Effective dose of HER2 bispecific antibody

Abstract

The present application provides a method for treating tumors, comprising administering a HER2 bispecific antibody at a dose of 15 mg / kg to 35 mg / kg, comprising a first light chain and a second light chain, a first heavy chain and a second heavy chain, wherein the variable region of the light chain has an amino acid sequence set forth in any of SEQ ID NOs: 1-6.

Description

[Background technology]

[0001] The HER2 protein is a type I transmembrane growth factor receptor tyrosine kinase. This HER2 protein mediates signaling pathways related to biological functions such as cell proliferation, apoptosis regulation, and angiogenesis and lymphotubulation. HER2 positivity accounts for approximately 15–20% of breast cancers. Nevertheless, patients are forced to endure progressive disease, necessitating the development of new drugs. The clinical application of bispecific antibodies can be challenging due to variations in target engagement and differences between preclinical and clinical tumors. Developing population pharmacokinetic (PK)-tumor growth models within a modeling framework helps understand the relationships between drug exposure, pharmacodynamics, and tumor response, providing a tool for optimizing clinical dose selection.

[0002] Furthermore, the search for the optimal dose of HER2 bispecific antibodies in humans is urgent and necessary. [Disclosure of the Invention]

[0003] The present invention provides a method for treating breast cancer or inhibiting breast tumor growth in subjects in need, comprising administering a bispecific antibody to such subjects in doses of 15 mg / kg to 35 mg / kg. The present invention also provides a formulation and a drug delivery device used in the above formulation.

[0004] On the other hand, the present invention provides a method for use in the prevention, palliative or therapeutic of tumors or the inhibition of tumor growth in a subject, comprising administering a HER2 bispecific antibody to the subject in a dose of approximately 15 mg / kg to approximately 35 mg / kg, wherein the HER2 bispecific antibody comprises a first light chain, a second light chain, a first heavy chain, and a second heavy chain, wherein the first light chain and the second light chain can assemble with the heavy chain of pertuzumab and the heavy chain of trastuzumab, respectively, and the variable region of the first light chain and / or the second light chain has an amino acid sequence represented by SEQ ID NO: 1-6.

[0005] In some embodiments, the variable region of the first light chain and / or the second light chain has an amino acid sequence represented by SEQ ID NO: 1.

[0006] In some embodiments, the first light chain and the second light chain are each selected from the light chain of pertuzumab or its variant and the light chain of trastuzumab or its variant.

[0007] In some embodiments, the first light chain has an amino acid sequence represented by any one of SEQ ID NOs: 7-12, and / or the second light chain has an amino acid sequence represented by any one of SEQ ID NOs: 7-12.

[0008] In some embodiments, the heavy chain variable regions are the heavy chain variable region of pertuzumab and the heavy chain variable region of trastuzumab, respectively.

[0009] In some embodiments, the variable region of the first heavy chain has an amino acid sequence represented by SEQ ID NO: 13, and the variable region of the second heavy chain has an amino acid sequence represented by SEQ ID NO: 14.

[0010] In some embodiments, the first heavy chain and the second heavy chain include a constant region derived from a human IgG constant region.

[0011] In some embodiments, the Fc fragment sequence of the first heavy chain or the second heavy chain has a sequence represented by any one of SEQ ID NOs: 19-49, 51-52.

[0012] In some embodiments, the first heavy chain or the second heavy chain has a sequence represented by any one of SEQ ID NOs: 15-18.

[0013] In some embodiments, the above dose is approximately 20 mg / kg to approximately 30 mg / kg.

[0014] In some embodiments, the above dose is approximately 20 mg / kg.

[0015] In some embodiments, the above dose is approximately 30 mg / kg.

[0016] In some embodiments, the above-mentioned HER2 bispecific antibody is administered once every two weeks or once every three weeks.

[0017] In some embodiments, the above-mentioned HER2 bispecific antibody is administered at a dose of approximately 20 mg / kg once every two weeks.

[0018] In some embodiments, the above-mentioned HER2 bispecific antibody is administered at a dose of approximately 30 mg / kg once every three weeks.

[0019] In some embodiments, the subjects described above do not respond to conventional therapies for HER2-related tumors.

[0020] In some embodiments, conventional therapies for the above-mentioned HER2-related tumors include administering HER2-ADCs, MBC hormones, taxanes, pirotinib, neratinib, tucatinib, trastuzumab, and / or pertuzumab.

[0021] In some embodiments, the conventional therapy for the above-mentioned HER2-related tumors includes administering docetaxel, capecitabine, and / or lapatinib.

[0022] In some embodiments, the above-mentioned tumor includes a solid tumor.

[0023] In some embodiments, the tumors described above include metastatic tumors, early-stage tumors, and / or locally advanced tumors.

[0024] In some embodiments, the tumors described above include HER2-positive tumors and / or HER2-low-expressing tumors.

[0025] In some embodiments, the tumors described above include breast cancer and / or gastric cancer.

[0026] In some embodiments, the breast cancers described above include HER2-positive breast cancers and / or HER2-low-expressing breast cancers.

[0027] In some embodiments, the breast cancer described above includes early-stage breast cancer, locally advanced breast cancer, and / or metastatic breast cancer, and / or the gastric cancer described above includes early-stage gastric cancer, locally advanced gastric cancer, and / or metastatic gastric cancer.

[0028] In some embodiments, the HER2 bispecific antibody described above is administered intravenously.

[0029] On the other hand, the present application provides a formulation for use in subjects requiring it to prevent, alleviate, or treat tumors or inhibit tumor growth, comprising at least 5 μg / mL of a HER2 bispecific antibody, wherein the HER2 bispecific antibody comprises a first light chain, a second light chain, a first heavy chain, and a second heavy chain, wherein the first and second light chains can assemble with the heavy chain of pertuzumab and the heavy chain of trastuzumab, respectively, and the variable regions of the first and / or second light chains have sequences represented by SEQ ID NO: 1-6.

[0030] In some embodiments, the variable region of the first light chain and / or second light chain in the above formulation has an amino acid sequence represented by SEQ ID NO: 1.

[0031] In some embodiments, the first and second light chains in the above formulation are selected from pertuzumab or a variant thereof, and trastuzumab or a variant thereof, respectively.

[0032] In some embodiments, the above formulation has an amino acid sequence represented by SEQ ID NO: 7-12, and / or the above second light chain has an amino acid sequence represented by SEQ ID NO: 7-12.

[0033] In some embodiments, the heavy chain variable regions in the above formulation are the heavy chain variable region of pertuzumab and the heavy chain variable region of trastuzumab, respectively.

[0034] In some embodiments, the above formulation has an amino acid sequence represented by SEQ ID NO: 13 in the variable region of the first heavy chain, and an amino acid sequence represented by SEQ ID NO: 14 in the variable region of the second heavy chain.

[0035] In some embodiments, the above-described formulation includes a constant region derived from the human IgG constant region, where the first and second heavy chains comprise a constant region.

[0036] In some embodiments, the above formulation has a heavy chain Fc fragment sequence represented by SEQ ID NO: 19-49 or 51-52.

[0037] In some embodiments, the above formulation has a sequence represented by SEQ ID NO: 15-18, where the first or second heavy chain is one of the sequences represented by SEQ ID NO: 15-18.

[0038] In some embodiments, the above formulation contains at least about 12 μg / mL of bispecific antibody.

[0039] In some embodiments, the above formulation contains at least about 20 μg / mL of bispecific antibody.

[0040] In some embodiments, the above-described formulation is packaged in a container. On the other hand, the present invention provides a drug delivery device for use in subjects requiring it to prevent, alleviate, or treat tumors or inhibit tumor growth, comprising a formulation containing at least 5 μg / mL of HER2 bispecific antibody, wherein the HER2 bispecific antibody comprises a first light chain, a second light chain, a first heavy chain, and a second heavy chain, the first and second light chains being able to assemble with the heavy chain of pertuzumab and the heavy chain of trastuzumab, respectively, and the variable regions of the first and / or second light chains having an amino acid sequence represented by SEQ ID NO: 1-6.

[0041] In some embodiments, in the drug delivery device described above, the variable region of the first light chain and / or the second light chain has an amino acid sequence represented by SEQ ID NO: 1.

[0042] In some embodiments, in the drug delivery device described above, the first light chain and the second light chain are selected from a light chain of pertuzumab or a variant thereof, and a light chain of trastuzumab or a variant thereof, respectively.

[0043] In some embodiments, in the drug delivery device described above, the first light chain has an amino acid sequence represented by SEQ ID NO: 7-12, and / or the second light chain has an amino acid sequence represented by SEQ ID NO: 7-12.

[0044] In some embodiments, in the drug delivery device described above, the heavy chain variable regions are the heavy chain variable region of pertuzumab and the heavy chain variable region of trastuzumab, respectively.

[0045] In some embodiments, the drug delivery device described above has an amino acid sequence represented by SEQ ID NO: 13 in the variable region of the first heavy chain, and an amino acid sequence represented by SEQ ID NO: 14 in the variable region of the second heavy chain.

[0046] In some embodiments, in the drug delivery device described above, the first and second heavy chains include a constant region derived from a human IgG constant region.

[0047] In some embodiments, the drug delivery device described above has a heavy chain Fc fragment sequence represented by SEQ ID NO: 19-49 or 51-52.

[0048] In some embodiments, the drug delivery device described above has a sequence represented by one of SEQ ID NO: 15-18.

[0049] In some embodiments, the above formulation contains at least about 12 μg / mL of bispecific antibody.

[0050] In some embodiments, the above formulation contains at least about 20 μg / mL of bispecific antibody.

[0051] In some embodiments, the above-described formulation is packaged in a container.

[0052] Additional aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present application. As realized, other different embodiments of the present application are possible, and some of their details can be modified in various obvious ways without departing from the present disclosure. Accordingly, the accompanying drawings and description are to be considered exemplary in nature and not restrictive. Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that individual publications, patents, and patent applications are specifically and individually represented so as to be incorporated by reference. [Brief explanation of the drawing]

[0053] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as “Figures” and “FIG”) illustrating exemplary embodiments in which the principles of the present invention are employed, among which, [Figure 1] Figure 1 shows the study of the HER2 bispecific antibody described above and the tumor volume dynamics in a xenograft model at different dose levels. [Figure 2] Figure 2 shows the goodness-of-fit (GOF) of tumor inhibition models for NCI-N87 and Calu-3 xenograft models. [Figure 3] Figures 3A-3D show the dynamics of tumor size in humans under different exposures to the HER2 bispecific antibodies described above, as predicted by translational models. [Figure 4] Figure 4A shows the dose-normalized concentrations of the above-mentioned HER2 bispecific antibody within administration cycle 1 in this application. Figure 4B shows the goodness-of-fit plot of the population PK model. [Figure 5] Figure 5 shows a comparison of the predicted change in SLD from baseline with different dosing regimens. [Figure 6] Figure 6 shows a structural model for constructing the exposure-SLD relationship in humans for the HER2 bispecific antibody described above. [Figure 7] Figure 7 shows the SLD data used in the intermediate ER analysis. [Figure 8] Figure 8 shows the tumor growth inhibition (TGI) study for constructing the above-mentioned HER2 bispecific antibody for PK-PD in this application. [Figure 9] Figure 9 shows the changes in tumor growth from baseline in the xenograft model obtained from the study. [Figure 10] Figure 10 shows the changes in tumor growth from baseline with respect to the administration level of the HER2 bispecific antibody described above in this application. [Figure 11]Figure 11 shows the changes in tumor growth from baseline with the above-described HER2 bispecific antibody administration regimen. [Figure 12] Figure 12 shows the tumor volume-dependent target concentrations of the HER2 bispecific antibody described above in this application in mice. [Figure 13] Figure 13 shows a comparison of Ctrough concentrations between candidate administration regimens. [Figure 14] Figure 14 shows the evaluable patient response and duration of response as shown in swimmer plots for each dose level. [Figure 15] Figures 15A-15B show the tumor response of all evaluable patients, as shown in waterfall plots and spider plots, by dose level. [Figure 16] Figures 16A–16F show pre- and post-treatment scans of patients with recurrent breast cancer who achieved a partial response. [Modes for carrying out the invention]

[0054] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are merely examples. Those skilled in the art will conceive of various modifications, alterations, and substitutions, provided they do not depart from the present invention. It should be understood that various substitutional forms may be applied to the embodiments of the present invention described herein.

[0055] In this application, the term “HER2” as used herein generally refers to a type I transmembrane protein belonging to the epidermal growth factor receptor family, also known as c-erbB2, ErbB2, or Neu. In the context of this application, the term “HER2” above also includes isoforms, including homologs, mutants, and splice isoforms of HER2. The term “HER2” above also includes proteins having one or more sequences of HER2 homologs, mutants, and isoforms and fragments of such sequences, provided that the mutant protein (including isoforms), homologous protein, and / or fragments are recognized by one or more HER2-specific antibodies such as pertuzumab, trastuzumab, and margetsuzumab. The HER2 above may be human HER2. The human HER2 gene above is mapped to chromosome position 17q12, and the genomic sequence of the HER2 gene above is listed in GenBank NG-007503.1. In humans, there are five HER2 isoforms: A, B, C, D, and E. The term "HER2" used herein is used to refer collectively to all HER2 isoforms.

[0056] In this application, the term “antibody” as used herein generally means immunoglobulin or a fragment or derivative thereof, encompassing any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, mutant, and transplant. For the purposes of this disclosure, unless the term “intact” is otherwise modified, such as “intact antibody,” the term “antibody” also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, and dAb, as well as antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind to HER2. Typically, such fragments include an antigen-binding domain.

[0057] In this application, the term “bispecific antibody” refers to an antibody capable of binding to two different antigens or their antigenic epitopes. For example, the above bispecific antibody may comprise at least one light chain or fragment thereof, and at least one heavy chain or fragment thereof. For example, the above bispecific antibody may comprise one light chain or fragment thereof capable of specifically binding to both a first antigen or its antigenic epitope and a second antigen or its antigenic epitope. For example, the above bispecific antibody may comprise two heavy chains or fragment thereof capable of binding to a first antigen or its antigenic epitope and a second antigen or its antigenic epitope, respectively. For example, the above first antigen or its antigenic epitope and the second antigen or its antigenic epitope may be two different HER2 antigens.

[0058] In this application, the terms “HER2-positive” or “HER2-enhanced” as used herein generally refer to tumors containing cells on which the HER2 protein is present on the cell surface. The HER2 protein may be overexpressed, for example, by gene amplification. Solid tumors that overexpress HER2 can be biochemically determined by an immunohistochemical score according to the copy number of HER2 molecules expressed per cell (see Hudziak et al., Proc. Natl. Acad. Sci. USA 84:7159-7163

[1987] ). For example, HER2-positive solid tumors may include HER2-positive breast cancer. The HER2-positive breast cancer may be estrogen receptor positive and may be HER2-non-amplified invasive breast cancer. The HER2-positive breast cancer may be progressive. The HER2-positive breast cancer may be metastatic.

[0059] In this application, the term "HER2 low expression" refers to tumors containing cells that express very low levels of HER2. The above HER2 low expression is IHC 1 + or 2 + and FISH -This may refer to HER2-negative tumors that are being tested for. HER2 expression levels can be measured by immunohistochemistry or FISH. For example, a group with low HER2 levels is likely to be high-grade EGFR-positive and ER / HER3 / HER4-negative.

[0060] In this application, the term "solid tumor" generally refers to an abnormal mass of tissue that does not contain a fluid area. Such solid tumors may be malignant or cancerous. Different types of solid tumors are named according to the type of cells that form them. For example, the above-mentioned solid tumors may include breast cancer.

[0061] In this application, the term “metastatic” means that a tumor has spread from its place of origin to another part of the body. Many types of tumors are also called stage IV (4) tumors. Metastatic tumors develop when tumor cells detach from the primary tumor and enter the bloodstream or lymphatic system. For example, breast cancer that has metastasized to the lungs is sometimes called metastatic breast cancer.

[0062] In this application, the term “early tumor” refers to a tumor that has not spread deeply into neighboring tissues. Such early tumors may also be called early cancers and / or stage I (1) tumors. These early tumors may not have spread very far.

[0063] In this application, the term "locally advanced tumor" refers to a tumor that has grown outside the site of origin and has not yet metastasized to other sites. For example, locally advanced breast cancer may be a subset of breast cancer characterized by the most advanced breast tumors without distant metastasis.

[0064] In this application, the term “treatment” as used herein generally refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment may include prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or mitigation of the disease state, remission, or improvement of prognosis. For example, the HER2 bispecific antibody described above is used to delay the onset of the disease or to slow the progression of the disease.

[0065] In this application, the term “prevent” as used herein generally means delaying the onset of, preventing the progression of, preventing the appearance of, defending against, suppressing or eliminating the appearance of, or reducing the incidence of any injury, effect, or symptom of a disease or disorder.

[0066] In this application, the term “alleviate” as used herein generally refers to the process by which the severity of the signs or symptoms of a disorder is reduced. Alleviation may include the alleviation, but not elimination, of the signs or symptoms of a disease or disorder.

[0067] In this application, the term “subject” as used herein generally refers to an animal, such as a human. For example, the above subjects may include “non-human animals” consisting of mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates.

[0068] In this application, the term “conventional treatments for HER2-related tumors” as used herein generally means administering any substance or agent that blocks HER2-related tumor growth. Conventional treatments for HER2-related tumors can interfere with the function of certain molecules involved in the growth and survival of HER2-related (e.g., HER2-positive and / or HER2-low-expressing) tumor cells. The above conventional treatments for HER2-related tumors may include any approved agents specific to the treatment of HER2-related tumors (e.g., HER2-related tumors may be solid tumors, and e.g., HER2-related tumors may be at any stage). The above conventional treatments for HER2-related tumors may include first-line and / or second-line agents approved for the treatment of HER2-related tumors (e.g., those approved for the treatment of HER2-positive breast cancer). Conventional treatments for HER2-related tumors include any approved agents suitable for the treatment of HER2-related tumors, such as agents used in general oncology treatments such as chemotherapy.

[0069] In this application, the term “trastuzumab” as used herein generally refers to a whole-human HER2 monoclonal antibody used to treat breast cancer and gastric cancer. Its trade names are Herceptin, Herzma, or Ogibri. The trastuzumab described above may be used particularly for HER2 receptor-positive cancers.

[0070] In this application, the term “MBC hormone” as used herein generally refers to hormone therapy for the treatment of breast cancer. In some embodiments, the hormone therapy may prevent hormones (e.g., estrogen or progesterone) from binding to receptors within breast cancer cells. For example, the hormone therapy may include administering tamoxifen and / or toremifene.

[0071] In this application, the term “taxane” as used herein generally refers to a type of diterpene. The taxane described above is also used in the treatment of metastatic breast cancer. The CAS number of the taxane described above may be 1605-68-1. The taxane may have the following formula:

[0072] [ka]

[0073] In this application, the term “HER2-ADC” as used herein generally refers to an antibody-drug conjugate that targets HER2 and is capable of binding to HER2 on the surface of tumor cells. For example, the above HER2-ADC may include trastuzumab emtansine (T-DM1), which may be indicated for the treatment of HER2-positive metastatic breast cancer. For example, the above HER2-ADC may include trastuzumab deruxtecan (Ds-8201a), which may be indicated for the treatment of adult patients with unresectable or metastatic HER2-positive breast cancer. For example, the above HER2-ADC may include SYD985, in which trastuzumab is linked via a cleavable linker to the duocalmycin prodrug seco-ducalmycin-hydroxybenzamide-azaindole or seco-duvá.

[0074] In this application, the term “pirotinib” as used herein generally refers to an irreversible bimodal pan-ErbB receptor tyrosine kinase inhibitor. The above-mentioned pirotinib may target EGFR, HER2, and HER4. The above-mentioned pirotinib may be used for the treatment of HER2-positive advanced solid tumors. Pilotinib brasemiform is a racemic mixture of pirotinib and is a compound having the following formula:

[0075] [ka]

[0076] In this application, the term “neratinib” as used herein generally refers to a tyrosine kinase inhibitor. The above-mentioned neratinib may be used as an adjuvant therapy for adults with early-stage hormone receptor-positive HER2-overexpressing / amplifying breast cancer. The above-mentioned neratinib is a compound having the following formula:

[0077] [ka]

[0078] In this application, the term "tucatinib" as used herein generally refers to a small molecule inhibitor of HER2. The tucatinib described above may be used for advanced, unresectable, or metastatic HER2-positive breast cancer. The tucatinib described above is a compound having the following formula:

[0079] [ka]

[0080] In this application, the term “pertuzumab” as used herein generally refers to a monoclonal antibody used for the treatment of HER2-positive breast cancer. The amino acid sequences of the variable light chain and variable heavy chain of the above-mentioned pertuzumab (OMNITARG®) can be found in WO2006033700A2. In this application, the term "trastuzumab" as used herein generally refers to a monoclonal antibody (trade names: Harcron, Herceptin) that interferes with the HER2 / neu receptor (Hudis, 2007, N. Engl. J. Med. 3577(1):39-51).

[0081] In this application, the term "docetaxel" as used herein generally refers to the active ingredient of TAXOTERE® or TAXOTERE® itself. The above-mentioned docetaxel is a compound having the following formula:

[0082] [ka]

[0083] In this application, the term “capecitabine” as used herein generally refers to a chemotherapeutic agent that is a prodrug converted to 5-FU in tissues. The chemical name of the capecitabine described above is pentyl[1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidine-4-yl]carbamate.

[0084] In this application, the term “lapatinib” as used herein generally refers to an orally active drug for breast cancer and other solid tumors. It is a bityrosine kinase inhibitor that blocks the HER2 / neu and epidermal growth factor receptor (EGFR) pathways. It functions as a bireversible TKI for these receptors, thus blocking the downstream MAPK / Erk1 / 2 and PI3K / AKT pathways. The lapatinib described above is a compound having the following formula:

[0085] [ka]

[0086] In this application, the term “formulation” as used herein generally refers to a composition comprising the HER2 bispecific antibody of this application. For example, the above formulation may further comprise one or more pharmaceutically acceptable excipients. For example, the above pharmaceutically acceptable excipients may include those described in Royal Pharmaceutical Society of Great Britain, Science & Practice Publishers 4th Edition or Remingtons: The Science and Practice of Pharmacy (19th Edition, Mack Publishing Company).

[0087] In this application, the term “drug delivery device” as used herein generally refers to a device comprising the formulation described herein. In this application, the drug delivery device is capable of delivering the HER2 bispecific antibody described above to tumor sites and / or other desired sites in a subject.

[0088] In this application, the term “one / kind” as used herein is not generally intended to be singular. In some embodiments, the term “one / kind” may refer to a plural. Where used throughout this disclosure, the singular “one / kind” and “the” include plural references unless the context explicitly indicates otherwise.

[0089] In this application, the term “about” as used herein generally means variation within the normal acceptable range in the art, and generally means variation within 10% of the stated value, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%. Unless otherwise evident from the context, all numerical values ​​provided herein are modified by the term “about.”

[0090] In one embodiment, the present invention provides a method for preventing, mitigating, or treating a tumor in a subject or inhibiting tumor growth, comprising administering to the subject about 15 mg / kg to about 35 mg / kg of a HER2 bispecific antibody, wherein the HER2 bispecific antibody comprises a first light chain, a second light chain, a first heavy chain, and a second heavy chain, wherein the first and second light chains can assemble with the heavy chain of pertuzumab and the heavy chain of trastuzumab, respectively, and the variable regions of the first and / or second light chains have an amino acid sequence represented by SEQ ID NO: 1-6.

[0091] For example, the HER2 bispecific antibody described above may be a bispecific antibody or its antigen-binding portion, and the bispecific antibody or its antigen-binding portion may include a first light chain and a second light chain having the same amino acid sequence. For example, the bispecific antibody or its antigen-binding portion may include a common light chain because the first and second light chains have the same amino acid sequence.

[0092] For example, the common light chain described above may be engineered from two different original monoclonal antibodies capable of binding to different epitopes of human HER2, respectively. In some cases, the common light chain described above may also be derived from the light chain of either of the two original monoclonal antibodies. In some cases, the common light chain described above may be modified based on the light chain of either of the two original monoclonal antibodies.

[0093] For example, the above modifications may include insertions, deletions, and / or substitutions at at least one amino acid position in the amino acid sequence of the light chain of either of the two original monoclonal antibodies. In some cases, the purpose of the modification is to maintain affinity between the bispecific antibody or its antigen-binding moiety and the corresponding epitope.

[0094] In this application, the constant light chain region of the bispecific antibody or its antigen-binding portion may be of the κ type, which may include various allotypes such as Km1, Km2, and Km3, or the λ type, which may include various allotypes such as CL1, CL2, CL3, CL6, and CL7.

[0095] In this application, the HER2 bispecific antibody may be a bispecific antibody or its antigen-binding moiety, and the bispecific antibody or its antigen-binding moiety may have a first heavy chain and a second heavy chain.

[0096] In this application, the first and second heavy chains described above can be correctly assembled with the light chain, respectively, under physiological conditions or during protein expression in vitro.

[0097] For example, the first and second light chains described above can be combined with the heavy chain of pertuzumab and the heavy chain of trastuzumab, respectively.

[0098] For example, the variable regions of the first and / or second light chains described above may have an amino acid sequence represented by SEQ ID NO:1.

[0099] For example, the first and second light chains described above may be selected from the light chains of pertuzumab or its variants, and the light chains of trastuzumab or its variants, respectively.

[0100] For example, the variable regions of the first light chain and the second light chain described above may be the variable regions of the trastuzumab light chain.

[0101] For example, the first and second light chains described above may have amino acid sequences represented by SEQ ID NO: 7 to 12. For example, the first and second light chains described above may have amino acid sequences represented by SEQ ID NO: 7.

[0102] For example, the variable region of the first heavy chain described above may be the variable region of the pertuzumab heavy chain, and the variable region of the second heavy chain described above may be the variable region of the trastuzumab heavy chain. For example, the variable region of the first heavy chain described above may have an amino acid sequence represented by SEQ ID NO:13, and the variable region of the second heavy chain described above may have an amino acid sequence represented by SEQ ID NO:14.

[0103] In this application, the first heavy chain and / or the second heavy chain may include a constant region. For example, the constant region may be derived from the human IgG constant region. For example, the constant regions of the first heavy chain and the second heavy chain may be identical or different. In some cases, the amino acid sequences of the variable regions and CH1 domains of the first and second heavy chains may be identical to the amino acid sequences of the original monoclonal antibody.

[0104] In this application, the above-described bispecific antibody or its antigen-binding moiety can block both ligand-dependent and ligand-independent HER2 signaling pathways. For example, the IgG1 Fc fragment of the above-described bispecific antibody or its antigen-binding moiety can bind to FcRγIIIa and mediate a potent ADCC effect. For example, the above-described bispecific antibody or its antigen-binding moiety may promote HER2 internalization and / or exhibit superior antitumor activity in preclinical models compared to the use of the original monoclonal antibody (e.g., trastuzumab and pertuzumab) alone.

[0105] In some cases, the light chain constant region and / or heavy chain constant region of the bispecific antibody or its antigen-binding moiety may include modifications to obtain better ADCC, CDC, endocytosis, stability, immunogenicity and / or half-life, and furthermore, such modifications may also promote the formation of heterodimeric proteins during antibody expression. In this application, techniques for modifying the Fc fragment of the heavy chain are known in the art.

[0106] For example, the Fc fragment of the first heavy chain described above may have an amino acid sequence represented by SEQ ID NO: 19-49 or 51-52; the Fc fragment of the second heavy chain described above may have an amino acid sequence represented by SEQ ID NO: 19-49 or 51-52.

[0107] For example, the Fc fragment of the first heavy chain described above may have an amino acid sequence represented by SEQ ID NO:19; and the Fc fragment of the second heavy chain described above may have an amino acid sequence represented by SEQ ID NO:20.

[0108] For example, the Fc fragment of the first heavy chain described above may have an amino acid sequence represented by SEQ ID NO: 51; and the Fc fragment of the second heavy chain described above may have an amino acid sequence represented by SEQ ID NO: 52.

[0109] For example, the first heavy chain described above may have an amino acid sequence represented by SEQ ID NO:17; the second heavy chain described above may have an amino acid sequence represented by SEQ ID NO:18.

[0110] For example, the first heavy chain described above may have an amino acid sequence represented by SEQ ID NO:15; the second heavy chain described above may have an amino acid sequence represented by SEQ ID NO:16.

[0111] In this application, the HER2 bispecific antibody may comprise a first light chain, a second light chain, a first heavy chain, and a second heavy chain, wherein the variable region of the first and / or second light chain may have a sequence represented by SEQ ID NO:1; the variable region of the first heavy chain may have an amino acid sequence represented by SEQ ID NO:13; and the variable region of the second heavy chain may have an amino acid sequence represented by SEQ ID NO:14. The first heavy chain may have an amino acid sequence represented by SEQ ID NO:15; and the second heavy chain may have an amino acid sequence represented by SEQ ID NO:16.

[0112] The amino acid sequence according to the present invention is also an amino acid sequence that is identical to at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 at least 100%) the amino acid sequence represented by any of the SEQ ID NOs: 1 to 52 of the sequence list. For example, the amino acid sequence according to the present invention may have an amino acid sequence in which one or more (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10 or more) amino acid deletions, insertions, and / or substitutions are made from the amino acid sequence represented by any of the SEQ ID NOs: 1 to 52 of the sequence list.

[0113] In this application, the above doses range from about 20 mg / kg to about 30 mg / kg (for example, a dose of at least about 20 mg / kg, at least about 20.5 mg / kg, at least about 21 mg / kg, at least about 21.5 mg / kg, at least about 22 mg / kg, at least about 22.5 mg / kg, at least about 23 mg / kg, at least about 23.5 mg / kg, at least about 24 mg / kg, at least about 24.5 mg / kg, at least about 25 mg / kg, at least about 25.5 mg / kg, at least about 26 mg / kg, at least about 26.5 mg / kg, at least about 27 mg / kg, at least about 27.5 mg / kg, at least about 28 mg / kg, at least about 28.5 mg / kg, at least about 29 mg / kg, at least about 29.5 mg / kg and at least about 30 mg / kg). The dose may be in mg / kg. For example, the above dose may be approximately 20 mg / kg. For example, the above dose may be approximately 30 mg / kg.

[0114] In this application, the above-mentioned HER2 bispecific antibody may be administered once every two weeks or once every three weeks. For example, the above-mentioned HER2 bispecific antibody may be administered at a dose of approximately 20 mg / kg once every two weeks. For example, the above-mentioned HER2 bispecific antibody may be administered at a dose of 30 mg / kg once every three weeks.

[0115] For example, the subjects described above may not respond to conventional therapies for HER2-related tumors. For example, conventional therapies for HER2-related tumors may include administering HER2-targeting specific drugs. For example, these may include HER2 antigen-binding proteins (e.g., anti-HER2 antibodies), their conjugates, and / or HER2-specific inhibitors. For example, conventional therapies for HER2-related tumors may include administering HER2-ADCs, MBC hormones, taxanes, pirotinib, neratinib, tucatinib, trastuzumab, and / or pertuzumab. For example, conventional therapies for HER2-related tumors may include administering drugs commonly used to treat tumors. For example, these may include any available chemotherapy agents. For example, conventional therapies for HER2-related tumors may include administering docetaxel, capecitabine, and / or lapatinib.

[0116] In this application, "not responding" may mean that the tumor syndrome in the subject has not been significantly alleviated after administration with conventional therapy for HER2-related tumors. For example, the syndrome may include a decrease in tumor volume. For example, the syndrome may include an expansion of OS, ORR, and / or PFS.

[0117] In this application, the subjects who require the above-mentioned treatment may have failed conventional therapies for HER2-related tumors, and the above-mentioned conventional therapies for HER2-related tumors may include the administration of trastuzumab, MBC hormone and / or taxanes.

[0118] For example, subjects with the above needs may have failed conventional therapies for HER2-related tumors. For example, conventional therapies for HER2-related tumors may include administration of trastuzumab, HER2 TKIs, and HER2 ADCs. For example, in subjects with the above needs, the median number of prior HER2-targeted therapy treatments may be 2 (range: 1-12).

[0119] For example, subjects who require the above treatment may have HER2-positive metastatic breast cancer that has progressed after treatment with trastuzumab and / or taxanes.

[0120] For example, subjects with the above needs may have received conventional hormonal therapy. For example, the above hormonal therapy may include administering drugs that block estrogen receptors. For example, the above hormonal therapy may include treatment with tamoxifen and / or toremifene. For example, the above taxanes may include paclitaxel (Taxol) and docetaxel (Taxotere).

[0121] For example, a subject with the above needs may have received hormone therapy first, followed by treatment with trastuzumab and / or taxanes. Another example is a subject with the above needs who may have received treatment with trastuzumab and / or taxanes first, followed by hormone therapy.

[0122] In this application, tumors may include solid tumors. For example, the above tumors may include metastatic tumors, early-stage tumors and / or locally advanced tumors. For example, the above tumors may include HER2-positive tumors and / or HER2-low-expressing tumors. For example, the tumors described above may include breast cancer and / or gastric cancer. For example, the breast cancer described above may include HER2-positive breast cancer and / or HER2-low-expressing breast cancer. For example, the breast cancer described above may include early-stage breast cancer, locally advanced breast cancer and / or metastatic breast cancer. For example, the gastric cancer described above may include early-stage gastric cancer, locally advanced gastric cancer and / or metastatic gastric cancer. For example, a subject requiring the above may have a histologically or cytologically proven diagnosis of HER2-positive breast cancer at the time of diagnosis of locally advanced, unresectable or metastatic disease.

[0123] For example, the breast cancer mentioned above is HR-negative (HR - ) or HR positive (HR + ) This could be breast cancer. For example, the above cancer is HR - It could be HER2-positive breast cancer. Also, for example, the above cancer is HER2-positive. + It could be HER2-positive breast cancer.

[0124] In this application, subjects who require the above treatment may be treated with a planned regimen of HER2 bispecific antibodies until disease progression, unacceptable toxicity, or withdrawal of informed consent occurs first.

[0125] In this application, treatment may result in a disease response. For example, the disease response may include a reduction in tumor volume. For example, tumor assessment based on RECIST 1.1 criteria is performed at baseline, within 12 months, every 8 weeks (QW and Q2W schedules), every 6 weeks (Q3W schedule), and thereafter every 12 weeks.

[0126] The HER2 bispecific antibody in this application may be administered via the same or different route of administration. For example, the HER2 bispecific antibody may be administered intravenously.

[0127] For example, the HER2 bispecific antibody described above may be administered as an initial dose via intravenous infusion over 90 minutes. For example, subsequent doses of the HER2 bispecific antibody may be administered via intravenous infusion over 60 minutes.

[0128] In this application, one cycle may be defined as 28 days for Q2W (once every two weeks) medication and 21 days for Q3W (once every three weeks) medication.

[0129] In this application, the formulation contains at least about 5 μg / mL (for example, at least about 5 μg / mL, at least about 5.5 μg / mL, at least about 6 μg / mL, at least about 6.5 μg / mL, at least about 7 μg / mL, at least about 7.5 μg / mL, at least about 8 μg / mL, at least about 8.5 μg / mL, at least about 9 μg / mL, at least about 9.5 μg / mL, at least about 10 μg / mL, at least about 10.5 μg / mL, at least about 11 μg / mL, at least about 11.5 μg / mL, at least about 12 μg / mL, at least about 12.5 μg / mL, at least about 13 μg / mL, at least about 13.5 μg / mL, at least about 14 μg / mL, at least about 14.5 μg / mL, at least about 15 μg / mL, at least about 15.5 μg / mL, at least about 16 μg / mL, at least about 17 The formulation may contain HER2 bispecific antibodies in amounts of μg / mL, at least about 16.5 μg / mL, at least about 17 μg / mL, at least about 17.5 μg / mL, at least about 18 μg / mL, at least about 18.5 μg / mL, at least about 19 μg / mL, at least about 19.5 μg / mL, at least about 20 μg / mL, at least about 27 μg / mL, at least about 78 μg / mL or more. For example, the formulation may contain at least about 5 μg / mL of bispecific antibody. For example, the formulation may contain at least about 20 μg / mL of bispecific antibody.

[0130] For example, the above formulation may be packaged in a container. For example, the above device may be a container. In this application, the above container may be a “container”, a “pen”, or a “syringe”. For example, the above container may be a pre-filled container, a pre-filled pen, or a pre-filled syringe. For example, intravenous administration is from a saline container. For example, the above container may be connected to a flow path including a tube and / or needle.

[0131] In this application, the above formulation may be a liquid formulation. For example, the above formulation may be prepared with an aqueous carrier. For example, a stabilizer may be added in an amount less than or equal to the amount that would produce a viscosity undesirable or unsuitable for intravenous administration. For example, the above liquid formulation may further contain one or more of a buffer, a surfactant, and a preservative.

[0132] For example, the above-mentioned formulation may be administered intravenously. [Examples]

[0133] The following examples are provided to those skilled in the art to give a complete disclosure and explanation of how the invention can be carried out and used, and are not intended to limit the scope of what the inventors consider to be the invention, nor to suggest that the following experiments are all or only experiments. While every effort has been made to ensure the accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise specified, parts refer to parts by weight, molecular weight to weight-average molecular weight, temperature to Celsius, and pressure to atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used, such as s or sec to seconds, min to minutes, h or hr to hours, im to intramuscular, ip to intraperitoneal, and sc to subcutaneous.

[0134] material HER2 bispecific antibody: The HER2 bispecific antibody comprises a common light chain having the amino acid sequence represented by SEQ ID No: 7, a first heavy chain having the amino acid sequence represented by SEQ ID No: 15, and a second heavy chain having the amino acid sequence represented by SEQ ID No: 16.

[0135] Human cancer cell lines: Calu-3 (human lung cancer cell line) and NCI-N87 (human gastric cancer cell line) were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cell lines were characterized by the vendor and no further cell line authentication was performed.

[0136] Preclinical xenograft models: The antitumor activity of the HER2 bispecific antibody was evaluated using NCI-N87 and Calu-3 xenograft models. In these experiments, 4 - 6 × 10 6 NCI-N87 or Calu-3 tumor cells were subcutaneously injected into the right flank of BALB / c mice. Treatment with the HER2 bispecific antibody was initiated 8 days after tumor cell transplantation when the first tumor was in the range of 100 - 150 mm 3 . BALB / c mice (n = 5 - 6 mice / group) were injected intraperitoneally (i.p.) with PBS, the HER2 bispecific antibody once a week or once every two weeks for 4 - 5 weeks. Tumor size was measured twice a week with calipers and tumor volume was calculated using the formula: tumor volume (mm 3 ) = (width 2 × length) × 0.5.

[0137] Mice were euthanized on day 28 - 35 or when the tumor size reached 2000 mm 3 . Before euthanasia, blood was collected from each animal subject at a certain time point for measurement of the concentration of the HER2 bispecific antibody in plasma. A total of 40 animal subjects in the placebo group and 50 animal subjects in the HER2 bispecific antibody treatment group, including 780 tumor volume observations (246 observations from the placebo group and 534 observations from the HER2 bispecific antibody group), were included in the analysis.

[0138] The mouse xenotransplantation study was conducted by Suzhou Alphamab in China, and the experimental protocol was designed in accordance with the 3 Rs of animal welfare.

[0139] Software: Dataset assembly was performed using SAS® (version 9.3). Graphical data exploration, model-based simulations, and additional data processing were performed using R (version 3.5.1). Nonlinear mixed-effects modeling was performed using NONMEM (version 7.4, ICON Development Solutions, Ellicott City, MD, USA). All models were estimated using the first-order conditional estimation method with η-ε interaction (FOCE-1).

[0140] Clinical trials Study design and patient selection This Phase I, multicenter, open-label, 3+3 dose-escalation study was designed to evaluate the safety, tolerance, pharmacokinetics (PK), and preliminary antitumor activity of a HER2 bispecific antibody in HER2-positive MBC patients, and to identify the recommended Phase II dose (RP2D) of the HER2 bispecific antibody. The study protocol was approved by an institutional review board prior to patient recruitment and was conducted in accordance with the International Conference on Harmonisation of Human Use (CIH) Guidelines E6 for the Conduct of Clinical Trials of Medicinal Products. Each patient provided signed informed consent prior to study enrollment.

[0141] Eligible patients were 18–75 years of age with histologically confirmed HER2-positive metastatic breast cancer. HER2 positivity was confirmed according to the ASCO / CAP 2018 guidelines. Patients had a metastatic setting, had received at least one prior anti-HER2 therapy, had at least one measurable disease per RECIST 1.1, and had a baseline left ventricular ejection fraction (LVEF) of 55% or higher. A history of unstable brain metastases, malignant meningitis, or symptomatic interstitial lung disease was also present. A 300 mg / m² dose was used. 2Patients with a history of cumulative doxorubicin administration exceeding or equivalent levels, or those with medically significant heart disease, were excluded. Information was collected if the patient had next-generation sequencing results from blood or archived tumor samples prior to the signing of informed consent.

[0142] In the 3+3 dose escalation phase, patients received HER2 bispecific antibody therapy at doses of 5 mg / kg QW, 10 mg / kg QW, 20 mg / kg Q2W, and 30 mg / kg Q3W. Eligible patients received HER2 bispecific antibodies intravenously in 21-day or 28-day cycles until disease progression, unacceptable toxicity, or withdrawal of informed consent. Three patients were initially assigned to the starting dose level of 5 mg / kg. Dose-limiting toxicity (DLT) was defined as a post-administration treatment study-related toxic reaction that was unacceptable due to severity and / or irreversibility, limiting further dose increases. The DLT evaluation period was 28 days for the QW and Q2W dose frequencies and 21 days for the Q3W dose. If an objective response (partial or complete response) was observed at a particular dose level, the dose level was expanded to enroll an additional 23–25 patients to explore HER2 bispecific antibody efficacy, safety, and resistance. The maximum tolerated dose (MTD) was defined as the highest dose level at which one or fewer patients out of six developed a dose-limiting trial (DLT). When the MTD could not be identified, the RP2D was determined using population pharmacokinetic and pharmacodynamic approaches.

[0143] Safety evaluation Safety evaluations were performed cycle-by cycle, including all adverse events (AEs), dose escalation stages (DLTs), laboratory parameters, electrocardiogram (ECG), ECOG performance status, vital signs, and physical examination. AEs were assessed according to NCI CTCAE, VERSION 4.03 and monitored until 90 days after the last dose.

[0144] clinical activity Tumor imaging assessments were performed every 6 weeks for the first 12 months, then every 12 weeks thereafter, and at the time of investigator review, according to the Solid Tumor Response Assessment Criteria (RECIST) guideline version 1.1, until disease progression, initiation of a new antitumor therapy, or withdrawal of informed consent. Patients who achieved an objective response were confirmed at least every 4 weeks. The above information was collected from 22 patients who had baseline tissue and peripheral blood ctDNA second-generation sequencing (NGS) information prior to informed consent in this study, and the correlation between NGS and efficacy was analyzed.

[0145] PK analysis Serum concentrations of HER2 bispecific antibodies were collected on day 1 of pre-administration cycles 1 and 2, 30 minutes post-infusion, at end of infusion (EOI), and 2, 6, 24, 72, and 168 hours after EOI, as well as on days 1 and 15 of pre-administration cycles 3, 4, 5, and 6 and at EOI (1 cycle = 28 days for Q2W administration, 1 cycle = 21 days for Q3W administration). Samples were shipped frozen with dry ice and stored at -80°C until analysis. The concentration of HER2 bispecific antibodies in human serum was measured by quantitative sandwich electrochemiluminescence (ECL) assay. An MSD Quickplex 120 was used for data acquisition, and a Watson LIMS was used for data processing. Dataset assembly was performed using SAS® (version 9.3). Graphical data exploration, model-based simulations, and additional data processing were performed using R (version 3.5.1). Non-compartmental analysis was performed using Phoenix WinNonlin (Pharsight, Mountain View, CA) version 8.0. Nonlinear mixed-effects modeling was performed using NONMEM (version 7.4, ICON Development Solutions, Ellicott City, MD, USA).

[0146] Determining sample size and statistical analysis The size of the dose-escalation cohort is based on the 3+3 Phase I trial design. The sample size of the expansion cohort was calculated using a one-sided exact test, assuming that approximately 60 subjects would be enrolled in each cohort during the dose-escalation phase. Assuming an ORR of 8–10% in HER2-positive MBC patients who have failed previous anti-HER2 therapy, a size of 60 patients can provide 80% power to detect an ORR increase at the 30% level (α=0.05). Safety analyses are based on the safety analysis set, and efficacy analyses are based on the full efficacy analysis set (FAS).

[0147] Descriptive statistics were used to summarize demographics, baseline characteristics, AEs, laboratory toxicity, and DLTs. ORR, DCR, and CBR were reported as point estimates and as 95% accurate binomial CIs based on the Clopper-Pearson method. Survival outcomes, including PFS and OS, were estimated by the Kaplan-Meier method. Descriptive statistics and graphical representations were performed for all PK endpoints. Tmax was described as median, 25th and 75th percentiles, minimum and maximum values. Statistical calculations were performed in SAS 9.4. Immunogenicity evaluation The clinical immunogenicity strategy followed a stepwise approach consistent with industry practices in biopharmaceuticals (FDA, Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (Silver Spring, MD, August 2014)). First, samples were screened with a validated in-solution cross-linked ELISA to detect potential positive reactions to different domains of the bispecific antibody. Next, samples showing a positive signal were confirmed with the same ELISA after a competitive binding step with the bispecific antibody. Then, the ADA domain specificity of the confirmed positive samples was analyzed by competitive binding with the bispecific antibody component. Finally, the relative level of ADA was determined by titer.

[0148] statistical analysis Safety analyses are based on a safety analysis set, and efficacy analyses are based on a complete efficacy analysis set (FAS). The safety analysis set includes patients who received at least one study treatment. The FAS includes patients who completed at least one study treatment and were evaluated for tumor response. The pharmacokinetic analysis population includes patients with evaluable pharmacokinetic data. Depending on data availability, analyses of different PK parameters may include different numbers of patients.

[0149] The primary endpoint in the dose escalation phase is the dose duration to treatment (DLT) in the first cycle of treatment. The primary endpoint in the dose expansion phase is the objective response rate (ORR), defined as the proportion of patients with complete or partial response as assessed by the investigator based on RECIST 1.1. Secondary endpoints include duration of response (DOR), defined as the time from initial enrollment in complete response (CR) or partial response (PR) to disease progression or death due to any cause; progression-free survival (PFS), defined as the time from initial study treatment to disease progression or death due to any cause; and clinical efficacy rate (CBR), defined as the proportion of patients with CR, PR, and SD for 24 weeks or longer. Safety evaluations include the type, incidence, severity, and laboratory abnormalities of treatment-related adverse events (TRAEs), PK parameters including but not limited to the total area under the concentration-time curve (AUC0-t), and peak plasma concentration (C). 最大 ), elimination half-life (T 1 / 2 ) includes.

[0150] Example 1: Clinical Study Clinical Study Design: This first-in-human (FIH) clinical trial (NCT03619681, protocol approved by the ethics committees of all participating clinical sites) is an ongoing Phase 1 study of HER2 bispecific antibodies in HER2-expressing breast cancer, gastric / gastroesophageal junction cancer, and other locally advanced / metastatic solid tumors. All patients provided written informed consent prior to participation in the study. All enrolled subjects were HER2-positive breast cancer patients who had failed available HER2-targeted therapies, including at least trastuzumab. The median number of prior HER2-targeted therapy treatments was 2 (range: 1–12). Patients undergoing dose escalation were treated with doses ranging from 5 mg / kg QW to 30 mg / kg Q3W. Patients undergoing dose expansion were treated with either 20 mg / kg Q2W or 30 mg / kg Q3W. The HER2 bispecific antibody was administered via intravenous infusion over 90 minutes for the initial dose, and then shortened to intravenous infusion over 60 minutes for subsequent doses. Serum concentration samples of HER2 bispecific antibody were collected on day 1 of cycles 1 and 2 prior to administration, 30 minutes post-infusion, at end of infusion (EOI), 2, 6, 24, 72, and 168 hours after EOI, and on days 1 and 5 of cycles 3, 4, 5, and 6 prior to administration and at EOI (1 cycle = 28 days for Q2W administration, 1 cycle = 21 days for Q3W administration). Samples were shipped frozen with dry ice and stored at -80°C until analysis. Subjects were treated with HER2 bispecific antibody in the planned regimen until disease progression, unacceptable toxicity, or withdrawal of informed consent (whichever occurred first). Tumor assessments according to RECIST 1.1 criteria were performed at baseline, within 12 months, every 8 weeks (QW and Q2W schedules), and every 6 weeks (Q3W schedule), and thereafter every 12 weeks. The sum of the longitudinal diameters of target lesions, determined by the RECIST 1.1 criteria, is used to construct a human tumor growth model.

[0151] Example 2: Inhibition of tumor growth Translational Tumor Growth Inhibition Modeling: Using longitudinal tumor volume data, including 780 observations from 87 mice in NCI-N87 and Calu-3 xenograft models, the relationship between Ctrough concentration of HER2 bispecific antibody and tumor volume dynamics was described. A tumor growth inhibition model was developed that considers both spontaneous tumor growth and tumor lethality induced by HER2 bispecific antibody in the xenograft model. Standard model evaluation was performed to validate the tumor growth inhibition model in mice.

[0152] To further correlate the antitumor effects of HER2 bispecific antibodies with human exposure, translational tumor models were then inferred from tumor growth inhibition models developed in mice. Next, different scenarios for initial tumor volume and tumor doubling time in humans under different HER2 bispecific antibody concentrations were explored by conducting simulations.

[0153] Tumor growth inhibition data and modeling Tumor volume data from mouse xenograft models show a plausible HER2 bispecific antibody dose-response relationship (Figure 1). Starting with the basic structural model adopted by established preclinical tumor models, various forms of tumor growth components were tested. Similar saturable tumor growth components were found to best explain the xenograft data. 最大 The drug efficacy model was found to better explain the antitumor effect of the HER2 bispecific antibody than a linear relationship. The final tumor growth inhibition model that explains the mouse xenograft data is as follows:

[0154]

number

[0155] Here, KG is the maximum spontaneous tumor growth rate, and KD is the maximum tumor killing rate associated with the maximum HER2 bispecific antibody effect. TV(t) is the tumor volume at time t, and Conc is the Ctrough concentration of the HER2 bispecific antibody. TG50 is the tumor volume when the tumor growth rate decreases to 50% of the maximum rate, and KC50 is the Ctrough concentration level of the HER2 bispecific antibody when the tumor killing rate decreases to 50% of the maximum tumor killing effect of the HER2 bispecific antibody.

[0156] Table 1 provides the estimated tumor growth model parameters. The goodness-of-fit plots demonstrate the good fit and minimal bias of the model (Figure 2).

[0157] [Table 1]

[0158] The final model shows that tumor growth in mice slows down as tumor volume increases, and as tumor volume decreases, higher concentrations are required to achieve the same tumor growth inhibition goal. Specifically, in a mouse xenograft model with a tumor volume of 200 mm², 3 In this case, a Ctrough concentration of 78.1 μg / mL of HER2 bispecific antibody is required to achieve 95% inhibition of tumor growth (Table 2).

[0159] [Table 2]

[0160] Translational tumor models for human prediction of HER2 bispecific antibodies In the developed mouse tumor growth inhibition model, the tumor growth component was replaced using the tumor growth equation described in the relevant literature, which is based on the tumor growth dynamics observed in breast cancer patients. The translational tumor model for human prediction of HER2 bispecific antibodies is as follows:

[0161]

number

[0162] Key parameters of tumor growth in breast cancer patients were determined by values ​​described in the literature. Specifically, λ0 represents the growth parameter from the exponential growth phase of the tumor and was calculated from the tumor doubling time during the 25-day exponential growth phase (CV% = 200%). λ1 represents the growth parameter from the linear growth phase of the tumor and was calculated from the tumor doubling time of 621 days associated with the linear growth phase (CV% = 85%). The maximum achievable tumor volume V 最大 Based on the maximum achievable tumor radius of 5 cm, the result is 523.8 cm. 3 It was set to ψ = 20 to reflect the empirical switching of shape parameters between exponential and linear growth. Finally, from the initial tumor lesion length of 19 mm (range 7-70 mm) and initial lesion width of 17 mm (range 7-80 mm), the initial tumor volume TV(0) = 2745.5 cm 3 The result was calculated.

[0163] On the other hand, the tumor-killing component and the model parameter specific to the HER2 bispecific antibody effect in tumor killing, namely K D and KC 50 These values ​​are assumed to be constant across species and are the same as those estimated from a mouse tumor growth inhibition model: KD = 0.106 / day and KC50 = 2.57 μg / mL.

[0164] Next, to predict the effective HER2 bispecific antibody exposure level in humans, simulations were performed to predict tumor size dynamics in humans under different Ctrough concentration levels of HER2 bispecific antibody or without treatment (Figures 3A-3D). In Figure 3, A represents no treatment and an exponential tumor doubling time of 25 days, B represents a trough HER2 bispecific antibody concentration of 5 μg / mL and an exponential tumor doubling time of 25 days, C represents a trough HER2 bispecific antibody concentration of 5 μg / mL and an exponential tumor doubling time of 250 days, and D represents a trough HER2 bispecific antibody concentration of 20 μg / mL and an exponential tumor doubling time of 25 days.

[0165] Simulation results of a translational tumor growth inhibition model showed that tumor stagnation could be achieved at Ctrough concentrations of HER2 bispecific antibody below 20 μg / mL, and that more aggressive tumors (i.e., an exponential doubling time of 25 days compared to less aggressive growth with a doubling time of 250 days) took longer to achieve tumor stagnation at given concentrations. A Ctrough concentration of 20 μg / mL significantly reduced the time required to construct the stagnation model compared to a Ctrough concentration of 5 μg / mL, but the improvement in efficacy above 20 μg / mL of HER2 bispecific antibody Ctrough appeared to be very limited.

[0166] Example 3: PK measurement Population PK Modeling: HER2 bispecific antibody concentration data, including 324 PK observations from 20 patients in the FIH study, and a nonlinear mixed-effects modeling approach were used for population PK analysis. A two-compartment model with linear exclusion was established to account for the PK profile of the HER2 bispecific antibody. Potential covariate effects were assessed for the relevant PK parameters. Individual differences were considered for all PK parameters, including central and peripheral volume, clearance, and intercompartmental clearance, as described below.

[0167] Pi =P POP ×exp(η i ), here, P i is the estimated individual parameter value for individual i, and P POP η is a typical population parameter estimate, i The mean is 0 and the squared deviation is ω 2 It was assumed that the data followed a normal distribution. Residual variability was explained by both proportional and dependent components.

[0168] C obs,ij =C pred,ij ×(1+ε p,ij )+ε a,ij Here, C obs,ij This represents the observed concentration of individual i and observation j, and C pred,ij ε represents the predicted concentration of the individual, p,ij ε is a proportional error. a,ij This represents the addictive error, and the different σ 2 A normal distribution N ~ (0, σ) 2 It follows 2). A baseline model evaluation was performed to certify the population PK model of HER2 bispecific antibodies.

[0169] To further determine exposure levels with different candidate dosing regimens of HER2 bispecific antibodies, simulations were performed for 1000 simulated patients for seven dosing regimens, each with a 30-week dosing time (no load: 5 mg / kg QW, 10 mg / kg QW, 20 mg / kg Q2W, 20 mg / kg Q3W; load in the first cycle: 20 mg / kg Q2W on days 1 and 8 with 20 mg / kg QW load, 30 mg / kg Q3W on days 1 and 8 with 20 mg / kg Qw load, 30 mg / kg Q2W on days 1 and 8 with 30 mg / kg Qw load). For each simulated patient, individual PK parameters were sampled from a distribution estimated from population PK analysis. Individual body weight (the only covariate in the population PK model) was sampled from a log-normal distribution, and the mean and standard deviation were calculated from the PK analysis dataset. Subsequently, for each administration regimen, the Ctrough concentration, maximum concentration, median mean concentration, and 90% prediction interval of the HER2 bispecific antibody were compiled and compared across all scenarios.

[0170] Population PK analysis in breast cancer patients Population PK analysis included observations of serum concentrations of HER2 bispecific antibodies after 324 doses from 20 patients. Baseline demographics and characteristics of these patients are summarized in Table 3. Overall, the concentration data exhibited a clear two-compartment configuration, and exposure was proportional to the dose within the study's dose range (Figure 4A).

[0171] [Table 3]

[0172] TIFF0007900289000012.tif103170

[0173] A two-compartment model with linear clearance from the central compartment represents the data well (Figure 4B). Body weight was found to be a significant covariate for both central volume and clearance. The parameter estimates for the final model were estimated within a reasonable range and with good accuracy (Table 4).

[0174] [Table 4]

[0175] Steady-state Ctrough concentrations for different dosing regimens were predicted by performing simulations based on a final population PK model of HER2 bispecific antibodies. As previously described, 1000 simulated subjects were parametrically sampled for each candidate dosing regimen from the estimated distribution of between-subject variability of PK parameters. The weight of the simulated subjects was sampled from patients in the population PK analysis dataset from a log-normal distribution with a linear scale mean of 58.7 kg and a log-scale standard deviation of 0.148. Table 5 provides the percentage of subjects with steady-state Ctrough concentrations above the 20 μg / mL threshold and steady-state peak concentrations above the 300 μg / mL threshold for each simulated dosing regimen.

[0176] [Table 5]

[0177] The results showed that over 98% of the simulated subjects were able to achieve a steady-state Ctrough concentration of over 20 μg / mL. On the other hand, approximately 80% of the simulated subjects were able to achieve a maximum concentration of over 400 μg / mL with the 20 mg / kg Q2W regimen, and over 95% of the simulated subjects were able to achieve a maximum concentration of over 400 μg / mL with the 30 mg / kg Q3W regimen. However, no simulated subjects were able to achieve the 5 mg / kg Qw regimen, and only about 20% of subjects were able to achieve this threshold with the 10 mg / kg Qw regimen.

[0178] Example 4 Preliminary ER analysis of the efficacy of HER2 bispecific antibodies in patients: A preliminary interim analysis of the relationship between exposure to HER2 bispecific antibodies and the response to tumor size (expressed as the sum of the longitudinal diameters of the target lesions) or SLD was performed when the first batch of SLD data became available, including 66 observations from 24 patients who underwent at least one SLD observation after administration. Of the 24 patients, 20 were included in the aforementioned population PK analysis, and exposure to HER2 bispecific antibodies was derived using individual post-hoc PK parameters. For the remaining 4 patients, exposure was derived using point estimates of PK parameters from the population PK analysis and individual body weight. Due to the limited availability of data, the tumor growth rate constant was set to an empirical value of 0.0228 per week by assuming a 20% increase in SLD within 8 weeks, i.e., if left untreated, the study population would develop progressive disease confirmed by RECIST 1.1 criteria within 6–8 weeks. The tumor killing rate constant by HER2 bispecific antibodies was estimated from individual differences. ER analysis uses different exposure metrics (C in a steady state). 最小 , C 最大 and C 平均 The following were tested: An ER model for SLD was developed and validated, and then simulations of the time course of SLD were performed under different candidate dosing regimens. For each dosing regimen, PK and ER parameters of 100 subjects were parametrically sampled from established population PK and intermediate ER models, and then resampled by bootstrapping to replace covariates of 24 subjects in the SLD dataset. Each subject was assumed to receive a total of 30 weeks of treatment with HER2 bispecific antibodies, and SLD values ​​were calculated every 6 weeks. After the simulation, the change in the percentage of SLD from baseline, the non-progression rate (change in SLD from baseline ≤) and the tumor reduction rate from baseline of ≥30% were summarized and compared between dosing regimens.

[0179] Interim ER analysis of human efficacy data In the ongoing FIH study, a preliminary ER analysis was performed using 66 post-administration SLD observations from 24 breast cancer patients (Figure 6). Of the 24 patients, 14 had only one post-administration SLD observation in addition to the baseline observation, while the rest had two or more post-administration SLD observations (Figure 7). Currently, individual patients in the ER dataset have up to five post-administration SLD observations.

[0180] Due to the sparse interim analysis data, it was not possible to reliably estimate the tumor growth rate constant. Meanwhile, HER2-positive breast cancer patients with at least one previous failure of trastuzumab-based treatment generally showed progressive disease as defined by RECIST 1.1 at intervals of 6–8 weeks if not treated with effective therapy. This indicates a 20% increase from baseline in approximately 8 weeks. Therefore, the tumor growth rate constant in the current model was determined to be 0.0228 per week, derived from the empirical growth of 20% within 8 weeks. (Steady state C) 最小 , C 最大 and C 平均 In ER analysis, it is tested as a predictor of tumor response exposure, and steady state C 最大 It was found that provides the best predictive power based on the current dataset. C is used as an exposure metric to promote the effectiveness of SLD. 最大 Using this method, the tumor killing rate constant was estimated to be 0.0943 mL / mg per week with acceptable precision (RSE = 22.6%). The squared deviation of the inter-individual variability of the tumor killing rate constant was estimated to be over 100%, indicating significant variability in the HER2 bispecific antibody effect among the 24 patients. Simulations based on the SLD intermediate ER model showed that more than half of the simulated individuals required doses of 20 or 30 mg / kg of HER2 bispecific antibody to achieve a 30% tumor reduction (Figure 5). Furthermore, more frequent initial loading doses have the advantage of maximizing initial tumor killing.

[0181] Dosage selection Translational tumor growth inhibition models indicate that Ctrough concentrations of HER2 bispecific antibody up to the predicted 20 μg / mL can significantly shorten the time to tumor cessation, but that further increases in tumor growth inhibition are very limited at Ctrough concentrations higher than 20 μg / mL. However, it has been acknowledged that current translational tumor growth inhibition models do not account for parameter variability or uncertainty. Therefore, this 20 μg / mL Ctrough concentration may not be the precise threshold for the antitumor activity of HER2 bispecific antibody, but rather a rough reference value. Based on this analysis, the antitumor activity of HER2 bispecific antibody is determined to be at the minimum target concentration (C 最小,ss Drive), Peak Level (C 最大,ss (Driven) or average concentration (C 平均,ss To explore whether it depends on maintaining (or AUC-driven) 20 μg / mL C, while giving a sufficient range, トラフ The clinical regimens for the initial human studies were selected to reach this target. Simulation results of the HER2 bispecific antibody population PK model using data from the FIH study (Table 4) show that all tested HER2 bispecific antibody doses (ranging from 5 mg / kg QW to 30 mg / kg Q3W) can achieve steady-state Ctrough concentrations of >20 μg / ml in almost all subjects (>98% of simulated subjects for each dosing regimen). High disease control rates (66.7%) and long-term clinical benefits were observed in patients at the lowest dose level of 5 mg / kg Qw, which is consistent with predictions from preclinical studies. At the same time, preliminary efficacy data from the FIH study appear to indicate that increasing the dose from 5 or 10 mg / kg Qw to 20 mg / kg Q2W or 30 mg / kg Q3W still increases the SLD response. This appears to mean that higher exposure levels are required to inhibit tumor growth in humans than in the xenograft model.

[0182] Furthermore, since the 10 mg / kg Qw regimen has essentially the same mean exposure as the 20 mg / kg Q2W and 30 mg / kg Q3W regimens, the increased efficacy at higher dose levels but lower frequencies appears to indicate that the SLD response is driven by peak concentration rather than trough or mean concentration. Indeed, even at the same total dose level, steady-state peak concentrations are much higher in the 20 or 30 mg / kg groups than in the 10 mg / kg groups (Table 4). However, in preclinical studies, only Ctrough concentrations were collected, so KCs related to peak or mean concentrations of HER2 bispecific antibodies are not available. 50 and K D This could not be directly estimated from preclinical or translational tumor growth inhibition models. Clinical exposure response analysis further optimized our understanding of effective dose selection for HER2 bispecific antibodies and complemented preclinical analyses that formed the entire course of translational PKPD evaluation.

[0183] Indeed, current population PK and exposure-response analyses for SLD are based on small datasets with limited patient data available for each dosing group. In particular, SLD data from only three patients is available for each of the 5 mg / kg QW, 10 mg / kg QW, and 30 mg / kg Q3W groups. Therefore, the current parameters of the SLD exposure-response analysis are estimated to have relatively high variability and uncertainty. This is reflected in the simulation results (Figure 6), which showed a wide range of SLD responses across each simulated dosing regimen. As more data becomes available for PK and efficacy endpoints, it is hoped that both models will be updated and refined to further evaluate the results of the current assumptions.

[0184] In short, the translational PKPD approach provided sufficient information for dose selection strategies for HER2 bispecific antibodies. 20 mg / kg Q2W and 30 mg / kg Q3W were selected as RP2D for future studies. Preliminary exposure-efficacy analyses in humans are needed to determine potential C 最大- It exhibits driving antitumor activity. This observation will be validated by future clinical efficacy data. The application of modeling and simulation techniques and translational PK-PD approaches proved to be a powerful tool for supporting drug development and improving dose selection strategies for novel bispecific antibodies.

[0185] Example 5: Results of the clinical trial 5.1 Patient characteristics and treatment Table 6 shows the baseline characteristics of patients for each schedule. A total of 63 female patients (median age 54 years, range 31 to 69 years) were enrolled between September 2018 and December 2019. Most patients had received severe prior treatment and had received a median of 3 previous treatment lines (range 1 to 12) and a median of 2 anti-HER2 treatment lines (range 1 to 10) in a metastatic situation. Of these, 57.1% of patients (36 / 63) received three or more palliative therapies. Trastuzumab had been previously used in almost all patients (61 / 63, 96.8%), and HER2 TKI and HER2 ADC treatments were also administered to 50.8% (32 / 63) and 23.8% (15 / 63), respectively. It should be noted that pertuzumab and T-DM1 were approved in China in December 2018 and February 2020, respectively. Therefore, patients who had not received prior treatment with pertuzumab or T-DM1 were permitted to enroll in this study. At baseline, 60 patients (95.2%) had visceral disease. Common sites of metastasis included the lungs (35 cases, 55.6%) and the liver (18 cases, 28.6%). By the data cutoff date for this report, May 22, 2020, 27 patients were still continuing with the study treatment, and 36 patients discontinued treatment due to disease progression (n=35) and treatment-related adverse events (TRAEs) (n=1) (Figure 14). In Figure 14, T represents trastuzumab, P represents pertuzumab, A represents anti-HER2 ADCs, and S represents small molecule anti-HER2 TKIs. The median duration of treatment across the entire population was 5.6 months (range: 1.0–18.5 months).

[0186] [Table 6]

[0187] TIFF0007900289000016.tif50170

[0188] 5.2 Safety Safety evaluations were performed in all patients (Table 7). No DLTs were observed at any of the four dose levels. HER2 bispecific antibody TRAEs of any grade were observed in 54 patients (85.7%) of the entire cohort. The most common (≧ 10%) TRAEs were fever (23.8%), diarrhea (22.2%), elevated aspartate aminotransferase (22.2%), elevated alanine aminotransferase (22.2%), decreased white blood cell count (15.9%), hypokalemia (12.7%), fluid-related reactions (12.7%), and decreased neutrophil count (12.7%). In the researchers' assessment, all fever events were considered infusion-related AEs. In addition, all patients with TRAEs less than grade 3 recovered well with symptomatic treatment. A total of four patients (6.3%) (two patients in the 20 mg / kg Q2W cohort and two patients in the 30 mg / kg Q3W cohort) reported grade 3 TRAEs, including infusion-related reactions, increased transaminases, ventricular arrhythmias, and cardiac myxoma. No grade 4 or 5 AEs were reported. A TRAE leading to treatment discontinuation occurred in one patient (1.5%) in the 20 mg / kg Q2W cohort. The patient had an abnormal ECG and a prior history of anthracycline and taxane use at the time of participation in the study. After two doses of HER2 bispecific antibody therapy, the ECG showed ventricular premature contractions (quadruplicate rhythm). This patient was hospitalized and HER2 bispecific antibody administration was discontinued. The patient recovered at 30 days of safe follow-up. No delays or dose reductions were reported.

[0189] [Table 7]

[0190] TIFF0007900289000018.tif220170

[0191] TIFF0007900289000019.tif220170

[0192] TIFF0007900289000020.tif192170

[0193] 5.3 Evaluation of PK and immunogenicity The pharmacokinetics and dose-proportionality of single and multiple doses of HER2 bispecific antibody after intravenous infusion were characterized by standard non-compartmental analysis based on intensive concentration data obtained at complete dosing intervals of 1, 2, or 3 weeks in 12 Chinese subjects treated in the dose-escalation cohort of the first human study of HER2 bispecific antibody-CHN-001. Maximum concentration after initial administration (C) 最大 ) and area under the concentration-time curve (AUC) 0-inf Exposure parameters for HER2 bispecific antibodies generally increased approximately proportionally with dose in the dose range of 5 mg / kg to 30 mg / kg. Total systemic clearance was 19.3 (± 5.7) and 14.6 (± 4.7) mL / h at doses of 20 mg / kg and 30 mg / kg, respectively. Weekend half-life increased with dose. Mean values ​​were 140 (± 23) hours and 242 (± 66) hours at doses of 20 mg / kg and 30 mg / kg, respectively. Table 8 shows the main PK parameters evaluated after initial and multiple doses of HER2 bispecific antibodies.

[0194] Of the 63 evaluable test subjects after administration of HER2 bispecific antibodies, two (3.2%) were confirmed to be positive for anti-drug antibodies. No differences were observed in the PK profiles, safety characteristics, or efficacy outcomes of these two patients (data not shown).

[0195] [Table 8]

[0196] 5.4 Clinical activity Response evaluations were performed for all patients. The median follow-up period was 8.2 months (range 4.9–19.8 months), and tumor reduction was observed in 46 (73.0%) of 63 patients with measurable lesions who underwent at least one post-baseline scan (Figures 15A–15B). Figure 15A shows the maximum change in tumor size from baseline according to RECIST v1.1 for patients who underwent radiographic evaluation after at least one treatment. The length of the bar represents the maximum reduction or minimum increase in the target lesion. Figure 15B shows the change in individual tumor burden over time from baseline, as assessed based on RECIST v1.1. Tumor response was evaluated pre-treatment, every 6 weeks for the first 12 months, and then every 12 weeks thereafter, until disease progression, initiation of a new antitumor therapy, or withdrawal of informed consent.

[0197] In a Phase 2 cohort of 57 patients at the recommended dose levels (20 mg / kg Q2W and 30 mg / kg Q3W), a total of 17 patients (29.8%) achieved the best partial response (PR), 25 patients (43.9%) had stable disease (SD), and 14 patients (24.6%) had progressive disease (PD). The overall response rate (ORR) was 29.8% (95% CI, 18.4–43.4), and the disease coverage rate (DCR) was 73.7% (95% CI, 60.3–84.5). The median duration of response (DOR) was 7.2 months (95% CI, 5.5, NE). The median progression-free survival (PFS) was 5.6 months (95% CI, 4.2–8.2), and the 6-month PFS was 44.6% (95% CI, 29.0–59.0). PFS and OS at 12 months were not achieved. In a cohort of 63 evaluable patients in total, 17 patients (27.0%) achieved the best response of PR, 28 patients (44.4%) were SD, and 17 patients (27.0%) were PD. The ORR was 27.0% (95% CI, 16.6–39.7), and the DCR was 71.4% (95% CI, 58.7–82.1). The median PFS was 5.5 months (95% CI, 4.1–7.0), and the 6-month PFS was 42.0% (95% CI, 27.8–55.6) (Figure 14 and Table 9). Interestingly, both patients previously treated with pertuzumab achieved PR after HER2 bispecific antibody treatment. For example, one responder with recurrent breast cancer had previously received adjuvant chemotherapy and radiotherapy (DFI=20 months), first-line docetaxel / trastuzumab / pertuzumab (PFS=10 months), and second-line capecitabine / lapatinib (PFS=7 months). The PFS for the third-line HER2 bispecific antibody (30 mg / kg Q3W) in a patient with a partial response was 6.77 months (Figures 16A-16F). In Figure 16, the patient had previously received adjuvant chemotherapy and radiotherapy, first-line docetaxel / trastuzumab / pertuzumab, and second-line capecitabine / lapatinib. In the fourth cycle of the third-line HER2 bispecific antibody (30 mg / kg q3w) of the present invention, tumor size was significantly reduced.Target lesions included the left lung (Figures 16A, 16B), anterior mediastinal lymph nodes (Figures 16C, 16D), and anterior mediastinal lymph nodes (Figures 16E, 16F). Left pleural effusion (Figures 16C, 16D) was a non-target lesion. The patient's PFS was 6.77 months.

[0198] Table 10 summarizes the efficacy in the recommended phase 2 dose level cohort, classified by the type of resistance to trastuzumab 6, hormone receptor status, and the presence or absence of pertuzumab, anti-HER2 TKI, or anti-HER2 ADC administration. Specifically, primary trastuzumab resistance was defined as disease progression at the first radiological reassessment within 8–12 weeks or 3 months after trastuzumab administration, regardless of the presence or absence of chemotherapy in a metastatic setting, or a new relapse diagnosed during or within 12 months after adjuvant trastuzumab administration. Secondary trastuzumab resistance was defined as disease progression after a trastuzumab-containing regimen that initially achieved a disease response or stabilization at the first radiological assessment. The classified efficacy across the entire cohort of 63 evaluable patients is summarized in Table 11.

[0199] [Table 9]

[0200] [Table 10]

[0201] TIFF0007900289000024.tif177170

[0202] [Table 11]

[0203] TIFF0007900289000026.tif173170

[0204] Based on the results of Example 5, the recommended phase 2 doses of the HER2 bispecific antibody of the present invention may be 20 mg / kg Q2W and 30 mg / kg Q3W, based on safety, clinical response, and pharmacokinetic parameters. The safety of the HER2 bispecific antibody of the present invention may have both similarities and differences with trastuzumab and pertuzumab. Furthermore, the HER2 bispecific antibody of the present invention may have antitumor effects comparable to treatment with trastuzumab and pertuzumab in combination. Promising results are possible, and all patients previously treated with pertuzumab achieved a partial response (PR). The HER2 bispecific antibody of the present invention may be well-tolerated and showed promising antitumor activity in HER2-positive breast cancer patients who had failed anti-HER2 therapy.

[0205] Even though preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that these embodiments are merely given as examples. The present invention is not limited to the embodiments described herein. Although the present invention has been described herein above, the description of embodiments and the drawings herein are not to be constrained. Those skilled in the art will be able to conceive of various changes, modifications and substitutions as long as they do not depart from the present invention. It should be understood that the present invention is not in any respect limited to the specific description, arrangement or relative proportions of the various conditions and variables described herein. It should be understood that various alternative forms of the embodiments described herein may be used when the present invention is put into practice. Therefore, it is expected that the present invention will cover all such alternatives, modifications, variations or equivalents. The following claims limit the scope of the present invention and include methods and structures within the scope of these claims and their equivalents.

Claims

1. A formulation for use in the prevention, palliative care, or treatment of tumors or the inhibition of tumor growth in subjects in need, The formulation contains a HER2 bispecific antibody, which is administered at a dose of 20 mg / kg once every two weeks or at a dose of 30 mg / kg once every three weeks. The HER2 bispecific antibody comprises a common light chain, a first heavy chain, and a second heavy chain, wherein the common light chain has an amino acid sequence represented by SEQ ID No: 7, the first heavy chain has an amino acid sequence represented by SEQ ID No: 15, and the second heavy chain has an amino acid sequence represented by SEQ ID No:

16. formulation.

2. The formulation comprises at least 5 μg / mL of the bispecific antibody, The formulation according to claim 1.

3. The formulation comprises at least 20 μg / mL of the bispecific antibody, The formulation according to claim 1.

4. The aforementioned formulation is packaged in a container. A preparation according to any one of claims 1 to 3.

5. A drug delivery device for use in the prevention, mitigation, or treatment of tumors or inhibition of tumor growth in subjects in need, comprising a formulation according to any one of claims 1 to 4.

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