Methods for 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 (ADC) targeting HER2 linked to an auristatin analog.

Description

[0001] Sequence Listing

[0002] 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, is named ZWI063sequencelisting.txt, and is 99,000 bytes in size. Background Art

[0003] Biliary tract cancer (BTC), including gallbladder cancer and bile duct cancer, 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 BTC are diagnosed in the late stage, and only about 25% are operable. 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).

[0004] First-line treatment options for inoperable BTC include systemic chemotherapy (gemcitabine plus cisplatin, which improves overall survival compared with gemcitabine alone [11.7 months vs. 8.1 months, respectively]). Alternative first-line treatments include pembrolizumab for MSI-H / dMMR tumors, fluoropyrimidine-based chemoradiation, radiation therapy without additional chemotherapy, investigational agents, or best supportive care. Second-line chemotherapy for BTC lacks supporting evidence, and clinical trials are recommended (NCCN Clinical Practice Guidelines: Hepatobiliary Cancers. 2nd edition. 2019).

[0005] There remains a need for treatments for biliary tract cancer.

[0006] International Patent Publication No. WO2015 / 077891 describes bispecific anti-HER2 antibodies directed against two different HER2 epitopes in ECD4 and ECD2, which are the same epitopes bound by trastuzumab and pertuzumab. Summary of the invention

[0007] Methods for treating biliary tract cancer using bispecific antigen binding constructs targeting HER2 are described herein. In one aspect of the present disclosure, a method for treating a subject with biliary tract cancer (BTC) is provided, comprising administering to the subject an effective amount of a bispecific anti-HER2 antigen binding construct or an antibody drug conjugate (ADC).

[0008] In some embodiments, the BTC is resectable, partially resectable, or unresectable.

[0009] In some embodiments, the BTC is late-stage.

[0010] In some embodiments, BTC is HER2 3+, HER2 2+, or HER2 1+, and is gene-amplified as measured by immunohistochemistry (IHC).

[0011] In some embodiments, BTC is HER2 3+, HER2 2+, or HER2 1+, without HER2 gene amplification, as measured by immunohistochemistry (IHC).

[0012] In some embodiments, the BTC is gallbladder cancer.

[0013] In some embodiments, the BTC is cholangiocarcinoma (CCA).

[0014] In some embodiments, 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 heavy chain L1 comprises the CDR sequences shown in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0015] In some embodiments, 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.

[0016] In some embodiments, the effective amount of the bispecific anti-HER2 antigen binding construct is 10 mg / kg per week.

[0017] In some embodiments, the effective amount of the bispecific anti-HER2 antigen binding construct is 20 mg / kg every two weeks.

[0018] In some embodiments, the effective amount of the bispecific anti-HER2 antigen binding construct is 30 mg / kg every three weeks.

[0019] In some embodiments, administration of the bispecific anti-HER2 antigen-binding construct to the subject results in a complete response (CR), a partial response (PR), or stable disease (SD) in the subject.

[0020] In some embodiments, 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%.

[0021] In some embodiments, 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%.

[0022] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered after at least one, two, or three first-line therapies.

[0023] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered as first-line monotherapy.

[0024] In some embodiments, the bispecific anti-HER2 antigen-binding construct is administered as adjuvant therapy or neoadjuvant therapy.

[0025] In some embodiments, the bispecific anti-HER2 antigen binding construct is administered in combination with one or more chemotherapeutic agents.

[0026] In some embodiments, the one or more chemotherapeutic agents are gemcitabine and / or cisplatin.

[0027] In another aspect of the present disclosure, there is provided use of a bispecific anti-HER2 antigen binding construct or antibody drug conjugate (ADC) in the preparation of a medicament for treating biliary tract cancer (BTC).

[0028] In yet another aspect of the present disclosure, provided is a use of an effective amount of a bispecific anti-HER2 antigen binding construct or ADC for treating BTC in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic representation of an exemplary bispecific anti-HER2 antigen-binding construct in Fab / scFv format is depicted.

[0030] Figure 2 Treatment duration and maximum reduction in sum of diameters (SOD) in subjects with BTC treated with v10000 are depicted. DETAILED DESCRIPTION

[0031] A method for treating biliary tract cancer (BTC) of a subject is described herein, including administering a bispecific antigen binding construct targeting HER2 to a patient. In some embodiments, the bispecific antigen binding construct targeting HER2 is connected to an auristatin analog (referred to herein as an antibody-drug conjugate or ADC). In some embodiments, the bispecific antigen binding construct targeting HER2 can be used in a method for treating gallbladder cancer or cholangiocarcinoma. In other embodiments, the bispecific antigen binding construct targeting HER2 can cause a tumor or lesion in a subject to be reduced in size when applied to a subject with BTC. In other embodiments, the administration of the bispecific antigen binding construct targeting HER2 can cause a complete response (CR), a partial response (PR) or stable disease (SD) in a subject, as measured by RECIST 1.1 guidelines.

[0032] Also described herein is a method of treating BTC comprising administering to a subject a bispecific antigen-binding construct targeting HER2 in combination with one or more chemotherapeutic agents.

[0033] definition

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0035] As used herein, unless otherwise indicated, the term "about" refers to a variation of approximately + / - 10% from a given value. It should be understood that such variation is always included in any given value provided herein, whether or not specifically mentioned.

[0036] The words "a" and "an" when used herein in conjunction with the term "comprising" may mean "one", but in certain embodiments are also consistent with the meaning of "one or more", "at least one" or "one or more than one".

[0037] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unlisted elements and / or method steps. The term "consisting essentially of" when used in conjunction with a composition, use, or method herein, indicates that additional elements and / or method steps may be present, but that these additions do not substantially affect the manner in which the listed 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. A composition, use, or method described herein as comprising certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and consist of those elements and / or steps in other embodiments, whether or not these embodiments are explicitly mentioned.

[0038] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use, or composition disclosed herein.

[0039] Certain features, structures, and / or characteristics described in connection with the embodiments disclosed herein may 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 further embodiments.

[0040] It should also be understood that the positive recitation of a feature in one embodiment serves as a basis for excluding the feature in an alternative embodiment. For example, where a list of options is provided for a given embodiment or claim, it should be understood that one or more options may be deleted from the list and the shortened list may form an alternative embodiment, whether or not such an alternative embodiment is explicitly mentioned.

[0041] Bispecific antigen binding constructs that bind HER2

[0042] Bispecific antigen-binding constructs that bind HER2 (also referred to as bispecific anti-HER2 antigen-binding constructs) are described below.

[0043] The term "antigen binding construct" refers to an agent, such as a polypeptide or polypeptide complex that can bind to 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 its antigen binding fragment; scFv, etc. An antigen binding construct can be a monospecific, bispecific, or multispecific polypeptide construct. In some aspects, an antigen binding construct can include, for example, one or more antigen binding components (e.g., Fab or scFv) connected to one or more Fcs. Other examples of antigen binding constructs are described below and provided in the Examples.

[0044] The term "bispecific" is intended to include any agent, such as an antigen binding construct, having two antigen binding moieties (e.g., antigen binding polypeptide constructs), each having a unique binding specificity. For example, a first antigen binding moiety binds an epitope on a first antigen, and a second antigen binding moiety binds an epitope on a second antigen. As used herein, the term "biparatope" refers to a bispecific antibody in which the first antigen binding moiety and the second antigen binding moiety bind to different epitopes on the same antigen. A biparatopic bispecific antibody can bind to two epitopes on the same antigen molecule, or it can bind to epitopes on two different antigen molecules.

[0045] A monospecific antigen-binding construct refers to an antigen-binding construct with one binding specificity. In other words, both antigen-binding moieties bind to the same epitope on the same antigen. Examples of monospecific antigen-binding constructs include trastuzumab and pertuzumab that bind to HER2.

[0046] Antigen binding constructs can be antibodies or antigen binding portions thereof. As used herein, "antibodies" or "immunoglobulins" refer to polypeptides that are substantially encoded by one or more immunoglobulin genes or fragments thereof, which specifically bind to and recognize an analyte (e.g., an antigen). Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as countless 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 can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

[0047] An exemplary immunoglobulin (antibody) structural unit consists 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 chain and heavy chain domains, respectively. The IgG1 heavy chain comprises the VH, CH1, CH2, and CH3 domains from the N-terminus to the C-terminus, respectively. The light chain comprises the VL and CL domains from the N-terminus to the C-terminus. The IgG1 heavy chain comprises a hinge between the CH1 and CH2 domains. In certain embodiments, the immunoglobulin construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked 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 include at least one immunoglobulin domain from a heavy chain antibody such as a camel antibody. In certain embodiments, the immunoglobulin constructs provided herein include at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, a human antibody, a camel antibody, a mouse antibody, or any chimeric antibody.

[0048] "Complementarity determining regions" or "CDRs" are amino acid sequences that contribute to antigen binding specificity and affinity. "Framework" regions (FRs) can help maintain the proper conformation of the CDRs to promote binding between the antigen binding region and the antigen. Structurally, the framework regions can be located in antibodies between the CDRs. The variable region typically exhibits the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (also called CDRs). The CDRs from the heavy and light chain variable domains are typically aligned by the framework regions, which allows for binding to a specific epitope. From the N-terminus to the C-terminus, the light and heavy chain variable domains typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Unless otherwise noted, the assignment of amino acids to each domain is typically in accordance with the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)). Typically, 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, and the three light chain CDRs are referred to as CDRL1, CDRL2 and CDRL3. Therefore, "CDR" as used herein may 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). CDRs provide most of the contact residues for the binding of an antibody to an antigen or epitope. Typically, three heavy chain CDRs and three light chain CDRs are necessary for binding to an antigen. However, in some cases, even a single variable domain may confer binding specificity to an antigen. In addition, as known in the art, in some cases, antigen binding may also occur by a combination of at least one or more CDRs (e.g., CDRH3) selected from a VH and / or VL domain.

[0049] 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 IMGT, AbM (University of Bath) and Contact (MacCallum RM and Martin ACR and Thornton JM, (1996), Journal of Molecular Biology, 262 (5), 732-745). For example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Therefore, as will be apparent to one skilled in the art, the exact numbering and position of the CDRs may differ based on the numbering system employed. However, it should be understood that the disclosure of VH herein includes the disclosure of the associated (intrinsic) heavy chain CDRs (HCDRs) defined by any known numbering system. Similarly, disclosure of a VL herein includes disclosure of the associated (intrinsic) light chain CDRs (LCDRs) as defined by any known numbering system.

[0050] Table 1: Common CDR definitions 1

[0051]

[0052] 1 Either the Kabat or Chothia numbering system may be used for all defined HCDR2, HCDR3, and light chain CDRs except for Contact, which uses Chothia numbering.

[0053] 2 Kabat numbering is used. The position in the Kabat numbering scheme that demarcates the ends of the Chothia and IMGT CDR-H1 loops varies depending on the loop length, as Kabat places insertions at positions 35A and 35B outside of these CDR definitions. However, the IMGT and Chothia CDR-H1 loops can be clearly defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0054] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly connected 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, scFv has a light chain variable domain (VL) connected to the N-terminus of a heavy chain variable domain (VH) from its C-terminus by a polypeptide chain. Alternatively, scFv can be a polypeptide chain in which the C-terminus of VH is connected to the N-terminus of VL by a polypeptide chain.

[0055] Antigen-binding polypeptide constructs

[0056] The bispecific anti-HER2 antigen-binding construct comprises two antigen-binding polypeptide constructs, each of which binds to a specific domain or epitope of HER2. In one embodiment, each antigen-binding polypeptide construct binds to an 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 scFv.

[0057] 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 a scFv-Fab form (i.e., one antigen-binding polypeptide construct is scFv and the other antigen-binding polypeptide construct is Fab, also referred to as a Fab-scFv form). In another embodiment, the bispecific anti-HER2 antigen-binding construct has a scFv-scFv form (i.e., both antigen-binding polypeptide constructs are scFv).

[0058] "Fab fragments" (also called antigen-binding fragments) contain the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain and the variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain the complementarity determining loops (CDRs, also called hypervariable regions) that are involved in antigen binding. Fab' fragments differ from Fab fragments in that a few residues are added at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

[0059] "Single-chain Fv" or "scFv" includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide also includes a polypeptide linker 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, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

[0060] Form and function of antigen-binding constructs

[0061] Provided herein are bispecific anti-HER2 antigen-binding constructs with two antigen-binding polypeptide constructs, wherein the first one specifically binds to HER2 ECD2, and wherein the second one specifically binds 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 a 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).

[0062] In certain embodiments, the bispecific anti-HER2 antigen-binding construct exhibits 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 and internalizing the HER2 antigen), and (iii) the ability to mediate antibody-directed effector cell killing (ADCC). These in vitro activities were observed with both naked bispecific anti-HER2 antigen-binding constructs and bispecific anti-HER2 antigen-binding constructs conjugated to an auristatin analog, and at different levels of HER2 expression (1+, 2+, and 3+).

[0063] As described in International Patent Publication No. WO2015 / 077891, the format (scFv / scFv, scFv / Fab or Fab / Fab) of the bispecific anti-HER2 antigen-binding construct 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 improved by increasing the affinity of one or both 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 a scFv, the construct is internalized to a greater extent than a construct of equal affinity in a Fab / Fab format, and is internalized to a similar extent to a construct of equal affinity in a scFv / scFv format by tumor cells that express both high and low HER2. The embodiment that is easy to internalize is a good candidate for an antibody-drug conjugate, which needs to be internalized by tumor cells to achieve killing. On the contrary, in certain embodiments, compared with the construct with equal affinity in the form of Fab / Fab, the bispecific anti-HER2 antigen-binding construct that is not easy to internalize shows increased efficacy in ADCC killing of tumor cells expressing low levels of HER2. In one embodiment, the bispecific anti-HER2 antigen-binding construct with Fab / scFv form is more effective than the anti-HER2 construct with Fab / Fab form in ADCC killing of tumor cells expressing low levels of HER2 (HER2 0-1+ or 1+), and the anti-HER2 construct with Fab / Fab form is more effective than the bispecific anti-HER2 antigen-binding construct with scFv / scFv form. The enhanced ADCC potency of some embodiments may be due to 1) their enhanced ability to bind with affinity to cells with low HER2 receptor density and subsequently cluster HER2 receptors on the target cell surface and mediate downstream cell-mediated killing; and / or 2) their enhanced ability to remain on the cell surface (rather than causing internalization); thus they are more available for cell-mediated effector killing.

[0064] HER2

[0065] The bispecific anti-HER2 antigen-binding constructs described herein comprise antigen-binding polypeptide constructs that bind to ECD2 and ECD4 of HER2.

[0066] The expressions "ErbB2" and "HER2" are used interchangeably herein and refer to the human HER2 protein described, for example, in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al., Nature 319:230-234 (1986) (Genebank Accession No. 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.

[0067] 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 EGFR, HER2, HER3, and HER4 receptors. A HER receptor will typically comprise an extracellular domain that can bind to a HER ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain with several tyrosine residues that can be phosphorylated. A "HER ligand" refers to a polypeptide that binds to and / or activates a HER receptor.

[0068] The extracellular (ecto) 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, no 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. 2012 Apr;38(2):133-42 (2012); or Plowman et al., Proc. Natl. Acad. Sci. 90:1746-1750 (1993).

[0069] 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.

[0070]

[0071] "Epitope 2C4" is the region in the extracellular domain of HER2 that binds to antibody 2C4. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER2. 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). In order to screen antibodies that bind to the 2C4 epitope, conventional cross-blocking assays can be performed, such as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed using methods known in the art to assess whether an antibody binds to the 2C4 epitope of HER2 and / or antibody-HER2 structures can be studied (Franklin et al., Cancer Cell 5:317-328 (2004)) to see which domains of HER2 are bound by the antibody.

[0072] "Epitope 4D5" is a region in the extracellular domain of HER2 that binds to antibody 4D5 (ATCC CRL 10463) and trastuzumab. The epitope is close to the transmembrane domain of HER2 and is within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, a conventional cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. 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 in the region from about residue 529 to about residue 625, inclusive, see U.S. Patent Publication No. 2006 / 0018899). Figure 1 ).

[0073] "Specifically bind", "specific binding" or "selective binding" refers to binding that is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of the 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) technology (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 unrelated 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 or antigen-binding molecule comprising the antigen-binding portion that binds to the antigen has a molecular weight 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 smaller, e.g. 10 ～8 M to 10" 13 M, e.g. 10" 9 M to 10" 13 The dissociation constant (K D ).

[0074] "Heregulin" (HRG) when used herein refers to a polypeptide encoded by the heregulin gene product as disclosed in US Pat. No. 5,641,869 or in Marchionni et al., Nature, 362:312-318 (1993). Examples of heregulins include heregulin-α, heregulin-β1, heregulin-β2, and heregulin-β3 (Holmes et al., Science, 256:1205-1210 (1992); and U.S. Pat. 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-hexahydrobenzyltransferase (Hydroxyprogesterone) (Schaefer et al., Oncogene 51:14524-14536 (1996)); 15:1385-1394 (1997)). The term includes biologically active fragments and / or amino acid sequence variants of a native sequence HRG polypeptide, such as an EGF-like domain fragment thereof (eg, HRGβ1177-244).

[0075] "HER activation" or "HER2 activation" refers to the activation or phosphorylation of any one or more HER receptors or HER2 receptors. Typically, HER activation results in signal transduction (e.g., caused by the intracellular kinase domain of the HER receptor phosphorylating tyrosine residues in the HER receptor or substrate polypeptide). HER activation can be mediated by the binding of a HER ligand to a HER dimer comprising a 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).

[0076] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies), wherein residues from the recipient's hypervariable regions are replaced by residues from non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates with desired specificity, affinity, and ability. In some cases, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may be included in residues not found in recipient antibodies or donor antibodies. These modifications are made in order to further improve antibody performance. In general, humanized antibodies will contain substantially all at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FRs are those of human immunoglobulin sequences. Humanized antibodies will optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further 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).

[0077] Fc of bispecific anti-HER2 antigen binding constructs.

[0078] In some embodiments, the bispecific anti-HER2 antigen-binding constructs described herein comprise an Fc, such as a dimeric Fc.

[0079] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a 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 Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that is capable of stably self-associating a C-terminal constant region of an immunoglobulin heavy chain. For example, the Fc polypeptide of a dimeric IgG Fc comprises IgG CH2 and IgG CH3 constant domain sequences.

[0080] The Fc domain comprises a CH3 domain or a CH3 and CH2 domain. The CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the Fc dimer. The CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the Fc dimer.

[0081] In some aspects, Fc comprises at least one or two CH3 sequences. In some aspects, Fc is coupled to the first antigen-binding polypeptide construct and / or the second antigen-binding polypeptide construct with or without one or more joints. In some aspects, Fc is human Fc. In some aspects, Fc is human IgG or IgG1 Fc. In some aspects, Fc is a heterodimer Fc. In some aspects, Fc comprises at least one or two CH2 sequences.

[0082] In some aspects, Fc comprises one or more modifications in at least one CH3 sequence. In some aspects, Fc comprises one or more modifications in at least one CH2 sequence. In some aspects, Fc is a single polypeptide. In some aspects, Fc is a plurality of peptides, such as two polypeptides.

[0083] In some aspects, the Fc is an Fc described in patent application PCT / CA2011 / 001238 filed on November 4, 2011 or PCT / CA2012 / 050780 filed on November 2, 2012, the entire disclosure of each of which is hereby incorporated by reference in its entirety for all purposes.

[0084] Modified CH3 domain

[0085] In some aspects, the bispecific anti-HER2 antigen binding construct described herein comprises a heterodimer Fc comprising a modified CH3 domain that has been asymmetrically modified. The heterodimer Fc may comprise two heavy chain constant domain polypeptides: a first Fc polypeptide and a second Fc polypeptide, which may be used interchangeably, provided that the Fc comprises a first Fc polypeptide and a second Fc polypeptide. Typically, the first Fc polypeptide comprises a first CH3 sequence and the second Fc polypeptide comprises a second CH3 sequence.

[0086] When two CH3 sequences are dimerized, the two CH3 sequences containing one or more amino acid modifications introduced in an asymmetric manner will generally produce a heterodimer Fc, rather than a homodimer. As used herein, "asymmetric amino acid modification" refers to any modification in which the amino acid at a specific position on the first CH3 sequence is different from the amino acid at the same position of the second CH3 sequence, and the first and second CH3 sequences preferentially pair to form a heterodimer, rather than a homodimer. This heterodimerization can be the result of modification of only one of the two amino acids at the same corresponding amino acid position on each sequence; or modification of two amino acids on each sequence at the same corresponding position on each of the first and second CH3 sequences. The first and second CH3 sequences of the heterodimer Fc may contain one or more asymmetric amino acid modifications.

[0087] Table 2 provides the amino acid sequence of the human IgGl Fc sequence, corresponding to amino acids 231 to 447 of the full-length human IgGl heavy chain. The CH3 sequence comprises amino acids 341-447 of the full-length human IgGl heavy chain.

[0088] Typically, Fc may include two continuous heavy chain sequences (A and B) capable of dimerization. In some aspects, one or two 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 mutation of variant 1A-B. In some aspects, Fc includes the mutation of variant 2A-B. In some aspects, Fc includes the mutation of variant 3A-B. In some aspects, Fc includes the mutation of variant 4A-B. In some aspects, Fc includes the mutation of variant 5A-B.

[0089] Table 2: IgG1 Fc sequences

[0090]

[0091] The first and second CH3 sequences may comprise amino acid mutations as described herein, with respect to amino acids 231 to 447 of a full-length human IgG1 heavy chain. In one embodiment, the heterodimer 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 amino acid modifications at position T394. In one embodiment, the heterodimer 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.

[0092] In one embodiment, the heterodimer 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 heterodimer 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 comprises an amino acid modification T350V.

[0093] In one embodiment, the heteromultimer 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 heteromultimer 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 amino acid modification T350V.

[0094] In one embodiment, the heteromultimer 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 an amino acid modification of T350V.

[0095] In one embodiment, the heterodimer Fc comprises a modified CH3 domain comprising the following amino acid modifications, wherein "A" represents an amino acid modification to a first CH3 sequence, and "B" represents an amino acid modification to a second CH3 sequence: A: L351Y_F405A_Y407V, B: T366L_K392M_T394W, A: L351Y_F405A_Y407V, B: T366L_K392L_T394W, A: T3 50V_L351Y_F405A_Y407V, B:T350V_T366L_K392L_T394W, A:T350V_L351Y_F405A_Y407V, B:T350V_T 366L_K392M_T394W, A:T350V_L351Y_S400E_F405A_Y407V and / or B:T350V_T366L_N390R_K392M_T394W.

[0096] One or more asymmetric amino acid modifications can promote the formation of heterodimeric Fc, wherein the heterodimeric CH3 domain has a stability comparable to that of a wild-type homodimeric CH3 domain. In one embodiment, one or more asymmetric amino acid modifications promote the formation of heterodimeric Fc domains, wherein the heterodimeric Fc domain has a stability comparable to that of a wild-type homodimeric Fc domain. In one embodiment, one or more asymmetric amino acid modifications promote the formation of heterodimeric Fc domains, wherein the heterodimeric Fc domain has a stability observed via a melting temperature (Tm) in a differential scanning calorimetry study, 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, Fc comprises one or more modifications in at least one CH3 sequence that promote the formation of heterodimeric Fc, and its stability is comparable to that of a wild-type homodimeric Fc.

[0097] Exemplary Bispecific Anti-HER2 Antigen Binding Constructs

[0098] In certain embodiments, the bispecific anti-HER2 antigen-binding construct is one of the biparatopic 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 Table 3, Table 4, Table 5, and Sequence Listing). In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence and a VL sequence of an 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 sequence and VL sequence 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 sequence and VL sequence of the ECD4 binding arm from one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717.

[0099] In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises CDR sequences from the ECD2 binding arm of 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 CDR sequences from the ECD2 binding arm of one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717, and the other antigen-binding polypeptide construct comprises CDR sequences from the ECD4 binding arm of one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717.

[0100] It will be appreciated by those skilled in the art that a limited number of amino acid substitutions may be introduced into the CDR sequences or VH or VL sequences of known antibodies without the antibody losing its ability to bind to its target. Candidate amino acid substitutions may be identified by computer modeling or by techniques known in the art such as alanine scanning, wherein the binding activity of the resulting variants is tested by standard techniques. Therefore, 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 a set of CDRs from one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717 ECD2 binding arms having 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD2. In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a variant of these CDR sequences comprising 1 to 10 amino acid substitutions across six CDRs (i.e., the CDRs can be modified by including up to 10 amino acid substitutions, wherein any combination of 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 to ECD2. 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 more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to a set of CDRs from the ECD2 binding arm of v10000, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD2.

[0101] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence that is 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% identical 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 to ECD2. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence that is 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% identical 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 to ECD2.

[0102] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence that is 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% identical to a VH sequence of the ECD2 binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD2. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence that is 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% identical to a VL sequence of the ECD2 binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD2.

[0103] 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 more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to a set of CDRs from an ECD4 binding arm of one of v5019, v5020, v7091, v10000, v6902, v6903, or v6717, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD4. In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a variant of these CDR sequences comprising 1 to 10 amino acid substitutions across six CDRs (i.e., the CDRs can be modified by including up to 10 amino acid substitutions, wherein any combination of 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 CDRs, wherein the variant retains the ability to bind to 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 greater, 95% or greater, 98% or greater, 99% or greater, or 100% sequence identity to a set of CDRs from the ECD4-binding arm of v10000, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD4.

[0104] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence that is 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% identical 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 to ECD4. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence that is 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% identical 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 to ECD4.

[0105] In certain embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VH sequence that is 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% identical to a VH sequence of the ECD4-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD4. In some embodiments, one of the antigen-binding polypeptide constructs of the bispecific anti-HER2 antigen-binding construct comprises a VL sequence that is 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% identical to a VL sequence of the ECD4-binding arm from v10000, wherein the antigen-binding polypeptide construct retains the ability to bind to ECD4.

[0106] Table 3: Exemplary bispecific anti-HER2 antigen binding constructs

[0107]

[0108]

[0109] *Fab or variable domain numbering according to Kabat (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)

[0110] § CH3 numbering according to the EU index in Kabat (Edelman et al., 1969, PNAS USA, 63:78-85)

[0111] Table 4: CDR sequences of the ECD2 binding arms of variants v5019, v5020, v7091, v10000, v6902, v6903 and v6717

[0112]

[0113] Table 5: CDR sequences of the ECD4 binding arms of variants v5019, v5020, v7091, v10000, v6902, v6903 and v6717

[0114] 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

[0115] Preparation of bispecific anti-HER2 antigen binding constructs

[0116] The bispecific anti-HER2 antigen-binding constructs described herein can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567 or International Patent Publication No. WO2015 / 077891.

[0117] In one embodiment, there is provided a nucleic acid encoding a separation of a bispecific anti-HER2 antigen-binding construct described herein. Such nucleic acids may encode an amino acid sequence of a VL comprising a bispecific anti-HER2 antigen-binding construct (e.g., a light chain and / or a heavy chain of an antigen-binding construct) and / or an amino acid sequence comprising its VH. In another embodiment, there is provided one or more vectors (e.g., expression vectors) comprising such nucleic acids. As known in the art, because many amino acids are encoded by more than one codon, multiple nucleic acids may encode a single polypeptide sequence. Exemplary nucleic acids for each polypeptide of a bispecific anti-HER2 antigen-binding construct are provided herein; however, it should be understood that other nucleic acids may be used to prepare a bispecific anti-HER2 antigen-binding construct described herein.

[0118] 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 such an embodiment, the host cell comprises the following (e.g., transformed by the following): (1) a vector comprising a nucleic acid encoding an amino acid sequence of the VL of a bispecific anti-HER2 antigen-binding construct and an amino acid sequence of the VH of an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence of the VL of an antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence of the VH of 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., a Y0, NS0, Sp20 cell). In one embodiment, a method for 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 expression of the bispecific anti-HER2 antigen-binding construct, and optionally recovering the bispecific anti-HER2 antigen-binding construct from the host cell (or host cell culture medium).

[0119] For the recombinant production of bispecific anti-HER2 antigen-binding constructs, nucleic acids encoding bispecific anti-HER2 antigen-binding constructs (e.g., as described above) are separated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of bispecific anti-HER2 antigen-binding constructs).

[0120] The term "substantially purified" refers to a construct or variant thereof described herein that is substantially or substantially free of components that normally accompany or interact with proteins found in their naturally occurring environment, i.e., native cells, or host cells in the case of recombinantly produced bispecific anti-HER2 antigen-binding constructs, which in certain embodiments are substantially free of cellular material, including protein preparations with 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 proteins. 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 cell dry weight. When the bispecific anti-HER2 antigen-binding construct is recombinantly produced by host cells, 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 cells. In certain embodiments, the "substantially purified" bispecific anti-HER2 antigen-binding constructs produced by the methods described herein have 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 a purity level of at least about 75%, 80%, 85%, more particularly a purity level of at least about 90%, at least about 95%, at least about 99% or more, as determined by appropriate methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0121] Suitable host cells for cloning or expressing bispecific anti-HER2 antigen binding construct encoding vectors include prokaryotic or eukaryotic cells described herein.

[0122] "Recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as 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-integrating vector, such as a plasmid, or may be integrated into the host genome.

[0123] As used herein, the term "eukaryote" refers to organisms 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.

[0124] As used herein, the term "prokaryote" refers to a prokaryotic organism. For example, the non-eukaryotic organism may belong to the phylogenetic domain of Eubacteria (including but not limited to Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.), or Archaea (including but not limited to Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Halobacteria such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobusfulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum agile, etc.). pernix) phylogenetic domain.

[0125] For example, bispecific anti-HER2 antigen binding constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For 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 (BKCLo ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the bispecific anti-HER2 antigen binding construct can be separated from the bacterial cell paste in a soluble portion and can be further purified.

[0126] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for bispecific anti-HER2 antigen binding construct encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce bispecific anti-HER2 antigen binding constructs with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0127] Suitable host cells 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 Spodopterafrugiperda cells.

[0128] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antigen-binding constructs in transgenic plants). TM technology).

[0129] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension 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, in Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals NY 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 antigen-binding construct production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0130] In one embodiment, the bispecific anti-HER2 antigen-binding construct described herein is produced in a stable mammalian cell by the following method, the method comprising: transfecting at least one stable mammalian cell with a nucleic acid encoding a 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 nucleic acids is determined in a transient transfection experiment to determine the relative ratio of input nucleic acids that results in the highest percentage of bispecific anti-HER2 antigen-binding constructs in the expression product.

[0131] In some embodiments, the bispecific anti-HER2 antigen-binding construct is produced in stable mammalian cells, wherein the expression product of at least one stable mammalian cell comprises a greater percentage of the desired glycosylated bispecific anti-HER2 antigen-binding construct compared to the monomeric heavy chain or light chain polypeptide or other antibody. In some embodiments, the identification of the glycosylated bispecific anti-HER2 antigen-binding construct is performed by one or both of liquid chromatography and mass spectrometry.

[0132] 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, performed at atmospheric pressure or high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoresis, immunoassay, precipitation, 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 constructs 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 a specific fusion partner. For example, if GST fusion is used, glutathione resin can be used to purify the antibody, and if a His tag is used, Ni can be used. +2Affinity chromatography, or if a flag tag is used, an immobilized anti-flag antibody can 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 fully incorporated by reference. The necessary degree of purification will vary depending on the use of the bispecific anti-HER2 antigen binding construct. In some cases, purification is not required.

[0133] 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.

[0134] In specific embodiments, the bispecific anti-HER2 antigen-binding constructs described herein are 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 equivalents and analogs thereof.

[0135] 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, WH Freeman & Co., NY and Hunkapiller et al., Nature, 310: 105-111 (1984)). For example, a polypeptide corresponding to a polypeptide fragment can be synthesized using a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions. 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-aminocaproic 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, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and general amino acid analogs. In addition, amino acids can be D (dextrorotatory) or L (levorotatory).

[0136] Post-translational modifications:

[0137] In certain embodiments, the bispecific anti-HER2 antigen-binding constructs described herein are differentially modified during or after translation.

[0138] As used herein, the term "modified" refers to any change made to a given polypeptide, such as changes to the polypeptide length, the amino acid sequence of the polypeptide, the chemical structure, the co-translational modification or the post-translational modification. The form "(modified)" term means that the polypeptide in question is optionally modified, i.e., the polypeptide of the bispecific anti-HER2 antigen binding construct may be modified or unmodified.

[0139] The term "post-translationally modified" refers to any modification that occurs to a natural or non-natural amino acid after it is incorporated into a polypeptide chain. 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.

[0140] In some embodiments, the modification is at least one of the following: glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, and connection to antibody molecules or bispecific anti-HER2 antigen binding constructs or other cellular ligands. In some embodiments, the bispecific anti-HER2 antigen binding constructs are 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.

[0141] Other post-translational modifications of the bispecific anti-HER2 antigen-binding constructs include, for example, N-linked or O-linked carbohydrate chains, processing of the N-terminus or C-terminus), attachment of chemical moieties to the amino acid backbone, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The bispecific anti-HER2 antigen-binding constructs described herein are modified with detectable labels such as enzyme labels, fluorescent labels, isotopic labels, or affinity labels to allow detection and separation of proteins. In certain embodiments, examples of suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin-biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, 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, platinum, xenon, fluorine.

[0142] In specific embodiments, the bispecific anti-HER2 antigen binding constructs described herein are attached to a macrocyclic chelator that is associated with a radioactive metal ion.

[0143] 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 in the same or varying degrees at several sites of a given polypeptide. In certain embodiments, the polypeptides from the bispecific anti-HER2 antigen binding constructs described herein are branched, for example, as a result of ubiquitination, and in some embodiments are cyclic, with or without branches. Circular, branched, and branched cyclic polypeptides are the result of post-translational natural 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 pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, e.g., PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd ed., Tecentron, WH Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, ed. BC Johnson, Academic Press, New York, pp. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992)).

[0144] Antibody Drug Conjugate (ADC)

[0145] 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 contained in the ADC is v10000.

[0146] In certain embodiments, the auristatin analog included 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 included in the ADC for use in the methods described herein is a compound of formula (I):

[0147]

[0148] Where R 1 Selected from:

[0149]

[0150] In certain embodiments, in the compounds of Formula (I), R 1 yes:

[0151]

[0152] In certain embodiments, in the compounds of Formula (I), R 1 yes:

[0153]

[0154] In certain embodiments, in the compounds of Formula (I), R 1 yes:

[0155]

[0156] In certain embodiments, the compound of formula (I) is selected from:

[0157]

[0158]

[0159] Compounds of general 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.

[0160] In certain embodiments, the ADC for use in the methods described herein comprises a bispecific anti-HER2 antigen-binding construct conjugated to an auristatin analog (toxin) via a linker (L), wherein the linker-toxin has the general formula (II):

[0161]

[0162] in:

[0163] R 1 Selected from:

[0164]

[0165] L is a cleavable linker, and

[0166] Represents the point of attachment of the linker-toxin to the bispecific anti-HER2 antigen-binding construct.

[0167] In some embodiments, in the linker-toxin of formula (II), R 1 yes:

[0168]

[0169] In some embodiments, in the linker-toxin of formula (II), R 1 yes:

[0170]

[0171] In some embodiments, in the linker-toxin of formula (II), R 1 yes:

[0172]

[0173] In some embodiments, in the linker-toxin of Formula (II), L is a peptide-containing linker.

[0174] In some embodiments, in the linker-toxin of Formula (II), L is a protease-cleavable linker.

[0175] In certain embodiments, the ADC for use in the methods described herein comprises a bispecific anti-HER2 antigen-binding construct conjugated to an auristatin analog (toxin) via a linker (L) and has the general formula (III):

[0176]

[0177] in:

[0178] R 1 and L are as defined for formula (II);

[0179] n is the average drug / antibody ratio (DAR) and is less than 3.9, and

[0180] Ab is a bispecific anti-HER2 antigen binding construct.

[0181] In some embodiments, in the ADC of formula (III), R 1 yes:

[0182]

[0183] In some embodiments, in the ADC of formula (III), R 1 yes:

[0184]

[0185] In some embodiments, in the ADC of formula (III), R 1 yes:

[0186]

[0187] In some embodiments, in the ADC of formula (III), L is a peptide-containing linker.

[0188] In some embodiments, in the ADC of general formula (III), L is a protease cleavable linker.

[0189] In some embodiments, in the ADC of formula (III), n is between 0.5 and 3.8.

[0190] In some embodiments, in the ADC of formula (III), n is between about 1.0 and 3.8, between about 1.0 and 3.5, between about 1.0 and 3.0, or between about 1.0 and 2.5.

[0191] In some embodiments, in the ADC of formula (III), n is between about 1.5 and 3.8, between about 1.5 and 3.5, between about 1.5 and 3.0, or between about 1.5 and 2.5.

[0192] In some embodiments, in the ADC of formula (III), n is between about 1.8 and 2.8, or between about 1.8 and 2.5.

[0193] In some embodiments, in the ADC of formula (III), Ab is v10000.

[0194] Combinations of any of the foregoing embodiments for the ADC of general formula (III) are also contemplated, and each combination forms a separate embodiment for purposes of this disclosure.

[0195] In the ADC described herein, the bispecific anti-HER2 antigen binding construct is connected to the auristatin analog (toxin) via a linker. A linker is a bifunctional or multifunctional moiety capable of connecting one or more toxin molecules to an antibody. A bifunctional (or monovalent) linker connects a single drug to a single site on an antibody, while a multifunctional (or multivalent) linker connects more than one toxin molecule to a single site on an antibody. A linker capable of connecting one toxin molecule to more than one site on an antibody can also be considered multifunctional.

[0196] Attachment of linkers 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 linkers to antibodies can be achieved by modifying the antibody to include additional cysteine ​​residues (see, e.g., U.S. Pat. Nos. 7,521,541; 8,455,622; and 9,000,130) or non-natural amino acids that provide a reactive handle, 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)).

[0197] 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 (GT Hermanson, 2013, Academic Press).

[0198] Non-limiting examples of functional groups for reacting with free cysteine ​​or thiol include maleimide, haloacetamide, haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this regard are "self-stabilizing" maleimides, such as Lyon et al., Nat. Biotechnol., 32: 1059-1062 (2014).

[0199] Non-limiting examples of functional groups for reacting with surface lysines on antibodies or free amines on toxins include activated esters such as N-hydroxysuccinamide (NHS) esters, sulfo-NHS esters, imino esters such as Traut's reagent, isothiocyanates, aldehydes, and anhydrides such as diethylenetriaminepentaacetic anhydride (DTPA). Other examples include succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).

[0200] Non-limiting examples of functional groups capable of reacting with an electrophilic group (eg, an aldehyde or ketone carbonyl) on an antibody or toxin include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, carboxylic acid hydrazide, and aryl hydrazide.

[0201] Other linkers include linkers with functional groups that allow bridging of two interchain cysteines on the 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) pyridazinedione-based linkers (Lee et al., Chem. Sci., 7: 799-802 (2016)), dibromopyridazinedione-based linkers (Maruani et al., Nat. Commun., 6: 6645 (2015)), and other linkers known in the art.

[0202] The joint can include various joint components. Usually, the joint will include two or more joint components. Exemplary joint components include functional groups for reacting with antibodies, functional groups for reacting with toxins, extensions, peptide components, self-degradable groups (self-immolative group), self-eliminating groups, hydrophilic parts, etc. Various joint components are known in the art, some of which are described below.

[0203] Certain useful linker components are available from various 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, MA, J. Org. Chem., 60: 5352-5355 (1995); Frisch et al., Bioconjugate Chem., 7: 180-186 (1996); U.S. Pat. Nos. 6,214,345 and 7,553,816, and International Patent Application Publication No. WO 02 / 088172).

[0204] The linker used in the ADC described herein is a cleavable linker. A cleavable linker is generally susceptible to cleavage under intracellular conditions, such as by lysosomal processes. Examples include protease-sensitive, acid-sensitive, reduction-sensitive or photolabile linkers.

[0205] Suitable cleavable linkers include, for example, linkers comprising a peptide component that includes two or more amino acids and can be cleaved by intracellular proteases such as lysosomal proteases or endosomal proteases. The peptide component can include 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 specific enzymes such as tumor-associated proteases, cathepsin B, C or D or plasmin proteases.

[0206] In certain embodiments, the linker included in the ADC may be a dipeptide-containing linker, such as a valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys) containing linker. Other examples of suitable dipeptides for inclusion 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 may 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.

[0207] 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 (sulfo-SPBD). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond to improve the extracellular stability of the linker, for example, including a geminal dimethyl group. Other suitable linkers include linkers that can be hydrolyzed at a specific pH or within a pH range, such as hydrazone linkers. Linkers comprising a combination of these functional groups may also be useful, for example, linkers comprising both hydrazones and disulfides are known in the art.

[0208] Another example of a cleavable linker is a linker comprising β-glucuronide, which can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., Curr. Pharm. Des., 8: 1391-1403 (2002)).

[0209] The cleavable linker may also optionally comprise one or more additional components, such as self-immolative and self-degradable groups, stretches, or hydrophilic moieties.

[0210] Self-decomposing and self-eliminating groups that can be used for joints include, for example, p-aminobenzyloxycarbonyl (PABC) and p-aminobenzyl ether (PABE) groups, and methylated ethylenediamine (MED). Other examples of self-decomposing 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 described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization when the amide bond is hydrolyzed, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 2: 223-227 (1995)) and 2-aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., 55: 5867-5877 (1990)).

[0211] Extensions that can be used for linkers of ADCs include, for example, alkylene and extensions based on fatty acids, diacids, amines or diamines, such as diglycolates, malonates, caproates and caproamides. Other extensions include, for example, glycine-based extensions, polyethylene glycol (PEG) extensions and monomethoxy polyethylene glycol (mPEG) extensions. PEG and mPEG extensions also act as hydrophilic moieties.

[0212] In certain embodiments, the linker comprised by the ADC for use in the methods described herein is a peptide-based linker having the general formula (IV):

[0213]

[0214] in:

[0215] Z is a functional group capable of reacting with a target group on the bispecific anti-HER2 antigen binding construct;

[0216] Str is the extension segment;

[0217] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites;

[0218] X is a self-decomposing group;

[0219] D is the point of attachment to the auristatin analog;

[0220] s is 0 or 1;

[0221] m is an integer between 1 and 4, and

[0222] o is 0, 1, or 2.

[0223] In some embodiments, in Formula (IV), Z is:

[0224]

[0225] In some embodiments, in Formula (IV), Str is selected from:

[0226]

[0227]

[0228] in:

[0229] R is H or C1-C6 alkyl;

[0230] p is an integer between 2 and 10, and

[0231] q is an integer between 1 and 10.

[0232] In some embodiments, in Formula (IV), Str is:

[0233]

[0234] wherein p and q are as defined above.

[0235] In some embodiments, in Formula (IV), Str is:

[0236]

[0237] where p is an integer between 2 and 6, and

[0238] q is an integer between 2 and 8.

[0239] In some embodiments, in Formula (IV), AA1-[AA2] mSelected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Al a. 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)L ys, Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu.

[0240] In some embodiments, in Formula (IV), m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0241] In some embodiments, in Formula (IV), AA1-[AA2] m It is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[0242] In some embodiments, in Formula (IV), m is 1, 2 or 3.

[0243] In some embodiments, in Formula (IV), s is 1.

[0244] In some embodiments, in Formula (IV), o is 0.

[0245] In some embodiments, in Formula (IV):

[0246] Z is

[0247] Str is wherein p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0248] m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit;

[0249] s is 1, and

[0250] o is 0.

[0251] In certain embodiments, the linker included in the ADC used in the methods described herein has the general formula (V):

[0252]

[0253] in:

[0254] AS - is the attachment point to the bispecific anti-HER2 antigen binding construct;

[0255] Y is one or more additional linker components, or is absent, and

[0256] D is the point of attachment to the auristatin analog.

[0257] In certain embodiments, the linker included in the ADC used in the methods described herein has the general formula (VI):

[0258]

[0259] in:

[0260] AS - is the attachment point to the bispecific anti-HER2 antigen binding construct;

[0261] Y is one or more additional linker components, or is absent, and

[0262] D is the point of attachment to the auristatin analog.

[0263] In certain embodiments, the ADC for use in the methods described herein comprises an auristatin analog of formula (I) conjugated to v10000 at a low average DAR via a linker having formula (IV), (V), or (VI).

[0264] In certain embodiments, an ADC for use in the methods described herein comprises v10000 conjugated to a linker-toxin of formula (II) at a low average DAR, wherein the linker (L) has formula (IV), (V), or (VI).

[0265] In certain embodiments, an ADC for use in the methods described herein comprises v10000 and has the general formula (III) as shown above, wherein linker (L) has the general formula (IV), (V), or (VI).

[0266] In certain embodiments, the ADC for use in the methods described herein comprises an auristatin analog conjugated to v10000 with a low average DAR via a linker having the general formula (IV), (V), or (VI), wherein the auristatin analog is Compound 16, Compound 17, or Compound 18.

[0267] In certain embodiments, an ADC for use in the methods described herein comprises a linker-toxin having the following structure:

[0268]

[0269] Where AS- is the attachment point to the bispecific anti-HER2 antigen binding construct.

[0270] Preparation of Antibody Drug Conjugates

[0271] ADCs for use in the methods described herein can be prepared by one of several routes known in the art, using organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, e.g., Bioconjugate Techniques (GT Hermanson, 2013, Academic Press, and the Examples provided herein). For example, conjugation can be achieved by: (1) reacting a nucleophilic or electrophilic group of an antibody with a bifunctional linker via a covalent bond to form an antibody-linker intermediate Ab-L, followed by reaction with an activated auristatin analog (D), or (2) reacting a nucleophilic or electrophilic group of an auristatin analog with a linker via a covalent bond to form a linker-toxin DL, followed by reaction with a nucleophilic or electrophilic group of an antibody.

[0272] As described above, auristatin analogs can be conjugated to various groups on antibodies via appropriate linkers to provide ADCs. For example, conjugation can be through surface lysines, through oxidized carbohydrates, or through cysteine ​​residues released by reduction of one or more interchain disulfide bonds. Alternatively, antibodies can be modified to include additional cysteine ​​residues or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine. Such modifications are well known in the art (see, e.g., U.S. Pat. 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)).

[0273] In certain embodiments, the ADC for use in the methods described herein comprises an auristatin analog conjugated via a suitable linker to a cysteine ​​residue on a bispecific anti-HER2 antigen-binding construct that has been released by reduction of one or more interchain disulfide bonds.

[0274] 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 an average DAR of less than 3.9 but greater than 0.5, such as in certain embodiments between about 1.5 and about 2.5.

[0275] Various methods are known in the art to prepare ADCs with low average DAR (see, e.g., reviews: McCombs and Owen, The AAPS Journal, 17(2):339-351 (2015) and references therein; Boutureira and Bernardes, Chem. Rev., 115:2174-2195 (2015)).

[0276] For example, in order to be conjugated with cysteine ​​residues, partial reduction of antibody interchain disulfide bonds can be performed, followed by conjugation with joint-toxin. Partial reduction can be achieved by limiting the amount of the reducing agent used in the reduction reaction (see, for example, Lyon et al., Methods in Enzymology, 502: 123-138 (2012) and embodiments therein, and embodiments 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 in addition, joint-toxins with less equivalents can be used to obtain low average DAR.

[0277] Alternatively, an engineered antibody can be used in which one or more cysteine ​​residues constituting interchain 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 to the linker-toxin.

[0278] Another method is to use a dithiol linker, which bridges two cysteines that usually constitute an interchain disulfide bond. If all four interchain disulfide bonds are reduced and replaced with a dithiol linker, using a dithiol linker carrying only one toxin molecule will produce an ADC with a maximum DAR4 for full-size antibodies. Partial reduction of interchain disulfide bonds and / or a linker with less equivalents can be used in combination with a dithiol linker to further reduce DAR. Various dithiol linkers are known in the art (see, for example, 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)).

[0279] Cysteine ​​engineering methods can also be used to generate ADCs with low average DAR. Such methods involve engineering solvent-accessible cysteines into antibodies to provide site-specific handles for conjugation. Many suitable sites for introducing cysteine ​​residues have been identified as having IgG structures, and are included in Junutula et al., J. Immunol Methods, 332 (1-2): 41-52 (2008); Junutula et al., Nat. Biotechnol., 26 (8), 925-932 (2008); and those described in U.S. Pat. Nos. 9,315,581; 9,000,130; 8,455,622; 8,507,654 and 7,521,541.

[0280] Low average DAR ADCs can also be prepared by lysine conjugation using a limited amount of activated linker-toxin. Selective reactions 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 the linker-toxin (see, e.g., Thompson et al., Bioconjug. Chem., 26(10): 2085-2096 (2015)). Similarly, the N-terminal cysteine ​​residue can selectively react with an aldehyde to obtain a thiazolidinone (see, e.g., Bernardes et al., Nature Protocols, 8: 2079-2089).

[0281] Other methods include engineering antibodies to include one or more unnatural amino acids, such as p-acetylphenylalanine (pAcPhe) or selenocysteine ​​(Sec). The keto group in pAcPhe can react 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 react with a linker-toxin containing maleimide or iodoacetamide to form a selenoether conjugate (see, e.g., Hofer et al., Biochemistry, 48: 12047-12057 (2009)).

[0282] Antibodies can also be engineered to include peptide tags recognized by certain enzymes to allow enzymatic conjugation. For example, sortase-A (SortA) recognizes the sequence LPXTG. The pentapeptide can be engineered to 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)). Transglutaminases have 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 appropriate consensus sequences into the antibodies and co-expressing the engineered antibodies with formylglycine generating enzymes (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)).

[0283] Another approach for generating DAR2 ADCs is to conjugate a linker-toxin to a native sugar on a glycosylated antibody. Conjugation to a glycosylated antibody can be achieved, for example, by periodate oxidation of a terminal sugar residue to produce an aldehyde, which can then be conjugated to an appropriate linker-toxin, or by a glycoengineering approach in which a native sugar is modified with a terminal sialic acid residue, which can then be oxidized to give an aldehyde for conjugation to a linker-toxin (Zhou et al., Bioconjug. Chem., 25(3): 510-520 (2014)).

[0284] It has also been reported that active moieties are conjugated to antibodies using UV crosslinking. The method uses a nucleotide binding site (NBS) to perform site-specific covalent functionalization of antibodies with reactive thiol moieties. Indole-3-butyric acid (IBA)-conjugated cysteine ​​forms are used to perform site-specific photocrosslinking of reactive thiol moieties to antibodies 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)).

[0285] Alternatively, ADCs with low average DARs can be separated from ADC preparations 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).

[0286] ADCs with low average DAR can also be produced by adding unconjugated (i.e., DAR0) antibodies to ADC preparations with average DAR≥3.9. As known in the art, most conjugation methods produce ADC preparations including various DAR species, wherein the reported DAR is the average value of each DAR species. In certain embodiments, it may be advantageous to include a certain proportion of DAR0 species of ADC. In some embodiments, the ADC with an average DAR of less than 3.9 for the methods described herein includes at least 5% of DAR0 species. In some embodiments, the ADC for the methods described herein includes at least 10% of DAR0 species, such as at least 15% of DAR0 species or at least 20% of DAR0 species. In some embodiments, the ADC for the methods described herein includes DAR0 species between about 5% and about 50%, such as DAR0 species between about 10% and about 50%, between about 10% and about 40%, or between about 10% and about 30% of DAR0 species.

[0287] The average DAR of 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 drug-linked forms (e.g., fractions of species such as DAR0, DAR1, DAR2, etc.) can also be analyzed by various techniques known in the art, including MS (with or without accompanying chromatographic separation steps), 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)).

[0288] In certain embodiments, the average DAR of the ADC is determined by hydrophobic interaction chromatography (HIC) technique.

[0289] After conjugation, the ADC can be purified and separated from unconjugated reactants and / or any conjugated aggregates by purification methods known in the art. Such methods include, but are not limited to, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, and combinations thereof.

[0290] Pharmaceutical composition

[0291] Also provided herein is a pharmaceutical composition comprising a bispecific anti-HER2 antigen binding construct as described herein. The pharmaceutical composition comprises a bispecific anti-HER2 antigen binding construct and a pharmaceutically acceptable carrier.

[0292] The term "pharmaceutically acceptable" refers to a pharmaceutical composition approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, more particularly for humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some respects, the carrier is an artificial carrier not found in nature. When the pharmaceutical composition is administered intravenously, water can be used as a carrier. Saline solutions and dextrose and glycerol aqueous 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of wetting agent or emulsifier, or a pH buffer. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of a bispecific anti-HER2 antigen binding construct, preferably in purified form, together with an appropriate amount of carrier to provide a form suitable for administration to a patient. The formulation should be suitable for the mode of administration.

[0293] In certain embodiments, the composition comprising a bispecific anti-HER2 antigen binding construct is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Typically, the components are provided separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or a pouch indicating the active dose. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the components can be mixed before administration.

[0294] In certain embodiments, the compositions described herein are formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0295] Treatment for biliary tract cancer (BTC)

[0296] Described herein are methods of treating biliary tract cancer (BTC) comprising administering to a subject with BTC a bispecific anti-HER2 antigen binding construct or ADC as described herein in an amount effective to treat, prevent, or ameliorate such a disease or condition. In specific embodiments 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 linked to an auristatin analog.

[0297] "Disorder" or "disease" refers to any condition 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 predispose a mammal to the disorder in question. In embodiments described herein, the disorder or disease is biliary tract cancer, which is described in more detail below.

[0298] The term "subject" or "patient" refers to an animal, in some embodiments a mammal, that is the object of treatment, observation or experiment. The animal can be a human, a non-human primate, a companion animal (e.g., a dog, a cat, etc.), a farm animal (e.g., a cow, a sheep, a pig, a horse, etc.), or a laboratory animal (e.g., a rat, a mouse, a guinea pig, etc.).

[0299] As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0300] "Treatment" refers to a clinical intervention that attempts to change the natural course of the treated individual or cell, and can be performed during clinical pathology. The ideal effects of treatment include, but are not limited to, preventing disease recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, a 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, a 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, a bispecific anti-HER2 antigen binding construct or ADC can be used to slow the progression of BTC.

[0301] As used herein, the term "effective amount" refers to the amount of the bispecific anti-HER2 antigen binding construct administered, which will achieve the goal of the method, such as alleviating one or more symptoms of the disease, illness or condition being treated to some extent. 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 used to help determine the optimal dosage range. The precise dose used in the formulation also depends on the route of administration and the severity of BTC, and should be determined according to the judgment of the practitioner and the circumstances of each patient. The effective dose can be inferred from the dose-response curve of an in vitro or animal model test system.

[0302] 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 cancer. The terms "second-line therapy" or "second-line treatment" are treatment regimens that are typically administered if first-line therapy fails to provide the desired effect.

[0303] The term "neoadjuvant therapy" refers to treatment given as a first step to shrink a tumor, before primary treatment (usually surgery) is given. Examples of neoadjuvant therapy include, but are not limited to, chemotherapy, radiation therapy, and hormone therapy. Neoadjuvant therapy may be considered first-line therapy.

[0304] The term "adjuvant therapy" refers to additional cancer treatment given after first-line treatment to reduce the risk of cancer recurrence. Adjuvant therapy may include, but is not limited to, chemotherapy, radiation therapy, hormone therapy, targeted therapy (small molecule drugs or antibodies that usually target specific types of cancer cells but not normal cells), or biological therapy (such as vaccines, cytokines, antibodies, or gene therapy).

[0305] "Advanced cancer" is cancer that has progressed to the point where it can't be safely removed or where a cure or long-term remission is extremely unlikely. Cancers become advanced by growing near structures that prevent their removal or by spreading from where they started, through tissue lines, or to other parts of the body (such as lymph nodes or other organs). Advanced cancers may be locally advanced, meaning they have spread beyond the organ where they started but have not yet spread to distant sites. Advanced cancers may also be metastatic, meaning cancer cells have spread from where the cancer started (the primary site) to other more distant parts of the body (secondary sites).

[0306] "Resectable" cancer is cancer that can be treated with surgery. "Unresectable" cancer is cancer that cannot be treated with surgery, usually because the cancer has spread to the tissues around the main tumor. Some cancers may be assessed by your doctor as "partially resectable" depending on how much it has spread to surrounding tissues.

[0307] Bispecific anti-HER2 antigen binding constructs or ADCs can be applied to subjects according to known methods. A variety of delivery systems are known and can be used to administer bispecific anti-HER2 antigen binding construct preparations described herein, such as encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing compounds, receptor-mediated endocytosis (see, for example, Wu and Wu, J.Biol.Chem.262:4429-4432 (1987)), construct nucleic acids as part of retroviruses or other vectors, etc. Introduction methods include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Bispecific anti-HER2 antigen binding constructs or ADCs can be administered by any suitable route, such as by infusion or push injection, absorbed by epithelial or mucocutaneous linings (such as oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local. In addition, in certain embodiments, it may be desirable to introduce the bispecific anti-HER2 antigen binding constructs described herein into the central nervous system by any suitable route, including intraventricular and intrathecal injections; intraventricular injections can be facilitated by intraventricular catheters, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by using an inhaler or nebulizer, and formulations containing aerosols. In specific embodiments, the bispecific anti-HER2 antigen binding constructs or ADCs may be administered intravenously (IV).

[0308] In a specific embodiment, it may be desirable to apply the bispecific anti-HER2 antigen binding constructs or ADCs described herein locally to the area in need of treatment; this may be accomplished, for example, but not limited to, by local infusion during surgery, local application such as postoperatively in conjunction with a wound dressing, by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being 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 the protein does not absorb.

[0309] In another embodiment, the bispecific anti-HER2 antigen binding construct or ADC can be delivered in a vesicle, particularly a liposome (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, supra, pp. 317-327; see generally supra).

[0310] 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, polymeric materials 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); see also 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, a controlled release system can be placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, Vol. 2, pp. 115-138 (1984)).

[0311] Bispecific anti-HER2 antigen binding constructs or ADCs can be administered alone or in combination with other types of treatment (e.g., radiotherapy, chemotherapy, hormone therapy, immunotherapy, and anti-tumor agents). Generally, it is preferred to administer products of the same species origin or species reactivity (in the case of antibodies) as the patient's species. Therefore, in one embodiment, human or humanized bispecific anti-HER2 antigen binding constructs, fragment derivatives, analogs, or nucleic acids are administered to human patients for treatment or prevention.

[0312] Biliary tract cancer (BTC, also known as "biliary 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 for treating BTC. In one embodiment, a bispecific anti-HER2 antigen binding construct or ADC described herein can be used in a method for treating advanced unresectable BTC. In other embodiments, a bispecific anti-HER2 antigen binding construct or ADC described herein can be used in a method for treating gallbladder cancer, ampullary cancer, cholangiocarcinoma, or cystic duct adenocarcinoma. In other embodiments, a bispecific anti-HER2 antigen binding construct or ADC described herein can be used in a method for treating intrahepatic CCA or extrahepatic CCA.

[0313] In one embodiment, the bispecific anti-HER2 antigen binding construct or ADC can be used to treat subjects with BTC that show HER2 expression, amplification or activation. "BTC that shows HER2 expression, amplification or activation" is a BTC that expresses (including overexpression) HER2 receptors, has amplified HER2 genes and / or otherwise exhibits HER2 receptor activation or phosphorylation in a diagnostic test.

[0314] A BTC that "shows HER2 activation" is a BTC that exhibits HER2 receptor activation or phosphorylation in a diagnostic test. This 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 with a BTC that shows HER2 expression.

[0315] A BTC with "HER2 receptor overexpression or amplification" is a BTC with significantly higher levels of HER2 receptor protein or gene compared to non-cancerous cells of the same tissue type. This 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 elevated levels of HER2 protein present on the surface of cells (e.g., via immunohistochemistry; IHC). In one embodiment, HER2 overexpression can be determined by IHC, e.g., using (Dako) analysis. Paraffin-embedded tissue sections from tumor biopsies were available for IHC assay and met the following criteria for HER2 protein staining intensity:

[0316] Score 0: No staining was observed or membrane staining was observed in less than 10% of tumor cells.

[0317] Score 1+: weak / barely perceptible membrane staining detected in more than 10% of tumor cells. Only part of the cell membrane is stained.

[0318] Score 2+: weak to moderate complete membrane staining was observed in more than 10% of tumor cells.

[0319] Score 3+: moderate to strong complete membrane staining was observed in more than 10% of tumor cells.

[0320] Those tumors with a score of 0 or 1+ for the HER2 overexpression assessment can be characterized as not overexpressing HER2, while those tumors with a score of 2+ or 3+ can be characterized as overexpressing HER2. In one embodiment, a bispecific anti-HER2 antigen-binding construct or ADC can be used to treat a subject with a BTC that exhibits HER2 overexpression and / or amplification.

[0321] Alternatively or additionally, the level of HER2 encoding nucleic acid in the cell can be measured, for example, via in situ hybridization (ISH), including fluorescent 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 blot, polymerase chain reaction (PCR) techniques such as quantitative real-time PCR (qRT-PCR) or next-generation sequencing (NGS). Evaluation of HER2 gene amplification using these methods is usually reported as positive (+) or negative (-), for example, FISH+ for HER2 gene amplified cancer or FISH- for HER2 gene non-amplified cancer. Evaluation of HER2 gene amplification by NGS can also be reported on the number of HER2 gene copies. In normal cells, there are two HER2 gene copies. Therefore, if a cancer has more than two copies of the HER2 gene, the cancer is considered to be a HER2 gene-amplified cancer.

[0322] Described herein is a method for treating a subject with BTC that exhibits HER2 expression, amplification, or activation, comprising providing the subject with an effective amount of a bispecific anti-HER2 antigen binding construct or ADC described herein. In some embodiments, a bispecific anti-HER2 antigen binding construct or ADC can be used to treat a subject with HER2 3+, genetically amplified BTC. In other embodiments, a bispecific anti-HER2 antigen binding construct or ADC can be used to treat HER2 2+, genetically amplified BTC. In other embodiments, a bispecific anti-HER2 antigen binding construct or ADC can be used to treat HER21+, genetically amplified BTC. In other embodiments, a bispecific anti-HER2 antigen binding construct or ADC can be used to treat BTC assessed as HER2 3+ without HER2 gene amplification. In other embodiments, a bispecific anti-HER2 antigen binding construct or ADC can be used to treat BTC assessed as HER2 2+ without HER2 gene amplification. In other embodiments, a bispecific anti-HER2 antigen binding construct or ADC can be used to treat BTC assessed as HER2 1+ without HER2 gene amplification.

[0323] In some embodiments, the subject receiving treatment may not have previous BTC treatment, and the bispecific anti-HER2 antigen binding construct or ADC is administered as a first-line treatment. In some embodiments, the bispecific anti-HER2 antigen binding construct or ADC can be used as an adjuvant or neoadjuvant therapy to treat subjects with resectable or partially resectable cancer. In other embodiments, the subject receiving treatment may have received one or more previous BTC treatments, and the bispecific anti-HER2 antigen binding construct or ADC is administered as a second-line treatment. One or more previous BTC treatments may include treatments selected from the following: systemic chemotherapy, such as gemcitabine alone or in combination with platinum-based chemotherapeutics, fluoropyrimidine-based chemoradiotherapy, radiotherapy without additional chemotherapy, antibodies (including but not limited to anti-HER2 targeted antibodies) and investigational agents (i.e., agents currently undergoing clinical trials but not yet approved by the FDA). Platinum-based chemotherapeutics may include cisplatin or oxaliplatin. In one embodiment, systemic chemotherapy includes gemcitabine and cisplatin, or gemcitabine and oxaliplatin.

[0324] An exemplary effective amount of a bispecific anti-HER2 antigen-binding construct or ADC that can be administered to a subject with BTC can be between 0.1 mg / kg and 100 mg / kg of the subject's 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 of body weight.

[0325] In some embodiments, the bispecific anti-HER2 antigen binding construct is administered weekly, every two weeks (Q2W), every three weeks (Q3W), or every 4 weeks (Q4W). An exemplary effective amount of a weekly administration of a bispecific anti-HER2 antigen binding construct is in the range of about 1 mg / kg to about 30 mg / kg. An exemplary effective amount of a bispecific anti-HER2 antigen binding construct administered every two weeks is in the range of about 10 mg / kg to about 50 mg / kg. An exemplary effective amount of a bispecific anti-HER2 antigen binding construct administered every three weeks is in the range of about 15 mg / kg to about 50 mg / kg. An exemplary effective amount of a bispecific anti-HER2 antigen binding construct administered every four weeks is in the range of about 40 mg / kg to about 70 mg / kg.

[0326] 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 a further embodiment, 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.

[0327] As is known in the art, the ADC can be administered to a subject at a dose lower than that used for a bispecific anti-HER2 antigen binding construct. In some embodiments, the ADC described herein (i.e., a bispecific anti-HER2 antigen binding construct linked 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 is between about 1 to about 15 mg / kg weekly, every two weeks, or every three weeks.

[0328] As described above, in specific 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 a related embodiment, the infusion rate may not exceed 250mL of normal saline per hour.

[0329] Also provided herein is a method for treating a subject with BTC, including the co-administration of an effective amount of a bispecific anti-HER2 antigen binding construct or ADC with additional anti-tumor therapy. Additional anti-tumor therapy can be selected from one or more BTC treatments, including systemic chemotherapy, such as gemcitabine alone or in combination with a platinum-based chemotherapeutic agent, a fluoropyrimidine-based chemoradiotherapy, radiotherapy without additional chemotherapy, and an investigational agent (i.e., an agent currently undergoing clinical trials but not yet approved by the FDA). In one embodiment, the method for treating a subject with BTC includes the co-administration of 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, a 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, a bispecific anti-HER2 antigen binding construct or ADC can be co-administered with 5-FU, oxaliplatin, and folinic acid. In other embodiments where the subject has BTC that is MSI-H / dMMR (high-level microsatellite instability / deficient mismatch repair), the bispecific anti-HER2 antigen-binding construct or ADC may be combined with an immune checkpoint inhibitor such as the anti-PD1 antibody pembrolizumab (Keytruda TM ) or the anti-PD-L1 antibody atezolizumab Combined administration.

[0330] As described in Table 2 of Simile et al., (2019) Medicina 55: 42, additional anti-tumor treatments for BTC are known in the art. One skilled in the art will be able to identify which of these treatments can be administered in combination with the bispecific anti-HER2 antigen-binding constructs or ADCs described herein.

[0331] The additional anti-tumor treatments described in the preceding paragraphs may be administered simultaneously with the bispecific anti-HER2 antigen-binding construct or ADC, or may be administered sequentially.

[0332] In some embodiments, the result of providing an effective amount of a bispecific anti-HER2 antigen binding construct to a subject with BTC is to reduce the lesion, inhibit the growth of the lesion, increase the time of lesion progression, prolong the disease-free survival of the subject, reduce metastasis, increase the progression-free survival of the subject, or increase the overall survival of the subject or increase the overall survival of a group of subjects treated. In a related embodiment, the result of providing an effective amount of a bispecific anti-HER2 antigen binding construct to a subject is a partial response (PR) or stable disease (SD) in the subject, as measured by the revised solid tumor efficacy evaluation criteria (Response Evaluation Criteria in Solid Tumors, RECIST) guidelines (version 1.1) [Eur JCa 45: 228-247, 2009]. In subjects with metastatic disease and CR or PR, the duration of response can also be measured.

[0333] 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 may be declared based on "unequivocal progression" in cases where 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 insufficient to qualify (especially in the presence of SD or PR in the target disease).

[0334] As used herein, the term "partial response" (PR) refers to a reduction of at least 30% in the sum of diameters of target lesions (including the short axis of any target lymph nodes) relative to the baseline sum diameter.

[0335] 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 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).

[0336] As used herein, the term "stable disease" (SD) refers to disease that has neither shrunk sufficiently to qualify as a PR nor increased sufficiently to qualify as a PD, with reference to the smallest summed diameter since the start of treatment.

[0337] As used herein, the term "objective response rate" (ORR) is the proportion of all randomized patients who received any amount of study drug and had a PR or CR according to RECIST v 1.1 from the start of treatment until disease progression / recurrence (taking PD as reference, the smallest measurement recorded since the start of treatment).

[0338] 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.

[0339] As used herein, the term "progression-free survival" (PFS) refers to the patient's survival without progression or deterioration of cancer. In one embodiment, PFS is defined as the time from randomization in the study until the first radiographic record of objective progression defined according to RECIST (version 1.1) or death from any cause. Patients who died without reporting previous progression will be considered to have progressed on the day of their death. Patients who did not progress or lost follow-up will be reviewed on the day of their last radiographic tumor assessment.

[0340] As used herein, the term "disease-free survival" (DFS) refers to the length of time a patient survives without any signs or symptoms of cancer after the initial treatment of the cancer is completed. DFS may also be referred to as "recurrence-free survival" (RFS).

[0341] As used herein, the term "time to progression" (TTP) refers to the length of time from the diagnosis or start of treatment of a cancer until the cancer begins to worsen or spread to other parts of the body.

[0342] 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 (PD patients are explicitly excluded) classified as CR, PR or SD, where the best overall response is the best response recorded from the start of treatment to PD.

[0343] As used herein, the term "duration of overall response" (DOR) refers to the period of time measured from the time the criteria for a complete or partial response are met (whichever status is recorded first) until the first date of objectively documented recurrent or progressive disease, with reference to the smallest measurement recorded since the start of treatment.

[0344] In some embodiments, the result of providing an effective amount of a bispecific anti-HER2 antigen binding construct or ADC to a subject with BTC is to improve the disease control rate (DCR) of a group of subjects. DCR can be used to measure the efficacy of a therapy with a tumor suppressive effect rather than a tumor killing effect. DCR is calculated as the percentage of BTC patients who show CR, PR or SD after treatment with a bispecific anti-HER2 or ADC. In one embodiment, administering an effective amount of a bispecific anti-HER2 antigen binding construct or 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, administration of an effective amount of a bispecific anti-HER2 antigen-binding construct or 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%.

[0345] PFS (progression-free survival) and ORR (overall response rate) can also be used to determine the efficacy of bispecific anti-HER2 antigen binding constructs or ADCs and are measured according to the above-mentioned revised RECIST 1.1 guidelines. 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 using a bispecific anti-HER2 antigen binding construct or ADC. ORR can be used as a measure of drug tumoricidal activity. In some embodiments, the result of providing an effective amount of a bispecific anti-HER2 antigen binding construct or ADC to a subject with BTC is to increase the progression-free survival (PFS) of a group of subjects. In some embodiments, the result of providing an effective amount of a bispecific anti-HER2 antigen binding construct or ADC to a subject with BTC is to improve the overall response rate (ORR). In one embodiment, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or 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 another embodiment, administering an effective amount of a bispecific anti-HER2 antigen-binding construct or 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%.

[0346] Overall survival, time to progression, duration of response (DOR) (can also be used to determine the efficacy of a bispecific anti-HER2 antigen binding construct or ADC.

[0347] When a bispecific anti-HER2 antigen-binding construct or ADC is administered as adjuvant or neoadjuvant therapy, disease-free survival may also be measured to determine the efficacy of the therapy.

[0348] Medicine boxes and products

[0349] Also described herein are kits containing 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 that manages the manufacture, use, or sale of drugs or biological products, which reflects the approval of the agency to manufacture, use, or sell. The kit may optionally include instructions or instructions outlining the method of use or administration regimen of the bispecific anti-HER2 antigen binding construct or ADC.

[0350] When one or more components of the kit are provided as a solution, e.g., 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 the other components of the kit.

[0351] The components of the kit may also be provided in a dried or lyophilized form and the kit may additionally contain a solvent suitable for reconstituting the lyophilized components. Regardless of the number or type of containers, the kits described herein may also contain an instrument for assisting in administering the composition to a patient. Such an instrument may be an inhaler, a nasal spray device, a syringe, a pipette, tweezers, a measuring spoon, an eye dropper, or a similar medically approved delivery vehicle.

[0352] In another aspect described herein, an article comprising materials useful for treating, preventing and / or diagnosing BTC is provided. The article comprises 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 may be formed of a variety of materials such as glass or plastic. The container is filled with a composition, which itself or in combination with another composition that effectively treats, prevents and / or diagnoses the disorder and may have a sterile access port (e.g., the container may 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 used to treat the selected disorder. In addition, the article may comprise (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 in this embodiment described herein may also include a package insert indicating that the composition can be used to treat BTC. Alternatively or additionally, the article of manufacture may further include a second (or third) container comprising 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 may also include other desirable materials, including other buffers, diluents, filters, needles, and syringes.

[0353] Peptides and polynucleotides

[0354] The bispecific anti-HER2 antigen binding constructs described herein comprise at least one polypeptide. Polynucleotides encoding the polypeptides described herein are also described. The bispecific anti-HER2 antigen binding constructs are typically isolated.

[0355] As used herein, "isolated" refers to an agent (e.g., a polypeptide or polynucleotide) that has been identified and separated and / or recovered from a component of its natural cell culture environment. The contaminant components of its natural environment are materials that interfere with the diagnostic or therapeutic use of the bispecific anti-HER2 antigen binding construct, and may include enzymes, hormones, and other proteins or non-protein solutes. Isolated also refers to an agent that has been synthetically produced, for example, via human intervention.

[0356] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0357] 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 function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are 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 refer to compounds having the same basic chemical structure (i.e., alpha carbon bound to hydrogen, carboxyl, amino and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. 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 having properties known in the art as amino acid characteristics. 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 with additional methylene groups in the side chains ("homo" amino acids), and amino acids in which the carboxylic acid functional groups in the side chains are replaced by sulfonic acid groups (e.g., cysteine). It may be advantageous to incorporate 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. Compared to their L-amino acid counterparts, peptides containing D-amino acids, etc., exhibit increased stability in vitro or in vivo. Therefore, when greater intracellular stability is desired or required, the construction of peptides incorporating D-amino acids, etc., may be particularly useful. More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule and, where such properties are desired, extended in vivo lifespan. In addition, D-peptides and the like cannot be efficiently processed for major histocompatibility complex class II-restricted presentation to T helper cells and are therefore less likely to induce a humoral immune response in the whole organism.

[0358] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0359] Also described herein are polynucleotides encoding polypeptides of bispecific anti-HER2 antigen binding constructs. The term "polynucleotide" or "nucleotide sequence" is intended to mean a continuous segment of two or more nucleotide molecules. The nucleotide sequence can be genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combination thereof.

[0360] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the term also refers to oligonucleotide analogs, including DNA analogs (phosphorothioates, phosphoramidates, etc.) used in PNA (peptide nucleic acids), antisense technology. Unless otherwise specified, specific nucleic acid sequences also implicitly encompass variants (including but not limited to degenerate codon substitutions) and complementary sequences of their conservative modifications and sequences explicitly specified. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base 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)).

[0361] "Conservatively modified variants" apply to amino acids and nucleic acid sequences. With respect to specific nucleic acid sequences, "conservatively modified variants" refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or, when the nucleic acid does not encode an amino acid sequence, to substantially identical sequences. Due to 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 each position where alanine is specified by a codon, the codon can be changed to any corresponding codon described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each 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 usually the only codon for methionine, and TGG is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0362] 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 alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence) are "conservatively modified variants," wherein the variation results in a deletion of an amino acid, an addition of an amino acid, or a substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to one of ordinary skill in the art. Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, interspecies homologs and alleles described herein.

[0363] Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservatively substituted for each other: 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

[0139] 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd Edition (December 1993)).

[0364] In the context of two or more nucleic acid or polypeptide sequences, the term "identity" or "identity" percentage refers to two or more identical sequences or subsequences. If when comparing and comparing maximum correspondence on a comparison window or a specified region as measured using one of the following sequence comparison algorithms (or other algorithms available to those of ordinary skill in the art) or by manual comparison and visual inspection, the sequence has the percentage of identical 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 on a specified region), then the sequence is "substantially identical". This definition also relates to the complementary sequence of a test sequence. Identity may be present in a region of at least about 50 amino acids or nucleotides in length, or a region of 75-100 amino acids or nucleotides in length, or, if unspecified, across the entire sequence of a polynucleotide or polypeptide. Polynucleotides encoding polypeptides described herein, including homologs from species other than humans, can be obtained by a method comprising the following steps: screening a library with a labeled probe having a polynucleotide sequence described herein or a fragment thereof under stringent hybridization conditions, and isolating full-length cDNA and genomic clones comprising the polynucleotide sequence. Such hybridization techniques are well known to technicians.

[0365] For sequence comparison, usually a sequence serves as a reference sequence for comparison with a test sequence. 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. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence according to the program parameters.

[0366] As used herein, "comparison window" includes reference to a segment of any one of a plurality of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence having the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be performed, 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 search by similarity method 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)).

[0367] An example of an algorithm suitable for determining percentages of sequence identity and 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 on the World Wide Web at 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) uses by default a word length (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses by default a word length of 3, and an expectation value (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) of 50, an expectation value (E) of 10, M=5, N=-4, and a comparison of both chains. The BLAST algorithm is typically performed with a "low complexity" filter turned off.

[0368] 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 similarity metric provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence 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.

[0369] The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing or hybridization of a molecule only to a specific nucleotide sequence under stringent hybridization conditions when the sequence is present in a complex mixture (including but not limited to total cellular or library DNA or RNA).

[0370] 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 as 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 cell or library DNA or RNA), but will not hybridize to other sequences in the complex mixture. Stringent conditions depend on the sequence and will vary in different situations. Longer sequences specifically hybridize at higher temperatures. An extensive guide to nucleic acid hybridization is 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).

[0371] As used herein, the term "engineering" is considered to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications to the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these methods. Engineered proteins are expressed and produced by standard molecular biology techniques.

[0372] "Isolated nucleic acid molecules or polynucleotides" refer to nucleic acid molecules, DNA or RNA that have been removed from their 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 heterologous host cells or polynucleotides purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that usually contain polynucleotide molecules, but the polynucleotide molecules are present in extrachromosomal or chromosomal locations that are different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts, as well as positive and negative strand forms, and double-stranded forms. The isolated polynucleotides or nucleic acids described herein also include such molecules produced synthetically, for example, via PCR or chemical synthesis. In addition, in certain embodiments, the polynucleotides or nucleic acids include regulatory elements such as promoters, ribosome binding sites or transcription terminators.

[0373] 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. Pat. No. 4,683, 195. In general, the PCR method involves repeated cycles of primer extension synthesis using oligonucleotide primers that are capable of preferentially hybridizing to a template nucleic acid.

[0374] For a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted 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 changes to the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, interspersed individually between residues in the reference sequence or interspersed in one or more consecutive 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 a 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).

[0375] If the amino acid sequence of a derivative or variant of a polypeptide has at least 50% identity with a 100 amino acid sequence from the original peptide, the derivative or variant of the polypeptide is said to have "homology" or "homologous" with the 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.

[0376] As used herein, the term "modification" refers to any change made to a given polypeptide, such as changes to the length of the polypeptide, the amino acid sequence of the polypeptide, the chemical structure, co-translational modification or post-translational modification. The form "(modified)" term means that the polypeptide in question is optionally modified, i.e., the polypeptide in question may be modified or unmodified.

[0377] 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 shown in the table or accession number disclosed herein, or a fragment thereof. 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 shown in the table or accession number disclosed herein, or a fragment thereof.

[0378] It should be understood that the present disclosure is not limited to a particular protocol; the cell lines, constructs, reagents, etc. described herein may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure.

[0379] In order to describe and disclose the construct and method described in for example publications, all publications and patents mentioned herein are incorporated herein by reference, and they can be used in combination with the construct described herein. The publications discussed herein are only provided with respect to the disclosures before the filing date of the present application. Anything herein should not be construed as admitting that the inventor has no right to carry out such disclosure in advance due to prior invention or any other reason.

[0380] Sequence Listing

[0381] Sequence Listing

[0382] Table 6: Clone numbers of variants v5019, v5020, v7091, v10000, v6903, v6902 and v6717

[0383] Variants H1 clone# H2 cloning# L1 clone# L2 cloning# 5019 3057 720 1811 -- 5020 719 3041 -- 1811 7091 3057 5244 1811 -- 10000 6586 5244 3382 -- 6903 5065 3468 5037 3904 6902 5065 3468 5034 3904 6717 3317 720 -- --

[0384] Table 7: Sequences of variants v5019, v5020, v7091, v10000, v6903, v6902 and v6717 listed by clone number

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400] Example

[0401] 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 present 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 errors and deviations should be allowed.

[0402] Unless otherwise indicated, the constructs and methods described herein can be prepared and implemented using conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. These techniques are fully explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALL Lehninger, Biochemistry (Worth Publishers, Inc., currently new); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan, ed., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd edition (Plenum Press) Volumes A and B (1992).

[0403] Example 1: Description and preparation of variant 10000 (v10000)

[0404] 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 homodimer formation. Figure 1 A schematic representation of the v10000 version is depicted, wherein heavy chain A and light chain A' form the ECD2 binding portion of the antibody, and heavy chain B comprises a scFv that forms the ECD4 binding portion of the antibody. Variant 10000 comprises a heavy chain H1 comprising the sequence set forth in SEQ ID NO:36 (corresponding to Figure 1 ), heavy chain H2 comprising the sequence shown in SEQ ID NO: 63 (corresponding to Figure 1 The preparation method of v10000 is described in detail in International Patent Publication No. WO 2015 / 077891.

[0405] v10000 is manufactured in accordance with regulatory requirements for human testing and is formulated at 15 mg / mL in a biocompatible aqueous buffer for IV infusion at ambient temperature. v10000 is supplied in vials containing 300 mg of v10000 in 20 mL of buffer. v10000 vials are shipped frozen and stored at -20°C (+ / -5°C) until ready for use. Vials are thawed at ambient temperature prior to use. The thawed solution in the vial may be stored at ambient temperature for up to 24 hours, or under refrigerated conditions (2°C to 8°C) for up to 72 hours and used before the labeled expiration date.

[0406] Example 2: Phase I trial of v10000 in patients with locally advanced (unresectable) and / or metastatic HER2-expressing cancer Clinical trials

[0407] This is an ongoing first-in-human study investigating 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.

[0408] Part 1 of the study was a 3+3 dose escalation, with 20 mg / kg Q2W single agent established as the recommended dose (RD). Part 2 is ongoing and 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 therapies known to provide clinical benefit. Tumor response was assessed by investigator review according to RECIST v1.1 Q8W.

[0409] Target

[0410] The primary objective of Part 1 of the clinical trial is to determine the MTD (maximum tolerated dose), OBD (optimal biological dose) or RD of v10000 monotherapy. The secondary objectives of Part 1 of the clinical trial are to (1) characterize the safety and tolerability of v10000; (2) characterize the serum PK profile of v10000; and (3) explore the potential anti-tumor effects of v10000 in eligible patients with HER2-expressing cancer.

[0411] 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 to (1) characterize the serum PK profile of v10000 monotherapy and (2) explore the potential anti-tumor effects of v10000 at MTD, OBD or RD in eligible patients with selected locally advanced (unresectable) and / or metastatic cancers expressing HER2.

[0412] patient

[0413] 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 according to the clinical investigator's opinion.

[0414] In Part 1, Cohorts 1-3 included patients with any locally advanced (unresectable) and / or metastatic HER2-expressing (HER2 1+, 2+, or 3+ by IHC) cancer (including but not limited to breast cancer, gastric cancer, ovarian cancer, colorectal cancer, and non-small cell lung cancer) who had progressed after all therapies known to provide clinical benefit. Cohorts 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. Cohorts 4-6 also include patients with any other HER2 IHC 3+ or FISH+ cancer, where the cancer is HER2 overexpressing (3+ by IHC) or HER2-2+, and FISH+ breast cancer must have progressed after prior treatment with trastuzumab, pertuzumab, and T-DM1; where the cancer is HER2 overexpressing (3+ by IHC) or HER2-2+, and FISH+GEA must have progressed after prior treatment with trastuzumab; where colorectal cancer patients are KRAS wild-type; or where NSCLC patients are ALK wild-type, EGFR wild-type, and ROS1 fusion negative, as determined by standard methods. Cohort 7 is recruiting at selected sites and includes patients with HER2 IHC 3+, HER2 IHC 2+ / FISH+, or HER2 IHC 2+ / FISH- breast cancer.

[0415] In Part 2, arm expansions from Part 1 of the study with v10000 administered at the MTD, OBD, or RD included patients with locally advanced (unresectable) and / or metastatic cancer that had progressed after all therapies known to provide clinical benefit (unless ineligible for specific therapy) as follows:

[0416] Group 1: HER2 IHC 2+ / FISH- breast cancer

[0417] Group 2: HER2 IHC 3+ or HER2 IHC 2+ / FISH+ breast cancer

[0418] Group 3: HER2 IHC 2+ / FISH-GEA

[0419] Group 4: HER2 IHC 3+ or HER2 IHC 2+ / FISH+GEA

[0420] Group 5: Any other HER2 IHC 3+ or IHC 2+ / FISH+ cancer, including the following:

[0421] Group 5a: HER2 IHC 3+ or IHC 2+ / FISH+ GI (gastrointestinal) cancers other than GEA, where colorectal cancer patients are KRAS wild-type

[0422] Group 5b: Any other solid tumor type that is not breast or gastrointestinal cancer and is HER2 IHC 3+ or IHC 2+ / FISH+, where NSCLC patients must be ALK wild-type, EGFR wild-type, and ROS1 fusion negative as determined by standard methods. Ovarian cancer patients must be KRAS wild-type.

[0423] Additional criteria for patients in Parts 1 and 2 included: (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 as determined by standard methods.

[0424] Patients were excluded from the study if one or more of the following criteria applied:

[0425] 1. Treatment with experimental therapy within 4 weeks prior to the first dose of V10000

[0426] 2. Treatment with other unspecified cancer therapies within 4 weeks prior to administration of V10000

[0427] 3. In the first v10000 dose or the lifetime total dose exceeds 300 mg / m 2 Treatment with anthracyclines within the previous 90 days with doxorubicin or equivalent

[0428] 4. Treatment with trastuzumab, pertuzumab, lapatinib, or T-DM1 within 3 weeks prior to the first dose of v10000

[0429] 5. Untreated brain metastases (patients with treated brain metastases who are off steroids and stable for at least 1 month at the time of screening are eligible). All breast cancer patients should be screened before starting treatment. Those found to have untreated brain metastases can be rescreened after appropriate therapy.

[0430] 6. Clinically assessed leptomeningeal disease (LMD). If LMD had been reported radiographically on the baseline MRI but was not clinically suspected by the investigator, the patient was eligible if he or she had no neurologic symptoms of LMD documented by the investigator

[0431] 7. Major surgery or radiotherapy within 3 weeks before the first administration of v10000

[0432] 8. Pregnant or breastfeeding women

[0433] 9. History of life-threatening hypersensitivity reaction to monoclonal antibodies or to recombinant proteins or excipients in drug preparations

[0434] 10. Any other cancer within 3 years prior to the first dose of v10000, except for contralateral breast cancer, adequately treated cervical cancer in situ, or adequately treated basal cell carcinoma or squamous cell carcinoma of the skin, or any other cancer that has received curative treatment, and approved by the sponsor's medical monitor.

[0435] 11. Acute or chronic uncontrolled renal disease, pancreatitis, or liver disease (except for patients with Gilbert's Syndrome, asymptomatic gallstones, liver metastases, or stable chronic liver disease as assessed by the investigator)

[0436] 12. Peripheral neuropathy: > Grade 2 NCI-CTCAE Version 4.03, July 14, 2010

[0437] 13. Clinically significant interstitial lung disease

[0438] 14. History of non-compliance with medical regimen

[0439] 15. Unwilling or unable to abide by the agreement

[0440] 16. Known active hepatitis B or C or known infection with human immunodeficiency virus (HIV)

[0441] 17. Use of corticosteroids at doses equivalent to >15 mg prednisone per day within 2 weeks of the first v10000 dose unless otherwise approved by the study medical monitor.

[0442] 18.QTc Fridericia(QTcF)>450ms.

[0443] 19. Any toxicity related to previous cancer therapy that has not resolved to ≤ Grade 1, except for the following: alopecia; neuropathy (must have resolved to ≤ Grade 2); and congestive heart failure (CHF), which must be ≤ Grade 1 in severity when it occurs and must resolve completely.

[0444] 20. Have clinically significant cardiac disease, such as ventricular arrhythmias requiring treatment, uncontrolled hypertension, or any history of symptomatic CHF.

[0445] 21. Known myocardial infarction or unstable angina within 6 months before the first dose of v10000.

[0446] treat

[0447] In Parts 1 and 2, v10000 was administered intravenously (IV) as a single agent (monotherapy). v10000 was administered by IV infusion in 0.9% saline over 120 to 150 minutes. If the first 2 administered doses were well tolerated for a particular patient, the infusion duration for that patient may have been reduced to 90 minutes. If the next 2 doses were well tolerated, the infusion duration may be reduced to 60 minutes. The infusion rate was not to exceed 250 mL of 0.9% saline / hour. (Example: If a dose of v10000 is diluted into a 250 mL bag of saline, the infusion should be administered over at least 60 minutes. If a dose of v10000 is 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 body weight on Cycle 1 Day 1. The dose was recalculated only if there was a 10% change in body weight based on the assessment on Cycle 1 Day 1.

[0448] In Part 1, dose escalation levels were 5, 10, and 15 mg / kg, administered once weekly (QW). Dosing every two weeks (Q2W) was also evaluated. The dose levels for Q2W dosing were 20, 25, or 30 mg / kg. Q2W dosing may have used an initial loading dose (not to exceed 20, 25, or 30 mg / kg), followed by administration of v10000 at a lower dose level recommended by the SMC. In addition, a dose of 30 mg / kg every 3 weeks (Q3W) was studied.

[0449] The dose level for Part 2 is the MTD, OBD, or RD determined in Part 1. The MTD is defined as the highest dose level at which no more than 1 of 6 patients experienced DLT during the first 4 weeks of treatment. The OBD is defined as the v10000 dose that results in a trough (7 days after dosing) serum concentration of v10000 that is at least 10 times higher than the maximum binding capacity of v10000 for a cell line representing HER2-3+ tumor histology. The RD is any other dose that does not exceed the MTD. Based on the results of Part 1, an RD of 20 mg / kg Q2W is used to treat most patients in Part 2. Some patients receive 10 mg / kg treatment per week.

[0450] Patients participated in a minimum of 2 cycles of 3 or 4 weeks each, depending on the part, group, or TG into which the patient was enrolled. Treatment could continue for additional cycles as long as there was no evidence of clinical progression, unacceptable toxicity, or evidence of progressive disease as defined by RECIST version 1.1. Clinical progression was defined as worsening or reappearance of pre-existing symptoms related to the underlying cancer, or the onset of new symptoms that could not be attributed to study drug toxicity or other causes. Patients who showed continued clinical benefit despite radiographic progression in the opinion of the clinical investigator may continue treatment after discussion with and approval from the Sponsor's Medical Monitor. For Part 3, patients may continue to receive v10000 treatment if chemotherapy was discontinued due to toxicity unrelated to v10000. Patients who discontinued v10000 treatment for any reason were withdrawn from the study.

[0451] Efficacy evaluation

[0452] Measures for evaluating antitumor activity based on efficacy assessments were based on new international standards proposed using the revised solid tumor efficacy evaluation criteria (RECIST) guidelines (version 1.1) [Eur J Ca 45:228-247, 2009]. Changes in the maximum diameter of tumor lesions (one-dimensional measurement) and the shortest diameter of malignant lymph nodes were used in the RECIST version 1.1 criteria. Clinical responses of CR, PR, SD, or progressive disease (PD) were 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 worsening or reappearance of pre-existing symptoms related to the underlying cancer, or the appearance of new symptoms that cannot be attributed to study drug toxicity or alternative causes.

[0453] Objective response rate (ORR) is defined as the percentage of patients with at least 1 CR or PR overall tumor response before any evidence of progression as defined by RECIST version 1.1. If the patient has a tumor response of CR, PR or SD according to RECIST version 1.1 standards, it is said that disease control has been achieved. Disease control rate is assessed every 8 weeks after the start of v10000 therapy. PFS time is defined as the time from the first dose of v10000 to the date of disease progression, clinical progression or death for any reason recorded according to RECIST version 1.1. Patients who are still alive and have not progressed at the time of analysis will be reviewed at the time of their last CR, PR or SD tumor assessment.

[0454] Tumor response was assessed based on CT and / or MRI scans of the chest, abdomen, and pelvis (using the same methodology for each scan of the same patient) plus additional areas of known or suspected tumor involvement (eg, brain, limbs).

[0455] Objective response and tumor progression were assessed locally. Scans were collected for all subjects for central review at the sponsor's discretion. Local assessment was used for all treatment-related decisions.

[0456] For some patients, tumor volume can be calculated centrally.

[0457] Adverse Reactions

[0458] AE (adverse reaction) is defined as any adverse medical event that occurs in clinical research patients with a pharmaceutical product that is not necessarily causally related to the treatment. Therefore, AE can be any adverse and unexpected sign (including abnormal laboratory findings), symptom or disease that is temporally related to the use of a drug (investigational) product, whether or not it is related to the drug (investigational) product. This includes a significant deterioration of a pre-existing condition or event, a concurrent disease, a drug interaction, or an indication being studied, which is not recorded in a specific efficacy assessment elsewhere in the CRF. Safety evaluations were conducted based on NCI-CTCAE Version 4.03 of July 14, 2010.

[0459] Early results:

[0460] In one patient with gallbladder cancer, v10000 treatment resulted in an approximately 40% reduction in the sum of the longest diameters (SLD) of target lesions as of June 2018. The patient later demonstrated an approximately 50% reduction in target lesions as measured by SOD (sum of diameters).

[0461] As of November 2018, in one CCA patient, an approximately 40% reduction in target lesions as measured by SOD was observed following treatment with v10000.

[0462] Interim results:

[0463] As of April 22, 2019, 89 patients across all indications had been enrolled and treated with single-agent v10000 in both Parts 1 and 2 of v10000 (23 in Part 1 and 66 in Part 2) (Table C). 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 BTC patients, the median number of prior systemic treatment regimens received was 4 (range 1-8), including trastuzumab in one patient.

[0464] In Parts 1 and 2 of the study, most AEs were Grade 1 or 2 in severity (Table D). v10000-related Grade 3 AEs included fatigue (n=3, including one event reported as a SAE), diarrhea (n=2), arthralgia (n=1), and hypophosphatemia (n=1).

[0465] Preliminary efficacy data in BTC patients are presented in Tables E, F, and Figure 2 .

[0466] Table C. Key demographics and baseline characteristics by cancer type in Parts 1 and 2

[0467]

[0468]

[0469] Table D. Summary of v10000-related adverse events by cancer type in Parts 1 and 2

[0470]

[0471] Table E. Summary of best responses in the measurable disease analysis set in Parts 1 and 2

[0472]

[0473] a Measurable disease analysis set—all patients in the safety analysis set with measurable disease according to RECIST 1.1;

[0474] b Safety analysis set—all patients who received at least one dose of study treatment.

[0475] CI = confidence interval, DCR = disease control rate, NE = not evaluable, ORR = overall response rate, PFS = progression-free survival, PR = partial response, SD = stable disease

[0476] Table F. Previous treatment regimens and disease response in BTC patients

[0477]

[0478] a All BTC patients were HER2+ (3+ or FISH+);

[0479] b Patients received trastuzumab concurrently with 6 of 8 systemic therapy regimens;

[0480] c Final DOR pending additional disease evaluation.

[0481] NA = Not Applicable NE = Not Evaluable, awaiting follow-up disease assessment

[0482] GBC = gallbladder cancer; CC = cholangiocarcinoma

[0483] These data showed that v10000 had a disease control rate of 83.3% and an ORR of 66.7% in patients with HER2 3+ or FISH+ BTC.

[0484] Example 3: Preparation of Linker-Toxin 001

[0485] Linker-toxin 001 was prepared as described below. Linker-toxin 001 can also be prepared as described in International Patent Application Publication No. WO 2016 / 041082.

[0486]

[0487] Connector-Toxin 001

[0488] A. Ethyl (2R,3R)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanoate (Compound 1)

[0489]

[0490] 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 anhydrous ethanol (27.0 mL) was added thionyl chloride (3.0 mL) dropwise at 0°C. The resulting solution was allowed to warm to room temperature and 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 under reduced pressure twice, then suspended in diethyl ether (5 mL) and concentrated under reduced pressure twice to give a white solid foam (3.78 g, quantitative yield %). MS m / z observed value = 216.5 (M+1).

[0491] B. (3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoic acid (Compound 3)

[0492]

[0493] Compound 2 was prepared as described in International Patent Application Publication No. WO 2016 / 041082.

[0494] Trifluoroacetic acid (5.0 mL) is added to a solution of stirred compound 2 (6.965 g, 14.14 mmol) in dichloromethane (20 mL). The reaction was monitored by HPLC-MS to complete, and no raw material remained after 40 hours. The reaction was concentrated under reduced pressure, co-evaporated with toluene (2 x 10 mL) and dichloromethane (2 x 10 mL), and a foamy white solid (6.2 g, quantitative yield, containing residual TFA) was obtained. The substance was dissolved in 200 mL of hot 1:3 EtOAc: hexane and allowed to cool to room temperature. During cooling, a precipitate and some small crystals were formed. 5 mL EtOAc was added and the suspension was heated again to completely dissolve the precipitate. More crystals were formed after cooling to room temperature, and the flask was placed at -30 ° C overnight. The mother liquor was decanted the next morning, and the crystals were rinsed with 2 x 50 mL hexane and dried under high vacuum. 5.67 g of crystalline products were recovered. MS m / z observations=405.7 (M+1).

[0495] C. (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid ethyl ester (Compound 4)

[0496]

[0497] To a stirred solution of compound 3 (6.711 g, 15.37 mmol, 1.025 eq) 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 eq) and N,N-diisopropylethylamine (7.84 mL, 3 eq). After stirring at room temperature for 30 minutes, a solution of compound 1 (3.776 g, 15.00 mmol, 1.0 eq) in a mixture of dichloromethane (1.0 mL) and N,N-dimethylformamide (1.0 mL) was added dropwise, and the residual compound 1 was rinsed 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 minutes. The reaction was concentrated under reduced pressure, diluted with ethyl acetate (~125 mL), and the organic phase was extracted with 1M HCl (2x 50 mL), 1x dH2O (1x 50 mL), saturated NaHCO3 (3x 50 mL), brine (25 mL). Both the acidic and basic aqueous layers were washed with 25 mL EtOAc. All organics were then combined and dried over MgSO4, filtered and concentrated to give a red oil. The residue was dissolved in a minimum amount of dichloromethane (~10 mL) and loaded onto SNAPUltra 360g silica gel column (Isolera TM Flash System; Biotage AB, Sweden) (20-100% EtOAc in hexanes over 10 column volumes). Fractions containing pure product were combined to recover 7.9 g of a foamy white solid. The impure fraction was purified a second time on a SNAP Ultra 100 g silica column and combined with the pure product to recover a white foam solid (8.390 g, 88.3%). MS m / z observed = 634.7 (M+1).

[0498] D. (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (Compound 5)

[0499]

[0500] To a stirred solution of compound 4 (8.390 g, 13.24 mmol) in 1,4-dioxane (158 mL) was added dHO (39.7 ml) and lithium hydroxide monohydrate (1M in H2O, 39.7 mL, 3 equivalents). The reaction was stirred at 4 ° C and the consumption of the starting material was monitored by HPLC-MS, which took 3 days until only traces of compound 4 remained. During the reaction, in addition to the desired material, a small percentage of new products was formed, corresponding to the loss of methanol (β-elimination, <2%). The reaction was acidified by adding 1M HCl aqueous solution (50 mL) and concentrated under reduced pressure to remove dioxane. The remaining reaction mixture was extracted with ethyl acetate (4x50 mL), and the organic phase was combined, washed with brine (15 mL+2 mL 2M HCl), dried over MgSO4, filtered and concentrated under reduced pressure to give a light oil. The oil was redissolved in diethyl ether (-50 mL) and concentrated under reduced pressure (3x) to facilitate removal of residual dioxane, affording the title product as a viscous oil (7.81 g, 97% yield, containing some residual dioxane and compound 4). MS m / z observed = 606.7 (M+1).

[0501] 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)

[0502]

[0503] Compound 6 was prepared as described in International Patent Application Publication No. WO 2016 / 041082.

[0504] 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 eq., dissolved in 3 mL DMF), N,N-lutidine (1.867 g, 1.3 eq.) and N,N-dimethylformamide (1.5 mL) to produce a light 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 eq.) was added in a single portion and the reaction was monitored by HPLC-MS. After 48 hours, the reaction did not proceed and an additional 400 mg of EDCI was added. After 18 hours, no residual starting material was observed and the reaction was concentrated under reduced pressure to give a yellow oil. The oil was dissolved in ethyl acetate (~150 mL) and 1M HCl (20 mL), and the organic phase was washed with cold 2M HCl (2 x 10 mL), saturated NaHCO3 (1 x 10 mL), brine (20 mL + 5 mL 2M HCl). The acidic and basic aqueous fractions were extracted with EtOAc (1 x 20 mL), and all organic fractions were combined, dried over MgSO4 and concentrated under reduced pressure to give an oily crude solid (13 g). The residue was dissolved in dichloromethane (~10 mL) and loaded onto Purification was performed on a SNAP Ultra 360 g silica column with a gradient of 10-100% EtOAc (2% AcOH) in hexanes over 12 column volumes, reaching a plateau at 50% EtOAc for 3 column volumes. Fractions containing pure product were combined, concentrated under reduced pressure, dissolved and concentrated from toluene (2 x 10 mL) and diethyl ether (2 x 10 mL) to give the desired product, 7.1 g of a white foamy solid. TM Use on the instrument Impure fractions were purified repeatedly on a SNAP Ultra 100 g silica gel column under shallower gradient conditions. All pure fractions were combined to recover the pure product (8.60 g, 86%) as a white foam solid. MS m / z observed = 856.7 (M+1).

[0505] 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)

[0506]

[0507] Compound 7 (3.71 g, 4.33 mmol) was dissolved in a 10% N,N-dimethylformamide ethyl acetate (30 mL) solution in a round-bottom flask containing a magnetic stirrer and equipped with a three-way gas line joint. The container was evacuated twice under reduced pressure and filled with nitrogen. 10% palladium / carbon (0.461 g, 0.1 equivalent) was added in a single portion, a three-way joint was installed on the flask, a hydrogen balloon was installed on the joint, and the container was evacuated twice under reduced pressure and filled with hydrogen. The reaction was stirred for 2 days, during which the hydrogen balloon was re-inflated from time to time. After about 48 hours, HPLC-MS analysis showed that no raw material remained. The reaction was diluted with methanol (20 mL) and filtered through a diatomaceous earth plug. Diatomaceous earth was washed with methanol (2x 50 mL). All 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%). MS m / z observed = 722.6 (M+1).

[0508] G. (S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-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-dimethylbutyramide (Compound 8)

[0509]

[0510] HATU (3.216 g, 8.46 mmol) and diisopropylethylamine (3.10 mL, 17.8 mmol) were added to a stirred solution of N,N-(L)-dimethylvaline (1.696 g, 9.35 mmol) in N,N-dimethylformamide (10 mL). A clear yellow solution was obtained after 5 minutes. Stirring was continued for another 10 minutes, and then compound 7a (3.213 g, 4.45 mmol) was added in a single portion. After stirring for another 1 hour, HPLC-MS showed that traces of compound 7a remained and the reaction lasted for 16 hours. The reaction was then concentrated under reduced pressure, diluted with ethyl acetate (120 mL) and 40 mL 1:1 NaHCO3 (saturated): 5% LiCl and transferred to a separatory funnel. The water layer was removed and the organic phase was washed with LiCl (1 x 20 mL), NaHCO3 (saturated, 2 x 20 mL). The water layers were combined and extracted with EtOAc (3 x 50 mL). The organic layers were combined and washed with brine (1 x 20 mL), dried over sodium sulfate, filtered and concentrated to give an oil laden with DMF, which was concentrated via rotary evaporator to remove residual DMF to give 7 g of a crude straw colored oil. The oil was dissolved in a small amount of 10% methanol in dichloromethane (~11 mL) and loaded onto Purification was performed on a SNAP Ultra 360 g silica column (2-20% MeOH in CH2Cl2 over 15 column volumes, product eluted at about 10-13%). Fractions containing the desired product were combined and concentrated under reduced pressure to give the title compound as a colorless foam. Impure fractions were combined, evaporated and concentrated in Isolera TM On the instrument The purification was repeated on a SNAP Ultra 100 g silica column and combined with the pure product from the first column to give a colorless foamy solid (3.78 g). MS m / z observed = 850.6 (M+1).

[0511] 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)

[0512]

[0513] 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 1M lithium hydroxide monohydrate (3 equivalents, 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 1M HCl and concentrated under reduced pressure to remove dioxane. The resulting aqueous phase was diluted with 60 mL of EtOAc and 5 mL of brine, then extracted with ethyl acetate (2 x 30 mL). The organic fractions were combined, dried over Na2SO4, filtered and evaporated to give the title compound (0.930 g) as a tan solid. f = 0.5 (8% MeOH in CH2Cl2). MS m / z observed = 753.7 (M+1).

[0514] I. 2,3,5,6-tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate (Compound 15)

[0515]

[0516] 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 hours, at which time it was cooled to 0°C and isocollidine (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 minutes, at which time isocollidine (2.407 mL, 4 eq) was added dropwise. The flask was stirred for another 15 minutes, and then the contents were added dropwise to the first flask via a syringe. The reaction was allowed to warm to room temperature and continued to stir under N2. The reaction was monitored for consumption of the starting materials by HPLC-MS. After 6 days, the reaction was complete and compound 14 was completely consumed, leaving only compound 15 and a small amount (~5%) of the bis-TFP maleamide intermediate. The reactants were transferred to a separatory funnel, diluted with diethyl ether (75 ml) and washed with 5% LiCl (1x 20 mL), 1M HCl (2x 20 mL), saturated NaHCO3 (5x 20 mL) and brine (1x 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 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. Pure fractions were combined and diluted with brine (20 mL) and then extracted with 3x 50 mL Et2O. The combined organics were dried over MgSO4, filtered and evaporated to recover a light yellow oil (1.34 g, 66% yield).

[0517] J. tert-Butyl ((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)sulfamoyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (Compound 12)

[0518]

[0519] Compound 11 was prepared as described in International Patent Application Publication No. WO 2016 / 041082.

[0520] 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.) were added to an empty 25 mL pear-shaped flask. These solids were dissolved in a mixture of N,N-dimethylformamide (0.5 mL) and dichloromethane (4.5 mL) at room temperature under stirring for 30 minutes. 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 a pear-shaped flask, rinsed with dichloromethane (1.0 mL). The stirring rate was increased to 1000 rpm to generate a vortex. Within 2 minutes of the addition of compound 9, copper (II) chloride (0.514 g, 3.83 mmol, 3.2 eq) was added in one portion directly through a narrow powder funnel into the center of the vortex. The initial light yellow solution became a dark brown suspension, which became a dark green suspension over 10 minutes. The completion of the reaction was monitored by HPLC-MS, and no change in the progress of the reaction was observed between samples collected at 30 minutes and 1 hour (~95% completion). The reaction was stirred overnight at room temperature, and then 2-(2-aminoethylamino)ethanol (0.483 mL, 4.781 mmol, 4 eq), EtOAc (10 mL) and dH2O (5 mL) were added to the stirred suspension, which experienced a color change to dark blue. The suspension was stirred vigorously for 4 hours as the suspended solids gradually dissolved into the biphasic mixture. 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 (4x50 mL). The organic layers were combined and 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 Purify on SNAP Ultra 100g silica column (2-20% MeOH in CH2Cl2 over 10 column volumes, followed by 8 column volumes at 20% MeOH to reach a plateau). The product eluted as a broad peak after 1-2 column volumes of ~20% MeOH in CH2Cl2. Fractions containing the desired material were combined and concentrated under reduced pressure to give the title compound (1.105 g, 83%) as a white solid. MS m / z observed = 555.9 ((M+2) / 2), 1109.8 (M+1).

[0521] K. (S)-2-((S)-2-amino-3-methylbutyramido)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-5-ureidopentanamide (Compound 13)

[0522]

[0523] A mixture of dichloromethane (10 mL) and trifluoroacetic acid (2.0 mL) was added to a solution of compound 12 (0.926 g, 0.834 mmol). The reaction was monitored by HPLC-MS for consumption of raw materials (~45 minutes). The reactant was co-evaporated with acetonitrile (2x 10 mL) and dichloromethane (2x 10 mL) under reduced pressure to remove excess trifluoroacetic acid. The resulting residue was dissolved in a minimum amount of dichloromethane and methanol (3:1, v / v, ~2 mL) and added dropwise to a stirred solution of diethyl ether (200 mL) and hexane (100 mL) via a pipette to produce a suspension of a light white solid. The solid was filtered and dried in vacuo to obtain the title compound as a trifluoroacetate in the form of a white powder (1.04 g, quantitative yield, containing some residual solvent). MS m / z observed value = 505.8 ((M+2) / 2).

[0524] L. (S)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-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-ureidopentanamide (Linker-Toxin 001)

[0525]

[0526] 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 eq) and diisopropylethylamine (0.305 mL, 3.0 eq). HPLC-MS analysis at 2 h indicated no residual starting material. The reaction was acidified with TFA (300 μL) and then diluted with diH2O + 0.1% TFA (9 mL). The resulting solution was loaded onto 120 g Purified on a SNAP Ultra C18 column (Biotage, Uppsala, Sweden) and 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. Pure fractions identified by LCMS were combined and lyophilized. A white powder solid was recovered from the lyophilizer. Lyophilization was repeated into vials at a higher concentration (approximately 50 mg / mL in 2:1 H2O / ACN) to produce a denser, less flocculated lyophilized solid (754.2 mg, 91%). MS m / z observed = 647.4 ((M+2) / 2), 1292.8 (M+1).

[0527] Example 4: Preparation of v10000 conjugated with linker-toxin 001

[0528] A solution of antibody v10000 (2.0 g) in 10 mM sodium acetate, 9% (w / v) sucrose pH 4.5 (138.9 mL) was pH adjusted by adding 200 mM Na2HPO4 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 disulfides was initiated by adding 10 mM TCEP aqueous 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. After 90 minutes, the conjugation reaction was quenched by adding an excess of 20 mM N-acetylcysteine ​​solution (4.81 mL; 6 eq).

[0529] Quenched Antibody Drug Conjugate (ADC) Solution at Millipore Labscale TM Used on tangential flow filtration instruments XL ultrafiltration module ( 30kDa 0.005m 2; Millipore Sigma) was purified with 9-15 volumes of 10 mM sodium acetate, 9% (w / v) sucrose pH 4.5. The eluted ADC was sterile filtered (0.22 um). Small-scale production of ADC was carried out on a 40 KDa MWCO Zeba TM The protein was purified on a column (ThermoFisher Scientific, Waltham, MA) preconditioned with PBS or 10 mM sodium acetate, 9% (w / v) sucrose, pH 4.5.

[0530] After purification, the concentration of ADC was determined by BCA assay with reference to the standard curve generated from v10000. Alternatively, the concentration of ADC was determined by measuring the 280 nm (ε = 195065 M -1 cm -1 ) to estimate the concentration.

[0531] Samples of ADC were evaluated by non-reducing and reducing SDS-PAGE. No extraneous bands were observed.

[0532] Antibodies and ADCs were analyzed by hydrophobic interaction chromatography (HIC) to estimate the drug / antibody ratio (DAR). Chromatography was performed on a HIC Ethyl column (7.8 x 50 mm, 5 μm) (Sepax Technologies Inc., Newark, DE) using a gradient of 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).

[0533] The average drug / antibody ratio (DAR) of an ADC can vary depending on the number of disulfide bonds released during antibody reduction. A single conjugation reaction that produces 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.

[0534] The results of HIC showed that the ADC containing v10000 conjugated with linker-toxin001 had an average DAR of 2.07. The individual contributions of 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 content % of the individual DAR species of each variant (determined by HIC) is shown in Table G.

[0535] Table G: DAR distribution of ADCs containing v10000 and linker-toxin001

[0536] DAR area% 0 23 2 56 4 17 6 4

Claims

1. Use of a bispecific anti-HER2 antigen-binding construct in the preparation of a medicament for treating biliary tract cancer (BTC), wherein the bispecific anti-HER2 antigen-binding construct comprises a heavy chain H1, a heavy chain H2 and a light chain L1, and 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.

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

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 a transfer BTC.

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

6. The use according to any one of claims 1 to 3, wherein the BTC is HER2 3+, HER2 2+ or HER2 1+ without HER2 gene amplification as measured by immunohistochemistry (IHC).

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 heavy chain H1 comprises the amino acid sequence shown in SEQ ID NO: 36, the heavy chain H2 comprises the amino acid sequence shown in SEQ ID NO: 63, and the light chain L1 comprises the amino acid sequence shown in SEQ ID NO:

24.

10. The use according to any one of claims 1, 2, 3, 7, 8 or 9, wherein the effective amount of the bispecific anti-HER2 antigen binding construct is 10 mg / kg per week.

11. The use according to any one of claims 1, 2, 3, 7, 8 or 9, wherein the effective amount of the bispecific anti-HER2 antigen binding construct is 20 mg / kg every two weeks.

12. The use according to any one of claims 1, 2, 3, 7, 8 or 9, wherein the effective amount of the bispecific anti-HER2 antigen binding construct is 30 mg / kg every three weeks.

13. The use of any one of claims 1, 2, 3, 7, 8 or 9, wherein administration of the bispecific anti-HER2 antigen-binding construct to the subject induces a complete response (CR), a partial response (PR) or stable disease (SD) in the subject.

14. The use of any one of claims 1, 2, 3, 7, 8 or 9, wherein the disease control rate in a group of subjects treated with the bispecific anti-HER2 antigen-binding construct is greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

15. The use of any one of claims 1, 2, 3, 7, 8 or 9, wherein the overall response rate in a group of subjects treated with the bispecific anti-HER2 antigen binding construct is greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

16. The use according to any one of claims 1, 2, 3, 7, 8 or 9, wherein the bispecific anti-HER2 antigen binding construct is administered after at least one, two or three prior therapies.

17. The use according to any one of claims 1, 2, 3, 7, 8 or 9, wherein the bispecific anti-HER2 antigen binding construct is administered as a first-line monotherapy.

18. The use according to any one of claims 1, 2, 3, 7, 8 or 9, wherein the bispecific anti-HER2 antigen binding construct is administered as adjuvant therapy or neoadjuvant therapy.

19. The use according to any one of claims 1, 2, 3, 7, 8 or 9, wherein the bispecific anti-HER2 antigen binding construct is administered in combination with one or more chemotherapeutic agents.

20. The use according to claim 19, wherein the one or more chemotherapeutic agents are gemcitabine and / or cisplatin.

21. The use according to any one of claims 1-3, wherein the BTC is FISH+ or FISH-.

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