Treatment of cancer with HER2xCD3 bispecific antibodies in combination with anti-HER2 MAB

Abstract

The present invention provides methods of treating HER2-positive cancers (such as HER2-positive breast cancer and HER2-positive gastric cancer) using a combination of a HER2 antibody, e.g., a HER2 T-cell-dependent bispecific antibody (TDB), with an additional HER2 antibody (e.g., trastuzumab).

Description

[Technical Field]

[0001] [Sequence table] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 4, 2020, is entitled 50474-197WO2_Sequence_Listing_3.4.20_ST25 and is 8,354 bytes in size.

[0002] The present invention relates to the treatment of HER2-positive cancers using a combination of a HER2 antibody, such as a HER2 T-cell dependent bispecific antibody (HER2 TDB), and another HER2 antibody. [Background technology]

[0003] Cancer is characterized by the uncontrolled proliferation of a subpopulation of cells. It is the leading cause of death in the developed world and the second leading cause of death in developing countries, with over 14 million new cancer cases diagnosed and over 8 million cancer deaths occurring annually. According to the American Cancer Society, an estimated 1,762,450 new cancer cases and 606,880 cancer deaths will occur in the United States in 2019. As the elderly population grows, so does the incidence of cancer, as the likelihood of developing cancer more than doubles after age 70. Therefore, cancer care represents a significant and growing societal burden.

[0004] Human epidermal growth factor receptor 2 (HER2)-positive cancers, such as breast and gastric cancers, represent some of the most common cancers worldwide. The majority of locally advanced and metastatic HER2-positive breast and gastric cancers remain untreatable, and most patients progress after receiving HER2-targeted therapy. Although significant progress has been achieved with the introduction of novel anticancer agents, overall survival rates have only modestly improved, and the long-term prognosis for HER2-positive cancer patients who experience disease progression during or after first-line treatment regimens remains bleak.

[0005] Thus, there is an unmet need in the art for the development of safe and effective treatment regimens for the treatment of HER2-positive cancers. Summary of the Invention

[0006] The present invention relates to methods of treating subjects with HER2-positive cancer using HER2-targeted T cell-dependent bispecific (TDB) antibodies.

[0007] In one aspect, the present invention provides a method of treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, the method comprising administering to the subject a treatment regimen comprising a HER2 antibody (e.g., a HER2 antibody that is not a HER2 T-cell dependent antibody (TDB), e.g., a monospecific HER2 antibody, e.g., a monospecific bivalent HER2 antibody, e.g., trastuzumab) and a HER2 TDB (e.g., BTRC4017A) comprising an anti-HER2 arm and an anti-CD3 arm, wherein the HER2 antibody and the HER2 TDB both bind domain IV of HER2, and the treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of the HER2 antibody. In some embodiments, the increased therapeutic index is accompanied by a decreased likelihood of experiencing on-target / off-tumor effects compared to treatment with the HER2 TDB in the absence of the HER2 antibody. In some embodiments, the on-target / off-tumor effect is a symptom of pulmonary toxicity (e.g., interstitial lung disease, acute respiratory distress syndrome, dyspnea, cough, fatigue, and pulmonary infiltrates), elevated liver enzyme levels, dry mouth, dry eyes, mucositis, esophagitis, or urinary symptoms. In some embodiments, the increased therapeutic index is accompanied by a reduced likelihood of experiencing immunogenic side effects compared to treatment with HER2 TDB in the absence of a HER2 antibody. The immunogenic side effects may include, for example, elevated levels of anti-drug antibodies, infusion / administration-related reactions (ARRs), cardiac dysfunction, pulmonary reactions, or cytokine release syndrome (CRS).

[0008] In some embodiments, the HER2 TDB and HER2 antibody competitively bind to domain IV of HER2. In some embodiments, the HER2 antibody comprises: (i) a complementarity determining region (CDR)-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the HER2 antibody comprises a variable heavy domain (V) having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 7. H ), and / or a variable light chain domain (V) having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:8. L In certain embodiments, V H comprises the amino acid sequence of SEQ ID NO: 7, and / or V L comprises the amino acid sequence of SEQ ID NO:8.

[0009] In some embodiments, the HER2 antibody (e.g., an additional HER2 antibody that is not HER2 TDB) is monospecific and / or bivalent for HER2. In some embodiments, the HER2 antibody is a full-length antibody (e.g., trastuzumab) comprising an Fc region. In some embodiments, the HER2 antibody is an Fc-modified trastuzumab variant, e.g., an Fc-modified trastuzumab variant having one or more amino acid modifications that reduce effector function (e.g., one or more substitution mutations, e.g., one or more substitution mutations at amino acid residues L234, L235, and / or P329 (EU numbering)). For example, in some embodiments, the one or more amino acid modifications include substitution mutations L234A, L235A, and P329G (LALAPG).

[0010] In some embodiments of any of the foregoing methods, the anti-HER2 arm of the HER2 TDB comprises a HER2 binding domain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the HER2 binding domain comprises a CDR-L1 having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 7. H and / or V having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:8. L In some embodiments, the V of the HER2 binding domain H comprises the amino acid sequence of SEQ ID NO: 7, and / or V of the HER2 binding domain L comprises the amino acid sequence of SEQ ID NO:8.

[0011] In some embodiments, the anti-CD3 arm of the HER2 TDB comprises a CD3-binding domain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD3-binding domain comprises a V CDR having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 15. Hand / or a variable V having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 16. L In some embodiments, the V of the CD3 binding domain H comprises the amino acid sequence of SEQ ID NO: 15, and / or V of the CD3 binding domain L comprises the amino acid sequence of SEQ ID NO:16.

[0012] In some embodiments, (i) the anti-HER2 arm of the HER2 TDB comprises (a) a V H and (b) V comprising the amino acid sequence of SEQ ID NO: 8 L and (ii) the anti-CD3 arm of the HER2 TDB comprises a HER2 binding domain comprising (a) a V comprising the amino acid sequence of SEQ ID NO: 15. H and (b) V comprising the amino acid sequence of SEQ ID NO: 16. L and a CD3 binding domain comprising:

[0013] In some embodiments of any of the methods described herein, the HER2 TDB is a full-length antibody comprising a modified Fc region. The modified Fc region can comprise one or more substitution mutations that reduce the effector function of the HER2 TDB. In some embodiments, the one or more substitution mutations comprise mutations at amino acid residues L234, L235, and / or D265 (EU numbering). In some embodiments, the one or more substitution mutations are L234A, L235A, and D265A. Additionally or alternatively, the one or more substitution mutations comprise an aglycosylation site mutation (e.g., an aglycosylation site mutation at amino acid residue N297 (EU numbering), e.g., an N297G or N297A aglycosylation site mutation). In some embodiments, the modified Fc region comprises N297G, L234A, L235A, and D265A substitution mutations. In some embodiments, the HER2 The TDB comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 (CH11) domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, a second CH1 (CH12) domain, a second CH2 (CH22) domain, and a second CH3 (CH32) domain. In some embodiments, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain, wherein (i) the CH31 domain and the CH32 domain each comprise a protuberance or cavity, and the protuberance or cavity in the CH31 domain can be positioned within the cavity or protuberance in the CH32 domain, respectively; or (ii) the CH21 domain and the CH22 domain each comprise a protuberance or cavity, and the protuberance or cavity in the CH21 domain can be positioned within the cavity or protuberance in the CH22 domain, respectively.

[0014] In another aspect, the present invention provides a method for treating or delaying the progression of a HER2-positive cancer (e.g., a HER2-positive breast cancer or a HER2-positive gastric cancer) in a subject in need thereof, the method comprising administering to the subject a treatment regimen comprising a HER2 antibody and a HER2 TDB, wherein (a) the HER2 antibody is trastuzumab or an Fc-modified trastuzumab variant; (b) the HER2 TDB comprises an anti-HER2 arm and an anti-CD3 arm, the anti-HER2 arm comprising a HER2 binding domain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; The CD3 arm comprises a CD3 binding domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:10, (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:12, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:13, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:14, wherein the treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of a HER2 antibody. The increased therapeutic index may be accompanied by a reduced likelihood of experiencing on-target / off-tumor effects compared to treatment with the HER2 TDB in the absence of a HER2 antibody. In some embodiments, the on-target / off-tumor effect is a symptom of pulmonary toxicity (e.g., interstitial lung disease, acute respiratory distress syndrome, dyspnea, cough, fatigue, and pulmonary infiltrates), elevated liver enzyme levels, dry mouth, dry eyes, mucositis, esophagitis, or urinary symptoms. In some embodiments, an increased therapeutic index is associated with a reduced likelihood of experiencing immunogenic side effects compared to treatment with HER2 TDB in the absence of a HER2 antibody.Immunogenic side effects can include, for example, elevated levels of anti-drug antibodies, infusion / administration-related reactions (ARR), cardiac dysfunction, pulmonary reactions, or cytokine release syndrome (CRS).

[0015] In some embodiments of any of the aforementioned aspects, the HER2 antibody is administered prior to administration of the HER2 TDB.

[0016] In some embodiments, the HER2 antibody is administered at a dose of about 5 mg / kg to about 10 mg / kg (e.g., 5 mg / kg to 10 mg / kg or 6 mg / kg to 8 mg / kg, e.g., about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg). In some embodiments, the HER2 antibody is administered about once every three weeks (Q3W).

[0017] In some embodiments, HER2 TDB is administered in an amount of 0.001 mg to 500 mg (e.g., 0.003 mg to 250 mg, 0.005 mg to 200 mg, 0.01 mg to 150 mg, 0.05 mg to 120 mg, 0.1 mg to 100 mg, 0.5 mg to 80 mg, or 1.0 mg to 50 mg, e.g., 0.001 mg to 0.005 mg, 0.005 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg) , 30mg to 40mg, 40mg to 50mg, 50mg to 60mg, 60mg to 70mg, 70mg to 80mg, 80mg to 90mg, 90mg to 100mg, 100mg to 120mg, 120mg to 150mg, 150mg to 200mg, 200mg to 250mg, 250mg to 300mg, 300mg to 350mg, 350mg to 400mg, 400mg to 450mg or 450mg to 500mg, for example, about 0.003mg, about 0.005mg, about 0.01mg, about 0.05mg, about 0.1mg, about 0.5mg, about 1.0 mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 11mg, about 12mg, about 13mg, about 14mg, about 15mg, about 16mg, about 17mg, about 18mg, about 19mg, about 20mg, about 21mg, about 22m g, approx. 23 mg, approx. 24 mg, approx. 25 mg, approx. 30 mg, approx. 35 mg, approx. 40 mg, approx. 45 mg, approx. 50 mg, approx. 55 mg, approx. 60 mg, approx. and / or administered in a fixed dose of 200 mg or about 250 mg, e.g., 0.003 mg, 0.009 mg, 0.027 mg, 0.081 mg, 0.24 mg, 0.72 mg, 1.08 mg, 1.51 mg, 2.2 mg, 2.3 mg, 4.0 mg, 4.6 mg, 6.6 mg, 8.0 mg, 9.2 mg, 12 mg, 13.2 mg, 14.8 mg, 18.4 mg, 19.8 mg, 26.4 mg, 36.8 mg, 51.5 mg, 52.8 mg, 61.3 mg, 72.1 mg, 105.6 mg, 147.8 mg, 176 mg, or 207 mg.In some embodiments, the HER2 TDB is administered about once every three weeks (Q3W).

[0018] In some embodiments of any of the above methods, the treatment regimen comprises: (a) a first dose of the HER2 antibody; (b) a first dosing cycle (C1) after the first dose of the HER2 antibody, wherein C1 comprises a first dose of HER2 TDB (C1D1) and a second dose of HER2 TDB (C1D2), where C1D2 is greater than C1D1; and (c) a second dosing cycle (C2) after C1, wherein C2 comprises: (i) the second dose of the HER2 antibody; and (ii) an additional dose of HER2 TDB (C2D1) after the second dose of the HER2 antibody, where C2D1 is equal to the highest dose of HER2 TDB in C1.

[0019] In another embodiment of the present invention, there is provided a method of treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, the method comprising administering to the subject a treatment regimen comprising a HER2 antibody and a HER2 TDB, wherein the HER2 TDB comprises an anti-HER2 arm and an anti-CD3 arm, and both the HER2 antibody and the HER2 TDB bind domain IV of HER2, the treatment regimen comprising: (a) a first dose of the HER2 antibody; (b) a first dosing cycle (C1) after the first dose of the HER2 antibody, wherein C1 comprises a first dose of HER2 TDB (C1D1) and a second dose of HER2 TDB (C1D2), wherein C1D2 is greater than C1D1; and (c) a second dosing cycle (C2) after C1, wherein C2 comprises: (i) a second dose of the HER2 antibody; and (ii) a second dose of the HER2 TDB after the second dose of the HER2 antibody. and a second dosing cycle (C2) comprising an additional dose of HER2 TDB (C2D1), wherein C2D1 is equal to the highest dose of HER2 TDB in C1.

[0020] In some embodiments, the first dose of the HER2 antibody is administered one day before C1D1, and the subject is monitored for a period of 30 minutes to 24 hours (e.g., 30 minutes to 2 hours, e.g., 30 minutes to 90 minutes, e.g., 30 minutes, 60 minutes, 90 minutes, or 120 minutes) between the first dose of the HER2 antibody and C1D1.

[0021] In some embodiments, the first dose of the HER2 antibody is 5 mg / kg to 10 mg / kg (e.g., about 6 mg / kg or about 8 mg / kg). In some embodiments, the first dose of the HER2 antibody is 6 mg / kg. In other embodiments, the first dose of the HER2 antibody is 8 mg / kg. In some embodiments, the second dose of the HER2 antibody is 5 mg / kg to 10 mg / kg (e.g., about 6 mg / kg). In some embodiments, the second dose of the HER2 antibody is 6 mg / kg. In some embodiments, the first and / or second dose of the HER2 antibody are administered by infusion over at least 30 minutes.

[0022] In some embodiments, the second dose of the HER2 antibody is administered on the same day as C2D1. In some embodiments, C1D2 is at least twice the dose of C1D1 (e.g., at least three times the dose of C1D1). In some embodiments, C1D1 is at a dose of 0.003 mg to 50 mg (e.g., 0.003 mg to 50 mg, 0.005 mg to 20 mg, 0.01 mg to 10 mg, 0.05 mg to 8 mg, or 0.1 mg to 5 mg, e.g., 0.001 mg to 0.005 mg, 0.005 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, or 40 mg). g to 50 mg, for example, about 0.003 mg, about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg). In some embodiments, C1D1 is 0.003 mg, 0.009 mg, 0.027 mg, 0.081 mg, 0.12 mg, 0.24 mg, 0.48 mg, 0.72 mg, 1.0 mg, 2.0 mg, 2.2 mg, 4.0 mg, 6.6 mg, 8.0 mg, 12 mg, 18 mg, 27 mg or 40.5 mg.

[0023] In some embodiments, C1D2 is administered in an amount of 0.009 mg to 200 mg (e.g., 0.01 mg to 150 mg, 0.05 mg to 100 mg, 0.1 mg to 50 mg, 0.5 mg to 20 mg, or 1 mg to 10 mg, e.g., 0.009 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, 90 mg to 100 mg, 100 mg to 120 mg, 120 mg to 150 mg, or 150 mg). mg to 200 mg, for example, about 0.009 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg or about 200 mg). In some embodiments, C1D2 is 0.009 mg, 0.027 mg, 0.081 mg, 0.24 mg, 0.4 mg, 0.72 mg, 0.08 mg, 1.6 mg, 2.2 mg, 2.3 mg, 3.2 mg, 4.6 mg, 6.4 mg, 6.6 mg, 9.2 mg, 12.8 mg, 14.8 mg, 18.4 mg, 19.8 mg, 25.6 mg, 36.8 mg, 38.4, 51.5 mg, 57.6 mg, 72.1 mg, 86.4 mg, 61.3 mg or 129.6 mg.

[0024] In some embodiments, for example in one-step fractionation, C2D1 and C1D2 are equal.

[0025] In some embodiments, C1 further comprises a third dose (C1D3) of HER2 TDB, where C1D3 is greater than C1D2. In some embodiments, C1D1, C1D2, and C1D3 are cumulatively greater than the highest cleared dose of HER2 TDB in the first dosing cycle of a single-step fractionated, dose-escalation dosing regimen (e.g., the highest cleared dose is about 0.01 mg to about 30 mg, e.g., 0.5 mg to 25 mg, 1 mg to 20 mg, or 2 mg to 10 mg). In some embodiments, C1D2 is 2-10 times the dose of C1D1 (e.g., about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold). In some embodiments, C1D3 is 2-3 times the dose of C1D2. In some embodiments, C2D1 and C1D3 are equal.

[0026] In some embodiments, C1D1 is administered in an amount of 0.01 mg to 20 mg (e.g., 0.05 mg to 15 mg, 0.1 mg to 10 mg, or 0.5 mg to 5 mg, e.g., 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, or 15 mg). g to 20 mg, for example, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg).

[0027] In some embodiments, C1D2 is administered in a dose of 0.1 mg to 100 mg (e.g., 0.1 mg to 80 mg, 0.5 mg to 50 mg, or 1 mg to 10 mg, e.g., 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, or 90 mg to 100 mg, e.g., about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg). g, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg or about 100 mg).

[0028] In some embodiments, C1D3 is administered in an amount of 1 mg to 400 mg (e.g., 10 mg to 300 mg, 20 mg to 200 mg, or 50 mg to 100 mg, for example, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, 90 mg to 100 mg, 100 mg to 120 mg, 120 mg to 150 mg, 150 mg to 200 mg, 200 to 250 mg, 250 mg to 300 mg, 300 mg to 350 mg, or 350 mg to 400 mg, for example, about 1.0 mg). g, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg or about 400 mg). In some embodiments, C1D3 is 1.1 mg, 2.2 mg, 4.4 mg, 6.6 mg, 8.8 mg, 13.2 mg, 17.6 mg, 26.4 mg, 35.2 mg, 52.8 mg, 70.4 mg, 105.6 mg, 147.8 mg, 158.4 mg, 176 mg, 207 mg, 237.6 mg or 356.4 mg.

[0029] In some embodiments, the method includes administering C1D1, C1D2, and C1D3 to a subject on, or about, days 1, 8, and 15 of C1, respectively. In some embodiments, C1 is administered for about 21 days. In some embodiments, C2 is administered for about 21 days.

[0030] In some embodiments, the method comprises administering C2D1 to the subject on day 1 of C2. In some embodiments, the treatment regimen comprises one or more additional dosing cycles (e.g., up to 15 additional dosing cycles, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional dosing cycles). In some embodiments, the length of each of the one or more additional dosing cycles is about 21 days. In some embodiments, each of the one or more additional dosing cycles comprises a single dose of the HER2 antibody and a single dose of the HER2 TDB (e.g., the HER2 antibody is administered before the HER2 TDB in each additional dosing cycle, e.g., on day 1 of each additional dosing cycle). In some embodiments, the method comprises administering a HER2 antibody and a HER2 TDB to the subject on day 1 of each of the one or more additional dosing cycles.

[0031] In another aspect, the present invention provides a method of treating or delaying the progression of HER2-positive cancer in a subject in need thereof, the method comprising administering to the subject a treatment regimen comprising HER2 TDB, the treatment regimen comprising: (a) a first cycle (C1) comprising a first dose of HER2 TDB (C1D1) and a second dose of HER2 TDB (C1D2), wherein C1D2 is greater than C1D1; and (b) a second cycle (C2) comprising an additional dose of HER2 TDB (C2D1), wherein C2D1 is equal to the highest dose of HER2 TDB in C1. In some embodiments, C1D2 is at least twice the dose of C1D1 (e.g., at least three times the dose of C1D1).

[0032] In some embodiments, C1D1 is 0.003 mg to about 10 mg (e.g., 0.005 mg to 9 mg, 0.01 mg to 8 mg, 0.05 mg to 7 mg, or 0.1 mg to 5 mg, e.g., 0.003 mg to 0.005 mg, 0.005 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, or 5 mg to 10 mg, e.g., about 0.003 mg, about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg).

[0033] In some embodiments, C1D2 is administered in an amount of 0.009 to about 20 mg (e.g., 0.01 mg to 15 mg, 0.05 mg to 10 mg, or 0.1 mg to 5 mg, e.g., 0.009 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, or is 15 mg to 20 mg, for example, about 0.009 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg).

[0034] In some embodiments, C2D1 and C1D2 are equal. In other embodiments, C1 further comprises a third dose of HER2 TDB (C1D3) that is greater than C1D2. In some embodiments, C1D1, C1D2, and C1D3 are cumulatively greater than the maximum clearing dose of HER2 TDB in the first dosing cycle of a single-step fractionated, dose-escalation dosing regimen. In some embodiments, the maximum clearing dose is about 0.01 mg to about 30 mg. In some embodiments, C1D2 is 2-10 times the dose of C1D1 (e.g., about 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold the dose of C1D1). In some embodiments, C1D3 is 2-3 times the dose of C1D2. In some embodiments, C2D1 and C1D3 are equal.

[0035] In some embodiments, C1D1 is administered in an amount of 0.01 mg to 20 mg (e.g., 0.01 mg to 15 mg, 0.05 mg to 10 mg, or 0.1 mg to 5 mg, e.g., 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, or 15 mg). mg to 20 mg, for example, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg).

[0036] In some embodiments, C1D2 is administered in a dose of 0.1 mg to 100 mg (e.g., 0.1 mg to 80 mg, 0.5 mg to 50 mg, or 1 mg to 10 mg, e.g., 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, or 90 mg to 100 mg, e.g., about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg). g, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg or about 100 mg).

[0037] In some embodiments, C1D3 is administered in an amount of 1 mg to 200 mg (e.g., 10 mg to 150 mg, 20 mg to 120 mg, or 50 mg to 100 mg, for example, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, 90 mg to 100 mg, 100 mg to 120 mg, 120 mg to 150 mg, or 150 mg to 200 mg, for example, about 1.0 mg, about 2 mg, about 3 mg, or about 4 mg). mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg or about 200 mg).

[0038] In some embodiments, the method comprises administering C1D1, C1D2, and C1D3 to the subject on, or about, days 1, 8, and 15 of C1, respectively. In some embodiments, C1 is for about 21 days. Additionally or alternatively, in some embodiments, C2 is for 21 days. In some embodiments, the method comprises administering C2D1 to the subject on day 1 of C2. The treatment regimen can include one or more additional dosing cycles (e.g., up to 15 additional dosing cycles, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional dosing cycles). In some embodiments, each additional dosing cycle is for about 21 days. In some embodiments, each additional dosing cycle comprises a single dose of HER2 TDB. In some embodiments, the method includes administering HER2 TDB to the subject on day 1 of each of one or more additional dosing cycles. In some embodiments of any of the above aspects, the HER2 antibody and / or HER2 TDB is administered by intravenous infusion (e.g., by IV bag). In some embodiments, the treatment regimen results in an increased therapeutic index of HER2 TDB compared to a control treatment regimen (e.g., treatment with HER2 TDB in the absence of the HER2 antibody or a treatment regimen without split dosing).

[0039] In some embodiments of any of the preceding aspects, the method further comprises administering one or more additional therapeutic agents. For example, the one or more additional therapeutic agents can be tocilizumab, a corticosteroid, a PD-1 axis antagonist, or an antibody-drug conjugate. In some embodiments, the PD-1 axis-binding antagonist is selected from the group consisting of a PD-L1-binding antagonist (e.g., MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, or MEDI4736), a PD-1-binding antagonist (e.g., MDX-1106 (nivolumab), MK-3475 (pembrolizumab), and AMP-224), and a PD-L2-binding antagonist (e.g., a PD-L1-binding antibody or immunoadhesin).

[0040] In some embodiments, the subject has received trastuzumab in a previous treatment regimen (eg, as a treatment for a HER2-positive cancer).

[0041] In some embodiments, the HER2-positive cancer is a HER2-positive solid tumor. Additionally or alternatively, the HER2-positive cancer can be a locally advanced or metastatic HER2-positive cancer. In some embodiments, the HER2-positive cancer is HER2-positive breast cancer or HER2-positive gastric cancer (e.g., HER2-positive gastroesophageal junction cancer or HER2-positive colorectal cancer. In some embodiments, the HER2-positive cancer is selected from the group consisting of HER2-positive gastroesophageal junction cancer, HER2-positive colorectal cancer, HER2-positive lung cancer (e.g., HER2-positive non-small cell lung carcinoma), HER2-positive pancreatic cancer, HER2-positive colorectal cancer, HER2-positive bladder cancer, HER2-positive salivary duct cancer, HER2-positive ovarian cancer (e.g., HER2-positive ovarian epithelial carcinoma), or HER2-positive endometrial cancer. [Brief explanation of the drawings]

[0042] [Figure 1]FIG. 1 is a rendering of the crystal structure of the HER2 extracellular domain (ECD) bound by the HER2 binding domains 4D5 (trastuzumab), 2C4 (pertuzumab), and 7C2. [Figure 2] FIG. 2 is an immunoblot showing the relative expression of HER2 protein by MCF7, HT55 and KPL4 cells. [Figure 3] Figure 3A is a graph showing the relative killing of KPL4 cells by BTRC4017A alone (red circles); BTRC4017A + 230 μg / mL trastuzumab (blue squares); and BTRC4017A + 60 μg / mL trastuzumab (brown triangles) as a function of BTRC4017A concentration (ng / mL). Figure 3B is a graph showing the relative killing of HT55 cells by BTRC4017A alone (red circles); BTRC4017A + 230 μg / mL trastuzumab (blue squares); and BTRC4017A + 60 μg / mL trastuzumab (brown triangles) as a function of BTRC4017A concentration (ng / mL). ND = not determined. [Figure 4] Figure 4 is a graph showing the binding of trastuzumab (red circles) and trastuzumab-LALAPG (blue squares) to HER2-expressing SKBR3 cells as a function of concentration. Binding was detected using a goat anti-human-FITC secondary antibody, and the presence of the secondary antibody was quantified by mean fluorescence intensity (MFI) using flow cytometry. [Figure 5]Figure 5A is a trellis plot showing KPL4 tumor volume during various treatments in a mouse model. The top row shows the effect of control treatment, the left graph shows tumor growth in response to vehicle administration, the middle graph shows tumor growth in response to trastuzumab (HERCEPTIN®) without peripheral blood mononuclear cells (PBMCs), and the right graph shows tumor growth in response to trastuzumab (HERCEPTIN®) with PBMCs. The middle and bottom rows show tumor growth during treatment with BTRC4017A alone and in combination with trastuzumab (HERCEPTIN®), respectively. In the middle and bottom rows, the left graph shows tumor growth in response to 0.05 mg / kg BTRC4017A, the middle graph shows tumor growth in response to 0.5 mg / kg BTRC4017A, and the right graph shows tumor growth in response to 5.0 mg / kg BTRC4017A. The thick solid lines represent the fitted tumor volume for each group. The dashed lines represent the fitted tumor volume of the vehicle control group. The gray lines represent individual animals. Figure 5B is a trellis plot showing HT55 tumor volume during various treatments in a mouse model. The top row shows the effect of control treatment; the left graph shows tumor growth in response to vehicle administration; the middle graph shows tumor growth in response to trastuzumab (HERCEPTIN®) without peripheral blood mononuclear cells (PBMCs); and the right graph shows tumor growth in response to trastuzumab (HERCEPTIN®) with PBMCs. The middle and bottom rows show tumor growth during treatment with BTRC4017A alone and in combination with trastuzumab (HERCEPTIN®), respectively. In the middle and bottom rows, the left graph shows tumor growth in response to 0.05 mg / kg BTRC4017A; the middle graph shows tumor growth in response to 0.5 mg / kg BTRC4017A; and the right graph shows tumor growth in response to 5.0 mg / kg BTRC4017A. The thick solid lines represent fitted tumor volumes for each group. The dashed line represents the fitted tumor volume of the vehicle control group. The grey lines represent individual animals. DETAILED DESCRIPTION OF THE INVENTION

[0043] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some instances, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, but the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0044] The term "about" as used herein refers to a normal error range for the respective value, which is readily understood by one of ordinary skill in the art. Reference herein to a value or parameter marked "about" includes (and describes) embodiments that are directed to that value or parameter itself.

[0045] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the reference to "an isolated peptide" means one or more isolated peptides.

[0046] It will be understood that throughout this specification and the claims the word "comprise" or variations such as "comprises" or "comprising" imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0047] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0048] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0049] "Antigen-binding portion" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Molecules comprising an antigen-binding portion include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and antibody VH and / or VL domains), receptors, ligands, aptamers, and other molecules with identified binding partners. An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs), which modifications result in improved affinity of the antibody for antigen compared to a parent antibody lacking such modifications.

[0050] "Binding domain" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. A binding domain can be a portion of a molecule such as an antibody (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), an antibody fragment or portion thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and the VH and / or VL domains of antibodies), receptor, ligand, aptamer, or other molecule with an identified binding partner.

[0051] As used herein, the term "complementarity determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to amino acid residues of an antibody variable domain, the presence of which is necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3. Each complementarity determining region is composed of amino acid residues from the "complementarity determining region" defined by Kabat (i.e., the light chain variable domain (V L ) and approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the heavy chain variable domain (V H 31-35 (H1), 50-65 (H2), and 95-102 (H3) in: Kabat et al., Sequences of Proteins of Immunological Interest, 5th

[0052] Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loops" (i.e., the light chain variable domain (V L ), approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the heavy chain variable domain (V H ) 26-32 (H1), 53-55 (H2), and 96-101 (H3); Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). In some examples, a complementarity-determining region can include amino acids from both the CDR regions and hypervariable loops as defined according to Kabat. For example, CDRH1 of the heavy chain of antibody 4D5 includes amino acids 26-35.

[0053] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., supra.

[0054] As used herein, the terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having a heavy chain containing an Fc region as defined herein.

[0055] "Effector function" refers to the biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0056] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0057] "Percent amino acid sequence identity" or "percent sequence identity" with respect to a base polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. However, in this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0058] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be written as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y

[0059] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0060] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0061] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called αα, δδ, εε, γγ, and μμ, respectively.

[0062] A "subject," "patient," or "individual" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject, patient, or individual is human.

[0063] The term "HER2-positive" cancer includes cancer cells with higher-than-normal levels of HER2. Examples of HER2-positive cancer include HER2-positive breast cancer and HER2-positive gastric cancer. In some embodiments, the HER2-positive cancer is selected from the group consisting of HER2-positive gastroesophageal junction cancer, HER2-positive colorectal cancer, HER2-positive lung cancer (e.g., HER2-positive non-small cell lung carcinoma), HER2-positive pancreatic cancer, HER2-positive colorectal cancer, HER2-positive bladder cancer, HER2-positive salivary duct cancer, HER2-positive ovarian cancer (e.g., HER2-positive ovarian epithelial cancer), or HER2-positive endometrial cancer. In some embodiments, the HER2-positive cancer is locally advanced or metastatic. Optionally, the HER2-positive cancer has an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification rate of ≧2.0. In some embodiments, HER2-positive breast cancer is defined according to the HER2 Testing in Breast Cancer Guideline: 2018 Focused Update (Wolff et al., J. Clin. Oncol. 2018, 36(20):2105-2122).

[0064] An "effective amount" of a compound, such as a HER2 antibody (e.g., HER2 TDB, trastuzumab, or a combination thereof), is at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder (e.g., a HER2-positive cancer, e.g., a HER2-positive breast cancer or a HER2-positive gastric cancer). The effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to induce a desired response in the individual. An effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifesting during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical results such as alleviating one or more symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs required for the treatment of the disease, enhancing the effect of other drugs, such as by targeting, delaying the progression of the disease, and / or prolonging survival.In the case of cancer or tumor, an effective amount of a drug can have the effect of reducing the number of cancer cells (e.g., HER2-positive cancer cells); reducing tumor size; inhibiting (i.e., delaying or preferably stopping) the infiltration of cancer cells into peripheral organs; inhibiting (i.e., delaying or preferably stopping) the metastasis of tumors, inhibiting tumor growth to some extent, and / or alleviating one or more of the symptoms associated with the disorder to some extent.An effective amount can be administered in one or more doses.In the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment. As is understood in the clinical arts, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.Thus, an "effective amount" may be considered in relation to the administration of one or more therapeutic agents, and if a desired result can be or is achieved in conjunction with one or more other agents, then a single agent may be considered to be given in an effective amount.

[0065] The term "therapeutic index" refers to the ratio of the dose of a therapeutic agent (e.g., a HER2 TDB, e.g., BTRC4017A) that induces a toxic effect (e.g., an off-tumor toxic effect) to the dose of the therapeutic agent (e.g., a HER2 TDB, e.g., BTRC4017A) sufficient to achieve a desired therapeutic effect. An increase in the therapeutic index can be achieved if (a) the dose sufficient to achieve the desired therapeutic effect is decreased compared to a reference treatment regimen, and / or (b) the dose at which the therapeutic agent induces a toxic effect (e.g., a maximum tolerated dose) is increased compared to a reference treatment regimen. In determining the therapeutic index, a dose sufficient to achieve a desired therapeutic effect can be determined according to the subject's objective response to the treatment regimen. In some embodiments, the objective response is a complete response (CR) or partial response (PR) according to RECIST v.1.1. Additionally or alternatively, a dose sufficient to achieve a desired therapeutic effect can be determined according to the subject's duration of response (DOR). In some embodiments, DOR is the time from the first occurrence of a documented objective response according to RECIST v.1.1 to the first documented disease progression or death from any cause, whichever occurs first. In determining the therapeutic index, the dose at which a therapeutic agent induces a toxic effect can be determined according to the presence of dose-limiting toxicities (DLTs) graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0, except that cytokine release syndrome (CRS) is graded according to the Modified Cytokine Release Syndrome Grading System (see Tables 1 and 2 in Section II - Treatment Methods).

[0066] " Survival time " refers to the subject remaining alive, and includes progression-free survival (PFS) and overall survival (OS).Survival time can be estimated by the Kaplan-Meier method, and any difference in survival time is calculated using stratified log-rank test.

[0067] " Progression-free survival (PFS) " refers to the time from treatment (or randomization) to first disease progression or death. For example, progression-free survival is the time that a subject survives without cancer recurrence for a specified period, such as, for example, about 1 month, 1.2 months, 2 months, 2.4 months, 2.9 months, 3 months, 3.5 months, 4 months, 6 months, 7 months, 8 months, 9 months, 1 year, about 2 years, about 3 years, etc., from the start of treatment or from the first diagnosis. In one embodiment of the present invention, PFS can be evaluated by Response Evaluation Criteria in Solid Tumors (RECIST v.1.1).

[0068] "Overall survival (OS)" refers to a subject surviving for a specified period of time, such as about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, etc., from the start of treatment or initial diagnosis.

[0069] As used herein, the term "on-target / off-tumor effect" refers to an effect associated with binding by a therapeutic agent to a disease-related target molecule expressed on a healthy cell (e.g., an effect associated with binding by a HER2 TDB (e.g., BTRC4017A) to a HER2 molecule expressed on a healthy cell, e.g., the effect is the result of T cell cytotoxicity directed against the healthy cell). In some embodiments, the on-target / off-tumor effect is a symptom of pulmonary toxicity (e.g., interstitial lung disease, acute respiratory distress syndrome, dyspnea, cough, fatigue, or the presence of pulmonary infiltrates), elevated liver enzyme levels, dry mouth, dry eyes, mucositis, esophagitis, or urinary symptoms. Additionally or alternatively, the on-target / off-tumor effect can be any effect caused by abnormal function of a healthy cell or tissue (i.e., a non-cancerous cell or tissue) that expresses HER2, where the abnormal function results from the administration of a HER2 antibody (e.g., a HER2 TDB antibody administered in the absence of an additional HER2 antibody (e.g., the HER2 TDB antibody and the additional HER2 antibody bind domain IV of HER2). In some embodiments, the on-target / off-tumor effect is an immunogenic effect such as CRS, which is graded according to the Modified Cytokine Release Syndrome Grading System (see Tables 1 and 2 in Section II - Methods of Treatment).

[0070] As used herein, the term "cluster of differentiation 3" or "CD3," unless otherwise specified, refers to any native CD3 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including, for example, the CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses "full-length" unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ) and any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length, and the human CD3γ protein (NCBI RefSeq No. NP_000064), which is 182 amino acids in length.

[0071] The term "HER2," as used herein, unless otherwise specified, refers to any native HER2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed HER2 and any form of HER2 resulting from intracellular processing. The term also encompasses naturally occurring variants of HER2, including, for example, splice variants or allelic variants. HER2 includes, for example, the human HER2 protein, which is 1240 amino acids in length (see, for example, NCBI RefSeq No. NP_001276865). Domain IV of HER2 is the extracellular protein region closest to the cell membrane. Domain IV has the amino acid sequence of SEQ ID NO: 17.

[0072] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention aimed at altering the natural course of the individual being treated and may be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0073] As used herein, "delaying the progression" of a disorder or disease refers to postponing, preventing, delaying, delaying, stabilizing, and / or postponing the onset of a disease or disorder (e.g., HER2-positive cancer, e.g., HER2-positive breast cancer or HER2-positive gastric cancer). This delay can be of various durations depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can actually encompass prevention, in that the individual does not develop the disease. For example, it can delay the development of late-stage cancer, such as the onset of metastasis.

[0074] "Reducing" or "inhibiting" refers to the ability to cause an overall decrease, e.g., a 20% or greater, 50% or greater, or a 75%, 85%, 90%, 95% or greater decrease. In certain embodiments, reducing or inhibiting can refer to a reduction or inhibition of undesirable events (e.g., on-target / off-tumor effects or immunogenic effects), such as cytokine-driven toxicity (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and / or central nervous system (CNS) toxicity, following treatment with HER2 TDB and an additional HER2 antibody (e.g., using a fractionated, dose-escalating dosing regimen of the present invention) compared to treatment with HER2 TDB in the absence of the additional HER2 antibody (e.g., with or without a fractionated dosing regimen). In other embodiments, reducing or inhibiting can refer to antibody effector functions mediated by the antibody Fc region, specifically including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0075] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0076] As used herein, one "week" is 7 days ± 2 days.

[0077] As used herein, "administering" refers to a method of providing a dosage of a compound (e.g., a HER2 antibody or an additional HER2 antibody) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a HER2 antibody) to a subject. The compounds and / or compositions utilized in the methods described herein can be administered, for example, intravenously (e.g., by intravenous infusion), subcutaneously, intramuscularly, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, injection, infusion, continuous infusion, by localized perfusion directly bathing target cells, by catheter, irrigation, in a cream, or in a lipid composition. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0078] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings pertaining to the use of such therapeutic product.

[0079] II. Treatment method The present invention provides improved methods of administering a HER2 antibody (e.g., a treatment regimen comprising administration of a HER2 TDB (e.g., BTRC4017A) and an additional HER2 antibody (e.g., a HER2 antibody that is not TDB, e.g., trastuzumab). Such methods can provide increased specificity for HER2-positive tumors, thereby reducing undesirable effects such as on-target / off-tumor effects. The present invention is based, in part, on the discovery that an increased therapeutic index can be achieved by co-treating a subject with a HER2 antibody (e.g., a bivalent, monospecific HER2 antibody, e.g., trastuzumab) and a HER2 TDB (e.g., BTRC4017) that binds the same HER2 domain (e.g., domain IV of HER2) as the HER2 antibody. The increased therapeutic index can be accompanied by a reduced likelihood of experiencing on-target / off-tumor effects compared to treatment with a HER2 TDB in the absence of the HER2 antibody. Additionally or alternatively, an increased therapeutic index can be accompanied by a reduced likelihood of experiencing immunogenic side effects compared to treatment with a second HER2 antibody in the absence of the first HER2 antibody.

[0080] On-target / off-tumor effects can occur as a result of the binding of HER2 TDB to HER2 expressed by non-tumor cells (e.g., healthy cells). On-target / off-tumor effects can be symptoms of pulmonary toxicity, such as interstitial lung disease, acute respiratory distress syndrome, dyspnea, cough, fatigue, or the presence of pulmonary infiltrates. Additionally or alternatively, on-target / off-tumor effects can be associated with the dysfunction of healthy cells or tissues with low or moderate HER2 expression, such as epithelial cells in the gastrointestinal tract, respiratory tract, reproductive tract, urinary tract, skin, breast, and placenta. Such on-target / off-tumor effects that can be reduced or inhibited by the methods described herein include, for example, elevated liver enzyme levels, dry mouth, dry eye, mucositis, esophagitis, or urinary symptoms.

[0081] In determining the therapeutic index, a dose sufficient to achieve a desired therapeutic effect can be determined according to the subject's objective response (OR) to the treatment regimen. In some embodiments, the OR is a complete response (CR) or partial response (PR) according to RECIST v.1.1. Additionally or alternatively, a dose sufficient to achieve a desired therapeutic effect can be determined according to the subject's duration of response (DOR). In some embodiments, the DOR is the time from the first occurrence of a documented objective response according to RECIST v.1.1 to the first documented disease progression or death from any cause, whichever occurs first. Thus, in some embodiments, the method of the present invention increases DOR and / or prolongs the subject's survival time (e.g., overall survival (OS) or progression-free survival (PFS)).

[0082] In some embodiments, the increased therapeutic index resulting from the treatment regimens of the present invention is accompanied by a reduced likelihood of experiencing immunogenic side effects (e.g., elevated levels of anti-drug antibodies, infusion / administration-related reactions (ARRs), cardiac dysfunction, pulmonary reactions, and cytokine release syndrome) compared to treatment.

[0083] In determining the therapeutic index, the dose at which a therapeutic agent induces toxic effects can be determined according to the presence of dose-limiting toxicities (DLTs), graded according to NCI CTCAE v5.0 (excluding cytokine release syndrome (CRS)). In some embodiments, a DLT is any of the following adverse events occurring during the evaluation period (e.g., the first dosing cycle):

[0084] (a) a decrease of 15% or more from baseline in left ventricular ejection fraction (LVEF) or a decrease of 10% or more to an LVEF less than 50%; (b) Abnormal liver function, for example, as determined by: (i) AST or ALT >3 times the upper limit of normal (ULN) and total bilirubin >2 times the ULN, except in the following cases: if this occurs in the setting of CRS of grade ≤2 (as defined by the criteria established by Lee et al., Blood 2014,124:188-195), resolves to grade ≤1 within 3 days, and none of the individual laboratory values ​​exceed grade 3, this is not considered a DLT; (ii) Any grade 3 elevation of AST or ALT, except in the following cases: if it occurs in the setting of grade 2 or less CRS (defined by the criteria established by Lee et al., Blood 2014, 124:188-195) and resolves to grade 1 or less in less than 3 days, it is not considered a DLT; in patients with metastatic liver disease, a grade 3 transient increase in bilirubin, transaminases, and / or gamma-glutamyltransferase (GGT) that begins after infusion and resolves to grade 2 or less or to baseline within 1 week is not considered a DLT; (c) Grade 3 or greater lymphopenia lasting for more than 7 days; (d) Grade 4 or greater neutropenia (ANC < 500 cells / μL) lasting for more than 7 days; (e) Grade 3 or higher febrile neutropenia; (f) grade 4 or higher anemia; (g) Grade 4 or greater thrombocytopenia or Grade 3 thrombocytopenia with clinically significant bleeding; and / or (h) Grade 3 or higher non-hematological non-hepatic adverse events not attributable to another clearly identifiable cause, except for: (i) Grade 3 nausea or vomiting that resolves to Grade 2 or less within 3 days with standard treatment; (ii) Grade 3 diarrhea, colitis, or enterocolitis that resolves to Grade 1 or less within 7 days with appropriate treatment; (iii) Grade 3 fatigue that resolves to Grade 2 or less within 7 days; (iv) Grade 3 fever (defined as >40°C for ≤24 hours); (v) Grade 3 laboratory abnormalities that are asymptomatic and considered by the investigator to be clinically insignificant; (vi) Grade 3 that resolves to Grade 2 or less within 7 days with treatment equivalent to prednisone 10 mg / day or less. (vii) Grade 3 arthralgia that can be adequately managed with symptomatic treatment or that resolves to Grade 2 or less within 7 days; (viii) Grade 3 tumor flare, defined as localized pain, irritation, or rash localized to the site of a known or suspected tumor that begins within 24 hours of infusion and resolves to Grade 2 or less within 7 days; (ix) Grade 3 hypoxia that begins within 24 hours of infusion and resolves to Grade 2 or less within 2 days after the onset of the event; or (x) in patients with metastatic pulmonary disease, Grade 3 dyspnea secondary to localized pulmonary edema that begins within 24 hours of infusion and resolves to Grade 1 or baseline within 2 days after the onset of the event and bronchospasm that resolves within 24 hours.

[0085] CRS is graded according to the Modified Cytokine Release Syndrome Grading System described in Russell et al., N. Engl. J. Med. 2008, 358:877-887 and Lee et al., Blood 2014, 124:188-195, and summarized in Tables 1 and 2 below. JPEG0007730761000001.jpg82170 a Low-dose vasopressors: A single vasopressor at a dose less than that shown in Table 2 below. b High-dose vasopressors: As defined in Table 2 below.

[0086] JPEG0007730761000002.jpg57170 a Norepinephrine equivalent dose = [norepinephrine (mcg / min)] + [dopamine (mcg / kg / min)] + [phenylephrine (mcg / min) / 10]

[0087] In some cases, treatment using the methods described herein by administering HER2 TDB (e.g., HER2 TDB in combination with an additional HER2 antibody and / or in the context of a fractionated, dose-escalating dosing regimen) is comparable to a control treatment regimen (e.g., HER2 TDB monotherapy (in the absence of an additional HER2 antibody) or HER2 TDB in a non-fractionated dosing regimen). TDB treatment), following a treatment regimen of the present invention results in a reduction or complete inhibition (100% reduction) of any one or more (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) of an undesirable event, e.g., an event described above.

[0088] In some examples, the increase in therapeutic index resulting from the methods of the invention is at least a 1% increase (e.g., a 1% to 1,000% (10-fold) increase, a 2% to 5,000% increase, a 3% to 4,000% increase, a 4% to 3,000% increase, a 5% to 2,000% increase, a 10% to 1,000% increase, a 20% to 500% increase, or a 50% to 100% increase, e.g., a 1% to 5% increase, a 5% to 10% increase) compared to a control group. increase, 10% to 20% increase, 20% to 30% increase, 30% to 40% increase, 40% to 50% increase, 50% to 60% increase, 60% to 70% increase, 70% to 80% increase, 80% to 90% increase, 90% to 100% increase, 100% to 150% increase, 150% to 200% increase, 200% to 300% increase, 300% to 400% increase, 400% to 500% increase, or 500% to 1,000% increase). HER2-positive cancer

[0089] The methods described herein can be used to treat HER2-positive cancer. In some examples, the HER2-positive cancer is a HER2-positive solid tumor. Additionally or alternatively, the HER2-positive cancer can be a locally advanced or metastatic HER2-positive cancer. In some examples, the HER2-positive cancer is a HER2-positive breast cancer or a HER2-positive gastric cancer. In some embodiments, the HER2-positive cancer is selected from the group consisting of HER2-positive gastroesophageal junction cancer, HER2-positive colorectal cancer, HER2-positive lung cancer (e.g., HER2-positive non-small cell lung carcinoma), HER2-positive pancreatic cancer, HER2-positive colorectal cancer, HER2-positive bladder cancer, HER2-positive salivary duct cancer, HER2-positive ovarian cancer (e.g., HER2-positive ovarian epithelial cancer), or HER2-positive endometrial cancer. Concurrent treatment with HER2 TDB and additional HER2 antibody

[0090] The methods described herein include administering to a subject with cancer (e.g., a HER2-positive cancer) a HER2 TDB (e.g., a TDB that binds to HER2 and CD3, e.g., BTRC4017) and a HER2 antibody (e.g., an additional antibody that binds to HER2, e.g., a HER2 antibody that is not HER2 TDB, e.g., a HER2 monospecific antibody (e.g., a monospecific bivalent HER2 antibody)). In some embodiments, the HER2 TDB and the HER2 antibody both bind domain IV of HER2. For example, the HER2 TDB and the HER2 antibody may competitively bind to domain IV of HER2. In some embodiments, the HER2 TDB and the HER2 antibody bind HER2 at the same epitope or overlapping epitopes (e.g., the same or overlapping epitopes of HER2). In some embodiments, the HER2 TDB has a lower HER2-binding avidity than the additional HER2 antibody, which may be due, at least in part, to a lower HER2-binding valency of the HER2 TDB compared to the additional antibody (e.g., the HER2 TDB binds monovalently to HER2, while the additional HER2 antibody binds bivalently to HER2). Additionally or alternatively, the HER2-binding domain of the HER2 TDB may have approximately the same HER2-binding affinity as the additional HER2 antibody (e.g., the HER2 TDB and the additional HER2 antibody may share one, two, three, four, five, or all six CDRs or one or both variable regions). In some embodiments, the HER2 TDB has a lower HER2-binding avidity than the additional HER2 antibody. H and / or V L V shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with H and / or V LIn some embodiments, the HER2 TDB and the HER2 antibody share the same HER2 binding domain (e.g., the HER2 binding domain of 4D5 (e.g., hu4D5), or an Fc-modified variant thereof, e.g., in an example where the HER2 TDB is BTRC4017A and the HER2 antibody is trastuzumab).

[0091] In some examples, any of the methods described herein may include administering a HER2 TDB comprising an anti-HER2 arm having a HER2 binding domain comprising at least one, two, three, four, five, or six complementarity determining regions (CDRs) selected from (a) CDR-H1 comprising the amino acid sequence of (SEQ ID NO: 1), (b) CDR-H2 comprising the amino acid sequence of (SEQ ID NO: 2), (c) CDR-H3 comprising the amino acid sequence of (SEQ ID NO: 3), (d) CDR-L1 comprising the amino acid sequence of (SEQ ID NO: 4), (e) CDR-L2 comprising the amino acid sequence of (SEQ ID NO: 5), and (f) CDR-L3 comprising the amino acid sequence of (SEQ ID NO: 6). In some examples, the HER2 TDB comprises (a) a heavy chain variable domain (V) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 7, or a sequence thereof. H ), (b) a light chain variable domain (V) comprising an amino acid sequence having SEQ ID NO: 8 or at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto). L ), or (c) an anti-HER2 arm comprising a HER2 binding domain comprising the VH domain in (a) and the VL domain in (b). Thus, in some examples, the HER2 binding domain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 7. H and V comprising the amino acid sequence of SEQ ID NO:8 LAn exemplary HER2 binding domain having the above CDR and variable region sequences is, for example, the HER2 binding domain of hu4D5, described in WO 2015 / 095392, which is incorporated herein by reference in its entirety.

[0092] In some examples, any of the methods described herein may include administering a HER2 TDB comprising an anti-CD3 arm having a CD3-binding domain comprising at least one, two, three, four, five, or six CDRs selected from (a) a CDR-H1 comprising the amino acid sequence of (SEQ ID NO: 9), (b) a CDR-H2 comprising the amino acid sequence of (SEQ ID NO: 10), (c) a CDR-H3 comprising the amino acid sequence of (SEQ ID NO: 11), (d) a CDR-L1 comprising the amino acid sequence of (SEQ ID NO: 12), (e) a CDR-L2 comprising the amino acid sequence of (SEQ ID NO: 13), and (f) a CDR-L3 comprising the amino acid sequence of (SEQ ID NO: 14). In some examples, the bispecific antibody comprises (a) a V arm comprising an amino acid sequence of SEQ ID NO: 15 or a sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15 or a sequence thereof. H (b) a V comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16 or a sequence thereof. L (c) an anti-CD3 arm comprising a CD3 binding domain comprising the VH domain in (a) and the VL domain in (b). Thus, in some examples, the CD3 binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15. H domain and V comprising the amino acid sequence of SEQ ID NO: 16 L An exemplary CD3 binding domain having the above CDR and variable region sequences is, for example, the CD3 binding domain of 40G5c, described in WO 2015 / 095392, which is incorporated herein by reference in its entirety.

[0093] In some examples, any of the methods described herein may involve (i) a HER2-binding domain comprising at least one, two, three, four, five, or six complementarity determining regions (CDRs) selected from (a) a CDR-H1 comprising the amino acid sequence of (SEQ ID NO: 1), (b) a CDR-H2 comprising the amino acid sequence of (SEQ ID NO: 2), (c) a CDR-H3 comprising the amino acid sequence of (SEQ ID NO: 3), (d) a CDR-L1 comprising the amino acid sequence of (SEQ ID NO: 4), (e) a CDR-L2 comprising the amino acid sequence of (SEQ ID NO: 5), and (f) a CDR-L3 comprising the amino acid sequence of (SEQ ID NO: 6). and (ii) an anti-CD3 arm having a CD3-binding domain comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of (SEQ ID NO: 9), (b) CDR-H2 comprising the amino acid sequence of (SEQ ID NO: 10), (c) CDR-H3 comprising the amino acid sequence of (SEQ ID NO: 11), (d) CDR-L1 comprising the amino acid sequence of (SEQ ID NO: 12), (e) CDR-L2 comprising the amino acid sequence of (SEQ ID NO: 13), and (f) CDR-L3 comprising the amino acid sequence of (SEQ ID NO: 14). In some examples, the HER2 TDB comprises: (i) a heavy chain variable domain (V) comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 7, or a sequence thereof; H ), (b) a light chain variable domain (V) comprising an amino acid sequence having SEQ ID NO: 8 or at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto). L ), or (c) an anti-HER2 arm comprising a HER2-binding domain comprising the VH domain in (a) and the VL domain in (b); and (ii) a VH arm comprising (a) SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a sequence thereof. H(b) a V comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16 or a sequence thereof. L or (c) an anti-CD3 arm comprising a CD3 binding domain comprising the VH domain in (a) and the VL domain in (b). Thus, in some examples, the HER2 binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7. H and V comprising the amino acid sequence of SEQ ID NO:8 L and the CD3 binding domain comprises V comprising the amino acid sequence of SEQ ID NO: 15. H domain and V comprising the amino acid sequence of SEQ ID NO: 16 L An exemplary such HER2 TDB is BTRC4017A, a full-length "knob-in-hole" antibody that has the hu4D5 HER2 binding domain in an anti-HER2 arm paired with an anti-CD3 arm that has the 40G5c CD3 binding domain.

[0094] In some examples, any of the methods described herein may include administering a HER2 TDB as described in WO 2015 / 063339. In some examples, any of the methods described herein may include administering a HER2 TDB GBR1302.

[0095] In some examples, any of the methods described herein may include administering a HER2 TDB having a monovalent arm and a bivalent arm. The monovalent arm may include a CD3 binding domain, and the bivalent arm may include two HER2 binding domains, each of which may have an Fc subunit that associates with the other Fc subunit to form an Fc domain (e.g., via a knob-in-hole configuration). In this embodiment, the C-terminus of the CD3 binding domain is fused to the N-terminus of the Fc subunit, the C-terminus of one HER2 binding domain is fused to the N-terminus of a second HER2 binding domain, and the C-terminus of the second HER2 binding domain is fused to the N-terminus of the other Fc subunit. In some examples, the HER2 TDB having a monovalent arm and a bivalent arm binds domain IV of HER2. For example, the HER2 binding domain may have a hu4D5 sequence (e.g., trastuzumab), and / or the CD3 binding domain may have a 40G5c sequence. Examples of such bivalent HER2 TDBs are described in International Patent Application No. PCT / US2019 / 17251.

[0096] HER2 antibodies for co-treatment with HER2 TDB include monospecific HER2 antibodies and multispecific (e.g., bispecific) HER2 antibodies (e.g., bispecific HER2 antibodies are not T cell-dependent bispecific antibodies). In some embodiments, the HER2 antibody is multivalent (e.g., bivalent) with respect to HER2. Additionally or alternatively, HER2 antibodies for use in the co-treatments described herein include full-length HER2 antibodies and HER2-binding fragments thereof. In examples involving full-length HER2 antibodies, the Fc region may contain one or more modifications, for example, to reduce effector function. Exemplary Fc modifications are further discussed in Section 5.c, below.

[0097] In some examples, any of the methods described herein may include administering (e.g., in addition to a HER2 TDB) a HER2 antibody comprising a HER2 binding domain comprising at least one, two, three, four, five, or six complementarity determining regions (CDRs) selected from: (a) a CDR-H1 comprising the amino acid sequence of (SEQ ID NO: 1); (b) a CDR-H2 comprising the amino acid sequence of (SEQ ID NO: 2); (c) a CDR-H3 comprising the amino acid sequence of (SEQ ID NO: 3); (d) a CDR-L1 comprising the amino acid sequence of (SEQ ID NO: 4); (e) a CDR-L2 comprising the amino acid sequence of (SEQ ID NO: 5); and (f) a CDR-L3 comprising the amino acid sequence of (SEQ ID NO: 6). In some examples, the HER2 antibody comprises (a) a heavy chain variable domain (V) comprising an amino acid sequence having SEQ ID NO: 7, or at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) thereto. H ), (b) a light chain variable domain (V) comprising an amino acid sequence having SEQ ID NO: 8 or at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto). L ), or (c) a HER2-binding domain comprising the VH domain in (a) and the VL domain in (b). Thus, in some examples, the HER2-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7. H and V comprising the amino acid sequence of SEQ ID NO:8 L An exemplary HER2 binding domain having the above CDR and variable region sequences is the HER2 binding domain of hu4D5. In some embodiments, the HER2 antibody is trastuzumab. In other embodiments, the HER2 antibody is an Fc-modified trastuzumab variant (e.g., trastuzumab-LALAPG).

[0098] HER2 TDB and / or additional HER2 antibodies may be produced using recombinant methods and compositions, such as those described in US Pat. No. 4,816,567, which is incorporated herein by reference in its entirety.

[0099] In some examples, the HER2 TDB and / or additional HER2 antibodies according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in sections 1-5 below.

[0100] 1. Antibody affinity In certain embodiments, the HER2 TDB and / or additional HER2 antibodies herein have a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 ~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D )

[0101] In one embodiment, K D is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by measuring the binding affinity of the Fab to the antigen at the lowest concentration ( 125 I) Fab is equilibrated with labeled antigen, followed by capturing the bound antigen on a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™; Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in competitive binding assays.

[0102] According to another embodiment, K Dis measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Prior to injection at a flow rate of 5 μl / min, the antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate, pH 4.8, to achieve approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μl / min in PBS supplemented with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C. By simultaneously fitting the association and dissociation sensorgrams, the association rate (k) was calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). on ) and dissociation rate (k off ) to calculate the equilibrium dissociation constant (K D ) is the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate by the surface plasmon resonance assay described above is 10 6 M- 1 s- 1above, the association rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer such as a spectrophotometer equipped with stopped-flow (Aviv Instruments) or an 8000 Series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0103] 2. Antibody fragment In certain embodiments, the HER2 TDB and / or additional HER2 antibodies are antibody fragments, e.g., antibody fragments of HER2 TDB bind to HER2 and CD3. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0104] Diabodies are antibody fragments that have two antigen binding sites, and can be bivalent or bispecific.See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al., Nat.Med.9:129-134(2003), and Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-6448(1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med.9:129-134(2003).

[0105] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0106] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0107] 3. Chimeric and humanized antibodies In certain embodiments, the HER2 TDB and / or additional HER2 antibodies used in the methods described herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0108] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0109] Humanized antibodies and methods for their production are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are also described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0110] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0111] 4. Design of Knob-in-Hole Bispecific Antibodies The HER2 TDB and / or additional HER2 antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for generating bispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)), and the "knob-in-hole" design (see, e.g., U.S. Pat. No. 5,731,168). The "knob-in-hole" design of bispecific antibodies can be used to generate a first arm containing a knob and a second arm containing a hole into which the knob of the first arm can bind. In one embodiment, the knob of the TDB can be present on the anti-CD3 arm. Alternatively, the knob of the bispecific antibody of the present invention can be present on the anti-HER2 arm. In one embodiment, the hole of the TDB of the present invention can be present on the anti-CD3 arm. Alternatively, the hole in the TDB of the present invention may be present on the anti-HER2 arm. In some examples, the HER2 TDB and / or additional HER2 antibodies produced using the knobs-in-holes technology may comprise one or more heavy chain constant domains, where the one or more heavy chain constant domains are selected from a first CH1 (CH11) domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, a second CH1 (CH12) domain, a second CH2 (CH22) domain, and a second CH3 (CH32) domain. In some examples, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some examples, the CH31 domain and the CH32 domain each comprise a protuberance or a cavity, and the protuberance or cavity in the CH31 domain can be positioned within the cavity or protuberance in the CH32 domain, respectively. In some examples, the CH31 and CH32 domains meet at the interface between the protuberance and the cavity. In some instances, the CH21 and CH22 domains each comprise a bulge or a cavity, and the bulge or cavity in the CH21 domain can be positioned within the cavity or bulge, respectively, in the CH22 domain.In some instances, the CH21 and CH22 domains meet at the interface between the bulge and the cavity.

[0112] Bispecific antibodies (e.g., TDB) can also be engineered using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see, e.g., WO 2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)). Bispecific antibodies can be engineered by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1), crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), or using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)). (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), using "diabody" technology to make bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and by using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and by preparing triabodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0113] HER2 TDB and / or additional HER2 antibodies or antibody fragments thereof may also include "dual acting FAbs" or "DAFs" that contain antigen binding sites that bind to targets other than HER2 in addition to HER2 (e.g., CD3 in the case of HER2 TDB) (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820, which is incorporated herein by reference in its entirety).

[0114] 5. Variants In some examples, amino acid sequence variants of the above-mentioned HER2 TDB and / or additional HER2 antibodies are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of one or both of the HER2 TDB and / or additional HER2 antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, such as antigen binding.

[0115] Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 3 under the heading "Preferred Substitutions." More substantial changes are provided in Table 3 under the heading "Exemplary Substitutions," and are as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. JPEG0007730761000003.jpg129170

[0116] Amino acids can be divided into groups according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0117] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0118] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will substantially retain a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibody is displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0119] For example, modifications (e.g., substitutions) may be made in CDRs to improve antibody affinity. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within residues that contact the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction of a secondary library and reselection from the secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0120] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such modifications do not substantially reduce the antibody's ability to bind antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may, for example, be outside the antigen-contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unmodified or contains no more than one, two, or three amino acid substitutions.

[0121] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues can be targeted as candidates for substitution or removed. Variants can be screened to determine whether they have the desired properties.

[0122] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the N- or C-terminal fusion of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0123] B glycosylation variants In some instances, the methods of the invention comprise administering to a subject (e.g., in the context of a fractionated, ascending-dose dosing regimen) a HER2 TDB and / or additional HER2 antibody variant that has been modified to increase or decrease the extent to which the bispecific antibody is glycosylated. Addition or deletion of glycosylation sites to the HER2 TDB and / or additional HER2 antibodies of the invention can be conveniently achieved by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0124] When the HER2 TDB and / or additional HER2 antibodies contain an Fc region, the carbohydrate attached to the Fc region can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention can be made to create antibody variants with specific improved properties.

[0125] In some instances, these methods include administering a variant of HER2 TDB and / or an additional HER2 antibody having a glycan structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, mixed, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to slight sequence variations in antibodies, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; WO 2002 / 0164328; WO 2004 / 0093621; WO 2004 / 0132140; WO 2004 / 0110704; WO 2004 / 0110282 ... No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol., 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng., 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially Example 11), as well as knockout cell lines, such as CHO cells knocked out of the α-1,6-fucosyltransferase gene FUT8 (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0126] In view of the above, in some examples, the methods of the present invention comprise administering to a subject (e.g., in the context of a split-dose escalation regimen) a HER2 TDB and / or additional HER2 antibody variant comprising an aglycosylation site mutation. In some examples, the aglycosylation site mutation reduces the effector function of the HER2 TDB and / or additional HER2 antibody. In some examples, the aglycosylation site mutation is a substitution mutation. In some examples, the bispecific antibody comprises a substitution mutation in the Fc region that reduces effector function. In some examples, the substitution mutation is at amino acid residues N297, L234, L235, and / or D265 (EU numbering). In some examples, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G. In some examples, the substitution mutation is at amino acid residue N297. In a preferred embodiment, the substitution mutation is N297A.

[0127] In another example, a variant having a bisected oligosaccharide, for example, a biantennary oligosaccharide attached to the Fc region of an antibody is separated into two parts by GlcNAc, is used according to the methods of the present invention. Such an antibody variant may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0128] c. Fc region variants In some examples, HER2 TDB and / or additional HER2 antibody variants (i.e., Fc region variants (see, e.g., U.S. Patent Application Publication No. 2012 / 0251531)) having one or more amino acid modifications introduced into the Fc region of the bispecific antibody can be administered to a subject with HER2-positive cancer according to the methods of the invention. The Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0129] In some instances, Fc region variants retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express Fc(RIII) only, whereas monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al., Proc.Nat'l Acad.Sci.USA 95:652-656 (1998).Also, C1q binding assay can be carried out to confirm that the antibody cannot bind C1q and lacks CDC activity.See, for example, the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood. 101:1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0130] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent Nos. 6,737,056 and 8,219,149). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0131] In a particular example, the proline at position 329 of the wild-type human Fc region in the antibody is substituted with glycine or arginine or an amino acid residue large enough to disrupt the proline sandwich within the Fc / Fcγ receptor interface formed between proline 329 of the Fc and tryptophan residues Trp87 and Trp110 of FcgRIII (Sondermann et al., Nature. 406, 267-273, 2000). In certain embodiments, the bispecific antibody comprises at least one additional amino acid substitution. In one embodiment, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S; in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A in the human IgG1 Fc region, or S228P and L235E in the human IgG4 Fc region (see, e.g., U.S. Patent Application Publication No. 2012 / 0251531); in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A and P329G in the human IgG1 Fc region.

[0132] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0133] In particular examples, the HER2 TDB and / or additional HER2 antibodies comprise an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 of the Fc region (EU numbering of residues).

[0134] In some instances, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0135] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include Fc variants having a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0136] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0137] d. Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered anti-HER2 TDB and / or additional HER2 antibodies, e.g., "thioMAbs," in which one or more residues of the bispecific antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the bispecific antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0138] Thus, immunoconjugates of HER2 TDB and / or additional HER2 antibodies conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes, are specifically contemplated.

[0139] In some examples, the immunoconjugate comprises a compound selected from the group consisting of maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0425235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin and doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and antibody-drug conjugates (ADCs) in which the bispecific antibody is conjugated to one or more drugs, including, but not limited to, CC1065.

[0140] In some examples, the immunoconjugate comprises HER2 TDB and / or an additional HER2 antibody conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, diansin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0141] In another embodiment, the immunoconjugate comprises HER2 TDB and / or an additional HER2 antibody conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When radioconjugates are used for detection, they can contain radioactive atoms for scintigraphy studies, such as TC99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0142] Conjugates of HER2 TDB and / or additional HER2 antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See International Publication No. 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0143] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0144] e. Other antibody derivatives In some instances, HER2 TDB and / or additional HER2 antibodies can be modified to contain additional non-proteinaceous moieties known in the art and readily available, and administered to a subject according to the methods described herein. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, the polymers can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0145] In some examples, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation.In one example, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc.Natl.Acad.Sci.USA 102:11600-11605(2005)).The radiation can be of any wavelength, including but not limited to, a wavelength that does not harm normal cells but heats the non-proteinaceous moiety to a temperature that kills cells that are close to the antibody-non-proteinaceous moiety.

[0146] dosage The HER2 TDB and the additional HER2 antibody are dosed and administered in a manner consistent with good medical practice. The treatment regimens provided herein include co-treatment of any of the HER2 TDBs described herein with an additional HER2 antibody (e.g., a HER2 antibody that is not TDB, e.g., trastuzumab), where the additional HER2 antibody is administered prior to administration of HER2 TDB (e.g., prior to the first administration of HER2 TDB and / or prior to administration of any subsequent administration of HER2 TDB).

[0147] In some embodiments, the HER2 antibody (e.g., a HER2 antibody that is not TDB, e.g., trastuzumab) can be administered at a dose of about 5 mg / kg to about 10 mg / kg (e.g., 5 mg / kg to 10 mg / kg or 6 mg / kg to 8 mg / kg, e.g., about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg). In some embodiments, the HER2 antibody (e.g., a HER2 antibody that is not TDB, e.g., trastuzumab) is administered about once every three weeks (Q3W). The HER2 antibody can be infused (e.g., intravenously) over at least about 30 minutes (e.g., 30 to 90 minutes). In some examples, for example, during the first administration of the HER2 antibody, the HER2 antibody can be infused (e.g., intravenously) over at least about 90 minutes, and the subject can be observed for adverse reactions from the HER2 antibody for 4 to 24 hours, for example, prior to administration of HER2 TDB. Alternatively, the HER2 antibody can be administered on the same day as the HER2 TDB injection (e.g., about 30 to 120 minutes after the HER2 antibody injection). In some embodiments, the period between the first dose of the HER2 antibody and the first dose of the HER2 TDB is longer in the first dosing cycle than in subsequent dosing cycles. For example, the first dose of the HER2 antibody can be administered 24 hours before initiating the first dosing cycle by administering the first dose of the HER2 TDB, but subsequent dosing cycles include a HER2 antibody dose on the same day as the HER2 TDB dose (e.g., 30 to 120 minutes before the HER2 TDB dose).

[0148] In some instances, the HER2 TDB (eg, BTRC4017A) is administered as a fixed dose.For example, HER2 TDB may be administered in a dose range of 0.001 mg to 500 mg (e.g., 0.003 mg to 250 mg, 0.005 mg to 200 mg, 0.01 mg to 150 mg, 0.05 mg to 120 mg, 0.1 mg to 100 mg, 0.5 mg to 80 mg, or 1.0 mg to 50 mg, e.g., 0.001 mg to 0.005 mg, 0.005 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30mg to 40mg, 40mg to 50mg, 50mg to 60mg, 60mg to 70mg, 70mg to 80mg, 80mg to 90mg, 90mg to 100mg, 100mg to 120mg, 120mg to 150mg, 150mg to 200mg, 200mg to 250mg, 250mg to 300mg, 300mg to 350mg, 350mg to 400mg, 400mg to 450mg or 450mg to 500mg, for example, about 0.003mg, about 0.005mg, about 0.01mg, about 0.05mg, about 0.1mg, about 0.5mg, about 1.0mg, About 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 11mg, about 12mg, about 13mg, about 14mg, about 15mg, about 16mg, about 17mg, about 18mg, about 19mg, about 20mg, about 21mg, about 22mg, about 23mg, about 24mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 150mg, about 200m g or about 250 mg, e.g., 0.003 mg, 0.009 mg, 0.027 mg, 0.081 mg, 0.24 mg, 0.72 mg, 1.08 mg, 1.51 mg, 2.2 mg, 2.3 mg, 4.0 mg, 4.6 mg, 6.6 mg, 8.0 mg, 9.2 mg, 12 mg, 13.2 mg, 14.8 mg, 18.4 mg, 19.8 mg, 26.4 mg, 36.8 mg, 51.5 mg, 52.8 mg, 61.3 mg, 72.1 mg, 105.6 mg, 147.8 mg, 176 mg, or 207 mg.In some embodiments, the HER2 TDB (eg, BTRC4017A) is administered about once every three weeks (Q3W).

[0149] The methods provided herein include a single-step fractionated treatment regimen. In a specific example, the single-step fractionated treatment regimen includes a first dose of a HER2 antibody (e.g., trastuzumab) followed by a first dosing cycle (C1). C1 includes a first dose of a HER2 TDB (e.g., BTRC2017A) (C1D1) and a second dose of the HER2 TDB (C1D2), where C1D2 is greater than C1D1 (e.g., at least twice as long as C1D1, e.g., about 2-5 times as long as C1D1, e.g., about 2- or 3-fold as long as C1D1). A second dosing cycle (C2) is administered after C1, and C2 includes a second dose of the HER2 antibody (e.g., on day 1 of C1), followed by an additional dose of HER2 TDB (C2D1) (e.g., about 30-120 minutes after the second dose of the HER2 antibody). In such a one-stage division, C2D1 can be made equal to C1D2.

[0150] In some examples, C1D1 is administered in a dose of 0.003 mg to 50 mg (e.g., 0.003 mg to 50 mg, 0.005 mg to 20 mg, 0.01 mg to 10 mg, 0.05 mg to 8 mg, or 0.1 mg to 5 mg, e.g., 0.001 mg to 0.005 mg, 0.005 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, or 40 mg). about 0.003 mg, about 0.005 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg). In some embodiments, C1D1 is 0.003 mg, 0.009 mg, 0.027 mg, 0.081 mg, 0.12 mg, 0.24 mg, 0.48 mg, 0.72 mg, 1.0 mg, 2.0 mg, 2.2 mg, 4.0 mg, 6.6 mg, 8.0 mg, 12 mg, 18 mg, 27 mg or 40.5 mg.C1D2 is 0.009 mg to 200 mg (e.g., 0.01 mg to 150 mg, 0.05 mg to 100 mg, 0.1 mg to 50 mg, 0.5 mg to 20 mg, or 1 mg to 10 mg, e.g., 0.009 mg to 0.01 mg, 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, 90 mg to 100 mg, 100 mg to 120 mg, 120 mg to 150 mg, or 150 mg to 200 mg , for example, about 0.009 mg, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg , about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg or about 200 mg). In some embodiments, C1D2 is 0.009 mg, 0.027 mg, 0.081 mg, 0.24 mg, 0.4 mg, 0.72 mg, 0.08 mg, 1.6 mg, 2.2 mg, 2.3 mg, 3.2 mg, 4.6 mg, 6.4 mg, 6.6 mg, 9.2 mg, 12.8 mg, 14.8 mg, 18.4 mg, 19.8 mg, 25.6 mg, 36.8 mg, 38.4, 51.5 mg, 57.6 mg, 72.1 mg, 86.4 mg, 61.3 mg, or 129.6 mg.

[0151] In a single-stage split treatment regimen, C1D1 and C1D2 are administered on different days within C1. In some embodiments, for example, when C1 is 21 days, C1D1 is administered on day 1 of C1 and C1D2 is administered on day 8 of C1.

[0152] Further provided herein is a two-stage fractionated treatment regimen comprising a third HER2 TDB dose (C1D3) in the first dosing cycle. C1D3 is greater than C1D2, which is greater than C1D1. In some embodiments, C1D1, C1D2, and C1D3 are cumulatively greater than the maximum HER2 TDB clearing dose in the first dosing cycle of the single-stage fractionated, dose-escalating dosing regimen. For example, in a study using a single-stage fractionated, dose-escalating dosing regimen in which the maximum clearing dose is determined to be 20 mg in C1, the sum of C1D1, C1D2, and C1D3 in the two-stage fractionated treatment regimen can exceed 20 mg (e.g., about 25 mg). In such a two-stage fractionated treatment regimen, C1D2 can be 2- to 10-fold (e.g., about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold) the dose of C1D1. Additionally or alternatively, C1D3 can be 2 to 3 times the dose of C1D2. In some instances, C2D1 is equal to C1D3.

[0153] In particular examples of two-stage split treatment regimens, C1D1 is administered at a dose of 0.01 mg to 20 mg (e.g., 0.05 mg to 15 mg, 0.1 mg to 10 mg, or 0.5 mg to 5 mg, e.g., 0.01 mg to 0.05 mg, 0.05 mg to 0.1 mg, 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, or The amount may be 15 mg to 20 mg, for example, about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg. Additionally or alternatively, C1D2 may be administered in an amount of 0.1 mg to 100 mg (e.g., 0.1 mg to 80 mg, 0.5 mg to 50 mg, or 1 mg to 10 mg, e.g., 0.1 mg to 0.5 mg, 0.5 mg to 1.0 mg, 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, or 90 mg to 100 mg, e.g., about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg). g, about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg).Therefore, C1D3 may be administered in the range of 1 mg to 400 mg (e.g., 10 mg to 300 mg, 20 mg to 200 mg, or 50 mg to 100 mg, e.g., 1.0 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, 90 mg to 100 mg, 100 mg to 120 mg, 120 mg to 150 mg, 150 mg to 200 mg, 200 to 250 mg, 250 mg to 300 mg, 300 mg to 350 mg, or 350 mg to 400 mg, e.g., about 1.0 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg. In some embodiments, C1D3 is 1.1 mg, 2.2 mg, 4.4 mg, 6.6 mg, 8.8 mg, 13.2 mg, 17.6 mg, 26.4 mg, 35.2 mg, 52.8 mg, 70.4 mg, 105.6 mg, 147.8 mg, 158.4 mg, 176 mg, 207 mg, 237.6 mg or 356.4 mg.

[0154] In a two-stage split treatment regimen, C1D1, C1D2, and C1D3 are administered on different days within C1. In some embodiments, for example, when C1 is 21 days, C1D1 is administered on day 1 of C1, C1D2 is administered on day 8 of C1, and C1D3 is administered on day 15 of C1.

[0155] In some examples of any of the aforementioned treatment regimens, the second and any subsequent dosing cycles are of the same duration as the first dosing cycle (e.g., 7 to 42 days, 14 to 35 days, or 21 to 28 days, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 days or longer). In some embodiments, C1, C2, C3, and all subsequent cycles (e.g., C4, C5, C6, etc.) are each about 21 days long. Both the HER2 TDB and the HER2 antibody may be administered on day 1 of each cycle after C1.

[0156] The duration of treatment can last as long as medically necessary or as long as the desired therapeutic effect (e.g., as described herein) is achieved. In certain embodiments, treatment continues for 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, or up to the subject's lifetime.

[0157] For all of the methods described herein, one or more HER2 antibodies are formulated, dosed, and administered in a manner consistent with good medical practice.Factors to consider in this regard include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of each patient, the cause of the disorder, the delivery site of the drug, the method of administration, the administration schedule, and other factors known to medical professionals.One or more HER2 antibodies do not need to be formulated together with one or more drugs currently used to prevent or treat the disorder, but can optionally be formulated together with these drugs.The effective amount of such other drugs depends on the amount of HER2 antibody present in the formulation, the type of disorder or treatment, and other factors mentioned above.One or more HER2 antibodies can be appropriately administered to patients over a course of treatment.

[0158] Additional therapeutic agents In some examples of any of the methods described herein for treating a subject with HER2-positive cancer, the treatment regimen may include administration of one or more additional therapeutic agents.

[0159] In one example, the additional therapeutic agent is a corticosteroid, which can be administered as a pretreatment prior to (e.g., about 1 hour before) administration of HER2 TDB or an additional HER2 antibody. Corticosteroid premedication can include administration of dexamethasone or methylprednisolone. Additionally or alternatively, the treatment regimen described herein can include administration of acetaminophen, paracetamol, or diphenhydramine (e.g., pretreatment with acetaminophen, paracetamol, or diphenhydramine).

[0160] In some examples, an IL-6R antagonist such as tocilizumab (ACTEMRA® / RoACTEMRA®) is administered as needed, for example, to manage CRS events. In certain embodiments, the IL-6R antagonist (e.g., tocilizumab) is administered intravenously as needed, for example, at a dose of 1 mg / kg to 25 mg / kg (e.g., 5 mg / kg to 10 mg / kg, e.g., about 8 mg / kg).

[0161] In some examples, any of the treatment regimens described herein includes administration of a PD-1 axis-binding antagonist (e.g., a PD-L1-binding antagonist, a PD-1-binding antagonist, or a PD-L2-binding antagonist).

[0162] In some examples, the PD-L1 binding antagonist is an anti-PD-L1 antibody selected from MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO 2010 / 077634. ​​MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874. MEDI4736 (durvalumab) is an anti-PD-L1 monoclonal antibody described in WO 2011 / 066389 and U.S. Patent Application Publication No. 2013 / 034559. Examples of anti-PD-L1 antibodies useful in the methods of the invention, and methods for making them, are described in WO 2010 / 077634, WO 2007 / 005874, WO 2011 / 066389, U.S. Patent No. 8,217,149, and U.S. Patent Application Publication No. 2013 / 034559, which are incorporated herein by reference.

[0163] In some examples, the PD-1 binding antagonist is another anti-PD-1 antibody, such as an anti-PD-1 antibody selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO 2006 / 121168. MK-3475, also known as pembrolizumab or lambrolizumab, is an anti-PD-1 antibody described in WO 2009 / 114335. In another example, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin that includes the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In another example, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor that is described in WO 2010 / 027827 and WO 2011 / 066342.

[0164] In other examples, the PD-L2 binding antagonist is an anti-PD-L2 antibody (e.g., a human, humanized, or chimeric anti-PD-L2 antibody). In some examples, the PD-L2 binding antagonist is an immunoadhesin.

[0165] In a further embodiment, the additional therapeutic agent is an additional chemotherapeutic agent and / or antibody-drug conjugate (ADC). In one embodiment, the HER2 TDB and / or HER2 antibody are co-administered with one or more additional chemotherapeutic agents selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP). In one embodiment, the HER2 TDB and / or HER2 antibody are co-administered with an ADC selected from an anti-CD79b antibody-drug conjugate (e.g., anti-CD79b-MC-vc-PAB-MMAE, or an anti-CD79b antibody-drug conjugate described in any one of U.S. Pat. No. 8,088,378 and / or U.S. Patent Application Publication No. 2014 / 0030280, or polatuzumab vedotin).

[0166] In some examples, the additional therapy includes an alkylating agent. In one example, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid and salts thereof. In one example, the alkylating agent is bendamustine.

[0167] In some examples, the additional therapy includes a BCL-2 inhibitor. In one embodiment, the BCL-2 inhibitor is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide and salts thereof. In one example, the BCL-2 inhibitor is venetoclax (CAS number: 1257044-40-8).

[0168] In some examples, the additional therapy includes a phosphoinositide 3-kinase (PI3K) inhibitor. In one example, the PI3K inhibitor inhibits the δ isoform PI3K (i.e., P110δ). In some examples, the PI3K inhibitor is 5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone and its salts. In some examples, the PI3K inhibitor is idelalisib (CAS number: 870281-82-6). In one example, the PI3K inhibitor inhibits the α and δ isoforms of PI3K. In some examples, the PI3K inhibitor is 2-{3-[2-(1-isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide and salts thereof.

[0169] In a further embodiment of the present invention, the additional therapy comprises a Bruton's tyrosine kinase (BTK) inhibitor. In one example, the BTK inhibitor is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one and salts thereof. In one example, the BTK inhibitor is ibrutinib (CAS number: 936563-96-1).

[0170] In some examples, the additional therapy includes thalidomide or a derivative thereof. In one example, the thalidomide or a derivative thereof is (RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione and its salts. In one example, the thalidomide or a derivative thereof is lenalidomide (CAS number: 191732-72-6).

[0171] Pharmaceutical Compositions and Formulations Pharmaceutical compositions and formulations of the HER2 TDB and / or HER2 antibodies can be prepared by mixing such agents having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0172] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0173] The preparations herein may contain more than one active ingredient, if necessary for the specific indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other.For example, it may be preferable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents and / or antihormonal agents, such as those mentioned herein above).These active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0174] The active ingredient can also be incorporated into microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0175] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0176] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0177] III.Product The present invention further provides an article of manufacture containing materials useful for treating or preventing HER2-positive cancer. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating or preventing a condition, alone or in combination with another composition, and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a HER2-positive derivative described herein. The label or package insert indicates that the composition is used to treat a selected HER2-positive cancer (e.g., HER2-positive breast cancer or HER2-positive gastric cancer) and further includes information related to at least one of the dosing regimens described herein. Additionally, the article of manufacture may include (a) a first container having a composition contained therein, the composition comprising HER2 TDB, and (b) a second container having a composition contained therein, the composition comprising an additional HER antibody (e.g., a multivalent (e.g., bivalent) HER2-binding antibody, e.g., trastuzumab). Alternatively or additionally, the article of manufacture may further include one or more additional containers containing (a) an additional therapeutic agent and / or (b) a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The containers may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0178] IV. Working Examples The following are examples of methods of the present invention: Given the general description above, it will be understood that various other embodiments may be practiced. Example 1 Preclinical Efficacy of BTRC4017A and Trastuzumab Cotreatment

[0179] Using a "knobs-in-holes" design (see, e.g., U.S. Pat. No. 5,731,168), we generated BTRC4017A, a full-length IgG1 TDB that binds both HER2 and CD3 and has an anti-HER2 arm containing a 4D5 HER2-binding site and an anti-CD3 arm containing a 40G5c CD3-binding site (see, e.g., WO 2015 / 095392). The 4D5 HER2-binding site of BTRC4017A is derived from trastuzumab (HERCEPTIN®) and binds the same epitope in domain IV of HER2, as shown in Figure 1. Trastuzumab competes with BTRC4017A for binding to HER2 and can therefore interfere with BTRC4017A activity.

[0180] In vitro pharmacology of BTRC4017A in combination with trastuzumab (Herceptin) The effect of trastuzumab on BTRC4017A activity was tested in vitro and in vivo using the HER2-amplified KPL4 cell line, which represents a HER2-positive cancer. The effect of this combination was also modeled using the HT55 cell line, which expresses low levels of HER2, similar to normal human tissue HER2 levels. HT55 tumors were used to model on-target activity against normal cells / tissues expressing low levels of HER2, while KPL4 tumor cells represent a HER2-overexpressing tumor. MCF7 is a HER2 IHC0 breast cancer cell line included as a negative control. HER2 expression levels in the model cell lines are shown in Figure 2.

[0181] To evaluate the effect of trastuzumab on the in vitro activity of BTRC4017A, a dose-response experiment was performed in the presence of 230 μg / mL and 60 μg / mL trastuzumab. These trastuzumab concentrations correspond to the clinical maximum serum concentrations (C) in breast cancer at the recommended doses (8 or 6 mg / kg every 3 weeks [Q3W]). max ) and minimum serum concentration (Cmin Purified human CD8 was used as an effector to minimize target cell killing by trastuzumab via cell-mediated ADCC. + T cells were used. Trastuzumab inhibited the activity of BTRC4017A in both cell lines (Figures 3A and 3B), and 600- to 2000-fold more BTRC4017A was required to reach a 50% effective concentration for KPL4 (Figure 3A) killing in the presence of trastuzumab. The inhibitory effect of trastuzumab on BTRC4017A activity was in a similar range when targeting HT55 cells, but at higher concentrations, BTRC4017A was able to overcome the inhibitory effect of trastuzumab in both cell lines.

[0182] In vivo pharmacology of BTRC4017A in combination with trastuzumab-LALAPG Fc receptor-mediated effector function plays a major role in the in vivo activity of trastuzumab and may interfere with in vivo experiments addressing the inhibitory effect of trastuzumab on BTRC4017A activity. Therefore, to generate the trastuzumab-LALAPG variant, we modified the Fc region of trastuzumab by introducing a set of amino acid substitutions that attenuate the effector function of human IgG1. The LALAPG mutations are L234A, L235A, and P329G. Because the anti-HER2 Fab in trastuzumab was not modified in the trastuzumab-LALAPG variant, this modification did not alter HER2 binding, as confirmed by flow cytometry HER2 binding assays (Figure 4).

[0183] To test the effect of trastuzumab / LALAPG on BTRC4017A activity, we used a dual-tumor mouse model based on severely immunodeficient NOD scid gamma (NSG) mice. NSG mice were supplemented with human T cells by intraperitoneal injection of human peripheral mononuclear cells (PBMCs). In each mouse, a KPL4 tumor was implanted in one flank and an HT55 tumor in the opposite flank. KPL4 is a HER2-amplified cell line and corresponds to a HER2-overexpressing tumor, whereas the HT55 tumor was used to model on-target / off-tumor activity against normal cells / tissues expressing low levels of HER2 (Figure 2).

[0184] As shown in Figure 5A, a single dose of BTRC4017A at 0.05 mg / kg or higher induced regression of KPL4 tumors. A 10-fold higher dose of BTRC4017A was required to induce regression of HT55 tumors (Figure 5B), demonstrating the therapeutic index can be increased based on the high expression of HER2 in HER2-positive tumors.

[0185] In the co-treatment group, 5.0 mg / kg of trastuzumab-LALAPG was administered 4 hours before BTRC4017A administration. Trastuzumab-LALAPG did not affect the efficacy of BTRC4017A in targeting KPL4 tumors at any of the BTRC4017A dose levels tested. In contrast, trastuzumab-LALAPG pretreatment abolished BTRC4017A activity in HT55 tumors at all BTRC4017A dose levels. These data demonstrate that the effect of trastuzumab-LALAPG pretreatment on BTRC4017A activity differs dramatically between tumors expressing different levels of HER2. Notably, activity was retained in KPL4 tumors (HER2 amplified) but lost in HT55 tumors, which express HER2 at levels similar to normal human tissues.

[0186] These data suggest that treatment with trastuzumab prior to BTRC4017A administration may reduce the risk of on-target and off-tumor toxicity of BTRC4017A against normal tissues expressing low levels of HER2, while maintaining its antitumor activity in HER2-overexpressing tumors. Coadministration of trastuzumab and BTRC4017A in an in vivo mouse efficacy study did not impair the antitumor activity of BTRC4017A against HER2-positive tumors, but completely abolished BTRC4017A antitumor activity against tumors expressing low HER2, which is representative of normal tissues. Without wishing to be bound by theory, trastuzumab may saturate HER2 in normal tissues expressing low levels of HER2 (thereby preventing BTRC4017A binding), but fails to saturate HER2 in tumors expressing higher HER2 levels. Therefore, coadministration of trastuzumab before each dose of BTRC4017A can mitigate the off-tumor / on-target toxicity of BTRC4017A in HER2-expressing normal tissues without significantly affecting the antitumor activity of BTRC4017A in patients with HER2-positive tumors. Thus, coadministration of trastuzumab can increase the therapeutic index of BTRC4017A.

[0187] Example 2: Fractionated, dose-escalating dosing regimen for the treatment of HER2-positive cancer with BTRC4017A and trastuzumab To reduce toxicity potentially caused by cytokines, BTRC4017A is administered in a split-dose regimen in cycle 1 (C1), where the first dose is less than the second dose. In a two-stage split-dose regimen, the second dose in C1 is less than the third dose. Cycle 2 and any necessary subsequent cycles include a single administration of a BTRC4017A dose equal to the highest dose of BTRC4017A in C1.

[0188] To adequately differentiate any infusion-related reactions (IRR) that may be associated with BTRC4017A versus trastuzumab, trastuzumab will be administered on day -1 of C1. All subsequent trastuzumab doses from cycle 2 (C2) onward will be administered on day 1 of the cycle, prior to administration of BTRC4017A. A summary of the trastuzumab administration procedure is provided in Table 4 below. JPEG0007730761000004.jpg64170C1-day-1 = cycle1-day-1; C1 day 1 = cycle 1 day 1; Day 1 of C2 = Day 1 of Cycle 2

[0189] BTRC4017A is administered by split dosing during the 21-day first cycle and on Day 1 of each subsequent cycle for up to 17 cycles. BTRC4017A is administered by intravenous infusion using a standard medical syringe and syringe pump or, as appropriate, an intravenous infusion bag at a fixed dose independent of body weight. The medication is delivered by syringe pump via an intravenous infusion set or intravenous infusion bag to which a final BTRC4017A volume, determined by the dose, is added. At the initial low dose, BTRC4017A is administered only via a peripheral catheter. The specific BTRC4017A dose determines the appropriate dosage concentration, volume, and infusion time, as well as the specific administration device to be used (e.g., peripheral catheter vs. syringe pump vs. intravenous infusion bag).

[0190] Beginning on day 1 of C1, subjects will receive escalating doses of BTRC4017A on days 1 and 8 or days 1, 8, and 15 for the one- and two-phase fractionated regimens, respectively. From cycle 2 onwards, BTRC4017A will be given as a single dose only on day 1 of each 21-day cycle. For logistical / scheduling reasons, BTRC4017A may be given up to ±2 days from the scheduled date (i.e., there will be a minimum of 19 days between doses).

[0191] Corticosteroid premedication consisting of dexamethasone (20 mg intravenous) or methylprednisolone (80 mg intravenous) will be administered 1 hour prior to the administration of each BTRC4017A dose in Cycle 1. Additionally, premedication with oral acetaminophen or paracetamol (e.g., 500–1000 mg) and / or 25–50 mg diphenhydramine will be administered according to standard institutional practice prior to the administration of BTRC4017A, unless contraindicated. Tocilizumab can be administered intravenously to patients at 8 mg / kg if needed.

[0192] In the absence of dose-limiting toxicity (DLT), unacceptable toxicity, or disease progression, patients experiencing clinical benefit will receive continuation of treatment with BTRC4017A as a single agent or in combination with trastuzumab every 21 days for up to 17 cycles until progression or intolerable toxicity, whichever occurs first.

[0193] Disease status is assessed using the Response Evaluation Criteria in Solid Tumors (RECIST v1.1). Patients undergo tumor evaluations at screening, throughout the study until treatment discontinuation, and at the end of the study. Immune-modified RECIST criteria are also used to characterize patterns of response associated with cancer immunotherapy. Immune-modified RECIST criteria can complement standard RECIST v1.1 criteria to enable a comprehensive assessment of patient benefit and risk. In the setting of BTRC4017A and bispecific antibodies in general, pseudoprogression may be observed. Therefore, patients experiencing clinical benefit despite radiological evidence of progressive disease as defined by standard RECIST v1.1 criteria may continue study treatment.

[0194] Adverse events will be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, Version 5.0 (NCI CTCAE v5.0), except for cytokine release syndrome (CRS), which will be graded according to the Modified Cytokine Release Syndrome Grading System (see Tables 1 and 2 above).

[0195] Other embodiments While the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 antibody, wherein the medicament is administered in a treatment regimen in combination with a HER2 T-cell dependent bispecific antibody (TDB), wherein the HER2 TDB comprises an anti-HER2 arm and an anti-CD3 arm, and wherein the HER2 antibody and the HER2 TDB competitively bind to domain IV of HER2; the HER2 antibody is (i) a complementarity determining region (CDR)-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 Including, the anti-HER2 arm of the HER2 TDB is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a HER2 binding domain comprising the anti-CD3 arm of the HER2 TDB (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a CD3 binding domain comprising wherein the HER2 antibody is administered first, simultaneously with or prior to administration of the HER2 TDB, and the treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

2. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 TDB, wherein the medicament is administered in a treatment regimen in combination with a HER2 antibody, the HER2 TDB comprising an anti-HER2 arm and an anti-CD3 arm, and wherein the HER2 antibody and the HER2 TDB competitively bind to domain IV of HER2; the HER2 antibody is (i) a complementarity determining region (CDR)-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 Including, the anti-HER2 arm of the HER2 TDB is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a HER2 binding domain comprising the anti-CD3 arm of the HER2 TDB (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a CD3 binding domain comprising wherein the HER2 antibody is administered first, simultaneously with or prior to administration of the HER2 TDB, and the treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

3. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 antibody and a HER2 TDB, wherein the medicament is administered in a treatment regimen, the HER2 TDB comprising an anti-HER2 arm and an anti-CD3 arm, and the HER2 antibody and the HER2 TDB competitively bind to domain IV of HER2; the HER2 antibody is (i) a complementarity determining region (CDR)-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 Including, the anti-HER2 arm of the HER2 TDB is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a HER2 binding domain comprising: the anti-CD3 arm of the HER2 TDB (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a CD3 binding domain comprising wherein the HER2 antibody is administered first, simultaneously with or prior to administration of the HER2 TDB, and the treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

4. The medicament of any one of claims 1 to 3, wherein the increased therapeutic index is accompanied by a reduced likelihood of experiencing on-target / off-tumor effects compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

5. The pharmaceutical of claim 4, wherein the on-target / off-tumor effect is a symptom of pulmonary toxicity.

6. The pharmaceutical composition of claim 5, wherein the symptoms of pulmonary toxicity are selected from the group consisting of interstitial lung disease, acute respiratory distress syndrome, dyspnea, cough, fatigue, and pulmonary infiltrates.

7. The medicament of claim 4, wherein the on-target / off-tumor effect is selected from the group consisting of elevated liver enzyme levels, dry mouth, dry eyes, mucositis, esophagitis and urinary symptoms.

8. The medicament of any one of claims 1 to 7, wherein the increased therapeutic index is accompanied by a reduced likelihood of experiencing immunogenic side effects compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

9. The pharmaceutical composition of claim 8, wherein the immunogenic side effect is selected from the group consisting of elevated levels of anti-drug antibodies, infusion / administration-related reactions (ARR), cardiac dysfunction, pulmonary reactions, and cytokine release syndrome.

10. The pharmaceutical according to any one of claims 1 to 9, wherein the HER2 antibody comprises a variable heavy chain domain (V H ) having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:7 and / or a variable light chain domain (V L ) having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

8.

11. The pharmaceutical according to any one of claims 1 to 10, wherein the HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and / or a VL comprising the amino acid sequence of SEQ ID NO:

8.

12. The V Ｈ comprises the amino acid sequence of SEQ ID NO: 7, and Ｌ The pharmaceutical composition of claim 11, wherein the amino acid sequence of SEQ ID NO: 8 is included.

13. The pharmaceutical according to any one of claims 1 to 12, wherein the HER2 antibody is monospecific.

14. The pharmaceutical according to any one of claims 1 to 13, wherein the HER2 antibody is a full-length antibody containing an Fc region.

15. The pharmaceutical agent according to any one of claims 1 to 14, wherein the HER2 antibody is trastuzumab.

16. The pharmaceutical according to any one of claims 1 to 14, wherein the HER2 antibody is an Fc-modified trastuzumab variant.

17. The medicament of claim 16, wherein the Fc-modified trastuzumab variant comprises one or more amino acid modifications that reduce effector function.

18. The pharmaceutical of claim 17, wherein the one or more amino acid modifications are substitution mutations.

19. The pharmaceutical of claim 18, wherein the substitution mutation is at amino acid residues L234, L235 and / or P329 (EU numbering).

20. 20. The medicament of claim 19, wherein the one or more amino acid modifications comprise substitution mutations L234A, L235A and P329G (LALAPG).

21. The HER2 binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

7. Ｈ and / or V having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:8 Ｌ The pharmaceutical composition according to any one of claims 1 to 20, comprising:

22. the V of the HER2 binding domain Ｈ comprises the amino acid sequence of SEQ ID NO: 7, and / or the V Ｌ The pharmaceutical composition of claim 21 , wherein the amino acid sequence of SEQ ID NO: 8 is included.

23. The CD3 binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

15. Ｈ and / or V having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 Ｌ The pharmaceutical composition according to any one of claims 1 to 22, comprising:

24. the V of the CD3-binding domain Ｈ comprises the amino acid sequence of SEQ ID NO: 15, and / or the V Ｌ The pharmaceutical composition of claim 23, wherein the amino acid sequence of SEQ ID NO: 16 is included.

25. (i) the anti-HER2 arm of the HER2 TDB comprises: (a) a V comprising the amino acid sequence of SEQ ID NO: 7; Ｈ and (b) V comprising the amino acid sequence of SEQ ID NO:

8. Ｌ and (ii) the anti-CD3 arm of the HER2 TDB comprises a HER2 binding domain comprising: (a) a V comprising the amino acid sequence of SEQ ID NO:

15. Ｈ and (b) V comprising the amino acid sequence of SEQ ID NO:

16. Ｌ The pharmaceutical according to any one of claims 1 to 24, comprising a CD3 binding domain comprising:

26. The pharmaceutical according to any one of claims 1 to 25, wherein the HER2 TDB is a full-length antibody comprising a modified Fc region.

27. The pharmaceutical of claim 26, wherein the modified Fc region comprises one or more substitution mutations that reduce the effector function of the HER2 TDB.

28. 28. The pharmaceutical of claim 27, wherein the one or more substitution mutations comprise mutations at amino acid residues L234, L235 and / or D265 (EU numbering).

29. The pharmaceutical of claim 28, wherein the one or more substitution mutations are L234A, L235A, and D265A.

30. The pharmaceutical of claim 27 , wherein the one or more substitution mutations comprise an aglycosylation site mutation.

31. The pharmaceutical agent of claim 30, wherein the aglycosylation site mutation is at amino acid residue N297 (EU numbering).

32. The pharmaceutical composition of claim 30, wherein the aglycosylation site mutation is N297G.

33. The pharmaceutical composition of claim 30, wherein the aglycosylation site mutation is N297A.

34. The pharmaceutical of any one of claims 26 to 32, wherein the modified Fc region comprises N297G, L234A, L235A and D265A substitution mutations.

35. The HER2 TDB comprises one or more heavy chain constant domains, and the one or more heavy chain constant domains comprise a first CH1 (CH1 １ ) domain, the first CH2 (CH2 １ ) domain, the first CH3 (CH3 １ ) domain, a second CH1 (CH1 ２ ) domain, the second CH2 (CH2 ２ ) domain and a second CH3 (CH3 ２ The pharmaceutical agent according to any one of claims 1 to 34, wherein the domain is selected from the group consisting of α- and β-glucan domains.

36. at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain; (i) the CH3 １ and CH3 ２ Each domain contains a protuberance or a cavity, and the CH3 １ The protuberance or cavity in the domain is the CH3 ２ or is disposable in said cavity or protuberance in the domain; or (ii) the CH2 １ and CH2 ２ Each domain contains a protuberance or a cavity, and the CH2 １ The protuberance or cavity in the domain is, respectively, the CH2 ２ can be located in said cavity or protuberance in the domain The pharmaceutical composition of claim 35.

37. 1. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 antibody, said medicament being administered in a treatment regimen in combination with a HER2 TDB, wherein (a) said HER2 antibody is trastuzumab or an Fc-modified trastuzumab variant, (b) said HER2 TDB comprises an anti-HER2 arm and an anti-CD3 arm, and said anti-HER2 arm is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a HER2 binding domain comprising: The anti-CD3 arm (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 and a CD3 binding domain comprising wherein the HER2 antibody is administered first, simultaneously with or prior to administration of the HER2 TDB, and the treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

38. 1. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 TDB, wherein the medicament is administered in a treatment regimen in combination with a HER2 antibody, wherein (a) the HER2 antibody is trastuzumab or an Fc-modified trastuzumab variant, and (b) the HER2 TDB comprises an anti-HER2 arm and an anti-CD3 arm, wherein the anti-HER2 arm is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a HER2 binding domain comprising: The anti-CD3 arm (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 and a CD3 binding domain comprising the HER2 antibody is administered first, simultaneously with or prior to administration of the HER2 TDB; The treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

39. 1. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 antibody and a HER2 TDB, wherein the medicament is administered in a treatment regimen, wherein (a) the HER2 antibody is trastuzumab or an Fc-modified trastuzumab variant, (b) the HER2 TDB comprises an anti-HER2 arm and an anti-CD3 arm, and the anti-HER2 arm is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a HER2 binding domain comprising: The anti-CD3 arm (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 and a CD3 binding domain comprising the HER2 antibody is administered first, simultaneously with or prior to administration of the HER2 TDB; The treatment regimen results in an increased therapeutic index of the HER2 TDB compared to treatment with the HER2 TDB in the absence of the HER2 antibody.

40. The pharmaceutical agent according to any one of claims 1 to 39, wherein the HER2 antibody is administered before the HER2 TDB is administered.

41. The pharmaceutical agent according to any one of claims 1 to 40, wherein the HER2 antibody is administered at a dose of 5 mg / kg to 10 mg / kg.

42. The pharmaceutical of any one of claims 1 to 41, wherein the HER2 antibody is administered approximately once every three weeks.

43. The pharmaceutical agent according to any one of claims 1 to 42, wherein the HER2 TDB is administered at a fixed dose of 0.001 mg to 500 mg.

44. The method of any one of claims 1 to 43, wherein the HER2 TDB is administered about once every three weeks.

45. the treatment regimen comprising: (a) a first dose of the HER2 antibody; (b) a first dosing cycle (C1) after the first dose of the HER2 antibody, wherein the C1 comprises a first dose of the HER2 TDB (C1D1) and a second dose of the HER2 TDB (C1D2), wherein the C1D2 is greater than the C1D1; (c) a second dosing cycle (C2) after C1, wherein C2 comprises: (i) a second dose of the HER2 antibody; and (ii) an additional dose of the HER2 TDB (C2D1) after the second dose of the HER2 antibody, wherein the C2D1 is equal in amount to the highest dose of the HER2 TDB in C1; and a second dosing cycle (C2) comprising: The pharmaceutical composition according to any one of claims 1 to 44, comprising:

46. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 antibody, wherein the medicament is administered in a treatment regimen in combination with a HER2 TDB, the HER2 TDB comprising an anti-HER2 arm and an anti-CD3 arm, and the HER2 antibody and the HER2 TDB competitively bind to domain IV of HER2; the HER2 antibody is (i) a complementarity determining region (CDR)-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 Including, the anti-HER2 arm of the HER2 TDB is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a HER2 binding domain comprising the anti-CD3 arm of the HER2 TDB (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a CD3 binding domain comprising The treatment regimen comprises: (a) a first dose of the HER2 antibody; (b) a first dosing cycle (C1) after the first dose of the HER2 antibody, wherein the C1 comprises a first dose of the HER2 TDB (C1D1) and a second dose of the HER2 TDB (C1D2), wherein the C1D2 is greater than the C1D1; (c) a second dosing cycle (C2) after C1, wherein C2 comprises: (i) a second dose of the HER2 antibody; and (ii) an additional dose of the HER2 TDB (C2D1) after the second dose of the HER2 antibody, wherein the C2D1 is equal in amount to the highest dose of the HER2 TDB in C1; and a second dosing cycle (C2) comprising: Pharmaceuticals, including

47. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 TDB, wherein the medicament is administered in a treatment regimen in combination with a HER2 antibody, the HER2 TDB comprising an anti-HER2 arm and an anti-CD3 arm, and wherein the HER2 antibody and the HER2 TDB competitively bind to domain IV of HER2; the HER2 antibody is (i) a complementarity determining region (CDR)-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 Including, the anti-HER2 arm of the HER2 TDB is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a HER2 binding domain comprising the anti-CD3 arm of the HER2 TDB (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a CD3 binding domain comprising The treatment regimen comprises: (a) a first dose of the HER2 antibody; (b) a first dosing cycle (C1) after the first dose of the HER2 antibody, wherein the C1 comprises a first dose of the HER2 TDB (C1D1) and a second dose of the HER2 TDB (C1D2), wherein the C1D2 is greater than the C1D1; (c) a second dosing cycle (C2) after C1, wherein C2 comprises: (i) a second dose of the HER2 antibody; and (ii) an additional dose of the HER2 TDB (C2D1) after the second dose of the HER2 antibody, wherein the C2D1 is equal in amount to the highest dose of the HER2 TDB in C1; and a second dosing cycle (C2) comprising: Pharmaceuticals, including

48. A medicament for treating or delaying the progression of a HER2-positive cancer in a subject in need thereof, comprising a HER2 antibody and a HER2 TDB, wherein the medicament is administered in a treatment regimen, the HER2 TDB comprising an anti-HER2 arm and an anti-CD3 arm, and the HER2 antibody and the HER2 TDB competitively bind to domain IV of HER2; the HER2 antibody is (i) a complementarity determining region (CDR)-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 Including, the anti-HER2 arm of the HER2 TDB is (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 and a HER2 binding domain comprising the anti-CD3 arm of the HER2 TDB (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a CD3 binding domain comprising The treatment regimen comprises: (a) a first dose of the HER2 antibody; (b) a first dosing cycle (C1) after the first dose of the HER2 antibody, wherein the C1 comprises a first dose of the HER2 TDB (C1D1) and a second dose of the HER2 TDB (C1D2), wherein the C1D2 is greater than the C1D1; (c) a second dosing cycle (C2) after C1, wherein C2 comprises: (i) a second dose of the HER2 antibody; and (ii) an additional dose of the HER2 TDB (C2D1) after the second dose of the HER2 antibody, wherein the C2D1 is equal in amount to the highest dose of the HER2 TDB in C1; and a second dosing cycle (C2) comprising: Pharmaceuticals, including

49. The pharmaceutical agent of any one of claims 45 to 48, wherein the first dose of the HER2 antibody is administered one day before the C1D1, and the subject is monitored for a period of 30 minutes to 24 hours between the first dose of the HER2 antibody and the C1D1.

50. The pharmaceutical agent of any one of claims 45 to 49, wherein the first dose of the HER2 antibody is 5 mg / kg to 10 mg / kg.

51. 51. The medicament of claim 50, wherein the first dose of the HER2 antibody is 6 mg / kg or 8 mg / kg.

52. The pharmaceutical agent of any one of claims 45 to 51, wherein the second dose of the HER2 antibody is 5 mg / kg to 10 mg / kg.

53. 53. The method of claim 52, wherein the second dose of the HER2 antibody is 6 mg / kg.

54. 54. The method of any one of claims 45 to 53, wherein the first and / or second dose of the HER2 antibody is administered by infusion over a period of at least 30 minutes.

55. The pharmaceutical agent of any one of claims 45 to 54, wherein the second dose of the HER2 antibody is administered on the same day as the C2D1.

56. The pharmaceutical agent of any one of claims 45 to 55, wherein the C1D2 is administered at a dose at least twice that of the C1D1.

57. 57. The method of claim 56, wherein the C1D2 is at least three times the dose of the C1D1.

58. The pharmaceutical composition according to any one of claims 45 to 57, wherein the C1D1 is 0.003 mg to 50 mg.

59. The pharmaceutical composition according to any one of claims 45 to 58, wherein the C1D2 is 0.009 mg to 200 mg.

60. The pharmaceutical composition of any one of claims 45 to 59, wherein the amounts of C2D1 and C1D2 are equal.

61. The pharmaceutical agent of any one of claims 45 to 59, wherein the C1 further comprises a third dose (C1D3) of the HER2 TDB, and the C1D3 is greater than the C1D2.

62. 62. The pharmaceutical agent of claim 61, wherein said C1D1, said C1D2 and said C1D3 are cumulatively greater than the maximum clearing dose of said HER2 TDB in the first dosing cycle of a single-step fractionated, dose-escalating dosing regimen.

63. The pharmaceutical composition of claim 62, wherein the maximum removal dose is from about 0.01 mg to about 30 mg.

64. The pharmaceutical agent according to any one of claims 61 to 63, wherein the C1D2 is administered at a dose 2 to 10 times that of the C1D1.

65. The pharmaceutical agent according to any one of claims 61 to 64, wherein the C1D3 is administered at a dose that is 2 to 3 times the dose of the C1D2.

66. The pharmaceutical composition of any one of claims 61 to 65, wherein the amounts of C2D1 and C1D3 are equal.

67. The pharmaceutical composition according to any one of claims 61 to 66, wherein the C1D1 is 0.01 mg to 20 mg.

68. The pharmaceutical composition according to any one of claims 61 to 67, wherein the C1D2 is 0.1 mg to 100 mg.

69. The pharmaceutical composition according to any one of claims 61 to 68, wherein the C1D3 is 1 mg to 200 mg.

70. The pharmaceutical agent according to any one of claims 61 to 69, wherein C1D1, C1D2 and C1D3 are administered to the subject on days 1, 8 and 15 of C1, respectively.

71. The pharmaceutical agent according to any one of claims 45 to 70, wherein the C1 length is 21 days.

72. The pharmaceutical agent according to any one of claims 45 to 71, wherein the length of C2 is 21 days.

73. The pharmaceutical agent according to any one of claims 45 to 72, wherein the C2D1 is administered to the subject on day 1 of the C2.

74. The method of any one of claims 45 to 73, wherein the treatment regimen comprises one or more additional dosing cycles.

75. 75. The method of claim 74, wherein the treatment regimen comprises up to 15 additional dosing cycles.

76. 76. The method of claim 74 or 75, wherein each of the one or more additional dosing cycles is 21 days in length.

77. The medicament of any one of claims 74 to 76, wherein each of the one or more additional dosing cycles comprises a single dose of the HER2 antibody and a single dose of the HER2 TDB.

78. The medicament of any one of claims 74 to 77, wherein the HER2 antibody is administered to the subject simultaneously with or prior to administration of the HER2 TDB on day 1 of each of the one or more additional dosing cycles.

79. 79. The method of claim 78, wherein the HER2 antibody is administered before the HER2 TDB on day 1 of each of the one or more additional dosing cycles.

80. The method of any one of claims 46 to 79, wherein the treatment regimen results in an increased therapeutic index for the HER2 TDB compared to a control treatment regimen.

81. The pharmaceutical agent of any one of claims 1 to 80, wherein the HER2 antibody and / or the HER2 TDB is administered by intravenous infusion.

82. 82. The medicament of any one of claims 1 to 81, wherein the medicament is administered in combination with one or more additional therapeutic agents.

83. 83. The medicament of claim 82, wherein the one or more additional therapeutic agents are selected from the group consisting of tocilizumab, corticosteroids, PD-1 axis binding antagonists, and antibody-drug conjugates.

84. The method of claim 83, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.

85. The pharmaceutical agent of claim 84, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

86. The method of claim 85, wherein the PD-L1 binding antagonist is selected from the group consisting of MPDL3280A (atezolizumab), MDX-1105, and MEDI4736.

87. The method of claim 84, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

88. 88. The pharmaceutical agent of claim 87, wherein the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), and AMP-224.

89. The method of claim 84, wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

90. 90. The method of claim 89, wherein the PD-L2 binding antagonist is an antibody or an immunoadhesin.

91. The method of any one of claims 1 to 90, wherein the subject has received trastuzumab in a previous treatment regimen.

92. The pharmaceutical agent according to any one of claims 1 to 91, wherein the HER2-positive cancer is a HER2-positive solid tumor.

93. The pharmaceutical agent of any one of claims 1 to 92, wherein the HER2-positive cancer is locally advanced or metastatic HER2-positive cancer.

94. The pharmaceutical agent according to any one of claims 1 to 93, wherein the HER2-positive cancer is HER2-positive breast cancer or HER2-positive gastric cancer.

95. The pharmaceutical agent of any one of claims 1 to 93, wherein the HER2-positive cancer is selected from the group consisting of HER2-positive gastroesophageal junction cancer, HER2-positive colorectal cancer, HER2-positive lung cancer, HER2-positive pancreatic cancer, HER2-positive bladder cancer, HER2-positive salivary duct cancer, HER2-positive ovarian cancer, or HER2-positive endometrial cancer.

96. The pharmaceutical agent of claim 95, wherein the HER2-positive lung cancer is HER2-positive non-small cell lung carcinoma.

97. The pharmaceutical agent of claim 95, wherein the HER2-positive ovarian cancer is HER2-positive ovarian epithelial cancer.

Images