Methods of treating biliary tract cancer using bispecific antigen binding constructs targeting HER2

Abstract

Described herein is a method of treating biliary tract cancer (BTC) comprising administering to a subject a bispecific antigen binding construct targeting HER2 or a bispecific antigen binding construct targeting HER2 (ADC) linked to an auristatin analogue.

Description

[0001] This application is a divisional application of the patent application with the filing date of May 31, 2019, application number 201980096969.4, and title "Methods of Treating Biliary Tract Cancer Using HER2-Targeted Bispecific Antigen-Binding Constructs".

[0002] Sequence Listing

[0003] This application contains a sequence listing that will be submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on May 31, 2019, named ZWI063sequencelisting.txt, and is 99,000 bytes in size. Background of the Invention

[0004] Biliary tract cancer (BTC), including gallbladder cancer and cholangiocarcinoma, is a rare malignancy with a poor prognosis. In the United States, the estimated annual incidence is 10,650 cases (Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: A Cancer Journal for Clinicians. 2014; 64(1):9 - 29). Most BTCs are diagnosed at an advanced stage, and only approximately 25% are surgically resectable. The 5-year overall survival rate is less than 10% (Anderson CD, Pinson CW, Berlin J, Chari RS. Diagnosis and treatment of cholangiocarcinoma. Oncologist. 2004; 9(1):43 - 57; de Groen PC, Gores GJ, LaRusso NF, Gunderson LL, Nagorney DM. Biliary Tract Cancers. N Engl J Med. 1999; 341(18):1368 - 78). HER2 is overexpressed in 3 - 25% of biliary tract cancers (Benavides M, Antón A, Gallego J, Gómez MA, Jiménez-Gordo A, La Casta A et al. Biliary tract cancers: SEOM clinical guidelines. Clinical and Translational Oncology. 2015; 17(12):982 - 7).

[0005] First-line treatment options for unresectable BTC include systemic chemotherapy (gemcitabline plus cisplatin, which provides a survival benefit [11.7 months vs. 8.1 months, respectively] compared to gemcitabline alone). Alternative first-line treatments include pembrolizumab for MSI-H / dMMR tumors, fluoropyrimidine-based chemoradiation, radiation without additional chemotherapy, investigational agents, or best supportive care. There is a lack of supportive evidence for second-line chemotherapy for BTC, and clinical trials are recommended (NCCN Clinical Practice Guidelines: Hepatobiliary Cancers. Version 2. 2019).

[0006] There is still a need for treatments for biliary tract cancer.

[0007] International Patent Publication No. WO2015 / 077891 describes bispecific anti-HER2 antibodies targeting two different HER2 epitopes in ECD4 and ECD2, which are the same epitopes as those bound by trastuzumab and pertuzumab. SUMMARY OF THE INVENTION

[0008] Described herein are methods of treating biliary tract cancer using HER2-targeted bispecific antigen-binding constructs. In one aspect of the present disclosure, provided is a method of treating a subject having biliary tract cancer (BTC), comprising administering to the subject an effective amount of a bispecific anti-HER2 antigen-binding construct or an antibody-drug conjugate (ADC).

[0009] In some embodiments, the BTC is resectable, partially resectable, or unresectable.

[0010] In some embodiments, the BTC is advanced.

[0011] In some embodiments, as measured by immunohistochemistry (IHC), the BTC is HER2 3+, HER2 2+, or HER2 1+ and is gene amplified.

[0012] In some embodiments, as measured by immunohistochemistry (IHC), the BTC is HER2 3+, HER2 2+, or HER2 1+ and there is no HER2 gene amplification.

[0013] In some embodiments, the BTC is gallbladder cancer.

[0014] In some embodiments, the BTC is cholangiocarcinoma (CCA).

[0015] In some embodiments, the bispecific anti-HER2 antigen-binding construct comprises heavy chain H1, heavy chain H2, and light chain L1, wherein: a) heavy chain H1 comprises the CDR sequences shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; b) heavy chain H2 comprises the CDR sequences shown in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72; and c) light chain L1 comprises the CDR sequences shown in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0016] In some embodiments, the bispecific anti-HER2 antigen-binding construct comprises heavy chain H1 comprising the amino acid sequence shown in SEQ ID NO:36, heavy chain H2 comprising the amino acid sequence shown in SEQ ID NO:63, and light chain L1 comprising the amino acid sequence shown in SEQ ID NO:24.

[0017] In some embodiments, an effective amount of the bispecific anti-HER2 antigen-binding construct is 10 mg / kg per week.

[0018] In some embodiments, an effective amount of the bispecific anti-HER2 antigen-binding construct is 20 mg / kg every two weeks.

[0019] In some embodiments, an effective amount of the bispecific anti-HER2 antigen-binding construct is 30 mg / kg every three weeks.

[0020] In some embodiments, administering the bispecific anti-HER2 antigen-binding construct to a subject elicits a complete response (CR), partial response (PR), or stable disease (SD) in the subject.

[0021] In some embodiments, the disease control rate of a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 60%, 70%, or 80%.

[0022] In some embodiments, the overall response rate of a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 50%, 60%, 70%, or 80%.

[0023] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered after at least one, two, or three first-line therapies.

[0024] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered as a first-line monotherapy.

[0025] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered as adjuvant therapy or neoadjuvant therapy.

[0026] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered in combination with one or more chemotherapeutic agents.

[0027] In some embodiments, the one or more chemotherapeutic agents are gemcitabine and / or cisplatin.

[0028] In another aspect of the present disclosure, there is provided the use of a bispecific anti-HER2 antigen-binding construct or an antibody-drug conjugate (ADC) in the preparation of a medicament for the treatment of biliary tract cancer (BTC).

[0029] In yet another aspect of the present disclosure, there is provided the use of an effective amount of a bispecific anti-HER2 antigen-binding construct or ADC for the treatment of BTC in a subject.

[0030] This application also includes the following embodiments.

[0031] 1. A method of treating a subject having biliary tract cancer (BTC), the method comprising administering to the subject an effective amount of a bispecific anti-HER2 antigen-binding construct or an antibody-drug conjugate (ADC).

[0032] 2. The method according to embodiment 1, wherein the BTC is resectable, partially resectable or non-resectable.

[0033] 3. The method according to embodiment 1, wherein the BTC is advanced.

[0034] 4. The method according to any one of embodiments 1 to 3, wherein the BTC is HER2 3+, HER2 2+ or HER2 1+ as measured by immunohistochemistry (IHC) and is gene amplification-positive.

[0035] 5. The method according to any one of embodiments 1 to 3, wherein the BTC is HER2 3+, HER2 2+ or HER2 1+ as measured by immunohistochemistry (IHC) and there is no HER2 gene amplification.

[0036] 6. The method according to any one of embodiments 1 to 5, wherein the BTC is gallbladder cancer.

[0037] 7. The method according to any one of embodiments 1 to 5, wherein the BTC is cholangiocarcinoma (CCA).

[0038] 8. The method according to any one of embodiments 1 to 7, wherein the bispecific anti-HER2 antigen-binding construct comprises heavy chain H1, heavy chain H2 and light chain L1, wherein: a) heavy chain H1 comprises the CDR sequences shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41; b) heavy chain H2 comprises the CDR sequences shown in SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72; and c) light chain L1 comprises the CDR sequences shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

[0039] 9. The method according to any one of embodiments 1 to 7, wherein the bispecific anti-HER2 antigen-binding construct comprises heavy chain H1 comprising the amino acid sequence shown in SEQ ID NO: 36, heavy chain H2 comprising the amino acid sequence shown in SEQ ID NO: 63 and light chain L1 comprising the amino acid sequence shown in SEQ ID NO: 24.

[0040] 10. The method according to embodiment 8 or 9, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 10 mg / kg per week.

[0041] 11. The method according to embodiment 8 or 9, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 20 mg / kg every two weeks.

[0042] 12. The method according to embodiment 8 or 9, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 30 mg / kg every three weeks.

[0043] 13. The method according to any one of embodiments 1 to 12, wherein administering the bispecific anti-HER2 antigen-binding construct to the subject elicits a complete response (CR), partial response (PR) or stable disease (SD) in the subject.

[0044] 14. The method according to any one of embodiments 1 to 12, wherein the disease control rate in a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 60%, 70% or 80%.

[0045] 15. The method according to any one of embodiments 1 to 12, wherein the overall response rate in a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 50%, 60%, 70% or 80%.

[0046] 16. A method according to any one of embodiments 1 to 12, wherein the bispecific anti-HER2 antigen-binding construct is administered after at least one, two or three first-line therapies.

[0047] 17. A method according to any one of embodiments 1 to 12, wherein the bispecific anti-HER2 antigen-binding construct is administered as a first-line monotherapy.

[0048] 18. A method according to any one of embodiments 1 to 17, wherein the bispecific anti-HER2 antigen-binding construct is administered as adjuvant therapy or neoadjuvant therapy.

[0049] 19. A method according to any one of embodiments 1 to 17, wherein the bispecific anti-HER2 antigen-binding construct is administered in combination with one or more chemotherapeutic agents.

[0050] 20. A method according to embodiment 19, wherein the one or more chemotherapeutic agents are gemcitabine and / or cisplatin.

[0051] 21. Use of a bispecific anti-HER2 antigen-binding construct or an antibody-drug conjugate (ADC) in the preparation of a medicament for the treatment of biliary tract cancer (BTC).

[0052] 22. Use according to embodiment 21, wherein the BTC is resectable, partially resectable or non-resectable.

[0053] 23. Use according to embodiment 21, wherein the BTC is advanced.

[0054] 24. Use according to any one of embodiments 21 to 23, wherein the BTC is HER2 3+, HER2 2+ or HER2 1+ as measured by immunohistochemistry (IHC) and is gene amplified.

[0055] 25. Use according to any one of embodiments 21 to 23, wherein the BTC is HER2 3+, HER2 2+ or HER2 1+ as measured by immunohistochemistry (IHC) and there is no HER2 gene amplification.

[0056] 26. Use according to any one of embodiments 21 to 25, wherein the BTC is gallbladder cancer.

[0057] 27. Use according to any one of embodiments 21 to 25, wherein the BTC is cholangiocarcinoma (CCA).

[0058] 28. The use according to any one of embodiments 21 to 27, wherein the bispecific anti-HER2 antigen-binding construct comprises a heavy chain H1, a heavy chain H2, and a light chain L1, wherein: a) the heavy chain H1 comprises the CDR sequences shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41; b) the heavy chain H2 comprises the CDR sequences shown in SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72; and c) the light chain L1 comprises the CDR sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.

[0059] 29. The use according to any one of embodiments 21 to 27, wherein the bispecific anti-HER2 antigen-binding construct comprises a heavy chain H1 comprising the amino acid sequence shown in SEQ ID NO: 36, a heavy chain H2 comprising the amino acid sequence shown in SEQ ID NO: 63, and a light chain L1 comprising the amino acid sequence shown in SEQ ID NO: 24.

[0060] 30. The use according to embodiment 28 or 29, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 10 mg / kg per week.

[0061] 31. The use according to embodiment 28 or 29, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 20 mg / kg every two weeks.

[0062] 32. The use according to embodiment 28 or 29, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 30 mg / kg every three weeks.

[0063] 33. The use according to any one of embodiments 21 to 32, wherein administering the bispecific anti-HER2 antigen-binding construct to the subject elicits a complete response (CR), a partial response (PR), or stable disease (SD) in the subject.

[0064] 34. The use according to any one of embodiments 21 to 32, wherein the disease control rate in a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 60%, 70%, or 80%.

[0065] 35. The use according to any one of embodiments 21 to 32, wherein the overall response rate in a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 50%, 60%, 70%, or 80%.

[0066] 36. Use according to any one of embodiments 21 to 32, wherein the bispecific anti-HER2 antigen-binding construct is administered after at least one, two or three first-line therapies.

[0067] 37. Use according to any one of embodiments 21 to 32, wherein the bispecific anti-HER2 antigen-binding construct is administered as a first-line monotherapy.

[0068] 38. Use according to any one of embodiments 21 to 37, wherein the bispecific anti-HER2 antigen-binding construct is administered as adjuvant therapy or neoadjuvant therapy.

[0069] 39. Use according to any one of embodiments 21 to 37, wherein the bispecific anti-HER2 antigen-binding construct is administered in combination with one or more chemotherapeutic agents.

[0070] 40. Use according to embodiment 39, wherein the one or more chemotherapeutic agents are gemcitabine and / or cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 A diagram depicting an exemplary bispecific anti-HER2 antigen-binding construct in Fab / scFv form.

[0072] Figure 2 Depicts the duration of treatment and the maximum reduction in the sum of diameters (SOD) in a subject with BTC treated with v10000. DETAILED DESCRIPTION

[0073] Described herein is a method of treating a subject with biliary tract cancer (BTC), comprising administering to the patient a bispecific antigen-binding construct that targets HER2. In some embodiments, the bispecific antigen-binding construct that targets HER2 is linked to an auristatin analogue (referred to herein as an antibody-drug conjugate or ADC). In some embodiments, the bispecific antigen-binding construct that targets HER2 can be used in a method of treating gallbladder cancer or cholangiocarcinoma. In other embodiments, administration of the bispecific antigen-binding construct that targets HER2 to a subject with BTC can result in a reduction in the size of a tumor or lesion in the subject. In other embodiments, administration of the bispecific antigen-binding construct that targets HER2 can result in a complete response (CR), partial response (PR) or stable disease (SD) in the subject, as measured by the RECIST 1.1 guidelines.

[0074] The present disclosure also describes a method of treating BTC, comprising administering to a subject a HER2-targeting bispecific antigen-binding construct in combination with one or more chemotherapeutic agents.

[0075] Definitions

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0077] As used herein, unless otherwise specified, the term "about" refers to a variation of approximately + / - 10% from a given value. It is to be understood that such variation is always included in any given value provided herein, whether or not specifically recited.

[0078] The word "a / an" as used herein in conjunction with the term "comprising" can mean "one", but in some embodiments also is consistent with the meaning of "one or more", "at least one", or "one or more than one".

[0079] As used herein, the terms "comprising", "having", "including", and "containing" and their grammatical variants are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. The term "consisting essentially of" when used in conjunction with a composition, use, or method herein means that additional elements and / or method steps may be present, but these additions do not materially affect the manner in which the recited composition, method, or use functions. The term "consisting of" when used in conjunction with a composition, use, or method herein excludes the presence of additional elements and / or method steps. Compositions, uses, or methods described herein as including certain elements and / or steps may in some embodiments also consist essentially of those elements and / or steps, and in other embodiments consist of those elements and / or steps, whether or not such embodiments are expressly recited.

[0080] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use, or composition disclosed herein.

[0081] Particular features, structures, and / or characteristics described in connection with an embodiment disclosed herein can be combined with features, structures, and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more other embodiments.

[0082] It should also be understood that an affirmative recitation of a feature in one embodiment serves as a basis for excluding that feature in alternative embodiments. For example, in the case where a list of options is provided for a given embodiment or claim, it should be understood that one or more of the options can be removed from the list and the shortened list can form an alternative embodiment, whether or not such alternative embodiment is expressly recited.

[0083] Bispecific antigen-binding construct that binds HER2

[0084] Bispecific antigen-binding constructs that bind HER2 (also referred to as bispecific anti-HER2 antigen-binding constructs) are described below.

[0085] The term "antigen-binding construct" refers to a reagent, such as a polypeptide or polypeptide complex, that is capable of binding an antigen. In some aspects, an antigen-binding construct is a polypeptide that specifically binds to an antigen of interest. An antigen-binding construct can be a monomer, dimer, multimer, protein, peptide, or protein or peptide complex; an antibody, antibody fragment, or antigen-binding fragment thereof; an scFv, etc. An antigen-binding construct can be a monospecific, bispecific, or multispecific polypeptide construct. In some aspects, an antigen-binding construct can comprise, for example, one or more antigen-binding moieties (e.g., Fab or scFv) linked to one or more Fc's. Other examples of antigen-binding constructs are described below and provided in the Examples.

[0086] The term "bispecific" is intended to include any reagent, such as an antigen-binding construct, that has two antigen-binding portions (e.g., an antigen-binding polypeptide construct), each with a distinct binding specificity. For example, a first antigen-binding portion binds an epitope on a first antigen, and a second antigen-binding portion binds an epitope on a second antigen. As used herein, the term "bifunctional" refers to a bispecific antibody in which the first antigen-binding portion and the second antigen-binding portion bind different epitopes on the same antigen. A bifunctional bispecific antibody can bind two epitopes on the same antigen molecule, or it can bind epitopes on two different antigen molecules.

[0087] A monospecific antigen-binding construct is an antigen-binding construct that has one binding specificity. In other words, both antigen-binding portions bind the same epitope on the same antigen. Examples of monospecific antigen-binding constructs include trastuzumab and pertuzumab, which bind HER2.

[0088] An antigen-binding construct can be an antibody or an antigen-binding portion thereof. As used herein, "antibody" or "immunoglobulin" refers to a polypeptide encoded, at least in part, by one or more immunoglobulin genes or fragments thereof, which specifically binds and recognizes an analyte (e.g., an antigen). Well-known immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as κ or λ. The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0089] Exemplary immunoglobulin (antibody) structural units are composed of two pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50 - 70 kD). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. An IgG1 heavy chain contains, from the N-terminus to the C-terminus, VH, CH1, CH2, and CH3 domains. A light chain contains VL and CL domains from the N-terminus to the C-terminus. An IgG1 heavy chain contains a hinge between the CH1 and CH2 domains. In certain embodiments, an immunoglobulin construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE attached to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antigen-binding constructs provided herein are from or derived from immunoglobulin-based constructs such as diabodies or nanobodies. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody such as a camelid antibody. In certain embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, a human antibody, a camelid antibody, a murine antibody, or any chimeric antibody.

[0090] "Complementary determining region" or "CDR" refers to amino acid sequences that contribute to antigen-binding specificity and affinity. The "framework" regions (FRs) help maintain the proper conformation of the CDRs to facilitate binding between the antigen-binding regions and the antigen. Structurally, the framework regions can be located in the antibody between the CDRs. The variable region typically exhibits the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (also known as CDRs). The CDRs from the variable domains of the heavy and light chains are typically aligned by the framework regions, which enables binding to a specific epitope. From the N-terminus to the C-terminus, the variable domains of the light and heavy chains typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Unless otherwise specified, the assignment of amino acids to each domain is typically according to the definition in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)). Generally, there are three heavy-chain and three light-chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. The three heavy-chain CDRs are referred to herein as CDRH1, CDRH2, and CDRH3, while the three light-chain CDRs are referred to as CDRL1, CDRL2, and CDRL3. Thus, as used herein, "CDR" can refer to all three heavy-chain CDRs or all three light-chain CDRs (or all heavy-chain CDRs and all light-chain CDRs, if appropriate). The CDRs provide most of the contact residues for the binding of the antibody to the antigen or epitope. Generally, three heavy-chain CDRs and three light-chain CDRs are required for antigen binding. However, in some cases, even a single variable domain can confer antigen-binding specificity. In addition, as is known in the art, in some cases, antigen binding can also occur through combinations of at least one or more CDRs (such as CDRH3) selected from the VH and / or VL domains.

[0091] Many different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369 - 847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901 - 917), and those defined and described by IMGT, AbM (University of Bath), and Contact (MacCallum R.M., Martin A.C.R., and Thornton J.M., (1996), Journal of Molecular Biology, 262(5), 732 - 745). For example, Table 1 below provides CDR definitions according to Kabat, Chothia, IMGT, AbM, and Contact. Thus, as will be apparent to those skilled in the art, the exact numbering and location of CDRs can vary based on the numbering system employed. However, it should be understood that the disclosure of VH herein includes the disclosure of the relevant (native) heavy - chain CDRs (HCDRs) defined by any known numbering system. Similarly, the disclosure of VL herein includes the disclosure of the relevant (native) light - chain CDRs (LCDRs) defined by any known numbering system.

[0092] Table 1: Common CDR Definitions 1

[0093]

[0094] 1 The Kabat or Chothia numbering system can be used for HCDR2, HCDR3, and light - chain CDRs for all definitions except Contact, which uses the Chothia numbering

[0095] 2 Using the Kabat numbering. The position in the Kabat numbering scheme that demarcates the end of the Chothia and IMGT CDR - H1 loops varies depending on the loop length, since Kabat places insertions at positions 35A and 35B outside these CDR definitions. However, the IMGT and Chothia CDR - H1 loops can be clearly defined using the Chothia numbering. CDR - H1 definition using Chothia numbering: Kabat H31 - H35, Chothia H26 - H32, AbM H26 - H35, IMGT H26 - H33, Contact H30 - H35.

[0096] As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen-binding polypeptide constructs is a single-chain Fv molecule (scFv). As described in more detail herein, the scFv has a light chain variable domain (VL) whose C-terminus is linked to the N-terminus of the heavy chain variable domain (VH) by a polypeptide chain. Alternatively, the scFv can be a polypeptide chain in which the C-terminus of the VH is linked to the N-terminus of the VL by a polypeptide chain.

[0097] Antigen-binding polypeptide construct

[0098] The bispecific anti-HER2 antigen-binding construct comprises two antigen-binding polypeptide constructs, each binding to a specific domain or epitope of HER2. In one embodiment, each antigen-binding polypeptide construct binds to the extracellular domain of HER2, such as ECD2 or ECD4. Depending on the application, the antigen-binding polypeptide construct can be, for example, a Fab or an scFv.

[0099] The form of the bispecific anti-HER2 antigen-binding construct determines the functional properties of the bispecific anti-HER2 antigen-binding construct. In one embodiment, the bispecific anti-HER2 antigen-binding construct has an scFv-Fab form (i.e., one antigen-binding polypeptide construct is an scFv and the other antigen-binding polypeptide construct is a Fab, also referred to as the Fab-scFv form). In another embodiment, the bispecific anti-HER2 antigen-binding construct has an scFv-scFv form (i.e., both antigen-binding polypeptide constructs are scFvs).

[0100] A "Fab fragment" (also referred to as an antigen-binding fragment) contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1) as well as the variable domains VL and VH on the light chain and heavy chain, respectively. The variable domains contain complementarity-determining loops (CDRs, also referred to as hypervariable regions) involved in antigen binding. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

[0101] "Single-chain Fv" or "scFv" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker located between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0102] Form and Function of Antigen-Binding Constructs

[0103] Provided herein are bispecific anti-HER2 antigen-binding constructs having two antigen-binding polypeptide constructs, wherein the first binds specifically to HER2 ECD2 and wherein the second binds specifically to HER2 ECD4. The form of the bispecific anti-HER2 antigen-binding construct is such that at least one of the first or second antigen-binding polypeptides is an scFv. The form of the bispecific anti-HER2 antigen-binding construct can be scFv-scFv, or Fab-scFv or scFv-Fab (first antigen-binding polypeptide construct - second antigen-binding polypeptide, respectively).

[0104] In certain embodiments, the bispecific anti-HER2 antigen-binding constructs exhibit anti-tumor activity in vitro, such as (i) the ability to inhibit cancer cell growth in the presence or absence of epidermal growth factor or heregulin stimulation, (ii) the ability to internalize in cancer cells (by binding to the HER2 antigen and internalizing it) and (iii) the ability to mediate antibody-directed effector cell killing (ADCC). These in vitro activities are observed with both naked bispecific anti-HER2 antigen-binding constructs and bispecific anti-HER2 antigen-binding constructs conjugated to auristatin analogs and at different levels of HER2 expression (1+, 2+ and 3+).

[0105] As described in International Patent Publication No. WO2015 / 077891, the format of the bispecific anti-HER2 antigen-binding construct (scFv / scFv, scFv / Fab, or Fab / Fab) is important for determining its functional profile. In certain embodiments, the anti-HER2 binding construct exhibits an increased ability to be internalized by tumor cells expressing HER2 compared to a reference antigen-binding construct in which both the ECD2- and ECD4-binding polypeptide constructs are Fabs. It is expected that the degree of internalization of the bispecific anti-HER2 antigen-binding construct can be further enhanced by increasing the affinity of one or both of the antigen-binding polypeptide constructs for ECD2 or ECD4. In one embodiment in which the ECD2-binding polypeptide is a Fab and the ECD4-binding polypeptide is an scFv, the construct is internalized to a greater extent compared to a construct of equal affinity having a Fab / Fab format, and is internalized to a similar extent by both high- and low-expressing HER2 tumor cells as a construct of equal affinity having an scFv / scFv format. Embodiments that are readily internalized are good candidates for antibody-drug conjugates, which require internalization by tumor cells to effect killing. In contrast, in certain embodiments, bispecific anti-HER2 antigen-binding constructs that are not readily internalized exhibit increased potency in ADCC killing of tumor cells expressing low levels of HER2. In one embodiment, a bispecific anti-HER2 antigen-binding construct having a Fab / scFv format is more effective than an anti-HER2 construct having a Fab / Fab format in ADCC killing of tumor cells expressing low levels of HER2 (HER2 0-1+ or 1+), while an anti-HER2 construct having a Fab / Fab format is in turn more effective than a bispecific anti-HER2 antigen-binding construct having an scFv / scFv format. The enhanced ADCC potency of some embodiments may be due to 1) their enhanced ability to bind avidly to cells with low HER2 receptor density and subsequently cluster HER2 receptors on the surface of the target cell and mediate downstream cell-mediated killing; and / or 2) their enhanced ability to remain on the cell surface (as opposed to causing internalization); and thus they are more useful for cell-mediated effector killing.

[0106] HER2

[0107] The bispecific anti-HER2 antigen-binding constructs described herein comprise antigen-binding polypeptide constructs that bind to ECD2 and ECD4 of HER2.

[0108] The terms "ErbB2" and "HER2" are used interchangeably herein and refer to the human HER2 protein as described, for example, in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al., Nature 319:230-234 (1986) (Genebank accession number X03363). The terms "erbB2" and "neu" refer to the gene encoding the human ErbB2 protein. p185 or p185neu refers to the protein product of the neu gene.

[0109] HER2 is a HER receptor. A "HER receptor" is a receptor protein tyrosine kinase that belongs to the human epidermal growth factor receptor (HER) family and includes the EGFR, HER2, HER3, and HER4 receptors. HER receptors generally will comprise an extracellular domain that can bind a HER ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signaling domain with several tyrosine residues that can be phosphorylated. A "HER ligand" refers to a polypeptide that binds and / or activates a HER receptor.

[0110] The extracellular domain of HER2 contains four domains, domain I (ECD1, amino acid residues approximately 1-195), domain II (ECD2, amino acid residues approximately 196-319), domain III (ECD3, amino acid residues approximately 320-488), and domain IV (ECD4, amino acid residues approximately 489-630) (residue numbering, without signal peptide). See Garrett et al., Mol. Cell. 11:495-505 (2003); Cho et al., Nature 421:756-760 (2003); Franklin et al., Cancer Cell 5:317-328 (2004); Tse et al., Cancer Treat Rev. April 2012; 38(2):133-42 (2012); or Plowman et al., Proc. Natl. Acad. Sci. 90:1746-1750 (1993).

[0111] The sequence of HER2 is as follows; the ECD boundaries are domain I: 1-165; domain II: 166-322; domain III: 323-488; domain IV: 489-607.

[0112]

[0113] "Epitope 2C4" is the region in the HER2 extracellular domain that binds to antibody 2C4. Epitope 2C4 contains residues from domain II in the HER2 extracellular domain. 2C4 and pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II, and III. Franklin et al., Cancer Cell 5:317-328 (2004). To screen for antibodies that bind to the 2C4 epitope, conventional cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be used to assess whether an antibody binds to the 2C4 epitope of HER2 and / or the antibody-HER2 structure can be studied (Franklin et al., Cancer Cell 5:317-328 (2004)) to see which domains of HER2 are bound by the antibody.

[0114] "Epitope 4D5" is the region in the HER2 extracellular domain that binds to antibody 4D5 (ATCC CRL 10463) and trastuzumab. This epitope is near the transmembrane domain of HER2 and is within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, conventional cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues (including the end values) in the region from about residue 529 to about residue 625, see Figure 1 ) of U.S. Patent Publication No. 2006 / 0018899.

[0115] "Specifically binds", "specific binding", or "selective binding" means binding that is selective for an antigen and distinguishable from unwanted or non-specific interactions. The ability of a bispecific anti-HER2 antigen-binding construct to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of the antigen-binding portion to an irrelevant protein is less than about 10% of the degree of binding of the bispecific anti-HER2 antigen-binding construct to the antigen, as measured, for example, by SPR. In certain embodiments, the bispecific anti-HER2 antigen-binding construct that binds to the antigen or an antigen-binding molecule comprising the antigen-binding portion has a dissociation constant (K D ) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 ～8 M or less, e.g., 10 ～8 M to 10" 13 M, e.g., 10" 9 M to 10" 13 M).

[0116] "Heregulin" (HRG), as used herein, refers to a polypeptide encoded by a heregulin gene product as disclosed in, for example, U.S. Patent No. 5,641,869 or Marchionni et al., Nature, 362:312-318 (1993). Examples of heregulin include heregulin-α, heregulin-β1, heregulin-β2, and heregulin-β3 (Holmes et al., Science, 256:1205-1210 (1992); and U.S. Patent No. 5,641,869); neu differentiation factor (NDF) (Peles et al., Cell 69:205-216 (1992)); acetylcholine receptor-inducing activity (ARIA) (Falls et al., Cell 72:801-815 (1993)); glial growth factor (GGF) (Marchionni et al., Nature, 362:312-318 (1993)); sensory and motor neuron-derived factor (SMDF) (Ho et al., J. Biol. Chem. 270:14523-14532 (1995)); γ-heregulin (Schaefer et al., Oncogene 15:1385-1394 (1997)). The term includes bioactive fragments and / or amino acid sequence variants of the native sequence HRG polypeptide, such as its EGF-like domain fragment (e.g., HRGβ1 177-244).

[0117] "HER activation" or "HER2 activation" refers to the activation or phosphorylation of any one or more HER receptors or HER2 receptors. Generally, HER activation results in signal transduction (e.g., caused by phosphorylation of tyrosine residues in the HER receptor or a substrate polypeptide by the intracellular kinase domain of the HER receptor). HER activation can be mediated by the binding of a HER ligand to a HER dimer containing the HER receptor of interest. The binding of a HER ligand to a HER dimer can activate the kinase domain of one or more HER receptors in the dimer, resulting in phosphorylation of tyrosine residues in one or more HER receptors and / or phosphorylation of tyrosine residues in other substrate polypeptides (e.g., Akt or MAPK intracellular kinases).

[0118] A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient hypervariable regions are replaced by residues from the hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, a humanized antibody will contain substantially all of at least one and usually two variable domains, in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody optionally will also contain at least a portion of the immunoglobulin constant region (Fc), usually the constant region of a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0119] Fc of bispecific anti-HER2 antigen-binding construct.

[0120] In some embodiments, the bispecific anti-HER2 antigen-binding constructs described herein contain an Fc, such as a dimeric Fc.

[0121] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The "Fc polypeptide" of a dimeric Fc as used herein refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide that contains the C-terminal constant region of an immunoglobulin heavy chain and is capable of stable self-association. For example, the Fc polypeptide of a dimeric IgG Fc contains IgG CH2 and IgG CH3 constant domain sequences.

[0122] The Fc domain comprises a CH3 domain or CH3 and CH2 domains. The CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc. The CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc.

[0123] In some aspects, the Fc comprises at least one or two CH3 sequences. In some aspects, the Fc is conjugated to a first antigen-binding polypeptide construct and / or a second antigen-binding polypeptide construct, with or without one or more linkers. In some aspects, the Fc is a human Fc. In some aspects, the Fc is a human IgG or IgG1 Fc. In some aspects, the Fc is a heterodimeric Fc. In some aspects, the Fc comprises at least one or two CH2 sequences.

[0124] In some aspects, the Fc comprises one or more modifications in at least one CH3 sequence. In some aspects, the Fc comprises one or more modifications in at least one CH2 sequence. In some aspects, the Fc is a single polypeptide. In some aspects, the Fc is multiple peptides, such as two polypeptides.

[0125] In some aspects, the Fc is the Fc described in patent application PCT / CA2011 / 001238 filed on November 4, 2011 or PCT / CA2012 / 050780 filed on November 2, 2012, the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0126] Modified CH3 domain

[0127] In some aspects, the bispecific anti-HER2 antigen-binding construct described herein comprises a heterodimeric Fc comprising a modified CH3 domain that has been asymmetrically modified. The heterodimeric Fc can comprise two heavy chain constant domain polypeptides: a first Fc polypeptide and a second Fc polypeptide, which can be used interchangeably provided that the Fc comprises one first Fc polypeptide and one second Fc polypeptide. Generally, the first Fc polypeptide comprises a first CH3 sequence and the second Fc polypeptide comprises a second CH3 sequence.

[0128] When two CH3 sequences dimerize, two CH3 sequences containing one or more amino acid modifications introduced in an asymmetric manner typically produce a heterodimeric Fc rather than a homodimer. As used herein, "asymmetric amino acid modification" refers to any modification in which the amino acid at a particular position on the first CH3 sequence is different from the amino acid at the same position on the second CH3 sequence, and the first and second CH3 sequences preferentially pair to form a heterodimer rather than a homodimer. Such heterodimerization can be the result of modification of only one of two amino acids at the same corresponding amino acid position on each sequence; or modification of each of the two amino acids at the same corresponding position on each of the first and second CH3 sequences. The first and second CH3 sequences of the heterodimeric Fc can contain one or more than one asymmetric amino acid modification.

[0129] Table 2 provides the amino acid sequence of the human IgG1 Fc sequence, corresponding to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence contains amino acids 341 - 447 of the full-length human IgG1 heavy chain.

[0130] Generally, Fc can include two contiguous heavy chain sequences (A and B) capable of dimerizing. In some aspects, one or both sequences of Fc include one or more mutations or modifications at the following positions: L351, F405, Y407, T366, K392, T394, T350, S400, and / or N390, using EU numbering. In some aspects, Fc includes the variant sequences shown in Table 2. In some aspects, Fc includes the mutations of variant 1A - B. In some aspects, Fc includes the mutations of variant 2A - B. In some aspects, Fc includes the mutations of variant 3A - B. In some aspects, Fc includes the mutations of variant 4A - B. In some aspects, Fc includes the mutations of variant 5A - B.

[0131] Table 2: IgG1 Fc Sequence

[0132]

[0133] The first and second CH3 sequences may contain amino acid mutations as described herein, with respect to amino acids 231 to 447 of the full-length human IgG1 heavy chain. In one embodiment, the heterodimeric Fc comprises a modified CH3 domain, wherein the first CH3 sequence has amino acid modifications at positions F405 and Y407, and the second CH3 sequence has an amino acid modification at position T394. In one embodiment, the heterodimeric Fc comprises a modified CH3 domain, wherein the first CH3 sequence has one or more amino acid modifications selected from L351Y, F405A, and Y407V, and the second CH3 sequence has one or more amino acid modifications selected from T366L, T366I, K392L, K392M, and T394W.

[0134] In one embodiment, the heterodimeric Fc comprises a modified CH3 domain, wherein the first CH3 sequence has amino acid modifications at positions L351, F405, and Y407, and the second CH3 sequence has amino acid modifications at positions T366, K392, and T394, and one of the first or second CH3 sequences further comprises an amino acid modification at position Q347, and the other CH3 sequence further comprises an amino acid modification at position K360. In another embodiment, the heterodimeric Fc comprises a modified CH3 domain, wherein the first CH3 sequence has amino acid modifications at positions L351, F405, and Y407, and the second CH3 sequence has amino acid modifications at positions T366, K392, and T394, one of the first or second CH3 sequences further comprises an amino acid modification at position Q347, and the other CH3 sequence further comprises an amino acid modification at position K360, and one or both of the CH3 sequences further comprise the amino acid modification T350V.

[0135] In one embodiment, the heterodimeric Fc comprises a modified CH3 domain, wherein the first CH3 sequence has amino acid modifications at positions L351, F405, and Y407, and the second CH3 sequence has amino acid modifications at positions T366, K392, and T394, and one of the first and second CH3 sequences further comprises an amino acid modification of D399R or D399K, and the other CH3 sequence comprises one or more of T411E, T411D, K409E, K409D, K392E, and K392D. In another embodiment, the heterodimeric Fc comprises a modified CH3 domain, wherein the first CH3 sequence has amino acid modifications at positions L351, F405, and Y407, and the second CH3 sequence has amino acid modifications at positions T366, K392, and T394, one of the first and second CH3 sequences further comprises an amino acid modification of D399R or D399K, and the other CH3 sequence comprises one or more of T411E, T411D, K409E, K409D, K392E, and K392D, and one or both of the CH3 sequences further comprises the amino acid modification T350V.

[0136] In one embodiment, the heterodimeric Fc comprises a modified CH3 domain, wherein the first CH3 sequence has amino acid modifications at positions L351, F405, and Y407, and the second CH3 sequence has amino acid modifications at positions T366, K392, and T394, wherein one or both of the CH3 sequences further comprises the amino acid modification T350V.

[0137] In one embodiment, the heterodimeric Fc includes a modified CH3 domain comprising the following amino acid modifications, wherein "A" represents an amino acid modification to the first CH3 sequence and "B" represents an amino acid modification to the second CH3 sequence: A: L351Y_F405A_Y407V, B: T366L_K392M_T394W, A: L351Y_F405A_Y407V, B: T366L_K392L_T394W, A: T350V_L351Y_F405A_Y407V, B: T350V_T366L_K392L_T394W, A: T350V_L351Y_F405A_Y407V, B: T350V_T366L_K392M_T394W, A: T350V_L351Y_S400E_F405A_Y407V, and / or B: T350V_T366L_N390R_K392M_T394W.

[0138] One or more asymmetric amino acid modifications can promote the formation of a heterodimeric Fc, wherein the heterodimeric CH3 domain has stability comparable to that of the wild-type homodimeric CH3 domain. In one embodiment, one or more asymmetric amino acid modifications promote the formation of a heterodimeric Fc domain, wherein the heterodimeric Fc domain has stability comparable to that of the wild-type homodimeric Fc domain. In one embodiment, one or more asymmetric amino acid modifications promote the formation of a heterodimeric Fc domain, wherein the heterodimeric Fc domain has stability as observed via the melting temperature (Tm) in differential scanning calorimetry studies, and wherein the melting temperature is within 4 °C of the melting temperature observed for the corresponding symmetric wild-type homodimeric Fc domain. In some aspects, the Fc contains one or more modifications in at least one CH3 sequence that promote the formation of a heterodimeric Fc with stability comparable to that of the wild-type homodimeric Fc.

[0139] Exemplary bispecific anti-HER2 antigen-binding construct

[0140] In certain embodiments, the bispecific anti-HER2 antigen-binding construct is one of the bispecific antibodies described in U.S. Patent Application Publication No. 2016 / 0289335 or International Patent Publication No. WO2015 / 077891. In some embodiments, the bispecific anti-HER2 antigen-binding construct is one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717 (see Tables 3, 4, 5, and the Sequence Listing). In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises the VH and VL sequences of the ECD2-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises the VH and VL sequences of the ECD2-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717, and the other antigen-binding polypeptide construct comprises the VH and VL sequences of the ECD4-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717.

[0141] In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises the CDR sequences of the ECD2-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903 or v6717. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises the CDR sequences of the ECD2-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903 or v6717, and the other antigen-binding polypeptide construct comprises the CDR sequences of the ECD4-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903 or v6717.

[0142] One of ordinary skill in the art will understand that a limited number of amino acid substitutions can be introduced into the CDR sequences or VH or VL sequences of a known antibody without the antibody losing its ability to bind its target. Candidate amino acid substitutions can be identified by computer modeling or by techniques known in the art such as alanine scanning, where the binding activity of the resulting variants is tested by standard techniques. Thus, in certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a set of CDRs (i.e., heavy-chain CDR1, CDR2, and CDR3, and light-chain CDR1, CDR2, and CDR3) having 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity to a set of CDRs of an ECD2-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717, wherein the antigen-binding polypeptide construct retains the ability to bind ECD2. In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises variants of these CDR sequences, the CDR sequences comprising 1 to 10 amino acid substitutions across the six CDRs (i.e., the CDRs can be modified by including up to 10 amino acid substitutions, where any combination of the CDRs is modified), e.g., 1 to 7 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 2 amino acid substitutions, or 1 amino acid substitution across the CDR sequences, wherein the variant retains the ability to bind ECD2. Generally, such amino acid substitutions will be conservative amino acid substitutions. In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a set of CDRs (i.e., heavy-chain CDR1, CDR2, and CDR3, and light-chain CDR1, CDR2, and CDR3) having 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity to a set of CDRs of an ECD2-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind ECD2.

[0143] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VH sequence of the ECD2-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903 or v6717, wherein the antigen-binding polypeptide construct retains the ability to bind ECD2. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VL sequence of the ECD2-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903 or v6717, wherein the antigen-binding polypeptide construct retains the ability to bind ECD2.

[0144] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VH sequence of the ECD2-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind ECD2. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VL sequence of the ECD2-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind ECD2.

[0145] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a set of CDRs (i.e., heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3) that have 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity with a set of CDRs of the ECD4-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717, wherein the antigen-binding polypeptide construct retains the ability to bind ECD4. In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises variants of these CDR sequences, the CDR sequence variants comprising 1 to 10 amino acid substitutions across the six CDRs (i.e., the CDRs can be modified by including up to 10 amino acid substitutions, wherein any combination of the CDRs is modified), e.g., 1 to 7 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 2 amino acid substitutions, or 1 amino acid substitution across the CDRs, wherein the variant retains the ability to bind ECD4. Typically, such amino acid substitutions will be conservative amino acid substitutions. In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a set of CDRs (i.e., heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3) that have 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity with a set of CDRs of the ECD4-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind ECD4.

[0146] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VH sequence of the ECD4-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903 or v6717, wherein the antigen-binding polypeptide construct retains the ability to bind ECD4. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VL sequence of the ECD4-binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903 or v6717, wherein the antigen-binding polypeptide construct retains the ability to bind ECD4.

[0147] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VH sequence of the ECD4-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind ECD4. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VL sequence of the ECD4-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind ECD4.

[0148] Table 3: Exemplary bispecific anti-HER2 antigen-binding constructs

[0149]

[0150]

[0151] *According to the Kabat numbering of the Fab or variable domains (Kabat et al., Sequences of proteins of immunological interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242, page 647, 1991)

[0152] § CH3 numbering according to the EU index in Kabat (Edelman et al., 1969, PNAS USA, 63:78-85)

[0153] Table 4: CDR sequences of the ECD2 binding arms of variants v5019, v5020, v7091, v10000, v6902, v6903 and v6717

[0154]

[0155] Table 5: CDR sequences of the ECD4 binding arms of variants v5019, v5020, v7091, v10000, v6902, v6903 and v6717

[0156] HC CDR SEQ ID NO LC CDR SEQ ID NO H1: GFNIKDTY 33 L1: QDVNTA 67 H2: IYPTNGYT 35 L2: SAS 68 H3: SRWGGDGFYAMDY 34 L3: QQHYTTPPT 69

[0157] Preparation of bispecific anti-HER2 antigen-binding construct

[0158] The bispecific anti-HER2 antigen-binding constructs described herein can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567 or International Patent Publication No. WO2015 / 077891.

[0159] In one embodiment, an isolated nucleic acid encoding the bispecific anti-HER2 antigen-binding construct described herein is provided. Such nucleic acids can encode the amino acid sequences of the VLs comprising the bispecific anti-HER2 antigen-binding construct (e.g., the light and / or heavy chains of the antigen-binding construct) and / or the amino acid sequences of the VHs comprising the same. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. As is known in the art, since many amino acids are encoded by more than one codon, multiple nucleic acids can encode a single polypeptide sequence. Exemplary nucleic acids for each polypeptide of the bispecific anti-HER2 antigen-binding construct are provided herein; however, it should be understood that other nucleic acids can be used to prepare the bispecific anti-HER2 antigen-binding constructs described herein.

[0160] In one embodiment, a nucleic acid is provided in a polycistronic vector. In another embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence of a VL comprising a bispecific anti-HER2 antigen-binding construct and an amino acid sequence of a VH comprising an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence of a VL encoding an antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence of a VH encoding an antigen-binding polypeptide construct. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of preparing a bispecific anti-HER2 antigen-binding construct is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a bispecific anti-HER2 antigen-binding construct as provided above under conditions suitable for expressing the bispecific anti-HER2 antigen-binding construct, and optionally recovering the bispecific anti-HER2 antigen-binding construct from the host cell (or the host cell culture medium).

[0161] For recombinant production of a bispecific anti-HER2 antigen-binding construct, a nucleic acid encoding the bispecific anti-HER2 antigen-binding construct (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the bispecific anti-HER2 antigen-binding construct).

[0162] The term "substantially purified" refers to a construct or variant thereof as described herein that is generally or substantially free of components that are normally associated with or interact with the protein in its native environment, i.e., natural cells, or host cells in the case of a recombinantly produced bispecific anti-HER2 antigen-binding construct, which in certain embodiments is substantially free of cellular material, including a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein. When the bispecific anti-HER2 antigen-binding construct is recombinantly produced by a host cell, in certain embodiments the protein is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cell. When the bispecific anti-HER2 antigen-binding construct is recombinantly produced by a host cell, in certain embodiments, the protein is present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the dry weight of the cell. In certain embodiments, the "substantially purified" bispecific anti-HER2 antigen-binding construct produced by the methods described herein has a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, particularly at least about 75%, 80%, 85%, more particularly at least about 90%, at least about 95%, at least about 99% or higher purity level, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0163] Suitable host cells for cloning or expressing the bispecific anti-HER2 antigen-binding construct-encoding vector include prokaryotic or eukaryotic cells as described herein.

[0164] A "recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art for producing recombinant host cells. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or may be integrated into the host genome.

[0165] As used herein, the term "eukaryote" refers to an organism belonging to the phylogenetic domain Eukaryota, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

[0166] As used herein, the term "prokaryote" refers to a prokaryotic organism. For example, a non-eukaryote may belong to the phylogenetic domain Bacteria (including but not limited to Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.), or Archaea (including but not limited to Methanococcus jannaschii, Methanobacterium thermoautotrophicum, halophilic archaea such as Haloferax volcanii and Halobacterium sp. NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.).

[0167] For example, a bispecific anti-HER2 antigen-binding construct can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For the expression of bispecific anti-HER2 antigen-binding construct fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli.) After expression, the bispecific anti-HER2 antigen-binding construct can be separated from the bacterial cell paste in a soluble fraction and further purified.

[0168] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding bispecific anti-HER2 antigen-binding constructs, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce bispecific anti-HER2 antigen-binding constructs with a partial or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006).

[0169] Host cells suitable for expressing glycosylated bispecific anti-HER2 antigen-binding constructs are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0170] Plant cell cultures can also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES TM technology for producing antigen-binding constructs in transgenic plants).

[0171] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension growth may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, as described, for example, by Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, by Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, by Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for the production of antigen-binding constructs, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0172] In one embodiment, the bispecific anti-HER2 antigen-binding construct described herein is produced in stable mammalian cells by a method comprising: transfecting at least one stable mammalian cell with a nucleic acid encoding the bispecific anti-HER2 antigen-binding construct at a predetermined ratio; and expressing the nucleic acid in the at least one mammalian cell. In some embodiments, the predetermined ratio of the nucleic acid is determined in a transient transfection experiment to determine the relative ratio of the input nucleic acid that results in the highest percentage of the bispecific anti-HER2 antigen-binding construct in the expression product.

[0173] In some embodiments, the bispecific anti-HER2 antigen-binding construct is produced in stable mammalian cells, wherein the expression product of the at least one stable mammalian cell comprises a greater percentage of the desired glycosylated bispecific anti-HER2 antigen-binding construct compared to monomeric heavy or light chain polypeptides or other antibodies. In some embodiments, the glycosylated bispecific anti-HER2 antigen-binding construct is identified by one or both of liquid chromatography and mass spectrometry.

[0174] If desired, the bispecific anti-HER2 antigen-binding construct can be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, size or gel filtration, and reverse phase, using systems such as FPLC and HPLC at atmospheric or high pressure. Purification methods also include electrophoresis, immunoprecipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, combined with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind to Fc and antibodies, and these proteins can be used to purify the bispecific anti-HER2 antigen-binding construct described herein. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can generally be achieved by specific fusion partners. For example, if GST fusion is used, the antibody can be purified using glutathione resin, and if His tag is used, Ni +2Affinity chromatography, or if a flag tag is used, immobilized anti-flag antibody may be used. For general guidance on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Edition, Scopes, Springer-Verlag, NY, 1994, which is incorporated by reference in its entirety. The degree of purification necessary will vary depending on the use of the bispecific anti-HER2 antigen-binding construct. In some cases, no purification is required.

[0175] In certain embodiments, the bispecific anti-HER2 antigen-binding construct is purified using anion exchange chromatography, including but not limited to chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q and DEAE columns.

[0176] In specific embodiments, the bispecific anti-HER2 antigen-binding construct described herein is purified using cation exchange chromatography, including but not limited to SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns and their equivalents and analogs.

[0177] In addition, the bispecific anti-HER2 antigen-binding constructs described herein can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a polypeptide fragment can be synthesized by using a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as substitutions or additions into the polypeptide sequence. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylalanine, phenylhexylalanine, β-alanine, fluoro-amino acids, designed amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids and general amino acid analogs. In addition, the amino acids can be D (right-handed) or L (left-handed).

[0178] Post-translational modifications:

[0179] In certain embodiments, the bispecific anti-HER2 antigen-binding constructs described herein are differentially modified during or after translation.

[0180] As used herein, the term "modified" refers to any alteration made to a given polypeptide, such as an alteration in polypeptide length, amino acid sequence of the polypeptide, chemical structure, co-translational modification or post-translational modification. The term in the form "(modified)" means that the polypeptide in question is optionally modified, i.e., the polypeptide of the bispecific anti-HER2 antigen-binding construct can be modified or unmodified.

[0181] The term "post-translationally modified" refers to any modification that occurs on such an amino acid after it has been incorporated into the polypeptide chain, whether the amino acid is natural or non-natural. By way of example only, the term encompasses co-translational in vivo modifications, co-translational in vitro modifications (e.g., in a cell-free translation system), post-translational in vivo modifications and post-translational in vitro modifications.

[0182] In some embodiments, the modification is at least one of the following: glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and conjugation with an antibody molecule or a bispecific anti-HER2 antigen-binding construct or other cellular ligands. In some embodiments, the bispecific anti-HER2 antigen-binding construct is chemically modified by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; and metabolic synthesis in the presence of tunicamycin.

[0183] Other post-translational modifications of the bispecific anti-HER2 antigen-binding construct include, for example, N-linked or O-linked carbohydrate chains, N-terminal or C-terminal processing), attachment of chemical moieties to the amino acid backbone, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of expression in a prokaryotic host cell. The bispecific anti-HER2 antigen-binding constructs described herein are modified with a detectable label such as an enzyme label, a fluorescent label, an isotopic label, or an affinity label to allow detection and isolation of the protein. In certain embodiments, examples of suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable cofactor complexes include streptavidin-biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, fluorine.

[0184] In a specific embodiment, the bispecific anti-HER2 antigen-binding construct described herein is attached to a macrocyclic chelator associated with a radioactive metal ion.

[0185] In some embodiments, the bispecific anti-HER2 antigen-binding constructs described herein are modified by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. In certain embodiments, the same type of modification may be present at several sites of a given polypeptide to the same or varying degrees. In certain embodiments, polypeptides from the bispecific anti-HER2 antigen-binding constructs described herein are branched, e.g., as a result of ubiquitination, and in some embodiments are cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides are the result of natural post-translational processes or are made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, polyethylene glycolylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of amino acids to proteins mediated by transfer RNA such as arginylation, and ubiquitination. (See, e.g., PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B.C. Johnson, ed., Academic Press, New York, pp. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

[0186] Antibody-drug conjugate (ADC)

[0187] Certain embodiments relate to methods of treating BTC using an antibody-drug conjugate (ADC) comprising a bispecific anti-HER2 antigen-binding construct conjugated to an auristatin analog at a low average drug / antibody ratio (DAR). As used herein, "low average DAR" refers to an average DAR of < 3.9. Particularly useful in the methods are ADCs comprising a bispecific anti-HER2 antigen-binding construct conjugated to an auristatin analog having an average DAR of about 2.5 or less, such as between about 1.8 and 2.5. In certain embodiments, the bispecific anti-HER2 antigen-binding construct comprised in the ADC is v10000.

[0188] In certain embodiments, the auristatin analog comprised in the ADC for use in the methods described herein may be an auristatin analog as described in International Patent Application Publication No. WO 2016 / 041082. In certain embodiments, the auristatin analog comprised in the ADC for use in the methods described herein is a compound of formula (I):

[0189]

[0190] Wherein R 1 is selected from:

[0191]

[0192] In certain embodiments, in the compound of formula (I), R 1 is:

[0193]

[0194] In certain embodiments, in the compound of formula (I), R 1 is:

[0195]

[0196] In certain embodiments, in the compound of formula (I), R 1 is:

[0197]

[0198] In certain embodiments, the compounds of formula (I) are selected from:

[0199]

[0200] The compounds of formula (I) can be prepared from commercially available starting materials by standard synthetic organic chemistry protocols. Exemplary synthetic methods are provided in International Patent Application Publication No. WO 2016 / 041082.

[0201] In certain embodiments, the ADCs for use in the methods described herein comprise a bispecific anti-HER2 antigen-binding construct conjugated via a linker (L) to an auristatin analog (toxin), wherein the linker-toxin has the general formula (II):

[0202]

[0203] Wherein:

[0204] R 1 Is selected from:

[0205]

[0206] L is a cleavable linker, and

[0207] Denotes the attachment point of the linker-toxin to the bispecific anti-HER2 antigen-binding construct.

[0208] In some embodiments, in the linker-toxin of general formula (II), R 1 Is:

[0209]

[0210] In some embodiments, in the linker-toxin of general formula (II), R 1 Is:

[0211]

[0212] In some embodiments, in the linker-toxin of general formula (II), R 1 Is:

[0213]

[0214] In some embodiments, in the linker-toxin of general formula (II), L is a peptide-containing linker.

[0215] In some embodiments, in the linker-toxin of general formula (II), L is a protease-cleavable linker.

[0216] In certain embodiments, the ADCs for use in the methods described herein comprise a bispecific anti-HER2 antigen-binding construct conjugated via a linker (L) to an auristatin analog (toxin) and having the general formula (III):

[0217]

[0218] Wherein:

[0219] R 1 And L are as defined with respect to general formula (II);

[0220] n is the average drug / antibody ratio (DAR) and is less than 3.9, and

[0221] Ab is a bispecific anti-HER2 antigen-binding construct.

[0222] In some embodiments, in the ADC of formula (III), R 1 is:

[0223]

[0224] In some embodiments, in the ADC of formula (III), R 1 is:

[0225]

[0226] In some embodiments, in the ADC of formula (III), R 1 is:

[0227]

[0228] In some embodiments, in the ADC of formula (III), L is a peptide-containing linker.

[0229] In some embodiments, in the ADC of formula (III), L is a protease-cleavable linker.

[0230] In some embodiments, in the ADC of formula (III), n ranges from 0.5 to 3.8.

[0231] In some embodiments, in the ADC of formula (III), n ranges from about 1.0 to 3.8, from about 1.0 to 3.5, from about 1.0 to 3.0, or from about 1.0 to 2.5.

[0232] In some embodiments, in the ADC of formula (III), n ranges from about 1.5 to 3.8, from about 1.5 to 3.5, from about 1.5 to 3.0, or from about 1.5 to 2.5.

[0233] In some embodiments, in the ADC of formula (III), n ranges from about 1.8 to 2.8, or from about 1.8 to 2.5.

[0234] In some embodiments, in the ADC of formula (III), Ab is v10000.

[0235] Also contemplated are combinations of any of the foregoing embodiments for the ADC of formula (III), and for the purposes of this disclosure, each combination forms a separate embodiment.

[0236] In the ADCs described herein, the bispecific anti-HER2 antigen-binding construct is linked to an auristatin analogue (toxin) via a linker. The linker is a bifunctional or multifunctional moiety capable of linking one or more toxin molecules to an antibody. A bifunctional (or monovalent) linker attaches a single drug to a single site on the antibody, while a multifunctional (or multivalent) linker attaches more than one toxin molecule to a single site on the antibody. A linker capable of attaching one toxin molecule to more than one site on an antibody can also be considered multifunctional.

[0237] Attachment of the linker to the antibody can be achieved in a variety of ways, such as through surface lysines on the antibody, reductive coupling to oxidized carbohydrates on the antibody, or through cysteine residues on the antibody released by reduction of interchain disulfide bonds. Alternatively, attachment of the linker to the antibody can be achieved by modifying the antibody to include additional cysteine residues (see, e.g., U.S. Patent Nos. 7,521,541; 8,455,622 and 9,000,130) or unnatural amino acids that provide reactive handles such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine (see, e.g., Hofer et al., Biochemistry, 48:12047-12057 (2009); Axup et al., PNAS, 109:16101-16106 (2012); Wu et al., PNAS, 106:3000-3005 (2009); Zimmerman et al., Bioconj. Chem., 25:351-361 (2014)) to allow site-specific conjugation.

[0238] The linker includes a functional group capable of reacting with one or more target groups on the antibody, and one or more functional groups capable of reacting with a target group on the toxin. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press).

[0239] Non-limiting examples of functional groups for reacting with free cysteine or thiol include maleimide, haloacetamide, haloacetyl, activated esters such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, acid anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this regard are "self-stabilizing" maleimides as described by Lyon et al., Nat. Biotechnol., 32:1059-1062 (2014).

[0240] Non-limiting examples of functional groups for reacting with surface lysines on antibodies or free amines on toxins include activated esters such as N-hydroxysuccinimide (NHS) esters, sulfo-NHS esters, imidoesters such as Traut's reagent, isothiocyanates, aldehydes, and acid anhydrides such as diethylenetriaminepentaacetic anhydride (DTPA). Other examples include succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).

[0241] Non-limiting examples of functional groups capable of reacting with electrophilic groups (such as aldehyde or ketone carbonyls) on antibodies or toxins include hydrazides, oximes, amines, hydrazines, thiosemicarbazones, carboxylic acid hydrazides, and aryl hydrazides.

[0242] Other linkers include linkers having functional groups that allow bridging of two interchain cysteines on an antibody, such as ThioBridge TM Linkers (Badescu et al., Bioconjug. Chem., 25:1124-1136 (2014)), dithiomaleimide (DTM) linkers (Behrens et al., Mol. Pharm., 12:3986-3998 (2015)), dithioaryl (TCEP) pyridazinone-based linkers (Lee et al., Chem. Sci., 7:799-802 (2016)), dibromopyridazinone-based linkers (Maruani et al., Nat. Commun., 6:6645 (2015)), and other linkers known in the art.

[0243] Linkers can include various linker components. Generally, a linker will contain two or more linker components. Exemplary linker components include functional groups for reacting with an antibody, functional groups for reacting with a toxin, spacer segments, peptide components, self-immolative groups, self-eliminating groups, hydrophilic moieties, and the like. Various linker components are known in the art, some of which are described below.

[0244] Certain useful linker components are available from a variety of commercial sources such as Pierce Biotechnology, Inc. (now Thermo Fisher Scientific, Waltham, MA) and Molecular Biosciences Inc. (Boulder, Colo.), or can be synthesized according to procedures described in the art (see, e.g., Toki et al., J. Org. Chem., 67:1866-1872 (2002); Dubowchik et al., Tetrahedron Letters, 38:5257-60 (1997); Walker, M.A., J. Org. Chem., 60:5352-5355 (1995); Frisch et al., Bioconjugate Chem., 7:180-186 (1996); U.S. Patent Nos. 6,214,345 and 7,553,816, and International Patent Application Publication No. WO 02 / 088172).

[0245] The linkers employed in the ADCs described herein are cleavable linkers. Cleavable linkers are generally susceptible to cleavage under intracellular conditions, e.g., by lysosomal processes. Examples include protease-sensitive, acid-sensitive, reduction-sensitive, or photolabile linkers.

[0246] Suitable cleavable linkers include, for example, linkers that contain a peptide component that includes two or more amino acids and is cleavable by intracellular proteases such as lysosomal proteases or endosomal proteases. The peptide component can contain naturally occurring amino acid residues and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. The peptide component can be designed and optimized for enzymatic cleavage by a specific enzyme such as a tumor-associated protease, cathepsin B, C, or D, or plasmin protease.

[0247] In certain embodiments, the linker included in the ADC can be a dipeptide-containing linker, such as a linker containing valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys). Other examples of suitable dipeptides included in the linker include Val-Lys, Ala-Lys, Me-Val-Cit, Phe-homoLys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, and Met-(D)Lys. The cleavable linker can also include longer peptide components, such as tripeptides, tetrapeptides, or pentapeptides. Examples include, but are not limited to, the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, and (D)Ala-Phe-Lys, and the tetrapeptides Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu.

[0248] Other examples of cleavable linkers include disulfide-containing linkers, such as N-succinyl-4-(2-pyridyldithio)butyrate (SPBD) and N-succinyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfonyl-SPBD). The disulfide-containing linker can optionally include additional groups to provide steric hindrance adjacent to the disulfide bond to improve the extracellular stability of the linker, for example, containing geminal dimethyls. Other suitable linkers include linkers that are hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers containing combinations of these functional groups can also be useful, for example, linkers containing both hydrazone and disulfide are known in the art.

[0249] Another example of a cleavable linker is a linker containing β-glucuronide, which can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, for example, De Graaf et al., Curr. Pharm. Des., 8:1391-1403 (2002)).

[0250] The cleavable linker can also optionally contain one or more additional components, such as self-degrading and self-eliminating groups, extensions, or hydrophilic moieties.

[0251] Self-cleaving and self-eliminating groups useful for linkers include, for example, p-aminobenzyloxycarbonyl (PABC) and p-aminobenzyl ether (PABE) groups, as well as methylated ethylenediamine (MED). Other examples of self-cleaving groups include, but are not limited to, aromatic compounds that are electronically similar to PABC or PABE groups, such as heterocyclic derivatives, such as 2-aminoimidazole-5-methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that cyclize upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyramide (Rodrigues et al., Chemistry Biology, 2:223-227 (1995)) and 2-aminophenylpropionamide (Amsberry et al., J. Org. Chem., 55:5867-5877 (1990)).

[0252] Spacer segments useful for linkers in ADCs include, for example, alkylene and fatty acid-, diacid-, amine- or diamine-based spacer segments, such as diglycolate, malonate, hexanoate, and hexanamide. Other spacer segments include, for example, glycine-based spacer segments, polyethylene glycol (PEG) spacer segments, and monomethoxypolyethylene glycol (mPEG) spacer segments. PEG and mPEG spacer segments also function as hydrophilic moieties.

[0253] In certain embodiments, the ADCs used in the methods described herein comprise a peptide-based linker having the general formula (IV):

[0254]

[0255] Wherein:

[0256] Z is a functional group capable of reacting with a target group on the bispecific anti-HER2 antigen-binding construct;

[0257] Str is a spacer segment;

[0258] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m forms a protease cleavage site;

[0259] X is a self-cleaving group;

[0260] D is an attachment point for an auristatin analog;

[0261] s is 0 or 1;

[0262] m is an integer between 1 and 4, and

[0263] o is 0, 1, or 2.

[0264] In some embodiments, in general formula (IV), Z is:

[0265]

[0266] In some embodiments, in general formula (IV), Str is selected from:

[0267]

[0268] wherein:

[0269] R is H or C1-C6 alkyl;

[0270] p is an integer between 2 and 10, and q is an integer between 1 and 10.

[0271] In some embodiments, in general formula (IV), Str is:

[0272]

[0273] where p and q are as defined above.

[0274] In some embodiments, in general formula (IV), Str is:

[0275]

[0276] where p is an integer between 2 and 6, and

[0277] q is an integer between 2 and 8.

[0278] In some embodiments, in general formula (IV), AA1-[AA2] m is selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu.

[0279] In some embodiments, in general formula (IV), m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0280] In some embodiments, in general formula (IV), AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit.

[0281] In some embodiments, in general formula (IV), m is 1, 2, or 3.

[0282] In some embodiments, in general formula (IV), s is 1.

[0283] In some embodiments, in general formula (IV), o is 0.

[0284] In some embodiments, in general formula (IV):

[0285] Z is

[0286] Str is where p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0287] m is 1 and AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit;

[0288] s is 1, and

[0289] o is 0.

[0290] In certain embodiments, the linker contained in the ADCs for the methods described herein has general formula (V):

[0291]

[0292] where:

[0293] A-S- is the attachment point to the bispecific anti-HER2 antigen-binding construct;

[0294] Y is one or more additional linker components, or is absent, and

[0295] D is the attachment point to the auristatin analog.

[0296] In certain embodiments, the linker contained in the ADCs for the methods described herein has the general formula (VI):

[0297]

[0298] Wherein:

[0299] A-S- is the attachment point to the bispecific anti-HER2 antigen-binding construct;

[0300] Y is one or more additional linker components, or is absent, and

[0301] D is the attachment point to the auristatin analog.

[0302] In certain embodiments, the ADCs for the methods described herein comprise an auristatin analog of general formula (I) conjugated to v10000 via a linker having general formula (IV), (V), or (VI) at a low average DAR.

[0303] In certain embodiments, the ADCs for the methods described herein comprise v10000 conjugated to a linker-toxin of general formula (II) at a low average DAR, wherein the linker (L) has general formula (IV), (V), or (VI).

[0304] In certain embodiments, the ADCs for the methods described herein comprise v10000 and have general formula (III) as shown above, wherein the linker (L) has general formula (IV), (V), or (VI).

[0305] In certain embodiments, the ADCs for the methods described herein comprise an auristatin analog conjugated to v10000 via a linker having general formula (IV), (V), or (VI) at a low average DAR, wherein the auristatin analog is Compound 16, Compound 17, or Compound 18.

[0306] In certain embodiments, the ADCs for the methods described herein comprise a linker-toxin having the following structure:

[0307]

[0308] Wherein A-S- is the attachment point to the bispecific anti-HER2 antigen-binding construct.

[0309] Preparation of antibody-drug conjugate

[0310] The ADCs for use in the methods described herein can be prepared by one of several routes known in the art, using organic chemical reactions, conditions, and reagents known to those of skill in the art (see, e.g., Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press), and the examples provided herein). For example, conjugation can be achieved by: (1) reaction of a nucleophilic or electrophilic group of the antibody with a bifunctional linker to form an antibody-linker intermediate Ab-L via a covalent bond, followed by reaction with an activated auristatin analog (D), or (2) reaction of a nucleophilic or electrophilic group of the auristatin analog with the linker to form a linker-toxin D-L via a covalent bond, followed by reaction with a nucleophilic or electrophilic group of the antibody.

[0311] As described above, auristatin analogs can be conjugated to various groups on the antibody via a suitable linker to provide an ADC. For example, conjugation can occur through surface lysines, through oxidized carbohydrates, or through cysteine residues released by reduction of one or more interchain disulfide bonds. Alternatively, the antibody can be modified to include additional cysteine residues or unnatural amino acids that provide a reactive handle, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine. Such modifications are well known in the art (see, e.g., U.S. Patent Nos. 7,521,541; 8,455,622; and 9,000,130; Hofer et al., Biochemistry, 48:12047-12057 (2009); Axup et al., PNAS, 109:16101-16106 (2012); Wu et al., PNAS, 106:3000-3005 (2009); Zimmerman et al., Bioconj. Chem., 25:351-361 (2014)).

[0312] In certain embodiments, the ADCs for use in the methods described herein comprise an auristatin analog conjugated via a suitable linker to a cysteine residue on a bispecific anti-HER2 antigen-binding construct, the cysteine residue having been released by reduction of one or more interchain disulfide bonds.

[0313] In the ADCs described herein, the bispecific anti-HER2 antigen-binding construct is conjugated to the toxin via a linker at a low average drug / antibody ratio (DAR), particularly less than 3.9 but greater than 0.5, e.g., between about 1.5 and about 2.5 in certain embodiments.

[0314] A variety of methods are known in the art for preparing ADCs with a low average DAR (see, e.g., the review: McCombs and Owen, The AAPS Journal, 17(2):339 - 351(2015) and references therein; Boutureira and Bernardes, Chem. Rev., 115:2174 - 2195(2015)).

[0315] For example, for conjugation to cysteine residues, partial reduction of the inter - chain disulfide bonds of the antibody can be carried out, followed by conjugation with a linker - toxin. Partial reduction can be achieved by limiting the amount of reducing agent used in the reduction reaction (see, e.g., Lyon et al., Methods in Enzymology, 502:123 - 138(2012) and examples therein, as well as the examples provided herein). Suitable reducing agents are known in the art and include, for example, dithiothreitol (DTT), tris(2 - carboxyethyl)phosphine (TCEP), 2 - mercaptoethanol, cysteamine, and many water - soluble phosphines. Alternatively or additionally, a lower equivalent amount of the linker - toxin can be used to obtain a low average DAR.

[0316] Alternatively, engineered antibodies can be used in which one or more cysteine residues that form the inter - chain disulfide bonds are replaced by serine residues, resulting in fewer available cysteine residues for conjugation (see McDonagh et al., Protein Eng. Des. Sel. PEDS, 19(7):299 - 307). The engineered antibody can then be treated with a reducing agent and conjugated with a linker - toxin.

[0317] Another approach is to use a dithiol linker that bridges two cysteines that normally form the inter - chain disulfide bonds. If all four inter - chain disulfide bonds are reduced and replaced with a dithiol linker, then using a dithiol linker that carries only one toxin molecule will produce an ADC with a maximum DAR of 4 for a full - size antibody. Partial reduction of the inter - chain disulfide bonds and / or a lower equivalent amount of the linker can be used in combination with the dithiol linker to further reduce the DAR. A variety of dithiol linkers are known in the art (see, e.g., Badescu et al., Bioconjug. Chem., 25(6):1124 - 1136(2014); Behrens et al., Mol. Pharm., 12:3986 - 3998(2015); Lee et al., Chem. Sci., 7:799 - 802(2016); Maruani et al., Nat. Commun., 6:6645(2015)).

[0318] A cysteine engineering approach can also be employed to generate ADCs with a low average DAR. Such methods involve engineering solvent-accessible cysteines into the antibody to provide a site-specific handle for conjugation. Many suitable sites for introducing cysteine residues have been identified in the IgG structure and include those described in Junutula et al., J. Immunol Methods, 332(1-2):41-52(2008); Junutula et al., Nat. Biotechnol., 26(8), 925-932(2008); and U.S. Patent Nos. 9,315,581; 9,000,130; 8,455,622; 8,507,654; and 7,521,541.

[0319] Low average DAR ADCs can also be prepared by lysine conjugation using a limited amount of an activated linker-toxin. Selective reaction of the N-terminal amino acid of the antibody can also be used. For example, the N-terminal serine can be oxidized to an aldehyde with periodate and then reacted with a linker-toxin (see, e.g., Thompson et al., Bioconjug. Chem., 26(10):2085-2096(2015)). Similarly, the N-terminal cysteine residue can be selectively reacted with an aldehyde to give a thiazolidinone (see, e.g., Bernardes et al., Nature Protocols, 8:2079-2089).

[0320] Other methods include engineering the antibody to contain one or more unnatural amino acids, such as p-acetylphenylalanine (pAcPhe) or selenocysteine (Sec). The ketone group in pAcPhe can be reacted with a linker-toxin containing a terminal alkoxyamine or hydrazide to form an oxime or hydrazone bond (see, e.g., Axup et al., PNAS USA, 109:16101-16106(2012)). Antibodies containing Sec can be reacted with a linker-toxin containing maleimide or iodoacetamide to form a selenoether conjugate (see, e.g., Hofer et al., Biochemistry, 48:12047-12057(2009)).

[0321] Antibodies can also be engineered to include peptide tags recognized by certain enzymes to allow enzyme-catalyzed conjugation. For example, sortase-A (SortA) recognizes the sequence LPXTG. This pentapeptide can be engineered into the N-terminus or C-terminus of the antibody to allow SortA-mediated conjugation (see, e.g., U.S. Patent Application Publication No. 2016 / 0136298; Kornberger and Skerra, mAbs, 6(2):354-366 (2014)). Transglutaminase has also been used to generate DAR2 ADCs by using antibodies deglycosylated at position N297 (exposing Q295 for enzyme conjugation) or by engineering antibodies to include a "glutamine tag" (LLQG) (Jeger et al., Angew. Chem., 49:9995-9997 (2010); Strop et al., Chem. Biol., 20(2):161-167 (2013)). In another approach, formylglycine residues can be introduced into antibodies by engineering an appropriate consensus sequence into the antibody and co-expressing the engineered antibody with formylglycine-generating enzyme (FGE). The aldehyde functional group of the introduced formylglycine can then be used as a handle for conjugating toxins (see, e.g., Drake et al., Bioconjug. Chem., 25(7):1331-1341 (2014)).

[0322] Another method for generating DAR2 ADCs is to conjugate the linker-toxin to the native sugars on the glycosylated antibody. Conjugation to the glycosylated antibody can be achieved, for example, by periodate oxidation of the terminal sugar residues to generate aldehydes, which can then be conjugated to an appropriate linker-toxin, or by a glycoengineering approach, where the native sugars are modified with terminal sialic acid residues, which can then be oxidized to yield aldehydes for conjugation to the linker-toxin (Zhou et al., Bioconjug. Chem., 25(3):510-520 (2014)).

[0323] Conjugation of the active moiety to the antibody using UV crosslinking has also been reported. This method uses the nucleotide binding site (NBS) for site-specific covalent functionalization of antibodies with a reactive thiol moiety. The indole-3-butyric acid (IBA)-conjugated cysteine form is used for site-specific photocrosslinking of the reactive thiol moiety to the antibody at the NBS. The thiol moiety can then be used to conjugate a linker-toxin with a thiol-reactive group (Alves et al., Bioconjug. Chem., 25(7):1198-1202 (2014)).

[0324] Alternatively, an ADC with a low average DAR can be isolated from an ADC formulation containing a mixture of DAR species using chromatographic separation techniques such as hydrophobic interaction chromatography (see, e.g., Hamblett et al., Clin. Cancer Res., 10:7063-7070 (2004); Sun et al., Bioconj Chem., 28:1371-81 (2017); U.S. Patent Application Publication No. 2014 / 0286968).

[0325] An ADC with a low average DAR can also be generated by adding an unconjugated (i.e., DAR0) antibody to an ADC formulation with an average DAR ≥ 3.9. As is known in the art, most conjugation methods produce an ADC formulation comprising a variety of DAR species, where the reported DAR is the average of the individual DAR species. In certain embodiments, it may be advantageous for the ADC to include a certain proportion of the DAR0 species. In some embodiments, the ADCs with an average DAR of less than 3.9 for use in the methods described herein include at least 5% of the DAR0 species. In some embodiments, the ADCs for use in the methods described herein include at least 10% of the DAR0 species, such as at least 15% of the DAR0 species or at least 20% of the DAR0 species. In some embodiments, the ADCs for use in the methods described herein include between about 5% and about 50% of the DAR0 species, such as between about 10% and about 50% of the DAR0 species, between about 10% and about 40%, or between about 10% and about 30% of the DAR0 species.

[0326] The average DAR of an ADC can be determined by standard techniques such as UV / VIS spectroscopy, ELISA-based techniques, chromatographic techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS), and MALDI-TOF MS. In addition, the distribution of the drug-linkage forms (e.g., the fractions of the DAR0, DAR1, DAR2, etc. species) can also be analyzed by various techniques known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reversed-phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, e.g., Sun et al., Bioconj Chem., 28:1371-81 (2017); Wakankar et al., mAbs, 3:161-172 (2011)).

[0327] In certain embodiments, the average DAR of the ADC is determined by hydrophobic interaction chromatography (HIC) techniques.

[0328] After conjugation, the ADC can be purified by purification methods known in the art and separated from unconjugated reactants and / or any conjugated aggregates. Such methods include, but are not limited to, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, chromatofocusing, ultrafiltration, diafiltration, and combinations thereof.

[0329] Pharmaceutical composition

[0330] Also provided herein are pharmaceutical compositions comprising the bispecific anti-HER2 antigen-binding constructs described herein. The pharmaceutical compositions comprise the bispecific anti-HER2 antigen-binding constructs and a pharmaceutically acceptable carrier.

[0331] The term "pharmaceutically acceptable" refers to being approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some aspects, the carrier is an artificial carrier not found in nature. When the pharmaceutical composition is administered intravenously, water can be used as the carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the bispecific anti-HER2 antigen-binding construct, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to a patient. The formulations should be suitable for the mode of administration.

[0332] In certain embodiments, a composition comprising a bispecific anti-HER2 antigen-binding construct is formulated, according to conventional procedures, into a pharmaceutical composition suitable for intravenous administration to humans. Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffers. Optionally, the composition may also include solubilizing agents and local anesthetics such as lidocaine to alleviate the pain at the injection site. Usually, the ingredients are provided individually or mixed together in unit dosage forms, for example, as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or a sachet indicating the active dose. When the composition is administered by infusion, it may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, ampoules of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

[0333] In certain embodiments, the compositions described herein are formulated in neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0334] Method of treating biliary tract cancer (BTC)

[0335] Methods of treating biliary tract cancer (BTC) are described herein, comprising administering to a subject having BTC a bispecific anti-HER2 antigen-binding construct or an ADC as described herein in an amount effective to treat, prevent or ameliorate the disease or disorder. In a specific embodiment of the methods described herein, the bispecific anti-HER2 antigen-binding construct is v10000. In other specific embodiments of the methods described herein, the ADC is v10000 conjugated to an auristatin analog.

[0336] "Disorder" or "disease" refers to any affliction that would benefit from treatment with the bispecific anti-HER2 antigen-binding constructs or methods described herein. This includes chronic and acute disorders or diseases, including pathological conditions that render a mammal susceptible to the disorder in question. In the embodiments described herein, the disorder or disease is biliary tract cancer, which is described in more detail below.

[0337] The term "subject" or "patient" refers to an animal that is the object of treatment, observation, or experimentation, which in some embodiments is a mammal. The animal can be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.) or a laboratory animal (e.g., rat, mouse, guinea pig, etc.).

[0338] As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, canines, felines, murine, bovines, equines, and porcines.

[0339] "Treatment" refers to a clinical intervention that attempts to alter the natural course of an individual or cell being treated and can be performed during a clinical pathologic process. Desirable effects of treatment include, but are not limited to, preventing disease recurrence, alleviating symptoms, reducing any direct or indirect pathologic consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to delay the development of the disease or slow the progression of the disease. In some embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to delay the development of BTC. In one embodiment, the bispecific anti-HER2 antigen-binding construct, ADC, and methods described herein can affect the inhibition of BTC tumor / cancer growth. In another embodiment, the bispecific anti-HER2 antigen-binding construct or ADC can be used to slow the progression of BTC.

[0340] As used herein, the term "effective amount" refers to the amount of the bispecific anti-HER2 antigen-binding construct administered that will achieve the objectives of the method, such as alleviating to some extent one or more symptoms of the disease, disorder, or condition being treated. The amount of the bispecific anti-HER2 antigen-binding construct effective in treating or inhibiting a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to assist in determining the optimal dosage range. The exact dosage used in the formulation also depends on the route of administration and the severity of the BTC and should be decided according to the judgment of the practitioner and the circumstances of each patient. The effective dose can be extrapolated from the dose-response curves of in vitro or animal model test systems.

[0341] The terms "first-line therapy", "first-line treatment", or "primary therapy" are treatment regimens that are generally accepted as the initial treatment for a patient, taking into account the type and stage of the cancer. The terms "second-line therapy" or "second-line treatment" are treatment regimens that are typically administered if the first-line therapy does not provide the desired efficacy.

[0342] The term "neoadjuvant therapy" refers to treatment given as the first step in shrinking a tumor prior to undergoing primary treatment (usually surgery). Examples of neoadjuvant therapy include, but are not limited to, chemotherapy, radiotherapy, and hormone therapy. Neoadjuvant therapy can be considered first-line therapy.

[0343] The term "adjuvant therapy" refers to additional cancer treatment given after primary treatment to reduce the risk of cancer recurrence. Adjuvant therapy may include, but is not limited to, chemotherapy, radiotherapy, hormone therapy, targeted therapy (small molecule drugs or antibodies that typically target specific types of cancer cells rather than normal cells), or biologic therapy (such as vaccines, cytokines, antibodies, or gene therapy).

[0344] "Advanced cancer" is cancer that has progressed to the point where it cannot be safely removed or is highly unlikely to be cured or in long-term remission. Cancer progresses to advanced stages by growing near structures that prevent its removal or by spreading from where it began, crossing tissue lines or to other parts of the body (such as lymph nodes or other organs). Advanced cancer may be locally advanced, meaning it has spread beyond the organ of the primary site but has not spread to distant sites. Advanced cancer may also be metastatic, meaning the cancer cells have spread from the site where the cancer began (primary site) to other more distant parts of the body (secondary sites).

[0345] "Resectable" cancer is cancer that can be treated by surgery. "Unresectable" cancer is cancer that cannot be treated by surgery, usually because the cancer has spread to the tissues around the main tumor. Depending on the extent of spread to the surrounding tissues, some cancers may be evaluated by doctors as "partially resectable".

[0346] The bispecific anti-HER2 antigen-binding construct or ADC can be administered to a subject according to known methods. A variety of delivery systems are known and can be used to administer the bispecific anti-HER2 antigen-binding construct formulations described herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of nucleic acids as part of a retrovirus or other vector, etc. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The bispecific anti-HER2 antigen-binding construct or ADC can be administered by any suitable route, such as by infusion or bolus injection, absorption through epithelial or mucosal linings (such as oral mucosa, rectal, and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Administration can be systemic or local. In addition, in certain embodiments, it may be necessary to introduce the bispecific anti-HER2 antigen-binding construct described herein into the central nervous system by any appropriate route, including intracerebroventricular and intrathecal injection; intracerebroventricular injection can be facilitated by an intracerebroventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, such as by using an inhaler or nebulizer, and formulations containing nebulizers. In a specific embodiment, the bispecific anti-HER2 antigen-binding construct or ADC can be administered intravenously (IV).

[0347] In one specific embodiment, it may be desirable to locally administer the bispecific anti-HER2 antigen-binding construct or ADC described herein to the area in need of treatment; this can be achieved, for example, but not limited to, local infusion during surgery, local application such as in combination with a wound dressing after surgery, by injection, via a catheter, via a suppository, or via an implant, which is a porous, non-porous, or gel-like material, including membranes such as sialic acid membranes or fibers. Preferably, when administering a protein such as a bispecific anti-HER2 antigen-binding construct, care must be taken to use materials that do not absorb the protein.

[0348] In another embodiment, the bispecific anti-HER2 antigen-binding construct or ADC can be delivered in vesicles, particularly liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0349] In yet another embodiment, the bispecific anti-HER2 antigen-binding construct or ADC can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, a polymeric material can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); also see Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, the controlled release system can be placed near the therapeutic target, such that only a fraction of the systemic dose is required (see, e.g., Goodson, in Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).

[0350] The bispecific anti-HER2 antigen-binding construct or ADC can be administered alone or in combination with other types of therapy (e.g., radiotherapy, chemotherapy, hormone therapy, immunotherapy, and anti-tumor agents). Generally, it is preferred to administer a product of the same species origin or species reactivity (in the case of an antibody) as the patient. Thus, in one embodiment, a human or humanized bispecific anti-HER2 antigen-binding construct, fragment derivative, analogue, or nucleic acid is administered to a human patient for treatment or prophylaxis.

[0351] Biliary tract cancer (BTC, also known as "bile duct cancer") includes gallbladder cancer, ampullary cancer, cholangiocarcinoma, and cystic duct adenocarcinoma. Cholangiocarcinoma (CCA) can also be divided into intrahepatic CCA or extrahepatic CCA. In one embodiment, a bispecific anti-HER2 antigen-binding construct or ADC can be used in a method of treating BTC. In one embodiment, the bispecific anti-HER2 antigen-binding construct or ADC described herein can be used in a method of treating advanced unresectable BTC. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC described herein can be used in a method of treating gallbladder cancer, ampullary cancer, cholangiocarcinoma, or cystic duct adenocarcinoma. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC described herein can be used in a method of treating intrahepatic CCA or extrahepatic CCA.

[0352] In one embodiment, a bispecific anti-HER2 antigen-binding construct or ADC can be used to treat a subject having BTC that exhibits HER2 expression, amplification, or activation. BTC that "exhibits HER2 expression, amplification, or activation" is BTC that expresses (including overexpresses) the HER2 receptor, has an amplified HER2 gene, and / or otherwise exhibits HER2 receptor activation or phosphorylation in a diagnostic test.

[0353] BTC that "exhibits HER2 activation" is BTC that exhibits HER2 receptor activation or phosphorylation in a diagnostic test. Such activation can be determined directly (e.g., by measuring HER2 phosphorylation by ELISA) or indirectly (e.g., by gene expression profiling). In one embodiment, a bispecific anti-HER2 antigen-binding construct or ADC can be used to treat a subject having BTC that exhibits HER2 expression.

[0354] BTC having "HER2 receptor overexpression or amplification" is BTC that has a significantly higher level of HER2 receptor protein or gene compared to non-cancerous cells of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. HER2 receptor overexpression or amplification can be determined in a diagnostic or prognostic assay by evaluating an elevated level of HER2 protein present on the cell surface (e.g., via immunohistochemical assay; IHC). In one embodiment, HER2 overexpression can be analyzed by IHC, such as using (Dako). Paraffin-embedded tissue sections from a tumor biopsy can be subjected to an IHC assay and meet the following HER2 protein staining intensity criteria:

[0355] Score 0: No staining is observed or membrane staining is observed in less than 10% of tumor cells.

[0356] Score 1+: Weak / almost imperceptible membrane staining is detected in more than 10% of tumor cells. Only part of the cell membrane is stained.

[0357] Score 2+: Weak to moderate complete membrane staining is observed in more than 10% of tumor cells.

[0358] Score 3+: Moderate to strong complete membrane staining is observed in more than 10% of tumor cells.

[0359] Those tumors with scores of 0 or 1+ in HER2 overexpression assessment can be characterized as not overexpressing HER2, while those with scores of 2+ or 3+ can be characterized as overexpressing HER2. In one embodiment, a bispecific anti-HER2 antigen-binding construct or an ADC can be used to treat a subject with BTC that exhibits HER2 overexpression and / or amplification.

[0360] Alternatively or additionally, the level of HER2-encoding nucleic acid in cells can be measured, for example, via in situ hybridization (ISH), including fluorescence in situ hybridization (FISH; see WO98 / 45479 published in October 1998) and chromogenic in situ hybridization (CISH; see, for example, Tanner et al., Am. J. Pathol. 157(5):1467-1472 (2000); Bella et al., J. Clin. Oncol. 26: (May 20 supplement; abstract 22147) (2008)), Southern blotting, polymerase chain reaction (PCR) techniques such as quantitative real-time PCR (qRT-PCR) or next-generation sequencing (NGS). Assessment of HER2 gene amplification using these methods is typically reported as positive (+) or negative (-), for example, FISH+ for HER2 gene-amplified cancers or FISH- for cancers without HER2 gene amplification. Assessment of HER2 gene amplification by NGS can also be reported in terms of the number of HER2 gene copies. In normal cells, there are two HER2 gene copies. Thus, if a cancer has more than two HER2 gene copies, the cancer can be considered a HER2 gene-amplified cancer.

[0361] The present disclosure describes methods of treating a subject with BTC that exhibits HER2 expression, amplification, or activation, including providing to the subject an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC described herein. In some embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to treat a subject with HER2 3+, gene-amplified BTC. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to treat HER2 2+, gene-amplified BTC. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to treat HER2 1+, gene-amplified BTC. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to treat BTC that is evaluated as HER2 3+ without HER2 gene amplification. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to treat BTC that is evaluated as HER2 2+ without HER2 gene amplification. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used to treat BTC that is evaluated as HER2 1+ without HER2 gene amplification.

[0362] In some embodiments, the subject being treated may have no prior BTC treatment and the bispecific anti-HER2 antigen-binding construct or ADC is administered as first-line treatment. In some embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be used as adjuvant or neoadjuvant therapy to treat a subject with resectable or partially resectable cancer. In other embodiments, the subject being treated may have received one or more prior BTC treatments and the bispecific anti-HER2 antigen-binding construct or ADC is administered as second-line treatment. The one or more prior BTC treatments can include treatments selected from the group consisting of: systemic chemotherapy, such as gemcitabine alone or in combination with a platinum-based chemotherapeutic agent, fluoropyrimidine-based chemoradiation, radiation therapy without additional chemotherapy, antibodies (including but not limited to anti-HER2-targeted antibodies), and investigational agents (i.e., agents that are currently in clinical trials but have not been approved by the FDA). Platinum-based chemotherapeutic agents can include cisplatin or oxaliplatin. In one embodiment, the systemic chemotherapy includes gemcitabine and cisplatin, or gemcitabine and oxaliplatin.

[0363] Exemplary effective amounts of the bispecific anti-HER2 antigen-binding construct or ADC that can be administered to a subject with BTC can range between 0.1 mg / kg and 100 mg / kg of subject body weight. In some embodiments, the bispecific anti-HER2 antigen-binding construct or ADC is administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg body weight.

[0364] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered weekly, every two weeks (Q2W), every three weeks (Q3W), or every four weeks (Q4W). Exemplary effective amounts for weekly dosing of the bispecific anti-HER2 antigen-binding construct range between about 1 mg / kg and about 30 mg / kg. Exemplary effective amounts for bi-weekly dosing of the bispecific anti-HER2 antigen-binding construct range between about 10 mg / kg and about 50 mg / kg. Exemplary effective amounts for every three-week dosing of the bispecific anti-HER2 antigen-binding construct range between about 15 mg / kg and about 50 mg / kg. Exemplary effective amounts for every four-week dosing of the bispecific anti-HER2 antigen-binding construct range between about 40 mg / kg and about 70 mg / kg.

[0365] In some embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is 5, 10, or 15 mg / kg per week. In some embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is 10 mg / kg per week. In some embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is 20, 25, or 30 mg / kg every two weeks. In other embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is 20 mg / kg every two weeks. In alternative embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is 20 mg / kg every three weeks. In other embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is 30 mg / kg every three weeks. In further embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is 40 mg / kg every four weeks. In some embodiments, the effective amount of the bispecific anti-HER2 antigen-binding construct is an initial dose of 20, 25, or 30 mg / kg, followed by a lower dose of the bispecific anti-HER2 antigen-binding construct.

[0366] As is known in the art, the ADC can be administered to a subject at a dose lower than the dose used for the bispecific anti-HER2 antigen-binding construct. In some embodiments, the ADC described herein (i.e., the bispecific anti-HER2 antigen-binding construct conjugated to an auristatin analog) is administered weekly, every two weeks (Q2W), every three weeks (Q3W), or every four weeks (Q4W). In some embodiments, the effective amount of the ADC that can be administered to a subject with BTC ranges between about 1 and about 15 mg / kg per week, every two weeks, or every three weeks.

[0367] As described above, in certain embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be administered intravenously. In one embodiment, the bispecific anti-HER2 antigen-binding construct can be administered by IV infusion in 0.9% saline over 120 to 150 minutes. In one embodiment, the bispecific anti-HER2 antigen-binding construct can be administered by IV infusion in 0.9% saline over 90 minutes. In one embodiment, the bispecific anti-HER2 antigen-binding construct can be administered by IV infusion in 0.9% saline over 60 minutes. In related embodiments, the infusion rate should not exceed 250 mL of saline per hour.

[0368] Also provided herein are methods of treating a subject having BTC, comprising co-administering an effective amount of a bispecific anti-HER2 antigen-binding construct or ADC with an additional anti-tumor therapy. The additional anti-tumor therapy can be selected from one or more BTC therapies, including systemic chemotherapy, such as gemcitabine alone or in combination with a platinum-based chemotherapeutic agent, fluoropyrimidine-based chemoradiation, radiation therapy without additional chemotherapy, and investigational agents (i.e., agents currently in clinical trials but not yet approved by the FDA). In one embodiment, the method of treating a subject having BTC comprises co-administering an effective amount of a bispecific anti-HER2 antigen-binding construct or ADC with gemcitabine and cisplatin or with gemcitabine and oxaliplatin. In one embodiment, the bispecific anti-HER2 antigen-binding construct or ADC can be co-administered with a fluoropyrimidine drug and a platinum-based drug. Examples of fluoropyrimidine drugs include, but are not limited to, fluorouracil (5-FU), capecitabine, or gemcitabine. Examples of platinum-based drugs include, but are not limited to, cisplatin or oxaliplatin. In other embodiments, the bispecific anti-HER2 antigen-binding construct or ADC can be co-administered with 5-FU, oxaliplatin, and leucovorin. In other embodiments where the subject has BTC that is MSI-H / dMMR (high microsatellite instability / deficient mismatch repair), the bispecific anti-HER2 antigen-binding construct or ADC can be co-administered with an immune checkpoint inhibitor such as the anti-PD1 antibody pembrolizumab TM (Keytruda ) or the anti-PD-L1 antibody atezolizumab

[0369] As described in Table 2 of Simile et al., (2019) Medicina 55:42, additional anti-tumor therapies for BTC are known in the art. Those skilled in the art will be able to identify which of these therapies can be co-administered with the bispecific anti-HER2 antigen-binding construct or ADC described herein.

[0370] The additional anti-tumor therapies described in the preceding paragraphs can be co-administered with the bispecific anti-HER2 antigen-binding construct or ADC simultaneously or can be administered sequentially.

[0371] In some embodiments, providing an effective amount of a bispecific anti-HER2 antigen-binding construct to a subject with BTC results in tumor shrinkage, inhibition of tumor growth, increased time to tumor progression, prolonged disease-free survival of the subject, reduced metastasis, increased progression-free survival of the subject, or increased overall survival of the subject or increased overall survival of a group of treated subjects. In related embodiments, providing an effective amount of a bispecific anti-HER2 antigen-binding construct to a subject results in partial response (PR) or stable disease (SD) in the subject, as measured by the Revised Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (version 1.1) [Eur J Ca 45:228-247, 2009]. In subjects with metastatic disease and CR or PR, duration of response can also be measured.

[0372] As used herein, the term "progressive disease" (PD) refers to the appearance of one or more new lesions and / or unequivocal progression of existing non-target lesions. PD can be declared based on "unequivocal progression" when the overall tumor burden has increased significantly enough to require a change in therapy; in most cases, a modest increase in the size of one or more non-target lesions is not sufficient to qualify (especially in the presence of SD or PR in the target disease).

[0373] As used herein, the term "partial response" (PR) refers to at least a 30% decrease in the sum of the diameters of target lesions (including the short axis of any target lymph nodes), referenced to the baseline sum of diameters.

[0374] As used herein, the term "complete response" (CR) refers to the disappearance of all non-target lesions, normalization of tumor marker levels (if tumor markers are measured and were initially above the upper limit of normal, those that must normalize for the patient are considered a complete clinical response). All lymph nodes must be <10 mm (short axis).

[0375] As used herein, the term "stable disease" (SD) refers to neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, referenced to the smallest sum of diameters since the start of treatment.

[0376] As used herein, the term "objective response rate" (ORR) is the proportion of all randomized patients who received any amount of study drug treatment and had a PR or CR from the start of treatment until disease progression / recurrence, as determined by RECIST v 1.1 (taking PD as the reference and the smallest measured value recorded since the start of treatment).

[0377] As used herein, the term "overall survival" (OS) refers to the time from the date of randomization to the date of death from any cause.

[0378] As used herein, the term "progression-free survival" (PFS) refers to a patient remaining alive without the cancer progressing or worsening. In one embodiment, PFS is defined as the time from randomization in the study until the first radiographic record of objective progression as defined by RECIST (version 1.1) or death from any cause. Patients who die without a prior reported progression will be considered to have progressed on the day of their death. Patients who have not progressed or been lost to follow-up will be censored on the day of their last radiographic tumor assessment.

[0379] As used herein, the term "disease-free survival" (DFS) refers to the length of time that a patient survives after the completion of initial treatment of the cancer without any signs or symptoms of that cancer. DFS may also be referred to as "recurrence-free survival" (RFS).

[0380] As used herein, the term "time to progression" (TTP) refers to the length of time from the diagnosis or start of treatment of the cancer until the cancer begins to worsen or spread to other parts of the body.

[0381] As used herein, the term "disease control rate" (DCR) refers to the absence of disease progression and its rate. It refers to the group of patients with the best overall response classified as CR, PR, or SD (explicitly excluding PD patients), where the best overall response is the best response recorded from the start of treatment until PD.

[0382] As used herein, the term "duration of overall response" (DOR) refers to the period measured from the time (whichever is recorded first) that the criteria for a complete or partial response are met until the first date of objectively recorded recurrent or progressive disease, with reference to the smallest measurement recorded since the start of treatment.

[0383] In some embodiments, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC to a subject with BTC results in an increased disease control rate (DCR) in a group of subjects. The DCR can be used to measure the efficacy of a therapy having an anti-tumor effect rather than a tumoricidal effect. The DCR is calculated as the percentage of BTC patients who exhibit CR, PR, or SD after treatment with the bispecific anti-HER2 or ADC. In one embodiment, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC to a subject results in a DCR greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In other embodiments, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC to a subject results in a DCR greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0384] Progression-free survival (PFS) and overall response rate (ORR) can also be used to determine the efficacy of a bispecific anti-HER2 antigen-binding construct or an ADC and are measured according to the revised RECIST 1.1 guidelines described above. PFS is defined as the time from randomization until objective tumor progression or death. ORR is defined as the proportion of BTC subjects who have a partial or complete response to a therapy utilizing a bispecific anti-HER2 antigen-binding construct or an ADC. The ORR can be used as a measure of the drug's tumoricidal activity. In some embodiments, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC to a subject with BTC results in an increased progression-free survival (PFS) in a group of subjects. In some embodiments, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC to a subject with BTC results in an increased overall response rate (ORR). In one embodiment, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC to a subject results in an ORR greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In yet another embodiment, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or an ADC to a subject results in an ORR greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0385] Overall survival, time to progression, duration of response (DOR) (which can also be used to determine the efficacy of a bispecific anti-HER2 antigen-binding construct or an ADC).

[0386] When a bispecific anti-HER2 antigen-binding construct or an ADC is administered as an adjuvant or neoadjuvant therapy, disease-free survival can also be measured to determine the efficacy of the therapy.

[0387] Kit and article of manufacture

[0388] Also described herein are kits comprising one or more bispecific anti-HER2 antigen-binding constructs or ADCs. The individual components of the kit will be packaged in separate containers, and associated with such containers may be a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency for manufacture, use, or sale. The kit may optionally include instructions or a label outlining the method of use or administration protocol of the bispecific anti-HER2 antigen-binding construct or ADC.

[0389] When one or more components of the kit are provided in solution, such as an aqueous solution or a sterile aqueous solution, the container device itself can be an inhaler, syringe, pipette, eye dropper, or other similar device from which the solution can be administered to a subject or applied to and mixed with other components of the kit.

[0390] The components of the kit can also be provided in dry or lyophilized form and the kit can additionally include a solvent suitable for reconstituting the lyophilized components. Irrespective of the number or type of containers, the kits described herein can also include an instrument for facilitating the administration of the composition to a patient. Such an instrument can be an inhaler, nasal spray device, syringe, pipette, forceps, measuring spoon, eye dropper, or similar medically approved delivery vehicle.

[0391] In another aspect described herein, there is provided an article of manufacture comprising materials useful for treating, preventing, and / or diagnosing BTC. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition which, by itself or in combination with another composition effective in treating, preventing, and / or diagnosing the disorder, can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is for treating the selected disorder. Additionally, the article of manufacture can include (a) a first container containing a composition, wherein the composition comprises a bispecific anti-HER2 antigen-binding construct or ADC described herein; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment described herein can also include a package insert indicating that the composition can be used to treat BTC. Alternatively or additionally, the article of manufacture can further include a second (or third) container that contains a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it can also include other desired materials, including other buffers, diluents, filters, needles, and syringes.

[0392] Polypeptides and polynucleotides

[0393] The bispecific anti-HER2 antigen-binding construct described herein comprises at least one polypeptide. Polynucleotides encoding the polypeptides described herein are also described. The bispecific anti-HER2 antigen-binding construct is generally isolated.

[0394] As used herein, "isolated" refers to a reagent (e.g., a polypeptide or polynucleotide) that has been identified and separated and / or recovered from the components of its natural cell culture environment. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the bispecific anti-HER2 antigen-binding construct and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated also refers to a reagent that has been produced synthetically, e.g., via human intervention.

[0395] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0396] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. References to amino acids include, for example, naturally occurring proteinogenic L-amino acids; D-amino acids, chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-proteinogenic amino acids such as β-alanine, ornithine, etc.; and chemically synthesized compounds that have properties known in the art as characteristic of amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methyl amino acids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, β-hydroxy-histidine, homohistidine), amino acids having an additional methylene group in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group (e.g., cysteine). Incorporating non-natural amino acids (including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids) into the proteins described herein in a variety of different ways may be advantageous. Peptides containing D-amino acids, for example, exhibit increased stability in vitro or in vivo compared to their L-amino acid-containing counterparts. Thus, the construction of peptides incorporating D-amino acids may be particularly useful when greater intracellular stability is desired or required. More specifically, D-peptides, etc., are resistant to endogenous peptidases and proteases, thereby providing improved molecular bioavailability and an extended in vivo lifetime when such properties are needed. In addition, D-peptides, etc., cannot be efficiently processed to present major histocompatibility complex class II-restricted epitopes to T helper cells and are thus less likely to induce a humoral immune response in the whole organism.

[0397] Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Similarly, nucleotides may be designated by their commonly accepted single-letter codes.

[0398] The present invention also describes polynucleotides encoding polypeptides of bispecific anti-HER2 antigen-binding constructs. The term "polynucleotide" or "nucleotide sequence" is intended to mean a contiguous segment of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.

[0399] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in either single-stranded or double-stranded form, and their polymers. Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically restricted, the term also refers to oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (including but not limited to degenerate codon substitutions) and complementary sequences as well as the explicitly recited sequence. Specifically, degenerate codon substitutions may be achieved by generating sequences in which one or more of the selected (or all) codons' third positions are substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0400] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, to substantially the same sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations" and they are one species of conservatively modified variation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG and TGG, AUG is typically the only codon for methionine and TGG is typically the only codon for tryptophan) can be modified to yield a functionally identical molecule. Thus, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0401] For amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence (which alter, add or delete a single amino acid or a small percentage of amino acids in the coding sequence) are "conservative modified variants", where the variant results in the deletion of an amino acid, the addition of an amino acid or the substitution of an amino acid with a chemically similar amino acid. Lists of conservative substitutions providing functionally similar amino acids are known to one of ordinary skill in the art. Such conservative modified variants are complementary to the polymorphic variants, interspecies homologs and alleles described herein and do not exclude these.

[0402] Lists of conservative substitutions providing functionally similar amino acids are known to one of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd ed. (December 1993)).

[0403] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or "identity" percent refers to two or more identical sequences or subsequences. Sequences are "substantially identical" if, when compared and aligned for maximum correspondence over a comparison window or specified region using one of the following sequence comparison algorithms (or other algorithms available to one of ordinary skill in the art) or by manual alignment and visual inspection, the sequences have the same percentage of amino acid residues or nucleotides (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% identity over the specified region). This definition also relates to the complementary sequences of a test sequence. Identity can exist over a region of at least about 50 amino acids or nucleotides in length, or over a region of 75 - 100 amino acids or nucleotides in length, or, if not specified, across the entire sequence of a polynucleotide or polypeptide. Polynucleotides encoding the polypeptides described herein, including homologs from species other than humans, can be obtained by a method comprising the steps of screening a library with a labeled probe having the polynucleotide sequence described herein or a fragment thereof under stringent hybridization conditions, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art.

[0404] For sequence comparison, typically one sequence serves as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence according to the program parameters.

[0405] As used herein, a "comparison window" includes a segment that refers to any one of a plurality of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, where after the two sequences are optimally aligned, the sequence can be compared to a reference sequence having the same number of contiguous positions. Methods of sequence alignment for comparison are known to those of ordinary skill in the art. Optimal alignment of the sequences for comparison can be conducted, including but not limited to by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the similarity method search of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 Supplement)).

[0406] An example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information at the World Wide Web (ncbi.nlm.nih.gov). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a wordlength (W) of 11, an expectation (E) of 10, M = 5, N = -4, and the comparison of both strands. For amino acid sequences, the BLASTP program defaults to a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, an expectation (E) of 10, M = 5, N = -4, and the comparison of both strands. The BLAST algorithm is typically performed with the "low complexity" filter turned off.

[0407] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences occurred by chance. For example, if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0408] The phrase "selectively (or specifically) hybridizes to" means that when a sequence is present in a complex mixture (including, but not limited to, total cellular or library DNA or RNA), the molecule binds, duplexes, or hybridizes only to a particular nucleotide sequence under stringent hybridization conditions.

[0409] The phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, or other nucleic acids, or combinations thereof, under conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe will hybridize to its target sequence in a complex nucleic acid mixture (including but not limited to total cellular or library DNA or RNA), but not to other sequences in the complex mixture. Stringent conditions depend on the sequence and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. General guidelines for nucleic acid hybridization are found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

[0410] As used herein, the term "engineer / engineered / engineering" is considered to include any manipulation of a peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, glycosylation pattern, or side chain group of an individual amino acid, as well as combinations of these methods. Engineered proteins are expressed and produced by standard molecular biology techniques.

[0411] "Isolated nucleic acid molecule or polynucleotide" refers to a nucleic acid molecule, DNA, or RNA that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or polynucleotides purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in a cell that normally contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include RNA transcripts in vivo or in vitro, as well as sense and antisense forms, and double-stranded forms. The isolated polynucleotides or nucleic acids described herein also include such molecules produced synthetically, e.g., via PCR or chemical synthesis. In addition, in certain embodiments, the polynucleotide or nucleic acid includes regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0412] The term "polymerase chain reaction" or "PCR" generally refers to a method for amplifying a desired nucleotide sequence in vitro, as described, for example, in U.S. Patent No. 4,683,195. Generally, the PCR method involves repeated cycles of primer extension synthesis using oligonucleotide primers that are capable of preferentially hybridizing to a template nucleic acid.

[0413] For a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to the reference nucleotide sequence of the present invention, it means that the nucleotide sequence of the polynucleotide is the same as the reference sequence, except that for every 100 nucleotides of the reference nucleotide sequence, the polynucleotide sequence may contain up to five point mutations. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or at any position between these terminal positions, singly dispersed among the residues in the reference sequence or dispersed in one or more contiguous groups within the reference sequence. In fact, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of the present invention can be routinely determined using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

[0414] A derivative or variant of a polypeptide is said to be "homologous" or "homologue" to the peptide if the amino acid sequence of the derivative or variant has at least 50% identity to a 100 amino acid sequence from the original peptide. In certain embodiments, the derivative or variant is at least 75% identical to a derivative or variant of a peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 85% identical to a derivative or variant of a peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of the derivative is at least 90% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95% identical to a peptide or peptide fragment having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 99% identical to a derivative or variant of a peptide or peptide fragment having the same number of amino acid residues as the derivative.

[0415] As used herein, the term "modified" refers to any change made to a given polypeptide, such as a change in polypeptide length, amino acid sequence of the polypeptide, chemical structure, co-translational modification, or post-translational modification. The form "(modified)" means that the polypeptide in question is optionally modified, i.e., the polypeptide in question may or may not be modified.

[0416] In some aspects, the bispecific anti-HER2 antigen-binding construct comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the relevant amino acid sequence or a fragment thereof shown in the tables or accession numbers disclosed herein. In some aspects, the isolated bispecific anti-HER2 antigen-binding construct comprises an amino acid sequence encoded by a polynucleotide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the relevant amino acid sequence or a fragment thereof shown in the tables or accession numbers disclosed herein.

[0417] It should be understood that the present disclosure is not limited to specific protocols; the cell lines, constructs, reagents, etc. described herein may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present disclosure.

[0418] For the purpose of describing and disclosing constructs and methods such as those described in publications, all publications and patents mentioned herein are incorporated herein by reference and can be used in conjunction with the constructs described herein. The publications discussed herein are provided only as to their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

[0419] Sequence Listing

[0420] Sequence Listing

[0421] Table 6: Clone numbers of variants v5019, v5020, v7091, v10000, v6903, v6902, and v6717

[0422]

[0423]

[0424] Table 7: Sequences of variants v5019, v5020, v7091, v10000, v6903, v6902, and v6717 listed by clone number

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440] Examples

[0441] The following are examples of specific embodiments for making and using the bispecific anti-HER2 antigen-binding constructs and ADCs described herein. These examples are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any way. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental error and deviation should be allowed.

[0442] Unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the skill of the art can be used to prepare and implement the constructs and methods described herein. These techniques are well explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th ed. (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd ed. (Plenum Press) Volumes A and B (1992).

[0443] Example 1: Description and preparation of variant 10000 (v10000)

[0444] v10000 is a humanized bispecific antibody that recognizes two non-overlapping epitopes of the ECD of the human HER2 antigen. The IgG1-like Fc region of v10000 contains complementary mutations in each CH3 domain that confer preferential pairing to generate heterodimeric molecules and correspondingly disfavor the formation of homodimers. Figure 1 A diagram depicting the form of v10000 is shown, where heavy chain A and light chain A' form the ECD2-binding portion of the antibody, and heavy chain B contains an scFv that forms the ECD4-binding portion of the antibody. Variant 10000 includes heavy chain H1 comprising the sequence shown in SEQ ID NO: 36 (corresponding to heavy chain A in Figure 1 ), heavy chain H2 comprising the sequence shown in SEQ ID NO: 63 (corresponding to heavy chain B in Figure 1 ), and light chain L1 comprising the sequence shown in SEQ ID NO: 24 (corresponding to light chain A'). The method for preparing v10000 is described in detail in International Patent Publication No. WO 2015 / 077891.

[0445] v10000 is manufactured in accordance with the relevant regulatory requirements for human trials and formulated at 15 mg / mL in a biocompatible aqueous buffer for IV infusion at ambient temperature. v10000 is supplied in vials containing 20 mL of buffer with 300 mg of v10000. The vials of v10000 are transported frozen and stored at -20 °C (+ / - 5 °C) until ready for use. The vials are thawed at ambient temperature before use. The thawed solution in the vial is stored at ambient temperature for up to 24 hours or at refrigerated conditions (2 °C to 8 °C) for up to 72 hours and used before the labeled expiration date.

[0446] Example 2: Phase I of v10000 in patients with locally advanced (unresectable) and / or metastatic HER2-expressing cancer Clinical trial

[0447] This is an ongoing first-in-human study to investigate the safety, tolerability, pharmacokinetics (PK), and preliminary antitumor activity of v10000 monotherapy in patients with locally advanced (unresectable) and / or metastatic human epidermal growth factor receptor 2 (HER2)-expressing cancers.

[0448] Part 1 of the study is a 3+3 dose escalation in which the single agent 20 mg / kg Q2W is determined as the recommended dose (RD). Part 2 is ongoing and the v10000 RD is being evaluated in additional patients, including HER2-high BTC patients. Eligible patients in Parts 1 and 2 must have had disease progression after all known clinically beneficial therapies. Tumor responses are assessed by investigator review according to RECIST v1.1 Q8W.

[0449] Objectives

[0450] The primary objective of Part 1 of the clinical trial is to determine the maximum tolerated dose (MTD), optimal biological dose (OBD), or RD of v10000 monotherapy. The secondary objectives of Part 1 of the clinical trial are (1) to characterize the safety and tolerability of v10000; (2) to characterize the serum PK profile of v10000; and (3) to explore the potential antitumor effects of v10000 in eligible HER2-expressing cancer patients.

[0451] The primary objective of Part 2 of the trial is to characterize the safety and tolerability of v10000 monotherapy in specific tumor types. The secondary objectives of Part 2 of the clinical trial are (1) to characterize the serum PK profile of v10000 monotherapy and (2) to explore the potential antitumor effects of v10000 at the MTD, OBD, or RD in eligible selected HER2-expressing locally advanced (unresectable) and / or metastatic cancer patients.

[0452] Patients

[0453] According to clinical investigators, male or female patients aged ≥ 18 years with an ECOG (Eastern Cooperative Oncology Group) performance status of 0 or 1 and a life expectancy of at least 3 months were included in the trial.

[0454] In Part 1, Groups 1 - 3 included patients with any locally advanced (unresectable) and / or metastatic HER2-expressing (HER2 1+, 2+, or 3+, according to IHC) cancers (including but not limited to breast, gastric, ovarian, colorectal, and non-small cell lung cancers) who had progressed after receiving all known clinically beneficial therapies. Groups 4 - 6 included patients with HER2 IHC 2+ / FISH-breast cancer or gastroesophageal adenocarcinoma (GEA); patients with HER2 IHC 3+ or HER2 IHC 2+ / FISH+ breast cancer or GEA. Groups 4 - 6 also included patients with any other HER2 IHC 3+ or FISH+ cancers, where the cancer was HER2 overexpressing (3+, according to IHC) or HER2 2+, and FISH+ breast cancer had to have progressed after prior treatment with trastuzumab, pertuzumab, and T-DM1; where the cancer was HER2 overexpressing (3+, according to IHC) or HER2 2+, and FISH+ GEA had to have progressed after prior treatment with trastuzumab; where colorectal cancer patients were KRAS wild-type; or where NSCLC patients were ALK wild-type, EGFR wild-type, and ROS1 fusion-negative, as determined by standard methods. Group 7 was recruited at selected sites and included patients with HER2 IHC 3+, HER2 IHC 2+ / FISH+, or HER2 IHC 2+ / FISH-breast cancer.

[0455] In Part 2, expansion of groups from Part 1 of the study using v10000 administered at the MTD, OBD, or RD included locally advanced (unresectable) and / or metastatic cancers (unless ineligible for specific therapies) that had progressed after receiving all known clinically beneficial therapies as follows:

[0456] Group 1: HER2 IHC 2+ / FISH-breast cancer

[0457] Group 2: HER2 IHC 3+ or HER2 IHC 2+ / FISH+ breast cancer

[0458] Group 3: HER2 IHC 2+ / FISH-GEA

[0459] Group 4: HER2 IHC 3+ or HER2 IHC 2+ / FISH+ GEA

[0460] Group 5: Any other HER2 IHC 3+ or IHC 2+ / FISH+ cancers, including the following:

[0461] Group 5a: HER2 IHC 3+ or IHC 2+ / FISH+ GI (gastrointestinal) cancers other than GEA, where colorectal cancer patients are KRAS wild-type

[0462] Group 5b: Any other HER2 IHC 3+ or IHC 2+ / FISH+ solid tumor type other than breast cancer or gastrointestinal cancer, where NSCLC patients must have ALK wild-type, EGFR wild-type, and ROS1 fusion-negative determined by standard methods. Ovarian cancer patients must be KRAS wild-type.

[0463] Other criteria for patients in Part 1 and Part 2 include: (1) HER2 IHC 3+ or IHC 2+ / FISH+ breast cancer must have progressed after prior treatment with trastuzumab, pertuzumab, and T-DM1; (2) HER2 IHC 3+ or IHC 2+ / FISH+ GEA must have progressed after prior treatment with trastuzumab; (3) colorectal cancer patients must be Kirsten rat sarcoma (KRAS) wild-type; and (4) NSCLC patients must have anaplastic lymphoma kinase (ALK) wild-type, EGFR wild-type, and receptor tyrosine kinase (ROS1) fusion-negative determined by standard methods.

[0464] If one or more of the following criteria apply, the patient is excluded from the study:

[0465] 1. Treatment with experimental therapy within 4 weeks before the first v10000 dose

[0466] 2. Treatment with other unspecified cancer therapies within 4 weeks before v10000 dosing

[0467] 3. Treatment with anthracyclines within 90 days before the first v10000 dose or a lifetime total dose exceeding 300 mg / m 2 Doxorubicin or equivalent

[0468] 4. Treatment with trastuzumab, pertuzumab, lapatinib, or T-DM1 within 3 weeks before the first v10000 dose

[0469] 5. Untreated brain metastases (patients with treated brain metastases are eligible only after discontinuing steroids and being stable for at least 1 month at screening). All breast cancer patients should be screened before starting treatment. Those found to have untreated brain metastases may be re-screened after appropriate therapy.

[0470] 6. Leptomeningeal disease (LMD) with clinical assessment. If LMD has been reported radiographically on baseline MRI but not clinically suspected by the investigator, the patient is eligible if he or she has no neurological symptoms of LMD recorded by the investigator.

[0471] 7. Major surgery or radiotherapy within 3 weeks prior to the first v10000 administration.

[0472] 8. Pregnant or lactating women.

[0473] 9. History of life-threatening hypersensitivity to monoclonal antibodies or to recombinant proteins or excipients in the drug formulation.

[0474] 10. Any other cancer within 3 years prior to the first v10000 administration, except for contralateral breast cancer, adequately treated carcinoma in situ of the cervix, adequately treated basal cell carcinoma or squamous cell carcinoma of the skin, or any other cancer that has received curative treatment, and subject to approval by the sponsor's medical monitor.

[0475] 11. Acute or chronic uncontrolled kidney disease, pancreatitis, or liver disease (except for patients with Gilbert's Syndrome, asymptomatic gallstones, liver metastases, or stable chronic liver disease as evaluated by the investigator).

[0476] 12. Peripheral neuropathy: > Grade 2 according to the National Cancer Institute - Common Terminology Criteria for Adverse Events (NCI - CTCAE), Version 4.03, July 14, 2010.

[0477] 13. Clinically significant interstitial lung disease.

[0478] 14. History of non - compliance with the medical protocol.

[0479] 15. Unwilling or unable to comply with the protocol.

[0480] 16. Known active hepatitis B or C or known infection with human immunodeficiency virus (HIV).

[0481] 17. Use of corticosteroids administered at a dose equivalent to > 15 mg of prednisone per day within 2 weeks prior to the first v10000 administration, unless otherwise approved by the study medical monitor.

[0482] 18. QTc Fridericia (QTcF) > 450 ms.

[0483] 19. Any toxicity related to previous cancer therapies that has not resolved to ≤ Grade 1, except for alopecia; neuropathy (must have resolved to ≤ Grade 2); and congestive heart failure (CHF), which must have been ≤ Grade 1 in severity at the time of occurrence and must have resolved completely.

[0484] 20. Having clinically significant heart disease, such as ventricular arrhythmias requiring treatment, uncontrolled hypertension, or any history of symptomatic CHF.

[0485] 21. Known myocardial infarction or unstable angina within 6 months prior to the first v10000 administration.

[0486] Treatment

[0487] In Parts 1 and 2, v10000 was administered intravenously (IV) as a single agent (monotherapy). v10000 was administered via IV infusion in 0.9% saline over 120 to 150 minutes. If the first 2 doses administered to a particular patient were well tolerated, the infusion duration for that patient may have been reduced to 90 minutes. If the next 2 doses were well tolerated, the infusion duration could be reduced to 60 minutes. The infusion rate was not to exceed 250 mL of 0.9% saline / hour. (For example: If one dose of v10000 was diluted into a 250 mL bag of saline, the infusion should be administered over at least 60 minutes. If one dose of v10000 was diluted into a 500 mL bag, the infusion should be administered over at least 120 minutes.) The study drug dose was calculated based on the patient's weight on Day 1 of Cycle 1. The dose was recalculated only if there was a 10% change in the evaluated weight from Day 1 of Cycle 1.

[0488] In Part 1, the dose escalation dose levels were 5, 10, and 15 mg / kg, administered once weekly (QW). Administration every two weeks (Q2W) was also evaluated. The dose levels for Q2W administration were 20, 25, or 30 mg / kg. Q2W administration may have used an initial loading dose (not exceeding 20, 25, or 30 mg / kg), followed by v10000 administered at a lower dose level recommended by the SMC. In addition, a dose of 30 mg / kg every 3 weeks (Q3W) was studied.

[0489] The dose levels in Part 2 were the MTD, OBD, or RD determined in Part 1. The MTD was defined as the highest dose level at which no more than 1 out of 6 patients experienced a DLT during the first 4 weeks of treatment. The OBD was defined as the v10000 dose that resulted in a trough (7 days after dosing) v10000 serum concentration at least 10 times higher than the maximum binding capacity of v10000 for a cell line representative of HER2-3+ tumor histology. The RD was any other dose not exceeding the MTD. Based on the results of Part 1, an RD of 20 mg / kg Q2W was used to treat most patients in Part 2. Some patients were treated with 10 mg / kg weekly.

[0490] Patients participate in a minimum of 2 cycles, each cycle being 3 or 4 weeks, depending on the patient's enrolled part, group, or TG. Treatment can continue for additional cycles as long as there is no evidence of clinical progression as defined by RECIST version 1.1, unacceptable toxicity, or evidence of progressive disease. Clinical progression is defined as the worsening or recurrence of pre-existing symptoms related to the underlying cancer, or the emergence of new symptoms not attributable to the toxicity of the study drug or other causes. Patients who show continued clinical benefit despite radiological progression, in the opinion of the clinical investigator, may continue treatment after discussion with and approval by the sponsor medical monitor. For Part 3, if chemotherapy is stopped due to toxicity unrelated to v10000, the patient may continue to receive v10000. Patients who discontinue v10000 treatment for any reason are withdrawn from the study.

[0491] Efficacy Assessment

[0492] Measures of anti-tumor activity are evaluated based on efficacy assessment according to the new international criteria proposed in the guidelines for the revised Response Evaluation Criteria in Solid Tumors (RECIST) (version 1.1) [Eur J Ca 45:228-247, 2009]. Changes in the greatest diameter of tumor lesions (one-dimensional measurement) and the shortest diameter of malignant lymph nodes are used in the RECIST version 1.1 criteria. Clinical responses of CR, PR, SD, or progressive disease (PD) are determined by the investigator at each assessment. PD includes progressive disease according to RECIST version 1.1 and clinical disease progression according to the investigator. Clinical progression is defined as the worsening or recurrence of pre-existing symptoms related to the underlying cancer, or the emergence of new symptoms not attributable to the toxicity of the study drug or alternative causes.

[0493] The objective response rate (ORR) is defined as the percentage of patients with at least 1 overall tumor response of CR or PR before any evidence of progression as defined by RECIST version 1.1. A patient is considered to have achieved disease control if they have a tumor response of CR, PR, or SD according to the RECIST version 1.1 criteria. The disease control rate is evaluated every 8 weeks after the start of v10000 therapy. Progression-free survival (PFS) time is defined as the time from the first dose of v10000 to the date of disease progression, clinical progression, or death due to any reason as recorded according to RECIST version 1.1. Patients who are alive and without progression at the time of analysis will be censored at their last CR, PR, or SD tumor assessment.

[0494] Tumor response is evaluated based on CT and / or MRI scans of the chest, abdomen, and pelvis (using the same method for each scan of the same patient) plus additional areas known or suspected to be involved by the tumor (e.g., brain, extremities).

[0495] Local assessment of objective response and tumor progression. Scans of all subjects were collected for central review at the sponsor's discretion. Local assessment was used for all treatment-related decisions.

[0496] For some patients, tumor volumes were calculated centrally.

[0497] Adverse events

[0498] An AE (adverse event) was defined as any untoward medical occurrence that occurred in patients in a clinical study following the administration of a medicinal product that did not necessarily have a causal relationship with this treatment. Thus, an AE could be any adverse and unintended sign (including abnormal laboratory findings), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not it was related to the medicinal (investigational) product. This included exacerbation of a pre-existing condition or event, concurrent disease, drug interaction, or significant worsening of the indication under study, which was not recorded elsewhere in the CRF in a specific efficacy assessment. Safety assessment was based on version 4.03 of the NCI-CTCAE dated July 14, 2010.

[0499] Early results:

[0500] As of June 2018, in a patient with gallbladder cancer, treatment with v10000 resulted in a decrease of approximately 40% in the sum of the longest diameters (SLD) of the target lesions. This patient later showed a decrease of approximately 50% in the target lesions measured by SOD (sum of diameters).

[0501] As of November 2018, in a patient with CCA, a decrease of approximately 40% in the target lesions was observed after treatment with v10000 as measured by SOD.

[0502] Intermediate results:

[0503] As of April 22, 2019, 89 patients with all indications had been enrolled in Parts 1 and 2 of v10000 (23 in Part 1 and 66 in Part 2) and treated with a single agent, v10000 (Table C). The tumor types evaluated included breast cancer (n = 42), gastroesophageal cancer (n = 20), colorectal cancer (n = 11), biliary tract cancer (n = 6), and other cancers (n = 10). In patients with BTC, the median number of prior systemic treatment regimens was 4 (range 1 - 8), including trastuzumab in one patient.

[0504] In Parts 1 and 2 of the study, the majority of AEs were grade 1 or 2 in severity (Table D). V10000-related grade 3 AEs included fatigue (n = 3, including one event reported as an SAE), diarrhea (n = 2), arthralgia (n = 1), and hypophosphatemia (n = 1).

[0505] Preliminary efficacy data for BTC patients are presented in Tables E, F, and Figure 2 .

[0506] Table C. Key demographics and baseline characteristics by cancer type in Parts 1 and 2

[0507]

[0508] Table D. Summary of v10000-related adverse events by cancer type in Parts 1 and 2

[0509]

[0510] Table E. Summary of best responses in the measurable disease analysis set in Parts 1 and 2

[0511]

[0512] a Measurable disease analysis set — all patients in the safety analysis set with measurable disease according to RECIST 1.1;

[0513] b Safety analysis set — all patients who received at least one dose of study treatment.

[0514] CI = confidence interval, DCR = disease control rate, NE = not evaluable, ORR = overall response rate, PFS = progression-free survival, PR = partial response, SD = stable disease

[0515] Table F. Prior treatment regimens and disease responses in BTC patients

[0516]

[0517] a All BTC patients were HER2+(3+ or FISH+);

[0518] b Patients received trastuzumab while on 6 of 8 systemic treatment regimens;

[0519] c Final DOR pending additional disease assessment.

[0520] NA = not applicable NE = not evaluable, pending follow-up disease assessment

[0521] GBC = gallbladder cancer; CC = cholangiocarcinoma

[0522] These data indicate that the disease control rate of v10000 in patients with HER2 3+ or FISH+ BTC is 83.3%, and the ORR is 66.7%.

[0523] Example 3: Preparation of linker-toxin 001

[0524] Prepare Linker-Toxin 001 as described below. Linker-Toxin 001 can also be prepared as described in International Patent Application Publication No. WO 2016 / 041082.

[0525]

[0526] Linker-Toxin 001

[0527] A. Ethyl (2R,3R)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propionate (Compound 1)

[0528]

[0529] To a stirred solution of (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (Boc-Dap-OH, 4.31 g, 15.0 mmol) in absolute ethanol (27.0 mL) at 0 °C was added thionyl chloride (3.0 mL) dropwise. The resulting solution was warmed to room temperature and the progress was monitored by HPLC-MS. After 18 hours, no remaining starting material was detected and the solution was concentrated to dryness under reduced pressure. The resulting oil was suspended in toluene (10 mL) and concentrated twice under reduced pressure, then suspended in diethyl ether (5 mL) and concentrated twice under reduced pressure to give a white solid foam (3.78 g, quantitative yield %). MS m / z observed = 216.5 (M+1).

[0530] B. (3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoic acid (Compound 3)

[0531]

[0532] Prepare Compound 2 as described in International Patent Application Publication No. WO 2016 / 041082.

[0533] Trifluoroacetic acid (5.0 mL) was added to a stirred solution of Compound 2 (6.965 g, 14.14 mmol) in dichloromethane (20 mL). The completion of the reaction was monitored by HPLC-MS and no starting material remained after 40 h. The reaction was concentrated under reduced pressure and co-evaporated with toluene (2 x 10 mL) and dichloromethane (2 x 10 mL) to afford a white solid foam (6.2 g, quantitative yield, containing residual TFA). This material was dissolved in 200 mL of hot 1:3 EtOAc:hexanes and cooled to room temperature. During cooling, a precipitate and some small crystals formed. 5 mL of EtOAc was added and the suspension was heated again to completely dissolve the precipitate. More crystals formed upon cooling to room temperature and the flask was placed at -30 °C overnight. The next morning, the mother liquor was decanted and the crystals were rinsed with 2 x 50 mL of hexanes and dried under high vacuum. 5.67 g of the crystalline product was recovered. MS m / z observed = 405.7 (M+1).

[0534] C. Ethyl (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionate (Compound 4)

[0535]

[0536] To a stirred solution of Compound 3 (6.711 g, 15.37 mmol, 1.025 equiv) in a mixture of dichloromethane (5.0 mL) and N,N-dimethylformamide (5.0 mL) at room temperature was added HATU (5.732 g, 15.07 mmol, 1.005 equiv) and N,N-diisopropylethylamine (7.84 mL, 3 equiv). After stirring at room temperature for 30 min, a solution of Compound 1 (3.776 g, 15.00 mmol, 1.0 equiv) in a mixture of dichloromethane (1.0 mL) and N,N-dimethylformamide (1.0 mL) was added dropwise, rinsing the residual Compound 1 with an additional 3 mL of 1:1 dichloromethane:N,N-dimethylformamide. The reaction was monitored by HPLC-MS and no residual Compound 1 was observed after 15 min. The reaction was concentrated under reduced pressure, diluted with ethyl acetate (~125 mL), and the organic phase was extracted with 1 M HCl (2 x 50 mL), 1 x dH2O (1 x 50 mL), saturated NaHCO3 (3 x 50 mL), and brine (25 mL). The acidic and basic aqueous layers were each washed with 25 mL of EtOAc. Then all of the organics were combined and dried over MgSO4, filtered, and concentrated to afford a red oil. The residue was dissolved in the minimum amount of dichloromethane (~10 mL) and loaded onto Purification was carried out on a SNAPUltra 360g silica column (Isolera TM Flash System; Biotage AB, Sweden) (hexane solution of 20 - 100% EtOAc, over 10 column volumes). The fractions containing the pure product were combined and 7.9 g of a white foamy solid was recovered. On a SNAP Ultra 100g silica column, the impure fractions were purified a second time and combined with the pure product to recover a white foamy solid (8.390 g, 88.3%). Observed MS m / z = 634.7 (M+1).

[0537] D. (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (Compound 5)

[0538]

[0539] To a stirred solution of Compound 4 (8.390 g, 13.24 mmol) in 1,4-dioxane (158 mL) was added dH2O (39.7 ml) and lithium hydroxide monohydrate (1 M in H2O, 39.7 mL, 3 eq). The reaction was stirred at 4 °C and monitored by HPLC-MS for consumption of the starting material, which took 3 days until only trace amounts of Compound 4 remained. During the reaction, in addition to the desired material, a small percentage of a new product was formed corresponding to the loss of methanol (β-elimination, <2%). The reaction was acidified by addition of 1 M aqueous HCl (50 mL) and concentrated in vacuo to remove the dioxane. The remaining reaction mixture was extracted with ethyl acetate (4 x 50 mL), and the combined organic phases were washed with brine (15 mL + 2 mL 2 M HCl), dried over MgSO4, filtered and concentrated in vacuo to give a light oil. The oil was redissolved in diethyl ether (~50 mL) and concentrated in vacuo (3x) to facilitate removal of residual dioxane, giving the title product as a viscous oil (7.81 g, 97% yield, containing some residual dioxane and Compound 4). Observed MS m / z = 606.7 (M+1).

[0540] E. ((S)-1-(((3R,4S,5S)-3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((4-(2,2,2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid benzyl ester (Compound 7)

[0541]

[0542] Compound 6 was prepared as described in International Patent Application Publication No. WO 2016 / 041082.

[0543] To a stirred solution of Compound 5 (7.12 g, 11.754 mmol) in dichloromethane (20 mL) was added 2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide (Compound 6, 4.095 g, 1.3 equiv, dissolved in 3 mL DMF), N,N-dimethylpyridine (1.867 g, 1.3 equiv) and N,N-dimethylformamide (1.5 mL) to form a pale yellow suspension. Further addition of 5 mL DMF did not clarify the solution. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI) (2.817 g, 1.25 equiv) was added in one portion and the reaction was monitored by HPLC-MS. After 48 h, the reaction had ceased and an additional 400 mg EDCI was added. After 18 h, no residual starting material was observed and the reaction was concentrated in vacuo to give a yellow oil. The oil was dissolved in ethyl acetate (~150 mL) and 1 M HCl (20 mL), and the organic phase was washed with cold 2 M HCl (2x 10 mL), saturated NaHCO3 (1x 10 mL), brine (20 mL + 5 mL 2 M HCl). The acidic and basic aqueous fractions were extracted with EtOAc (1x20 mL), all the organic fractions were combined, dried over MgSO4 and concentrated in vacuo to give a crude solid oil (13 g). The residue was dissolved in dichloromethane (~10 mL), loaded onto a SNAP Ultra 360 g silica gel column and purified using a gradient of hexane solution of 10 - 100% EtOAc (2% AcOH) over 12 column volumes, reaching a stable level at 3 column volumes at 50% EtOAc. The fractions containing the pure product were combined, concentrated in vacuo, dissolved and concentrated from toluene (2x 10 mL) and diethyl ether (2x 10 mL) to give the desired product, 7.1 g of a white foam solid. On an Isolera TM instrument using The SNAP Ultra 100g silica gel column was used to repeatedly purify the impure fraction under a relatively gentle gradient condition. All the pure fractions were combined to recover the pure product (8.60 g, 86%) as a white foamy solid. Observed MS m / z = 856.7 (M+1).

[0544] F. (S)-2-Amino-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((4-(2,2,2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N,3-dimethylbutanamide (Compound 7a)

[0545]

[0546] Compound 7 (3.71 g, 4.33 mmol) was dissolved in a solution of ethyl acetate (30 mL) in 10% N,N-dimethylformamide in a round-bottom flask equipped with a magnetic stirrer and a three-way gas line adapter. The vessel was evacuated twice under reduced pressure and flushed with nitrogen. 10% Palladium / carbon (0.461 g, 0.1 equivalent) was added in one portion, the three-way adapter was attached to the flask, a hydrogen balloon was attached to the adapter, and the vessel was evacuated twice under reduced pressure and flushed with hydrogen. The reaction was stirred for 2 days, during which the hydrogen balloon was refilled from time to time. After approximately 48 hours, HPLC-MS analysis showed no residual starting material. The reaction was diluted with methanol (20 mL) and filtered through a Celite plug. The Celite was washed with methanol (2 x 50 mL). All the filtrates were combined and concentrated under reduced pressure, and the resulting oil was dissolved and concentrated from dichloromethane. After drying under reduced pressure, the title compound was isolated as a colorless powder (3.10 g, 99%). Observed MS m / z = 722.6 (M+1).

[0547] G. (S)-2-((S)-2-(Dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((4-(2,2,2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N,3-dimethylbutanamide (Compound 8)

[0548]

[0549] To a stirred solution of N,N-(L)-dimethylvaline (1.696 g, 9.35 mmol) in N,N-dimethylformamide (10 mL) was added HATU (3.216 g, 8.46 mmol) and diisopropylethylamine (3.10 mL, 17.8 mmol). A clear yellow solution was obtained after 5 minutes. Stirring was continued for 10 minutes, then compound 7a (3.213 g, 4.45 mmol) was added in one portion. After stirring for 1 hour, HPLC-MS indicated trace amounts of compound 7a remaining and the reaction was continued for 16 hours. The reaction was then concentrated under reduced pressure, diluted with ethyl acetate (120 mL) and 40 mL of 1:1 NaHCO3 (saturated):5% LiCl and transferred to a separatory funnel. The aqueous layer was removed and the organic phase was washed with LiCl (1x 20 mL), NaHCO3 (saturated, 2x 20 mL). The aqueous layers were combined and extracted with EtOAc (3x 50 mL). The organic layers were combined, washed with brine (1x 20 mL), dried over sodium sulfate, filtered and concentrated to give an oil filled with DMF, which was concentrated via a rotary evaporator to remove residual DMF, giving 7 g of a crude straw-colored oil. The oil was dissolved in a small amount of dichloromethane solution of 10% methanol (~11 mL) and loaded onto a SNAP Ultra 360 g silica gel column for purification (2-20% MeOH in CH2Cl2 solution, over 15 column volumes, the product eluted at about 10-13%). The fractions containing the desired product were combined and concentrated under reduced pressure to give the title compound as a colorless foam. The impure fractions were combined, evaporated and purified repeatedly on a TM SNAP Ultra 100 g silica gel column on an Isolera instrument and combined with the pure product from the first column to give a colorless foam solid (3.78 g). MS m / z observed = 850.6 (M+1).

[0550] H.(S)-N-((3R,4S,5R)-1-((S)-2-((1R,2R)-3-((4-aminophenyl)sulfonamido)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide (Compound 9)

[0551]

[0552] To a stirred solution of compound 8 (0.980 g, 1.154 mmol) in 1,4-dioxane (15 mL) was added water (3.5 mL) and 1 M lithium hydroxide monohydrate (3 eq., 3.46 mL). The resulting light suspension was stirred at 4 °C and the consumption of the starting material was monitored by HPLC-MS. When the conversion was complete (∼5 days), the reaction was neutralized with 3.46 mL of 1 M HCl and concentrated in vacuo to remove dioxane. The resulting aqueous phase was diluted with 60 mL of EtOAc and 5 mL of brine, and then extracted with ethyl acetate (2 x 30 mL). The combined organic fractions were dried over Na2SO4, filtered and evaporated to give the title compound as a brown solid (0.930 g). R f = 0.5 (CH2Cl2 solution with 8% MeOH). MS m / z observed = 753.7 (M+1).

[0553] I. 3-(2-(2-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoic acid 2,3,5,6-tetrafluorophenyl ester (Compound 15)

[0554]

[0555] In a dry 50 mL conical flask, 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid (Compound 14, 1.000 g, 4.52 mmol) and maleic anhydride (0.443 g, 4.52 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL). The reaction was stirred at room temperature under N2 for 6 h, at which point it was cooled to 0 °C and N,N,N-trimethylpyridin-1-ium iodide (1.263 mL, 2.1 eq) was added dropwise. In a separate dry 50 mL conical flask, tetrafluorophenol (3.002 g, 4 eq) was dissolved in anhydrous N,N-dimethylformamide (10 mL). The flask was cooled to 0 °C in an ice bath and trifluoroacetic anhydride (2.548 mL, 4 eq) was added dropwise. The flask was stirred for 15 min, at which point N,N,N-trimethylpyridin-1-ium iodide (2.407 mL, 4 eq) was added dropwise. The flask was stirred for a further 15 min, then the contents were added dropwise to the first flask via syringe. The reaction was warmed to room temperature and stirring was continued under N2. The reaction was monitored by HPLC-MS for consumption of the starting materials. After 6 days, the reaction was complete, with all of Compound 14 consumed, leaving only Compound 15 and a small amount (∼5%) of the bis-TFP maleamide intermediate. The reaction mixture was transferred to a separatory funnel, diluted with diethyl ether (75 mL) and washed with 5% LiCl (1 x 20 mL), 1 M HCl (2 x 20 mL), saturated NaHCO3 (5 x 20 mL) and brine (1 x 20 mL). The organic layer was dried over Na2SO4, filtered and evaporated to give a brown crude oil containing residual DMF. The crude oil was dissolved in 8 mL of 1:1 DMF:H2O + 0.1% TFA and loaded onto a 60 g SNAP Ultra C18 column (Biotage AB, Uppsala, Sweden) and purified under a linear 30-100% ACN / H2O + 0.1% TFA gradient over 8 column volumes. The pure fractions were combined and diluted with brine (20 mL), then extracted with 3 x 50 mL Et2O. The combined organics were dried over MgSO4, filtered and evaporated to recover a pale yellow oil (1.34 g, 66% yield).

[0556] J. ((S)-1-(((S)-1-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)aminosulfonyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate tert-butyl ester (Compound 12)

[0557]

[0558] Compound 11 was prepared as described in International Patent Application Publication No. WO 2016 / 041082.

[0559] To an empty 25 mL pear-shaped flask were added Compound 11 (1.342 g, 3.58 mmol, 3.0 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.664 g, 3.46 mmol, 2.9 equiv), and 7-hydroxy-azabenzotriazole (HOAT) (0.472 g, 3.46 mmol, 2.9 equiv). These solids were dissolved in a mixture of N,N-dimethylformamide (0.5 mL) and dichloromethane (4.5 mL) with stirring over 30 min at room temperature. Separately, Compound 9 (0.900 g, 1.20 mmol) was dissolved in a mixture of N,N-dimethylformamide (0.2 mL) and dichloromethane (1.8 mL) and added to the pear-shaped flask, rinsing with dichloromethane (1.0 mL). The stirring rate was increased to 1000 rpm to create a vortex. Within 2 min after addition of Compound 9, copper(II) chloride (0.514 g, 3.83 mmol, 3.2 equiv) was added in one portion directly into the center of the vortex through a narrow powder funnel. The initial pale yellow solution turned into a dark brown suspension and then into a dark green suspension over 10 min. Completion of the reaction was monitored by HPLC-MS, and no change in the reaction progress was observed between samples taken at 30 min and 1 h (~95% complete). The reaction was stirred overnight at room temperature, then 2-(2-aminoethylamino)ethanol (0.483 mL, 4.781 mmol, 4 equiv), EtOAc (10 mL), and dH2O (5 mL) were added to the stirred suspension, which underwent a color change to dark blue. As the suspended solids gradually dissolved into the biphasic mixture, the suspension was stirred vigorously for 4 h. The mixture was transferred to a separatory funnel and diluted with EtOAc (100 mL) and brine (10 mL), and the aqueous layer was extracted with 10% IpOH / EtOAc (4 x 50 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and evaporated to give a pale blue crude solid. This crude solid was dissolved in a mixture of methanol (0.5 mL) and dichloromethane (6 mL), and at Purification on a SNAP Ultra 100 g silica gel column (2 - 20% MeOH in CH₂Cl₂ solution, over 10 column volumes, followed by 8 column volumes at 20% MeOH until a steady state was reached). After 1 - 2 column volumes of the CH₂Cl₂ solution with ∼20% MeOH, the product eluted as a broad peak. The fractions containing the desired material were combined and concentrated under reduced pressure to give the title compound as a white solid (1.105 g, 83%). MS m / z observed = 555.9 ((M + 2) / 2), 1109.8 (M + 1).

[0560] K.(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)aminosulfonyl)phenyl)-5-ureidopentanamide (Compound 13)

[0561]

[0562] To a solution of Compound 12 (0.926 g, 0.834 mmol) was added a mixture of dichloromethane (10 mL) and trifluoroacetic acid (2.0 mL). The reaction was monitored by HPLC - MS for consumption of the starting material (∼45 minutes). The reaction mixture was co - evaporated with acetonitrile (2 x 10 mL) and dichloromethane (2 x 10 mL) under reduced pressure to remove the excess trifluoroacetic acid. The resulting residue was dissolved in the minimum amount of dichloromethane and methanol (3:1, v / v, ∼2 mL) and added dropwise via pipette to a stirred solution of diethyl ether (200 mL) and hexane (100 mL), resulting in a suspension of a light - white solid. The solid was filtered and dried in vacuo to give the title compound as the trifluoroacetate salt in the form of a white powder (1.04 g, quantitative yield, containing some residual solvent). MS m / z observed = 505.8 ((M + 2) / 2).

[0563] L.(S)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-2-((S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-14-isopropyl-12-oxo-3,6,9-trioxa-13-azapentadecanamido)-5-ureidovaleramide (Linker-Toxin 001)

[0564]

[0565] To a stirred solution of compound 13 (0.722 g, 0.584 mmol) in N,N-dimethylformamide (4 mL) was added compound 15 (0.314 g, 1.2 equiv) and diisopropylethylamine (0.305 mL, 3.0 equiv). HPLC-MS analysis at 2 h showed no residual starting materials. The reaction was acidified with TFA (300 μL) and then diluted with diH2O + 0.1% TFA (9 mL). The resulting solution was loaded onto a 120 g SNAP Ultra C18 column (Biotage, Uppsala, Sweden) and purified under an ACN / H2O + 0.1% TFA gradient: 20 - 60% ACN over 10 column volumes, 60 - 100% ACN over 5 column volumes. The product eluted near 40% ACN. The pure fractions identified by LCMS were combined and lyophilized. The white powder solid was recovered from the lyophilizer. Lyophilization was repeated into vials at a higher concentration (ca. 50 mg / mL, in 2:1 H2O / ACN) to produce a denser, less flocculent lyophilized solid (754.2 mg, 91%). MS m / z observed = 647.4 ((M+2) / 2), 1292.8 (M+1).

[0566] Example 4: Preparation of v10000 conjugated with linker-toxin 001

[0567] The solution of antibody v10000 (2.0 g) in 10 mM sodium acetate, 9% (w / v) sucrose at pH 4.5 (138.9 mL) was adjusted in pH by adding 200 mM Na2HPO4 at pH 8.9 (15.4 mL). After adding DTPA solution (44 mL in PBS, pH 7.4, final concentration 1.0 mM), the reduction of interchain disulfide bonds was initiated by adding 10 mM aqueous TCEP solution (1.68 mL, 1.05 eq). After 90 minutes at 37 °C, the reaction was cooled on ice and then an excess of linker-toxin 001 (4.81 mL; 6 eq) was added from a 20 mM DMSO stock solution. The conjugation reaction was quenched after 90 minutes by adding an excess of 20 mM N-acetylcysteine solution (4.81 mL; 6 equivalents).

[0568] The quenched antibody-drug conjugate (ADC) solution was used on a Millipore Labscale TM tangential flow filtration instrument XL ultrafiltration module ( 30 kDa 0.005 m 2 ; Millipore Sigma) and purified with 9 - 15 volumes of 10 mM sodium acetate, 9% (w / v) sucrose at pH 4.5. The eluted ADC was sterile filtered (0.22 um). The small-scale produced ADC was purified on a 40KDa MWCO Zeba TM column (ThermoFisher Scientific, Waltham, MA) which was preconditioned with PBS or 10 mM sodium acetate, 9% (w / v) sucrose at pH 4.5.

[0569] After purification, the concentration of the ADC was determined by BCA assay with reference to a standard curve generated from v10000. Alternatively, the concentration was estimated by measuring the absorbance at 280 nm (ε = 195065 M -1 cm -1 −1

[0570] Samples of the ADC were evaluated by non-reducing and reducing SDS-PAGE. No extra bands were observed.

[0571] The antibody and ADC were analyzed by hydrophobic interaction chromatography (HIC) to estimate the drug / antibody ratio (DAR). At Chromatography was performed on a HIC Ethyl column (7.8 x 50 mm, 5 μm) (Sepax Technologies Inc., Newark, DE) using a gradient from 80% MPA / 20% MPB to 35% MPA / 65% MPB over a period of 13.5 minutes at a flow rate of 1 mL / min (MPA = 1.5 M (NH4)2SO4, 25 mM Na x PO4, and MPB = 75% 25 mM Na x PO4, 25% isopropanol).

[0572] The average drug / antibody ratio (DAR) of the ADC can vary depending on the number of disulfide bonds released during antibody reduction. A single conjugation reaction to produce an ADC with a specific average DAR contains a mixture of multiple species. For v10000 conjugated to linker-toxin 001, a mixture of four species was produced: unconjugated antibody, ADC with a DAR of 2, ADC with a DAR of 4, and ADC with a DAR of 6.

[0573] The results of HIC showed that the ADC containing v10000 conjugated to linker-toxin 001 had an average DAR of 2.07. The individual contributions of the DAR0, DAR2, DAR4, and DAR6 species to the average DAR of the purified ADC were evaluated by integration of the HPLC-HIC chromatogram. Each peak in the HIC chromatogram was separated by preparative chromatography, and the identity of the peak was verified by LC-MS. The percentage content of the individual DAR species of each variant (determined by HIC) is shown in Table G.

[0574] Table G: DAR distribution of ADCs containing v10000 and linker-toxin 001

[0575] DAR Area % 0 23 2 56 4 17 6 4

Claims

1. Use of a bispecific anti-HER2 antigen-binding construct or an antibody-drug conjugate (ADC) in the preparation of a medicament for the treatment of biliary tract cancer (BTC).

2. The use according to claim 1, wherein the BTC is resectable, partially resectable or non-resectable.

3. The use according to claim 1, wherein the BTC is locally advanced.

4. The use according to any one of claims 1 to 3, wherein the BTC is metastatic BTC.

5. The use according to any one of claims 1 to 3, wherein the BTC is HER2 3+, HER2 2+ or HER2 1+ as measured by immunohistochemistry (IHC) and is HER2 gene amplified.

6. The use according to any one of claims 1 to 3, wherein the BTC is HER2 3+, HER2 2+ or HER2 1+ as measured by immunohistochemistry (IHC) and has no HER2 gene amplification.

7. The use according to claim 1, wherein the BTC is gallbladder cancer.

8. The use according to claim 1, wherein the BTC is cholangiocarcinoma (CCA).

9. The use according to claim 1, wherein the bispecific anti-HER2 antigen-binding construct comprises a heavy chain H1, a heavy chain H2 and a light chain L1, wherein: a) The heavy chain H1 comprises the CDR sequences shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41; b) The heavy chain H2 comprises the CDR sequences shown in SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72; and c) The light chain L1 comprises the CDR sequences shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO:

29.

10. The use according to claim 1, wherein the bispecific anti-HER2 antigen-binding construct comprises a heavy chain H1 comprising the amino acid sequence shown in SEQ ID NO: 36, a heavy chain H2 comprising the amino acid sequence shown in SEQ ID NO: 63 and a light chain L1 comprising the amino acid sequence shown in SEQ ID NO:

24.

11. The use according to claim 9 or 10, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 10 mg / kg per week.

12. The use according to claim 9 or 10, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 20 mg / kg every two weeks.

13. The use according to claim 9 or 10, wherein the effective amount of the bispecific anti-HER2 antigen-binding construct is 30 mg / kg every three weeks.

14. The use according to claim 1, wherein administering the bispecific anti-HER2 antigen-binding construct to the subject elicits a complete response (CR), a partial response (PR) or stable disease (SD) in the subject.

15. Use according to claim 1, wherein the disease control rate in a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 60%, 70% or 80%.

16. Use according to claim 1, wherein the overall response rate in a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 50%, 60%, 70% or 80%.

17. Use according to claim 1, wherein the bispecific anti-HER2 antigen-binding construct is administered after at least one, two or three first-line therapies.

18. Use according to claim 1, wherein the bispecific anti-HER2 antigen-binding construct is administered as a first-line monotherapy.

19. Use according to claim 1, wherein the bispecific anti-HER2 antigen-binding construct is administered as adjuvant therapy or neoadjuvant therapy.

20. Use according to claim 1, wherein the bispecific anti-HER2 antigen-binding construct is administered in combination with one or more chemotherapeutic agents.

21. Use according to claim 20, wherein the one or more chemotherapeutic agents are gemcitabine and / or cisplatin.

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