ANTI-DENGUE VIRUS ANTIBODIES, POLYPEPTIDES CONTAINING VARIANT Fc REGIONS, AND METHODS FOR USE THEREOF

Abstract

To provide anti-DENV (dengue virus) antibodies and methods of making the same, and methods for treating DENV infection with the anti-DENV antibodies.SOLUTION: The present disclosure provides: an isolated antibody that binds to E protein of DENV and comprises a specific amino acid sequence; an anti-DENV antibody comprising a variant Fc region that has a decreased FcγR-binding activity and does not have a decreased C1q-binding activity when compared to the parent Fc region; a method of making the anti-DENV antibody; and a method for treating DENV infection.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to anti-dengue virus antibodies and methods of using the same. The present invention also relates to polypeptides comprising a mutant Fc region and methods of using the same.

Background Art

[0002] Background Dengue fever is the most common arthropod-borne viral disease worldwide. The virus that causes dengue fever (referred to herein as DENV) can be classified into four different infectious serotypes, such as DENV-1, DENV-2, DENV-3, and DENV-4. Symptoms of dengue fever infection include fever, muscle pain, headache, decreased platelet count, decreased white blood cell count, blood coagulation disorders, bleeding, vascular leakage, etc., which can lead to dengue shock syndrome. When a person is exposed to the dengue virus after previously being infected with dengue fever, antiviral antibodies may promote the uptake of the virus into host cells, and that patient is at a higher risk of developing severe dengue fever. However, severe dengue fever can also occur during the first infection.

[0003] Although it is the most common arthropod-borne viral disease, to date, there are no drugs available for treating dengue fever. Approaches to dengue fever as a disease have mainly been directed towards the prevention of infection and / or treatment to relieve symptoms.

[0004] Therefore, there is a need to provide an agent that can neutralize and / or bind to at least one dengue serotype. Recently, several groups have reported on anti-DENV neutralizing antibodies (see, for example, WO2012 / 082073, WO2013 / 089647, WO2013 / 151764, WO2013 / 173348, WO2014 / 025546, WO2015 / 123362, WO2015 / 122995, and WO2016 / 012800 (Patent Documents 1-8)). However, antibodies with excellent therapeutic properties are still needed.

[0005] Vaccines and antibody therapeutics are currently under development for the prevention and treatment of viral infections. However, antibody-based therapies are not without risk. One such risk is antibody-dependent enhancement (ADE), which occurs when non-neutralizing antiviral antibodies promote viral entry into host cells, resulting in an increase in infectivity within the cell (Expert Rev Anti Infect Ther (2013) 11, 1147-1157 (Non-Patent Document 1)). The most common mechanism of ADE is the interaction between the virus-antibody complex via the Fc portion of the antibody and the Fc receptor (FcR) on the cell surface. Usually mild viral infections are enhanced by ADE and become life-threatening diseases. Anti-DENV antibodies having mutations in the Fc region that prevent binding to FcγR have been reported not to enhance DENV infection (WO2010 / 043977 (Patent Document 9)). However, there is still a need for antibody therapeutics that do not increase the risk of antibody-dependent enhancement of infection.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Non-Patent Document

[0007]

Non-Patent Document 1

Summary of the Invention

[0008] Summary The present invention provides anti-DENV antibodies, polypeptides comprising a mutant Fc region, and methods of using them.

[0009] In some embodiments, the isolated anti-DENV antibodies of the invention bind to the DENV E protein. In some embodiments, the anti-DENV antibodies of the invention comprise: (a) (i) Amino acid sequence: TIFF2025108688000001.tif4128 (where X1 is R or E, X2 is R or F, X3 is G, D or L) (SEQ ID NO: 42) comprising HVR-H3, (ii) Amino acid sequence: TIFF2025108688000002.tif4128 (where X1 is D, S or E, X2 is D or A) (SEQ ID NO: 45) comprising HVR-L3, and (iii) Amino acid sequence: HVR-H2 containing TIFF2025108688000003.tif4128 (where X1 is T or R and X2 is A or R) (SEQ ID NO: 41); (b) (i) Amino acid sequence: SX1YX2H HVR-H1 containing (where X1 is N or Y and X2 is I or M) (SEQ ID NO: 40), (ii) Amino acid sequence: HVR-H2 containing TIFF2025108688000004.tif4128 (where X1 is T or R and X2 is A or R) (SEQ ID NO: 41), and (iii) Amino acid sequence: HVR-H3 containing TIFF2025108688000005.tif4128 (where X1 is R or E, X2 is R or F, and X3 is G, D or L) (SEQ ID NO: 42); (c) (i) Amino acid sequence: SX1YX2H HVR-H1 containing (where X1 is N or Y and X2 is I or M) (SEQ ID NO: 40), (ii) Amino acid sequence: HVR-H2 containing TIFF2025108688000006.tif4128 (where X1 is T or R and X2 is A or R) (SEQ ID NO: 41), (iii) Amino acid sequence: HVR-H3 containing TIFF2025108688000007.tif4128 (where X1 is R or E, X2 is R or F, and X3 is G, D or L) (SEQ ID NO: 42), (iv) Amino acid sequence: HVR-L1 containing TIFF2025108688000008.tif4128 (where X1 is D or E and X2 is K or Q) (SEQ ID NO: 43), (v) Amino acid sequence: HVR-L2 containing TIFF2025108688000009.tif4128 (where X1 is N or E and X2 is T or F) (SEQ ID NO: 44), and (vi) Amino acid sequence: HVR-L3 containing TIFF2025108688000010.tif4128 (where X1 is D, S or E and X2 is D or A) (SEQ ID NO: 45); or (d) (i) Amino acid sequence: HVR-L1 containing TIFF2025108688000011.tif4128 (where X1 is D or E and X2 is K or Q) (SEQ ID NO: 43), (ii) Amino acid sequence: HVR-L2 containing TIFF2025108688000012.tif4128 (where X1 is N or E and X2 is T or F) (SEQ ID NO: 44), and (iii) Amino acid sequence: HVR-L3 containing TIFF2025108688000013.tif4128 (where X1 is D, S or E and X2 is D or A) (SEQ ID NO: 45). In some embodiments, the antibody of the invention is not an antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0010] In some embodiments, the isolated anti-DENV antibody of the invention comprises: (a) (i) An HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (ii) an HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8 to 10, and (iii) an HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6; (b) (i) An HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (ii) an HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, and (iii) an HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6; (c) (i) An HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (ii) an HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (iii) an HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (iv) an HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 8 to 10, (v) an HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 8 to 10, and (vi) an HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8 to 10; (d) (i) An HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 8 to 10, (ii) an HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 8 to 10, and (iii) an HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8 to 10; or (e) (i) An HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (ii) an HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (iii) an HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6, (iv) an HVR-L1 derived from any one of the VL sequences of SEQ ID NO: 7, (v) an HVR-L2 derived from any one of the VL sequences of SEQ ID NO: 7, and (vi) an HVR-L3 derived from any one of the VL sequences of SEQ ID NO: 7. In some embodiments, the antibody of the invention is not an antibody comprising (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 1, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 1, (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 1, (iv) HVR-L1 derived from the VL sequence of SEQ ID NO: 7, (v) HVR-L2 derived from the VL sequence of SEQ ID NO: 7, and (vi) HVR-L3 derived from the VL sequence of SEQ ID NO: 7.

[0011] In some embodiments, the isolated anti-DENV antibody of the invention further comprises an FR1 comprising the amino acid sequence of SEQ ID NO: 31 of the heavy chain variable domain framework, an FR2 comprising the amino acid sequence of SEQ ID NO: 32, an FR3 comprising the amino acid sequence of SEQ ID NO: 33 or 34, and an FR4 comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the isolated anti-DENV antibody of the invention further comprises an FR1 comprising the amino acid sequence of SEQ ID NO: 36 of the light chain variable domain framework, an FR2 comprising the amino acid sequence of SEQ ID NO: 37, an FR3 comprising the amino acid sequence of SEQ ID NO: 38, and an FR4 comprising the amino acid sequence of SEQ ID NO: 39.

[0012] In some embodiments, the isolated anti-DENV antibody of the invention comprises (a) a VH sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 2-6; (b) a VL sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 8-10; (c) any one of the VH sequences of SEQ ID NOs: 2-6 and any one of the VL sequences of SEQ ID NOs: 8-10; or (d) any one of the VH sequences of SEQ ID NOs: 2-6 and any one of the VL sequences of SEQ ID NO: 7.

[0013] In some embodiments, the isolated anti-DENV antibody of the present invention is a monoclonal antibody. In some embodiments, the isolated anti-DENV antibody of the present invention is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the isolated anti-DENV antibody of the present invention is an antibody fragment that binds to DENV or DENV E protein. In some embodiments, the isolated anti-DENV antibody of the present invention is a full-length IgG antibody.

[0014] In some embodiments, the Fc region of the anti-DENV antibody of the present invention contains Ala at position 234 and Ala at position 235 according to EU numbering. In some embodiments, the Fc region of the anti-DENV antibody of the present invention can be selected from the mutant Fc regions described herein.

[0015] The present invention also provides an isolated nucleic acid encoding the anti-DENV antibody of the present invention. The present invention also provides a host cell containing the nucleic acid of the present invention. The present invention also provides a method for producing an antibody, which includes culturing the host cell of the present invention so that the antibody is produced.

[0016] The present invention also provides a pharmaceutical formulation containing the anti-DENV antibody of the present invention and a pharmaceutically acceptable carrier.

[0017] The anti-DENV antibody of the present invention can be for use as a medicament. In some embodiments, the anti-DENV antibody of the present invention can be for use in the treatment of DENV infection.

[0018] The anti-DENV antibody of the present invention can be used in the manufacture of a medicament. In some embodiments, the medicament is for the treatment of DENV infection.

[0019] The present invention also provides a method for treating an individual suffering from DENV infection. In some embodiments, the method includes administering an effective amount of the anti-DENV antibody of the present invention to the individual. In some embodiments, the method further includes administering an additional therapeutic agent to the individual, such as those described hereinafter.

[0020] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid modification in the parental Fc region. In further embodiments, the variant Fc region has substantially reduced FcγR binding activity when compared to the parental Fc region. In further embodiments, the variant Fc region does not have substantially reduced C1q binding activity when compared to the parental Fc region.

[0021] In some embodiments, the variant Fc region of the present invention comprises Ala at position 234 and Ala at position 235 according to EU numbering. In further embodiments, the variant Fc region comprises any one of the following amino acid modifications (a)-(c): (a) positions 267, 268, and 324; (b) positions 236, 267, 268, 324, and 332; and (c) positions 326 and 333; according to EU numbering.

[0022] In some embodiments, the variant Fc region of the present invention comprises an amino acid selected from the group consisting of: (a) Glu at position 267; (b) Phe at position 268; (c) Thr at position 324; (d) Ala at position 236; (e) Glu at position 332; (f) Ala, Asp, Glu, Met, or Trp at position 326; and (g) Ser at position 333; according to EU numbering.

[0023] In some embodiments, the variant Fc region of the present invention further comprises an amino acid selected from the group consisting of: (a) Ala at position 434; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; and (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440; according to EU numbering.

[0024] In some embodiments, the variant Fc region of the invention comprises any one or combination of the amino acid modifications as described in Table 4. In some embodiments, the parental Fc region described in the invention is derived from human IgG1. The invention provides a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 51 to 59.

[0025] In some embodiments, the polypeptide comprising the variant Fc region of the invention is an antibody. In a further embodiment, the antibody is an antiviral antibody. In a further embodiment, the antibody comprises a variable region derived from the anti-DENV antibody described herein.

[0026] In a further embodiment, the antibody comprises the following: (a) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0027] In a further aspect, the antibody comprises: (a) (i) HVR-H3 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-L3 derived from the VL sequence of SEQ ID NO: 10, and (iii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; (b) (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, and (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; (c) (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6, (iv) HVR-L1 derived from the VL sequence of SEQ ID NO: 10, (v) HVR-L2 derived from the VL sequence of SEQ ID NO: 10, and (vi) HVR-L3 derived from the VL sequence of SEQ ID NO: 10; (d) (i) HVR-L1 derived from the VL sequence of SEQ ID NO: 10, (ii) HVR-L2 derived from the VL sequence of SEQ ID NO: 10, and (iii) HVR-L3 derived from the VL sequence of SEQ ID NO: 10; or (e) (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6, (iv) HVR-L1 derived from the VL sequence of SEQ ID NO: 7, (v) HVR-L2 derived from the VL sequence of SEQ ID NO: 7, and (vi) HVR-L3 derived from the VL sequence of SEQ ID NO: 7.

[0028] The present invention also provides an isolated nucleic acid encoding a polypeptide comprising the variant Fc region of the present invention. The present invention also provides a host cell comprising the nucleic acid of the present invention. The present invention also provides a method for producing the polypeptide, which comprises culturing the host cell of the present invention such that a polypeptide comprising the variant Fc region is produced.

[0029] The present invention also provides a pharmaceutical preparation containing a polypeptide comprising the variant Fc region of the present invention and a pharmaceutically acceptable carrier.

[0030] The polypeptide comprising the variant Fc region of the present invention can be for use as a medicament. In some embodiments, the polypeptide comprising the variant Fc region of the present invention can be for use in the treatment of viral infections.

[0031] The polypeptide comprising the variant Fc region of the present invention can be used in the manufacture of a medicament. In some embodiments, the medicament is for the treatment of viral infections.

[0032] The present invention also provides a method for treating an individual suffering from a viral infection. In some embodiments, the method comprises administering to the individual an effective amount of a polypeptide comprising the variant Fc region of the present invention.

[0033] The present invention also provides the anti-DENV antibody described herein, which further comprises a polypeptide comprising the variant Fc region of the present invention.

[0034] In one aspect, the present invention provides a method for preparing an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, the method comprising the following steps: (a) A step of combining a VH variant sequence selected from the group consisting of 3CH1047 (SEQ ID NO: 6) and 3CH1049 (SEQ ID NO: 95) with a human IgG1 CH sequence selected from the group consisting of SG182 (SEQ ID NO: 46), SG1095 (SEQ ID NO: 54), and SG1106 (SEQ ID NO: 59); (b) A step of combining a VL variant sequence selected from the group consisting of 3CL (SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98) with a human CL sequence SK1 (SEQ ID NO: 60); (c) A step of cloning each of the combinations into an expression vector; (d) A step of expressing the obtained expression vector in co-transfected cells (host cells); and (e) A step of purifying the antibody obtained from step (d). [Invention 1001] An isolated antibody that binds to the dengue virus (DENV) E protein, (a) (i) Amino acid sequence: HVR-H3 containing TIFF2025108688000014.tif4128 (where X1 is R or E, X2 is R or F, and X3 is G, D, or L) (SEQ ID NO: 42), (ii) Amino acid sequence: HVR-L3 containing TIFF2025108688000015.tif4128 (where X1 is D, S, or E, and X2 is D or A) (SEQ ID NO: 45), and (iii) Amino acid sequence: HVR-H2 containing TIFF2025108688000016.tif4128 (where X1 is T or R, and X2 is A or R) (SEQ ID NO: 41); (b) (i) Amino acid sequence: SX1YX2H (where X1 is N or Y, and X2 is I or M) (SEQ ID NO: 40) containing HVR-H1, (ii) Amino acid sequence: HVR-H2 containing TIFF2025108688000017.tif4128 (where X1 is T or R and X2 is A or R) (SEQ ID NO: 41), and (iii) Amino acid sequence: HVR-H3 containing TIFF2025108688000018.tif4128 (where X1 is R or E, X2 is R or F, and X3 is G, D, or L) (SEQ ID NO: 42); (c) (i) Amino acid sequence: SX1YX2H HVR-H1 containing (where X1 is N or Y and X2 is I or M) (SEQ ID NO: 40), (ii) Amino acid sequence: HVR-H2 containing TIFF2025108688000019.tif4128 (where X1 is T or R and X2 is A or R) (SEQ ID NO: 41), (iii) Amino acid sequence: HVR-H3 containing TIFF2025108688000020.tif4128 (where X1 is R or E, X2 is R or F, and X3 is G, D, or L) (SEQ ID NO: 42), (iv) Amino acid sequence: HVR-L1 containing TIFF2025108688000021.tif4128 (where X1 is D or E and X2 is K or Q) (SEQ ID NO: 43), (v) Amino acid sequence: HVR-L2 containing TIFF2025108688000022.tif4128 (where X1 is N or E and X2 is T or F) (SEQ ID NO: 44), and (vi) Amino acid sequence: HVR-L3 containing TIFF2025108688000023.tif4128 (where X1 is D, S, or E and X2 is D or A) (SEQ ID NO: 45); or (d) (i) Amino acid sequence: HVR-L1 containing TIFF2025108688000024.tif4128 (where X1 is D or E and X2 is K or Q) (SEQ ID NO: 43), (ii) Amino acid sequence: HVR-L2 containing TIFF2025108688000025.tif4128 (where X1 is N or E and X2 is T or F) (SEQ ID NO: 44), and (iii) Amino acid sequence: HVR-L3 containing TIFF2025108688000026.tif4128 (where X1 is D, S or E and X2 is D or A) (SEQ ID NO: 45) and does not include (i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (vi) the antibody containing the amino acid sequence of SEQ ID NO: 27. [Invention 1002] The heavy chain variable domain framework FR1 containing the amino acid sequence of SEQ ID NO: 31, FR2 containing the amino acid sequence of SEQ ID NO: 32, FR3 containing the amino acid sequence of SEQ ID NO: 33 or 34, FR4 containing the amino acid sequence of SEQ ID NO: 35 The antibody of Invention 1001 (b) further comprising [Invention 1003] The light chain variable domain framework FR1 containing the amino acid sequence of SEQ ID NO: 36, FR2 containing the amino acid sequence of SEQ ID NO: 37, FR3 containing the amino acid sequence of SEQ ID NO: 38, FR4 containing the amino acid sequence of SEQ ID NO: 39 The antibody of Invention 1001 (d) further comprising [The present invention 1004] (a) A VH sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 2 to 6; (b) A VL sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 8 to 10; or (c) Any one of the VH sequences of SEQ ID NOs: 2 to 6 and any one of the VL sequences of SEQ ID NOs: 8 to 10 An isolated antibody comprising the same. [The present invention 1005] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27 An isolated antibody that binds to the dengue virus (DENV) E protein comprising the same. [The present invention 1006] A pharmaceutical preparation comprising any one of the antibodies of the present inventions 1001 to 1005 and a pharmaceutically acceptable carrier. [The present invention 1007] A method for treating DENV infection in an individual, comprising the step of administering to the individual an effective amount of any one of the antibodies of the present inventions 1001 to 1005. [The present invention 1008] A polypeptide comprising a mutant Fc region comprising at least one amino acid modification in the parental Fc region, wherein the mutant Fc region has substantially reduced FcγR binding activity and does not have substantially reduced C1q binding activity when compared to the parental Fc region, Said polypeptide. [The present invention 1009] The mutant Fc region, according to EU numbering, Ala at position 234, Ala at position 235, and the following (a) to (c): (a) positions 267, 268, and 324; (b) positions 236, 267, 268, 324, and 332; and (c) positions 326 and 333 The polypeptide of the present invention 1008, comprising any one further amino acid modification thereof. [The present invention 1010] The mutant Fc region, according to EU numbering, (a) Glu at position 267; (b) Phe at position 268; (c) Thr at position 324; (d) Ala at position 236; (e) Glu at position 332; (f) Ala, Asp, Glu, Met or Trp at position 326; and (g) Ser at position 333 The polypeptide of the present invention 1009, comprising an amino acid selected from the group consisting of. [The present invention 1011] The mutant Fc region, according to EU numbering, (a) Ala at position 434; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; and (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 The polypeptide of any one of the present inventions 1008 to 1010, further comprising an amino acid selected from the group consisting of. [The present invention 1012] A polypeptide comprising any one amino acid sequence of SEQ ID NOs: 51 to 59. [The present invention 1013] The polypeptide of any one of the present inventions 1008 to 1012, which is an antibody. [The present invention 1014] Said antibody is (a) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27 The polypeptide of the present invention 1013, comprising: [The present invention 1015] A pharmaceutical formulation comprising any polypeptide of the present invention 1008 - 1014 and a pharmaceutically acceptable carrier. [The present invention 1016] An antibody of any of the present invention 1001 - 1005, further comprising any polypeptide of the present invention 1008 - 1012. [The present invention 1017] (a) An HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) a method for preparing an antibody comprising an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, wherein (a) combining a VH variant sequence selected from the group consisting of 3CH1047 (SEQ ID NO: 6) and 3CH1049 (SEQ ID NO: 95) with a human IgG1 CH sequence selected from the group consisting of SG182 (SEQ ID NO: 46), SG1095 (SEQ ID NO: 54), and SG1106 (SEQ ID NO: 59); (b) combining a VL variant sequence selected from the group consisting of 3CL (SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98) with a human CL sequence SK1 (SEQ ID NO: 60); (c) cloning each of the combinations into an expression vector; (d) expressing the resulting expression vector in co-transfected cells; and (e) purifying the antibody obtained from step (d) The method comprising. BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

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Figure 6G

Figure 6H

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 8

Figure 9

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Modes for Carrying Out the Invention

[0036] Detailed Description of Aspects of the Invention The methods and procedures described or cited in this specification are generally well understood. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C.are commonly used by those skilled in the art using conventional techniques such as those widely used techniques described in Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

[0037] I. Definitions Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992) provide general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0038] For the purposes of interpreting this specification, the following definitions apply, and whenever appropriate, terms used in the singular also include the plural and vice versa. It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. In the event of any conflict between any of the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0039] As used herein, an "acceptor human framework" is a framework that includes the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework that is derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework that is "derived from" a human immunoglobulin framework or a human consensus framework may include the same amino acid sequence thereof, or may include changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0040] "Affinity" refers to the total strength of non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the binding partner of the molecule (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific exemplary and illustrative embodiments for measuring binding affinity are described below.

[0041] An "affinity matured" antibody is one that has one or more modifications in one or more hypervariable regions (HVRs) that result in an improvement in the affinity of the antibody for an antigen, as compared to the unmodified parental antibody.

[0042] The terms "anti-DENV antibody" and "antibody that binds to DENV" refer to antibodies that can bind to DENV with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting DENV. The antibody can bind to the E protein of DENV. The terms "anti-DENV E protein antibody" and "antibody that binds to DENV E protein" refer to antibodies that can bind to DENV E protein with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting DENV. In one aspect, the degree of binding of an anti-DENV E protein antibody to an irrelevant protein that is not the DENV E protein is, for example, measured by radioimmunoassay (RIA) and is less than about 10% of the binding of the antibody to the DENV E protein. In certain aspects, an antibody that binds to DENV and / or DENV E protein has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 13 M, 10 -9 M to 10 -13 M). In certain aspects, the anti-DENV antibody binds to an epitope of DENV that is conserved among DENVs from different serotypes. In certain aspects, the anti-DENV E protein antibody binds to an epitope of DENV E protein that is conserved among DENV E proteins from different serotypes.

[0043] The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures including, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity.

[0044] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cell injury in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) specifically binds these cytotoxic effector cells to antigen-bearing target cells, followed by killing of the target cells by cytotoxins. Primary NK cells, which are the effector cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay as described in U.S. Patent Nos. 5,500,362, 5,821,337, or 6,737,056 (Presta) can be performed. Effector cells useful in such assays include PBMC cells and NK cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0045] "Antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that can bind to an 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; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0046] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen in a competitive assay. Exemplary competitive assays are provided herein.

[0047] "C1q" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. C1q forms a complex C1, which is the first component of the complement-dependent cytotoxicity (CDC) pathway, together with two serine proteases, C1r and C1s. Human C1q can be commercially purchased, for example, from Quidel Corporation (San Diego, CA).

[0048] 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 and the remainder of the heavy and / or light chain is derived from a different source or species.

[0049] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0050] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) bound to a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with an altered amino acid sequence in the Fc region (polypeptides having a mutant Fc region) and having an increased or decreased ability to bind C1q are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0051] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P , 212 Pb, and radioisotopes of Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nuclease and fragments thereof; antibiotics; toxins such as, for example, low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor or anticancer agents disclosed hereinafter.

[0052] "Effector function" refers to the biological activities that vary depending on the isotype of an antibody and are attributable to the Fc region of the antibody. Examples of effector functions of antibodies include the following: 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.

[0053] An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to the amount at the required dosage and for the required period of time that is effective to achieve the desired therapeutic or prophylactic result.

[0054] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is the region of an antigen that is bound by an antibody targeting the antigen and includes specific amino acids that come into direct contact with the antibody. Epitope determinants can include chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, an antibody specific for a particular target antigen preferentially recognizes the epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0055] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and forms due to alternative splicing of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also encompassed by the term "FcR" herein.

[0056] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for 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)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)). Binding to human FcRn in vivo and the serum half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice expressing human FcRn or transfected human cell lines or in primates administered polypeptides with mutated Fc regions. WO2000 / 42072 (Presta) describes antibody variants with improved or decreased binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0057] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one aspect, the 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) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0058] The term "Fc region-containing antibody" refers to an antibody that includes an Fc region. The C-terminal lysine (residue 447) or glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant manipulation of the nucleic acid encoding the antibody. Accordingly, a composition comprising an antibody having an Fc region according to the invention can include an antibody with G446-K447, an antibody with G446 and without K447, an antibody with G446-K447 completely removed, or a mixture of the three types of antibodies.

[0059] "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. Accordingly, the sequences of HVRs and FRs generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0060] The terms "full-length antibody", "complete antibody", and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0061] A "functional Fc region" has the "effector functions" of the native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor: BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays disclosed, for example, within the definitions herein.

[0062] The terms "host cell", "host cell line", "cotransfected cell", and "host cell culture" are used interchangeably and refer to a cell into which foreign nucleic acid has been introduced (including progeny of such cells). Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from such cells regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as that used when the original transformed cell was screened or selected are also included herein. In certain embodiments, the host cell or cotransfected cell is CHO-DXB11, CHO-K1, or CHO-DG44. Such host cells or cotransfected cells may be cells that express the taurine transporter and can be obtained by introducing DNA encoding the taurine transporter (WO2007 / 119774).

[0063] A "human antibody" is an antibody comprising an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or an antibody derived from a non-human source using a human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0064] A "human consensus framework" is a framework that represents the amino acid residues that occur most commonly in a selected group of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is the subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al. supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al. supra.

[0065] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein in said variable regions all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may also include at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to the antibody that has been humanized.

[0066] As used herein, the term "hypervariable region" or "HVR" refers to each region of the variable domain of an antibody that is hypervariable in sequence ("complementary determining region" or "CDR"), and / or forms structurally defined loops ("hypervariable loops"), and / or contains antigen - contacting residues ("antigen contact"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) Hypervariable loops occurring at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987)); (b) CDRs occurring at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contacts occurring at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732 - 745 (1996)); and (d) Combinations of (a), (b), and / or (c) including HVR amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1), 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3).

[0067] Unless otherwise indicated, HVR residues and other residues in the variable domains (e.g., FR residues) are numbered herein according to Kabat et al. as described above.

[0068] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (heterologous molecules include, but are not limited to, cytotoxic agents).

[0069] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, farm animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0070] An "isolated" antibody is separated from the components of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). See, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007) for a review of methods for assessing antibody purity.

[0071] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its original environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its native chromosomal location.

[0072] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including nucleic acid molecules carried on one vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0073] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies making up the population are identical and / or bind to the same epitope, except for variant antibodies that may arise (e.g., variant antibodies that include naturally occurring mutations or mutations that occur during the production of a monoclonal antibody preparation; such variants are typically present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody that is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that include all or part of the human immunoglobulin locus. Such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0074] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. A naked antibody may be present in a pharmaceutical formulation.

[0075] "Natural antibody" refers to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0076] "Native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0077] The term "package insert" is used to refer to the instructions for use that are usually included in the commercial package of a therapeutic product and contain information about indications, usage, dosage, administration method, combination therapy, contraindications, and / or warnings regarding the use of such a therapeutic product.

[0078] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after the sequences are aligned to obtain the maximum percent sequence identity and gaps are introduced if necessary, and when any conservative substitutions are not considered part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0079] The ALIGN-2 sequence comparison computer program is the work of Genentech, Inc., and its source code has been filed with the U.S. Copyright Office (Washington D.C., 20559) together with user documentation and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), and may also be compiled from the source code. The ALIGN-2 program is 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. 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 against a given amino acid sequence B (or, equivalently, a given amino acid sequence A having or containing a % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y. Where X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that where 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 specifically stated, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0080] The term "pharmaceutical formulation" refers to a preparation in a form in which the biological activity of the active ingredient contained therein can exert an effect and which does not contain additional elements that are toxic to an unacceptable degree to the subject to which the formulation is administered.

[0081] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical preparation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0082] As used herein, the term "DENV E protein" refers to the native DENV E protein from any DENV serotype, including DENV-1, DENV-2, DENV-3, and DENV-4, unless otherwise indicated. The DENV genome encodes three structural proteins (capsid (C), pre-membrane / membrane (prM / M), and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The E protein is a glycoprotein of approximately 55 kDa and exists as a heterodimer with the PrM protein prior to virion maturation. X-ray crystallographic studies of the extracellular domain of the E protein have revealed three distinct β-barrel domains linked to the viral membrane by a helical stem-anchor and two antiparallel transmembrane domains. Domain III (EDIII) adopts an immunoglobulin-like fold and is suggested to play an important role in receptor interaction. Domain II (EDII) is an elongated domain consisting of two long finger-like structures and contains a highly conserved 13-amino acid fusion loop (EDII-FL) at its tip and is involved in membrane fusion and dimerization of the E protein. The central domain (domain I; EDI) is a nine-stranded β-barrel linked to EDIII and EDII by one and four flexible linkers, respectively. The E protein is important for virus assembly, receptor binding, entry, virus fusion, and possibly immune evasion in the viral life cycle and is thus a dynamic protein required to adopt several different conformations and arrangements on the virus particle. This term encompasses not only the unprocessed "full-length" DENV E protein but also any form of the DENV E protein resulting from intracellular processing. This term also encompasses naturally occurring variants of the DENV E protein, such as serotype variants or quasispecies.

[0083] As used herein, the terms "substantially decreased", "substantially increased", or "substantially different" refer to a sufficiently high degree of difference between two numerical values (generally, a numerical value associated with a molecule and another numerical value associated with a reference / comparative molecule) such that one of ordinary skill in the art would consider the difference between these two values to be statistically significant in the context of the biological characteristic measured by said value (e.g., Kd value).

[0084] As used herein, "treatment" (and its grammatical derivatives, e.g., "treat", "treating", etc.) means a clinical intervention that is intended to modify the natural course of an individual being treated and can be performed both for prophylaxis and during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological effects of the disease, preventing metastasis, reducing the rate of disease progression, restoring or alleviating the disease state, and having a remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset or slow the progression of a disease.

[0085] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) One VH or VL domain would be sufficient to confer antigen-binding specificity. Further, an antibody that binds a particular antigen may be isolated by screening a complementary library of VL or VH domains using the VH or VL domain from an antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0086] A "mutated Fc region" includes an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the mutated Fc region has at least one amino acid substitution in the native sequence Fc region or the Fc region of the parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, as compared to the native sequence Fc region or the Fc region of the parent polypeptide. The mutated Fc regions herein preferably have at least about 80% homology, most preferably at least about 90% homology, and more preferably at least about 95% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide.

[0087] As used herein, the term "vector" refers to a nucleic acid molecule that can amplify another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which they are introduced. A vector can effect the expression of a nucleic acid operably linked thereto. Such a vector is also referred to herein as an "expression vector".

[0088] II. Compositions and Methods In one aspect, the invention is based in part on anti-DENV antibodies and their use. In certain embodiments, antibodies that bind to DENV and / or DENV E protein are provided. The antibodies of the invention are useful, for example, for the diagnosis or treatment of DENV infection.

[0089] In one aspect, the invention is based in part on polypeptides comprising a mutant Fc region and their use. In certain embodiments, polypeptides comprising a mutant Fc region having substantially reduced FcγR binding activity are provided. In certain embodiments, polypeptides comprising a mutant Fc region having substantially no C1q binding activity are provided. In certain embodiments, the polypeptides of the invention are antibodies. The polypeptides of the invention comprising a mutant Fc region are useful, for example, for the diagnosis or treatment of viral infections.

[0090] A. Exemplary Anti-DENV Antibodies and Polypeptides Comprising a Mutant Fc Region In one aspect, the present invention provides an isolated antibody that binds to DENV and / or DENV E protein. In certain embodiments, the anti-DENV antibody blocks the binding of DENV and / or DENV E protein to host cells. In certain embodiments, the anti-DENV antibody inhibits DENV entry into host cells. In certain embodiments, the anti-DENV antibody binds to whole DENV particles. In a further aspect, the antibody binds to whole DENV particles more than it binds to monomeric DENV E protein. In certain embodiments, the anti-DENV antibody of the present invention binds to and / or neutralizes at least 1, at least 2, at least 3, or all 4 DENV serotypes selected from the group consisting of DENV serotype 1 (DENV-1), DENV serotype 2 (DENV-2), DENV serotype 3 (DENV-3), and DENV serotype 4 (DENV-4). In certain embodiments, the anti-DENV antibody binds to and / or neutralizes DENV E protein derived from at least 1, at least 2, at least 3, or all 4 DENV serotypes selected from the group consisting of DENV-1, DENV-2, DENV-3, and DENV-4. "Serotype" refers to clearly distinguishable variation within a virus species.

[0091] In one aspect, the present invention provides an anti-DENV antibody comprising at least 1, 2, 3, 4, 5, or 6 HVRs selected from: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 11-12; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 13-15; (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20; (d) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 21-23; (e) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 24-26; and (f) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30.

[0092] In another aspect, the present invention provides an anti-DENV antibody comprising at least 1, 2, 3, 4, 5, or 6 HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0093] In another aspect, the present invention provides an anti-DENV antibody comprising at least 1, 2, 3, 4, 5, or 6 HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0094] In another aspect, the present invention provides an anti-DENV antibody comprising at least 1, 2, 3, 4, 5, or 6 HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0095] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 11-12; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 13-15; and (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20. In one embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20. In another embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20 and HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30. In a further embodiment, the antibody comprises HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20, HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30, and HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 13-15. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 11-12; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 13-15; and (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20.

[0096] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42.

[0097] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20.

[0098] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 21-23; (b) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 24-26; and (c) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30. In one embodiment, the antibody comprises: (a) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 21-23; (b) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 24-26; and (c) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30.

[0099] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0100] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0101] In another aspect, the antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 11-12, (ii) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 13-15, and (iii) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 21-23, (ii) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 24-26, and (iii) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30.

[0102] In another aspect, the antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0103] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0104] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0105] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 11-12; (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 13-15; (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20; (d) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 21-23; (e) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 24-26; and (f) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30.

[0106] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0107] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0108] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0109] In certain embodiments, one or more amino acids of any of the anti-DENV antibodies described above are substituted at the following HVR positions: (a) at positions 2 and 4 in HVR-H1 (SEQ ID NO: 11); (b) at positions 9 and 10 in HVR-H2 (SEQ ID NO: 13); (c) at positions 3, 14, and 16 in HVR-H3 (SEQ ID NO: 16); (d) at positions 5 and 8 in HVR-L1 (SEQ ID NO: 21); (e) at positions 4 and 7 in HVR-L2 (SEQ ID NO: 24); and (f) at positions 4 and 5 in HVR-L3 (SEQ ID NO: 27).

[0110] In certain embodiments, one or more amino acid substitutions of the anti-DENV antibody are conservative substitutions as provided herein. In certain embodiments, any one or more of the following substitutions can be made in any combination: (a) in HVR-H1 (SEQ ID NO: 11), N2Y; I4M; (b) in HVR-H2 (SEQ ID NO: 13), T9R; A10R; (c) in HVR-H3 (SEQ ID NO: 16), R3E; R14F; G16D or L; (d) in HVR-L1 (SEQ ID NO: 21), D5E; K8Q; (e) in HVR-L2 (SEQ ID NO: 24), N4E; T7F; and (f) in HVR-L3 (SEQ ID NO: 27), D4S or E; D5A.

[0111] All possible combinations of the above substitutions are encompassed by the consensus sequences of SEQ ID NO: 40, 41, 42, 43, 44 and 45 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively.

[0112] In a further aspect, the anti-DENV antibodies of the invention are not antibodies that comprise (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0113] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (b) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (c) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (d) HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 8-10; (e) HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 8-10; and (f) HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8-10.

[0114] In another aspect, the present invention provides an anti-DENV antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (b) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (c) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (d) HVR-L1 derived from any one of the VL sequences of SEQ ID NO: 7; (e) HVR-L2 derived from any one of the VL sequences of SEQ ID NO: 7; and (f) HVR-L3 derived from any one of the VL sequences of SEQ ID NO: 7.

[0115] In another aspect, the present invention provides an anti-DENV antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 10; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 10; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 10.

[0116] In another aspect, the present invention provides an anti-DENV antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 7; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 7; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 7.

[0117] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (b) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6; and (c) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6. In one embodiment, the antibody comprises HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6. In another embodiment, the antibody comprises HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6 and HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8-10. In another embodiment, the antibody comprises HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6 and HVR-L3 derived from any one of the VL sequences of SEQ ID NO: 7. In a further embodiment, the antibody comprises HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6, HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8-10, and HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6. In a further embodiment, the antibody comprises HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6, HVR-L3 derived from any one of the VL sequences of SEQ ID NO: 7, and HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6. In a further embodiment, the antibody comprises: (a) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (b) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6; and (c) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6.

[0118] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three of the following VH HVR sequences selected from: (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises HVR-H3 derived from the VH sequence of SEQ ID NO: 6. In another embodiment, the antibody comprises HVR-H3 derived from the VH sequence of SEQ ID NO: 6 and HVR-L3 derived from the VL sequence of SEQ ID NO: 10. In another embodiment, the antibody comprises HVR-H3 derived from the VH sequence of SEQ ID NO: 6 and HVR-L3 derived from the VL sequence of SEQ ID NO: 7. In a further embodiment, the antibody comprises HVR-H3 derived from the VH sequence of SEQ ID NO: 6, HVR-L3 derived from the VL sequence of SEQ ID NO: 10, and HVR-H2 derived from the VH sequence of SEQ ID NO: 6. In a further embodiment, the antibody comprises HVR-H3 derived from the VH sequence of SEQ ID NO: 6, HVR-L3 derived from the VL sequence of SEQ ID NO: 7, and HVR-H2 derived from the VH sequence of SEQ ID NO: 6. In a further embodiment, the antibody comprises: (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; and (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 6.

[0119] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 8-10; (b) HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 8-10; and (c) HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8-10. In one embodiment, the antibody comprises (a) HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 8-10; (b) HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 8-10; and (c) HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8-10.

[0120] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 10; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 10; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises (a) HVR-L1 derived from the VL sequence of SEQ ID NO: 10; (b) HVR-L2 derived from the VL sequence of SEQ ID NO: 10; and (c) HVR-L3 derived from the VL sequence of SEQ ID NO: 10.

[0121] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2-6, (ii) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6, and (iii) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 8-10, (ii) HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 8-10, and (iii) HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8-10.

[0122] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2-6, (ii) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6, and (iii) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 7, (ii) HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 7, and (iii) HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 7.

[0123] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, and (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 derived from the VL sequence of SEQ ID NO: 10, (ii) HVR-L2 derived from the VL sequence of SEQ ID NO: 10, and (iii) HVR-L3 derived from the VL sequence of SEQ ID NO: 10.

[0124] In another aspect, the antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, and (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 derived from the VL sequence of SEQ ID NO: 7, (ii) HVR-L2 derived from the VL sequence of SEQ ID NO: 7, and (iii) HVR-L3 derived from the VL sequence of SEQ ID NO: 7.

[0125] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (b) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (c) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2-6; (d) HVR-L1 derived from any one of the VL sequences of SEQ ID NOs: 8-10; (e) HVR-L2 derived from any one of the VL sequences of SEQ ID NOs: 8-10; and (f) HVR-L3 derived from any one of the VL sequences of SEQ ID NOs: 8-10.

[0126] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6; (b) HVR-H2 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6; (c) HVR-H3 derived from any one of the VH sequences of SEQ ID NOs: 2 to 6; (d) HVR-L1 derived from any one of the VL sequences of SEQ ID NO: 7; (e) HVR-L2 derived from any one of the VL sequences of SEQ ID NO: 7; and (f) HVR-L3 derived from any one of the VL sequences of SEQ ID NO: 7.

[0127] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 10; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 10; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 10.

[0128] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 derived from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; (c) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; (d) HVR-L1 derived from the VL sequence of SEQ ID NO: 7; (e) HVR-L2 derived from the VL sequence of SEQ ID NO: 7; and (f) HVR-L3 derived from the VL sequence of SEQ ID NO: 7.

[0129] In a further aspect, the anti-DENV antibody of the present invention is not an antibody comprising: (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 1; (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 1; (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 1; (iv) HVR-L1 derived from the VL sequence of SEQ ID NO: 7; (v) HVR-L2 derived from the VL sequence of SEQ ID NO: 7; and (vi) HVR-L3 derived from the VL sequence of SEQ ID NO: 7.

[0130] In any of the above aspects, the anti-DENV antibody can be humanized. In one aspect, the anti-DENV antibody comprises the HVRs in any of the above aspects and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another aspect, the anti-DENV antibody comprises the HVRs in any of the above aspects and further comprises a VH or VL comprising an FR sequence. In a further aspect, the anti-DENV antibody comprises the following heavy-chain and / or light-chain variable domain FR sequences. In the case of the heavy-chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO: 31, FR2 comprises the amino acid sequence of SEQ ID NO: 32, FR3 comprises the amino acid sequence of SEQ ID NO: 33 or 34, and FR4 comprises the amino acid sequence of SEQ ID NO: 35. In the case of the light-chain variable domain, FR1 comprises the amino acid sequence of SEQ ID NO: 36, FR2 comprises the amino acid sequence of SEQ ID NO: 37, FR3 comprises the amino acid sequence of SEQ ID NO: 38, and FR4 comprises the amino acid sequence of SEQ ID NO: 39.

[0131] In another aspect, the anti-DENV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 2-6. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DENV antibody comprising such a sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 2-6. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., the FRs). Optionally, the anti-DENV antibody comprises a VH sequence of any one of SEQ ID NOs: 2-6, including post-translational modifications. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising any one of the amino acid sequences of SEQ ID NOs: 11-12, (b) HVR-H2 comprising any one of the amino acid sequences of SEQ ID NOs: 13-15, and (c) HVR-H3 comprising any one of the amino acid sequences of SEQ ID NOs: 16-20. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0132] In another aspect, the anti-DENV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DENV antibody comprising the sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 6. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., FRs). Optionally, the anti-DENV antibody comprises a VH sequence of SEQ ID NO: 6, including post-translational modifications. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0133] In another aspect, the anti-DENV antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 8-10. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DENV antibody comprising the sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 8-10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., FRs). Optionally, the anti-DENV antibody comprises a VL sequence of any one of SEQ ID NOs: 8-10, including post-translational modifications. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising any one of the amino acid sequences of SEQ ID NOs: 21-23, (b) HVR-L2 comprising any one of the amino acid sequences of SEQ ID NOs: 24-26, and (c) HVR-L3 comprising any one of the amino acid sequences of SEQ ID NOs: 27-30. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0134] In another aspect, there is provided an anti-DENV antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DENV antibody comprising such a sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., FRs). Optionally, the anti-DENV antibody comprises a VL sequence of SEQ ID NO: 10 including post-translational modifications. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0135] In another aspect, there is provided an anti-DENV antibody comprising a VH as in any of the above embodiments and a VL as in any of the above embodiments. In one embodiment, the antibody comprises a VH sequence and a VL sequence of any one of SEQ ID NOs: 2-6 and any one of SEQ ID NOs: 8-10, respectively, including post-translational modifications. In one embodiment, the antibody comprises a VH and a VL sequence of any one of SEQ ID NOs: 2-6 and SEQ ID NO: 7, respectively, including post-translational modifications. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0136] In another aspect, there is provided an anti-DENV antibody comprising VH as in any of the above aspects and VL as in any of the above aspects. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 6 and SEQ ID NO: 10, respectively, including post-translational modifications. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, including post-translational modifications. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0137] In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-DENV antibody provided herein. In certain aspects, there is provided an antibody that binds to an epitope comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all of the amino acids selected from the group consisting of G100, W101, K122, I162, S274, K310, W391, and F392 on the DENV-2 E protein. In certain aspects, there is provided an antibody that binds to an epitope comprising at least 1, at least 2, or all of the amino acids selected from the group consisting of K122, I162, and S274 on the DENV-2 E protein. In certain aspects, when the epitope comprises at least 1, at least 2, or all of the amino acids selected from the group consisting of K122, I162, and S274, there is provided an antibody that binds to the epitope further comprising at least 1 amino acid selected from the group consisting of G100, W101, K310, W391, and F392.

[0138] In a further aspect of the invention, the anti-DENV antibody according to any of the above aspects is a monoclonal antibody, including chimeric antibodies, humanized antibodies or human antibodies. In one aspect, the anti-DENV antibody is an antibody fragment, for example, Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another aspect, the antibody is a full-length antibody, for example, an intact IgG1 antibody, or other antibody classes or isotypes defined herein.

[0139] In a further aspect, the anti-DENV antibody of the present invention may have a modification that abolishes the binding of the antibody to the Fc gamma receptor (FcγR). Without being bound by theory, a modification that abolishes the binding of the antibody to the Fc gamma receptor may be advantageous because it can avoid the antibody-dependent enhancement (ADE) phenomenon of infection, where the decrease in binding to FcR is thought to be mainly mediated by the interaction with FcR. In one aspect, the Fc region of the anti-DENV antibody of the present invention contains Ala at position 234 and Ala at position 235 according to EU numbering.

[0140] In a further aspect, the anti-DENV antibody according to any of the above aspects may incorporate, alone or in combination, any of the features described in items 1 to 7 below.

[0141] 1. Antibody affinity In certain aspects, the antibodies provided herein have a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0142] In one aspect, Kd is measured by a radiolabeled antigen binding assay (RIA). In one aspect, the RIA is performed using the 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 equilibrating the Fab with a minimum concentration of ( 125 I) radiolabeled antigen and then capturing the bound antigen with a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To construct the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbing plates (Nunc #269620), 100 pM or 26 pM of 125 I]-antigen is mixed with serial dilutions of the Fab of interest (similar to 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, although this incubation can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is achieved. The mixture is then transferred to the capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 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 gives less than 20% of the maximum binding is selected for use in the competitive binding assay.

[0143] In another aspect, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25° C. using a CM5 chip with approximately 10 response units (RU) of antigen immobilized thereon. In one aspect, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8 before injection at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RU) of protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For measurement of the reaction rate, two-fold dilutions (0.78 nM to 500 nM) of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25° C. at a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (k off ) are calculated by simultaneously fitting the binding and dissociation sensorgrams using a simple 1:1 Langmuir binding model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio of k off / k on . See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The association rate by the above surface plasmon resonance assay is 10 6 M -1 s -1When exceeding, the on-rate can be determined using fluorescence quenching techniques that measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25 °C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0144] 2. Antibody fragments In certain embodiments, the antibodies provided herein are antibody fragments. 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 hereinafter. For a review of particular antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab’)2 fragments that contain salvage receptor binding epitope residues and have an extended half-life in vivo, see U.S. Patent No. 5,869,046.

[0145] A diabody is an antibody fragment with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0146] A single-domain antibody is an antibody fragment that includes all or a portion of the heavy-chain variable domain of an antibody, or all or a portion of the light-chain variable domain. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0147] Antibody fragments can be made by a variety of methods including, but not limited to, proteolytic digestion of a full antibody as described herein, and production by recombinant host cells (e.g., E. coli or phage).

[0148] 3. Chimeric and Humanized Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Specific 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 includes 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 a further example, a chimeric antibody is a "class-switch" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include their antigen-binding fragments.

[0149] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while maintaining the specificity and affinity of the parental non-human antibody. Usually, a humanized antibody contains 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. A humanized antibody optionally includes at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) to, for example, restore or improve the specificity or affinity of the antibody.

[0150] Humanized antibodies and methods of making them are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. 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 "guide selection" approach for FR shuffling).

[0151] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of certain subgroups of the light or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992) and Presta et al. J. Immunol. 151:2623 (1993)); human mature (somatic 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 of FR libraries (see Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0152] 4. Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by a variety of methods known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0153] Human antibodies may be prepared by administering an immunogen to a transgenic animal modified to produce a fully human antibody or a complete antibody with human variable regions in response to an antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, which may replace the endogenous immunoglobulin locus or be present extrachromosomally or randomly integrated within the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. See, for example, Lonberg, Nat. Biotech. 23:1117-1125 (2005) for a review of methods for obtaining human antibodies from transgenic animals. Also see, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Pat. No. 5,770,429, which describes HUMAB® technology; U.S. Pat. No. 7,041,870, which describes K-M MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0154] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0155] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Methods for selecting human antibodies from antibody libraries are described below.

[0156] 5. Antibodies from Libraries The antibodies of the present invention may be isolated by screening a combinatorial library for antibodies with one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies with desired binding properties are known in the art. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37; O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0157] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and the phage library can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phage display antibody fragments typically as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization. Finally, naive libraries can also be made synthetically as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning V-gene segments from stem cells prior to rearrangement and using PCR primers that encode the hypervariable CDR3 region and contain random sequences to achieve recombination in vitro. Patent documents describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0158] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0159] 6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for the DENV E protein and the other is for any other antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of the DENV E protein. The bispecific antibody may be used to localize a cytotoxic agent to cells expressing the DENV E protein. The bispecific antibody can be prepared as a full-length antibody or as an antibody fragment.

[0160] Methods for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having 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" technology (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); creating antibodies having two specificities using leucine zippers (see Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); making bispecific antibody fragments using the "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see Gruber et al., J. Immunol. 152:5368 (1994)); and may also be made by preparing trispecific antibodies, for example, as described in Tutt et al., J. Immunol. 147: 60 (1991).

[0161] Also included herein are modified antibodies with three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576 A1).

[0162] As used herein, an antibody or fragment also includes a "dual acting Fab" or "DAF" that includes one antigen binding site that binds to a DENV E protein and a different, distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0163] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may 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 possesses the desired characteristics, e.g., antigen binding.

[0164] a) Substitution, Insertion, and Deletion Variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for introduction of substitutional mutations include the HVRs and FRs. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 1. More substantial changes are provided under the heading of "exemplary substitutions" in Table 1 and are detailed below while referring to the classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest and the products screened for the desired activity such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0165] (Table 1) TIFF2025108688000027.tif158169

[0166] Amino acids can be grouped according to common side-chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) Neutral hydrophilic: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) Residues affecting chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the replacement of one member of these classes with another member of a different class.

[0167] One type of substitution variant involves substitution of one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Typically, the resulting variant, selected for further study, will have a modification (e.g., improvement) (e.g., increased affinity, decreased immunogenicity) in certain biological properties compared to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which can be made as appropriate using, for example, phage display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated and the mutated antibody is displayed on a phage and screened for a particular biological activity (e.g., binding affinity).

[0168] Modifications (e.g., substitutions) can be made in the HVRs, for example, to improve the affinity of an antibody. Such modifications can be made at the “hot spots” of the HVRs, i.e., residues encoded by codons that mutate at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or at residues that contact the antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects 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 created. This library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. The 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.

[0169] In certain aspects, substitutions, insertions, or deletions can be made within one or more HVRs so long as such modifications do not substantially decrease the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially decrease binding affinity can be made in the HVRs. Such modifications can be, for example, outside of the antigen contact residues of the HVRs. In certain aspects of the mutant VH and VL sequences described above, each HVR is unmodified or contains only one, two, or three amino acid substitutions.

[0170] A method useful for identifying residues or regions of an antibody that can be targeted for mutagenesis is that described by Cunningham and Wells (1989), Science, 244:1081-1085, called "alanine scanning mutagenesis." In this method, one residue or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified, replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that showed functional sensitivity to this initial substitution. Alternatively or in addition, the crystal structure of the antigen-antibody complex can be analyzed to identify the contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or excluded from substitution candidates. Variants can be screened to determine whether they contain the desired properties.

[0171] Insertions of amino acid sequences include fusions in the range of the length of a polypeptide containing from 1 residue to over 100 residues at the amino terminus and / or carboxyl terminus, as well as insertions of single or multiple amino acid residues into the interior of the sequence. Examples of terminal insertions include antibodies with a methionyl residue at the N-terminus. Other insertion mutants of antibody molecules include those in which an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody is fused to the N- or C-terminus of the antibody.

[0172] b) Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be readily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0173] If the antibody contains an Fc region, the carbohydrates added thereto may be modified. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are usually attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and also include fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the invention may be performed to create antibody variants with certain improved properties.

[0174] In one aspect, provided is an antibody variant having a carbohydrate structure lacking fucose (directly or indirectly) added 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 sugar chain at Asn297 relative to the sum of all sugar structures added to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry as described, for example, in WO 2008 / 077546. Asn297 represents the asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence diversity among 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, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 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 afucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11) and knockout cell lines such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (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).

[0175] For example, there is further provided an antibody variant having a bisected oligosaccharide, in which the bisected oligosaccharide added to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide added to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0176] c) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variants may comprise a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0177] In certain embodiments, antibody variants that possess some, but not all, effector functions are also contemplated by the present invention, where the effector functions make the antibody a desirable candidate for applications where its in vivo half-life is important but certain effector functions, such as ADCC, are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to measure CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be performed to determine whether an antibody has FcγR binding ability (and thus is likely to have ADCC activity) and / or FcRn binding ability. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs 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 for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent 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); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and, CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model such as that described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Also, C1q binding measurements may be performed to confirm whether the antibody can bind to C1q and thus has CDC activity. See, for example, the C1q and C3c binding ELISAs of WO2006 / 029879 and WO2005 / 100402. Also, CDC measurements may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie Blood 103:2738-2743 (2004)). Further, FcRn binding and determination of clearance / half-life in vivo can also be performed using methods known in the art (see, for example, Petkova, et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0178] Antibodies with modified effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0179] Certain antibody variants with altered binding to FcRs have been described. (See U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0180] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that modify ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residues in EU numbering) of the Fc region).

[0181] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, WO2011 / 091078, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0182] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn: which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994))) have been described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region that contains one or more substitutions that increase its binding to FcRn in the Fc region. Such Fc variants include those with substitutions at one or more of the 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., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0183] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260 and U.S. Patent No. 5,624,821; and WO94 / 29351.

[0184] In another aspect, the antibody may comprise a variant Fc of the invention as detailed hereinbelow.

[0185] d) Cysteine-modified antibody variants In certain embodiments, it may be desirable to create cysteine-modified antibodies (e.g., “thioMAbs”) in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the residues to be replaced occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, and those reactive thiol groups may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates as further detailed herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine-modified antibodies may be generated, for example, as described in U.S. Patent No. 7,521,541.

[0186] e) Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties that are known in the art and are readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / prolylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in manufacture because of its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary widely and if more than one polymer is attached they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative is to be used in therapy under defined conditions, and the like.

[0187] In another aspect, a conjugate is provided that includes an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one aspect, the non-protein 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 wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature that kills cells in proximity to the antibody-non-protein moiety, although not limited thereto.

[0188] In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region having substantially reduced FcγR binding activity. In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region having substantially reduced C1q binding inactivity. In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region having substantially reduced FcγR binding activity and substantially reduced C1q binding inactivity. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the mutant Fc region comprises at least one amino acid residue modification (e.g., substitution) compared to the corresponding sequence of the Fc region of the native sequence or reference variant sequence (collectively referred to herein as the "parent" Fc region). In certain embodiments, the mutant Fc region of the present invention has substantially reduced FcγR binding activity compared to the parent Fc region. In certain embodiments, the mutant Fc region of the present invention has substantially reduced C1q binding inactivity compared to the parent Fc region. In certain embodiments, the FcγR is a human FcγR, a monkey FcγR (e.g., cynomolgus monkey, rhesus monkey, marmoset, chimpanzee, or baboon FcγR), or a mouse FcγR.

[0189] In one aspect, the variant Fc region of the present invention has substantially reduced binding activity to one or more human FcγRs, including but not limited to, for example, FcγRIa, FcγRIIa (including allelic variants 167H and 167R), FcγRIIb, FcγRIIIa (including allelic variants 158F and 158V), and FcγRIIIb (including allelic variants NA1 and NA2), as compared to the parental Fc region. In a further aspect, the variant Fc region of the present invention has substantially reduced binding activity to human FcγRIa, FcγRIIa (including allelic variants 167H and 167R), FcγRIIb, FcγRIIIa (including allelic variants 158F and 158V), and FcγRIIIb (including allelic variants NA1 and NA2), as compared to the parental Fc region.

[0190] In one aspect, the variant Fc region of the present invention has substantially reduced binding activity to one or more mouse FcγRs, including but not limited to, FcγRI, FcγRIIb, FcγRIII, and FcγRIV, as compared to the parental Fc region. In a further aspect, the variant Fc region of the present invention has substantially reduced binding activity to mouse FcγRI, FcγRIIb, FcγRIII, and FcγRIV, as compared to the parental Fc region.

[0191] "Fcγ receptor" (as used herein, Fcγ receptor, FcγR or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and in fact means any member of the family of proteins encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to: FcγRI (CD64), such as isoforms FcγRIa, FcγRIb, FcγRIc, etc.; FcγRII (CD32), such as isoforms FcγRIIa (including allotypes H131 (type H) and R131 (type R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), FcγRIIc, etc.; and FcγRIII (CD16), such as isoforms FcγRIIIa (including allotypes V158 and F158), FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), etc.; as well as human FcγRs, isoforms or allotypes of FcγR that have not yet been discovered. FcγRIIb-1 and FcγRIIb-2 have been reported as splicing variants of human FcγRIIb. Furthermore, a splicing variant named FcγRIIb-3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human FcγRIIb includes all splicing variants registered in NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb includes not only FcγRIIb, but also all previously reported genetic polymorphisms (Arthritis Rheum. 48:3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46:1242-1254 (2002)), and all genetic polymorphisms that will be reported in the future.

[0192] FcγRIIa has two allotypes, one with histidine at amino acid position 167 of FcγRIIa (type H), and the other with arginine substituted for the amino acid at position 167 (type R) (Warrmerdam, J. Exp. Med. 172:19-25 (1990)).

[0193] FcγRs include, but are not limited to, those derived from humans, mice, rats, rabbits, and monkeys, and may be from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV (CD16-2), as well as any mouse FcγR or FcγR isoform.

[0194] The amino acid sequence of human FcγRIa is shown in SEQ ID NO: 69; the amino acid sequence of human FcγRIIa (167H) is shown in SEQ ID NO: 70; the amino acid sequence of human FcγRIIa (167R) is shown in SEQ ID NO: 71; the amino acid sequence of human FcγRIIb is shown in SEQ ID NO: 72; the amino acid sequence of human FcγRIIIa (158F) is shown in SEQ ID NO: 73; the amino acid sequence of human FcγRIIIa (158V) is shown in SEQ ID NO: 74; the amino acid sequence of human FcγRIIIb (NA1) is shown in SEQ ID NO: 75; the amino acid sequence of human FcγRIIIb (NA2) is shown in SEQ ID NO: 76.

[0195] The amino acid sequence of mouse FcγRI is shown in SEQ ID NO: 77; the amino acid sequence of mouse FcγRIIb is shown in SEQ ID NO: 78; the amino acid sequence of mouse FcγRIII is shown in SEQ ID NO: 79; the amino acid sequence of mouse FcγRIV is shown in SEQ ID NO: 80.

[0196] In one aspect, the variant Fc region of the present invention has substantially reduced FcγR binding activity, and the FcγR binding activity is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% as a function of the FcγR binding activity of the parental Fc region. In one aspect, the variant Fc region of the present invention has substantially reduced FcγR binding activity, which means that the ratio of [the difference in RU values of the sensorgram before and after the interaction between FcγR and the variant Fc region] / [the difference in RU values of the sensorgram before and after capturing FcγR on the sensor chip] is less than 1, less than 0.8, less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05, less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002, or less than 0.001.

[0197] In one aspect, the variant Fc region of the present invention does not have substantially reduced C1q binding activity, which means that the difference in C1q binding activity between the variant Fc region and the parental Fc region of the present invention is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% as a function of the C1q binding activity of the parental Fc region.

[0198] In one aspect, the present invention provides an isolated polypeptide comprising a variant Fc region having substantially reduced ADCC activity. In one aspect, the present invention provides an isolated polypeptide comprising a variant Fc region having substantially reduced CDC activity. In one aspect, the present invention provides an isolated polypeptide comprising a variant Fc region having substantially reduced ADCC activity and not having substantially reduced CDC activity.

[0199] In one aspect, the variant Fc region of the present invention has substantially reduced ADCC activity, which is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% as a function of the ADCC activity of the parental Fc region.

[0200] In one aspect, the variant Fc region of the present invention has substantially no reduced CDC activity, which means that the difference in CDC activity between the variant Fc region of the present invention and the parental Fc region is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% as a function of the CDC activity of the parental Fc region.

[0201] In one aspect, the present invention provides an isolated polypeptide comprising a variant Fc region that has substantially reduced FcγR binding activity and substantially no reduced C1q binding activity as compared to a polypeptide comprising a parental Fc region. In a further aspect, the polypeptide of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of 234, 235, 236, 267, 268, 324, 326, 332, and 333 according to EU numbering.

[0202] In one aspect, a variant Fc region that has substantially reduced FcγR binding activity and substantially no reduced C1q binding activity comprises Ala at position 234, Ala at position 235, and at least one amino acid modification at at least one position selected from the group consisting of 236, 267, 268, 324, 326, 332, and 333 according to EU numbering.

[0203] In one aspect, a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains Ala at position 234, Ala at position 235, and one additional amino acid modification of any one of the following (a) to (c) according to EU numbering: (a) positions 267, 268, and 324; (b) positions 236, 267, 268, 324, and 332; and (c) positions 326 and 333.

[0204] In a further aspect, a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains an amino acid selected from the group consisting of (a) Glu at position 267; (b) Phe at position 268; (c) Thr at position 324; (d) Ala at position 236; (e) Glu at position 332; (f) Ala, Asp, Glu, Met, or Trp at position 326; and (g) Ser at position 333 according to EU numbering.

[0205] In one aspect, a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, Ala at position 235, Ala at position 326, and Ser at position 333. In one aspect, a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, Ala at position 235, Asp at position 326, and Ser at position 333. In one aspect, a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, Ala at position 235, Glu at position 326, and Ser at position 333. In one aspect, a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, Ala at position 235, Met at position 326, and Ser at position 333. In one aspect, a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains the following amino acids according to EU numbering: Ala at position 234, Ala at position 235, Trp at position 326, and Ser at position 333.

[0206] In another aspect, the mutant Fc region of the present invention can further contain at least one amino acid modification at at least one position selected from the group consisting of 428, 434, 436, 438, and 440 according to EU numbering.

[0207] In a further aspect, the variant Fc region can further comprise an amino acid selected from the group consisting of: (a) Ala at position 434; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to the EU numbering; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; and (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (see also WO2016 / 125495 which describes the relationship between the amino acid modifications and the FcRn binding activity of the variant Fc region).

[0208] In another aspect, the variant Fc region of the present invention comprises the following amino acids according to the EU numbering: Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440. In another aspect, the variant Fc region of the present invention comprises the following amino acids according to the EU numbering: Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440.

[0209] In one aspect, it is preferred that the variant Fc region of the present invention does not have a substantially increased FcRn binding activity, particularly at pH 7.4, compared to the parental Fc region.

[0210] "FcRn" is structurally similar to the polypeptide of the major histocompatibility complex (MHC) class I and shows 22% - 29% sequence identity with the MHC class I molecule. FcRn is expressed as a heterodimer consisting of a transmembrane α-chain or heavy chain and a soluble β-chain or light chain (β2-microglobulin) that forms a complex. Similar to MHC, the α-chain of FcRn contains three extracellular domains (α1, α2, and α3), and its short cytoplasmic domain anchors them to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain of the antibody Fc region. The polynucleotide sequence and amino acid sequence of human FcRn can be derived from the precursors (including the signal sequence) shown in NM_004107.4 and NP_004098.1, respectively.

[0211] The amino acid sequence of human FcRn (α-chain) is shown in SEQ ID NO: 81; the amino acid sequence of human β2-microglobulin is shown in SEQ ID NO: 82.

[0212] In one aspect, the mutant Fc region of the present invention preferably does not have a substantially increased FcRn-binding activity, particularly at pH 7.4, which is less than 1000-fold, less than 500-fold, less than 200-fold, less than 100-fold, less than 90-fold, less than 80-fold, less than 70-fold, less than 60-fold, less than 50-fold, less than 40-fold, less than 30-fold, less than 20-fold, less than 10-fold, less than 5-fold, less than 3-fold, or less than 2-fold compared to the FcRn-binding activity of the parental Fc region. In one aspect, the mutant Fc region of the present invention does not have a substantially increased FcRn-binding activity, particularly at pH 7.4, which means that the ratio of [the difference in RU values of the sensorgram before and after the interaction between FcRn and the mutant Fc region] / [the difference in RU values of the sensorgram before and after capturing FcRn on the sensor chip] is less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05, less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002, or less than 0.001.

[0213] In another aspect, the mutant Fc region of the present invention comprises any one or a combination of the amino acid modifications described in Table 4. In another aspect, the mutant Fc region of the present invention comprises at least any one of the amino acid modifications described in Table 4. In another aspect, the present invention provides a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 51 to 59.

[0214] In some embodiments, the polypeptide comprising the mutant Fc region of the present invention is the constant region of an antibody heavy chain. In some embodiments, the polypeptide comprising the mutant Fc region of the present invention further comprises an antigen-binding domain. In a further embodiment, the polypeptide is an antibody heavy chain. In a further embodiment, the polypeptide is an antibody. In a particular embodiment, the antibody is a chimeric antibody or a humanized antibody. The origin of the antibody is not particularly limited, and examples include human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, and the like. In a further embodiment, the polypeptide is an Fc fusion protein.

[0215] Two or more polypeptides comprising the mutant Fc region described herein can be included in one molecule, in which case the two polypeptides comprising the mutant Fc region are associated like an antibody. The type of antibody is not limited, and IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM can be used, among others.

[0216] In some embodiments, the polypeptide comprising the mutant Fc region of the present invention is an antibody. In a further embodiment, the polypeptide comprising the mutant Fc region of the present invention is an antiviral antibody.

[0217] In a further embodiment, the polypeptide comprising the mutant Fc region of the present invention comprises an antibody variable region comprising: (a) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0218] In a further aspect, the polypeptide comprising the variant Fc region of the invention comprises an antibody variable region comprising: (a) (i) HVR-H3 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-L3 derived from the VL sequence of SEQ ID NO: 10, and (iii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6; (b) (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, and (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6; (c) (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6, (iv) HVR-L1 derived from the VL sequence of SEQ ID NO: 10, (v) HVR-L2 derived from the VL sequence of SEQ ID NO: 10, and (vi) HVR-L3 derived from the VL sequence of SEQ ID NO: 10; (d) (i) HVR-L1 derived from the VL sequence of SEQ ID NO: 10, (ii) HVR-L2 derived from the VL sequence of SEQ ID NO: 10, and (iii) HVR-L3 derived from the VL sequence of SEQ ID NO: 10; or (e) (i) HVR-H1 derived from the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 derived from the VH sequence of SEQ ID NO: 6, (iii) HVR-H3 derived from the VH sequence of SEQ ID NO: 6, (iv) HVR-L1 derived from the VL sequence of SEQ ID NO: 7, (v) HVR-L2 derived from the VL sequence of SEQ ID NO: 7, and (vi) HVR-L3 derived from the VL sequence of SEQ ID NO: 7.

[0219] In a further aspect, the polypeptide comprising the variant Fc region of the present invention comprises a VH region comprising the amino acid sequence of SEQ ID NO: 1 and a VL region comprising the amino acid sequence of SEQ ID NO: 7. In a further aspect, the present invention provides an antibody comprising a VH region comprising the amino acid sequence of SEQ ID NO: 1 in the heavy chain and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 54, and a VL region comprising the amino acid sequence of SEQ ID NO: 7 in the light chain. In a further aspect, the present invention provides an antibody comprising a VH region comprising the amino acid sequence of SEQ ID NO: 1 in the heavy chain and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 58, and a VL region comprising the amino acid sequence of SEQ ID NO: 7 in the light chain. In a further aspect, the present invention provides an antibody comprising a VH region comprising the amino acid sequence of SEQ ID NO: 1 in the heavy chain and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 59, and a VL region comprising the amino acid sequence of SEQ ID NO: 7 in the light chain.

[0220] The present invention also provides the anti-DENV antibodies described herein, which further comprise a polypeptide comprising a variant Fc region of the present invention. In some embodiments, the anti-DENV antibody of the present invention comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6 in the heavy chain and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 54, and a VL region comprising the amino acid sequence of SEQ ID NO: 10 in the light chain. In some embodiments, the anti-DENV antibody of the present invention comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6 in the heavy chain and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 58, and a VL region comprising the amino acid sequence of SEQ ID NO: 10 in the light chain. In some embodiments, the anti-DENV antibody of the present invention comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6 in the heavy chain and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 59, and a VL region comprising the amino acid sequence of SEQ ID NO: 10 in the light chain.

[0221] In some embodiments, the anti-DENV antibody of the present invention comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6 in the heavy chain and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 59, and a VL region comprising the amino acid sequence of SEQ ID NO: 7 in the light chain.

[0222] As used herein, the "parent Fc region" refers to the Fc region before introduction of the amino acid modification(s) described herein. Preferred examples of the parent Fc region include Fc regions derived from natural antibodies. Examples of antibodies include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. The antibody can be derived from human or monkey (e.g., cynomolgus monkey, rhesus monkey, marmoset, chimpanzee, or baboon). Natural antibodies may contain naturally occurring mutations. "Sequences of proteins of immunological interest", NIH Publication No. 91-3242 describes multiple allotype sequences of IgG due to genetic polymorphisms, any of which can be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356-358 (EU numbering) can be either DEL or EEM. Preferred examples of the parent Fc region include Fc regions derived from the heavy chain constant regions of human IgG1 (SEQ ID NO: 83), human IgG2 (SEQ ID NO: 84), human IgG3 (SEQ ID NO: 85), and human IgG4 (SEQ ID NO: 86). Another preferred example of the parent Fc region is the Fc region derived from the heavy chain constant region SG1 (SEQ ID NO: 87). Another preferred example of the parent Fc region is the Fc region derived from the heavy chain constant region SG182 (SEQ ID NO: 46). Furthermore, the parent Fc region may be an Fc region resulting from addition of amino acid modification(s) other than the amino acid modifications described herein to an Fc region derived from a natural antibody.

[0223] Furthermore, amino acid modifications made for other purposes can be combined in the mutant Fc region described herein. For example, amino acid substitutions that improve FcRn binding activity (Hinton et al., J. Immunol. 176(1):346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12):1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159 (2010); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320), and amino acid substitutions for improving antibody heterogeneity or stability (WO 2009 / 041613) can be added. Alternatively, a polypeptide having properties that promote antigen clearance (described in WO 2011 / 122011, WO 2012 / 132067, WO 2013 / 046704 or WO 2013 / 180201), a polypeptide having properties that specifically bind to a target tissue (described in WO 2013 / 180200), a polypeptide having properties that repeatedly bind to multiple antigen molecules (described in WO 2009 / 125825, WO 2012 / 073992 or WO 2013 / 047752) can be combined with the mutant Fc region described herein. Alternatively, for the purpose of conferring binding ability to other antigens, the amino acid modifications disclosed in EP1752471 and EP1772465 can be combined in the CH3 of the mutant Fc region described herein. Alternatively, for the purpose of increasing plasma retention, amino acid modifications that decrease the pI of the constant region (WO 2012 / 016227) can be combined in the mutant Fc region described herein. Alternatively, for the purpose of promoting cellular uptake, amino acid modifications that increase the pI of the constant region (WO 2014 / 145159) can be combined in the mutant Fc region described herein.Alternatively, amino acid modifications that increase the pI of the constant region (WO2016 / 125495 and WO2016 / 098357) can be combined in the mutant Fc regions described herein for the purpose of promoting the excretion of the target molecule from the plasma.

[0224] Amino acid modifications that enhance human FcRn binding activity under acidic pH can also be combined in the mutant Fc regions described herein. Specifically, examples of such modifications include: substitution of Met at position 428 with Leu and substitution of Asn at position 434 with Ser according to EU numbering (Nat Biotechnol, 2010, 28: 157-159); substitution of Asn at position 434 with Ala (Drug Metab Dispos, 2010 Apr; 38(4): 600-605); substitution of Met at position 252 with Tyr, substitution of Ser at position 254 with Thr, and substitution of Thr at position 256 with Glu (J Biol Chem, 2006, 281: 23514-23524); substitution of Thr at position 250 with Gln and substitution of Met at position 428 with Leu (J Immunol, 2006, 176(1): 346-356); substitution of Asn at position 434 with His (Clin Pharmacol Ther, 2011, 89(2): 283-290); and modifications described in WO2010 / 106180, WO2010 / 045193, WO2009 / 058492, WO2008 / 022152, WO2006 / 050166, WO2006 / 053301, WO2006 / 031370, WO2005 / 123780, WO2005 / 047327, WO2005 / 037867, WO2004 / 035752, WO2002 / 060919, etc. In another embodiment, such modifications can include at least one modification selected from the group consisting of, for example, substitution of Met at position 428 with Leu, substitution of Asn at position 434 with Ala, and substitution of Tyr at position 436 with Thr. These modifications can further include substitution of Gln at position 438 with Arg and / or substitution of Ser at position 440 with Glu (WO2016 / 125495).

[0225] In the present invention, amino acid modification means any one of substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, amino acid modification can be paraphrased as amino acid mutation.

[0226] Amino acid modification is brought about by various methods known to those skilled in the art. Such methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh et al., Gene 152:271-275 (1995); Zoller, Meth. Enzymol. 100:468-500 (1983); Kramer et al., Nucleic Acids Res. 12: 9441-9456 (1984)); Kramer and Fritz, Methods Enzymol. 154: 350-367 (1987); and Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCR mutagenesis, and cassette mutagenesis.

[0227] The number of amino acid modifications introduced into the Fc region is not limited. In certain embodiments, it can be one, two or less, three or less, four or less, five or less, six or less, eight or less, ten or less, twelve or less, fourteen or less, sixteen or less, eighteen or less, or twenty or less.

[0228] Furthermore, the polypeptide containing the mutant Fc region of the present invention can be chemically modified using various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Chemical modification methods for such polypeptides are established in the art.

[0229] In some embodiments, the polypeptide comprising the variant Fc region of the invention is an antibody or an Fc fusion protein comprising a domain(s) capable of binding to any antigen. Examples of antigens that can be bound by such antibodies and Fc fusion proteins include, but are not limited to, ligands (such as cytokines, chemokines, etc.), receptors, cancer antigens, viral antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes that partially contain immunoglobulins.

[0230] B. Recombinant Methods and Compositions In one example, a process for preparing the antibodies described herein is referred to, where the process comprises the following steps: (a) combining the VH variant sequence described herein with the human IgG1 CH sequence described herein; (b) combining the VL variant sequence described herein with the human CL sequence SK1; (c) cloning each of the combinations into an expression vector; (d) expressing the resulting expression vector in cotransfected cells (host cells); and (e) purifying the antibody obtained from step (d). In certain examples, the host cell is CHO-DXB11, CHO-K1, or CHO-DG44. Such host cells may be cells that express a taurine transporter and can be obtained by introducing DNA encoding the taurine transporter (WO2007 / 119774). Vectors that can be used for the production of such antibodies are known in the art. Generally, antibodies can be made using, for example, the recombinant methods and compositions described in U.S. Patent No. 4,816,567. In one aspect, an isolated nucleic acid encoding an anti-DENV antibody described herein is provided. In another aspect, an isolated nucleic acid encoding a polypeptide comprising a mutated Fc region or a parental Fc region described herein is provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light chain and / or heavy chain of the antibody). In a further aspect, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further aspect, a host cell comprising such nucleic acids is provided. In one such aspect, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one aspect, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphocyte cell (e.g., Y0, NS0, Sp2 / 0 cells). In one aspect, a method for producing an anti-DENV antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for the expression of the antibody, and optionally, recovering the antibody from the host cell (or host cell culture medium).In another aspect, provided is a method for producing a polypeptide comprising a mutant Fc region or a parental Fc region, the method comprising culturing a host cell comprising a nucleic acid encoding a polypeptide such as an antibody, an Fc region, or a mutant Fc region as provided above under conditions suitable for expression of the polypeptide, and optionally, recovering the polypeptide from the host cell (or the host cell culture medium).

[0231] To recombinantly produce an anti-DENV antibody, for example, a nucleic acid encoding the antibody is isolated as described above and inserted into one or more vectors for further cloning and / or expression in a host cell. To recombinantly produce an Fc region, a nucleic acid encoding the Fc region is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of an antibody).

[0232] Host cells suitable for cloning or expressing a vector encoding an antibody include prokaryotic or eukaryotic cells as described herein. For example, an antibody may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the soluble fraction of the bacterial cell paste and further purified.

[0233] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeast whose glycosylation pathways have been "humanized" to result in the production of antibodies with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0234] Those derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjunction with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0235] Plant cell cultures can also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants.

[0236] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension would be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7); human fetal kidney lines (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; and FS4 cells, etc. Other useful mammalian host cell lines include DHFR - Chinese hamster ovary (CHO) cells including DHFR

[0237] It should be noted that there seems to be an incomplete sentence in the original text for where it says "Other useful mammalian host cell lines include DHFR" without further elaboration. Also, in the translation of , it's not clear what the "DHFR" in the brackets is supposed to mean in the context. It might be a typo or some specialized term that requires more context to accurately translate and integrate.Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. The relevant antigen is conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional substance or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR (where R and R 1 are different alkyl groups) may be useful for conjugation.

[0238] Animals (usually non-human mammals) are immunized against the antigen, immunogenic conjugate, or derivative by, for example, intradermally injecting a solution of 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) in combination with three volumes of Freund's complete adjuvant at multiple sites. One month later, the animal is boost-immunized with 1 / 5 to 1 / 10 of the initial amount of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animal is bled and the serum is assayed for antibody titer. The animal is boost-immunized until the titer reaches a plateau. Preferably, the animal is boost-immunized with conjugates that are the same antigen but conjugated to a different protein and / or conjugated via a different cross-linking reagent. The conjugate can also be prepared as a protein fusion in recombinant cell culture. Also, aggregating agents such as alum are preferably used to enhance the immune response.

[0239] A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies making up the population are identical except for naturally occurring potential mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates the characteristic of an antibody that it is not a mixture of distinct antibodies.

[0240] For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described hereinabove to induce lymphocytes capable of producing or having the ability to produce antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0241] The immunizing agent typically includes the antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes (PBLs) are used when cells of human origin are desired, and spleen cells or lymph node cells are used when a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0242] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are utilized. The hybridoma cells thus produced are seeded and grown in a suitable culture medium preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT-deficient cells.

[0243] Preferred immortalized myeloma cells are cells that fuse efficiently, assist in the stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, mouse myeloma strains, such as those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, and the SP-2 cells (and its derivatives, such as X63-Ag8-653) available from the American Type Culture Collection in Manassas, Virginia, USA, are preferred. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described (Kozbor et al., J Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0244] The culture medium in which hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, the binding affinity can be determined by Scatchard analysis as described in Munson, Anal Biochem. 107(1):220-239 (1980).

[0245] After hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity have been identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Also, hybridoma cells can be grown in vivo as tumors in mammals.

[0246] Monoclonal antibodies secreted by the subclones can be appropriately separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0247] The Fc region can be obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a protease such as pepsin and then eluting the fraction adsorbed to a protein A column again. The protease is not particularly limited as long as it can digest the full-length antibody and thereby produce Fab and F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions such as pH, and examples include pepsin and papain.

[0248] Furthermore, the present invention provides a method for producing a polypeptide comprising a mutant Fc region with substantially reduced FcγR binding activity and substantially reduced C1p binding activity compared to a polypeptide comprising a parental Fc region, the method comprising introducing at least one amino acid modification into the parental Fc region. In some aspects, the produced polypeptide is an antibody. In certain embodiments, the antibody is a chimeric antibody or a humanized antibody. In some aspects, the produced polypeptide is an Fc fusion protein.

[0249] In one aspect, in the above method for preparing a polypeptide comprising a mutant Fc region having substantially reduced FcγR binding activity and substantially reduced C1q binding activity, at least one amino acid at at least one position selected from the group consisting of 234, 235, 236, 267, 268, 324, 326, 332, and 333 is modified according to EU numbering.

[0250] In another aspect, in the above method for preparing a polypeptide comprising a mutant Fc region having substantially reduced FcγR binding activity and substantially reduced C1q binding activity, two amino acids are modified at positions 234 and 235.

[0251] In another aspect, in the above method for preparing a polypeptide comprising a mutant Fc region having substantially reduced FcγR binding activity and substantially reduced C1q binding activity, the amino acid is modified, the modification comprising: according to EU numbering, (a) two amino acid modifications at positions 234 and 235, and (b) at least one amino acid modification at at least one position selected from the group consisting of 236, 267, 268, 324, 326, 332, and 333.

[0252] In another aspect, in the above method for preparing a polypeptide comprising a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity, the amino acids are modified, and the modification includes the following: according to EU numbering, (a) two amino acid modifications at positions 234 and 235, and (b) at least one amino acid modification of any one of the following (i)-(iii): (i) positions 267, 268, and 324; (ii) positions 236, 267, 268, 324, and 332; (iii) positions 326 and 333.

[0253] In a further aspect, the amino acid modification in the above method for preparing a polypeptide comprising a mutant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity is selected from the group consisting of, at each position: according to EU numbering, (a) Ala at position 234; (b) Ala at position 235; (c) Glu at position 267; (d) Phe at position 268; (e) Thr at position 324; (f) Ala at position 236; (g) Glu at position 332; (h) Ala, Asp, Glu, Met, Trp at position 326; and (i) Ser at position 333.

[0254] In a further aspect, the amino acid modifications in the above method for preparing a polypeptide comprising a variant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity are Ala at position 234, Ala at position 235, Ala at position 326, and Ser at position 333, according to EU numbering. In a further aspect, the amino acid modifications in the above method for preparing a polypeptide comprising a variant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity are Ala at position 234, Ala at position 235, Asp at position 326, and Ser at position 333, according to EU numbering. In a further aspect, the amino acid modifications in the above method for preparing a polypeptide comprising a variant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity are Ala at position 234, Ala at position 235, Glu at position 326, and Ser at position 333, according to EU numbering. In a further aspect, the amino acid modifications in the above method for preparing a polypeptide comprising a variant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity are Ala at position 234, Ala at position 235, Met at position 326, and Ser at position 333, according to EU numbering. In a further aspect, the amino acid modifications in the above method for preparing a polypeptide comprising a variant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity are Ala at position 234, Ala at position 235, Trp at position 326, and Ser at position 333, according to EU numbering.

[0255] In another aspect, in the above method, at least one amino acid is further modified at at least one position selected from the group consisting of 428, 434, 436, 438, and 440, according to EU numbering.

[0256] In a further aspect, the amino acid modification in the above method is further selected from the following (a) to (d): according to EU numbering, (a) Ala at position 434; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; and (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (see also WO2016 / 125495, which describes the relationship between the amino acid modification and the FcRn binding activity of the mutant Fc region).

[0257] In a further aspect, the amino acid modification in the above method is, according to EU numbering, Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440. In a further aspect, the amino acid modification in the above method is, according to EU numbering, Ala at position 234, Ala at position 235, Ala at position 326, Ser at position 333, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440.

[0258] In one aspect, it is preferred that the mutant Fc region of the present invention does not have a substantially increased FcRn binding activity, particularly at pH 7.4, compared to the parental Fc region.

[0259] In a further aspect, the amino acid modification in the above production method is selected from any single modification, a combination of single modifications, or a combined modification described in Table 4.

[0260] Also included in the present invention are polypeptides containing a mutant Fc region produced by any of the above methods or other methods known in the art.

[0261] C. Assay The anti-DENV antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0262] The mutant Fc regions provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0263] 1. Binding Assays and Other Assays In one aspect, the antibodies of the invention are tested for their antigen-binding activity by known methods such as ELISA, Western blot, etc. In one aspect, polypeptides comprising the mutant Fc region of the invention are tested for their antigen-binding activity by known methods such as ELISA, Western blot, etc.

[0264] In another aspect, a competition assay can be used to identify antibodies that compete with any of the anti-DENV antibodies described herein with respect to binding to DENV and / or the DENV E protein. In certain embodiments, if such competing antibodies are present in excess, they inhibit (e.g., reduce) the binding of a reference antibody to DENV and / or the DENV E protein by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some instances, the binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as that bound by the anti-DENV antibodies described herein. Detailed, exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) 「Epitope Mapping Protocols」Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0265] In an exemplary competitive assay, immobilized DENV or DENV E protein is incubated in a solution containing a first labeled antibody that binds to the DENV and / or DENV E protein and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to the DENV or DENV E protein. The second antibody can be present in a hybridoma supernatant. As a control, the immobilized DENV or DENV E protein is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that permit binding of the first antibody to the DENV or DENV E protein, excess unbound antibody is removed and the amount of label bound to the immobilized DENV or DENV E protein is measured. If the amount of label bound to the immobilized DENV or DENV E protein is substantially decreased in the test sample as compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the DENV or DENV E protein. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0266] Assays for determining the binding activity of a polypeptide comprising a mutant Fc region to one or more FcR family members are described herein or are otherwise known in the art. Such binding assays include, but are not limited to, the BIACORE® assay, which utilizes surface plasmon resonance (SPR) phenomena, amplified luminescence proximity homogeneous assay (ALPHA) screening, ELISA, and fluorescence-activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103(11): 4005-4010).

[0267] In one aspect, the BIACORE® assay can be used to determine whether the binding activity of a polypeptide comprising a mutant Fc region is enhanced, maintained, or reduced with respect to a particular FcR family member by observing whether the dissociation constant (Kd) value obtained from the analysis of a sensorgram subjected to the interaction of various FcRs as analytes with a polypeptide comprising a mutant Fc region immobilized or captured on a sensor chip using known methods and reagents (e.g., Protein A, Protein L, Protein A / G, Protein G, anti-λ chain antibody, anti-κ chain antibody, antigen peptide, antigen protein) is decreased or increased. The change in binding activity can also be determined by comparing the change in resonance unit (RU) values in the sensorgram before and after subjecting one or more types of FcRs as analytes to the interaction with a polypeptide comprising a captured mutant Fc region. Alternatively, the FcR can be immobilized or captured on the sensor chip, and the polypeptide comprising the mutant Fc region is used as the analyte.

[0268] In BIACORE® analysis, one of the substrates (ligand) in the observation of interaction is immobilized on the gold thin film on the sensor chip, and total reflection occurs at the interface between the gold thin film and the glass by irradiating light from the back side of the sensor chip, whereby in a part of the reflected light, a portion where the reflection intensity decreases is formed (SPR signal). When another one of the substrates (analyte) in the observation of interaction flows onto the sensor chip surface and the ligand binds to the analyte, the mass of the immobilized ligand molecules increases and the refractive index of the solvent on the sensor chip surface changes. As a result of this change in refractive index, the position of the SPR signal shifts (on the other hand, when this binding dissociates, the signal position returns). The BIACORE® system indicates the amount of the above shift or, more specifically, indicates the time variable of mass by plotting the change in mass of the sensor chip surface as measurement data (sensorgram) on the vertical axis. The amount of analyte bound to the ligand trapped on the sensor chip surface is determined from the sensorgram. Kinetic parameters such as the association rate constant (ka) and the dissociation rate constant (kd) are determined from the curve of the sensorgram, and the dissociation constant (Kd) is determined from the ratio of these constants. In the BIACORE® method, a method for measuring inhibition is preferably used. An example of a method for measuring inhibition is described in Lazar et al., Proc. Natl. Acad. Sci. USA 103(11):4005-4010 (2006).

[0269] ALPHA screening is carried out by the ALPHA technology using two types of beads, donor and acceptor, based on the following principle. When the molecule bound to the donor bead physically interacts with the molecule bound to the acceptor bead, a luminescence signal is detected only when the two beads are in very close proximity to each other. The laser-excited photosensitizer in the donor bead converts the surrounding oxygen into singlet oxygen in the excited state. The singlet oxygen is dispersed around the donor bead, and when it reaches the adjacent acceptor bead, a chemiluminescence reaction is induced in the bead, and finally light is emitted. When the molecule bound to the donor bead does not interact with the molecule bound to the acceptor bead, the singlet oxygen generated by the donor bead does not reach the acceptor bead, so the chemiluminescence reaction does not occur.

[0270] For example, a biotinylated polypeptide complex is bound to the donor bead, and an Fc receptor tagged with glutathione S-transferase (GST) is bound to the acceptor bead. In the absence of a competing polypeptide complex containing a mutant Fc region, the polypeptide complex containing the parental Fc region interacts with the Fc receptor to produce a signal at 520 - 620 nm. A polypeptide complex containing a tagless mutant Fc region competes with the polypeptide complex containing the parental Fc region for interaction with the Fc receptor. The relative binding activity can be measured by quantifying the decrease in fluorescence observed as a result of the competition. Biotinylation using, for example, sulfo-NHS-biotin of a polypeptide complex such as an antibody is well known. A method of expressing the Fc receptor and GST in a cell carrying a fusion gene prepared by in-frame fusion of a polynucleotide encoding the Fc receptor and a polynucleotide encoding GST in an expressible vector and purifying using a glutathione column is suitable for use as a method for tagging the Fc receptor with GST. The obtained signals are preferably analyzed by fitting them to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0271] A mutant Fc region having a reduced FcR binding activity refers to an Fc region that binds to FcR with a binding activity that is substantially weaker than that of the corresponding parental Fc region when assays are performed using substantially the same amounts of the corresponding parental Fc region and the mutant Fc region. Further, a mutant Fc region having an increased FcR binding activity refers to an Fc region that binds to FcR with a binding activity that is substantially stronger than that of the corresponding parental Fc region when assays are performed using substantially the same amounts of the parental Fc region and the mutant Fc region. A mutant Fc region that maintains FcR binding activity refers to an Fc region that binds to FcR with a binding activity that is equivalent to or not substantially different from that of the parental Fc region when assays are performed using a polypeptide containing substantially the same amounts of the corresponding parental Fc region and the mutant Fc region.

[0272] Whether the binding activity of the Fc region to various FcRs has increased or decreased can be determined from the increase or decrease in the amount of binding of various FcRs to the Fc region, measured according to the above method. Here, the amount of binding of various FcRs to the Fc region can be evaluated as a value obtained by dividing the difference in the RU values of the sensorgrams that changed before and after the interaction between the various FcRs as the analyte and the Fc region by the difference in the RU values of the sensorgrams that changed before and after the Fc region was captured on the sensor chip. The binding activity of the Fc region to FcγR or FcRn can be measured by the method described in Example 5 of this specification.

[0273] In the present invention, substantially reduced FcγR binding activity preferably means, for example, that the binding activity of the mutant Fc region to FcγR is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% as a function of the FcγR binding activity of the parental Fc region. It also preferably means, for example, that the ratio of [difference in RU value of the sensorgram before and after interaction between FcγR and the mutant Fc region] / [difference in RU value of the sensorgram before and after capturing FcγR on the sensor chip] is less than 1, less than 0.8, less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05, less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002, or less than 0.001.

[0274] In the present invention, having substantially increased FcRn binding activity, particularly at pH 7.4, preferably means, for example, that the binding activity of the mutant Fc region to FcRn is less than 1000-fold, less than 500-fold, less than 200-fold, less than 100-fold, less than 90-fold, less than 80-fold, less than 70-fold, less than 60-fold, less than 50-fold, less than 40-fold, less than 30-fold, less than 20-fold, less than 10-fold, less than 5-fold, less than 3-fold, or less than 2-fold compared to the FcRn binding activity of the parental Fc region. It also preferably means, for example, that the ratio of [difference in RU value of the sensorgram before and after interaction between FcRn and the mutant Fc region] / [difference in RU value of the sensorgram before and after capturing FcRn on the sensor chip] is less than 0.5, less than 0.3, less than 0.2, less than 0.1, less than 0.08, less than 0.05, less than 0.03, less than 0.02, less than 0.01, less than 0.008, less than 0.005, less than 0.003, less than 0.002, or less than 0.001.

[0275] To measure the binding activity of a polypeptide containing a mutant Fc region to C1q, a C1q binding ELISA can be performed. Briefly, the assay plate is coated overnight at 4 °C with a polypeptide containing a mutant Fc region or a polypeptide containing a parental Fc region (control) in coating buffer. The plate is then washed and blocked. After washing, an aliquot of human C1q is added to each well and incubated at room temperature for 2 hours. After further washing, 100 μl of sheep anti-complement C1q peroxidase-conjugated antibody is added to each well and incubated at room temperature for 1 hour. The plate is washed again with wash buffer, and 100 μl of substrate buffer containing OPD (o-phenylenediamine dihydrochloride (Sigma)) is added to each well. The oxidation reaction observed by the yellow color development is allowed to proceed for 30 minutes and stopped by the addition of 100 μl of 4.5N H2SO4. Thereafter, the absorbance is read at (492 - 405) nm. The binding activity of the Fc region to C1q can be measured by the method described in Example 4 of the present specification.

[0276] In one aspect, the difference in C1q binding activity between the mutant Fc region and the parental Fc region of the present invention is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% as a function of the C1q binding activity of the parental Fc region.

[0277] 2. Activity assay In one aspect, an assay for identifying an anti-DENV antibody having biological activity is provided. Biological activities include, for example, blocking the binding of DENV E protein to host cells, preventing DENV entry into host cells, preventing and / or preventing DENV infection of host cells, and the like. Also provided are antibodies having such biological activities in vivo and / or in vitro.

[0278] In certain embodiments, the antibodies of the invention are tested for such biological activity. In certain embodiments, a plaque reduction neutralization test (PRNT) assay can be utilized to measure the activity or neutralizing ability of the test antibody. In some embodiments, an animal host can be used to measure anti-DENV activity in vivo.

[0279] In certain embodiments, cells can be directly assayed for binding of DENV and the test antibody. Immunohistochemical techniques, confocal techniques, and / or other techniques for evaluating binding are well known to those of skill in the art. A variety of cell lines can be utilized in such screening assays, including specially engineered cells for this purpose. Examples of cells used in screening assays include mammalian cells, fungal cells, bacterial cells, or viral cells. The cells can be stimulated cells, e.g., cells stimulated with a growth factor. Those of skill in the art will understand that the invention disclosed herein contemplates a wide variety of assays for measuring the ability of a test antibody to bind DENV.

[0280] Depending on the assay, cell and / or tissue culture may be required. The cells can be tested using any of a number of different physiological assays. Alternatively, or in addition, molecular analysis can be performed, including but not limited to Western blotting for monitoring protein expression and / or testing for protein-protein interactions, mass spectrometry for monitoring other chemical modifications, etc.

[0281] In some embodiments, such methods utilize an animal host. For example, animal hosts suitable for the present invention can be mammalian hosts such as primates, ferrets, cats, dogs, cows, horses, and rodents such as mice, hamsters, rabbits, rats, etc. In some embodiments, the animal host is inoculated with, infected with, or otherwise exposed to a virus before or simultaneously with the administration of the test antibody. Naïve animals and / or inoculated animals can be used in various studies. For example, such animal models are used in virus transmission studies as known in the art. To determine the effectiveness of a test antibody in blocking the binding and / or infectivity of a virus to an animal host, the test antibody can be administered to a suitable animal host before, during, or after a virus transmission study.

[0282] In one aspect, an assay is provided for identifying a polypeptide comprising a mutant Fc region having biological activity. Biological activities include, for example, ADCC activity and CDC activity. Further provided are polypeptides comprising a mutant Fc region having such biological activity in vivo and / or in vitro.

[0283] In certain embodiments, the polypeptides comprising a mutant Fc region of the present invention are tested for such biological activity. In certain aspects, the polypeptides comprising a mutant Fc region of the present invention modulate effector function as compared to a polypeptide comprising a parental Fc region. In certain aspects, this modulation is modulation of ADCC and / or CDC.

[0284] To confirm CDC activity and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, an Fc receptor (FcR) binding assay can be conducted to confirm that the antibody has FcγR binding (and thus is likely to have ADCC activity) and retains FcRn binding ability. NK cells, which are primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu Rev Immunol (1991) 9, 457-492. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom et al, Proc Natl Acad Sci USA (1986) 83, 7059-7063) and Hellstrom et al, Proc Natl Acad Sci USA (1985) 82, 1499-1502; U.S. Patent No. 5,821,337 (see Bruggemann et al, J Exp Med (1987) 166, 1351-1361). Alternatively, non-radioactive assay methods can be used (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al, Proc Natl Acad Sci USA (1998) 95, 652-656. Also, a C1q binding assay can be performed to confirm whether the antibody has CDC activity due to binding to C1q.See, for example, the C1q and C3c binding ELISAs described in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al, J Immunol Methods (1997) 202, 163-171; Cragg et al, Blood (2003) 101, 1045-1052; and Cragg and Glennie, Blood (2004) 103, 2738-2743). FcRn binding and in vivo clearance / half-life measurements can also be performed using methods known in the art (see, for example, Petkova et al, Int Immunol (2006) 18, 1759-1769).

[0285] D. Immunoconjugates In some embodiments, the invention also provides an immunoconjugate comprising an anti-DENV antibody described herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin derived from bacteria, fungi, plants or animals, or fragments thereof), or a radioisotope. In some embodiments, the invention also provides an immunoconjugate comprising a polypeptide comprising a variant Fc region described herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin derived from bacteria, fungi, plants or animals, or fragments thereof), or a radioisotope.

[0286] In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, examples of such drugs include, but are not limited to: maytansinoids (see U.S. Patent Nos. 5,208,020 and 5,416,064; European Patent EP 0 425 235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Patent 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 or doxorubicin (see 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; trichothecene; and CC1065.

[0287] In another aspect, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, and such toxins include, but are not limited to: diphtheria A chain, 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, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0288] In another aspect, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioisotopes of Pb and Lu. When using a radioconjugate for detection, it can include a radioactive atom for scintigraphy, such as Tc-99m or 123 I, or a spin label 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 in this case as well.

[0289] Conjugates of antibodies and cytotoxic agents can be prepared using various bifunctional protein coupling agents such as the following: N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 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 WO94 / 11026. The linker can be a "cleavable linker" that promotes the release of the cytotoxic drug intracellularly. For example, an acid-labile linker, a peptidase-sensitive linker, a light-labile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0290] The immunoconjugates or ADCs described herein specifically contemplate, but are not limited to, such conjugates prepared using crosslinking reagent(s); crosslinking reagents include, but are 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, as well as SVSB ((4-vinylsulfone) succinimidyl benzoate), which are commercially available (e.g., Pierce Biotechnology, Rockford, IL, USA).

[0291] E. Methods and Compositions for Diagnosis and Detection In certain embodiments, the anti-DENV antibodies provided herein are useful for detecting the presence of DENV and / or DENV E protein in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, breast secretion, lymph fluid, urine, sweat, tear fluid, gastric juice, synovial fluid, ascites, eye lens fluid, or mucus.

[0292] In one aspect, anti-DENV antibodies for use in a diagnostic or detection method are provided. In a further aspect, a method for detecting the presence of DENV in a biological sample is provided. In certain aspects, the method comprises contacting the biological sample with an anti-DENV antibody described herein under conditions that permit binding of the anti-DENV antibody to DENV, and detecting whether a complex is formed between the anti-DENV antibody and DENV. Such methods can be in vitro or in vivo methods. In a further aspect, a method for detecting the presence of DENV E protein in a biological sample is provided. In certain aspects, the method comprises contacting the biological sample with an anti-DENV antibody described herein under conditions that permit binding of the anti-DENV antibody to DENV E protein, and detecting whether a complex is formed between the anti-DENV antibody and DENV E protein. Such methods can be in vitro or in vivo methods. In one aspect, for example, when DENV or DENV E protein is a biomarker for selecting a patient, the anti-DENV antibody is used to select a subject eligible for treatment with the anti-DENV antibody.

[0293] Exemplary diseases that can be diagnosed using the antibodies of the invention include DENV infection, and diseases and / or symptoms caused by or associated with DENV infection, such as dengue fever, dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS), and the like.

[0294] In certain aspects, labeled anti-DENV antibodies are provided. Labels include, but are not limited to, labels or components that are directly detectable (fluorescent labels, chromophore labels, high electron density labels, chemiluminescent labels, radioactive labels, etc.), as well as components that are indirectly detectable, such as enzymes or ligands, via, for example, enzymatic reactions or intermolecular interactions. Exemplary labels include, but are not limited to: radioisotopes 32 P, 14 C, 125 I, 3 H, and 131I. Fluorophores, such as rare earth element chelates, or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidase, such as uricase and xanthine oxidase, an enzyme that uses hydrogen peroxide to oxidize a pigment precursor (coupled with HRP, lactoperoxidase, or microperoxidase, etc.), biotin / avidin, spin label, bacteriophage label, stable free radical, etc.

[0295] In one aspect, an antibody comprising the mutant Fc region of the present invention can be used as an affinity purifying agent. In this process, the antibody mutant is immobilized on a solid phase such as Sephadex resin or filter paper using methods well known in the art. The immobilized antibody mutant is contacted with a sample containing the antigen to be purified, and then the support is washed with a suitable solvent that removes substantially all substances in the sample except the antigen to be purified that is bound to the immobilized antibody mutant. Finally, the support is washed with another suitable solvent, such as glycine buffer pH 5.0, that releases the antigen from the antibody mutant.

[0296] The antibody mutant can also be useful in diagnostic assays, for example, for detecting the expression of a target antigen in a particular cell, tissue, or serum.

[0297] Antibody variants can be used in known assay methods such as, for example, competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, (1987) pp. 147-158, CRC Press, Inc.

[0298] F. Pharmaceutical Formulations The pharmaceutical formulations of the anti-DENV antibodies described herein are prepared in the form of lyophilized formulations or aqueous solution formulations by mixing such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)).

[0299] The pharmaceutical formulations of the polypeptides containing the variant Fc region described herein are prepared in the form of lyophilized formulations or aqueous solution formulations by mixing such polypeptides having the desired purity with one or more optional pharmaceutically acceptable carriers.

[0300] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and examples of such carriers include, but are not limited to: buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants such as ascorbic acid, methionine, etc.; preservatives (e.g., octadecyl dimethyl benzyl ammonium 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, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, immunoglobulins, etc.; hydrophilic polymers such as polyvinyl pyrrolidone, etc.; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine, etc.; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, dextrin, etc.; chelating agents such as EDTA, etc.; sugars such as sucrose, mannitol, trehalose, sorbitol, etc.; salt-forming counterions such as sodium, etc.; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), etc. Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International). Some exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0301] 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 WO2006 / 044908, the latter of which contains a histidine - acetate buffer.

[0302] The formulations described herein may also contain, if necessary for the particular indication being treated, two or more active ingredients, preferably those having complementary activities that do not adversely affect each other. For example, but not limited to, it may be desirable to provide antiviral agents such as: interferons (e.g., interferon α - 2b, interferon γ, etc.), anti - DENV monoclonal antibodies, anti - DENV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, small interfering RNAs, small hairpin - type RNAs, microRNAs, RNA aptamers, ribozymes, and combinations thereof. Such active ingredients are present in appropriate combinations in effective amounts for the intended purpose.

[0303] The active ingredients can be encapsulated, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxy - methylcellulose or gelatin - microcapsules and poly(methyl methacrylate) microcapsules, respectively, or enclosed in 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).

[0304] It is possible to prepare sustained - release formulations. Suitable examples of sustained - release formulations include semi - permeable matrices of solid hydrophobic polymers containing the antibody, the matrix being in the form of shaped articles such as films or microcapsules.

[0305] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through a sterilizing membrane.

[0306] G. Treatment Methods and Compositions Any of the anti-DENV antibodies provided herein can be used in a treatment method.

[0307] In one aspect, anti-DENV antibodies for use as a medicament are provided. In a further aspect, anti-DENV antibodies for use in the treatment of DENV infection are provided. In certain embodiments, anti-DENV antibodies for use in a treatment method are provided. In certain embodiments, the present invention provides anti-DENV antibodies for use in a method of treating an individual infected with DENV, the method comprising administering to the individual an effective amount of the anti-DENV antibody. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In a further embodiment, the present invention provides anti-DENV antibodies for use in preventing the binding of DENV E protein to host cells and / or DENV entry into host cells. In certain embodiments, the present invention provides anti-DENV antibodies for use in a method of preventing the binding of DENV E protein to host cells and / or DENV entry into host cells in an individual, the method comprising administering to the individual an effective amount of the anti-DENV antibody to prevent the binding of DENV E protein to host cells and / or DENV entry into host cells. The "individual" according to any of the above embodiments is preferably a human.

[0308] In a further aspect, the present invention provides the use of an anti-DENV antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating DENV infection. In a further aspect, the medicament is for use in a method of treating DENV infection comprising the step of administering to an individual infected with DENV an effective amount of the medicament. In such an aspect, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent as described below. In a further aspect, the medicament is for preventing the binding of DENV E protein to host cells and / or the entry of DENV into host cells. In a further aspect, the medicament is for use in a method of preventing the binding of DENV E protein to host cells and / or the entry of DENV into host cells in an individual, the method comprising the step of administering to the individual an amount of the medicament effective to prevent the binding of DENV E protein to host cells and / or the entry of DENV into host cells. The "individual" according to any of the above aspects can be a human.

[0309] In a further aspect, the present invention provides a method for treating DENV infection. In one aspect, the method comprises the step of administering to an individual infected with DENV an effective amount of an anti-DENV antibody. In such an aspect, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent as described below. The "individual" according to any of the above aspects can be a human.

[0310] In a further aspect, the present invention provides a method for preventing the binding of DENV E protein to host cells and / or the entry of DENV into host cells in an individual. In one aspect, the method comprises the step of administering to the individual an amount of an anti-DENV antibody effective to prevent the binding of DENV E protein to host cells and / or the entry of DENV into host cells. In one aspect, the "individual" is a human.

[0311] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-DENV antibodies provided herein for use, for example, in any of the above-described treatment methods. In one aspect, the pharmaceutical formulation contains any of the anti-DENV antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical formulation comprises any of the anti-DENV antibodies provided herein and at least one additional therapeutic agent, such as those described below.

[0312] In a further aspect, the pharmaceutical formulation is for treating DENV infection. In a further aspect, the pharmaceutical formulation is for blocking the binding of DENV E protein to host cells and / or the entry of DENV into host cells. In one aspect, the pharmaceutical formulation is administered to an individual infected with DENV. The "individual" according to any of the above aspects is preferably a human.

[0313] In certain aspects, DENV infection may include diseases and / or symptoms caused by or associated with DENV infection, such as dengue fever, dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS), etc.

[0314] Any polypeptide comprising the variant Fc region described herein can be used in a treatment method.

[0315] In one aspect, a polypeptide comprising a variant Fc region is provided for use as a medicament. In certain aspects, a polypeptide comprising a variant Fc region is provided for use in a treatment method. In certain aspects, the present invention provides a polypeptide comprising a variant Fc region for use in a method of treating an individual having a disease, the method comprising administering to the individual an effective amount of the polypeptide comprising a variant Fc region. In such an aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one aspect, the disease is a viral infection. In one aspect, the "individual" is a human.

[0316] In a further aspect, the present invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating a disease. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In a further aspect, the medicament is for use in a method of treating a disease comprising the step of administering to an individual having the disease to be treated an effective amount of the medicament. In such an aspect, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent. In one aspect, the disease is a viral infection. In one aspect, the "individual" is a human.

[0317] In a further aspect, the present invention provides a method for treating a disease. In one aspect, the method comprises the step of administering to an individual having such a disease an effective amount of a polypeptide comprising a variant Fc region. In such an aspect, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent. In one aspect, the disease is a viral infection. In one aspect, the "individual" is a human.

[0318] In a further aspect, the present invention provides a pharmaceutical formulation containing a polypeptide comprising a variant Fc region as described herein for use in a treatment method, for example any of the treatment methods described herein. In one aspect, the pharmaceutical formulation contains a polypeptide comprising a variant Fc region as described herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical formulation contains a polypeptide comprising a variant Fc region as described herein and at least one additional therapeutic agent.

[0319] In a further aspect, the pharmaceutical formulation is for treating a disease. In one aspect, the pharmaceutical formulation is administered to an individual having the disease. In one aspect, the disease is a viral infection. In one aspect, the "individual" is a human.

[0320] The antiviral antibody comprising the mutant Fc region of the present invention can suppress antibody-dependent enhancement of infection (ADE) observed with conventional antiviral antibodies. ADE is a phenomenon in which a virus bound to an antibody is phagocytosed via activated FcγR, resulting in enhanced viral infection of cells. Fc modifications that reduce the interaction with activated FcγR are thought to reduce the risk of ADE. Mutations at positions 234 and 235 from leucine to alanine to form the LALA variant have been shown to reduce the risk of ADE of dengue infection in vivo (Cell Host Microbe (2010) 8, 271-283). However, such modifications reduce other effector immune functions mediated by antibodies, such as ADCC and CDC. In particular, CDC is expected to play an important role in suppressing ADE, and therefore, complement component C1q binding of the Fc region should not be reduced for therapeutic effects. Furthermore, the half-life of an antibody can be extended by engineering the Fc region to change its binding affinity to the salvage receptor FcRn, which may lead to the prophylactic use of antibodies to defend against viral infections.

[0321] The virus is preferably selected from adenovirus, astrovirus, hepadnavirus, herpesvirus, papovavirus, poxvirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, orthomyxovirus, paramyxovirus, picornavirus, reovirus, retrovirus, rhabdovirus, or togavirus.

[0322] In preferred embodiments, the adenovirus includes, but is not limited to, human adenovirus. In preferred embodiments, the astrovirus includes, but is not limited to, mamastrovirus. In preferred embodiments, the hepadnavirus includes, but is not limited to, hepatitis B virus. In preferred embodiments, the herpesvirus includes, but is not limited to, herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, roseolovirus, and Kaposi's sarcoma-associated herpesvirus. In preferred embodiments, the papovavirus includes, but is not limited to, human papillomavirus and human polyomavirus. In preferred embodiments, the poxvirus includes, but is not limited to, variola virus, vaccinia virus, cowpox virus, monkeypox virus, smallpox virus, pseudocowpox virus, orf virus, tanapox virus, yaba tumor virus, and molluscum contagiosum virus. In preferred embodiments, the arenavirus includes, but is not limited to, lymphocytic choriomeningitis virus, Lassa virus, Machupo virus, and Junin virus. In preferred embodiments, the bunyavirus includes, but is not limited to, hantavirus, nairovirus, orthobunyavirus, and phlebovirus. In preferred embodiments, the calicivirus includes, but is not limited to, besivirus, norovirus, such as Norwalk virus, and sapovirus. In preferred embodiments, the coronavirus includes, but is not limited to, human coronavirus (the pathogen of severe acute respiratory syndrome (SARS)). In preferred embodiments, the filovirus includes, but is not limited to, Ebola virus and Marburg virus.In preferred embodiments, flaviviruses include, but are not limited to, yellow fever virus, West Nile virus, dengue viruses (DENV-1, DENV-2, DENV-3, and DENV-4), hepatitis C virus, tick-borne encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis virus, Saint Louis encephalitis virus, Russian spring-summer encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Kyasanur Forest disease virus, and Powassan encephalitis virus. In preferred embodiments, orthomyxoviruses include, but are not limited to, influenza A virus, influenza B virus, and influenza C virus. In preferred embodiments, paramyxoviruses include, but are not limited to, parainfluenza viruses, rubulavirus (mumps), morbillivirus (measles), pneumovirus, such as human RSV, and subacute sclerosing panencephalitis virus. In preferred embodiments, picornaviruses include, but are not limited to, poliovirus, rhinovirus, coxsackievirus A, coxsackievirus B, hepatitis A virus, echovirus, and enterovirus. In preferred embodiments, reoviruses include, but are not limited to, Colorado tick fever virus and rotavirus. In preferred embodiments, retroviruses include, but are not limited to, lentiviruses, such as human immunodeficiency virus, and human T-lymphotropic virus (HTLV). In preferred embodiments, rhabdoviruses include, but are not limited to, lyssavirus, such as rabies virus, vesicular stomatitis virus, and infectious hematopoietic necrosis virus. In preferred embodiments, togaviruses include, but are not limited to, alphaviruses, such as Ross River virus, O'nyong'nyong virus, Sindbis virus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus, as well as rubella virus.

[0323] In a further aspect, the present invention provides a method for preparing a medicament or pharmaceutical formulation for use, for example, in any of the above-described treatment methods, the method comprising mixing any of the anti-DENV antibodies described herein with a pharmaceutically acceptable carrier. In one aspect, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0324] The antibodies of the present invention can be used alone or in combination with other agents in a treatment method. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In certain aspects, the additional therapeutic agent is an antiviral agent such as, but not limited to, interferons (e.g., interferon α-2b, interferon γ, etc.), anti-DENV monoclonal antibodies, anti-DENV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, small interfering RNAs, small hairpin RNAs, microRNAs, RNA aptamers, ribozymes, and combinations thereof.

[0325] In a further aspect, the present invention provides a method for preparing a medicament or pharmaceutical formulation for use, for example, in any of the above-described treatment methods, the method comprising mixing any of the polypeptides comprising a mutant Fc region described herein with a pharmaceutically acceptable carrier. In one aspect, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0326] The polypeptide comprising the variant Fc region of the present invention can be used alone or in combination with other agents in a treatment method. For example, the polypeptide comprising the variant Fc region of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an antiviral agent such as, but not limited to, interferon (e.g., interferon α-2b, interferon γ, etc.), antiviral monoclonal antibody, antiviral polyclonal antibody, RNA polymerase inhibitor, protease inhibitor, helicase inhibitor, immunomodulatory agent, antisense compound, small interfering RNA, small hairpin RNA, microRNA, RNA aptamer, ribozyme, and combinations thereof.

[0327] Such combination therapies as described above include co-administration (when two or more therapeutic agents are included in the same formulation or separate formulations) and separate administration. In the case of separate administration, the administration of the antibody or polypeptide comprising the variant Fc region of the present invention can be carried out before, simultaneously with, and / or following the administration of the additional therapeutic agent(s). In one embodiment, the administration of the anti-DENV antibody and the administration of the additional therapeutic agent are carried out within about 1 month, within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. In another embodiment, the administration of the polypeptide comprising the variant Fc region and the administration of the additional therapeutic agent are carried out within about 1 month, within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0328] The polypeptide comprising the antibody or mutant Fc region of the present invention (and any additional therapeutic agent) can be administered by any suitable means, for example, parenterally, intranasally, and intratracheally, and, if local treatment is desired, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection such as intravenous or subcutaneous injection, depending in part on whether its administration is short-term or long-term. A variety of dosing schedules are contemplated herein, such as single or multiple administrations at various times, bolus dosing, pulse infusion, and the like.

[0329] The polypeptide comprising the antibody or mutant Fc region of the present invention will be formulated and dosed in a manner that is consistent with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site to which the agent is to be delivered, the method of administration, the dosing regimen, and other factors known to the medical practitioner. The agent may optionally but not necessarily be formulated with one or more agents currently being used to prevent or treat the disease in question. The effective amount of such other agents will be determined by the amount of agent present in the formulation, the type of disease or treatment, and other factors as described above. These are generally used at the same dosage and route as described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and by any route that has been determined empirically / clinically to be appropriate.

[0330] When preventing or treating a disease, the appropriate dosage of the antibody or polypeptide comprising a mutant Fc region of the invention (when used alone or in combination with one or more other additional therapeutic agents) is determined by the following factors: the type of disease being treated, the type of antibody, the type of polypeptide comprising a mutant Fc region, the severity and course of the disease, whether the antibody or polypeptide comprising a mutant Fc region is administered for prophylactic purposes or for therapeutic purposes, previous therapies, the patient's medical history and response to the antibody or polypeptide comprising a mutant Fc region, and the judgment of the attending physician. The antibody or polypeptide comprising a mutant Fc region may be administered to the patient once or appropriately administered over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the antibody or polypeptide comprising a mutant Fc region may be an initial candidate dosage for administration to the patient, whether by, for example, one or more separate administrations or by continuous infusion. One typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the above factors. Depending on the disease, for repeated administrations over several days or more, the treatment is generally continued until a desirable suppression of the disease symptoms occurs. One exemplary dosage of the antibody or polypeptide comprising a mutant Fc region ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient one or more times. Such dosages can be administered intermittently, e.g., once a week or once every three weeks (e.g., such that the patient receives the antibody or polypeptide comprising a mutant Fc region about 2 to about 20 times, e.g., about 6 times). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of this therapy can be readily monitored by conventional techniques and assays.

[0331] It is understood that any of the above formulations or treatment methods can be carried out using the immunoconjugates of the present invention, instead of or in addition to anti-DENV antibodies. Similarly, it is understood that any of the above formulations or treatment methods can be carried out using the immunoconjugates of the present invention, instead of or in addition to the polypeptides containing the variant Fc region described herein.

[0332] H. Manufactured article In another aspect of the present invention, there is provided a manufactured article comprising a substance or material useful for the treatment, prevention and / or diagnosis of the above-mentioned diseases. The manufactured article includes a container and a label on the container or an accompanying document enclosed in the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, etc. The container can be formed from various materials such as glass or plastic. The container contains the composition, either alone or in combination with other compositions effective for treating, preventing and / or diagnosing the disease, and may be provided with a sterile access port (for example, the container can be an intravenous infusion bag or a vial having a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an antibody or a polypeptide containing a variant Fc region of the present invention. The label or the accompanying document indicates that the composition is used for the treatment of a predetermined disease. Further, the manufactured article can include (a) a first container containing a composition containing an antibody or a polypeptide containing a variant Fc region of the present invention; and (b) a second container containing a composition containing a further cytotoxic agent or other therapeutic agent. The manufactured article in this aspect of the present invention may further include an accompanying document indicating that the composition can be used for the treatment of a specific disease. Alternatively, or in addition, the manufactured article may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, dextrose solution, etc. It can further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, etc.

[0333] It is understood that any of the above-mentioned manufactured products may contain the immunoconjugates of the present invention, either instead of or in addition to anti-DENV antibodies. Similarly, it is understood that any of the above-mentioned manufactured products may contain the immunoconjugates of the present invention, either instead of or in addition to polypeptides containing a mutant Fc region.

Example

[0334] III. Example The following are examples of the methods and compositions of the present invention. It will be understood that various other embodiments can be implemented in view of the general description above.

[0335] Example 1: Preparation of Antigens and Antibodies Expression and purification of recombinant soluble E proteins derived from DENV-1, DENV-2, DENV-3, and DENV-4 Recombinant soluble E proteins (0.8E-His) derived from DENV-1, DENV-2, DENV-3, and DENV-4 having an 8×histidine tag at the carboxy terminus (SEQ ID NOs: 65-68, respectively) were transiently expressed using the FreeStyle293-F cell line or the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). By expressing prM0.8E-His (SEQ ID NOs: 61-64, respectively) derived from DENV-1, DENV-2, DENV-3, and DENV-4, prM0.8E-His was expressed as a single polypeptide within the cells; this polypeptide was then processed intracellularly and cleaved between prM and 0.8E-His. As a result, 0.8E-His was secreted into the cell culture medium. The conditioned medium containing 0.8E-His was applied to a column packed with immobilized metal affinity chromatography (IMAC) resin to which nickel or cobalt was added, and then eluted with imidazole. The fractions containing 0.8E-His were pooled and applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). The fractions containing 0.8E-His were pooled and stored at -80°C.

[0336] Expression and purification of recombinant human FcγR The extracellular domain of human FcγR was prepared by the following method. First, the gene for the extracellular domain of FcγR was synthesized by methods well known to those skilled in the art. At that time, the sequence of each FcγR was prepared based on the information registered in NCBI. Specifically, FcγRIa was prepared based on the sequence of NCBI accession number NM_000566 (version number NM_000566.3), FcγRIIa was prepared based on the sequence of NCBI accession number NM_001136219 (version number NM_001136219.1), FcγRIIb was prepared based on the sequence of NCBI accession number NM_004001 (version number NM_004001.3), FcγRIIIa was prepared based on the sequence of NCBI accession number NM_001127593 (version number NM_001127593.1), FcγRIIIb was prepared based on the sequence of NCBI accession number NM_000570 (version number NM_000570.3), and a His tag was attached to the C-terminus of each FcγR construct. Furthermore, the presence of polymorphisms is known for FcγRIIa, FcγRIIIa, and FcγRIIIb. For FcγRIIa, refer to Warmerdam et al. (J Exp Med (1990) 172, 19-25), for FcγRIIIa, refer to Wu et al. (J Clin Invest (1997) 100, 1059-1070), and for FcγRIIIb, refer to Ory et al. (J Clin Invest (1989) 84, 1688-1691) to create the polymorphic sites.

[0337] An expression vector was constructed by inserting the obtained gene fragment into an animal cell expression vector. The constructed expression vector was transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells to express the target protein. The culture supernatant obtained from the culture medium of the cells that had undergone transient introduction was filtered through a 0.22 μm filter, and the resulting liquid was purified in principle by the following four steps: (i) cation exchange column chromatography (SP Sepharose FF); (ii) affinity column chromatography for His tag (HisTrap HP); (iii) gel filtration column chromatography (Superdex200); and (iv) sterile filtration. Anion exchange column chromatography using Q Sepharose FF was used in step (i) to purify FcγRI. The absorbance of the purified protein was measured at 280 nm using a spectrophotometer. Based on the measured value, the concentration of the purified protein was calculated using the extinction coefficient determined by methods such as PACE (Protein Science (1995) 4, 2411-2423).

[0338] Expression and purification of recombinant mouse FcγR The extracellular domain of mouse FcγR (mFcγR) was prepared by the following method. First, the gene for the extracellular domain of FcγR was synthesized by a method generally known to those skilled in the art. In this synthesis, the sequences of each FcγR were prepared based on the information registered in NCBI. Specifically, mFcγRI was prepared based on the sequence of NCBI reference sequence: NP_034316.1, mFcγRIIb was prepared based on the sequence of NCBI reference sequence: NP_034317.1, mFcγRIII was prepared based on the sequence of NCBI reference sequence: NP_034318.2, and mFcγRIV was prepared based on the sequence of NCBI reference sequence: NP_653142.2. A His tag was added to the C-terminus of each of these sequences.

[0339] Each of the obtained gene fragments was inserted into a vector for expression in animal cells to prepare an expression vector. The prepared expression vector was transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells to express the target protein. After collecting the obtained culture supernatant, the culture supernatant was passed through a 0.22 μm filter to obtain a culture supernatant. The obtained culture supernatant was purified in principle by the following four steps: (i) ion exchange column chromatography; (ii) affinity column chromatography for His-tag (HisTrap HP); (iii) gel filtration column chromatography (Superdex200); and (iv) sterile filtration. The ion exchange column chromatography in step (i) was performed using Q Sepharose HP for mFcγRI, SP Sepharose FF for mFcγRIIb and mFcγRIV, and SP Sepharose HP for mFcγRIII. D-PBS(-) was used as the solvent after step (iii), but D-PBS(-) containing 0.1 M arginine was used for mFcγRIII. The absorbance of each purified protein was measured at 280 nm using a spectrophotometer, and the concentration of the purified protein was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science (1995) 4, 2411-2423) from the obtained value.

[0340] Expression and purification of recombinant human FcRn FcRn is a heterodimer of the FcRn α-chain and β2-microglobulin. Based on the published human FcRn gene sequence (J Exp Med (1994) 180, 2377-2381), oligonucleotide DNA primers were prepared. Using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers, a DNA fragment encoding the entire gene was prepared by PCR. Using the obtained DNA fragment as a template, a DNA fragment encoding the extracellular domain (Met1-Leu290) containing the signal region was amplified by PCR and inserted into a mammalian cell expression vector. Similarly, based on the published human β2-microglobulin gene sequence (Proc Natl Acad Sci USA (2002) 99, 16899-16903), oligonucleotide DNA primers were prepared. Using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers, a DNA fragment encoding the entire gene was prepared by PCR. Using the obtained DNA fragment as a template, a DNA fragment encoding the entire protein containing the signal region (Met1-Met119) was amplified by PCR and inserted into a mammalian cell expression vector.

[0341] Soluble human FcRn was expressed by the following procedure. Plasmids constructed for the expression of the human FcRn α-chain (SEQ ID NO: 81) and β2-microglobulin (SEQ ID NO: 82) were introduced into cells of the human embryonic kidney cancer-derived cell line HEK293H (Invitrogen) by the lipofectamine method using PEI (Polyscience). The obtained culture supernatant was collected, and FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) and then further purified using HiTrap Q HP (GE Healthcare) (J Immunol (2002) 169, 5171-5180).

[0342] Expression and purification of recombinant antibodies Either the FreeStyle293-F cell line or the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) was used to transiently express a recombinant antibody. Purification from the conditioned culture medium expressing the antibody was performed by a conventional method using Protein A. Gel filtration was further performed as needed.

[0343] Expression and purification of recombinant soluble human CD154 The human CD154 gene was synthesized based on the published protein sequence (NP_000065.1). A DNA fragment encoding soluble human CD154 (shCD154) with a FLAG tag was prepared by PCR using the synthesized DNA as a template, and the resulting DNA fragment was inserted into a mammalian cell expression vector, thereby generating a FLAG-shCD154 (SEQ ID NO: 106) expression vector. The nucleotide sequence of the obtained expression vector was determined using conventional methodologies known to those skilled in the art. FLAG-shCD154 was expressed using the FreeStyle 293 cell line (Invitrogen) as described in the protocol provided by the manufacturer. After transfection, the cells were grown for an appropriate time and then the conditioned culture medium was collected. The conditioned culture medium was subjected to cation exchange chromatography using 25 mM MES (pH 6.0), and FLAG-shCD154 was eluted with a continuous gradient of 25 mM MES, 1 M NaCl (pH 6.0). The peak fractions were pooled, concentrated using AmiconUltra Ultracel, and subjected to gel filtration chromatography using phosphate buffered saline (Wako). The peak fractions were pooled again, concentrated using Amicon Ultra Ultracel, and then filtered through a 0.22 μm PVDF membrane filter for sterilization. To determine the concentration of purified FLAG-shCD154, the absorbance was measured at 280 nm using a spectrophotometer. The protein concentration was calculated using the extinction coefficient calculated by the method described in Protein Science (1995) 4: 2411-2423 from the measured value.

[0344] Example 2: Preparation of an antibody variant with improved affinity for DENV E protein Genes encoding the VH (3CH, SEQ ID NO: 1) and VL (3CL, SEQ ID NO: 7) of the anti-DENV E protein antibody were synthesized and combined with human IgG1 CH (SG182, SEQ ID NO: 46) and human CL (SK1, SEQ ID NO: 60), respectively, and both constructs were cloned into a single expression vector. This antibody is referred to herein as DG_3CH-SG182 / 3CL-SK1 or 3C.

[0345] To identify mutations and their combinations that improve the binding properties of 3C, many mutations and their combinations were examined. Next, multiple mutations were introduced into the variable regions to enhance the binding affinity for the E protein. Thus, optimized VH mutants 3CH912 (SEQ ID NO: 2), 3CH953 (SEQ ID NO: 3), 3CH954 (SEQ ID NO: 4), 3CH955 (SEQ ID NO: 5), 3CH1047 (SEQ ID NO: 6), 3CH987 (SEQ ID NO: 90), 3CH989 (SEQ ID NO: 91), 3CH992 (SEQ ID NO: 92), 3CH1000 (SEQ ID NO: 93), 3CH1046 (SEQ ID NO: 94), 3CH1049 (SEQ ID NO: 95), and optimized VL mutants 3CL499 (SEQ ID NO: 8), 3CL563 (SEQ ID NO: 9), 3CL658 (SEQ ID NO: 10), 3CL012 (SEQ ID NO: 96), 3CL119 (SEQ ID NO: 97), 3CL633 (SEQ ID NO: 98), 3CL666 (SEQ ID NO: 99), 3CL668 (SEQ ID NO: 100) were obtained. The gene encoding VH was combined with one of human IgG1 CH (either SG182, SEQ ID NO: 46; SG1095, SEQ ID NO: 54; or SG1106, SEQ ID NO: 59), and the gene encoding VL was combined with human CL (SK1, SEQ ID NO: 60). Each of them was cloned into an expression vector. The amino acid sequences of the antibody mutants are shown in Table 2. One of the mutants, DG_3CH1047-SG182 / 3CL658-SK1 is also referred to as 3Cam herein, and another mutant, DG_3CH1047-SG182 / 3CL-SK1 is referred to as 3Cam2 herein.

[0346] Antibodies were expressed in HEK293 cells co-transfected with a mixture of heavy chain and light chain expression vectors and purified by Protein A.

[0347] (Table 2) Amino acid sequences of 3C and 3C variants TIFF2025108688000028.tif189144

[0348] The affinity of anti-DENV E protein antibodies that bind to the E proteins of DENV-1, DENV-2, DENV-3, and DENV-4 was measured at 25 °C using a Biacore T200 instrument (GE Healthcare). Anti-histidine antibody (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in PBS at pH 7.4 containing 20 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The E proteins of DENV-1, DENV-2, DENV-3, and DENV-4 with C-terminal His tags were captured in flow cell 2 or 3, and flow cell 1 was used as the reference flow cell. The capture level of the E protein was targeted at 200 resonance units (RU). The anti-DENV E protein antibody was injected at 250 nM for 180 s over the entire sensor surface, followed by dissociation for 300 s. After each cycle, the sensor surface was regenerated using 10 mM Gly-HCl pH 1.5. Binding affinity was measured by processing the data using Biacore T200 analysis software, version 2.0 (GE Healthcare) and fitting to a 1:1 binding model.

[0349] Tables 3a and 3b show the affinity (K) of anti-DENV E protein antibodies that bind to the E proteins of DENV-1 (designated DV1 in the table), DENV-2 (designated DV2 in the table), DENV-3 (designated DV3 in the table), and DENV-4 (designated DV4 in the table). d) is shown. Each 3C variant showed increased binding affinity for all four DENV serotypes compared to the parental 3C. As an example, the sensorgrams of the parental antibody 3C (DG_3CH-SG182 / 3CL-SK1) and one variant antibody 3Cam (DG_3CH1047-SG182 / 3CL658-SK1) are shown in Figure 1. Also, the sensorgrams of the parental antibody 3C (DG_3CH-SG182 / 3CL-SK1) and one variant antibody 3Cam2 (DG_3CH1047-SG182 / 3CL-SK1) are shown in Figure 10.

[0350] (Table 3a) Kd values of 3C and 3C variants for four DENV serotypes TIFF2025108688000029.tif67128 Note: * Strong binder, slow off-rate <1E-05, KD cannot be uniquely determined.

[0351] (Table 3b) Kd values of 3C and 3C variants for DENV1 and DENV3 serotypes TIFF2025108688000030.tif247100 Note: * Strong binder, slow off-rate <1E-05, KD cannot be uniquely determined.

[0352] Example 3: Generation of antibody CH variants for improved properties Multiple mutations were introduced into the human IgG1 heavy chain constant region CH (SG182, SEQ ID NO: 46). As a result, human IgG1 CH variants SG192 (SEQ ID NO: 47), SG1085 (SEQ ID NO: 48), SG1086 (SEQ ID NO: 49), SG1087 (SEQ ID NO: 50), SG1088 (SEQ ID NO: 51), SG1089 (SEQ ID NO: 52), SG1090 (SEQ ID NO: 53), SG1095 (SEQ ID NO: 54), SG1096 (SEQ ID NO: 55), SG1097 (SEQ ID NO: 56), SG1098 (SEQ ID NO: 57), SG1105 (SEQ ID NO: 58), SG1106 (SEQ ID NO: 59), SG1109 (SEQ ID NO: 107), SG1044 (SEQ ID NO: 108), and SG1045 (SEQ ID NO: 109) were generated. The genes encoding the CH variants were combined with the VH of 3C (3CH, SEQ ID NO: 1), the VH of one 3C variant (3CH1047, SEQ ID NO: 6), or the VH of an anti-CD154 antibody (SLAPH0336a, SEQ ID NO: 88). The VL gene of the anti-CD154 antibody (SLAPL0336a, SEQ ID NO: 89) was combined with the human CL (SK1, SEQ ID NO: 60). Each of them was cloned into an expression vector. The details of the CH variants are summarized in Table 4. To evaluate the affinity binding of the CH variants to each Fc receptor, the variable regions SLAPH0336a / SLAPL0366a of an anti-CD154 antibody that can form large immune complexes in the presence of the trimeric antigen CD154 were used.

[0353] Antibodies were expressed in HEK293 cells cotransfected with a mixture of a heavy chain expression vector and a light chain expression vector and purified by Protein A.

[0354] (Table 4) Amino acid sequences of mutant Fc regions TIFF2025108688000031.tif64139

[0355] Example 4: Binding Affinity of Antibodies with Fc Variants to Complement C1q Human C1q binding assay An anti-CD154 antibody with an Fc variant (an in-house antibody prepared using the method described in Example 3) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left standing overnight at 4°C. After washing with PBST, the plate was blocked with TBST containing 0.5% BSA and 1× Block Ace: blocking agent (DS Pharma) at room temperature for 2 hours. After washing the plate, human C1q (Calbiochem) was dispensed onto the plate and left standing at room temperature for 1 hour. The plate was washed, HRP-labeled anti-human C1q antibody (Bio Rad) was added, and the reaction was carried out at room temperature for 1 hour, followed by washing. Subsequently, TMB substrate (Invitrogen) was added. The signal was measured with a plate reader at wavelengths of 450 nm (test wavelength) and 570 nm (reference wavelength). The binding affinity of the antibody with a wild-type Fc region (WT) to human C1q decreased by introducing the LALA mutation into the Fc region, and the decrease in binding affinity to human C1q was restored by further introducing KWES, EFT, or EFT+AE in addition to the LALA mutation (Figure 2). The LALA+KMES mutation slightly increased the binding affinity, while the LALA+KWES, LALA+KAES, and LALA+KEES variants bound to C1q with an affinity comparable to that of WT (Figure 3). The binding characteristics of the above LALA or LALA+KAES variants were not affected by further introducing the ACT3 or ACT5 mutation (Figure 4).

[0356] Mouse C1q binding assay An anti-CD154 antibody having an Fc variant (a company antibody prepared using the method described in Example 3) was dispensed into Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4°C. After washing with PBST, the plate was blocked with TBST containing 0.5% BSA and 1×Block Ace: blocking agent (DS Pharma) at 4°C for 7 hours. After washing the plate, 10% mouse plasma (Innovative Research) was dispensed into the plate and allowed to stand overnight at 4°C. The plate was washed, biotinylated anti-mouse C1q antibody (Hycult Biotech) was added, and the reaction was carried out at room temperature for 1 hour, followed by washing. Streptavidin-HRP (Pierce) was added and the reaction was carried out at room temperature for 1 hour, followed by washing. Subsequently, ABTS ELISA HRP substrate (KPL) was added. The signal was measured with a plate reader at a wavelength of 405 nm. The binding affinity of the antibody with a wild-type Fc region (WT) to mouse C1q was decreased by introducing the LALA mutation into the Fc region, and the decrease in the binding affinity to mouse C1q was restored by further introducing KAES in addition to the LALA mutation. The binding characteristics of the above LALA or LALA+KAES variants were not affected by further introducing the ACT3 or ACT5 mutation (Figure 5).

[0357] Example 5: Biacore analysis of Fc variants binding to FcγR and FcRn Binding of Fc variants to human or mouse FcγR and human FcRn at pH 7.4 was measured at 25 °C using a Biacore T200 instrument (GE Healthcare). All antibodies and FcγR or FcRn were prepared in PBS-P at pH 7.4 containing 50 mM Na phosphate, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. In the FcγR binding assay, anti-histidine antibody (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Each of the FcγRs was captured with the anti-histidine antibody in flow cell 2, 3, or 4, and flow cell 1 was used as the reference flow cell. The capture level of FcγR was targeted at 400 resonance units (RU). All antibodies were injected at 100 nM into all flow cells. Immune complexes were prepared by mixing the antibody and trimeric CD154 at a 1:1 molar ratio and incubating at room temperature for 1 hour. The sensor surface was regenerated after each cycle with 10 mM glycine-HCl pH 1.5.

[0358] In the FcRn binding assay, a Biotin CAPture reagent (GE Healthcare) was immobilized on both flow cells 1 and 2 of a CAP sensor chip using a Biotin CAPture kit (GE Healthcare). Using flow cell 1 as the reference flow cell, biotinylated FcRn was captured in flow cell 2. The capture level of FcRn was targeted at 400 RU. All antibodies were injected at 100 nM into flow cells 1 and 2. Immune complexes were prepared by mixing the antibody and trimeric CD154 at a 1:1 molar ratio and incubating at room temperature for 1 hour. The sensor surface was regenerated after each cycle with 8 M guanidine-HCl, 1 M NaOH (3:1 vol / vol).

[0359] The binding levels were normalized to the corresponding capture levels of FcγR or FcRn. The binding of antibodies alone or immune complexes (antibody and trimeric CD154 antigen) to human or mouse FcγRs and human FcRn was monitored based on the binding response. The immune complexes were used to evaluate the enhanced binding to FcγR or FcRn due to the avidity effect. The results for human FcγR1a, FcγR2a 167H and 167R, FcγR2b, FcγR3a 158F and 158V, FcγR3b NA1 and NA2 are shown in FIGS. 6(a)-(h). The results for mouse FcγR1, FcγR2b, FcγR3, FcγR4 are shown in FIGS. 7(a)-(d). The binding of the wild-type Fc region (designated as WT IgG) was significantly reduced for each of the FcγRs tested by introducing the LALA, LALA+KAES or LALA+KWES mutations into the Fc region. This trend was almost the same between the assays using antibodies alone (designated as Ab alone) and immune complexes (designated as CD154 IC). The binding of the KAES or KWES mutants was not significantly reduced compared to the binding of WT IgG for most of the FcγRs tested. The results for human FcRn are shown in FIG. 8. The binding of the wild-type Fc region (designated as hIgG1) to human FcRn did not seem to be affected even after introducing the LALA or LALA+KAES mutations into the Fc region. The binding was slightly enhanced by further introducing the ACT3 or ACT5 mutations, but still remained relatively low (FIG. 8).

[0360] Example 6: In Vivo Efficacy of Anti-DENV Antibodies against DENV Infection AG129 mice at 6-8 weeks of age were given 10 6Mice were intraperitoneally infected with DENV-2 strain D2Y98P at plaque-forming units (pfu). After 48 hours, the mice were treated with 25 μg of antibody in PBS. The antibody was injected intravenously via the retro-orbital route. After an additional 24 hours, i.e., 72 hours after the first infection, blood was collected. Previous studies (Zust et al, J Virol (2014) 88, 7276-7285; Tan et al, PLOS Negl Trop Dis (2010) 4, e672) have demonstrated that the peak of viremia after D2Y98P infection is reached 3 to 4 days after infection. Viral RNA was extracted from plasma from each mouse, and quantitative PCR was performed and compared to a DENV-2 standard with known infectivity in a plaque assay. Both the 3C antibody and the 3Cam antibody significantly reduced viremia in this mouse model compared to the PBS control, and the effectiveness of both antibodies was comparable. Antibodies with the LALA+KAES mutation in the Fc region showed stronger effectiveness compared to antibodies with the LALA mutation alone. This was true for both the 3C antibody and the 3Cam antibody (Figure 9). This result indicates that the restoration of C1q binding activity by adding the KAES mutation to the LALA mutation contributes to the antiviral effect of the antibody.

[0361] For the purpose of clarity of understanding, the above invention has been described in some detail by way of explanation and examples, which should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety.

[0362] Sequence Information SEQUENCE LISTING <110> Chugai Seiyaku Kabushiki Kaisha Agency for Science, Technology and Research <120> Anti-Dengue Virus Antibodies, Polypeptides Containing Variant Fc Regions, And Methods Of Use <150> SG 10201607778X <151> 2016-09-16 <160> 109 <170> PatentIn version 3.5 <210> 1 <211> 132 <212> PRT <213> Homo sapiens <400> 1 Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Arg Gly Gly Ser Thr Ala Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Arg 100 105 110 Asp Gly Gly Ser Trp Trp Phe Asp Pro Trp Gly Gln Gly Ser Leu Val 115 120 125 Thr Val Ser Ser 130 <210> 2 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 2 Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Arg Gly Gly Ser Arg Ala Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Phe 100 105 110 Asp Gly Gly Ser Trp Trp Phe Asp Pro Trp Gly Gln Gly Ser Leu Val 115 120 125 Thr Val Ser Ser 130 <210> 3 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 3 Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Arg Gly Gly Ser Arg Arg Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Phe 100 105 110 Asp Gly Gly Ser Trp Trp Phe Asp Pro Trp Gly Gln Gly Ser Leu Val 115 120 125 Thr Val Ser Ser 130 <210> 4 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 4 Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Arg Gly Gly Ser Arg Arg Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Arg 100 105 110 Asp Asp Gly Ser Trp Trp Phe Asp Pro Trp Gly Gln Gly Ser Leu Val 115 120 125 Thr Val Ser Ser 130 <210> 5 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 5 Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Arg Gly Gly Ser Arg Arg Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Arg 100 105 110 Asp Leu Gly Ser Trp Trp Phe Asp Pro Trp Gly Gln Gly Ser Leu Val 115 120 125 Thr Val Ser Ser 130 <210> 6 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 6 Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Arg Gly Gly Ser Arg Arg Ser Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Glu Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Phe 100 105 110 Asp Gly Gly Ser Trp Trp Phe Asp Pro Trp Gly Gln Gly Ser Leu Val 115 120 125 Thr Val Ser Ser 130 <210> 7 <211> 107 <212> PRT <213> Homo sapiens <400> 7 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Gln Ala Ser Gln Asp Ile Arg Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Lys Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Asp Asp Leu Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Gln Ile Lys 100 105 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 8 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Gln Ala Ser Gln Asp Ile Arg Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Lys Phe Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Asp Ala Leu Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Gln Ile Lys 100 105 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 9 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Gln Ala Ser Gln Asp Ile Arg Gln Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Lys Phe Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Ala Leu Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Gln Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 10 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Gln Ala Ser Gln Glu Ile Arg Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Glu Leu Lys Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Asp Leu Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Gln Ile Lys 100 105 <210> 11 <211> 5 <212> PRT <213> Homo sapiens <400> 11 Ser Asn Tyr Ile His 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1 <400> 12 Ser Tyr Tyr Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Homo sapiens <400> 13 Val Ile Asn Pro Arg Gly Gly Ser Thr Ala Ser Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HVR-H2 <400> 14 Val Ile Asn Pro Arg Gly Gly Ser Arg Ala Ser Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HVR-H2 <400> 15 Val Ile Asn Pro Arg Gly Gly Ser Arg Arg Ser Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 16 <211> 23 <212> PRT <213> Homo sapiens <400> 16 Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Arg Asp Gly 1 5 10 15 Gly Ser Trp Trp Phe Asp Pro 20 <210> 17 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <400> 17 Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Phe Asp Gly 1 5 10 15 Gly Ser Trp Trp Phe Asp Pro 20 <210> 18 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <400> 18 Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Arg Asp Asp 1 5 10 15 Gly Ser Trp Trp Phe Asp Pro 20 <210> 19 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <400> 19 Gly Gly Arg Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Arg Asp Leu 1 5 10 15 Gly Ser Trp Trp Phe Asp Pro 20 <210> 20 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <400> 20 Gly Gly Glu Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Phe Asp Gly 1 5 10 15 Gly Ser Trp Trp Phe Asp Pro 20 <210> 21 <211> 11 <212> PRT <213> Homo sapiens <400> 21 Gln Ala Ser Gln Asp Ile Arg Lys Tyr Leu Asn 1 5 10 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HVR-L1 <400> 22 Gln Ala Ser Gln Asp Ile Arg Gln Tyr Leu Asn 1 5 10 <210> 23 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HVR-L1 <400> 23 Gln Ala Ser Gln Glu Ile Arg Lys Tyr Leu Asn 1 5 10 <210> 24 <211> 7 <212> PRT <213> Homo sapiens <400> 24 Asp Ala Ser Asn Leu Lys Thr 1 5 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <400> 25 Asp Ala Ser Asn Leu Lys Phe 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <400> 26 Asp Ala Ser Glu Leu Lys Thr 1 5 <210> 27 <211> 9 <212> PRT <213> Homo sapiens <400> 27 Gln Gln Phe Asp Asp Leu Pro Ile Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 28 Gln Gln Phe Asp Ala Leu Pro Ile Thr 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 29 Gln Gln Phe Ser Ala Leu Pro Ile Thr 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 30 Gln Gln Phe Glu Asp Leu Pro Ile Thr 1 5 <210> 31 <211> 30 <212> PRT <213> Homo sapiens <400> 31 Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 32 <211> 14 <212> PRT <213> Homo sapiens <400> 32 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 33 <211> 32 <212> PRT <213> Homo sapiens <400> 33 Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 34 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR-H3 <400> 34 Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 35 <211> 11 <212> PRT <213> Homo sapiens <400> 35 Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser 1 5 10 <210> 36 <211> 23 <212> PRT <213> Homo sapiens <400> 36 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys 20 <210> 37 <211> 15 <212> PRT <213> Homo sapiens <400> 37 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 38 <211> 32 <212> PRT <213> Homo sapiens <400> 38 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys 20 25 30 <210> 39 <211> 10 <212> PRT <213> Homo sapiens <400> 39 Phe Gly Gln Gly Thr Arg Leu Gln Ile Lys 1 5 10 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1 <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is Asn or Tyr <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is Ile or Met <400> 40 Ser Xaa Tyr Xaa His 1 5 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HVR-H2 <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa is Thr or Arg <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa is Ala or Arg <400> 41 Val Ile Asn Pro Arg Gly Gly Ser Xaa Xaa Ser Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 42 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (14)..(14) <223> Xaa is Arg or Phe <220> <221> MISC_FEATURE <222> (16)..(16) <223> Xaa is Gly, Asp or Leu <400> 42 Gly Gly Xaa Ala Leu Phe Tyr Asp Ser Tyr Thr Thr Pro Xaa Asp Xaa 1 5 10 15 Gly Ser Trp Trp Phe Asp Pro 20 <210> 43 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HVR-L1 <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is Asp or Glu <220> <221> MISC_FEATURE <222> (8)..(8) <223> Xaa is Lys or Gln <400> 43 Gln Ala Ser Gln Xaa Ile Arg Xaa Tyr Leu Asn 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is Asn or Glu <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa is Thr or Phe <400> 44 Asp Ala Ser Xaa Leu Lys Xaa 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is Asp, Ser or Glu <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is Asp or Ala <400> 45 Gln Gln Phe Xaa Xaa Leu Pro Ile Thr 1 5 <210> 46 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> CH <400> 46 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 <210> 47 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> CH <400> 47 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 <210> 48 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> CH <400> 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Trp Ala Leu Pro Ala Pro Ile Ser Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 <210> 49 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> CH <400> 49 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn 195 200 205 Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 <210> 50 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> CH <400> 50 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 <210> 51 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> CH <400> 51 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Trp Ala Leu Pro Ala Pro Ile Ser Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro 325 <210> 52 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> CH <400> 52 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Ala Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu...

Claims

1. An isolated antibody that binds to a dengue virus (DENV) E protein, (a) (i) Amino acid sequence: (Here, X 1 is R or E, and X 2 is R or F, and X 3 is G, D or L)(SEQ ID NO: 42), including HVR-H3, (ii) Amino acid sequence: (Here X 1 is D, S or E, and X 2 is D or A)(SEQ ID NO: 45), and HVR-L3 containing (iii) Amino acid sequence: (Here X 1 is T or R, and X 2 is A or R)(SEQ ID NO: 41)-containing HVR-H2; (b) (i) Amino acid sequence: SX 1 YX 2 H (Here X 1 is N or Y, and X 2 is I or M)(SEQ ID NO: 40) comprising HVR-H1, (ii) Amino acid sequence: (Here X 1 is T or R, and X 2 is A or R)(SEQ ID NO: 41) containing HVR-H2, and (iii) Amino acid sequence: (Here, X 1 is R or E, and X 2 is R or F, and X 3 is G, D or L)(SEQ ID NO: 42)-containing HVR-H3; (c) (i) Amino acid sequence: SX 1 YX 2 H (Here X 1 is N or Y, and X 2 is I or M)(SEQ ID NO: 40) comprising HVR-H1, (ii) Amino acid sequence: (Here, X 1 is T or R, and X 2 is A or R)(SEQ ID NO: 41), an HVR-H2 containing (iii) Amino acid sequence: (Here X 1 is R or E, and X 2 is R or F, and X 3 is G, D or L)(SEQ ID NO: 42) containing HVR-H3, (iv) Amino acid sequence: (Here X 1 is D or E, and X 2 is K or Q)(SEQ ID NO: 43) containing HVR-L1, (v) Amino acid sequence: (Here X 1 is N or E, and X 2 is T or F)(SEQ ID NO: 44), and (vi) Amino acid sequence: (Here X 1 is D, S or E, and X 2 is D or A)(SEQ ID NO: 45); or (d) (i) Amino acid sequence: (Here X 1 is D or E, and X 2 is K or Q)(SEQ ID NO: 43) including HVR-L1, (ii) Amino acid sequence: (Here X 1 is N or E, and X 2 is T or F)(SEQ ID NO: 44), and HVR-L2 containing (iii) Amino acid sequence: (Here, X 1 is D, S or E, and X 2 is D or A)(SEQ ID NO: 45) and contains HVR-L3 comprising, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and not being the antibody described above.

2. A heavy chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO: 31, an FR2 comprising the amino acid sequence of SEQ ID NO: 32, an FR3 comprising the amino acid sequence of SEQ ID NO: 33 or 34, an FR4 comprising the amino acid sequence of SEQ ID NO: 35 The antibody according to claim 1 (b), further comprising the above.

3. A light chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO: 36, an FR2 comprising the amino acid sequence of SEQ ID NO: 37, an FR3 comprising the amino acid sequence of SEQ ID NO: 38, an FR4 comprising the amino acid sequence of SEQ ID NO: 39 The antibody according to claim 1 (d), further comprising the above.

4. (a) A VH sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 2 to 6; (b) A VL sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 8 to 10; or (c) Any one of the VH sequences of SEQ ID NOs: 2 to 6 and any one of the VL sequences of SEQ ID NOs: 8 to 10 An isolated antibody comprising the above.

5. (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27 An isolated antibody that binds to the dengue virus (DENV) E protein, comprising **Claim 6** A pharmaceutical formulation comprising the antibody according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier. **Claim 7** A method for treating DENV infection in an individual, comprising the step of administering to the individual an effective amount of the antibody according to any one of claims 1 to 5. **Claim 8** A polypeptide comprising a mutant Fc region comprising at least one amino acid modification in the parental Fc region, wherein the mutant Fc region has substantially reduced FcγR binding activity and does not have substantially reduced C1q binding activity when compared to the parental Fc region, said polypeptide. **Claim 9** The polypeptide according to claim 8, wherein the mutant Fc region comprises Ala at position 234, Ala at position 235, and the following (a) to (c) according to EU numbering: (a) positions 267, 268, and 324; (b) positions 236, 267, 268, 324, and 332; and (c) positions 326 and 333 and one further amino acid modification of any one thereof. **Claim 10** The polypeptide according to claim 9, wherein the mutant Fc region comprises, according to EU numbering, (a) Glu at position 267; (b) Phe at position 268; (c) Thr at position 324; (d) Ala at position 236; (e) Glu at position 332; (f) Ala, Asp, Glu, Met, or Trp at position 326; and (g) Ser at position 333 and comprises an amino acid selected from the group consisting of. **Claim 11** The polypeptide according to claim 9, wherein the mutant Fc region comprises, according to EU numbering, (a) Ala at position 434; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440; and (d) A polypeptide according to any one of claims 8 to 10, further comprising an amino acid selected from the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440. A polypeptide according to any one of claims 8 to 10, further comprising an amino acid selected from the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440. **Claim 12** A polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 51 to 59. **Claim 13** A polypeptide according to any one of claims 8 to 12, which is an antibody. **Claim 14** The antibody is (a) (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and (iii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27 A polypeptide according to claim 13, comprising the same. **Claim 15** A pharmaceutical formulation comprising a polypeptide according to any one of claims 8 to 14 and a pharmaceutically acceptable carrier. **Claim 16** An antibody according to any one of claims 1 to 5, further comprising a polypeptide according to any one of claims 8 to 12. **Claim 17** A method for preparing an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, (a) combining a VH variant sequence selected from the group consisting of 3CH1047 (SEQ ID NO: 6) and 3CH1049 (SEQ ID NO: 95) with a human IgG1 CH sequence selected from the group consisting of SG182 (SEQ ID NO: 46), SG1095 (SEQ ID NO: 54), and SG1106 (SEQ ID NO: 59); (b) combining a VL variant sequence selected from the group consisting of 3CL (SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98) with the human CL sequence SK1 (SEQ ID NO: 60); (c) cloning each of the combinations into an expression vector; (d) expressing the resulting expression vector in cotransfected cells; and (e) purifying the antibody obtained from step (d) The method comprising the above steps.

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