METHODS USING MONOVALENT ANTIGEN BINDING CONSTRUCTS TARGETING HER2

1 . A method of treating a subject comprising, administering an effective amount of a first monovalent antigen-binding construct or a combination of a first and a second monovalent antigen-binding construct to the subject,

a) wherein the first and second monovalent antigen-binding constructs each comprise an antigen-binding polypeptide construct and a dimeric Fc coupled, with or without a linker, to the antigen-binding polypeptide construct;

b) each antigen-binding polypeptide construct specifically binds a extracellular domain 2 (ECD2) of human epidermal growth factor receptor 2 (HER2), a ECD4 of HER2, or a ECD1 of HER2;

c) the first monovalent antigen-binding construct and the second monovalent antigen-binding construct bind to non-overlapping epitopes and do not compete with each other for binding to HER2,

d) wherein the first monovalent antigen-binding construct comprises v1040 and the second monovalent antigen-binding construct comprises v4182, and

e) wherein treating the subject is treating a HER2+ cancer that expresses HER2 at the 2+ level or lower as determined by immunohistochemistry (IHC).

2 . A method of treating a subject comprising, administering an effective amount of a first monovalent antigen-binding construct or a combination of a first and a second monovalent antigen-binding construct to the subject,

a) wherein the first and second monovalent antigen-binding construct each comprise an antigen-binding polypeptide construct and a dimeric Fc coupled, with or without a linker, to the antigen-binding polypeptide construct;

b) each antigen-binding polypeptide construct specifically binds a extracellular domain 2 (ECD2) of human epidermal growth factor receptor 2 (HER2), a ECD4 of HER2, or a ECD1 of HER2; and

c) the first monovalent antigen-binding construct and the second monovalent antigen-binding construct bind to non-overlapping epitopes and do not compete with each other for binding to HER2.

3 . The method of claim 1 or 2 , wherein treating a subject is inhibiting growth of a HER2+ tumor, delaying progression of a HER2+ tumor, treating a HER2+ cancer or preventing a HER2+ cancer.

4 . The method of claim 3 , wherein the HER2+ tumor or cancer is selected from breast, ovarian, stomach, gastroesophageal junction, endometrial, salivary gland, head and neck, lung, brain, kidney, colon, colorectal, thyroid, pancreatic, prostate or bladder.

5 . The method of claim 4 , wherein the HER2+ tumor or cancer is selected from breast, ovarian, stomach, lung, or brain.

6 . The method of claim 4 , wherein the HER2+ tumor or cancer expresses HER2 at a 2+ level or lower, as determined by immunohistochemistry (IHC).

7 . The method of claim 4 , wherein the HER2+ tumor or cancer is an ovarian cancer that expresses HER2 at a 2+ or 3+ level, as determined by immunohistochemistry (IHC).

8 . The method of claim 4 , wherein the HER2+ tumor or cancer is a breast cancer.

9 . The method of claim 8 , wherein the breast cancer expresses HER2 at a 2+ level or lower, as determined by immunohistochemistry (IHC).

10 . The method of claim 8 , wherein the breast cancer is a trastuzumab-resistant breast cancer, a chemotherapy-resistant breast cancer, a triple-negative breast cancer, an estrogen receptor-negative breast cancer, or a estrogen receptor-positive breast cancer.

11 . The method of claim 1 or 2 , wherein the treating is treating or preventing a HER2+ metastatic cancer.

12 . The method of claim 11 , wherein the HER2+ metastatic cancer is a metastatic breast cancer, metastatic brain cancer or a metastatic lung cancer.

13 . The method of claim 11 , wherein the HER2+ cancer is an established primary and metastatic breast cancer, or a lung metastasis or brain metastasis of a breast cancer.

14 . The method of claim 1 , wherein the subject is a human.

15 . The method of any of the above claims, the first monovalent antigen-binding construct comprising v1040 and the second monovalent antigen-binding construct comprising v4182.

16 . The method of any one of claims 1 to 15 , wherein the dimeric Fc is a heterodimeric Fc comprising at least two CH3 sequences and the dimerized CH3 sequences have a melting temperature (Tm) of about 68° C. or higher.

17 . The method of any one of claims 1 to 16 , wherein each monovalent antigen-binding construct selectively and/or specifically binds HER2 with a greater maximum binding (Bmax) as compared to a monospecific bivalent antigen-binding construct that specifically binds HER2, and wherein at a monovalent antigen-binding construct to target ratio of 1:1 the increase in Bmax relative to the monospecific bivalent antigen-binding construct is observed at a concentration greater than the observed equilibrium constant (KD) of the constructs up to saturating concentrations.

18 . The method of any one of claims 2 to 17 , wherein the antigen-binding polypeptide construct of the first monovalent antigen-binding construct comprises the v1040 antigen-binding polypeptide amino acid sequence and the antigen-binding polypeptide construct of the second monovalent antigen-binding construct comprises the v4182 antigen-binding polypeptide construct amino acid sequence.

19 . The method of any one of claims 2 to 17 , wherein the antigen-binding polypeptide construct of the first monovalent antigen-binding construct comprises an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the v1040 antigen-binding polypeptide construct and the antigen-binding polypeptide construct of the second monovalent antigen-binding construct comprises an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the v1040 antigen-binding polypeptide construct.

20 . The method of any one of claims 2 to 17 , wherein the first monovalent antigen-binding construct comprises a heterodimeric Fc of v1040 and the second monovalent antigen-binding construct comprises a heterodimeric Fc of v4182.

21 . The method of any one of claims 2 to 17 , wherein the first monovalent antigen-binding construct and the second monovalent antigen-binding construct are selected from v1041, v1041, v4182, v630, v878, v4442, v4443, v4444, and v4445.

22 . The method of any one of claims 2 to 17 , wherein the combination of first monovalent antigen-binding construct and second monovalent antigen-binding construct is v1040 and v4182.

23 . The method of any one of claims 2 to 17 , wherein only a first monovalent antigen-binding construct is administered and the first monovalent antigen-binding construct is selected from v1041, v1041, v4182, v630, v878, v4442, v4443, v4444, and v4445.

24 . The method of claim 16 , wherein each heterodimeric Fc

a. is a human Fc; and/or

b. is a human IgG1 Fc; and/or

c. comprises one or more modifications in at least one of the CH3 domains; and/or

d. comprises one or more modifications in at least one of the CH3 domains that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc; and/or

e. comprises one or more modifications in at least one of the CH3 domains as described in Table A2

f. further comprises at least one CH2 domain; and/or

g. further comprises at least one CH2 domain comprising one or more modifications; and/or

h. further comprises at least one CH2 domain comprising one or more modifications in at least one of the CH2 domains as described in Table A2; and/or

i. comprises one or more modifications to promote selective binding of Fc-gamma receptors.

25 . The method of claim 24 , wherein the dimerized CH3 domains have a melting temperature (Tm) of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, 83, 84, or 85° C. or higher.

26 . The method of claim 24 , wherein each heterodimeric Fc domain is fused to the antigen-binding polypeptide construct by a linker.

27 . The method of claim 26 , wherein the linker is a polypeptide linker.

28 . The method of claim 26 , wherein the linker comprises an IgG1 hinge region.

29 . The method of any one of claims 2 to 28 , wherein the first and/or second monovalent antigen-binding construct is conjugated to a drug.

30 . The method of any one of claims 2 to 29 , wherein the first and/or second monovalent antigen-binding construct is conjugated to maytansine (DM1).

31 . The method of any one of claims 2 to 30 , comprising administering the first and second monovalent antigen-binding constructs in a pharmaceutical composition.

32 . The method of any one of claims 2 to 30 , comprising administering the first and second monovalent antigen-binding constructs in a pharmaceutical composition comprising a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an amino acid, a carbohydrate, a lipid, a chelating agent, a stabilizer, or an excipient.

33 . The method of any one of claims 1 to 32 , wherein the first and second monovalent antigen-binding constructs are co-administered.

34 . The method of any one of claims 1 to 33 , further comprising administering an additional agent.

35 . The method of one of claims 1 to 34 , wherein administering is orally or through injection.

36 . A pharmaceutical composition for use in the method of any one of claims 1 to 35 .

37 . A method for

a. shrinking a tumor in a subject and/or

b. increasing overall survival in the subject wherein the subject has a tumor; and/or

c. treating a disorder characterized by HER2 expression in the subject; and/or

d. treating a disorder characterized by HER2 expression in the breast, colon, ovarian, gastro-intestinal, or brain tissue of the subject; and/or

e. treating a disorder characterized by HER2 expression in the subject wherein the subject is refractory or resistant to anti-HER2 treatments comprising trastuzumab and/or pertuzumab and/or trastuzumab emtansine (T-DM1); and/or

f. treating a cancer in the subject; and/or

g. treating a cancer in the subject wherein the subject is refractory to chemotherapy Standard of Care (SoC); and/or

h. treating a breast cancer in the subject;

the method comprising administering an effective amount of a first monovalent antigen-binding construct or a combination of a first and a second monovalent antigen-binding construct to the subject,

a) wherein the first and second monovalent antigen-binding construct each comprise at least one antigen-binding polypeptide construct and a dimeric Fc coupled, with or without a linker, to the antigen-binding polypeptide construct;

b) each antigen-binding polypeptide construct specifically binds a extracellular domain 2 (ECD2) of human epidermal growth factor receptor 2 (HER2), a ECD4 of HER2, or a ECD1 of HER2; and

c) the first monovalent antigen-binding construct and the second monovalent antigen-binding construct bind to non-overlapping epitopes and do not compete with each other for binding to HER2.

38 . The method of claim 37 , wherein the dimeric Fc comprises at least two CH3 domains and the dimerized CH3 domains have a melting temperature (Tm) of about 68° C. or higher.

39 . The method of claim 37 , wherein each monovalent antigen-binding construct selectively and/or specifically binds HER2 with a greater maximum binding (Bmax) as compared to monospecific bivalent antigen-binding construct that specifically binds HER2, and wherein at a construct to target ratio of 1:1 the increase in Bmax relative to the monospecific bivalent antigen-binding construct is observed at a concentration greater than the observed equilibrium constant (KD) of the constructs up to saturating concentrations.

40 . The method of claim 37 , wherein the first and second monovalent antigen-binding constructs are characterized by:

a. higher cell surface decoration in SKOV3 cells as determined by FACS and/or confocal microscopy when contacting cells with both constructs compared to contacting cells with each monovalent antigen-binding construct alone and/or an bivalent antigen-binding construct that specifically binds HER2; and/or

b. increased growth inhibition in BT-474 cells when contacting the cells with both constructs compared to contacting the cells with each monovalent antigen-binding construct alone; and/or

c. internalization of both monovalent antigen-binding constructs in SKOV3 cells when contacting the cells with both constructs; and/or

d. mediation of antibody-dependent cellular toxicity (ADCC) of SKOV3 cells when contacting the cells with both constructs; and/or

e. increased potency as measured by cell toxicity in SKOV3 and/or JIMT-1 cells when contacting the cells with both constructs compared to contacting the cell with each monovalent antigen-binding construct alone and/or

f. comparable potency as measured by cell toxicity in Herceptin resistant JIMT-1 cells when contacting the cells with both constructs compared to the cell with each monovalent antigen-binding construct alone.

41 . A method of inhibiting growth of a HER2+ cancer cell, comprising contacting the HER2+ cancer cell with a first monovalent antigen-binding construct or a combination of a first and a second monovalent antigen-binding construct to the subject,

a. wherein the first and second monovalent antigen-binding construct each comprise at least one antigen-binding polypeptide construct and a dimeric Fc coupled, with or without a linker, to the antigen-binding polypeptide;

b. each antigen-binding polypeptide construct specifically binds a extracellular domain 2 (ECD2) of human epidermal growth factor receptor 2 (HER2), a ECD4 of HER2, or a ECD1 of HER2; and

c. the first monovalent antigen-binding construct and the second monovalent antigen-binding construct bind to non-overlapping epitopes and do not compete with each other for binding to HER2.

42 . A method of killing HER2+ cancer cells, comprising contacting the HER2+ cancer cells with a first monovalent antigen-binding construct or a combination of a first and a second monovalent antigen-binding construct to the subject,

a. wherein the first and second monovalent antigen-binding construct each comprise at least one antigen-binding polypeptide construct and a dimeric Fc coupled, with or without a linker, to the antigen-binding polypeptide construct;

b. each antigen-binding polypeptide construct specifically binds a extracellular domain 2 (ECD2) of human epidermal growth factor receptor 2 (HER2), a ECD4 of HER2, or a ECD1 of HER2; and

c. the first monovalent antigen-binding construct and the second monovalent antigen-binding construct bind to non-overlapping epitopes and do not compete with each other for binding to HER2.

43 . The method of claim 41 or 42 , wherein the dimeric Fc comprises at least two CH3 domains and the dimerized CH3 domains have a melting temperature (Tm) of about 68° C. or higher.

44 . The method of claim 41 or 42 , wherein each monovalent antigen-binding construct selectively and/or specifically binds HER2 with a greater maximum binding (Bmax) as compared to an, monospecific bivalent antigen-binding construct that specifically binds HER2, and wherein at a construct to target ratio of 1:1 the increase in Bmax relative to the monospecific bivalent antigen-binding construct is observed at a concentration greater than the observed equilibrium constant (KD) of the constructs up to saturating concentrations.

45 . The method of claim 42 , wherein the first monovalent antigen-binding or the combination of first and second monovalent antigen-binding constructs mediate killing of HER2+ cancer cells by ADCC, ADCP, or CDC.

46 . The method of claim 42 , wherein the first monovalent antigen-binding or the combination of first and second monovalent antigen-binding constructs are conjugated to a drug.