antibody K _D (M) (ForteBio) ¹ k _on (1/M/c) (ForteBio) ¹ k Off (1/c) ( _ForteBio ) ¹ K _D (M) (MSD) ² XMT 1517 8.7 xlO' ^{8 2.3xlO5 2.0xlO'2} 2.4 xlO' ⁸ XMT 1518 3.3 xlO' ^{9 2.4xlO5 7.9xlO'4} 8.5 xlO' ¹⁰ XMT 1519 3.5 xlO' ^{8 2.7xlO5 9.5xlO'3} 1.4 xlO' ⁸ XMT 1520 2.1 xlO' ⁹ 1.4 xlO ^{5 3.1xlO'4} 6.1 xlO' ¹⁰

¹ Measured with ForteBio. IgG on the tip, and human HER2 monomer in solution.

² Measured on the Meso Scale Discovery. IgG versus biotinylated human HER2 monomer.

Example 5 Epitope-Specific Sort Octet Red384.

Epitope-specific antibody sorting was performed on a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park, CA) using a standard sandwich sort assay. Control anti-target IgG was applied to AHQ sensors and unoccupied Fc binding sites on the sensor were blocked with irrelevant human IgG1 antibody. The sensors were then exposed to 100 nM of the target antigen followed by exposure to a second anti-target antibody. Data were processed using ForteBio data analysis software version 7.0. Additional binding by a second antibody after antigen association indicates the presence of an unoccupied epitope (non-competitor), while no binding indicates blocking of the epitope (competitor). This process was repeated for 4 control antibodies: (i) trastuzumab, which binds to domain IV of HER2 (Cho et al., Nature. 2003 Feb 13;421(6924):756-60); (ii) pertuzumab, which binds to HER2 domain II (Franklin et al., Cancer Cell. 2004 Apr; 5(4):317-28); (iii) Fab37, which binds to HER2 domain III (Fisher et al., J. Mol. Biol. 2010 Sep 10; 402(1):217-29), and (iv) chA21, which binds to HER2 domain I ( Zhou et al., J. Biol. Chem. 2011 Sep 9;286(36):31676-83; Cheng et al., Cell Res. 2003 Feb;13(1):35-48). Control antibody sequences are shown below.

>Trastuzumab, heavy chain variable region amino acid sequence 1 15 3045 □ IQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK

60 7590

105 120135

HyTTPPTFGQGTKVElKRTVAAPSVFIFPPSDEQLKSGTASVVCL

136 150 165180

LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

181 195 210214

LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40).

>Trastuzumab, light chain variable region amino acid sequence

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15 3045

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL

60 7590

EWVARIYPTNGYTRYAQSVKGRFTISADTSKNTAYLQMNSLRAED

105 ISO135

TftVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAFSS

136 150 165iao

KSTSGGTAALGCLVKDYFPBPVTVSWNSGALTSGVHTFPAVLQSS

101 195 3102Ξ5

GLYSLSSVVIVPSSSLGTQTYiCNVNHKPSNTKVDKKVEPKSCDK

226 240 255270

THTCPPCPAPELLGGPEVFLFPPKPKDTLMISRTPEVTCWVDVS

271 265 30D315

HEDPEVKFNWYVDaVEVHHAKTKPREEQYNSTYRVVSVLjTVLHQD

316 33D 345360

WLNGKEYKCKV£HJiALPAPIEKTISKAKGQPRE₽QVYTLPP£REE

361 375 390405

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

406 420 435449

5NUEKTUVK5II(EICG¥E5C£UMNEDNKNUCK315b£PC (SEQ ID NO:41). >Pertuzumab, heavy chain variable region amino acid sequence 1 10 20 30 40 S0 60

DIQMTQS PS SLSASVGDRVTITCKASQDVSIGVAWYQQKPGKA PKLLIYSASYRYTGV PS

SO 90 100 110120

RE SGSG SGTDFTLTISS LQ PEDFΑΤΎYCQQYYTYPYTFGQGTKVEIKRTVAAPSVFIFPP

130 140 150 160 170ISO

I I I I I I ] I I I 1I

50E0KZSTA5UUSSI"1EU₽YEAKU0IKUPTSAYESK[V0E¥TE0P$KO8TUYAZTTT

190 200210

IrSKADY EKHKVY ACEVTHQGbS Ξ PVTKS FNRQEC (SEQ ID NO: 42).

>Pertuzumab, light chain variable region amino acid sequence 1 10 20 30 40 5060

E VQbVb SlK^b AAiGFTTTDYTMDWVKyAPGKGbEW VADVKFWSCiGSI Ϊ

80 90 100 110120

MQRF^RFTLSWRSKI^rLYLQMNSLRAEDTAVYYCARNLGPSFYTOTWGOGTLVTVSSA

130 140 150 160 170180

STKGPSVFPLAPS SK STSGGTAALGCLVKDYF?E P VTVS WNSGAbT SGVHTF PAVLOS SG 190 200 210 220 230240

I I I I I I I I I I I I LYS L S Ξ WTVPS Ξ SLQTQTYICNVNHKP SMTKVDKKVZPK.SCDKTHTC P PC PAPE LLGGP

25ΰ 260 270 2B0 290300

SVFL F P PKPKDTLMr SRTP Ξ VTC VWDV SHEDPE VKFNWY VDG VE VHM AKTKPREEQ ΥΝΞ

310 3ΞΟ 330 340 350360

T YRVVS VLrVLHQDWLNGKE YKC KVSNKAL PAPIEKTIS KAKGQ PRE PQVYTL P P EREEM

370 380 390 400 410420

TKMdU5NTSEUKAR¥RZO1AUEIIEBYUD?EM#KTRRUOZPS5RGYU5KGTTOKZKI0

430 44044B

Illi II QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 43).

- 78 039374 >Fab37, heavy chain variable region amino acid sequence EVOLVESGGGLVOPGGSLRLSCAASGFSIWWSWIHWVRQAPGKGLEWVASIS

PSSGWTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARWWSSA

MDYWGQGTLVTVSS (SEQ ID NO: 44) >Fab37, light chain variable region amino acid sequence

DIOMTOSPSSLSASVGDRVTITCRASOSVSSAVAWYOQKPGKAPKLLIYSASSL

YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCOQWWWWPSTFGQGTKVEIK (SEQ ID NO: 45) >chA21, heavy chain variable region amino acid sequence

EVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNSGKSPEWIGHISSS

YATSTYNQKFKNKAAFTVDTSSSTAFMQLNSLTSEDSAVYYCVRSGNYEEYA

MDYWGQGTSVTVSS (SEQ ID NO: 46) >chA21, light chain variable region amino acid sequence DIVLTOTPSSLPVVSVGEKVTMTCKSSOTLLYSNNOKNYLAWYOQKPGOSPKL

LISWAFTRKSGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQQYSNYPWTFG

GGTRLEIK (SEQ ID NO: 47)

Antibodies XMT 1519, XMT 1520, XMT 1517, XMT 1518 with the highest affinity were selected, which did not compete with the four control antibodies.

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

Example 6 Measurements of Antibody Binding Constants to JIMT-1 Cells.

Binding of anti-HER2 antibodies to the cell surface of JIMT-1 cells was assessed using a Macsquant flow pytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). JIMT-1 cells grown overnight to approximately 90% complete cultures in DMEM (ATCC, Manassas, VA) with 10% PBS (Gibco®, Life Technologies, Grand Island, NY) , were removed from the plate surface with Accutase Cell Separation Solution (Innovative Cell Technologies, San Diego, CA). The separated cells were washed once with ice-cold medium containing 6% goat serum and resuspended in the same medium. Aliquots of 100,000 cells per V-bottom well were transferred to a 96-well plate and incubated in a range of anti-HER2 antibody concentrations (0.05-100 nM) in 150 µl DMEM with 6% goat serum on ice for 3 h. Then cells were washed once with ice-cold PBS and resuspended in 100 μl DMEM with 2% goat serum and 6 μg/ml secondary fluorescently labeled antibody, Alexa Fluor® 647-labeled goat anti-human IgG (Life Technologies cat. no. A-21445) for 1 hour on ice. Cells were washed once with ice-cold PBS (Gibco®, Life Technologies, cat. no. 10010049) and suspended in 200 μl of ice-cold PBS with 1% paraformaldehyde. The amount of bound fluorescence per cell was determined for each treatment by analyzing 5000 cells on a flow meter. The median fluorescence value for each treatment was plotted and the dissociation constant, Kd, for each antibody was calculated using Graphpad Prism software by non-linear regression using a single site specific binding model. In FIG. 2 shows the binding of anti-HER2 antibodies to JIMT-1 cells. The calculated K _d 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. Measurement of the affinity of antibodies by ELISA.

The affinity of antibodies to recombinant human and cynomolgus HER2 was determined by ELISA analysis. Purified recombinant HER2 extracellular domain, either human-derived (amino acids 23-652, Aero Biosystems cat. no. HE2-H5225) or cynomolgus monkey-derived (amino acids 1-652, Sino Biological, cat. no. 90295-C08H) was applied to 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 solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.05% Tween 20). The wells were blocked by incubation for 1 h with 50 μl of TTBS containing 5% bovine serum albumin. A range of dilutions (0.005 to 10 nM, 3-fold serial dilutions) of each test antibody or Example 16H was then added to incubate for 2 hours at room temperature in 50 μl of TTBS with 3% BSA. Unbound test antibodies were removed with TTBS washes (4x). HRP-conjugated secondary anti-human IgG (Bethyl Laboratories, #A80-115P) at 0.2 μg/ml in TTBS with 3% BSA was incubated in each well for 1 hour. Unbound secondary antibody was removed by 4 washes with TTBS. HRP substrate (TMB, Bethyl Laboratories No. E102) was added to each well and incubated until a yellow color was visible. The reaction was stopped by adding 50 µl of 0.2N sulfuric acid. The 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 non-linear regression using a single site specific binding model.

In FIG. 3a shows the results of binding of anti-HER2 antibodies XMT 1517, XMT 1518 to human HER2 and cynomolgus monkey HER2. In FIG. 3b shows the results of binding of anti-HER2 antibodies XMT 1519, XMT 1529 to human HER2 and cynomolgus monkey HER2. The calculated values of Kd are given in Table. II.

Table II

Researched antibody HER2 human Qi(nM) HER2 cynomolgus monkey Ka(nM) HMT 1517 1.2 0.6 HMT 1518 0.3 0.3 HMT 1519 0.1 one XMT 1520 0.1 one

The binding affinities of the 4 antibodies tested for human HER2 and cynomolgus monkey HER2 were similar.

In FIG. 3c and 3d show the results of binding of anti-HER2 antibodies XMT 1519 and Example 16H, respectively, to human HER2 and cynomolgus monkey HER2. The calculated Kd values are given in Table 1 below. IIA.

Table IIA

researched an object HER2 human CD(nM) HER2 cynomolgus monkey Ki(nM) HMT 1519 0.03 0.03 Example 16H 0.05 0.05

The binding affinities of XMT 1519 and Example 16H to human HER2 and cynomolgus monkey HER2 were similar.

Example 8 Competitive Assay for Antibody Binding to JIMT-1 Cells.

To confirm that XMT 1518 and XMT 1519 antibodies bind to overlapping epitopes, the antibodies were analyzed in a competitive binding assay on JIMT-1 cells. JIMT-1 cells were grown as described in Example 6. Aliquots of 50,000 cells in 50 μl per well were transferred to a 96-well V-bottom plate. Then 30 nM XMT 1519 antibody conjugated to Alexa Fluor® 647 in 25 µl of medium was added alone or mixed with a second unlabeled antibody (XMT 1518, XMT 1519 or trastuzumab) diluted to a concentration range (0.05-100 nM) and incubated on ice for 3 h. 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. No. A-21445) in for 1 h on ice. Cells were washed once with ice-cold PBS and suspended in 200 μl of ice-cold PBS with 1% paraformaldehyde. The amount of bound fluorescence per cell was determined for each treatment by analyzing 5000 cells on a MacsQuant flow meter (Miltenyi Biotec, Bergisch Gladbach, Germany). The median fluorescence value for each treatment was plotted and the dissociation constant, K _D , for each antibody was calculated using Graphpad Prism by non-linear regression using a single site specific binding model. As shown in FIG. 4, XMT 1518 antibody competed for binding of Alexa Fluor® 647-labeled XMT 1519 to a similar extent as unlabeled XMT 1519, indicating that both antibodies recognize overlapping epitopes on HER2, while trastuzumab did not compete for binding of Alexa Fluor® 647-labeled HMT 1519.

Example 9 Antibody-Dependent Cell-Mediated Cytotoxicity Assay.

Antibody-dependent cell-mediated cytotoxic (ADCC) activity of anti-HER2 antibodies was quantified on BT474 cells. 5000 cells were placed per well of a 96-well plate in DMEM containing 10% FBS and grown overnight at 37° in 5% CO ₂ . Concentrations of XMT 1518, XMT 1519, XMT 1520, or trastuzumab antibodies ranging from 0.001 to 100 nM (8 serial 5-fold dilutions) were added to cells and ADCC activity was measured with the Promega Biological Assay Kit (Cat. No. G7018), which uses effector cells engineered to produce the enzyme luciferase as a reporter for ADCC activity. Luciferase activity was measured on a spectrophotometer (Molecular Devices, Spectramax M5). Values (expressed as the ratio of luciferase activity for each sample of antibody-treated cells to the activity of a sample of cells that were not treated with antibody) were plotted by non-linear regression using agonist log values versus response, variable by clone factor in a four-parameter model with using the GraphPad Prism software.

In FIG. 5 is a graph showing ADCC values. XMT 1519 and XMT 1520 show maximal activity similar to that of trastuzumab: the ratio of signal obtained with antibody to signal obtained without antibody is 30.1 for XMT 1519 and 29.7 for XMT 1520, while the ratio for trastuzumab is 34 ,6. XMT 1518 has a maximum activity of 11.0. The EC ₅₀ values (half maximum binding concentration) for ADCC activation are respectively 0.16 nM for XMT 1518, 0.18 nM for XMT 1519, 0.54 nM for XMT 1520 and 0.07 nM for trastuzumab.

Example 10 Ligand-Dependent HER2 Signaling in MCF7 Cells.

The effect of anti-HER2 antibodies on HER2 ligand-dependent signaling in MCF7 (ATCC, HTB-22) cells was assessed by measuring the amount of phosphorylated AKT after treatment of cells with the HER3 ligand, neuregulin-β 1. 300,000 MCF7 cells in MEM medium with 0.01 mg/ml bovine insulin and 10% FBS and grown overnight at 37° in 5% CO ₂ atmosphere. The medium was removed and replaced with fresh medium containing 10 μg/ml of the test antibody and incubated for 1 hour, followed by a 15-minute treatment with 40 pM neuregulin-31 (R&D Systems, Minneapolis, Minnesota, Cat. No. 377-HB- 050). Cells were washed once with ice-cold PBS and lysed with 200 µl of buffer solution containing 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, cOmplete protease inhibitor kit tablets (Roche, Indianapolis, Ind.) and PhosSTOP phosphatase inhibitor kit tablets (Roche, Indianapolis, Ind.). The lysates were centrifuged at 15,000 rpm for 15 min at 4°C to remove insoluble debris. Phosphorylated AKT levels were determined using the PathScan® Phospho-Aktl Cell Signaling Sandwich ELISA Kit (Ser473) following the manufacturer's instructions.

In 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

Absence Trastuzumab Pertuzumab HMT 1517 HMT 1518 HMT 1519 HMT 1520 ACT 522 ± 86 369±9 127 ± 22 564 ± 55 441± 23 381± 21 439± 49

Neuregulin treatment increased the levels of phosphorylated AKT five-fold (522% compared to unstimulated cells). XMT 1517, XMT 1518, XMT 1519, and XMT 1520 caused very little or no reduction in the neuregulin-induced increase in AKT phosphorylation to 564%, 441%, 381%, and 439%, respectively, which is similar to the increase induced by trastuzumab (369%). Pertuzumab, which has been shown to inhibit ligand-dependent signaling (Franklin et al., Cancer Cell. 2004 April 19; 5:317-28), reduced neuregulin stimulation to a similar value obtained for unstimulated cells (127%). These results suggest that the tested antibodies do not act by inhibiting HER2 and HER3 heterodimerization, which is stimulated by neuregulin and inhibited by pertuzumab.

Example 11 Ligand-Dependent HER2 Signaling in MCF7, SKBR3 and JIMT-1 Cells.

The effect of anti-HER2 antibodies on HER2 signaling in MCF7 (ATCC, NTV-22), SKBR3 (ATCC, NTV-30), JIMT-1 (ATCC, NTV-30) cells was assessed by measuring changes in the amount of phosphorylated ACT after a 4-hour exposure. antibody treatment. In each well of 6-well culture dishes, cells were placed in a special medium with 10% FBS and grown overnight at 37°C in an atmosphere of 5% CO ₂ . The medium was removed and replaced with fresh medium containing 10 μg/ml of the test antibody and cultured for 4 h. 1% Triton X-100, cOmplete protease inhibitor kit tablets (Roche, Indianapolis, IN) and PhosSTOP phosphatase inhibitor kit tablets (Roche, Indianapolis, IN). The lysates were centrifuged at 15,000 rpm for 15 min at 4°C to remove insoluble debris. Phosphorylated AKT levels were determined using the PathScan® Phospho-Aktl Cell Signaling Sandwich ELISA Kit (Ser473) following the manufacturer's instructions.

In table. IV shows inhibition of HER2 ligand-independent signaling in MCF7, SKBR3 and JIMT-1 cells as indicated by the amount of AKT phosphorylation. The results are presented relative to the percentage of untreated cells.

Table IV

cell line Trastuzumab HMT 1517 HMT 1518 HMT 1519 HMT 1520 SKBR3 64±1 43±5 59±2 34±2 29±5

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LMT-1 56±4 66±4 62±1 84±2 69±4 MCF7 98±4 79±5 103 ± 17 76±3 66±3

The tested aHmu-HER2 antibodies induced strong inhibition of HER2 signaling in SKBR3 cells, in which XMT 1517, XMT 1518, XMT 1519, and XMT 1520 reduced levels of phosphorylated AKT to 43%, 59%, 34%, and 29% of the levels occurring in untreated cells, according to compared with a reduction of up to 64% caused by trastuzumab.

Ahtu-HER2 antibodies inhibited HER2 signaling in JIMT-1 cells as shown by a reduction in phosphorylated AKT to 66%, 62%, 84%, and 69% of levels occurring in untreated cells, respectively, for XMT 1517, XMT 1518, XMT 1519, and XMT 1520 , which is similar to the 56% reduction caused by trastuzumab.

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 the levels occurring in untreated cells.

Example 12 Ligand-independent HER2 signaling in BT474 cells.

The effect of anti-HER2 antibodies on HER2 signaling in BT474 cells (ATCC, HTB-20) was assessed by measuring changes in the amount of phosphorylated AKT after 4 hours of antibody treatment. BT474 cells (300,000 cells) were placed in each well of 6-well culture dishes in DMEM with 10% FBS and grown overnight at 37°C in 5% CO ₂ atmosphere. The medium was removed and replaced with fresh medium containing 10 μg/ml of the test antibody and cultured for 4 h. 1% Triton X-100, cOmplete protease inhibitor kit tablets (Roche, Indianapolis, IN) and PhosSTOP phosphatase inhibitor kit tablets (Roche, Indianapolis, IN). The lysates were centrifuged at 15,000 rpm for 15 min at 4°C to remove insoluble debris. Phosphorylated AKT levels were determined by Western analysis. Mix 20 µl of each extract with 7 µl of NUPAGE loading dye (Life Technologies, cat. no. NP0007) and 2 µl of 10x reducing agent (Life Technologies, cat. no. , cat. no. NP0341), which was analyzed in MOPS running buffer (Life Technologies cat. no. NP000102) for 90 min at 120 V. Individual proteins were transferred to a nitrocellulose membrane in a semi-dry electrophoretic transfer system (Bio-Rad, Transblot system) in for 30 min at 10 V in transfer buffer (Life Technologies, cat. no. NP0006) containing 10% methanol. The membrane was incubated for 1 h in blocking buffer (Li-cor, cat. no. 927-40000), and then with a mouse antibody that recognizes the AKT protein (Cell Signaling Technology, no. 2920) diluted 1:1000, a rabbit antibody, which recognizes phosphorylated AKT on serine 473 (Cell Signaling Technology, no. 4060), and a rabbit antibody that recognizes actin (LiCor, cat. no. 926-42210) diluted 1:5000 in the same blocking buffer for 1 h. After incubation the membrane was washed 3 times with 10 ml TTBS and then incubated for 1 h with secondary antibodies: goat anti-rabbit IgG conjugated with IRdye® 800CW (Li-Cor, cat. no. 926-32211) and goat anti-mouse IgG conjugated with IRdye® 680RD (LiCor cat. no. 926-68070), both diluted 1:10000 in blocking buffer. The membrane was washed again 3 times with 10 ml TTBS and scanned on a Li-Cor Odyssey scanner. The intensity of the bands corresponding to ACT, phospho-ACT and actin was quantified using the scanner software. Each phospho-ACT band was normalized to the total AKT band and expressed as a percentage of the phospho-AKT protein obtained from cells that were not treated with any anti-HER2 antibodies.

In the same experiment, the effect of anti-HER2 antibodies on HER3 phosphorylation was also investigated. The nitrocellulose blots described above were also probed for 1 hour with a rabbit antibody that recognizes 1289 tyrosine phosphorylated HER3 (Cell Signaling Technology, No. 4791) diluted 1:1000 in blocking buffer. After the incubation, the membrane was washed 3 times with 10 ml TTBS and then incubated for 1 hour with 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 TTBS and scanned on a Li-Cor Odyssey scanner. The intensity of the bands corresponding to phospho-HER3 and actin was 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 derived from cells that were not treated with any anti-HER2 antibodies.

In table. V is the inhibition of ligand-independent HER2 signaling in BT474 cells, as determined by the amount of AKT phosphorylation or Her3 phosphorylation. The results are presented relative to the percentage of unstimulated cells.

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Table V

Trastuzumab Pertuzumab HMT 1519 XMT 1520 ACT 31±2 44±3 21 ± 8 30±5 HER3 80 85 71 76

Ahtu-HER2 antibodies XMT 1519 reduced AKT phosphorylation to about 21% and HT 19B to about 30%, as shown in untreated BT474 cells, compared to trastuzumab, which caused a decrease to about 31%, and pertuzumab, which caused a decrease to about 44%, relative to untreated cells. HER3 phosphorylation was moderately affected by all antibodies: 71%, 76%, 80%, and 85% of that observed for untreated cells under XMT 1519, XMT 1520, trastuzumab, and pertuzumab, respectively.

Example 13. Measurements of internalization rates.

The rate at which each XMT 1518, XMT 1519, XMT 1520 and trastuzumab antibodies are internalized from the cell surface of SKBR3 cells was determined by fluorescence in a 96-well plate assay. SKBR3 cells were seeded in 96-well plates and allowed to attach by growing overnight in DMEM (ATCC, Manassas, VA cat. no. 30-2002) with 10% fetal bovine serum (FBS) (Gibco®, Life Technologies, Grand Island, NY Cat No. 16140-071) at 37° in 5% CO ₂ . Cells were incubated in medium containing 10 µg/ml antibody on ice for 1 h, then washed with ice-cold medium and incubated on ice with 75 µl of medium containing 1 µg/ml goat anti-human IgG Fab fragment (Rockland Immunochemicals, Gilbertsville, PA Cat 809-1102) conjugated with Alexa Fluor® 647 for one hour. After washing, the cells were transferred to a 37° incubator to allow for internalization for varying periods of time. The plates were scanned on an Odyssey scanner (Li-Cor Biosciences, Lincoln, Neb.) using the 700 nm band. The plates were then acid washed by incubation with a solution of 100 mM glycine, 20 mM MgSO ₄ , 50 mM KCl, pH 2.2, on ice to remove antibodies bound to the cell surface, washed with ice-cold PBS, and scanned again to determine the amount of fluorescence internalized cells. In FIG. Figure 6 shows the rate of antibody internalization over 4 hours. The rate of internalization of all antibodies is nearly identical to that of trastuzumab, all having about 50% total surface binding at time 0, internalizing at 4 hours (Figure 6).

Example 13A. Internalization of HER2 into SKBR3 cells.

The effect of a combination of anti-HER2 antibodies on HER2 internalization into SKBR3 cells was examined by fluorescence microscopy. Trastuzumab was conjugated to Alexa Fluor-647 and used to visualize the location of trastuzumab and preferably HER2 within cells. SKBR3 cells were seeded on 4-chamber microscope slides and allowed to attach by growing overnight in DMEM (ATCC, Manassas, VA, cat. no. 30-2002) with 10% fetal bovine serum (FBS) (Gibco ®, Life Technologies, Grand Island, NY Cat No. 16140-071) at 37° in 5% CO ₂ . SKRB3 cells were incubated for 90 min in medium containing either (i) a combination of trastuzumab-Alexa-647 and pertuzumab, or (i) a combination of trastuzumab-Alexa-647, pertuzumab, and anti-HER2 antibody XMT 1519. Localization of trastuzumab was visualized using a fluorescent microscope . As shown in FIG. 7, since most of trastuzumab-Alexa Fluor-627, in combination with pertuzumab alone, co-localized with cell mask orange, this suggests localization in the cytoplasmic membrane, and most of trastuzumab-Alexa Fluor-647, together with pertuzumab and XMT 1519, co-localized with dye Lysotracker, which suggests localization in lysosomes. This result suggests that the presence of XMT 1519 in combination with trastuzumab and pertuzumab promotes HER2 internalization and migration into lysosomes.

Example 13B. Internalization rate measurements with a combination of anti-HER2 antibodies.

The effect of combining multiple anti-HER2 antibodies that recognize different epitopes on the rate of HER2 internalization was determined by performing a 96-well plate assay similar to the assay described in Example 13. In this experiment, cells were treated with a single test anti-HER2 antibody, in combination with trastuzumab or with trastuzumab and pertuzumab, and measured the amount of anti-HER2 antibody internalized at different time points.

For the assay, 40,000 SKBR3 cells in DMEM containing 10% PBS were placed in each well of three 96-well plates (stained black with a clear deep bottom) and allowed to adhere overnight at 37°C in 5% CO ₂ . The next day, the cells were treated with antibodies as follows. In each well, the medium was replaced with fresh ice-cold medium containing 5 μg/ml of one of the tested anti-HER2 antibodies, or trastuzumab, and incubated on ice for 1 h. Unbound antibody was removed by washing the cells twice with ice-cold medium. For detection, 5 μg/ml 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 antibody was removed by washing each well 2 times with ice-cold medium. Next, medium containing either 5 µg/ml trastuzumab, 5 µg/ml trastuzumab and pertuzumab, or no additional antibodies was added to each well. Each addition was performed in triplicate. One set of wells was treated with one Alexa 647 labeled monovalent anti-human IgG Fab fragment and the fluorescence measured from these wells was used to subtract background fluorescence.

One plate was left on ice to determine the values at time 0 for each treatment, and the other plates were incubated at 37°C. After 1.5 hours, one 37° C. plate and the time 0 plate were washed 2 times with ice-cold PBS and scanned using the 680 nm band of a Licor Odyssey scanner. The fluorescence measured in each well was used as the total fluorescence of each treatment. Cells were then acid washed 2 times with 100 mM glycine, 50 mM KCl and 20 mM MgSO ₄ , pH2.2, then washed twice with ice-cold PBS to remove surface-bound antibodies. The plates were scanned again to determine the amount of internalized fluorescence in each well. The next day, the plates aged for 24 h were analyzed in the same way.

The internalized percentage of each HER2 antibody (or trastuzumab in control wells) was calculated by subtracting the background value averaged over each of the 3 repeats, then dividing the internalized fluorescence by the total fluorescence for each treatment and multiplying by 100.

As shown in Table. VA, 80-90% XMT 1518, XMT 1519 and XMT 1520 are internalized within one and a half hours when combined with trastuzumab and pertuzumab. XMT 1517 shows 60% internalization, probably due to its very high dissociation rate, which causes it to diffuse out of the cells during incubation. When combined with trastuzumab, 20-40% of each test antibody is internalized within one and a half hours and about 15% is internalized when each test antibody is presented alone. Trastuzumab alone shows about 15% internalization within 1.5 hours and about 30% when it is combined with pertuzumab.

Thus, the combination of three anti-HER2 antibodies, any of the tested anti-HER2 antibodies, trastuzumab and pertuzumab, increases the rate of antibody internalization most likely by increasing the rate of HER2 endocytolysis.

VA table

Percentage of internalization Only Trust. Trust. + Perth. On one's own With trastuzumab HMT 1517 HMT 1518 HMT 1519 HMT 1520 HMT 1517 HMT 1518 HMT 1519 HMT 1520 2 one 4 3 2 2 3 2 2 2 thirteen 28 eleven sixteen fourteen fourteen 17 41 35 23 88 94 83 88 83 78 106 106 107 100

With trastuzumab and pertuzumab HMT 1517 HMT 1518 HMT 1519 HMT 1520 4 2 2 3 63 90 83 89 107 103 88 101

Example 14 Destruction of HER2.

The effect of anti-HER2 antibodies on HER2 degradation in SKBR3 cells after antibody treatment was determined by Western analysis. 300,000 SKBR3 cells were placed in each well of 6-well culture dishes in DMEM with 10% FBS and grown overnight at 37° in 5% CO ₂ . The medium was removed and replaced with fresh medium containing 10 μg/ml of each antibody and cultured for 4 h. 1% Triton X-100, cOmplete protease inhibitor kit tablets (Roche, Indianapolis, IN) and PhosSTOP phosphatase inhibitor kit tablets (Roche, Indianapolis, IN). The lysates were centrifuged at 15,000 rpm for 15 min at 4°C to remove insoluble debris. 84 039374 Mix 20 µl of each extract with 7 µl of NUPAGE loading dye (Life Technologies, cat. no. NP0007) and 2 µl of 10x reducing agent (Life Technologies, cat. no. NP0004) and run on a polyacrylamide gel with 4-12% bis tris (Life Technologies, cat. no. NP0341), which was analyzed in MOPS running buffer (Life Technologies cat. no. NP000102) for 90 min at 120 V. Individual proteins were transferred to a nitrocellulose membrane on a semi-dry electrophoretic transfer system (Bio-Rad, Transblot system) for 30 min at 10 V in transfer buffer (Life Technologies, cat. no. NP0006) containing 10% methanol. The membrane was incubated for 1 hour in blocking buffer (Li-cor, cat. no. 927-40000) and then with rabbit antibody that recognizes the HER2 protein (Cell Signaling Technology, no. 2165) diluted 1:1000 and rabbit antibody, which recognizes actin (LiCor, cat. no. 926-42210), diluted 1:5000, in the same blocking buffer for 1 h. After incubation, the membrane was washed 3 times with 10 ml TTBS and then incubated for 1 h with secondary antibodies: IgG goat anti-rabbit conjugated to IRdye® 800CW (Li-Cor, cat. no. 926-32211) and goat anti-mouse IgG conjugated to IRdye® 680RD (Li-Cor, cat. no. 926-68070), both diluted 1:10000 in blocking buffer. The membrane was washed again 3 times with 10 ml TTBS and scanned on a Li-Cor Odyssey scanner. The intensity of the bands corresponding to the full-length HER2 protein was quantified using the scanner software. Each HER2 band was normalized for actin and expressed as a percentage of HER2 protein derived from cells that were not treated with any anti-HER2 antibodies.

In FIG. 8 shows that treatment of SKBR3 cells with 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 degradation products with lower molecular weights. However, the triple combination of trastuzumab, pertuzumab, and one of the investigated anti-HER2 antibodies caused extensive destruction of HER2, as well as the appearance of HER2 degradation products. The largest reductions were seen when XMT 1518 or XMT 1520 were combined with trastuzumab and pertuzumab, with 20% or 31% remaining HER2. When the XMT 1517 or XMT 1519 antibody is combined with trastuzumab and pertuzumab, the reduction is, respectively, 40 or 42% of the remaining HER2 of the normal amount.

Example 15 Mapping of epitopes.

Epitope mapping of XMT 1517 and XMT 1519 antibodies was performed at Integral Molecular Inc., 3711 Market Street, Suite 900, Philadelphia, PA using shotgun mutagenesis technology. Shotgun mutagenesis employs a proprietary 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 undergoes a separate mutation, usually resulting in a variety of other amino acids in order to analyze changes in function. Proteins are expressed in standard mammalian cell lines, so even complex proteins that require eukaryotic translational or post-translational processing can be mapped.

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 regions at the C-terminus of domains III and the N-terminus of domain IV of HER2 . Two secondary critical mutations at H456 and G496 may also be involved in XMT 1517 binding to HER2.

Three critical amino acids for XMT 1519 binding (E521, L525 and R530) have been identified, indicating that XMT 1519 binds the N-terminus of domain IV of Her2.

Example 16 Synthesis of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)))

HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates were prepared using the procedure described in US Application Serial No. 14/512316, filed Oct. 10, 2014. VI, the features of antibody-polymer drug conjugates are given.

Table VI

Example No. Antibody DAR (Ratio. lek. in-in: antibody) 16A Trastuzumab About 10:1 to about 15:1 16V Trastuzumab From about 16:1 to about 21:1 16С Trastuzumab About 5:1 to about 10:1 16D HMT 1519 About 11:1 to about 16.5:1 16E HMT 1519 From about 13:1 to about 20:1 16F HMT 1519 About 12:1 to about 18:1 16G HMT 1517 About 7:1 to about 10.5:1 16N HMT 1519 About 11.8:1 to about 17.5:1 16J HMT 1519 About 8.9:1 to about 13.3:1

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. Synthesized/measured polymer and polymer conjugates typically have a polydispersity of <1.5. Polymer-drug conjugates (i.e., drug-bearing polymer chain attached to the antibody) contained about 27 mole % to about 33 mole % β-alanine, about 6.4 mole % 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)))

Rituximab-(EG2-M1-(10 kDa PHF-BA-(AF-HPA-Ala))) (non-binding control) conjugates were prepared using the procedure described in U.S. Application Serial No. 14/512316, filed October 10, 2014. In table. VII the features of antibody-polymer drug conjugates are given.

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Table VII

Example No. Antibody DAR (Drug Ratio: Antibody) 17A Rituximab About 10:1 to about 15:1 17V Rituximab From about 16:1 to about 21:1 17С Rituximab About 10:1 to about 15:1

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. Synthesized/measured polymer and polymer conjugates typically have a polydispersity of <1.5. Polymer-drug conjugates (i.e., drug-bearing polymer chain attached to the antibody) contained about 27 mole % to about 33 mole % β-alanine, about 6.4 mole % 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-HPAAla))) conjugates.

The antiproliferative properties of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))) conjugates were evaluated on tumor cell lines in vitro using the Cell Titer-Glo method (Promega Corp). JIMT-1 cells (cells with an average expression of HER2, DSMZ, Braunschweig, Germany, cat. no. ACC589), MDA-MB-361 (breast cancer cells with an average expression of HER2, ATCC, cat. no. HTB-27), MDA-MB-453 (triple negative breast cancer cell line, ATCC, cat. no. HTB-131) was cultured in DMEM (ATCC, Manassas, VA, cat. no. 30-2002) with 10% PBS (Gibco ®, Life Technologies, Grand Island, NY, Cat No. 16140-071). NCI-H522 (non-amplifying non-small cell lung carcinoma cell line, ATCC, cat. no. CRL-5810) and NCI-H2170 ((non-small cell lung carcinoma cell line, ATCC, cat. no. CRL-5928) cells were cultured in RPMI-1640 medium. (ATCC cat. no. 30-2001) NCI-H1581 cells (non-small cell lung carcinoma cell line with medium expression, non-amplifying, ATCC cat. no. CRL-5878) were cultured in DMEM:F12 medium (ATCC cat. no. 30 -2006) SNU5 (non-amplifying gastric carcinoma cell line, ATCC, cat. no. CRL-5973) was cultured in Iskov's modified Dulbecco's medium (Invitrogen Life Technologies, cat. no. 12440053) with 20% PBS. OVCAR3 (adenocarcinoma cell line MDA-MB-175-VII (human breast cancer cell line, non-amplifying, ATCC, Cat. No. NTV-25) were cultured in RPMI medium with 20% PBS. cultured in Leibovitz L-15 medium with 20% FBS CAMA-1 (human breast cancer cell line, non-amplifying, ATCC, cat. no. NTV-21 ) were cultured in 90% Eagle's minimal essential medium prepared by ATCC. ZR75-1 (human breast cancer cell line, non-amplifying, ATCC, cat. no. CRL-1500) was cultured in RPMI-1640 medium. HCC1187 (human breast cancer cell line, non-amplifying, ATCC, cat. no. CRL-2322) was cultured in RPMI-1640 medium. HCC38 (human breast cancer cell line, non-amplifying, ATCC, cat. no. CRL-2314) was cultured in RPMI-1640 medium. T47D (human breast cancer cell line, non-amplifying, ATCC, cat. no. HTB-133) were cultured in RPMI-1640 medium. HCC70 (human breast cancer cell line, non-amplifying, ATCC, cat. no. CRL-2315) was cultured in RPMI-1640 medium. MDA-MB-231 (human breast cancer cell line, non-amplifying, ATCC, cat. no. HTB-26) was cultured in DMEM. CALU3 (non-amplifying human lung adenocarcinoma cell line, ATCC, cat. no. HTB-55) was cultured in ATCC-prepared Minimal Essential Eagle's Medium. A549 (human lung carcinoma cell line, non-amplifying, ATCC, cat. no. CCL-185) was cultured in F12K medium. NCI-H2122 (human lung adenocarcinoma cell line, non-amplifying, ATCC, cat. no. CRL-5985) was cultured in RPMI1640 medium, (ATCC, cat. no. 30-2001). NCI-H460 (human lung carcinoma cell line, non-amplifying, ATCC, cat. no. HTB-177) was cultured in RPMI-1640 medium, (ATCC, cat. no. 30-2001). SHP-77 (human small cell lung cancer cell line, non-amplifying, ATCC, cat. no. CRL-2195) was cultured in RPMI-1640 medium, (ATCC, cat. no. 30-2001). CATO III (non-amplifying human gastric carcinoma cell line, ATCC, Cat. No. NTV-103) was cultured in Iskov's Dulbecco's medium with 20% FBS. MKN-45 III (non-amplifying human gastric adenocarcinoma cell line, DSMZ, Braunschweig, Germany, cat. no. ACC409) was cultured in RPMI-1640 medium, (ATCC, cat. no. 30-2001). SKOV3 (human ovarian adenocarcinoma cell line, ATCC, cat. no. HTB77) was cultured in McCoy's 5a medium. TOV-21G (non-amplifying human ovarian adenocarcinoma cell line, ATCC, cat. no. CRL-11730) was cultured in a 1:1 mixture of MCDB 105 medium containing a final concentration of 1.5 g/l sodium bicarbonate and 199 medium containing a final concentration of 2.2 g/l sodium bicarbonate. BT474 (Human Breast Cancer Cells Highly Expressing HER2, ATCC, Cat. No. HTB-20) were cultured in DMEM with 10% PBS. NCI-N87 (high HER2-expressing gastric cancer cell line, ATCC, cat. no. CRL-5822) was grown in RPMI-1640 medium with

10% FBS.

For cytotoxicity assays, cells were seeded at a density of 3000 cells per well in 96-well plates and allowed to adhere overnight by incubation at 37° C. in the presence of 5% CO ₂ . The medium was then replaced with fresh medium containing HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates in the concentration range (100 nM to 0.1 pM) or Kadcyla (Genentech) , and the cells were incubated for 72 h or 6 days at 37°C in the presence of 5% CO ₂ . Cell viability was measured using the CellTiter-Glo® Fluorescent Cell Viability Assay (Promega, Madison, WI) as described in the kit instructions. Cell viability was normalized to untreated control and expressed as a percentage. Values were plotted and IC ₅₀ values calculated using Graphpad Prism software (San Diego, CA) using a 4-parameter variable slope dose-response curve fitting algorithm. On the table VIII, illustrative results of the cytotoxicity of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and Kadcyla conjugates are given.

Table VIII

1S5v(nM)

cell line K-bo molecules HFR2 on cage Expression Neg2 Example 16G Example 16D Example 16B Example 16E Kadcyla JIMT-1 80000 2+ one 0.6 o o ^o BUT 17.5 IEA-MVL53 125000 2+ but 0.04 0.04/0.0 eight 0.04 -100 MDA-MB-361 135000 2+ but 0.02 0.005/ 0.001 but 0.27/0.3 3 MDA-MB- 175VII 51000 1+ but but 0.1 0.3 1.2 CAMA-1 50000 1+ but but 0.02 0.1 7.8 ZR75-1 40000 1+ but but 1.1 2.7 >100 HCC1187 38000 2+ but but 0.8 1.7 thirty HCC38 36000 2+ but but 0.8 4 >100 T47D 20000 1+ but but 4.3 1.2 82.3 HCC70 10000 1+ but but but 4 ^e 83.4 MDA-MB-231 5400 1+ but but eleven 6.3 ^e 27 NCI-H522 25000 1+ but 0.8 0.18 BUT -100 NCI-H1581 13000 1+ but 5.6 2.3 but -100 NCI-H2170 660000 3+ but but 0.07 0.08 0.4 CALU3 330000 3+ but but 0.25 0.15 >100 NCI-H2122 12000 1+ but but 0.5 2.5 nineteen A549 6000 1+ but but 20 10.8 ^e >100 NCI-H460 4000 1+ but but sixteen ^20th 68

BUT - not determined;

b - Example 16C was used in the analysis instead of Example 16B;

c is the activity equivalent to the relevant antibody control.

As shown in Table. VIII, all anti-HER2 antibody-polymer-drug conjugates are more active than Kadcyla for all cell lines tested.

Example 19 Cytotoxicity assays of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)) conjugates in a mutant HER2 cell line.

The antiproliferative properties of HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))) conjugates were evaluated on tumor cell lines in vitro using the Cell Titer-Glo method (Promega Corp.). NCIH1781 (Human lung cancer cells expressing mutant HER2, ATCC, cat. no. CRL-5894) were cultured in RPMI-1640 medium containing 10% PBS.

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For cytotoxicity assays, cells were seeded at a density of 3000 cells per well in 96-well plates and allowed to attach overnight by incubation at 37°C in the presence of 5% CO2. The medium was then replaced with fresh medium containing HER2-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates in the concentration range (100 nM to 0.1 pM), Example 17A (rituximab( EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) or Kadcyla (Genentech) and cells were incubated for 72 h at 37°C in the presence of 5% CO _2. Cell viability was measured using fluorescent cell viability assay (Promega, Madison, WI) as described in the kit instructions Cell viability was normalized to untreated control and expressed as a percentage Values were plotted and IC ₅₀ values calculated using Graphpad Prism software ( San Diego, California) using a 4-parameter variable-slope dose-response curve fitting algorithm Table IX summarizes the results of these analyses.

Table IX ,____________,____________IiC _S0 (hM)

cell line Number of molecules HER2 per cell Example 16E Example 16J Example 17A Kadcyla NCHI1781 8500 0.71 0.75 1.8 5.8

As shown in Table. IX, all anti-HER2 antibody-polymer-drug conjugates are more active than Kadcyla for all cell lines tested.

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

Female CB-17 SCID mice were subcutaneously inoculated 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), Kadcyla-resistant NCI-N87 cells (n=10 for each group), Kadsile cells NCIN87-MSA (n=10 for each group) or fragments of BT474 tumors (n=10 for each group). NCr nu/nu females were subcutaneously inoculated with TOV-21G cells (n=10 for each group). Doses of test compounds or vehicle were administered IV as a single dose on day 1 or as indicated. Tumor size was measured at the time points indicated in FIG. 8-18 using digital compasses. Tumor volume was calculated and used to determine tumor growth retardation. Mice were sacrificed when the tumors reached a size of 800 to 1500 mm ³ . Tumor volumes for each group are reported as mean ± SEM.

In FIG. 9 shows the results of tumor response in mice subcutaneously inoculated with NCI-N87 cells (n=10 for each group) after IV administration of vehicle; example 16A, trastuzumab (EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at a dose of 0.67 mg/kg, or example 16D, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at a dose of 0.67 mg/kg, as a single dose on day 1. For vehicle 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 assay for day 29.

In FIG. 10 shows the results of tumor response in mice subcutaneously inoculated with JIMT-1 cells (n=10 for each group) after IV administration of vehicle; Kadsil, at a dose of 20 mg / kg; example 16A, trastuzumab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at a dose of 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. Vehicle and Kadcyla showed an increase in tumor volume. For conjugates of trastuzumab-(EG2-MI(10 kDa PHF-BA-(AF-HPA-Ala))) and XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) therapeutic potential, and they provided, respectively, 40 and 70% regressions. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) provided 5 partial regressions and 2 complete regressions that left tumor-free survivors at the end of the study on day 69.

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

In FIG. 12 shows the results of tumor response in mice subcutaneously inoculated with NCI-N87 cells (n=10 for each group) after IV administration of vehicle; example 16F, XMT 1519-(EG2MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at a dose of 0.67 mg/kg as a single dose on day 1; combinations

- 89 039374 trastuzumab 15 mg/kg and pertuzumab 15 mg/kg administered weekly for 3 weeks (i.e. on days 1, 8 and 15), or a triple combination of trastuzumab 15 mg/kg, and pertuzumab, at 15 mg/kg, were each administered weekly for 3 weeks (i.e., days 1, 8, and 15) along with Example 16F, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at a dose of 0.67 mg/kg, as a single dose on day 1. An increase in tumor volume has been shown for the vehicle. All, XMT alone 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) or the combination, showed tumor reduction, and the triple combination of trastuzumab; pertuzumab and example 16F was the most effective and resulted in a 100% partial regression, while example 16F alone or the combination of trastuzumab and pertuzumab alone resulted in one partial response in ten. This triplet quantified a higher partial response rate and resulted in a significantly greater reduction in tumor volume (p<0.05, Mann-Whitney test) compared to either Example 16F alone or the trastuzumab + pertuzumab doublet. A partial response is defined as a regression to less than 50% of the original tumor volume maintained for at least 3 consecutive tumor measurements.

The overall survival benefit for XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))) in combination with pertuzumab and trastuzumab is significantly different compared to XMT 1519-(EG2-MI-(10 kDa of PHF-BA-(AF-HPA-Ala))) (P < 0.011, log-rank test) and exceeded the outcomes achieved with the combination of pertuzumab and trastuzumab (P < 0.273, log-range test) or the triple combination of XMT-1519 antibodies, pertuzumab and trastuzumab (P < 0.05, log-rank test).

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

In FIG. 14 shows the results of tumor response in mice subcutaneously inoculated with TOV-21G cells (n=10 for each group) after IV administration of vehicle; Kadcyla, at a dose of 10 mg/kg as a single dose; Example 16E, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at 5 or 2 mg/kg, as a single dose on Day 1. For XMT 1519-( EG2-MI-(10 kDa PHF-BA-(AFHPA-Ala))) was shown to delay tumor development and was more effective than Kadcyla, which did not reach the threshold of therapeutic activity.

In FIG. 15 shows the results of tumor response in mice subcutaneously inoculated with H522 cells (n=10 for each group) after IV administration of vehicle; Kadcyla, at a dose of 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 a dose of 5 mg/kg, as a single dose on day 1. For XMT conjugate 1519-(EG2-MI-(10 kDa PHF-BA-(AFHPA -Ala))) was shown to delay tumor development and was more effective than Kadcyla and rituximab(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), which did not reach the threshold of therapeutic activity.

In FIG. 16 shows the results of tumor response in mice subcutaneously inoculated with SKOV3 cells (n=10 for each group) after IV administration of vehicle; Kadcyla, at a dose of 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 a dose of 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 effective than Kadcyla and rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), which did not reach the threshold value of therapeutic activity.

In FIG. 17 shows the results of tumor response in mice subcutaneously inoculated with Calu-3 cells (n=10 for each group) after IV administration of vehicle or Example 16F, XMT 1519-(EG2MI-(10 kDa PHF-BA-(AF-HPA-Ala ))) at a dose of 5 mg/kg, as a single dose on day 1. XMT 1519-(EG2MI-(10 kDa PHF-BA-(AF-HPA-Ala))) resulted in 100% regressions consisting of two partial responses and eight complete responses, six of which retained the status of tumor-free on day 60.

In FIG. 18 shows the results (n=8 for each group) of tumor response in the BRE-0333 HER21+ xenograft model obtained from a patient after IV administration of vehicle; Kadcyla, at a dose of 10 mg/kg as a single dose; example 16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at 3 mg/kg as a single dose, or example 17B, rituximab-(EG2-MI- (10 kDa PHF-BA-(AFHPA-Ala))), at a dose of 3 mg/kg on day 1. For XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))), delayed tumor development and was more effective than Kadcyla or rituximab-(EG2-90 039374

MI-(10 kDa PHF-BA-(AF-HPA-Ala))).

In FIG. 19 shows the results (n=10 for each group) of tumor response in the MAXF1162 HER23+ xenograft model obtained from a patient after IV administration of vehicle; Kadcyla, at a dose of 10 mg/kg as a single dose; example 16H, XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AFHPA-Ala))) at 1 or 3 mg/kg, or example 17B, rituximab-(EG2-MI-(10 kDa PHF- BA-(AF-HPAAla))), at a dose of 3 mg/kg as a single dose on day 1. XMT 1519-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))) as in dose 1 , and 3 mg/kg resulted in 100% regression responses and tumor-free survival, and was more effective than Kadcyla or rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at a dose 3 mg/kg.

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

Single doses of 1 or 0.67 mg/kg XMT conjugate 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) showed complete regression in HER2 low-expressing breast and gastric cancer models in which Ado-trastuzumab emtansine (Kadcyla) was inactive at doses of 10 mg/kg and above. In HER2-mediated tumor models, XMT conjugate 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala))) shows synergistic efficacy in combination with the widely used anti-HER2 drugs trastuzumab and pertuzumab. The XMT conjugate 1519-(EG2-MI-(PHF-BA-(AF-HPA-Ala)) showed an excellent pharmacokinetic profile and was well tolerated by animal primates at therapeutic doses.

Preclinical data suggest that the XMT 1519-(EG2MI-(PHF-BA-(AF-HPA-Ala))) conjugate has the potential to increase the number of patients who may benefit from HER2-targeted therapies. This conjugate provides effective delivery to cancerous foci with as few as 10,000 HER2 receptors where other therapies are inactive.

Other embodiments of the invention.

Since the invention has been described in conjunction with its detailed description, the following description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications of the invention are within the scope of the following claims.

CLAIM

Claims (43)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, wherein the isolated antibody or antigen-binding fragment thereof contains a complementarity determining region (CDRH1) of the heavy chain variable domain containing the amino acid sequence FTFSSYSMN (SEQ ID NO: 25) ; a complementarity determining region (CDRH2) of a heavy chain variable domain containing the amino acid sequence YISSSSTIYYADSVKG (SEQ ID NO: 26); a complementarity determining region (CDRH3) of a heavy chain variable domain containing the amino acid sequence GGHGYFDL (SEQ ID NO: 27); a complementarity determining region (CDRL1) of a light chain variable domain containing the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 28); complementarity determining region (CDRL2) of a light chain variable domain containing the amino acid sequence GASSRAT (SEQ ID NO: 21) and a complementarity determining region (CDRL3) of a light chain variable domain containing the amino acid sequence QQYHHSPLT (SEQ ID NO: 29) . 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment thereof contains a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: fourteen. 3. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that the antibody or its antigen-binding fragment contains a heavy chain containing the amino acid sequence of SEQ ID NO: 5, and a light chain containing the amino acid sequence of SEQ ID NO: 6. 4. An isolated antibody or antigen-binding fragment thereof according to any of the preceding claims, characterized in that the antibody or antigen-binding fragment thereof is a monoclonal antibody, a domain antibody, a single-chain antibody, a Fab fragment, an F(ab')2 fragment, one - 91 039374 no chain variable fragment (scFv), scFv-Fc fragment, single chain antibody (scAb), domain antibody (dAb), heavy chain single domain antibody, or light chain single domain antibody. 5. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that the antibody or antigen-binding fragment thereof is a rabbit, mouse, chimeric, humanized or fully human monoclonal antibody. 6. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that the antibody or antigen-binding fragment thereof is an IgG isotype. 7. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that the antibody or antigen-binding fragment thereof is an IgG1 isotype. 8. An isolated nucleic acid molecule encoding an isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims. 9. An antibody expression vector according to any one of claims 1 to 7, containing the isolated nucleic acid molecule according to claim 8. 10. A method for producing an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 by culturing the cell under conditions that lead to the expression of said isolated antibody or antigen-binding fragment thereof, in which the cell contains the vector according to claim 9. 11. Conjugate for treating, preventing, slowing down the progression 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, esophageal cancer, laryngeal cancer , mesothelioma, skin cancer, myeloma, 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, cancer of the kidneys and cancer of the gastrointestinal tract, containing an isolated antibody or its antigen-binding fragment according to any one of claims 1-7 and one or more therapeutic agents (D), wherein the conjugate contains one or more polymer scaffolds connected to the antibody or its antigen-binding fragment , wherein one or more Ds are independently linked to the antibody or antigen-binding fragment thereof via one or more polymer backbones. 12. The conjugate of claim 11, wherein each of the one or more polymer backbones independently comprises a poly(1-hydroxymethylethylene hydroxymethylformal) (PHF) having a molecular weight ranging from about 2 kDa to about 40 kDa. 13. The conjugate according to claim 12, characterized in that each of the one or more polymer backbones is independently represented by formula (Ic) where L ^D1 represents a carbonyl-containing fragment; -c(=o)-1?C_ -c(=o)-l ^d, -|_ _d each presence of ^ς in ^ς is independently the first linker that contains a biodegradable bond such that when the bond is broken D is released in an active form for the manifestation of its intended therapeutic effect; and when T- - C (= °) - ^lD 4-D, _u _ the absence of ^ς in between L and D denotes a direct or indirect connection of D to L ^D1 ; ---C(=O)-L ^di —£-L ^p2 each presence of ^ζ is independently a second linker not yet connected to the isolated antibody or its antigen-binding fragment, in which LP ² is a fragment containing a functional group that all it will have to form a covalent bond with the functional group of the isolated antibody or its antigen-binding - 92 039374 fragment, and $ between L ^D1 and L ^P2 denotes direct or indirect attachment of L ^P2 to L ^d1 and each presence of the second linker does not coincide with each presence of the first linker; ---C(=O)-L ^d1 —|-l ^P2 —^— each presence of ^ς is independently a third linker that connects each D-bearing polymer scaffold to an isolated antibody or its antigen-binding fragment, in which the terminal , attached to L ^p2 denotes the direct or indirect attachment of L ^P2 to the selected antibody or antigen-binding fragment in the formation of a covalent bond between the functional group L ^P2 and the functional group of the selected antibody or antigen-binding fragment; and each presence of the third linker is not the same as each presence of the first linker; m is an integer from 1 to about 300; mi is an integer from 1 to about 140; m ₂ is an integer from 1 to about 40; m ₃ is an integer from 0 to about 18; m ₄ is an integer from 1 to about 10; the sum of m, m _b m ₂ , m ₃ and m ₄ is in the range from about 15 to about 300; and the total number of L ^p2 coupled to the isolated antibody or antigen-binding fragment thereof is 10 or less. 14. The conjugate according to claim 13, characterized in that the sum of m, m1, m ₂ , m ₃ and m ₄ is in the range from 15 to 150, m1 is an integer from 1 to 70, m ₂ is an integer from 1 to 20, m3 is an integer from 0 to 10, and PHF has a molecular weight in the range of about 2 kDa to about 20 kDa. 15. The conjugate according to claim 13, characterized in that the sum of m, m1, m ₂ , m3 and m ₄ is in the range from 20 to 110, m1 is an integer from 2 to 50, m ₂ is an integer from 2 up to 15, m3 is an integer from 0 to 8; a PHF has a molecular weight in the range of about 3 kDa to about 15 kDa. 16. The conjugate according to claim 13, characterized in that the sum of m, m ₁₅ m ₂ , m3 and m ₄ is in the range from 40 to 75, m1 is an integer from 2 to 35, m ₂ is an integer from 2 up to 10, m3 is an integer from 0 to 5; a PHF has a molecular weight in the range of about 5 kDa to about 10 kDa. 17. The conjugate according to claim 13, characterized in that the functional group L ^P2 is selected from -SR ^p , -S-So xA\\ LG, o and halogen, wherein LG is a substitutable group, R ^p is H or a sulfur protecting group, one of X _a and X _b is H and the other is a water-soluble maleimido blocking moiety, or X _a and X _b Together with the carbon atoms to which they are attached, they form a carbon-carbon double bond. 18. The conjugate according to claim 13, characterized in that L contains -X-(CH ₂ ) _v -C(=O)- with X directly ---C(=O)-L ^D1 -£_ connected to the carbonyl group h in which X represents CH ₂ , O or NH, a v is an integer from 1 to 6. ---C(=O)-L ^di -£-L ^p2 19. Conjugate according to claim 13, characterized in that each presence of ^ζ is independently -C(=O)-X-(CH ₂ ) _v -C(=O)-NH-(CH ₂ ) _u -NHC (=O)-(CH ₂ ) _w -(OCH ₂ CH ₂ ) _x -NHC(=O)(CH ₂ )yM, in which X represents CH ₂ , O or NH, each of v, u, w, x and y are independently is an integer from 1 to 6, and M is o, where one of X _a and X _b is H and the other is a water-soluble maleimido blocking moiety, or X _a and X _b together with the carbon atoms to which they attached, form a carbon-carbon double bond. 20. Conjugate according to claim 19, characterized in that each of v, u, w, x and y is equal to 2. 21. A conjugate according to any one of claims 11-20, characterized in that each of the one or more D's is a therapeutic agent having a molecular weight of <5 kDa. 22. The conjugate of claim 11, further comprising one or more D-bearing polymeric backbones coupled to the isolated antibody, each of the one or more polymeric backbones being independently represented by formula (Id) - 93 039374 (Id), where m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, and terminal denotes the direct attachment of one or more polymer scaffolds to the isolated antibody or antigen-binding fragment, and the ratio between D-bearing polymer scaffolds and antibody is 10 or less. 23. A conjugate according to any one of claims 11-20, characterized in that the isolated antibody or antigen-binding fragment thereof has a molecular weight of 40 kDa or more. 24. A conjugate according to claim 11 or 22, wherein each of the one or more Ds is independently linked to the antibody or antigen-binding fragment thereof via one or more non-polymeric binding fragments. 25. The conjugate of claim 11, further comprising one or more D-bearing polymeric backbones coupled to the isolated antibody, each of the one or more polymeric backbones being independently represented by the formula (If) he\ ^x he ^x he\>n\>> ° >O > _O HN HN ΗΝ HN ig / W\l BUT ΗΝ ΝΗ \ _n , C C c S \ A L /-o a Sm Em ° ° n ₂ yN ъ ъ coon (If), where sh. is an integer from 1 to about 300; m1 is an integer from 1 to about 140; w ₂ is an integer from 1 to about 40; w _3a is an integer from 0 to about 17; m _3b is an integer from 1 to about 8; TE 'on ^h o ² >= o HN HN 0=8 O . oM |1 V O Ome O OMa O Μθ - 94 039374 the sum of m _3a and m _3b ranges from 1 to about 18; and the sum of m, m _b m2, m _3a and m _3b is in the range from about 15 to about 300; terminal refers to the attachment of one or more polymer backbones to an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor, and wherein the ratio between D-bearing polymer backbones and antibody is 10 or less. 26. The conjugate according to claim 25, characterized in that the PHF in the formula (If) has a molecular weight in the range from about 2 kDa to about 20 kDa, the sum of m, m _b m2, m _3a and m _3b is in the range from about 15 to about 150, m ₁ is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m _3a is an integer from 0 to about 9, m _3b is an integer from 1 to about 8 , the sum of m _3a and m _3b ranges from 1 to about 10, and the ratio between PHF and isolated anti-HER2 antibody is an integer from 2 to about 8. 27. The conjugate according to claim 25, characterized in that the PHF in the formula (If) has a molecular weight in the range from about 3 kDa to about 15 kDa, the sum of m, m _b m ₂ , m _3a and m _3b is in the range from about 20 to about 110 m1 is an integer from 2 to about 50 m2 is an integer from 2 to about 15 m _3a is an integer from 0 to about 7 m _3b is an integer from 1 to about 8 , the sum of m _3a and m _3b ranges from 1 to about 8, and the ratio between PHF and isolated anti-HER2 antibody or antigen-binding fragment thereof is an integer from 2 to about 8. 28. The conjugate according to claim 25, characterized in that the PHF in the formula (If) has a molecular weight in the range from about 5 kDa to about 10 kDa, the sum of m, m _b m ₂ , m _3a and m _3b is in the range from about 40 to about 75 m1 is an integer from 2 to about 35 m2 is an integer from 2 to about 10 m _3a is an integer from 0 to about 4 m _3b is an integer from 1 to about 5 , the sum of m _3a and m _3b ranges from 1 to about 5, and the ratio between PHF and isolated anti-HER2 antibody is an integer from 2 to about 8. 29. The conjugate according to claim 25, characterized in that the PHF in the formula (If) has a molecular weight in the range from about 5 kDa to about 10 kDa, the sum of m, m _b m ₂ , m _3a and m _3b is in the range from about 40 to about 75, m1 is an integer from 2 to about 35, m2 is an integer from 2 to about 10, m3a is an integer from 0 to about 4, m3b is an integer from 1 to about 5, sum m _3a and m _3b range from 1 to about 5, and the ratio between PHF and isolated anti-HER2 antibody is an integer from 2 to about 6. 30. A method for producing a conjugate according to any one of claims 11 to 29, comprising the step of reacting an isolated antibody or an antigen-binding fragment thereof that specifically binds to an epitope of the human HER2 receptor with a polymer backbone of formula (Ia) to form a conjugate (1a), where L ^d1 represents a carbonyl-containing fragment; ---C(=O)-L ^d1 -|_ ---C(=O)-L ^D1 -|_ _D each present ⁱⁿ independently is the first linker that contains a biodegradable bond, such that when the bond is broken , then D is released in active form to manifest its intended therapeutic effect; and when> _C (= O). C4- _O D1 ________ __________ __________ the presence in ⁴ between L and D denotes a direct or indirect connection of D to L ^D1 ; ---C(=O)-L ^D1 —£-L ^p2 each presence of ^ζ is independently a second linker not yet connected to the isolated antibody or its antigen-binding fragment, in which L ^P2 is a fragment containing a functional group that all will have to form - 95 039374 covalent bond with the functional group of the isolated antibody or its antigen-binding fragment, and between L ^D1 and L ^P2 denotes a direct or indirect attachment of L ^P2 to L ^d1 and each presence of the second linker does not match each presence of the first linker; m is an integer from 1 to about 300; m1 is an integer from 1 to about 140; m ₂ is an integer from 1 to about 40; m3 is an integer from 1 to about 18; and the sum of m, m1, m ₂ and m ₃ ranges from about 15 to about 300. 31. A method of treating a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, esophageal cancer, laryngeal cancer, mesothelioma, skin cancer , myeloma, 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 gastrointestinal cancer. intestinal tract, in a subject in need, wherein the subject is administered a conjugate according to any one of claims 11-29 in a therapeutically effective amount. 32. The method of claim 31, wherein the subject is a human. 33. The method of claim 31 wherein the cancer is selected from the group consisting of breast cancer, gastrointestinal cancer, non-small cell lung cancer (NSCLC), and ovarian cancer. 34. The method of claim 31, further comprising the step of administering to the subject a second agent, wherein the second agent is at least a second antibody or antigen-binding fragment thereof that specifically binds HER2, wherein the second antibody or antigen-binding fragment thereof is an antibody against HER2, a HER2 dimerization inhibitor antibody, or a combination of an anti-HER2 antibody and a HER2 dimerization inhibitor antibody. 35. The method of claim 34, wherein the anti-HER2 antibody, HER2 dimerization inhibitor antibody, or combination of the anti-HER2 antibody and the HER2 dimerization inhibitor antibody comprises trastuzumab or pertuzumab, or a combination thereof. 36. The method of claim 31, wherein the subject is identified as having low HER2 expression. 37. The method according to claim 31, characterized in that the subject is identified as having an expression index of HER2 1+ or 2+, as detected by immunohistochemical (IHC) analysis performed on the studied cell population, while in the studied cell population the HER2 gene is not amplified. 38. The method of claim 31 wherein the subject is refractory to chemotherapy. 39. The method of claim 31 wherein the subject is not responsive to treatment with Kadcyla. 40. The method of claim 31 wherein the subject is responsive to treatment with Kadcyla. 41. The method according to claim 31, characterized in that the subject is identified as having an expression index of HER2 2+ or 3+, as detected by immunohistochemical (IHC) analysis performed on the studied cell population, while in the studied cell population the HER2 gene is amplified or is mutated. 42. The use of a conjugate according to any one of claims 11-29 for the manufacture of a medicament for the treatment of a cancer in a subject selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, esophageal cancer, laryngeal cancer, mesothelioma, skin cancer, myeloma, 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 gastrointestinal cancer. 43. Use according to claim 42, wherein the cancer is selected from the group consisting of breast cancer, gastrointestinal cancer, non-small cell lung cancer (NSCLC) and ovarian cancer.