RU2811697C2 - Antibodies to dengue virus with cross-reactivity with zika virus - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: following is proposed: the use of an antibody containing the VH sequence with SEQ ID NO: 6, VL sequence with SEQ ID NO: 7 and SEQ ID NO: 59, for the production of medicinal products for the treatment or prevention of infection caused by the Zika virus. The antibody is specific for the dengue virus and is cross-reactive with the Zika virus.

EFFECT: invention helps to effectively neutralize the Zika virus.

2 cl, 20 dwg, 10 ex

Description

Field of technology to which the invention relates

The present invention provides antibodies to the Zika virus and methods for their use.

Prerequisites for creating the invention

Dengue fever is the most widespread arthropod-borne viral disease in the world. The dengue fever virus (or the virus referred to herein as DENV) can be divided into four different infectious serotypes, such as DENV-1, DENV-2, DENV-3 and DENV-4. Symptoms of dengue virus infection include fever, muscle pain, headache, low platelet count and low white blood cell count, coagulopathy, bleeding and vascular leakage, which can lead to dengue shock syndrome. When an individual is exposed to the dengue virus after a previous dengue infection, antibodies to the virus may enhance the uptake of the virus into host cells, and the patient has a higher risk of developing severe dengue fever. However, severe forms of dengue fever can also occur during the first infection.

Although dengue fever is the most common arthropod-borne viral disease, there is currently no drug available to treat it. Approaches to dengue fever as a disease primarily focus on preventing infection and/or treating it to relieve symptoms.

Antibody-based vaccines and therapeutics are currently being developed to prevent and treat viral infections. However, antibody-based treatment options are not without risks. One such risk is antibody-dependent enhancement (ADE), which occurs when non-neutralizing antiviral antibodies facilitate virus entry into host cells, resulting in increased infectivity in the cells (Expert Rev Anti Infect Ther 11, 2013, cc. 1147-1157). The most common mechanism of ADE is the interaction of the virus-antibody complex through the Fc region of the antibody with Fc receptors (FcRs) on the cell surface. A common mild viral infection can be aggravated by ADE, turning into a life-threatening illness. An anti-DENV antibody with mutations in the Fc region that prevents binding to the FcγR has been described, thereby preventing the enhancement of DENV infection (WO 2010/043977). However, there is still a need for antibody-based therapeutics that do not carry the risk of antibody-dependent enhancement of infection.

Cross-reactivity of DENV-specific antibodies with Zika virus is described in the literature [JCI Insight. 2017; 2(8). doi: 10.1172/jci.insight.92428.]. It has also been demonstrated that DENV-specific Abs can enhance Zika virus infection [Nature. 2016; 536(7614): 48-53. doi: 10.1038/nature 18938, Nat Immunol. 2016; 17(9): 1102-8.].

Summary of the invention

The invention provides antibodies to DENV, polypeptides containing Fc region variants, and methods for their use.

In some embodiments, the isolated anti-DENV antibody of the present invention binds to the DENV E protein. In some embodiments, the anti-DENV antibody of the invention comprises:

(a) (I) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42), (II) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₂ is D or A (SEQ ID NO: 45), and (III) HVR- H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41);

(b) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X1 is N or Y, X ₂ is I or M (SEQ ID NO: 40), (II) HVR-H2 containing the amino acid the sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41), and (III) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in in which X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42);

(c) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X ₁ is N or Y, X ₂ is I or M (SEQ ID NO: 40), (II) HVR-H2 containing amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41), (III) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in in which X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42), (IV) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ is D or E, X ₂ is K or Q (SEQ ID NO: 43); (V) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ is N or E, X ₂ is T or F (SEQ ID NO: 44); and (VI) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X1 is D, S or E, X ₂ is D or A (SEQ ID NO: 45); or

(d) (I) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ is D or E, X ₂ is K or Q (SEQ ID NO: 43); (II) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ is N or E, X ₂ is T or F (SEQ ID NO: 44); and (III) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₂ is D or A (SEQ ID NO: 45). In some embodiments, the antibody of the invention is not an antibody that contains (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) HVR-L3 containing the amino acid sequence SEQ ID NO: 27.

In some embodiments, the anti-DENV antibody of the invention comprises:

(a) (I) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6, (II) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10, and (III) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6;

(b) (I) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6, and (III) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6;

(c) (I) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6, ( III) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6, (IV) HVR-L1 from the VL sequence presented in any of SEQ ID NO: 8-10; (V) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8-10; and (VI) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10;

(d) (I) HVR-L1 from the VL sequence presented in any of SEQ ID NO: 8-10; (II) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8-10; and (III) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10; or

(e) (I) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6, ( III) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6, (IV) HVR-L1 from the VL sequence presented in SEQ ID NO: 7; (V) HVR-L2 from the VL sequence shown in SEQ ID NO: 7; and (VI) HVR-L3 from the VL sequence shown in SEQ ID NO: 7.

In some embodiments, the antibody of the invention is not an antibody that contains (I) HVR-H1 from the VH sequence SEQ ID NO: 1, (II) HVR-H2 from the VH sequence SEQ ID NO: 1, (III) HVR-H3 from VH sequence SEQ ID NO: 1, (IV) HVR-L1 from VL sequence SEQ ID NO: 7, (V) HVR-L2 from VL sequence SEQ ID NO: 7 and (VI ) HVR-L3 from VL sequence SEQ ID NO: 7.

In some embodiments, the isolated antibody of the present invention also comprises a heavy chain variable domain framework region FR1 comprising 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, comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the isolated anti-DENV antibody of the present invention also comprises an FR1 light chain variable domain framework region comprising 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.

In some embodiments, the isolated anti-DENV antibody of the present invention contains (a) a VH sequence that is at least 95% identical to any of the amino acid sequences of SEQ ID NOs: 2-6; (b) a VL sequence that is at least 95% identical to the amino acid sequence of any of SEQ ID NO: 8-10; (c) the VH sequence presented in any of SEQ ID NO: 2-6, and the VL sequence presented in any of SEQ ID NO: 8-10; or (d) the VH sequence presented in any of SEQ ID NO: 2-6, and the VL sequence presented in SEQ ID NO: 7.

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

In some embodiments, the Fc region of an 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 may be selected from the Fc region options set forth herein.

The invention also provides isolated nucleic acids that encode the anti-DENV antibody of the present invention. The invention also provides host cells that contain the nucleic acid of the present invention. The invention also provides a method for producing an antibody, which includes culturing a host cell according to the present invention so as to produce the antibody.

The invention also provides a pharmaceutical composition that contains an anti-DENV antibody of the present invention and a pharmaceutically acceptable carrier.

Antibodies to DENV proposed in the present invention can be used as a medicine. In some embodiments, the anti-DENV antibodies of the present invention can be used to treat a DENV infection.

Antibodies to DENV proposed in the present invention can be used for the production of drugs. In some embodiments, the medicament is for treating a DENV infection.

The invention also provides a method for treating an individual having a DENV infection. In some embodiments, the method comprises administering to an individual an effective amount of an anti-DENV antibody of the present invention. In some embodiments, the method also includes administering to the individual an additional therapeutic agent, such as those described below.

In some embodiments, the Fc region variant of the present invention contains at least one amino acid change in the parent Fc region. In other embodiments, the variant Fc region has substantially reduced FcγR binding activity compared to the parent Fc region. In other embodiments, the variant Fc region does not have significantly reduced C1q binding activity compared to the parent Fc region.

In some embodiments, the variant Fc region of the present invention contains Ala at position 234, Ala at position 235 according to EU numbering. In other embodiments, the variant Fc region contains any of the amino acid changes at the positions specified in the following subparagraphs (a)-(c): (a) positions 267, 268 and 324, (b) positions 236, 267, 268, 324 and 332 and (c) provisions 326 and 333; according to EU numbering.

In some embodiments, the variant Fc region provided by the present invention contains amino acids 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.

In some embodiments, the variant Fc region provided by the present invention also contains amino acids selected from the group consisting of: (a) Ala at position 434, (b) Ala at position 434, Thr at position 436, Arg at 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.

In some embodiments, the variant Fc region provided by the present invention contains any of the amino acid changes, individually or in combination, that are presented in Table 4. In some embodiments, the parent Fc region specified in the present invention is derived from human IgG1. The invention provides a polypeptide containing the amino acid sequence presented in any of SEQ ID NO: 51-59.

In some embodiments, the polypeptide that contains the Fc region variant of the present invention is an antibody. In other embodiments, the antibody is an antiviral antibody. In other embodiments, the antibody comprises a variable region derived from the anti-DENV antibody described herein.

In other embodiments, the antibody comprises:

(a) (I) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 16, (II) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27, and (III) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 13;

(b) (I) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 11, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 13, and (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 16;

(c) (I) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 11, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 13, (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 16, (IV) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21; (V) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24; and (VI) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27, or

(d) (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21; (II) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24; and (III) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27.

In other embodiments, the antibody comprises:

(a) (I) HVR-H3 from VH sequence SEQ ID NO: 6, (II) HVR-L3 from VL sequence SEQ ID NO: 10 and (III) HVR-H2 from VH sequence SEQ ID NO: 6 ;

(b) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6 and (III) HVR-H3 from VH sequence SEQ ID NO: 6 ;

(c) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6, (III) HVR-H3 from VH sequence SEQ ID NO: 6 , (IV) HVR-L1 from VL sequence SEQ ID NO: 10; (V) HVR-L2 from the VL sequence SEQ ID NO: 10 and (VI) HVR-L3 from the VL sequence SEQ ID NO: 10;

(d) (I) HVR-L1 from VL sequence SEQ ID NO: 10; (II) HVR-L2 from the VL sequence SEQ ID NO: 10 and (III) HVR-L3 from the VL sequence SEQ ID NO: 10, or

(e) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6, (III) HVR-H3 from VH sequence SEQ ID NO: 6 , (IV) HVR-L1 from VL sequence SEQ ID NO: 7; (V) HVR-L2 from the VL sequence SEQ ID NO: 7 and (VI) HVR-L3 from the VL sequence SEQ ID NO: 7.

The invention also provides isolated nucleic acids encoding a polypeptide that contains a variant of the Fc region proposed in the present invention. The invention also provides host cells that contain the nucleic acid of the present invention. The invention also provides a method for producing a polypeptide that contains a variant Fc region, comprising culturing a host of the present invention to produce the polypeptide.

The invention also provides a pharmaceutical composition containing a polypeptide that contains a variant of the Fc region proposed in the present invention, and a pharmaceutically acceptable carrier.

Polypeptides that contain variants of the Fc regions proposed in the present invention can be used as a medicine. In some embodiments, polypeptides that contain Fc region variants of the present invention can be used to treat a viral infection.

Polypeptides that contain variants of the Fc regions proposed in the present invention can be used for the production of a drug. In some embodiments, the medicament is for treating a viral infection.

The invention also provides a method for treating an individual having a viral infection. In some embodiments, the method comprises administering to an individual an effective amount of a polypeptide that contains an Fc region variant of the present invention.

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

One object of the invention is methods for treating or preventing infection caused by the Zika virus.

In some embodiments, a method of treating or preventing an infection caused by the Zika virus provided by the present invention comprises administering an antibody that binds to the Zika virus.

In some embodiments, an antibody of the present invention that binds to the Zika virus comprises:

(a) (I) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42), (II) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₂ is D or A (SEQ ID NO: 45), and (III) HVR- H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41);

(b) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X ₁ is N or Y, X ₂ is I or M (SEQ ID NO: 40), (II) HVR-H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41), and (III) HVR-H3, containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42);

(c) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X ₁ is N or Y, X ₂ is I or M (SEQ ID NO: 40), (II) HVR-H2 containing amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₂ is A or R (SEQ ID NO: 41), (III) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in in which X ₁ is R or E, X ₂ is R or F, X ₃ is G, D or L (SEQ ID NO: 42), (IV) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ is D or E, X ₂ is K or Q (SEQ ID NO: 43); (V) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ is N or E, X ₂ is T or F (SEQ ID NO: 44); and (VI) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₂ is D or A (SEQ ID NO: 45); or

(d) (I) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ is D or E, X ₂ is K or Q (SEQ ID NO: 43); (II) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ is N or E, X ₂ is T or F (SEQ ID NO: 44); and (III) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₂ is D or A (SEQ ID NO: 45). In some embodiments, the antibody of the invention is not an antibody that contains (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) HVR-L3 containing the amino acid sequence SEQ ID NO: 27.

In some embodiments, an antibody that binds to the Zika virus of the present invention also comprises an FR1 heavy chain variable domain framework region comprising 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. In some embodiments, the antibody that binds to the Zika virus of the present invention also contains a framework region of the FR1 light chain variable domain, 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.

In some embodiments, an antibody that binds to the Zika virus of the present invention comprises (a) a VH sequence that is at least 95% identical to any of the amino acid sequences of SEQ ID NOs: 2-6; (b) a VL sequence that is at least 95% identical to the amino acid sequence of any of SEQ ID NO: 8-10; (c) the VH sequence presented in any of SEQ ID NO: 2-6, and the VL sequence presented in any of SEQ ID NO: 8-10; or (d) the VH sequence presented in any of SEQ ID NO: 2-6.

The invention also provides a method for treating an infection caused by the Zika virus. In some embodiments, the method comprises administering to an individual an effective amount of an anti-Zika virus antibody of the present invention.

In some embodiments, the variant Fc region of an antibody that binds to the Zika virus of the present invention contains Ala at position 234, Ala at position 235 according to EU numbering. In other embodiments, the variant Fc region contains any of the amino acid changes at the positions specified in the following subparagraphs (a)-(c): (a) positions 267, 268 and 324, (b) positions 236, 267, 268, 324 and 332 and (c) provisions 326 and 333; according to EU numbering.

In some embodiments, the variant Fc region of an antibody that binds to the Zika virus of the present invention contains amino acids that are 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 Tgr at position 326, and (g) Ser at position 333; according to EU numbering.

In some embodiments, the variant Fc region of an antibody that binds to the Zika virus of the present invention contains amino acids that are 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.

The invention provides a polypeptide that contains the amino acid sequence presented in any of SEQ ID NO: 51-59.

In other embodiments, an antibody of the present invention that binds to the Zika virus comprises:

(a) (I) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 16, (II) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27, and (III) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 13;

(b) (I) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 11, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 13, and (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 16;

(c) (I) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 11, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 13, (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 16, (IV) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21; (V) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24; and (VI) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27; or

(d) (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21; (II) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24; and (III) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27.

In other embodiments, the antibody of the present invention that binds to the Zika virus contains a VH sequence variant 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 SGI 106 (SEQ ID NO: 59); and a VL sequence variant selected from the group consisting of: 3CL (SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98), with the human CL sequence SKI (SEQ ID NO: 60).

Brief description of drawings

The drawings show:

in fig. 1 (A)-(D) - illustration of BIACORE -sensograms of antibodies to DENV 3C and 3Cam to DENV-1 E protein (A), DENV-2 E protein (B), DENV-3 E protein (C) and E-protein DENV-4 E (G), obtained according to the method described in example 2;

in fig. 2 data on the binding affinity of antibodies containing various Fc variants to human C1q, obtained according to the method described in example 4. Binding activity was measured using ELISA. The following Fc variants were tested: WT, LALA+KWES, LALA+EFT+AE, LALA+EFT and LALA;

in fig. 3 data on the binding affinity of antibodies containing various Fc variants to human C1q, obtained according to the method described in example 4. Binding activity was measured using ELISA. The following Fc variants were tested: WT, LALA, LALA+KWES, LALA+KAES, LALA+KDES, LALA+KEES and LALA+KMES;

in fig. 4 shows the binding affinity of antibodies containing various Fc variants to human C1q, obtained according to the method described in Example 4. Binding activity was measured using ELISA. The following Fc variants were tested: WT, LALA, LALA+AST3, LALA+AST5, LALA+KAES, LALA+AST3+KAES and LALA+AST5+KAES;

in fig. 5 shows the binding affinity of antibodies containing various Fc variants to human C1q, obtained according to the method described in Example 4. Binding activity was measured using ELISA. The following Fc variants were tested: WT, LALA, LALA+ACT3, LALA+ACTS, LALA+KAES, LALA+ACT3+KAES and LALA+ACT5+KAES;

in fig. 6 (a)-(h) - Biacore analysis data characterizing the binding of Fc variants to human FcγR, obtained according to the method described in example 5. The following Fc variants were tested: WT (in the drawing designated as WT IgG), KWES, EFT+ AE, EFT, KAES, LALA+KWES, LALA+EFT+AE, LALA+EFT, LALA, LALA+ACT3, LALA+ACT5, LALA+KAES, LALA+KAES+ACT3 and LALA+KAES+ACT5. For the indicated Fc variants, binding to FcyRs was assessed, including: (a) human FcγR1a, (b) human FcγR2a, allelic variant 167H, (c) human FcγR2a, allelic variant 167R, (d) human FcγR2b, (e) human FcγR3a allelic variant 158F, (f) human FcγR3a allelic variant 158V, (g) human FcγR3b allelic variant NA1 and (h) human FcγR3b allelic variant NA2. Each Fc variant was examined both in the form of an individual antibody with the Fc variant (designated “Ab only”) and in the form of an immune complex formed by the antibody and the trimeric CD154 antigen (designated “CD154 IC”);

in fig. 7 (a)-(d) Biacore analysis data characterizing the binding of Fc variants to mouse FcγR, obtained according to the method described in example 5. The following Fc variants were tested: WT (in the drawing designated as WT IgG), KWES, EFT+AE , EFT, KAES, LALA+KWES, LALA+EFT+AE, LALA+EFT, LALA, LALA+ACT3, LALA+ACT5, LALA+KAES, LALA+KAES+ACT3 and LALA+KAES+ACTS. For the indicated Fc variants, binding to FcγRs was assessed, including: (a) mouse FcγR1, (b) mouse FcγR2b, (c) mouse FcγR3, and (d) mouse FcγR4. Each Fc variant was examined both in the form of an individual antibody with the Fc variant (designated “Ab only”) and in the form of an immune complex formed by the antibody and the trimeric CD154 antigen (designated CD154 IC);

in fig. 8 - Biacore analysis data characterizing the binding of Fc variants to human FcRn, obtained according to the method described in example 5. The following Fc variants were tested: WT (in the drawing designated as hIgG1), LALA, LALA+AST3, LALA+AST5, LALA+ KAES, LALA+KAES+AST3 and LALA+KAES+AST5. Each Fc variant was examined both in the form of an individual antibody with the Fc variant (designated “Ab only”) and in the form of an immune complex formed by the antibody and the trimeric CD154 antigen (designated CD154 IC);

in fig. 9 data on viremia on day 3 after infection with the DENV-2 virus in mice of the AG129 line, obtained according to the method described in example 6. Antibodies to DENV 3C and 3Cam in combination with Fc variants WT (designated hIgG1), LALA and LALA+KAES or PBS as a negative control was administered on day 2 after virus infection;

in fig. 10 data on viremia on day 3 after infection with DENV-1 - DENV-4 viruses of mice of the AG129 line, obtained according to the method described in example 6. Antibodies to DENV 3C and 3Cam, having the same Fc variant (LALA+KAES), or P1- buffer as a negative control was administered on day 2 after virus infection. LOD: limit of detection. Each symbol indicates the level of viremia in one mouse. One-way analysis of variance was used to calculate the significance of differences between groups;

in fig. 11 - data on viremia on day 3 after infection of AG129 mice with the DENV-2 virus, obtained according to the method described in example 6. Antibodies to DENV 3Cam2-LALA and 3Cam2-LALA+KAES or P1 buffer as a negative control were administered on day 2 after infection with the virus;

in fig. 12 (A)-(D) - illustration of BIACORE® sensograms of antibodies to DENV 3C and 3Cam2 to DENV-1 E protein (A), DENV-2 E protein (B), DENV-3 E protein (C) and E-protein DENV-4 E (G), obtained according to the method described in example 2;

in fig. 13 (A)-(B) Results of Zika virus neutralization assay performed using BHK-21-DC-SIGN cells. The Zika virus strain used was KF993678. Each experimental point corresponded to one measurement. The NT ₅₀ value for 3Cam2-LALA+KAES+ACT5 was approximately 0.33 μg/ml and for 3C-LALA+KAES+ACT5 5.33 μg/ml in the first experiment (A). The NT ₅₀ value for 3Cam2-LALA+KAES+ACT5 was approximately 0.93 μg/ml in the second experiment (B);

in fig. 14 (A)-(B) - results of ADE analysis on K562 cells for the Zika virus. The Zika virus strain used was KF993678. A) Enhancement was detected with the wild-type version of 3Cam2, but not with the Fc variants 3Cam2-LALA+KAES and 3Cam2-LALA+KAES+ACT5. Infected (indicated as “inf” in the drawing): without added antibodies. Data are presented as means ± error (range) at n=2. B) The experiment was repeated with a new batch of 3Cam2-LALA+KAES+ACT5 using the wild type version of 3Cam2 as a control. Data are presented as means ± error (range) at n=2;

in fig. 15 - In vivo efficacy data against Zika virus infection. Mice were infected i.p. Zika virus and two days after infection were treated i.v. 100 µg 3Cam2-LALA+KAES+ACT5 (3Cam2-SG1106) or 3C-LALA+KAES+ACT5 (3C-SG1106). P1 buffer was used as a control. Each point corresponds to one mouse and results are presented as means ±SD. The Kruskal-Wallis test was used to assess significance, *: p<0.05, **: p<0.005;

in fig. 16 - data on the survival of Zika virus-infected IFNAR mice after treatment with antibodies. Mice were infected i.p. Zika virus and two days after infection were treated i.v. 100 μg 3Cam2-LALA+KAES+ACT5 or 3C-LALA+KAES+ACT5. P1 buffer was used as a control. Data are presented on the percentage of survival per group 40 days after infection;

in fig. 17 data on the binding activity of monoclonal antibodies 3C, 3C-LALA and 3Cam2-LALA+KAES+ACT5 to the Zika virus (ZIKV). Shown are ELISA curves for purified ZIKV virions;

in fig. 18 - results of in vitro neutralizing activity assay obtained using human antibodies 3C, 3C-LALA and 3Cam2-LALA+KAES+ACT5 against ZIKV. Data are presented on the relative infectivity of the virus when incubated with antibodies compared to the variant in which only the virus was used. Two different batches of 3C and 3C-LALA (old and new) were evaluated;

in fig. 19(A)-(B) Results demonstrating that antibody treatment prevented ZIKV-induced congenital developmental failure in IFNαR KO mice. (A) Images of representative fetuses of mock-infected mice (a), ZIKV-infected mice treated with isotype control antibody (b), ZIKV-infected mice treated with 3C-LALA (c), and ZIKV-infected mice treated with 3Cam2-LALA+ KAES+ACT5 (d). (B) Data on the weight of the fetuses in each group (n): whole body weight (a) and head weight (b). Each dot corresponds to 1 fruit;

in fig. 20(a)-(d) are results demonstrating that the treatment significantly reduced maternal-to-fetal transmission of ZIKV. Data on the viral load in the amniotic fluid (a), placenta (b), fetal brain (c) and fetal liver (d) are presented. Each point corresponds to 1 fetus, n≥7 in each group.

Detailed Description of Embodiments of the Invention

The technologies and procedures that are described or referenced herein are generally well known to those skilled in the art and have been widely used using conventional methodologies such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2001; Current Protocols in Molecular Biology (edited by B.M. Ausubel et al., 2003); series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (ed. M.J. MacPherson, B.D. Hames and G.R. Taylor, 1995), Antibodies, A Laboratory Manual, ed. Harlow and Lane, 1988 and Animal Cell Culture (ed. R.I. Freshney, 1987); Oligonucleotide Synthesis (ed. M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J.E. Cellis, 1998) from Academic Press; Animal Cell Culture (ed. R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998), Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. Griffiths and D.G. Newell, 1993-8), J. Wiley and Sons; Handbook of Experimental Immunology (edited by D.M. Weir and S.S. Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by J.M. Miller and M.P. Calos, 1987); PCR: The Polymerase Chain Reaction, (ed. Mullis et al., 1994); Current Protocols in Immunology (ed. J.E. Coligan et al., 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 (ed. D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and S. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (ed. M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by V.T. DeVita et al., J.B. Lippincott Company, 1993).

I. Definitions

Unless otherwise stated, technical and scientific terms used herein have meanings well known to one of ordinary skill in the art to which the present invention relates. For those skilled in the art, interpretations of many of the terms used herein are generally provided in 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. Bee references cited herein, including patent applications and patent publications, are incorporated by reference in their entirety.

The following definitions should apply to the interpretation of this specification and, whenever possible, terms used in the singular include their use in the plural and vice versa. It should be understood that the terminology used herein is presented for the purpose of describing specific embodiments of the invention only and is not intended to limit its scope. To the extent that any definition below conflicts with that contained in any document incorporated by reference, the definition below shall apply.

For purposes of this specification, "acceptor human framework" means a framework that contains the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. listed below. An acceptor human framework region "derived from" a human immunoglobulin framework region or a human consensus framework region may have the same amino acid sequence or may contain 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 human VL acceptor framework sequence is identical to the human immunoglobulin VL framework sequence or the human consensus framework sequence.

The concept of "affinity" refers to the total strength of all non-covalent interactions between one binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). As used herein, unless otherwise noted, “binding affinity” refers to the inherent binding affinity of a molecule, reflecting a 1:1 interaction between the components of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be characterized using a dissociation constant (Kd). Affinity can be assessed using conventional methods known in the art, including those presented herein. Specific options for measuring binding affinity are presented below for illustration.

An “affinity matured” antibody means an antibody with one or more changes in one or more hypervariable regions (HVRs) compared to a parent antibody that does not contain the changes, the changes resulting in increased affinity of the antibody for the antigen.

The terms “anti-DENV antibody” and “antibody that binds to DENV” refer to an antibody that has the ability to bind to DENV with sufficient affinity to enable the antibody to be used as a diagnostic and/or therapeutic agent that targets DENV . The antibody can bind to the DENV E protein. The terms "antibody to DENV E protein" and "antibody that binds to DENV E protein" refer to an antibody that has the ability to bind to DENV E protein with sufficient affinity to enable the antibody to be used diagnostically and /or a therapeutic agent that targets DENV. In one embodiment, the level of binding of an antibody to the DENV E protein to an unrelated non-DENV E protein is less than about 10% of the binding of the antibody to the DENV E protein as measured, for example, by radioimmunoassay (RIA). ). In some embodiments, 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 than 0.01 nM or less or 0.001 nM or less (eg 10 ^-8 M or less, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M). In some embodiments, the anti-DENV antibody binds to a DENV epitope that is conserved across different DENV serotypes. In some embodiments, an anti-DENV E protein antibody binds to an epitope of the DENV E protein that is conserved among the E proteins of various DENV serotypes.

The terms “anti-Zika virus antibody,” “anti-Zika antibody,” “anti-ZIKV antibody,” and “antibody that binds to the Zika virus” refer to an antibody that has the ability to bind to the Zika virus with sufficient affinity that the antibody can be used as a diagnostic and/or therapeutic agent targeting the Zika virus. The terms “anti-ZIKV antibody” and “anti-DENV antibody” can be used interchangeably to refer to an antibody that has the ability to bind to both ZIKV and DENV.

As used herein, the term “antibody” is used in its broadest sense and refers to various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that that they have the required antigen-binding activity.

Antibody-mediated cell-dependent cytotoxicity or ADCC refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) that are present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages) activates the ability of these cytotoxic effector cells to specifically bind to antigen-bearing target cells and then destroy the target cells using cytotoxins. Primary cells mediating ADCC, i.e. NK cells express only Fc-gamma RIII, while monocytes express Fc-gamma RI, Fc-gamma RII and Fc-gamma RIII. Data on FcR expression on hematopoietic cells are summarized in Table 3 on p. 464 from Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, cc. 457-492. To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, for example, as described in US No. 5,500,362 or 5,821,337 or US No. 6,737,056 (to Presta). Suitable effector cells for such analysis include PBMCs and NK cells. In an alternative or additional embodiment, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model, for example, as described in Clynes et al., PNAS (USA) 95, 1998, pp. 652-656.

The term "antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to: Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; dimeric antibodies (diabody); linear antibodies; single chain antibody molecules (eg scFv) and multispecific antibodies formed from antibody fragments.

The term "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of a reference antibody to its antigen when assessed by a competition assay and/or, conversely, a reference -Antibody blocks the binding of an antibody to its antigen when assessed using a competition assay. An example of a competitive analysis is presented in this description.

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

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.

The term "class" of an antibody refers to the type of constant domain or constant region that is included in its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further subdivided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains that correspond to different classes of immunoglobulins are designated alpha, delta, epsilon, gamma, and mu (α, δ, ε, γ, and μ), respectively.

The term "complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell 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 antibodies (of the corresponding subclass) that are associated with their cognate antigen. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202, 1996, pp. 163-171). Polypeptide variants with altered Fc region amino acid sequences (Fc region variant polypeptides) and increased or decreased C1q binding capacity are described, for example, in US No. 6194551 B1 and WO 1999/51642 (see also, for example, Idusogie et al. , J. Immunol. 164, 2000, pp. 4178-4184).

The term "cytotoxic agent" as used herein refers to a substance that inhibits or interferes with cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (eg, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu radioactive isotopes); chemotherapeutic agents or drugs (eg, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antineoplastic or anticancer agents listed below.

The term "effector functions" refers to those biological activities associated with the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-mediated cell-dependent cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B-cell receptor) and B-cell activation.

The term "effective amount" of an agent, for example, a pharmaceutical composition, means an amount effective in doses and for a period of time necessary to achieve the desired therapeutic or prophylactic result.

The term "epitope" includes any determinant that has the ability to be bound by an antibody. An epitope is a region of an antigen that binds to an antibody that targets that antigen and includes specific amino acids that directly contact the antibody. Epitope determinants may include reactive surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structure characteristics and/or specific charge characteristics. In general, antibodies specific for a particular target antigen should preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

"Fc receptor" or "FcR" means 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 a receptor that binds to an IgG antibody (gamma receptor) and includes receptors of the Fc-γRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains immunoreceptor tyrosine-based activating motifs (ITAMs) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains in its cytoplasmic domain an immunoreceptor tyrosine inhibitory motif (ITIM) (see, for example, Daeron, Annu. Rev. Immunol. 15, 1997, pp. 203-234). An overview of FcR information is provided, for example, by Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, cc. 457-492; Capel et al., Immunomethods 4, 1994, cc. 25-34 and de Haas et al., J. Lab. Clin. Med. 126, 1995, cc. 330-341. The term "FcR" as used herein includes other FcRs, including those that will be identified in the future.

The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the embryo (Guyer et al., J. Immunol. 117, 1976, pp. 587-593 and Kim et al., J . Immunol. 24, 1994, pp. 2429-2434) and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward., Immunol. Today 18(12), 1997, pp. 592-598; Ghetie et al., Nature Biotechnology 15(7), 1997, pp. 637 -640; Hinton et al., J. Biol. Chem. 279(8), 2004, pp. 6213-6216; WO 2004/92219 (to Hinton et al.) Binding to human FcRn in vivo and half-life in vivo serum human FcRn having high affinity binding to polypeptides can be assessed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with a variant Fc region. name Presta) antibody variants with increased or decreased ability to bind to FcR are described (see, for example, also Shields et al., J. Biol. Chem. 9(2), 2001, pp. 6591-6604).

The term "Fc region" as used herein refers to the C-terminal region of an immunoglobulin heavy chain that contains at least part of a constant region. The concept includes native sequence Fc regions and variant Fc regions. In one embodiment, the Fc region of the human IgG heavy chain extends from Cys226 or 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 specifically stated otherwise, the numbering of amino acid residues in the Fc region or constant region corresponds to the EU numbering system, which is also referred to as the EU index, described by Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term “Fc region-containing antibody” refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region may be removed, for example, during antibody purification or during recombinant construction of the nucleic acid encoding the antibody. Thus, a composition that contains an antibody having the Fc region of the present invention may contain an antibody with G446-K447, with G446 and without K447, with all G446-K447 removed, or a mixture of the three types of antibodies described above.

"Framework region" or "FR" means variable domain residues other than hypervariable region residues (HVR). The FR variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4.

Thus, the HVR and FR sequences typically have the following arrangement in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably as used herein to refer to an antibody having a structure substantially similar to the native antibody structure or having heavy chains that contain an Fc region as defined herein.

A “functional Fc region” has the “effector function” inherent in a native sequence Fc region. Examples of "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (eg, B-cell receptor; BCR), etc. The expression of these effector functions generally requires that the Fc region be combined with a binding domain (eg, an antibody variable domain) and can be assessed using various assays, such as those presented in the Definitions section herein.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of said cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and its progeny, regardless of the number of passages. The offspring may not be completely identical in nucleic acid composition to the parent cell, but may contain mutations. The invention includes mutant progeny that have the same function or biological activity as found by screening or selection in the original transformed cell.

A “human antibody” is an antibody having an amino acid sequence that corresponds to that of an antibody produced in a human body or in a human cell, or originating from a non-human source that utilizes the human antibody spectrum or other human antibody coding sequences. This definition of a human antibody specifically excludes a humanized antibody containing non-human antigen-binding moieties.

A “human consensus framework” is a framework that includes the most frequently occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable domain sequences. Typically, the subset of sequences is the subset described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH publication 91-3242, Bethesda MD, vol. 1-3, 1991. In one embodiment of the VL invention, the subgroup is kappa I according to Kabat et al., supra. In one embodiment of the VH invention, the subgroup is subgroup III according to Kabat et al., supra.

The term "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody may contain substantially all of at least one and typically two variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to regions of the non-human antibody, and all or substantially all of the FRs correspond to regions human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The term "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has been humanized.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain whose sequences are hypervariable (“complementarity determining regions” or “CDRs”) and/or form loops of a specific structure (“hypervariable loops”). , and/or contain antigen-contacting residues (“antigen contacts”). Typically, antibodies contain six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Examples of HVR include

(a) hypervariable loops including 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, 1987, cc. 901-917);

(b) CDRs including amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (HI), 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) areas of contact with the antigen, including 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, 1996, pp. 732-745); And

(d) combinations of residues specified in subparagraphs (a), (b) and/or (c), including amino acid residues HVR 46-56 (L2), 47-56 (L2), 48-56 (L2), 49- 56 (L2), 26-35 (H1), 26-35b (HI), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

Unless specifically stated otherwise, residues in HVR and other residues in the variable domain (eg, residues in FR) are numbered according to Kabat et al., supra.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs and horses), primates (such as humans and non-human primates such as monkeys), rabbits and rodents (such as mice and rats ). In some embodiments of the invention, the individual or subject is a human.

An "isolated" antibody is an antibody that is separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic assays (such as SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic assays (such as such as ion exchange chromatography or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chrom. In 848, 2007, pp. 79-87.

An "isolated" nucleic acid is a nucleic acid molecule that is separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains a nucleic acid molecule, but in which the nucleic acid molecule is present outside the chromosome or in which its location on the chromosome differs from its naturally occurring location on the chromosome.

The term “isolated antibody-encoding nucleic acid” refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including said nucleic acid molecule(s) in the same vector or in different vectors, and specified nucleic acid molecule(s) present at one or more positions in the host cell.

The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. individual antibodies included in the population are identical and/or bind to the same epitope, with the exception of possible antibody variants, for example, containing mutations that occur naturally or arise during the production of a monoclonal antibody preparation, these variants are usually present in minor quantities. Unlike polyclonal antibody preparations, which typically include different antibodies to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the adjective "monoclonal" defines the characteristic of an antibody, characterizing it as being derived from a substantially homogeneous population of antibodies, rather than being engineered to meet the requirements of producing the antibody using any particular method. For example, monoclonal antibodies that can be used according to the present invention can be generated using a variety of technologies, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and techniques based on the use of transgenic animals that contain all or part of the human immunoglobulin loci, these methods and other exemplary methods for producing monoclonal antibodies are presented in the present description.

The term “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or radiolabel. The naked antibody may be present in the pharmaceutical composition.

The term “native antibodies” refers to naturally occurring immunoglobulin molecules with different structures. For example, native antibodies in the form of IgG are heterotetrameric glycoproteins with a molecular weight of approximately 150,000 Da, consisting of two identical light chains and two identical heavy chains linked by disulfide bridges. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Likewise, in the direction from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL). The light chain of an antibody, depending on the amino acid sequence of its constant domain, can belong to one of two types, designated kappa (κ) and lambda (λ).

A “native sequence Fc region” contains an amino acid sequence identical to the amino acid sequence of an Fc region that occurs in nature. The native sequence of human Fc regions includes the native sequence of the human IgGl Fc region (non-A and A allotypes); the native sequence of the Fc region of human IgG2; the native human IgG3 Fc region sequence and the native human IgG4 Fc region sequence, as well as naturally occurring variants thereof.

The term “package insert” refers to instructions typically included in commercial packages of therapeutic products containing information regarding the indications, use, dosage, route of administration, combination therapy, contraindications and/or precautions associated with the use of the indicated therapeutic products.

“Percentage (%) amino acid sequence identity” relative to a polypeptide reference sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the polypeptide reference sequence, after sequence alignment and, if necessary, the introduction of gaps to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as a component in assessing sequence identity. Comparative analysis for the purpose of determining percent amino acid sequence identity can be carried out in various ways known in the art, using, for example, publicly available computer programs such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or GENETYX® (Genetyx Co.) ., Ltd.) Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

The ALIGN-2 sequence comparison computer program is owned by Genentech, Inc. and the source code has been made available with user documentation in the U.S. Copyright Office, Washington D.C. 20559, where it is registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, NY. California, or it can be compiled from source code. ALIGN-2 can be compiled for use on UNIX operating systems, including UNIX Digital System V4.0D. All parameters required for sequence comparison are set by ALIGN-2 and should not be changed. In situations in which ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A compared to or relative to a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A that has or contains a certain % The identity of an amino acid sequence compared to or relative to a given amino acid sequence B) is calculated as follows:

100 × fraction X/Y,

where X denotes the number of amino acid residues identified as identical matches when evaluated using the sequence analysis program ALIGN-2, where the program compared A and B, and where Y denotes the total number of amino acid residues in B. It should be obvious that , if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % identity of amino acid sequences of A relative to B cannot be equal to the % identity of amino acid sequence B relative to A. Unless otherwise specifically stated, all % amino acid sequence identity values specified herein description, were obtained using the ALIGN-2 computer program described in the previous paragraph.

The term "pharmaceutical composition" refers to a preparation that is in a form that provides the biological activity of its constituent active substance to be effective and that does not contain additional components that are unacceptable toxic to the individual to whom the composition is to be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, a buffer, excipient, stabilizer or preservative.

The term “DENV E protein” as used herein refers to any native DENV E protein from any DENV serotype, including, unless otherwise noted, DENV-1, DENV-2, DENV-3 and DENV-4. The DENV genome encodes three structural (capsid (C), (pre)membrane/membrane (prM/M), and envelope (E)) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The E protein, a glycoprotein with a molecular mass of approximately 55 kDa, is present as a heterodimer with the PrM protein prior to virion maturation. X-ray crystallographic studies of the E protein ectodomain revealed the presence of three different beta-barrel (beta-pleated) domains connected to the viral membrane by a coiled-coil anchor and two antiparallel transmembrane domains. Domain III (EDIII) has an immunoglobulin-like fold and is thought to play a major role in receptor interactions. 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 the apex and is involved in membrane fusion and E protein dimerization. The central domain (domain I; EDI) is a nine-strand β-barrel connected to EDIII and EDII by one and four flexible linkers, respectively. E proteins are essential for viral assembly, receptor binding, entry, viral fusion, and possibly immune escape during the viral life cycle and are therefore dynamic proteins required to adopt several different conformations and arrangements on the viral particle. The concept includes the “full-length” full-length DENV E protein, as well as any form of DENV E protein that is the result of processing in the cell. The concept includes naturally occurring variants of the DENV E protein, such as serotype variants or quasispecies.

“Zika virus (ZIKV)” as used herein refers to a member of the Flaviviridae family of viruses. It is transmitted by day-active Aedes mosquitoes such as A. aegypti and A. albopictus. The ZIKV genome encodes a single polyprotein consisting of 3419 amino acids, which is cleaved by the viral serine protease and the cellular prosthesis furin to form two functional domains: capsid (C), precursor membrane (pgM), envelope (E), and 7 nonstructural proteins (NS).

The term “substantially decreases,” “substantially increases,” or “substantially differs” as used herein refers to a significantly high degree of difference between two numerical values (one of which is typically associated with a molecule and the other is associated with a reference molecule/molecule with comparison) such that one skilled in the art can consider the difference between two values to be statistically significant in the context of the biological characteristic that is measured by the specified values (eg, Kd values).

As used herein, the concept of “treatment” (and its grammatical variations such as “to treat” or “the process of treatment”) refers to a clinical intervention with the purpose of changing the natural history of a disease in the individual being treated, and can be carried out either for prevention or during the development of clinical pathology. The desired treatment actions include, but are not limited to, prevention of onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastases, reduction in the rate of disease progression, alleviation or temporary amelioration of the disease state, and remission or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt and al., Kuby Immunology, 6th ed., W.H. Freeman and Co., 2007, p. 91).

A single VH or VL domain may be sufficient to provide antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated by using the VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano et al. , J. Immunol. 150, 1993, pp. 880-887; Clarkson et al., Nature 352, 1991, pp. 624-628).

An "Fc region variant" contains an amino acid sequence that differs from the native Fc region sequence due to the presence of at least one amino acid modification(s), preferably one or more amino acid substitution(s). Preferably, the Fc region variant has at least one amino acid substitution relative to the native Fc region sequence of the parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the native Fc region sequence or in the Fc region of the parent polypeptide. The Fc region variant should preferably have at least about 80% homology to the native sequence of the Fc region and/or the Fc region of the parent polypeptide, and most preferably at least about 90% homology thereto, more preferably at least approximately 95% homology with it.

The term "vector" as used herein refers to a nucleic acid molecule that has the ability to propagate another nucleic acid to which it is linked. The concept includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector included in the genome of the host cell into which it is introduced. Some vectors have the ability to control the expression of the nucleic acids to which they are operably linked. These vectors are referred to as “expression vectors” in the context of the present description.

II. Compositions and methods

One object of the invention is, in particular, antibodies to DENV and their uses. Some embodiments of the invention are antibodies that bind to DENV and/or DENV E protein. The antibodies of the invention can be used, for example, to diagnose or treat DENV infection.

One object of the invention is, in particular, polypeptides containing Fc region variants and their uses. Some embodiments of the invention are polypeptides containing Fc region variants with substantially reduced FcyRIIb binding activity. Some embodiments of the invention are polypeptides containing Fc region variants without significantly reduced C1q binding activity. In specific embodiments, the polypeptides of the invention are antibodies. Polypeptides containing variants of the Fc regions proposed in the invention can be used, for example, to diagnose or treat a viral infection.

One object of the invention is, in particular, antibodies to ZIKV and their uses. Some embodiments of the invention are antibodies that bind to ZIKV. Some embodiments of the invention are anti-ZIKV antibodies that are cross-reactive with DENV.

One object of the invention is, in particular, methods of treating or preventing infection caused by the Zika virus, including the administration of antibodies that bind to ZIKV. One object of the invention is, in particular, methods of inhibiting the transmission of Zika virus from a pregnant mother to the fetus, including the administration of antibodies that bind to ZIKV. One object of the invention is, in particular, methods for preventing congenital syndrome caused by the Zika virus, including the administration of antibodies that bind to ZIKV. One aspect of the invention is, in particular, methods of inhibiting the reduction of fetal weight (eg, total body weight of the child, head weight, etc.), including the administration of antibodies that bind to ZIKV.

In one example, the term “congenital Zika virus syndrome” refers to a special pattern of birth defects unique to fetuses and infants infected with the Zika virus before birth. As will be apparent to one skilled in the art, individuals with congenital Zika virus syndrome may have syndromes such as, but not limited to, mental retardation, microcephaly, severe microcephaly in which there is partial reduction in skull size, tissue loss brain with a specific pattern of brain damage, including subcortical calcification, damage to the posterior part of the eye, including macular scars and patchy retinal pigmentation, congenital contractures such as clubfoot or arthrogryposis, hypertension limiting body movement soon after birth, etc. It is likely that administration of the antibodies provided herein can prevent at least one, or at least two, or at least three, or at least four, or all symptoms of the congenital syndrome caused by the Zika virus. For example, an antibody provided herein prevents Zika virus infection of a fetus or infant before birth, thereby inhibiting the decrease in fetal weight (eg, infant's total body weight, head weight, etc.).

In one aspect of the invention, the term "anti-DENV antibody" as defined in this section (Compositions and Methods) can be used to refer to an antibody that has the ability to bind to ZIKV ("anti-ZIKV antibody") if the antibody can also bind to DENV , and with ZIKV. One aspect of the invention is methods of treating or preventing infection caused by the Zika virus, including the administration of antibodies that bind to the Zika virus (“anti-ZIKV antibody”). One aspect of the invention is a method of inhibiting transmission of the Zika virus from a pregnant mother to a fetus, comprising administering antibodies that bind to the Zika virus.

A. Examples of DENV Antibodies and Polypeptides Containing Fc Region Variants

One object of the invention is isolated antibodies that bind to DENV and/or DENV E protein. In some embodiments, an anti-DENV antibody blocks the binding of DENV and/or DENV E protein to a host cell. In some embodiments, the anti-DENV antibody inhibits the entry of DENV into a host cell. In some embodiments, the anti-DENV antibody binds to the entire DENV particle. In other embodiments, the antibody binds to the whole DENV particle better than to the monomeric DENV E protein. In some embodiments, the anti-DENV antibody of the present invention binds to and/or neutralizes at least one, at least two, at least three, or all four 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 some embodiments, an anti-DENV antibody binds to and/or neutralizes the DENV E protein originating from at least one, at least two, at least three, or all four DENV serotypes selected from the group consisting of DENV-1, DENV-2, DENV-3 and DENV-4. The term "serotype" refers to different variations of one type of virus.

One object of the invention is isolated antibodies that bind to ZIKV. In some embodiments, an anti-ZIKV antibody blocks the binding of ZIKV to a host cell. In some embodiments, the anti-ZIKV antibody inhibits the entry of ZIKV into a host cell. In some embodiments, the anti-ZIKV antibody is cross-reactive with DENV. One aspect of the invention is an anti-DENV antibody that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1, which contains the amino acid sequence presented in any of SEQ ID NO: 11 -12; (b) HVR-H2, which contains the amino acid sequence presented in any of SEQ ID NO: 13-15; (c) HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20; (d) HVR-L1, which contains the amino acid sequence presented in any of SEQ ID NO: 21-23; (e) HVR-L2, which contains the amino acid sequence presented in any of SEQ ID NO: 24-26; and (f) HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NO: 27-30.

Another aspect of the invention is an anti-DENV antibody that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 45.

Another aspect of the invention is an anti-DENV antibody that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 30.

Another aspect of the invention is an anti-DENV antibody that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27.

One aspect of the invention is an antibody that contains at least one, at least two or all three HVR sequences VH selected from (a) HVR-H1, which contains the amino acid sequence presented in any of SEQ ID NO: 11- 12; (b) HVR-H2, which contains the amino acid sequence presented in any of SEQ ID NO: 13-15; and (c) HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20. In one embodiment, the antibody contains HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20. In another embodiment, the antibody contains HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20, and HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NO: 27-30. In a further embodiment of the invention, the antibody comprises HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20, HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NO: 27-30, and HVR-H2 which contains the amino acid sequence presented in any of SEQ ID NO: 13-15. In the following embodiment, the antibody contains (a) HVR-H1, which contains the amino acid sequence presented in any of SEQ ID NO: 11-12; (b) HVR-H2, which contains the amino acid sequence presented in any of SEQ ID NO: 13-15; and (c) HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20.

Another object of the invention is an antibody that contains at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 41; and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 42. In another embodiment of the invention, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 42, and HVR-L3, which contains the amino acid sequence of SEQ ID NO: 45. In another embodiment, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 42, HVR-L3, which contains the amino acid sequence of SEQ ID NO: 45, and HVR-H2, which contains the amino acid sequence of SEQ ID NO: 41. In the following embodiment, the antibody contains (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 41; and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 42.

Another object of the invention is an antibody that contains at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15; and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20. In another embodiment, the antibody contains HVR-H3 which contains the amino acid sequence of SEQ ID NO: 20, and HVR-L3, which contains the amino acid sequence of SEQ ID NO: 30. In another embodiment of the invention, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20, and HVR- L3, which contains the amino acid sequence of SEQ ID NO: 27. In a further embodiment of the invention, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20, HVR-L3, which contains the amino acid sequence of SEQ ID NO: 30, and HVR- H2, which contains the amino acid sequence of SEQ ID NO: 15. In a further embodiment of the invention, the antibody contains HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20, HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27, and HVR- H2, which contains the amino acid sequence of SEQ ID NO: 15. In the next embodiment of the invention, the antibody contains (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15; and (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20.

Another object of the invention is an antibody that contains at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1, which contains the amino acid sequence presented in any of SEQ ID NO: 21-23 ; (b) HVR-L2, which contains the amino acid sequence presented in any of SEQ ID NO: 24-26; and (c) HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NO: 27-30. In one embodiment, the antibody contains (a) HVR-L1, which contains the amino acid sequence presented in any of SEQ ID NO: 21-23; (b) HVR-L2, which contains the amino acid sequence presented in any of SEQ ID NO: 24-26; and (c) HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NO: 27-30.

Another object of the invention is an antibody that contains at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 45. In one embodiment, the antibody contains (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 45.

Another object of the invention is an antibody that contains at least one, at least two or all three VL HVR sequences selected from (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antibody contains (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 30.

In another aspect of the invention, the antibody of the invention contains (a) a VH domain that contains at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1, which contains an amino acid sequence represented in any of SEQ ID NOs: 11-12, (II) HVR-H2, which contains the amino acid sequence presented in any of SEQ ID NOs: 13-15, and (III) HVR-H3, which contains the amino acid sequence, presented in any of SEQ ID NO: 16-20; and (b) a VL domain that contains at least one, at least two, or all three VL HVR sequences selected from (I) HVR-L1 that contains the amino acid sequence presented in any of SEQ ID NO: 21 -23, (II) HVR-L2, which contains the amino acid sequence presented in any of SEQ ID NO: 24-26, and (III) HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NO: 27- thirty.

In another aspect of the invention, the antibody of the invention contains (a) a VH domain that contains at least one, at least two, or all three VH HVR sequences selected from (I) HVR-H1, which contains an amino acid sequence SEQ ID NO: 40, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 41, and (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 42; and (b) a VL domain that contains at least one, at least two, or all three VL HVR sequences selected from (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 43, (II) HVR -L2, which contains the amino acid sequence presented in any of SEQ ID NO: 44, and (III) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 45.

In another aspect of the invention, the antibody of the invention contains (a) a VH domain that contains at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1, which contains an amino acid sequence SEQ ID NO: 12, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15, and (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20; and (b) a VL domain that contains at least one, at least two, or all three VL HVR sequences selected from (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 23, (II) HVR -L2, which contains the amino acid sequence presented in any of SEQ ID NO: 26, and (III) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 30.

In another aspect of the invention, the antibody of the invention contains (a) a VH domain that contains at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1, which contains an amino acid sequence SEQ ID NO: 12, (II) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15, and (III) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20; and (b) a VL domain that contains at least one, at least two, or all three VL HVR sequences selected from (I) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21, (II) HVR -L2, which contains the amino acid sequence presented in any of SEQ ID NO: 24, and (III) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27.

Another object of the invention is an antibody containing (a) HVR-H1, which contains the amino acid sequence presented in any of SEQ ID NO: 11-12; (b) HVR-H2, which contains the amino acid sequence presented in any of SEQ ID NO: 13-15; (c) HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20; (d) HVR-L1, which contains the amino acid sequence presented in any of SEQ ID NO: 21-23; (e) HVR-L2, which contains the amino acid sequence presented in any of SEQ ID NO: 24-26; and (f) HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NO: 27-30.

Another object of the invention is an antibody containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 45.

Another object of the invention is an antibody containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 30.

Another object of the invention is an antibody containing (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24; and (e) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 27.

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

In some embodiments, one or more amino acid substitutions in the anti-DENV antibody are conservative substitutions as defined herein. In some embodiments, any one or more of the following substitutions may 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 (e) in HVR-L3 (SEQ ID NO: 27), D4S or E; D5A.

All possible combinations of the above substitutions are covered by the consensus sequences 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.

In another embodiment, the anti-DENV antibody of the invention is not an antibody that contains

(I) HVR-H1 containing the amino acid sequence SEQ ID NO: 11,

(II) HVR-H2 containing the amino acid sequence SEQ ID NO: 13,

(III) HVR-H3 containing the amino acid sequence SEQ ID NO: 16,

(IV) HVR-L1 containing the amino acid sequence SEQ ID NO: 21,

(V) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24, and (VI) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27.

Another aspect of the invention is an anti-DENV antibody that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6 ; (b) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6; (c) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6; (d) HVR-L1 from the VL sequence presented in any of SEQ ID NO: 8-10; (e) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8-10; and (f) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10.

Another aspect of the invention is an anti-DENV antibody that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6 ; (b) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6; (c) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6; (d) HVR-L1 from VL sequence SEQ ID NO: 7; (e) HVR-L2 from VL sequence SEQ ID NO: 7; and (f) HVR-L3 from the VL sequence SEQ ID NO: 7.

Another object of the invention is an antibody to DENV that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 from the VH sequence presented in SEQ ID NO: 6; (b) HVR-H2 from VH sequence SEQ ID NO: 6; (c) HVR-H3 from the VH sequence shown in SEQ ID NO: 6; (d) HVR-L1 from VL sequence SEQ ID NO: 10; (e) HVR-L2 from VL sequence SEQ ID NO: 10; and (e) HVR-L3 from the VL sequence SEQ ID NO: 10.

Another aspect of the invention is an anti-DENV antibody that contains at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 from the VH sequence of SEQ ID NO: 6; (b) HVR-H2 from VH sequence SEQ ID NO: 6; (c) HVR-H3 from VH sequence SEQ ID NO: 6; (d) HVR-L1 from VL sequence SEQ ID NO: 7; (e) HVR-L2 from VL sequence SEQ ID NO: 7; and (f) HVR-L3 from the VL sequence SEQ ID NO: 7.

One aspect of the invention is an antibody that contains at least one, at least two or all three HVR VH sequences selected from (a) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6 ; (b) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6; and (c) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6. In one embodiment, the antibody comprises HVR-H3 from the VH sequence set forth in any of SEQ ID NOs: 2-6. In another embodiment, the antibody comprises HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6, and HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10. In another embodiment of the invention, the antibody contains HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6, and HVR-L3 from the VL sequence SEQ ID NO: 7. In a further embodiment of the invention, the antibody contains HVR- H3 from the VH sequence presented in any of SEQ ID NO: 2-6, and HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10, and HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6. In a further embodiment, the antibody comprises HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6, and HVR-L3 from the VL sequence SEQ ID NO: 7, and HVR-H2 from the VH sequence, presented in any of SEQ ID NO: 2-6. In a further embodiment, the antibody comprises (a) HVR-H1 from the VH sequence set forth in any of SEQ ID NOs: 2-6; (b) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6; and (c) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6.

One aspect of the invention is an antibody that contains at least one, at least two or all three VH HVR sequences selected from (a) HVR-H1 from the VH sequence SEQ ID NO: 6; (b) HVR-H2 from VH sequence SEQ ID NO: 6; and (c) HVR-H3 from VH sequence SEQ ID NO: 6. In one embodiment, the antibody contains HVR-H3 from VH sequence SEQ ID NO: 6. In another embodiment, the antibody contains HVR-H3 from VH -sequences SEQ ID NO: 6 and HVR-L3 from the VL sequence SEQ ID NO: 10. In another embodiment, the antibody comprises HVR-H3 from the VH sequence SEQ ID NO: 6 and HVR-L3 from the VL sequence SEQ ID NO: 7. In a further embodiment, the antibody comprises HVR-H3 from the VH sequence SEQ ID NO: 6, HVR-L3 from the VL sequence SEQ ID NO: 10, and HVR-H2 from the VH sequence SEQ ID NO: 6. In a further embodiment, the antibody comprises HVR-H3 from the VH sequence SEQ ID NO: 6, HVR-L3 from the VL sequence SEQ ID NO: 7, and HVR-H2 from the VH sequence SEQ ID NO: 6. In a further embodiment The antibody of the invention contains (a) HVR-H1 from the VH sequence SEQ ID NO: 6; (b) HVR-H2 from VH sequence SEQ ID NO: 6; and (c) HVR-H3 from the VH sequence SEQ ID NO: 6.

Another object of the invention is an antibody that contains at least one, at least two or all three VL HVR sequences selected from (a) HVR-V1 from the VL sequence presented in any of SEQ ID NO: 8-10; (b) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8-10; and (c) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10. In one embodiment, the antibody comprises (a) HVR-L1 from the VL sequence shown in any of SEQ ID NO: 8-10; (b) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8-10; and (c) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10.

Another aspect of the invention is an antibody that contains at least one, at least two or all three VL HVR sequences selected from (a) HVR-V1 from the VL sequence SEQ ID NO: 10; (b) HVR-L2 from VL sequence SEQ ID NO: 10; and (c) HVR-L3 from the VL sequence SEQ ID NO: 10. In one embodiment, the antibody comprises (a) HVR-L1 from the VL sequence SEQ ID NO: 10; (b) HVR-L2 from VL sequence SEQ ID NO: 10; and (c) HVR-L3 from the VL sequence SEQ ID NO: 10.

Another aspect of the invention is an antibody of the invention which comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (i) HVR-H1 of the VH sequence, presented in any of SEQ ID NO: 2-6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6, and (III) HVR-H3 from the VH sequence presented in any from SEQ ID NO: 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 from the VL sequence presented in any of SEQ ID NO: 8-10 , (II) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8-10, and (III) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10.

Another aspect of the invention is an antibody of the invention which comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (i) HVR-H1 of the VH sequence, presented in any of SEQ ID NO: 2-6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6, and (III) HVR-H3 from the VH sequence presented in any from SEQ ID NO: 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 from the VL sequence SEQ ID NO: 7, (II) HVR-L2 from the VL sequence SEQ ID NO: 7 and (III) HVR-L3 from the VL sequence SEQ ID NO: 7.

Another aspect of the invention is an antibody of the invention which comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (I) HVR-H1 from the VH sequence SEQ ID NO:6, (II) HVR-H2 from the VH sequence SEQ ID NO: 6, and (III) HVR-H3 from the VH sequence SEQ ID NO: 6; and (6) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (I) HVR-L1 from the VL sequence SEQ ID NO: 10, (II) HVR-L2 from the VL sequence SEQ ID NO: 10 and (III) HVR-L3 from the VL sequence SEQ ID NO: 10.

Another aspect of the invention is an antibody of the invention which comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from (I) HVR-H1 from the VH sequence SEQ ID NO:6, (II) HVR-H2 from the VH sequence SEQ ID NO: 6, and (III) HVR-H3 from the VH sequence SEQ ID NO: 6; and (6) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (I) HVR-L1 from the VL sequence SEQ ID NO: 7, (II) HVR-L2 from the VL sequence SEQ ID NO: 7 and (III) HVR-L3 from the VL sequence SEQ ID NO: 7.

Another object of the invention is an antibody that contains (a) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6; (b) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6; (c) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6; (d) HVR-L1 from the VL sequence presented in any of SEQ ID NO: 8-10; (e) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8-10; and (f) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8-10.

Another object of the invention is an antibody that contains (a) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2-6; (6) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2-6; (c) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2-6; (d) HVR-L1 from VL sequence SEQ ID NO: 7; (e) HVR-L2 from VL sequence SEQ ID NO: 7; and (e) HVR-L3 from the VL sequence SEQ ID NO: 7.

Another object of the invention is an antibody that contains (a) HVR-H1 from the VH sequence SEQ ID NO: 6; (b) HVR-H2 from VH sequence SEQ ID NO:6; (c) HVR-H3 from VH sequence SEQ ID NO: 6; (d) HVR-L1 from VL sequence SEQ ID NO: 10; (e) HVR-L2 from VL sequence SEQ ID NO: 10; and (e) HVR-L3 from the VL sequence SEQ ID NO: 10.

Another object of the invention is an antibody that contains (a) HVR-H1 from the VH sequence SEQ ID NO: 6; (b) HVR-H2 from VH sequence SEQ ID NO:6; (c) HVR-H3 from VH sequence SEQ ID NO: 6; (d) HVR-L1 from VL sequence SEQ ID NO: 7; (e) HVR-L2 from VL sequence SEQ ID NO: 7; and (f) HVR-L3 from the VL sequence SEQ ID NO: 7.

In yet another embodiment, the anti-DENV antibody of the invention is not an antibody that contains (I) HVR-H1 from the VH sequence SEQ ID NO: 1, (II) HVR-H2 from the VH sequence SEQ ID NO: : 1, (III) HVR-H3 from VH sequence SEQ ID NO: 1, (IV) HVR-L1 from VL sequence SEQ ID NO: 7, (V) HVR-L2 and VL sequence SEQ ID NO: 7 , and (VI) HVR-L3 from the VL sequence SEQ ID NO: 7.

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

In another aspect of the invention, an anti-DENV antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identical to the amino acid sequence presented in any of SEQ ID NO: 2-6. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions ), insertions or deletions relative to the reference sequence, but an anti-DENV antibody containing the specified sequence retains the ability to bind to DENV. In some embodiments, a total of 1 to 10 amino acids are replaced, inserted, and/or deleted by deletion in any of SEQ ID NOs: 2-6. In some embodiments, the substitutions, insertions, or deletions involve regions outside the HVR (ie, FR). Optionally, the anti-DENV antibody contains a VH sequence set forth in any of SEQ ID NOs: 2-6, including post-translational modifications to said sequence. In a specific embodiment of the invention, VH contains one, two or three HVRs selected from: (a) HVR-H1, which contains the amino acid sequence presented in any of SEQ ID NO: 11-12, (b) HVR-H2, which contains the amino acid sequence presented in any of SEQ ID NO: 13-15, and (c) HVR-H3, which contains the amino acid sequence presented in any of SEQ ID NO: 16-20. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

In another aspect of the invention, an anti-DENV antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical, 98% or 99%, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but the DENV antibody containing the specified sequence retains the ability to bind to DENV. In some embodiments, a total of 1 to 10 amino acids are replaced, inserted, and/or deleted by deletion in SEQ ID NO:6. In some embodiments, the substitutions, insertions, or deletions involve regions outside the HVR (ie, FR). Optionally, the anti-DENV antibody contains the VH sequence of SEQ ID NO:6, including post-translational modifications of the specified sequence. In a specific embodiment, the VH contains one, two or three HVRs selected from: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15 , and (c) HVR-H3, which contains the amino acid sequence SEQ ID NO: 20. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid .

In another aspect of the invention, an anti-DENV antibody comprises a light chain variable domain (VL) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identical to the amino acid sequence presented in any of SEQ ID NO: 8-10. In some embodiments, a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions ), insertions or deletions relative to the reference sequence, but an anti-DENV antibody containing the specified sequence retains the ability to bind to DENV. In some embodiments, a total of 1 to 10 amino acids are replaced, inserted, and/or deleted by deletion in any of SEQ ID NO: 8-10. In some embodiments, the substitutions, insertions, or deletions involve regions outside the HVR (ie, FR). Optionally, the anti-DENV antibody contains a VL sequence set forth in any of SEQ ID NOs: 8-10, including post-translational modifications to said sequence. In a specific embodiment, VL contains one, two or three HVRs selected from: (a) HVR-L1, which contains the amino acid sequence presented in any of SEQ ID NO: 21-23, (b) HVR-L2, which contains the amino acid sequence presented in any of SEQ ID NOs: 24-26, and (c) HVR-L3, which contains the amino acid sequence presented in any of SEQ ID NOs: 27-30. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

In another aspect, the anti-DENV antibody comprises a light chain variable domain (VL) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical, 98% or 99%, contains substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but the DENV antibody containing the specified sequence retains the ability to bind to DENV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted by deletion in SEQ ID NO: 10. In some embodiments, the substitutions, insertions, or deletions involve regions outside the HVR (ie, FR). Optionally, the anti-DENV antibody contains the sequence VL of SEQ ID NO: 10, including post-translational modifications to said sequence. In a specific embodiment, VL contains one, two or three HVRs selected from: (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 23, (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 26 , and (c) HVR-L3, which contains the amino acid sequence SEQ ID NO: 30. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

Another object of the invention is an antibody to DENV, where the antibody contains VH specified in any of the above embodiments of the invention, and VL specified in any of the above embodiments of the invention. In one embodiment, the antibody comprises the VH and VL sequences set forth in any of SEQ ID NOs: 2-6 and any of SEQ ID NOs: 8-10, respectively, including post-translational modifications to said sequences. In one embodiment, the antibody comprises the VH and VL sequences set forth in any of SEQ ID NO: 2-6 and SEQ ID NO: 7, respectively, including post-translational modifications to said sequences. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

Another object of the invention is an antibody to DENV, where the antibody contains VH specified in any of the above embodiments of the invention, and VL specified in any of the above embodiments of the invention. In one embodiment, the antibody contains the VH and VL sequences of SEQ ID NO: 6 and SEQ ID NO: 10, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, including post-translational modifications to said sequences. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

Another aspect of the invention is an antibody that binds to the same epitope as the anti-DENV antibody provided herein. Some embodiments of the invention is an antibody that binds to an epitope consisting of at least one, at least two, at least three, at least four, at least five, at least six, of at least seven or all amino acids selected from the group consisting of G100, W101, K122, 1162, S274, K310, W391 and F392 of the DENV-2 E protein. In some embodiments, the invention is an antibody that binds to an epitope consisting of at least one, at least two, or all of the amino acids selected from the group consisting of K122, 1162, and S274 of the DENV-2 E protein. Some embodiments of the invention is an antibody that binds to an epitope further comprising at least one of the amino acids selected from the group consisting of G100, W101, K310, W391 and F392, where the epitope contains at least one, at least two or all amino acids selected from the group consisting of K122, 1162 and S274.

One object of the invention is methods for treating or preventing infection caused by the Zika virus. One object of the invention is a method for inhibiting transmission of the Zika virus from a pregnant mother to her fetus. One of the objects of the invention is methods for preventing congenital syndrome caused by the Zika virus (for example, mental development deficiency, microcephaly, etc.). One object of the invention is methods of inhibiting the reduction of fetal weight (for example, the weight of the child's whole body, the weight of the head, etc.).

In one example, the term “congenital Zika virus syndrome” refers to a special pattern of birth defects unique to fetuses and infants infected with the Zika virus before birth. As will be apparent to one skilled in the art, individuals with congenital Zika virus syndrome may have syndromes such as, but not limited to, mental retardation, microcephaly, severe microcephaly in which there is partial reduction in the size of the skull, decreased brain tissue with a specific pattern of brain damage, including subcortical calcification, damage to the posterior part of the eye, including macular scars and patchy retinal pigmentation, congenital contractures such as clubfoot or arthrogryposis, hypertension limiting body movement soon after birth, etc. . It is likely that administration of the antibodies provided herein can prevent at least one, or at least two, or at least three, or at least four, or all symptoms of the congenital syndrome caused by the Zika virus. For example, an antibody provided herein prevents Zika virus infection of a fetus or infant before birth, thereby inhibiting the decrease in fetal weight (eg, infant's total body weight, head weight, etc.).

In some embodiments, a method of treating or preventing an infection caused by the Zika virus provided by the present invention comprises administering an antibody that binds to the Zika virus. In some embodiments, a method of inhibiting transmission of the Zika virus from a pregnant mother to a fetus includes administering an antibody that binds to the Zika virus. In some embodiments, a method of preventing a congenital syndrome caused by the Zika virus (eg, mental retardation, microcephaly, etc.) provided by the present invention includes administering an antibody that binds to the Zika virus. In some embodiments, methods of inhibiting fetal weight loss (eg, total body weight, head weight, etc.) provided by the present invention include administering an antibody that binds to the Zika virus. In one example, the term “congenital Zika virus syndrome” refers to a special pattern of birth defects unique to fetuses and infants infected with the Zika virus before birth. As will be apparent to one skilled in the art, individuals with congenital Zika virus syndrome may have syndromes such as, but not limited to, mental retardation, microcephaly, severe microcephaly in which there is a partial reduction in the size of the skull, decreased brain tissue with a specific pattern of brain damage, including subcortical calcification, damage to the back of the eye, including macular scars and patchy retinal pigmentation, congenital contractures such as clubfoot or arthrogryposis, hypertension limiting movement soon after birth, etc. . It is likely that administration of the antibodies provided herein can prevent at least one, or at least two, or at least three, or at least four, or all symptoms of the congenital syndrome caused by the Zika virus. For example, an antibody provided herein prevents Zika virus infection of a fetus or infant before birth, thereby inhibiting the decrease in fetal weight (eg, infant's total body weight, head weight, etc.).

In some embodiments, an antibody that binds to the Zika virus of the present invention is cross-reactive with the dengue virus.

In some embodiments, the anti-Zika virus antibody of the present invention comprises:

(a) (I) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ is a charged amino acid, X ₃ is a charged amino acid or a hydrophobic amino acid, X ₃ is a charged amino acid or a hydrophobic amino acid, (II) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ represents a charged amino acid or a polar amino acid, X ₃ represents a charged amino acid or a polar amino acid, and (III) HVR-H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ represents a charged amino acid or a polar amino acid, X ₃ represents a charged amino acid or a hydrophobic amino acid;

(b) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X ₁ denotes a polar amino acid, X ₃ denotes a hydrophobic amino acid, (II) HVR-H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in in which X ₁ denotes a charged amino acid or a polar amino acid, X ₃ denotes a charged amino acid or a hydrophobic amino acid, and (III) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ denotes a charged amino acid, X ₃ denotes a charged an amino acid or a hydrophobic amino acid, X ₃ denotes a charged amino acid or a hydrophobic amino acid;

(c) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X ₁ denotes a polar amino acid, X ₃ denotes a hydrophobic amino acid, (II) HVR-H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in in which X ₁ denotes a charged amino acid or a polar amino acid, X ₃ denotes a hydrophobic amino acid or a charged amino acid, (III) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ denotes a charged amino acid, X ₃ denotes a charged amino acid or a hydrophobic amino acid, X ₃ is a charged amino acid or a hydrophobic amino acid, (IV) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ is a charged amino acid, X ₃ is a charged amino acid or a polar amino acid; (V) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ represents a charged amino acid or a polar amino acid, X ₃ represents a polar amino acid or a hydrophobic amino acid; and (VI) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ represents a charged amino acid or a polar amino acid, X ₃ represents a charged amino acid or a hydrophobic amino acid; or

(d) (I) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ represents a charged amino acid, X ₃ represents a charged amino acid or a polar amino acid; (II) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ is a polar amino acid or a charged amino acid, X ₃ is a polar amino acid or a hydrophobic amino acid; and (III) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is a charged amino acid or a polar amino acid, X ₃ is a charged amino acid or a hydrophobic amino acid.

As will be apparent to one skilled in the art, the charged amino acid is arginine (R, Arg), lysine (K, Lys), aspartic acid (D, Asp), glutamic acid (E, Glu); polar amino acid is glutamine (Q, Gln), asparagine (N, Asn), histidine (H, His), ser (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine (C, Cys) and tryptophan (W, Tgr); hydrophobic amino acid is alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), valine (Val, V), proline (Pro, P) and glycine (Gly, G).

In some embodiments, an antibody of the present invention that binds to the Zika virus comprises:

(a) (I) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ is R or E, X ₃ is R or F, X3 is G, D or L (SEQ ID NO: 42 ), (II) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₃ is D or A (SEQ ID NO: 45), and (III) HVR-H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₃ is A or R (SEQ ID NO: 41);

(b) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X ₁ is N or Y, X ₃ is I or M (SEQ ID NO: 40), (II) HVR-H2 containing the amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₃ is A or R (SEQ ID NO: 41), and (III) HVR-H3, containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in which X ₁ is R or E, X ₃ is R or F, X ₃ is G, D or L (SEQ ID NO: 42);

(c) (I) HVR-H1 containing the amino acid sequence SX ₁ YX ₂ H, in which X ₁ is N or Y, X ₃ is I or M (SEQ ID NO: 40), (II) HVR-H2 containing amino acid sequence VINPRGGSX ₁ X ₂ SAQKFQG, in which X ₁ is T or R, X ₃ is A or R (SEQ ID NO: 41), (III) HVR-H3 containing the amino acid sequence GGX ₁ ALFYDSYTTPX ₂ DX ₃ GSWWFDP, in in which X ₁ is R or E, X ₃ is R or F, X3 is G, D or L (SEQ ID NO: 42), (IV) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ is D or E, X ₃ is K or Q (SEQ ID NO: 43); (V) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ is N or E, X ₃ is T or F (SEQ ID NO: 44); and (VI) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₃ is D or A (SEQ ID NO: 45); or

(d) (I) HVR-L1 containing the amino acid sequence QASQX ₁ IRX ₂ YLN, in which X ₁ is D or E, X ₃ is K or Q (SEQ ID NO: 43); (II) HVR-L2 containing the amino acid sequence DASX ₁ LKX ₂ in which X ₁ is N or E, X ₃ is T or F (SEQ ID NO: 44); and (III) HVR-L3 containing the amino acid sequence QQFX ₁ X ₂ LPIT, in which X ₁ is D, S or E, X ₃ is D or A (SEQ ID NO: 45).

In some embodiments, an antibody of the present invention that binds to the Zika virus comprises:

(a) (I) HVR-H3 containing the amino acid sequence SEQ ID NO: 20, (II) HVR-L3 containing the amino acid sequence SEQ ID NO: 27, and (III) HVR-H2 containing the amino acid sequence SEQ ID NO: 15;

(b) (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 12, (II) HVR-H2 containing the amino acid sequence SEQ ID NO: 15, and (III) HVR-H3 containing the amino acid sequence SEQ ID NO: 20;

(c) (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 12, (II) HVR-H2 containing the amino acid sequence SEQ ID NO: 15, (III) HVR-H3 containing the amino acid sequence SEQ ID NO: 20 , (IV) HVR-L1 containing the amino acid sequence SEQ ID NO: 21; (V) HVR-L2 containing the amino acid sequence SEQ ID NO: 24; and (VI) HVR-L3 containing the amino acid sequence SEQ ID NO: 27; or

(d) (I) HVR-L1 containing the amino acid sequence SEQ ID NO: 21; (II) HVR-L2 containing the amino acid sequence SEQ ID NO: 24; and (III) HVR-L3 containing the amino acid sequence SEQ ID NO: 27.

In some embodiments, the antibody of the invention is not an antibody that contains (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 11, (II) HVR-H2 containing the amino acid sequence 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) HVR- L3 containing the amino acid sequence SEQ ID NO: 27.

In some embodiments, an antibody of the present invention that binds to the Zika virus comprises:

(a) (I) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, (II) HVR-L3 from the VL sequence presented in any of SEQ ID NO : 8, 9 or 10, and (III) HVR-H2 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6;

(b) (I) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO : 2, 3, 4, 5 or 6, and (III) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6;

(c) (I) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO : 2, 3, 4, 5 or 6, (III) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, (IV) HVR-L1 from the VL sequence represented in any of SEQ ID NO: 8, 9 or 10; (V) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8, 9 or 10; and (VI) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8, 9 or 10;

(d) (I) HVR-L1 from the VL sequence presented in any of SEQ ID NO: 8, 9 or 10; (II) HVR-L2 from the VL sequence presented in any of SEQ ID NO: 8, 9 or 10; and (III) HVR-L3 from the VL sequence presented in any of SEQ ID NO: 8, 9 or 10; or

(e) (I) HVR-H1 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, (II) HVR-H2 from the VH sequence presented in any of SEQ ID NO : 2, 3, 4, 5 or 6, (III) HVR-H3 from the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, (IV) HVR-L1 from the VL sequence presented in SEQ ID NO: 7; (V) HVR-L2 from the VL sequence shown in SEQ ID NO: 7; and (VI) HVR-L3 from the VL sequence shown in SEQ ID NO: 7.

In some embodiments, the antibody of the invention is not an antibody that contains (I) HVR-H1 from the VH sequence SEQ ID NO: 1, (II) HVR-H2 from the VH sequence SEQ ID NO: 1, (III) HVR-H3 from VH sequence SEQ ID NO: 1, (IV) HVR-L1 from VL sequence SEQ ID NO: 7, (V) HVR-L2 from VL sequence SEQ ID NO: 7 and (VI ) HVR-L3 from VL sequence SEQ ID NO: 7.

In some embodiments, an antibody of the present invention that binds to the Zika virus also comprises an FR1 heavy chain variable domain framework region comprising the amino acid sequence of SEQ ID NO: 31, FR2 containing the amino acid sequence of SEQ ID NO: 32, FR3 containing amino acid sequence of SEQ ID NO: 33 or 34, FR4, containing the amino acid sequence of SEQ ID NO: 35. In some embodiments, an antibody of the present invention that binds to the Zika virus also contains an FR1 light chain variable domain framework region containing the amino acid sequence 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.

In some embodiments, an antibody of the present invention that binds to the Zika virus contains (a) a VH sequence that is at least 95% identical to the amino acid sequence set forth in any of SEQ ID NO: 2, 3, 4 , 5 or 6; (b) a VL sequence, the sequence of which is at least 95% identical to the amino acid sequence presented in any of SEQ ID NO: 8, 9 or 10; (c) a VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, and a VL sequence presented in any of SEQ ID NO: 8, 9 or 10; or (d) the VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, and the VL sequence presented in SEQ ID NO: 7.

In another aspect of the invention, an anti-ZIKV antibody contains a heavy chain variable domain (VH) sequence identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% of the amino acid sequence presented in any of SEQ ID NO: 2-6. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitution), insertion or deletion compared to the reference sequence, but an anti-ZIKV antibody containing such a sequence retains the ability to bind to ZIKV. In some embodiments, a total of 1 to 10 amino acids are replaced, inserted, and/or deleted by deletion in any of SEQ ID NOs: 2-6. In some embodiments, the substitutions, insertions, or deletions involve regions outside the HVR (ie, FR). Optionally, the anti-ZIKV antibody contains a VH sequence presented in any of SEQ ID NOs: 2-6, including post-translational modifications of the specified sequence. In a specific embodiment, the VH contains one, two or three HVRs selected from: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15 , and (c) HVR-H3, which contains the amino acid sequence SEQ ID NO: 20. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

In another aspect of the invention, an anti-ZIKV antibody contains a heavy chain variable domain (VH) sequence identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% of the amino acid sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6. In some embodiments, a VH sequence that is at least 90%, 91%, 92%, 93% identical , 94%, 95%, 96%, 97%, 98%, or 99%, contains substitutions (eg, conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-ZIKV antibody containing such a sequence retains the ability to bind with ZIKV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted by deletion in any of SEQ ID NO: 2, 3, 4, 5, or 6. In some embodiments, the substitutions, insertions, or deletions involve regions outside HVR (i.e. FR). Optionally, the anti-ZIKV antibody contains a VH sequence presented in any of SEQ ID NO: 2, 3, 4, 5 or 6, including post-translational modifications of the specified sequence. In a specific embodiment, the VH contains one, two or three HVRs selected from: (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 15 , and (c) HVR-H3, which contains the amino acid sequence SEQ ID NO: 20. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

Another aspect of the invention is an anti-ZIKV antibody that contains a light chain variable domain (VL) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical. , 99%, or 100% of the amino acid sequence presented in any of SEQ ID NO: 8, 9, or 10. In some embodiments, a VL sequence that is at least 90%, 91%, 92%, 93%, 94 identical %, 95%, 96%, 97%, 98% or 99%, contains substitutions (for example, conservative

Translation of application materials for consideration in the national replacement phase), insertion or deletion compared to the reference sequence, but the anti-ZIKV antibody containing such a sequence retains the ability to bind to ZIKV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted by deletion in any of SEQ ID NO: 8, 9, or 10. In some embodiments, the substitutions, insertions, or deletions involve regions outside the HVR (i.e. i.e. FR). Optionally, the anti-ZIKV antibody contains a VL sequence presented in any of SEQ ID NO: 8, 9 or 10, including post-translational modifications of the specified sequence. In a specific embodiment, VL contains one, two or three HVRs selected from: (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21, (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24 , and (c) HVR-L3, which contains the amino acid sequence SEQ ID NO: 27. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

Another aspect of the invention is an anti-ZIKV antibody that contains a light chain variable domain (VL) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical. , 99% or 100% of the amino acid sequence of SEQ ID NO: 10. In some embodiments, a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 identical %, 98% or 99%, contains substitutions (eg, conservative substitutions), insertions or deletions compared to the reference sequence, but an anti-ZIKV antibody containing such a sequence retains the ability to bind to ZIKV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted by deletion in SEQ ID NO: 10. In some embodiments, the substitutions, insertions, or deletions involve regions outside the HVR (ie, FR). Optionally, the anti-ZIKV antibody contains the VL sequence set forth in SEQ ID NO: 10, including post-translational modifications to said sequence. In a specific embodiment, VL contains one, two or three HVRs selected from: (a) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 21, (b) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 24 , and (c) HVR-L3, which contains the amino acid sequence SEQ ID NO: 27. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

Another object of the invention is an antibody to ZIKV, where the antibody contains VH specified in any of the above embodiments of the invention, and VL specified in any of the above embodiments of the invention. In one embodiment, the antibody comprises VH and VL sequences that appear in any of SEQ ID NOs: 2, 3, 4, 5 or any of SEQ ID NOs: 8, 9 or 10, respectively, including post-translational modifications thereof sequences. In one embodiment, the antibody comprises VH and VL sequences as set forth in any of SEQ ID NO: 2, 3, 4, 5 or SEQ ID NO: 7, respectively, including post-translational modifications to said sequences. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

Another aspect of the invention is an anti-ZIKV antibody, wherein the antibody comprises a VH as defined in any of the above embodiments and a VL as defined in any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 7, respectively, including post-translational modifications to said sequences. In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: and SEQ ID NO: 7, respectively, including post-translational modifications to said sequences. Post-translational modifications include, but are not limited to, modification by pyroglutamylation of glutamine or glutamate in the N-terminal region of the heavy chain or light chain to pyroglutamic acid.

In some embodiments, the antibody contains a heavy chain constant region (i.e., CH) sequence of SEQ ID NO: 59. In some embodiments, the antibody contains a light chain constant region (i.e., CL) sequence of SEQ ID NO: 60. In some embodiments, the antibody comprises the heavy chain constant region and light chain constant region sequences of SEQ ID NO: 59 and SEQ ID NO: 60, respectively.

In some embodiments, the antibody of the invention that binds to the Zika virus is a monoclonal antibody. In some embodiments, the antibody of the invention that binds to the Zika virus is a human, humanized, or chimeric antibody. In some embodiments, the antibody of the invention that binds to the Zika virus is a full-length IgG antibody.

In some embodiments, the Fc region of an antibody of the invention that binds to the Zika virus contains Ala at position 234 and Ala at position 235 according to EU numbering. In some embodiments, the Fc region of an antibody of the invention that binds to the Zika virus can be selected from the Fc region options set forth herein.

The invention also provides a pharmaceutical composition containing an antibody to the Zika virus according to the present invention and a pharmaceutically acceptable carrier.

Antibodies to the Zika virus according to the present invention can be used as a medicine. In some embodiments, anti-Zika virus antibodies of the present invention can be used to treat or prevent Zika virus infection.

Antibodies to the Zika virus proposed in the present invention can be used for the preparation of a medicinal product. In some embodiments, the medicament is for treating or preventing Zika virus infection.

The invention also provides a method of treating an individual having a Zika virus infection. In some embodiments, the method comprises administering to an individual an effective amount of an anti-Zika virus antibody of the present invention. In some embodiments, the method also includes administering to the individual an additional therapeutic agent, such as those described below.

In some embodiments, a variant Fc region of an antibody of the present invention that binds to the Zika virus contains at least one amino acid change in the parent Fc region. In other embodiments, the variant Fc region has substantially reduced FcyR binding activity compared to the parent Fc region. In other embodiments, the variant Fc region does not have significantly reduced C1q binding activity compared to the parent Fc region.

In some embodiments, a variant Fc region of an antibody of the present invention that binds to the Zika virus contains Ala at position 234, Ala at position 235 according to EU numbering. In other embodiments, the variant Fc region contains amino acid changes specified in any of the following positions specified in subparagraphs (a)-(c): (a) positions 267, 268 and 324, (b) positions 236, 267, 268 , 324 and 332 and (c) provisions 326 and 333; according to EU numbering.

In some embodiments, a variant Fc region of an antibody of the present invention that binds to the Zika virus contains amino acids 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.

In some embodiments, a variant Fc region of an antibody of the present invention that binds to the Zika virus also contains amino acids 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.

In some embodiments, a variant Fc region of an antibody of the present invention that binds to the Zika virus contains any of the amino acid changes, individually or in combination, that are presented in Table 4. In some embodiments, the parent Fc region specified herein invention, is derived from human IgG ₁ . The invention provides a polypeptide containing the amino acid sequence presented in any of SEQ ID NO: 51-59.

In other embodiments, an antibody of the present invention that binds to the Zika virus comprises:

(a) (I) HVR-H3 containing the amino acid sequence of SEQ ID NO: 16, (II) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27, and (III) HVR-H2 containing the amino acid sequence of SEQ ID NO: 13;

(b) (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 11, (II) HVR-H2 containing the amino acid sequence SEQ ID NO: 13, and (III) HVR-H3 containing the amino acid sequence SEQ ID NO: 16;

(c) (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 11, (II) HVR-H2 containing the amino acid sequence SEQ ID NO: 13, (III) HVR-H3 containing the amino acid sequence 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) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27, or

(d) (I) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21; (II) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24; and (III) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(e) (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 12, (II) HVR-H2 containing the amino acid sequence SEQ ID NO: 15, and (III) HVR-H3 containing the amino acid sequence SEQ ID NO: 20.

In other embodiments, an antibody of the present invention that binds to the Zika virus comprises:

(a) (I) HVR-H3 from VH sequence SEQ ID NO: 6, (II) HVR-L3 from VL sequence SEQ ID NO: 10 and (III) HVR-H2 from VH sequence SEQ ID NO: 6 ;

(b) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6 and (III) HVR-H3 from VH sequence SEQ ID NO: 6 ;

(c) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6, (III) HVR-H3 from VH sequence SEQ ID NO: 6 , (IV) HVR-L1 from VL sequence SEQ ID NO: 10; (V) HVR-L2 from the VL sequence SEQ ID NO: 10 and (VI) HVR-L3 from the VL sequence SEQ ID NO: 10;

(d) (I) HVR-L1 from VL sequence SEQ ID NO: 10, (II) HVR-L2 from VL sequence SEQ ID NO: 10 and (III) HVR-L3 from VL sequence SEQ ID NO: 10 ; or

(e) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6, (III) HVR-H3 from VH sequence SEQ ID NO: 6 , (IV) HVR-L1 from the VL sequence SEQ ID NO: 7, (V) HVR-L2 from the VL sequence SEQ ID NO: 7, and (VI) HVR-L3 from the VL sequence SEQ ID NO: 7.

In other embodiments, an antibody of the present invention that binds to the Zika virus comprises (I) a VH containing the amino acid sequence of SEQ ID NO:6, (II) a VL containing the amino acid sequence of SEQ ID NO:7, (III ) CH containing the amino acid sequence of SEQ ID NO: 59, and (IV) CL containing the amino acid sequence of SEQ ID NO: 60. In other embodiments, the antibody of the present invention that binds to the Zika virus contains a variant VH sequence , selected from the group which consists of: 3CH1047 (VH from 3Cam2-LALA+KAES+ACT5; SEQ ID NO: 6) and 3CH1049 (SEQ ID NO: 95), with the CH sequence of human IgG ₁ selected from the group which consists of: SG182 (WT; SEQ ID NO: 46), SG1095 (SEQ ID NO: 54) and SGI 106 (CH 3Cam2-LALA+KAES+ACT5; SEQ ID NO: 59); contains a VL sequence variant selected from the group consisting of: 3CL (VL from 3Cam2-LALA+KAES+ACT5; SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98), with the human CL sequence SKI (CL from 3Cam2-LALA+KAES+ACT5 SEQ ID NO: 60). The sequence variants and mutations proposed in the present invention are indicated and described in detail in tables 2 and 4.

In some examples, an antibody of the present invention that binds to the Zika virus is described, for example, in the Examples section herein. For example, the antibody described herein may be the antibody described in Example 7, such as, but not limited to, the following variants: (a) 3Cat2, which is DG3CH1047 (SEQ ID NO: 6)-SG182 ( SEQ ID NO: 46)/3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO: 60), (6) 3Cam2-LALA+KAES, which is DG3CH1047 (SEQ ID NO: 6)-SG1095 (SEQ ID NO: 54)/3CL (SEQ ID NO: 7) SKI (SEQ ID NO: 60), (c) 3Cam2-LALA+KAES+ACT5, which is DG3CH1047 (SEQ ID NO: 6)-SG1106 (SEQ ID NO: 59) /3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO: 60), (d) ZS, which is DG 3CH (SEQ ID NO: 1)-SG182 (SEQ ID NO: 46)/3CL (SEQ ID NO: 7) SKI (SEQ ID NO: 60), (e) 3C-LALA, which is DG 3CH (SEQ ID NO: 1)-SG192 (SEQ ID NO: 47)/3CL (SEQ ID NO: 7) SKI (SEQ ID NO: 60), (e) 3C-LALA+KAES+ACT5, which is DG3CH (SEQ ID NO: 1)-SG1106 (SEQ ID NO: 59)/3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO : 60), etc.

In a further aspect of the invention, the anti-DENV antibody or the anti-ZIKV antibody specified in any of the above aspects of the invention is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-DENV antibody or anti-ZIKV antibody is an antibody fragment, for example, an Fv, Fab, Fab', scFv, dimeric antibody (diabody) or E(ab') ₂ antibody fragment. In another embodiment, the antibody is a full-length antibody, for example, an intact IgG ₁ subclass antibody or an antibody of another class or isotype as defined herein.

In a further aspect, the anti-DENV antibody or anti-ZIKV antibody of the invention may have a modification that abolishes binding of the antibodies to Fc gamma receptors (FcyRs). Without being bound by theory, it is conceivable that a modification that abolishes antibody binding to Fc-gamma receptors may be valuable because reduced FcR binding may prevent ADE (antibody-dependent enhancement) of infection, which is likely due primarily to interaction with FcR . In one embodiment, the Fc region of the anti-DENV antibody of the invention contains Ala at position 234 and Ala at position 235 according to EU numbering.

In a further aspect, the anti-DENV antibody or anti-ZIKV antibody specified in any of the above embodiments may have any of the features, individually or in combination, described below in sections 1-7:

1. Antibody affinity

In some embodiments, an antibody described herein has a dissociation constant (Kd) value 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 (eg 10 ^-8 M or less, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M).

In one embodiment, the Kd value is measured using a radiolabeled antigen binding assay (RIA). In one embodiment of the invention, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the binding affinity of a Fab to an antigen is measured in solution by equilibrating the Fab with a minimum concentration of ¹²⁵ I-labeled antigen in the presence of titrated dilutions of unlabeled antigen, then capturing the bound antigen on an anti-Fab antibody-coated plate (see, e.g., Chen et al. ., J. Mol Biol 293, 1999, pp. 865-881). To provide assay conditions, MICROTITER ^® multiwell plates (Thermo Scientific) are coated overnight with 5 μg/mL anti-Fab capture antibody (Capel 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 a non-adsorbent plate (Nunc, No. 269620), 100 or 26 pM ¹²⁵ I-labeled antigen is mixed with serial dilutions of the Fab of interest (for example, the anti-VEGF antibody suitable for evaluation, Fab-12, see Presta et al. , Cancer Res. 57, 1997, pp. 4593-4599). The Fab of interest is then incubated overnight; however, incubation may be carried out for a longer period of time (eg 65 hours) to ensure that equilibrium is achieved. The mixtures are then transferred to a prepared capture plate and incubated at room temperature (eg, 1 hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20) in PBS. After drying the plates, add 150 µl/well of scintillation liquid (MICROSCINT-20™; Packard) and read the plates using a TOPCOUNT™ gamma counter (Packard) for 10 minutes. To perform competition binding assay select concentrations of each Fab that provide a level of binding less than or equal to 20% of the maximum.

In another embodiment of the invention, Kd is measured by surface plasmon resonance using a BIACORE® device. For example, the assay is performed using a BIACORE®-2000 or BIACORE®-3000 device (GE Healthcare Inc., Piscataway, NJ) at 25°C using antigen immobilized on CM5 chips with a level of immobilization corresponding to - 10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, GE Healthcare Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions . The antigen is diluted with 10 mM sodium acetate, pH 4.8 to a concentration of 5 μg/ml (~0.2 μM) before injection at a flow rate of 5 μl/min to achieve a level of immobilization corresponding to approximately 10 response units (RU) of cross-linked protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (from 0.78 to 500 nM) are injected in PBS supplemented with 0.05% polysorbate 20 (TWEEN-20) surfactant (PSRT) at 25°C with a flow rate of approximately 25 µl/min. The rates of the association reaction (k _on ) and dissociation reaction (k _off ) are calculated using a simple 1:1 Langmuir binding model (Evaluation software version 3.2, BIACORE®) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium constant of the dissociation reaction (Kd) is calculated as the ratio k _off /k _on (see, for example, Chen et al., J Mol Biol 293, 1999, pp. 865-881). If the association reaction rate exceeds 106M-1 s-1 as assessed by the surface plasmon resonance method described above, then the association reaction rate can be determined using the fluorescence quenching method, which measures the increase or decrease in fluorescence emission intensity (excitation wavelength 295 nm; emission wavelength 340 nm, bandwidth 16 nm) at 25°C of 20 nM antibody to the antigen (Fab format) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, measured using a spectrometer such as spectrophotometer equipped with a flow stop block (Aviv Instruments), or SLM-AMINCO™ 8000 series spectrophotometer (ThermoSpectronic), while mixing the contents of the cuvette.

2. Antibody fragments

In some embodiments, an antibody described herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments and other fragments described below. For a review of some antibody fragments, see Hudson et al., Nat. Med. 9, 2003, pp. 129-134. For a review of scFv fragments, see, for example, Plueckthun in: The Pharmacology of Monoclonal Antibodies, ed. Rosenburg and Moore, Springer, New York, vol. 113, 1994, cc. 269-315; see also WO 93/16185; and US Nos. 5,571,894 and 5,587,458. For discussion regarding Fab and F(ab') ₂ fragments that contain salvage receptor binding epitope residues and have an extended half-life in vivo, see US No. 5,869,046.

Dimeric antibodies are antibody fragments with two antigen-binding sites, which can be divalent or bispecific (see, for example, EP 0404097; WO 1993/01161; Hudson et al., Nat. Med. 9, 2003, pp. 129-134; and Holliger et al., Proc. Natl. Acad. Sci. USA 90, 1993, pp. 6444-6448). Trimeric and tetrameric antibodies are also described in Hudson et al., Nat. Med. 9, 2003, pp. 129-134.

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain, or all or part of the light chain variable domain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, for example, US No. 6,248,516 B1).

Antibody fragments can be created by various techniques, including, but not limited to, proteolytic cleavage of an intact antibody, and can also be produced using recombinant host cells (eg, E. coli or phage) as described herein.

3. Chimeric and humanized antibodies

In some embodiments, an antibody described herein is a chimeric antibody. Some chimeric antibodies are described, for example, in US No. 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81, 1984, ss. 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region from an antibody from mice, rats, hamsters, rabbits, or non-human primates such as monkeys) and a human constant region. In another example, a chimeric antibody is a "class switched" antibody, the class or subclass of which has been changed from the parent antibody. Chimeric antibodies include their antigen-binding fragments.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains, in which the HVRs, eg, CDRs (or regions thereof), are derived from a non-human antibody, and the FRs (or regions thereof) are derived from human antibody sequences. The humanized antibody may optionally also contain at least a portion of the human constant region. In some embodiments, certain FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which the HVR residues are derived), for example, to maintain or improve the specificity or affinity of the antibody.

Information about humanized antibodies and methods for their creation is summarized, for example, in Almagro and Fransson, Front. Biosci. 13, 2008, pp. 1619-1633, and also described in Riechmann et al., Nature 332, 1988, pp. 323-329; Queen et al., Proc. Natl. Acad. Sci. USA 86, 1989, ss. 10029-10033; US Nos. 5821337, 7527791, 6982321 and 7087409; Kashmiri et al., Methods 36, 2005, pp. 25-34 (SDR transplant description (a-CDR)); Padlan, Mol. Immunol. 28, 1991, pp. 489-498 (describing “re-coating”); Dall'Acqua et al., Methods 36, 2005, pp. 43-60 (description of "FR permutation"); and Osbourn et al., Methods 36, 2005, pp. 61-68 and Klimka et al., Br. J. Cancer 83, 2000, pp. 252-260 (describing the "directed selection" approach to FR permutation).

Human scaffolds that can be used for humanization include, but are not limited to: scaffolds selected using “best fit” techniques (see, for example, Sims et al., J. Immunol. 151, 1993, pp. 2296-2308; framework regions derived from the consensus sequence of a particular subset of the variable regions of the light and heavy chains of human antibodies (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA 89, 1992, pp. 4285 -4289 and Presta et al., J. Immunol. 151, 1993, pp. 2623-2632); human mature (with somatic mutations) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13, 2008, pp. 1619-1633); and framework regions obtained by screening FR libraries (see, for example, Vasa et al., J. Biol. Chem. 272, 1997, pp. 10678-10684 and Rosok et al., J. Biol. Chem. 271, 1996, pp. 22611-22618) 4. Human antibodies

In some embodiments, the antibody described herein is a human antibody. Human antibodies can be obtained using various techniques known in the art. Human antibodies are described in general by van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5, 2001, cc. 368-374 and Lonberg, Curr. Opin. Immunol. 20, 2008, cc. 450-459.

Human antibodies can be produced by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with human variable regions in response to challenge with the antigen. These animals typically contain all or part of the human immunoglobulin loci instead of endogenous immunoglobulin loci that are either present outside the chromosomes or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for producing human antibodies in transgenic animals, see Lonberg, Nat. Biotech. 23, 2005, pp. 1117-1125 (see, for example, also in US No. 6075181 and US No. 6150584 for a description of XENOMOUSE™ technology; in US No. 5770429 for a description of HUMAB® technology; in US No. 7041870 for a description of K-M MOUSE® technology and in the publication of a US patent application 2007/0061900 description of VELOCIMOUSE® technology). Human variable regions from intact antibodies produced using these animals can be further modified, for example, by combining with another human constant region.

Human antibodies can be created using hybridoma-based methods. Human myeloma and murine human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor, J. Immunol. 133, 1984, pp. 3001-3005; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, pp. 51-63 and Boerner et al., J. Immunol. 147, 1991, pp. 86-95). Human antibodies generated using human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103, 2006, cc. 3557-3562. Additional methods include those described, for example, in US No. 7189826 (describing the production of monoclonal human antibodies of the IgM type from hybridoma cell lines), and in Ni, X ₁ andai Mianyixue 26, 2006, pp. 265-268 (description of human-human hybridomas). The technology of human hybridomas (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology 20, 2005, cc. 927-937 and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27, 2005, cc. 185-191.

Human antibodies can also be created by isolating the variable domain sequences of Fv clones selected from phage display libraries for the creation of which human antibodies were used. These variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

5. Antibodies obtained from libraries

Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening said libraries for antibodies having the desired binding characteristics. These methods are summarized, for example, by Hoogenboom et al. in: Methods in Molecular Biology 178, 2001, cc. 1-37 (ed. O'Brien et al., Human Press, Totowa, NJ, 2001), and are also described, for example, in McCafferty et al., Nature 348, 1990, pp. 552-554; Clackson et al., Nature 352, 1991, pp. 624-628; Marks et al., J. Mol. Biol. 222, 1992, pp. 581-597; Marks and Bradbury in: Methods in Molecular Biology 248, 2003, cc. 161-175 (ed. Lo, Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338, 2004, cc. 299-310; Lee et al., J. Mol. Biol. 340, 2004, pp. 1073-1093; Fellowes, Proc. Natl. Acad. Sci. USA 101, 2004, cc. 12467-12472 and Lee et al., J. Immunol. Methods 284, 2004, cc. 119-132.

In some phage display methods, VH and VL gene populations are cloned separately by polymerase chain reaction (PCR) and recombined randomly into phage libraries, which are then screened for antigen-binding phage according to the method described in Winter et al., Ann. . Rev. Immunol. 12, 1994, pp. 433-455. Phage typically displays antibody fragments as either single-chain Fv fragments (scFv) or Fab fragments. Libraries obtained from immunized sources include antibodies with high affinity for the immunogen without the need to create hybridomas. Alternatively, it is possible to clone an untreated (“naïve”) population (e.g. from a human), obtaining a single source of antibodies to a wide range of foreign as well as self antigens without any immunization, as described in Griffiths et al., EMBO J. 12, 1993, pp. 725-734. Finally, naïve libraries can also be obtained by synthetic methods by cloning unrearranged V gene segments from stem cells and using PCR primers containing a random sequence to encode hypervariable CDR3 regions and to carry out in vitro rearrangement according to the method described in Hoogenboom and Winter, J. Mol. Biol. 227, 1992, cc. 381-388. Phage libraries of human antibodies are described, for example, in the following patent publications: US No. 5750373 and US patent application publications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/ 0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments.

6. Multispecific antibodies

In some embodiments, an antibody as defined herein is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have specificities that bind to at least two different sites. In some embodiments, one of the binding specificities provides binding to the DENV E protein and the other to any other antigen. In some embodiments, bispecific antibodies can bind to two different epitopes of the DENV E protein. Bispecific antibodies can also be used to localize cytotoxic agents in cells that express DENV E protein. Bispecific antibodies can be produced as full-length antibodies or antibody fragments.

Techniques for creating 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, 1983, pp. 537-540, WO 93/08829 and Traunecker et al., EMBO J. 10,1991, pp. 3655-3659), and “knob-in-hole” technology (providing “protrusion-in-hole” interaction, see, for example, US No. 5731168). Multispecific antibodies can also be obtained by creating controlled electrostatic forces to produce antibody molecules with heterodimeric Fc (WO 2009/089004); cross-linking of two or more antibodies or fragments (see, for example, US No. 4676980 and Brennan et al., Science 229, 1985, pp. 81-83); the use of leucine zippers for the production of bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148, 1992, pp. 1547-1553); the use of "dimeric antibody" technology to create bispecific antibody fragments (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90, 1993, pp. 6444-6448); and the use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol. 152, 1994, pp. 5368-5374); and preparing trispecific antibodies according to the method described, for example, in Tutt et al., J. Immunol. 147, 1991, pp. 60-69).

Also included within the scope of the present invention are engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, for example, US 2006/0025576 A1).

The antibody or fragment thereof may also include a “dual action Fab” or “DAF” containing an antigen binding site that binds to the DENV E protein as well as to another antigen other than it (see, for example, US 2008/0069820).

7. Antibody variants

Some embodiments of the invention relate to variants of the amino acid sequences of the antibodies presented in the present description. For example, it may be desirable to increase the binding affinity and/or other biological properties of the antibody. Variants of the amino acid sequence of an antibody can be obtained by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by synthesizing peptides. These modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions and substitutions can be used to produce the final construct, as long as the final construct has the required characteristics, such as the ability to bind antigen. a) Substitution, Insertion and Deletion Variants Some embodiments of the invention are antibody variants that have one or more amino acid substitutions. Sites of interest for replacement mutagenesis include HVR and FR. Conservative substitutions are presented below in Table 1 under the heading “preferred substitutions” and are described below with reference to amino acid side chain classes. More significant substitutions are presented below in Table 1 under the heading “exemplified substitutions” and are described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for desired activity, eg, retained/increased antigen binding capacity, decreased immunogenicity, or increased ADCC or CDC.

Amino acids can be grouped based on common side chain properties as follows:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro;

(6) aromatic: Tgr, Tyr, Phe.

Non-conservative substitutions mean replacing a representative of one of the specified classes with a representative from another class.

One type of resulting substitution variant involves replacing one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, the resulting variant(s) selected for additional study must have modifications (eg, improvements) in some biological properties (eg, increased affinity, decreased immunogenicity) relative to the parent antibody, and /or must have substantially retained certain biological properties of the parent antibody. An example of a resulting replacement variant is an affinity matured antibody, which can be generated, for example, using phage display-based affinity maturation technologies, such as those described herein. In general, the method is as follows: one or more HVR residues are mutated and antibody variants are displayed on phage and screened for specific biological activity (eg, binding affinity).

Changes (eg, substitutions) can be made in the HVR, for example, to increase the affinity of the antibody. These changes can be made in “hot (active) spots” in HVR, i.e. residues encoded by codons that undergo mutation at a high rate during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207, 2008, pp. 179-196), and/or in residues that contact the antigen, followed by testing the resulting VH or VL variant for binding affinity. Affinity maturation by generation and re-selection from secondary libraries is described, for example, by Hoogenboom et al. in: Methods in Molecular Biology 178, 2002, cc. 1-37 (ed. O'Brien et al., Human Press, Totowa, NJ, 2001). In some embodiments of the affinity maturation process, diversity is introduced into the variable region genes selected for maturation using any of a wide variety of methods (eg, reduced fidelity PCR, strand shuffling, or site-directed mutagenesis using oligonucleotides). Then a secondary library is created. The library is then screened to identify any antibody variants with the required affinity. Another method used to introduce diversity involves HVR-targeting approaches in which multiple HVR residues are randomized (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Often the targets are primarily CDR-H3 and CDR-L3.

In some embodiments, substitutions, insertions, or deletions may affect one or more HVRs as long as the changes do not significantly reduce the ability of the antibody to bind the antigen. For example, conservative changes can be made to HVR (eg, conservative substitutions as described herein) that do not significantly reduce binding affinity. These changes, for example, may be outside the HVR residues involved in contact with the antigen. In some embodiments, in the VH and VL variant sequences noted above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A valuable method for identifying residues or regions of an antibody that can be mutagenized is the so-called “alanine scanning mutagenesis” described in Cunningham and Wells, Science 244, 1989, pp. 1081-1085. In this method, a target residue or group of residues (eg, charged residues such as arg, asp, his, lys and glu) is identified and replaced with a neutral or negatively charged amino acid (eg, alanine or polyalanine) to determine whether whether this will affect the interaction of the antibody with the antigen. Additional substitutions can be introduced at those amino acid positions for which functional sensitivity to the initial substitutions has been demonstrated.

Alternatively or additionally, the crystal structure of the antigen-antibody complex can be analyzed to identify points of contact between the antibody and the antigen. These contacting residues and adjacent residues can be considered as targets or excluded from consideration as candidates for replacement. Variants can be screened to determine whether they have the desired properties.

Insertions into amino acid sequences include amino- and/or carboxy-terminal fusions ranging in length from a single residue to polypeptides containing a hundred or more residues, as well as insertions of one or more amino acid residues within a sequence. An example of the result of terminal insertions is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion of the N- or C-termini of the antibody with an enzyme (for example, for ADEPT (antibody-directed enzyme prodrug therapy) or polypeptide that extends the half-life of the antibody in plasma.

b) Glycosylation options

In some embodiments, an antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites in an antibody can be easily accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

If the antibody contains an Fc region, the carbohydrate attached to it can be modified. Native antibodies that are produced by mammalian cells typically contain a branched biantennary oligosaccharide, which is typically N-linked to Asn297 of the CH2 domain of the Fc region (see, for example, Wright et al., TIBTECH 15, 1997 , pp. 26-32). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications to the oligosaccharide in the antibody of the invention can be made to create antibody variants with certain improved properties.

One embodiment of the invention is antibody variants having a carbohydrate structure in which the content of fucose attached (directly or indirectly) to the Fc region is reduced. For example, the fucose content in said Fc region may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. Fucose content is determined by calculating the average fucose content of the sugar chain on Asn297 relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry according to the method described, for example, in WO 2008/077546. Asn297 denotes an asparagine residue present at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor variations in the antibody sequence, Asn297 can also be located at a position approximately ±3 amino acids forward or reverse from position 297, i.e. between positions 294 and 300. These fucosylated variants may have improved ADCC function (see, for example, US 2003/0157108 (Presta L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications regarding "defucosylated" antibody variants or "fucose deficient" antibody variants are: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/013214 0; 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 2 002/031140; Okazaki et al. J. Mol. Biol. 336, 2004, pp. 1239-1249; Yamane-Ohnuki et al., Biotech. Bioeng. 87, 2004, pp. 614-622). Examples of cell lines that have the ability to produce defucosylated antibodies are Lecl3 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249, 1986, pp. 533-545; US 2003/0157108 (to Presta L.) and WO 2004/056312 (to Adams et al., see first example 11), and gene-silenced cell lines, for example, CHO cells with the alpha-1,6-gene knocked out fucosyltransferase, FUT8 (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87, 2004, pp. 614-622); Kanda et al., Biotechnol. Bioeng., 94(4), 2006, pp. 680-688; and WO 2003/085107).

In addition, variants of antibodies with bisectional oligosaccharides are described, for example, in which the biantennary oligosaccharide attached to the Fc region of the antibody is bisectioned by GlcNAc. These antibody variants may differ in reduced fucosylation and/or increased ADCC function. Examples of these antibody variants are described, for example, in WO 2003/011878 (to Jean-Mairet et al.); US No. 6602684 (to Umana et al.) and US 2005/0123546 (to Umana et al.). Variants of antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region are also proposed. These antibody variants may have increased CDC function. These antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (to Raju S.); and WO 1999/22764 (to Raju S.).

c) Variants of the Fc region

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby creating a variant Fc region. The Fc region variant may comprise a human Fc region sequence (eg, human IgG1 IgG2, IgG3, or IgG4 Fc region) including an amino acid modification (eg, substitution) at one or more amino acid positions.

Some embodiments of the invention provide an antibody variant that has some, but not all, effector functions, making it a promising candidate for applications where the in vivo half-life of the antibody is important, but some effector functions (eg, ADCC) are not essential. or are harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm changes in CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody has the ability to bind to Fc-gamma R (and therefore is likely to have ADCC activity) and the ability to bind to FcRn. Primary cells mediating ADCC, i.e. NK cells express only Fc-gamma RIII, while monocytes express Fc-gamma RI, Fc-gamma RII and Fc-gamma RIII. Data on FcR expression on hematopoietic cells are summarized in Table 3 on p. 464 from Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, cc. 457-492. Examples of in vitro assays for ADCC activity of a molecule of interest include, but are not limited to, the assays described in US No. 5,500,362 (see, for example, Hellstrom et al., Proc. Natl. Acad. Sci. USA 83, 1986, cc 7059-7063 and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82, 1985, pp. 1499-1502); US No. 5821337 (see Bruggemann et al., J. Exp. Med. 166, 1987, cc. 1351-1361). Alternatively, non-radioactive assays may be used (see, for example, the ACT1™ Flow Cytometry-Based Non-Radioactive Cytotoxicity Assay (CellTechnology, Inc. Mountain View, Calif.); and the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, CA) Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model, for example, as described in Clynes et al., Proc. Natl. Acad. Sci. USA 95, 1998, pp. 652-656. C1q binding assays can also be performed to confirm that the antibody can bind to C1q and therefore has CDC- activity (see, for example, the description of ELISA for assessing the binding of C1q and C3c 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 202, 1996, pp. 163-171; Cragg et al., Blood 101, 2003, pp. 1045-1052; and Cragg, Blood 103, 2004, pp. 2738-2743). Determination of FcRn binding and clearance/half-life in vivo can also be carried out using methods known in the art (see, for example, Petkova et al., Int'l. Immunol. 18(12), 2006, pp. 1759- 1769).

Antibodies with modified effector function include antibodies with replacement of one or more of the following residues of the Fc region, such as 238, 265, 269, 270, 297, 327 and 329 (see, for example, US No. 6,737,056). These Fc mutants include Fc mutants with substitutions at two or more of the following amino acid positions: 265, 269, 270, 297 and 327, including the so-called “DANA” mutant of the Fc region, carrying a substitution of residues 265 and 297 to alanine (US No. 7332581).

Several variants of antibodies with altered ability to bind to FcR have been described (see, for example, US No. 6737056; WO 2004/056312 and Shields et al., J. Biol. Chem. 276, 2001, pp. 6591-6604).

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that alter ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU residue numbering).

In some embodiments, changes are made to the Fc region that result in altered (i.e., either increased or decreased) C1q binding ability and/or complement dependent cytotoxicity (CDC), as described, for example, in US No. 6,194,551, WO 1999/51642 and Idusogie et al., J. Immunol. 164, 2000, pp. 4178-4184.

Antibodies with an extended half-life and increased ability to bind to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the embryo (Guyer et al., J. Immunol. 117, 1976, pp. 587-593 and Kim et al. , J. Immunol. 24, 1994, pp. 2429-2434), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that increase the binding of the Fc region to FcRn. These Fc region variants include variants with substitutions of one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311,312, 317,340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, substitution of residue 434 in the Fc region (US No. 7371826).

Additional examples regarding other Fc region variants are also described in Duncan, Nature 322, 1988, pp. 738-740; in US No. 5648260; US No. 5624821 and WO 1994/29351.

In another embodiment, the antibody may contain a variant of the Fc region proposed in the present invention, described in more detail below.

d) Antibody variants constructed using cysteine

In some embodiments, it may be desirable to create antibodies constructed using cysteine, for example, "thioMAbs", in which one or more residues in the antibody are replaced with cysteine residues. In certain embodiments of the invention, the replaced residues are at accessible sites in the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or drug linker moieties, creating the immunoconjugate indicated below in this description. In some embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Cabot numbering) light chain; A118 (EU numbering) of the heavy chain and S400 (EU numbering) of the Fc region of the heavy chain. Antibodies constructed using cysteine can be created according to the method, for example, described in US No. 7521541.

e) Antibody derivatives

In some embodiments, the antibody provided herein can be further modified to contain additional non-protein fragments known in the art and readily available. Fragments suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene copolymer /maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have an advantage in production due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary and, if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for derivatization can be determined based on, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is intended to be used in therapy under certain conditions, and etc.

Another embodiment of the invention are conjugates of an antibody and a non-protein fragment that can be selectively heated by exposing it to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102, 2005, pp. 11600-11605). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells, but which heat the non-protein moiety to a temperature that kills the cells closest to the antibody-non-protein moiety conjugate.

One object of the invention is an isolated polypeptide containing a variant of the Fc region having significantly reduced FcγR-binding activity. One object of the invention is an isolated polypeptide containing a variant of the Fc region that does not have significantly reduced C1q-binding activity. One object of the invention is an isolated polypeptide containing a variant of the Fc region that has significantly reduced FcγR binding activity, and which does not have significantly reduced C1q binding activity. In some aspects of the invention, the polypeptide is an antibody. In some aspects of the invention, the polypeptide is an Fc protein (Fc fusion protein). In some embodiments, the Fc region variant comprises at least one amino acid residue change (e.g., substitution) relative to the corresponding native Fc region sequence or reference variant sequence (in some cases referred to generally as the "parent" Fc region ). In some embodiments, the variant Fc region of the invention has substantially reduced FcγR binding activity compared to the parent Fc region. In some embodiments, the variant Fc region of the invention does not have significantly reduced C1q binding activity compared to the parent Fc region. In some embodiments, the FcγR is a human FcγR, a monkey FcγR (eg, a cynomolgus monkey, rhesus monkey, marmoset, chimpanzee, or baboon FcγR), or a mouse FcγR.

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

In one aspect, the variant Fc region of the invention has substantially reduced binding activity to one or more murine FcγRs, including but not limited to FcγRI, FcγRIIb, FcγRIII, and FcγRIV, compared to the parent Fc region . In another aspect of the invention, the variant Fc region of the invention has significantly reduced binding activity for murine FcγRI, FcγRIIb, FcγRIII and FcγRIV compared to the parent Fc region.

"Fcγ receptors" (referred to herein as Fcγ receptors, FcγR or FcgR) are receptors that can bind to the Fc region of monoclonal antibodies of the IgG1, IgG2, IgG3 and IgG4 isotypes, and are primarily any member of the protein family , encoded by Fcγ receptor genes. In humans, this family includes, but is not limited to, FcγRI (CD64), including the FcγRIa, FcγRIb, and FcγRIc isoforms; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 (type H) and R131 (type R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including FcγRIIIa isoforms (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and any human FcγR, isoforms or allotypes of FcγR that have not yet been discovered. FcγRIIb 1 and FcγRIIb2 are described as splice variants of human FcγRIIb. In addition, a splice variant designated FcγRIIb3 has been described (J Exp Med, 170, 1989, pp. 1369-1385). In addition to these splice variants, human FcγRIIb includes all splice variants reported in NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992.3. In addition, human FcγRIIb includes each of the previously described gene polymorphisms as well as FcγRIIb (Arthritis Rheum. 48, 2003, pp. 3242-3252; Kono et al., Hum. Mol. Genet. 14, 2005, pp. 2881-2892 and Kyogoju et al., Arthritis Rheum. 46, 2002, pp. 1242-1254), and each gene polymorphism that will be described in the future.

There are two allotypes of FcγRIIa, one in which the amino acid at position 167 of FcγRIIa is a histidine (type H) and one in which the amino acid at position 167 is replaced by arginine (type R) (Warrmerdam, J. Exp. Med. 172, 1990, pp. 19-25).

FcγR includes FcγRs having origin in humans, mice, rats, rabbits and monkeys (but is not limited to these), and it can be originating from any organism. Murine FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2) and any mouse FcγR or FcγR isoforms.

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

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

In one aspect of the invention, the Fc region variant of the invention has significantly reduced FcγR binding activity, which is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less 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% of the FcγR binding activity of the parent Fc region. In one aspect of the invention, the Fc region variant of the invention has significantly reduced FcγR binding activity, which means that the ratio [difference in RU values on sensorgrams that is detected before and after interaction of the FcγR with the Fc region variant]/[ the difference in RU values on sensorgrams, which was detected before and after the “capture” of FcγR by sensor chips] 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.

In one aspect of the invention, the Fc region variant of the invention does not have significantly reduced C1q binding activity, meaning that the difference in C1q binding activity between the Fc region variant and the parent Fc region of the invention is less than 50%, less than 45%, less than 40%, less than n 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% compared with the C1q-binding activity of the parental Fc region.

One object of the invention is an isolated polypeptide containing a variant of the Fc region with significantly reduced ADCC activity. One object of the invention is an isolated polypeptide containing a variant of the Fc region that does not have significantly reduced CDC activity. One object of the invention is an isolated polypeptide containing a variant of the Fc region with significantly reduced ADCC activity and not having significantly reduced CDC activity.

In one aspect of the invention, the Fc region variant of the invention has a substantially reduced ADCC activity of 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% of the ADCC activity of the parent Fc region.

In one aspect of the invention, the Fc region variant of the invention does not have significantly reduced CDC activity, meaning that the difference in CDC activity between the Fc region variant and the parent Fc region of the invention is less than 50% , less than 45%, less than 40%, less than n 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% compared to CDC -activity of the parent Fc region.

One object of the invention is an isolated polypeptide that contains a variant Fc region that has substantially reduced FcγR binding activity and does not have significantly reduced C1q binding activity compared to the polypeptide containing the parent Fc region. In other aspects of the invention, the polypeptide of the invention contains at least one amino acid change 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.

In one aspect of the invention, a variant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity comprises Ala at position 234, Ala at position 235, and at least one amino acid change 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.

In one aspect of the invention, a variant Fc region having significantly reduced FcγR binding activity and not having significantly reduced C1q binding activity contains Ala at position 234, Ala at position 235, and additional amino acid changes specified in one of the following subclauses (a )-(c): (a) provisions 267, 268 and 324; (b) provisions 236, 267, 268, 324 and 332; and (c) provisions 326 and 333 according to EU numbering.

In a further aspect of the invention, a variant Fc region having substantially reduced FcγR binding activity and not having substantially reduced C1q binding activity contains amino acids 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.

In one aspect of the invention, a variant Fc region having significantly reduced FcγR-binding activity and not having significantly reduced C1q-binding activity contains the amino acids: Ala at position 234, Ala at position 235, Ala at position 326 and Ser at position 333 according to EU numbering. In one aspect of the invention, a variant Fc region having significantly reduced FcγR-binding activity and not having significantly reduced C1q-binding activity contains the amino acids: Ala at position 234, Ala at position 235, Asp at position 326 and Ser at position 333 according to EU numbering. In one aspect of the invention, a variant Fc region having significantly reduced FcγR-binding activity and not having significantly reduced C1q-binding activity contains the amino acids: Ala at position 234, Ala at position 235, Glu at position 326 and Ser at position 333 according to EU numbering. In one aspect of the invention, a variant Fc region having significantly reduced FcγR-binding activity and not having significantly reduced C1q-binding activity contains the amino acids: Ala at position 234, Ala at position 235, Met at position 326 and Ser at position 333 according to EU numbering. In one aspect of the invention, a variant Fc region having significantly reduced FcγR-binding activity and not having significantly reduced C1q-binding activity contains the amino acids: Ala at position 234, Ala at position 235, Trp at position 326 and Ser at position 333 according to EU numbering.

According to another aspect of the invention, the Fc region variant of the invention may further comprise at least one amino acid change at at least one position selected from the group consisting of: 428, 434, 436, 438 and 440 according to EU numbering .

In a further aspect of the invention, the variant Fc region may further comprise amino acids 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 (see also WO 2016/125495 for a description of the relationship between amino acid changes and FcRn binding activity of an Fc region variant ).

In another aspect of the invention, the variant Fc region proposed in the invention contains the amino acids: 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 according to EU numbering. In another aspect of the invention, the variant Fc region proposed in the invention contains the amino acids: 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 according to EU numbering.

In one aspect of the invention, it is preferred that the Fc region variant of the invention does not have significantly increased FcRn binding activity, especially at pH 7.4, compared to the parent Fc region.

"FcRn" is a structural analogue of major histocompatibility complex (MHC) class I polypeptides and is characterized by 22-29% sequence identity with MHC class I molecules. FcRn is expressed as a heterodimer consisting of a soluble β- or light chain (β2-microglobulin) in complex with the transmembrane α- or heavy chain. Like MHC, the FcRn α chain contains three extracellular domains (α1, α2, and α3) and is attached to the cell surface through its short cytoplasmic domain. The α1 and α2 domains interact with the FcRn-binding domain of the Fc region of the antibody. Polynucleotide and amino acid sequences of human FcRn can be obtained, for example, from the precursors registered under numbers NM_004107.4 and NP_004098.1 (which contain a signal sequence), respectively.

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

According to one aspect of the invention, it is preferable that the Fc region variant proposed in the invention does not have significantly increased FcRn-binding activity, especially at pH 7.4, which would be less than 1000 times, less than 500 times, less less than 200 times, less than 100 times, less than 90 times, less than 80 times, less than 70 times, less than 60 times, less than 50 times, less than 40 times, 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 the FcRn-binding activity of the parent Fc region. According to one aspect of the invention, the variant Fc region proposed in the invention does not have significantly increased FcRn-binding activity, especially at pH 7.4, which means that the ratio [difference in RU values in the sensorgrams that was detected before and after the interaction FcRn with Fc-region variant]/[difference in RU values on sensorgrams, which was detected before and after “capture” of FcRn by sensor chips] 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.

In another aspect of the invention, the Fc region variant of the invention contains any of the amino acid changes, individually or in combination, that are presented in Table 4. In another aspect of the invention, the Fc region variant of the invention contains any of the amino acid changes presented in Table 4. in table 4. Another object of the invention is a polypeptide containing the amino acid sequence presented in any of SEQ ID NO: 51-59.

In some embodiments, the polypeptide that contains a variant Fc region of the present invention is an antibody heavy chain constant region. In some embodiments, the polypeptide; which contains the Fc region variant of the present invention further comprises an antigen binding domain. In another embodiment of the invention, the polypeptide is an antibody heavy chain. In a further embodiment of the invention, the polypeptide is an antibody. In some embodiments, the antibody is a chimeric antibody or a humanized antibody. The origin of the antibody is not limited to a specific origin, but examples include human antibody, mouse antibody, rat antibody and rabbit antibody. In another embodiment of the invention, the polypeptide is an Fc fusion protein.

Two or more polypeptides that contain an Fc region variant as defined herein can be included in a single molecule, wherein the two polypeptides that contain Fc region variants are in an association that closely resembles that in an antibody.

The type of antibody is not limited to a particular type, and IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) and IgM or the like can be used.

In some embodiments, the polypeptide that contains the Fc region variant of the present invention is an antibody. In other embodiments, the polypeptide that contains the Fc region variant of the present invention is an antiviral antibody.

In other embodiments, a polypeptide that contains a variant Fc region of the present invention contains an antibody variable region that contains:

(a) (I) HVR-H3 containing the amino acid sequence SEQ ID NO: 16, (II) HVR-L3 containing the amino acid sequence SEQ ID NO: 27, and (III) HVR-H2 containing the amino acid sequence SEQ ID NO: 13;

(b) (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 11, (II) HVR-H2 containing the amino acid sequence SEQ ID NO: 13, and (III) HVR-H3 containing the amino acid sequence SEQ ID NO: 16;

(c) (I) HVR-H1 containing the amino acid sequence SEQ ID NO: 11, (II) HVR-H2 containing the amino acid sequence SEQ ID NO: 13, (III) HVR-H3 containing the amino acid sequence 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) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27, or

(d) (I) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21; (II) HVR-L2 containing the amino acid sequence of SEQ ID NO: 24; and (III) HVR-L3 containing the amino acid sequence of SEQ ID NO: 27.

In other embodiments, a polypeptide that contains a variant Fc region of the present invention contains an antibody variable region that contains:

(a) (I) HVR-H3 from VH sequence SEQ ID NO: 6, (II) HVR-L3 from VL sequence SEQ ID NO: 10 and (III) HVR-H2 from VH sequence SEQ ID NO: 6 ;

(b) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6 and (III) HVR-H3 from VH sequence SEQ ID NO: 6 ;

(c) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6, (III) HVR-H3 from VH sequence SEQ ID NO: 6 , (IV) HVR-L1 from VL sequence SEQ ID NO: 10; (V) HVR-L2 from the VL sequence SEQ ID NO: 10 and (VI) HVR-L3 from the VL sequence SEQ ID NO: 10;

(d) (I) HVR-L1 from VL sequence SEQ ID NO: 10; (II) HVR-L2 from the VL sequence SEQ ID NO: 10 and (III) HVR-L3 from the VL sequence SEQ ID NO: 10; or

(e) (I) HVR-H1 from VH sequence SEQ ID NO: 6, (II) HVR-H2 from VH sequence SEQ ID NO: 6, (III) HVR-H3 from VH sequence SEQ ID NO: 6 , (IV) HVR-L1 from VL sequence SEQ ID NO: 7; (V) HVR-L2 from the VL sequence SEQ ID NO: 7 and (VI) HVR-L3 from the VL sequence SEQ ID NO: 7.

In other embodiments, a polypeptide that comprises a 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 other embodiments The invention is an antibody that contains a VH region containing the amino acid sequence SEQ ID NO: 1, and a variant Fc region containing the amino acid sequence SEQ ID NO: 54, in the heavy chain, and a VL region containing the amino acid sequence SEQ ID NO: 7, in light chain. In other embodiments, the invention is an antibody that contains a VH region containing the amino acid sequence of SEQ ID NO: 1, and a variant Fc region containing the amino acid sequence of SEQ ID NO: 58, in the heavy chain, and a VL region containing the amino acid sequence of SEQ ID NO: 7, light chain. In other embodiments, the invention is an antibody that contains a VH region containing the amino acid sequence of SEQ ID NO: 1, and a variant Fc region containing the amino acid sequence of SEQ ID NO: 59, in the heavy chain, and a VL region containing the amino acid sequence of SEQ ID NO: 7, light chain.

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

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

As used herein, the term “parental Fc region” refers to the Fc region prior to the introduction thereof of the amino acid change(s) specified herein. Preferred examples of the parent Fc region are Fc regions from native antibodies. Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) and IgM or the like. Antibodies may be of human or monkey origin (eg, cynomolgus, rhesus monkeys, marmosets, chimpanzees, or baboons). Native antibodies may also include naturally occurring mutations. A variety of IgG allotype sequences associated with genetic polymorphisms are described in “Sequences proteins of immunological interest,” NIH publication No. 91-3242, and any of them can be used in the present invention. In particular, in the case of human IgG1, the amino acid sequence at positions 356-358 (EU numbering) can be either DEL or EEM. Preferable examples of the parent Fc region include Fc regions from the heavy chain constant region 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 a parent Fc region is the Fc region from the SG1 heavy chain constant region (SEQ ID NO: 87). Another preferred example of a parent Fc region is the Fc region from the heavy chain constant region of SG182 (SEQ ID NO: 46). In addition, the parent Fc region may be an Fc region obtained by adding amino acid change(s) different from the amino acid change(s) specified herein for the Fc- regions from the native antibody.

In addition, amino acid changes that are made for other purpose(s) can be combined in a variant Fc region as defined herein. For example, amino acid substitutions can be added that increase FcRn binding activity (Hinton et al., J. Immunol. 176(1), 2006, pp. 346-356; Dall'Acqua et al., J. Biol. Chem. 281( 33), 2006, pp. 23514-23524; Petkova et al., Intl. Immunol. 18(12), 2006, pp. 1759-1769; Zalevsky et al., Nat. Biotechnol. 28(2), 2010, cc 157-159; WO 2006/019447; WO 2006/053301; and WO 2009/086320), and amino acid substitutions that increase the heterogeneity or stability of the antibody (WO 2009/041613). Alternatively, polypeptides having the ability to enhance antigen clearance, which are described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides having the ability to specifically bind to a target tissue, which are described in WO 2013 /180200, polypeptides having the ability to rebind to multiple antigen molecules, which are described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, can be combined with a variant of the Fc region specified in the present description. Alternatively, for the purpose of imparting the ability to bind to other antigens, the amino acid changes described in EP 1752471 and EP 1772465 can be combined in the CH3 region of the variant Fc region specified herein. Alternatively, in order to increase plasma retention, amino acid changes that lower the pI of the constant region (WO 2012/016227) can be combined in the Fc region variant described herein. Alternatively, in order to increase cellular uptake, amino acid changes that increase the pI of the constant region (WO 2014/145159) can be combined in the Fc region variant described herein. Alternatively, amino acid changes that increase the pI of the constant region (WO 2016/125495 and WO 2016/098357) can be combined in the Fc region variant described herein to enhance elimination of the target molecule from plasma.

Amino acid changes that increase binding activity to human FcRn at acidic pH values can also be combined in the Fc region variant described herein. In particular, said changes may include, for example, a replacement with Leu Met at position 428 and a replacement with Ser Asn at position 434 according to EU numbering (Nat Biotechnol, 28, 2010, pp. 157-159); replacement with Ala Asn at position 434 (Drug Metab Dispos, 38(4), April 2010, pp. 600-605); a Tyr Met substitution at position 252, a Thr Ser substitution at position 254, and a Glu Thr substitution at position 256 (J Biol Chem, 281, 2006, pp. 23514-23524); replacement with Gln Thr at position 250 and replacement with Leu Met at position 428 (J Immunol, 176(1), 2006, pp. 346-356); substitution to His Asn at position 434 (Clin Pharmacol Ther, 89(2), 2011, pp. 283-290), and changes described in WO 2010/106180, WO 2010/045193, WO 2009/058492, WO 2008/022152 , WO 2006/050166, WO 2006/053301, WO 2006/031370, WO 2005/123780, WO 2005/047327, WO 2005/037867, WO 2004/035752, WO 2002/060919 or etc. n. In another embodiment of the invention such changes may include, for example, at least one change selected from the group consisting of a change to Leu Met at position 428, a change to Ala Asn at position 434, and a change to Thr Tyr at position 436. Such changes may also include a change to Arg Gln at position 438 and/or replacement with Glu Ser at position 440 (WO 2016/125495).

According to the present invention, an amino acid change means any substitution, deletion, addition, insertion and modification, or a combination thereof. According to the present invention, an amino acid change can be considered an amino acid mutation.

Amino acid changes are obtained using various methods known to those skilled in the art. These methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh et al., Gene 152, 1995, pp. 271-275; Zoller, Meth. Enzymol. 100, 1983, pp. 468-500; Kramer and al., Nucleic Acids Res. 12, 1984, pp. 9441-9456; Kramer and Fritz, Methods Enzymol. 154, 1987, pp. 350-367 and Kunkel, Proc. Natl. Acad. Sci. USA 82, 1985, pp. 488-492), a method based on PCR mutagenesis and cassette mutagenesis.

The number of amino acid changes introduced into the Fc region is not limited. In some embodiments, it may be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less or 20 or less.

In addition, the polypeptide that contains the Fc region variant of the present invention can be modified chemically with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Methods for such chemical modification of a polypeptide are known in the art.

In some embodiments, the polypeptide that contains the Fc region variant of the present invention is an antibody or Fc fusion protein containing domain(s) that can bind to any antigen. Examples of antigens to which these antibodies and Fc fusion proteins can bind include, but are not limited to, ligands (cytokines, chemokines, etc.), receptors, cancer antigens, viral antigens, MHC antigens, differentiation antigens, immunoglobulins and immune complexes and immune complexes, of which immunoglobulins are a part.

B. Recombination and composition methods

Antibodies can be produced using recombination and composition methods, for example, described in US No. 4816567. One embodiment of the invention is an isolated nucleic acid encoding a DENV antibody as provided herein. In one embodiment, the anti-DENV antibody is cross-reactive with ZIKV. Another embodiment of the invention is an isolated nucleic acid that encodes a polypeptide containing a variant Fc region or a parent Fc region as specified herein. Said nucleic acid may encode an amino acid sequence containing a VL and/or an amino acid sequence containing a VH of an antibody (eg, antibody light and/or heavy chains). Another embodiment of the invention is one or more vectors (eg, expression vectors) that contain said nucleic acid. Another embodiment of the invention is a host cell that contains said nucleic acid. In one of these embodiments, the host cell contains (for example, as a result of transformation with these vectors): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing a VL antibody and an amino acid sequence containing a VH antibody, or (2 ) a first vector containing a nucleic acid that encodes an amino acid sequence containing a VL antibody, and a second vector containing a nucleic acid that encodes an amino acid sequence containing a VH antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, a Y0, NS0, Sp20 cell). One embodiment of the invention is a method of producing an antibody to DENV, comprising culturing a host cell containing a nucleic acid that encodes the antibody described above under conditions suitable for expression of the antibody, and optionally isolating the antibody from the host cell (or culture medium host cell). Another embodiment of the invention is a method of producing a polypeptide that contains a variant Fc region or a parent Fc region, comprising culturing a host cell that contains nucleic acid(s) encoding the polypeptide, such as an antibody, Fc- region or variant of the Fc region as defined above, under conditions suitable for expressing the polypeptide, and optionally isolating the polypeptide from the host cell (or host cell culture medium).

To recombinantly produce an anti-DENV antibody, nucleic acids encoding the antibody, such as those described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. To recombinantly produce the Fc region, the 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. The nucleic acid can be easily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that have the ability to specifically bind to the genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc-linked effector function are not required. For information on the expression of antibody fragments and polypeptides in bacteria, see, for example, US No. 5648237, 5789199 and 5840523 (see also Charlton in: Methods in Molecular Biology, ed. Lo V.K.S., Humana Press, Totowa, NJ, v. 248, 2003, pp. 245-254 description of the expression of antibody fragments in E. coli.) Once expressed, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.

In addition to prokaryotic organisms, eukaryotic microorganisms such as filamentous fungi or yeast can be used as hosts suitable for cloning or expression of vectors that encode antibodies, including strains of fungi and yeast whose glycosylation pathways have been “humanized”, allowing the production of the antibody with partial or a completely human glycosylation pattern (see Gerngross, Nat. Biotech. 22, 2004, pp. 1409-1414 and Li et al., Nat. Biotech. 24, 2006, pp. 210-215).

Host cells that can be used to express a glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells are insect cells, and plant cells can also be used. Numerous strains of baculoviruses and corresponding insect cells suitable for them as hosts have been identified, primarily for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts (see, for example, US Nos. 5959177, 6040498, 6420548, 7125978 and 6417429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to growth in suspension can be used. Other examples of suitable mammalian host cell lines include the OB40-transformed monkey kidney cell line CV1 (COS-7); a human embryonic kidney cell line (HEK 293 cells or line 293 cells, described, for example, in Graham et al., J. Gen. Virol., 36, 1977, pp. 59); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, described, for example, in Mather, Biol. Reprod., 23, 1980, pp. 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76,); human cervical carcinoma (HELA) cells; canine kidney cells (MDCK); bovine rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells described, for example, by Mather et al., Annals NY Acad. Sci., 383, 1982, pp. 44-68); MRC 5 cells and FS4 cells. Other valuable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFRα ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 77, 1980, pp. 4216); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of specific mammalian host cell lines that can be used to produce antibodies, see, for example, Yazaki and Wu, in Methods in Molecular Biology, eds. V.K.S. Lo, Humana Press, Totowa, NJ, vol. 248, 2003, pp. 255-268.

Polyclonal antibodies are preferably produced in animals after multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It is possible to conjugate the relevant antigen to a protein immunogenic for the species to be immunized, for example, snail hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), N -hydroxysuccinimide (conjugation via lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ or R ¹ N=C=NR, where R and R ¹ denote different alkyl groups.

Animals (typically non-human mammals) are immunized with the antigen, immunogenic conjugates or derivatives by combining, for example, 100 or 5 μg of the protein or conjugate (for rabbits and mice, respectively) with 3 volumes of Freund's complete adjuvant, and administering the solution intradermally into several places. After 1 month, the animals are subjected to a booster vaccination (re-vaccination) using 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at several sites. After 7-14 days, animal blood samples are obtained and the antibody titer in the serum is assessed. Animals are revaccinated until the titer reaches a plateau. Preferably, animals are revaccinated using a conjugate that includes the same antigen but conjugated to a different protein and/or via a different cross-linking reagent. Conjugates can also be produced in recombinant cell culture in the form of protein fusions. In addition, aggregating agents such as alum can be used to enhance the immune response.

Monoclonal antibodies are obtained from a population of practically homogeneous antibodies, i.e. The individual antibodies within a population are identical except for possible naturally occurring mutations and/or post-translational modifications (eg, isomerization, amidation), which may be present in minor quantities. The adjective “monoclonal” indicates that the antibody is not present in a mixture of different antibodies.

For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature 256(5517), 1975, pp. 495-497. In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized according to the method described above to produce lymphocytes that produce or have the ability to produce antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent will generally comprise an antigenic protein or a fusion thereof. Typically, either peripheral blood lymphocytes (PBLs) are used if cells of human origin are required, or spleen cells or lymph node cells are used if the sources are non-human mammals. The lymphocytes are then fused to the immortalized cell line using a suitable fusion agent such as polyethylene glycol to produce a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, pp. 59-103).

Immortalized cell lines are typically transformed mammalian cells, primarily rodent, bovine and human myeloma cells. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the culture medium for hybridomas, as a rule, should include hypoxanthine, aminopterin and thymidine (HAT medium), i.e. substances that inhibit the growth of HGPRT-deficient cells.

Preferred immortalized myeloma cells are cells that can be efficiently fused, maintain a sustained high level of antibody production by the selected antibody-producing cells, and are sensitive to a medium, such as HAT medium. Among them, preferred are murine myeloma lines, such as those derived from murine tumors MOPC-21 and MPC-11, which are available from Salk Institute Cell Distribution Center, San Diego, NY. California, USA, and SP-2 cells (and their derivatives, e.g. X63-Ag8-653), which are available from the American Type Culture Collection, Manassas, CA. Virginia, USA. Human myeloma and murine-human heteromyeloma cell lines that can be used for the production of human monoclonal antibodies have also been described (Kozbor et al., J. Immunol. 133(6), 1984, pp. 3001-3005; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, pp. 51-63).

The culture medium in which hybridoma cells are grown is assessed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined using immunoprecipitation or an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). These techniques and assays are known in the art. For example, binding affinity can be determined using the Scatchard assay described in Munson, Anal. Biochem. 107(1), 1980, cc. 220-239.

Once hybridoma cells have been determined to produce antibodies of the required specificity, affinity, and/or activity, clones can be subcloned using serial dilution procedures and grown using standard methods (Coding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 media. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

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

The Fc region can be obtained by re-eluting the fraction adsorbed on the Protein A column after partial digestion of monoclonal antibodies in the form of IgG1, IgG2, IgG3, IgG4 or the like with a protease such as pepsin. A protease is not limited to a particular protease as long as it can cleave a full-length antibody to form Fab and F(ab')2 in a limited manner when appropriate enzymatic reaction conditions such as pH are created, and examples thereof include pepsin and papain.

In addition, the present invention provides a method for producing a polypeptide containing a variant Fc region that has significantly reduced FcγR binding activity and does not have significantly reduced C1q binding activity compared to a polypeptide containing a parental Fc region that includes the introduction of at least at least one amino acid change into the parent Fc region. In some aspects of the invention, the resulting polypeptide is an antibody. In some embodiments, the antibody is a chimeric antibody or a humanized antibody. In some aspects of the invention, the resulting polypeptide is an Fc fusion protein.

In one aspect of the invention, when carrying out the above method for producing a polypeptide containing a variant Fc region that has significantly reduced FcγR binding activity and does not have significantly reduced C1q binding activity, at least one amino acid is changed 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.

In another aspect of the invention, when carrying out the above method for producing a polypeptide containing a variant Fc region that has significantly reduced FcγR binding activity and does not have significantly reduced C1q binding activity, two amino acids at positions 234 and 235 are changed.

In another aspect of the invention, amino acids are changed in the above method for producing a polypeptide containing a variant Fc region that has significantly increased FcγR binding activity and does not have significantly reduced C1q binding activity, where the changes include: (a) two amino acid position changes 234 and 235, and (b) at least one amino acid change at at least one position selected from the group consisting of: 236, 267, 268, 324, 326, 332 and 333 according to EU numbering.

In another aspect of the invention, amino acids are changed in the above method for producing a polypeptide containing a variant Fc region that has substantially reduced FcγR binding activity and does not have substantially reduced C1q binding activity, wherein the changes include: (a) two amino acid position changes 234 and 235; and (b) at least one amino acid change specified in one of the following subclauses (I)-(III): (I) provisions 267, 268 and 324; (II) provisions 236, 267, 268, 324 and 332; and (III) provisions 326 and 333 according to EU numbering.

In a further aspect of the invention, when carrying out the above method of producing a polypeptide containing a variant Fc region that has significantly reduced FcγR binding activity and does not have significantly reduced C1q binding activity, the amino acid change at each position is selected from the group consisting of: ( 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; (e) Ala at position 236; (g) Glu at position 332; (h) Ala, Asp, Glu, Met, Trp at position 326; and/or Ser at position 333 according to EU numbering.

In a further aspect of the invention, the amino acid changes when carrying out the above method for producing a polypeptide containing a variant of the Fc region having significantly reduced FcγR binding activity and not having significantly 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 of the invention, the amino acid changes when carrying out the above method for producing a polypeptide containing a variant of the Fc region having significantly reduced FcγR binding activity and not having significantly 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 of the invention, the amino acid changes when carrying out the above method for producing a polypeptide containing a variant Fc region that has significantly reduced FcγR binding activity and does not have significantly 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 of the invention, the amino acid changes when carrying out the above method for producing a polypeptide containing a variant Fc region that has significantly reduced FcγR binding activity and does not have significantly 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 of the invention, the amino acid changes when carrying out the above method for producing a polypeptide containing a variant Fc region that has significantly reduced FcγR binding activity and does not have significantly 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.

In another aspect of the invention, when carrying out the above method, at least one amino acid is further changed at at least one position selected from the group consisting of 428, 434, 436, 438 and 440 according to EU numbering.

In a further aspect of the invention, when carrying out the above method, the amino acid change is also selected from the following changes specified in subparagraphs (a) to (d): (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 (see also WO 2016/125495 for a description of the relationship between amino acid changes and FcRn binding activity of the Fc region variant ).

In a further aspect of the invention, when carrying out the above method, the amino acid changes are: 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 according to EU numbering. In a further aspect of the invention, when carrying out the above method, the amino acid changes are: 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, according to EU numbering.

According to one aspect of the invention, it is preferable that the variant Fc region proposed in the invention does not have significantly increased FcRn binding activity, especially at pH 7.4, compared to the parent Fc region.

In a further aspect of the invention, when carrying out the above method, the amino acid changes are selected from a single change, a combination of individual changes, or a combination of changes, which are presented in Table 4.

Polypeptides containing a variant Fc region obtained using any of the above methods or other methods known in the art are included in the present invention.

B. Analyzes

Anti-DENV antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities using a variety of assays known in the art.

The Fc region variants provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities using a variety of assays known in the art.

1. Binding and other assays

According to one aspect of the invention, the antibody of the invention is tested for antigen binding activity using, for example, known methods such as ELISA, Western blotting, etc. In one aspect of the invention, a polypeptide containing an Fc region variant of the invention is tested for its antigen binding activity using known methods such as ELISA, Western blotting, etc.

In another aspect of the invention, competition assays can be used to identify an antibody that competes for binding to DENV and/or DENV E protein with any anti-DENV antibody provided herein. In some embodiments, when said competitive antibody is present in excess, it blocks (e.g., reduces) binding of the reference antibody to DENV and/or DENV E protein by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In some embodiments, said competitive antibody binds to the same epitope (eg, linear or conformational epitope) to which the anti-DENV antibody provided herein binds. For a detailed description of exemplary methods for mapping the epitope to which an antibody binds, see Morris, Epitope Mapping Protocols, in: Methods in Molecular Biology, vol. 66, Humana Press, Totowa, NJ, 1996.

As an example of a competition assay, immobilized DENV or DENV E protein is incubated in a solution containing a first labeled antibody that binds to DENV or DENV E protein and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to DENV or DENV E protein. The second antibody may be present in the supernatant of the hybridoma. As a control, 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 suitable for binding of the first antibody to DENV or DENV E protein, excess unbound antibody is removed and the amount of label associated with immobilized DENV or DENV E protein is measured. If the amount of tag associated with immobilized DENV or DENV E protein is significantly reduced in the test sample compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to DENV or DENV E protein (see eg, Harlow and Lane, Antibodies: A Laboratory Manual, chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Assays to determine the binding activity of a polypeptide containing an Fc region variant to one or more members of the FcR family are provided herein or are known in the art. Such binding assays include, but are not limited to, the BIACORE® assay , which utilizes the phenomenon of surface plasma resonance (SPR), screening based on a homogeneous proximity-enhanced luminescence assay (Amplified Luminescent P roximity Homogeneous A ssay ). ALPHA), ELISA, and fluorescence intensity cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA 103(11), 2006, ee. 4005-4010).

In one embodiment, the BIACORE® assay can be used to determine whether the binding activity of a polypeptide containing a variant Fc region is increased, maintained at the same level, or decreased for a particular member of the FcR family. For example, for this purpose, it is determined whether the dissociation constant (KD) value, which is determined by sensorgram analysis, decreases or increases when various FcRs are reacted as an analyte with polypeptides containing a variant Fc region immobilized or “captured” on a sensor chip, using known methods and reagents such as protein A, protein L, protein A/G, protein G, lambda chain antibodies, kappa chain antibodies, antigenic peptides, antigenic proteins. Changes in binding activity can also be determined by comparing changes in the number of resonance units (RU) in a sensorgram before and after one or more FcR types are reacted as analytes with captured polypeptides containing the Fc region variant. Alternatively, the FcR can be immobilized or “captured” on sensor chips, and polypeptides containing the variant Fc region can be used as the analyte.

In BIACORE® assays, one of the substances (ligand) to be studied is immobilized on a thin layer of gold on a sensor chip and illuminated with light from the back surface of the sensor chip so that there is total reflection at the interface between the thin layer of gold. and glass, an area with reduced intensity (SPR signal) is formed in the part of the reflected light. Another substance (analyte), intended for studying the interaction, is prepared for injection onto the surface of the sensor chip; and when the ligand binds to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the surface of the sensor chip changes. As a result of this change in refractive index, the position of the SPR signal is shifted (and vice versa, the position of the signal returns to its original position if the specified binding dissociates). Using the BIACORE® system, the level of the shift described above, or more specifically the change in mass as a function of time, is determined by plotting the change in mass on the surface of the sensor chip along the vertical axis, and thus obtaining quantitative data (sensogram). The amount of analyte bound to the ligand immobilized on the surface of the sensor chip is determined from the sensogram. Kinetic parameters, such as the rate constant of the association reaction (ka) and the rate constant of the dissociation reaction (kd), are determined from the sensorgrams presented in the form of curves, and the affinity (KD) is determined as the ratio of these constants. The BIACORE® method is preferably used as the inhibition assay method. Examples of such a method for inhibition assays are described in Lazar et al., Proc. Natl. Acad. Sci. USA 103(11), 2006, ss. 4005-4010.

ALPHA-based screening is carried out using ALPHA technology, which is based on the principle described below, using two types of beads, donors and acceptors. Luminescent signals are only detectable when molecules bound to donor beads physically interact with molecules bound to acceptor beads, and when both beads are in close proximity to each other. The photosensitizer in the donor granules, excited by a laser beam, converts ambient oxygen into excited singlet oxygen. When singlet oxygen diffuses from donor granules and reaches acceptor granules located in close proximity, a chemiluminescent reaction is induced in the granules, which ultimately leads to the emission of light. If the molecules associated with the donor beads do not interact with the molecules associated with the acceptor beads, then the chemiluminescent reaction does not occur because the singlet oxygen that is produced by the donor beads does not reach the acceptor beads.

For example, a biotinylated polypeptide complex is coupled to donor beads and a glutathione S-transferase (GST)-tagged Fc receptor is coupled to acceptor beads. In the absence of a competing polypeptide complex containing the variant Fc region, the polypeptide complex containing the parent Fc region interacts with the Fc receptor and generates signals with a wavelength of 520 to 620 nm. A polypeptide complex containing an untagged variant of the Fc region competes with a polypeptide complex containing the parent Fc region for binding to the Fc receptor. Relative binding activity can be assessed by quantifying the decrease in fluorescence due to competition. Methods for biotinylation of polypeptide complexes such as antibodies using sulfo-NHS-biotin or similar agents are well known. The method of expressing the Fc receptor and GST in a cell carrying a fusion gene obtained by fusing a polynucleotide that encodes the Fc receptor in frame with a polynucleotide that encodes GST in an expression vector and purifying using a glutathione-containing column is suitably adapted , obtaining the Fc receptor tagging method with GST. The induced signals can be analyzed, for example, by fitting a single-site competition model based on nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

The term "variant Fc region with reduced FcR binding activity" refers to an Fc region that binds to FcR with substantially weaker binding activity than the parent Fc region when performed in assays using substantially equal amounts of the corresponding parent Fc region and variant of the Fc region. In addition, the term "Fc region variant with increased FcR binding activity" refers to an Fc region that binds to an FcR with substantially greater binding activity than the parent Fc region when performed in assays using substantially equal amounts of the corresponding parent Fc -region and variant Fc region. The term "variant Fc region with retained FcR binding activity" refers to an Fc region that binds to an FcR with activity equivalent to or substantially the same as that of the parent Fc region when performed in assays using substantially equal amounts of the corresponding parent Fc region and a polypeptide containing a variant of the Fc region.

Whether the binding activity of the Fc region to various FcRs increases or decreases can be determined by the increase or decrease in the level of binding of various FcRs to the Fc region when determined using the above-described measurement method. In the context of the present description, the level of binding of various FcRs to the Fc region can be estimated as a value obtained by dividing the difference in RU levels in the sensorgrams before and after the interaction of various FcRs that are used as an analyte with the Fc region by the difference in RU levels by sensorgrams, which changed before and after the “capture” of Fc regions on sensor chips. The binding activity of the Fc region to FcγR or FcRn can be determined using the method presented herein in Example 5.

In the present invention, substantially reduced FcγR binding activity preferably means, for example, that the FcγR binding activity of the Fc region variant 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% of the FcγR binding activity of the parent Fc region. This preferably also means that, for example, the ratio [difference in RU values on sensorgrams that is detected before and after interaction of FcγR with an Fc region variant]/[difference in RU values on sensorgrams that is detected before and after “capture” of FcγR by sensory chips] 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 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.

In the present invention, a not significantly increased FcRn binding activity, especially at pH 7.4, preferably means, for example, that the binding activity of the Fc region variant for FcRn is less than 1000-fold, less than 500-fold, less than 200 times, less than 100 times, less than 90 times, less than 80 times, less than 70 times, less than 60 times, less than 50 times, less than 40 times, less than 30 times , less than 20-fold, less than 10-fold, less than 5-fold, less than 3-fold, or less than 2-fold the FcRn-binding activity of the parent Fc region. This preferably also means, for example, that the ratio [difference in RU values on sensorgrams that is detected before and after interaction of FcRn with an Fc region variant]/[difference in RU values on sensorgrams that is detected before and after “capture” of FcRn by sensory chips] 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.

To determine the binding activity of a polypeptide containing an Fc region variant to C1q, a C1q binding assay can be performed using an ELISA. In general, the method is as follows: assay plates can be coated overnight at 4° C. with a polypeptide that contains a variant Fc region, or a polypeptide that contains a parent Fc region (control), in sensitization buffer. The plates can then be washed and blocked. After washing, an aliquot of human C1q can be added to each well and incubated for 2 hours at room temperature. After an additional wash, 100 µl of sheep anti-complement C1q peroxidase-conjugated antibody can be added to each well and incubated for 1 hour at room temperature. The plate can be washed again with wash buffer and 100 μl of substrate buffer containing OPD (o-phenylenediamine dihydrochloride (Sigma)) can be added to each well. The oxidation reaction, which is detected by the appearance of a yellow color, can be allowed to proceed for 30 minutes and the reaction can be stopped by adding 100 µl of 4.5 N H ₂ SO ₄ . Absorbance can then be determined at a wavelength of (492-405) nm. Fc region binding activity to C1q can be determined using the method presented herein in Example 4.

In one aspect of the invention, the difference in C1q binding activity between the variant Fc region and the parent Fc region of the 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% of the C1q-binding activity of the parent Fc region.

2. Activity tests

One object of the invention is assays designed to identify antibodies to DENV that have biological activity. The biological activity may include, for example, blocking DENV E protein binding to a host cell, inhibiting DENV entry into a host cell, inhibiting and/or preventing DENV infection of a host cell, etc. Antibodies having the specified biological activity in vivo and/or in vitro are also proposed.

In some embodiments, an antibody of the invention is tested for said biological activity. In some embodiments, a plaque neutralization/reduction test (PRNT) can be used to measure the activity or neutralizing ability of a test antibody. In some embodiments, animal hosts can be used to measure anti-DENV activity in vivo.

In some embodiments, cells can be directly assayed for binding between DENV and a test antibody. Immunohistochemical techniques, confocal microscopy techniques, and/or other techniques for assessing binding are well known to those skilled in the art. For these screening assays, you can use a variety of cell lines, including cells specifically created for this purpose. Examples of cells that are used in screening assays include mammalian cells, fungal cells, bacterial cells or viral cells. The cell may be a stimulated cell, such as a growth factor stimulated cell. It will be apparent to one skilled in the art that a wide variety of assays designed to measure the ability of a test antibody to bind to DENV fall within the scope of this invention.

One object of the invention is assays designed to identify antibodies to ZIKV that have biological activity. The biological activity may include, for example, blocking ZIKV binding to a host cell, inhibiting ZIKV entry into a host cell, inhibiting and/or preventing ZIKV infection of a host cell, etc. Antibodies having the specified biological activity in vivo and/or in vitro are also proposed.

In some embodiments, an antibody of the invention is tested for said biological activity. In some embodiments, a plaque reduction neutralization test (PRNT) or a focus reduction neutralization test (FRNT) can be used to measure the activity or neutralizing ability of the test antibody. In some embodiments, animal hosts can be used to measure anti-ZIKV activity in vivo.

In some embodiments, cells can be directly assayed for binding between ZIKV and a test antibody. Immunohistochemical techniques, confocal microscopy techniques, and/or other techniques for assessing binding are well known to those skilled in the art. For these screening assays, you can use a variety of cell lines, including cells specifically created for this purpose. Examples of cells that are used in screening assays include mammalian cells, fungal cells, bacterial cells or viral cells. The cell may be a stimulated cell, such as a growth factor stimulated cell. It will be apparent to one skilled in the art that a wide variety of assays designed to measure the ability of a test antibody to bind to ZIKV fall within the scope of this invention.

Depending on the assay, cell and/or tissue cultures may be required. Any of a variety of different physiological assays can be used for cellular assessment. Alternatively or additionally, molecular assays can be performed including, but not limited to, Western blotting to monitor protein expression and/or protein-protein interactions; mass spectrometry for monitoring other chemical modifications, etc.

In some embodiments, these methods use an animal host. For example, suitable animal hosts in the context of the invention may be mammalian hosts, including primates, ferrets, cats, dogs, cows, horses and rodents such as mice, hamsters, rabbits and rats. In some embodiments, the host animal is inoculated, infected, or otherwise exposed to the virus prior to or simultaneously with administration of the test antibody. Naive and/or inoculated animals can be used in any of a variety of studies. For example, these animal models can be used in various viral transmission experiments known in the art. The test antibody may be administered to a suitable animal host before, during, or after viral transmission experiments to determine the effectiveness of the test antibody in blocking virus binding and/or infectivity in the host animal.

One object of the invention is assays designed to identify polypeptides that contain Fc region variants that have biological activity. The biological activity may include, for example, ADCC activity and CDC activity. Also proposed are polypeptides that contain variants of the Fc region that have the specified biological activity in vivo and/or in vitro.

In some embodiments, a polypeptide comprising an Fc region variant of the invention is tested for said biological activity. In some aspects of the invention, a polypeptide containing a variant Fc region of the invention modulates effector function compared to a polypeptide that contains a parent Fc region. In some aspects of the invention, the modulation is ADCC and/or CDC modulation.

In vitro and/or in vivo cytotoxicity assays can be performed to confirm the presence of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody has the ability to bind to FcγR (and therefore is likely to have ADCC activity) but retains the ability to bind to FcRn. Primary cells mediating ADCC, i.e. NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. Data on FcR expression on hematopoietic cells are summarized in Table 3 on p. 464 from Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, pp. 457-492. Examples of in vitro assays for ADCC activity of a molecule of interest include, but are not limited to, the assays described in US No. 5,500,362 (see, for example, Hellstrom et al., Proc. Nail. Acad. Sci. USA 83, 1986, ss 7059-7063 and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82, 1985, pp. 1499-1502); US No. 5821337 (see Bruggemann et al., J. Exp. Med. 166, 1987, pp. 1351-1361). Alternatively, non-radioactive assays may be used (see, for example, the ACT1™ Flow Cytometry-Based Non-Radioactive Cytotoxicity Assay (CellTechnology, Inc. Mountain View, Calif.); and the CytoTox ^96® Non-Radioactive Cytotoxicity Assay (Promega, Madison , Wisconsin. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model , for example, as described in Clynes et al., Proc. Natl. Acad. Sci. USA 95, 1998, pp. 652-656. C1q binding assays can also be performed to confirm that the antibody binds to C1q and therefore has CDC activity (See, for example, the description of ELISA for assessing the binding of C1q and C3c 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 202, 1996, cc. 163-171; Cragg et al., Blood 101, 2003, cc. 1045-1052; and Cragg and Glennie, Blood 103, 2004, cc. 2738-2743). Determination of FcRn binding and clearance/half-life in vivo can also be carried out using methods known in the art (see, for example, Petkova et al., Int'l. Immunol. 18(12), 2006, cc. 1759- 1769).

D. Immunoconjugates

Some embodiments of the invention also include immunoconjugates comprising an anti-DENV antibody as provided herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active bacterial toxins, , fungal, plant or animal origin or their fragments) or radioactive isotopes.

Some embodiments of the invention also provide immunoconjugates comprising a polypeptide that contains a variant Fc region as described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins , toxins of bacterial, fungal, plant or animal origin or their fragments with enzymatic activity) or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see US Nos. 5208020, 5416064 and EP 0425235 IN 1); auristatin, for example, monomethyl auristatin DE and DF fragments (MMAE and MMAF) (see US Nos. 5635483, 5780588 and 7498298); dolastatin; calicheamicin or its derivatives (see US Nos. 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001 and 5877296; Hinman et al., Cancer Res. 53, 1993, pp. 3336-3342; and Lode and etc., Cancer Res. 58, 1998, pp. 2925-2928); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Curr. Med. Chem. 13, 2006, pp. 477-523; Jeffrey et al., Bioorg. Med. Chem. Lett. 16, 2006, pp. 358-362; Torgov et al., Bioconjug. Chem. 16, 2005, pp. 717-721; Nagy et al., Proc. Natl. Acad. Sci. USA 97, 2000, pp. 829-834; Dubowchik et al. ., Bioorg. & Med. Chem. Letters 12, 2002, pp. 1529-1532; King et al., J. Med. Chem. 45, 2002, pp. 4336-4343; and US No. 6630579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecene and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as provided herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-5), inhibitor from Momordica charantia, curcin, crotin, inhibitor from Sapaonaria officinalis, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

In another embodiment of the invention, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A wide variety of radioactive isotopes can be used to obtain radioconjugates. Examples include ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as TC ^99m or I ¹²³ , 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.

Antibody-cytotoxic agent conjugates can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) , iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate × HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide derivatives (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and dual-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the immunotoxin ricin can be prepared according to the method described in Vitetta et al., Science 238, 1987, pp. 1098-1104. C-labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent used to conjugate a radionucleotide to an antibody (see WO 94/11026). The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug into the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52, 1992, pp. 127-131; US No. 5,208,020).

As used herein, immunoconjugates or ADCs include, but are not limited to, those conjugates produced using cross-linking reagents, which 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, and SVSB (succinimidyl(4-vinylsulfone) benzoate) commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL, USA).

D. Methods and compositions for diagnosis and detection

In some embodiments, any of the anti-DENV antibodies provided herein can be used to detect the presence of DENV and/or DENV E protein in a biological sample. In some embodiments, any of the anti-ZIKV antibodies provided herein can be used to detect the presence of ZIKV in a biological sample. The concept of "detection" in the context of the present description provides quantitative or qualitative detection. In some embodiments, the biological sample comprises a cell or tissue, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites fluid, milk , colostrum, mammary secretions, lymph, urine, sweat, tear fluid, gastric juice, synovial fluid, peritoneal fluid, eye lens fluid or mucus.

One embodiment of the invention is an anti-DENV antibody as provided herein for use in a diagnostic or detection method. Another aspect of the invention relates to a method for detecting the presence of DENV in a biological sample. In some embodiments, the method comprises contacting a biological sample with an anti-DENV antibody as provided herein under conditions such that the anti-DENV antibody binds to DENV and detecting the formation of a complex between the anti-DENV antibody and DENV. Said method may be an in vitro or in vivo method. A further aspect of the invention relates to a method for detecting the presence of DENV E protein in a biological sample. In some embodiments, the method comprises contacting a biological sample with an anti-DENV antibody as provided herein under conditions that allow the anti-DENV antibody to bind to the DENV E protein and detecting the formation of a complex between the anti-DENV antibody and the E protein. -DENV protein. Said method may be an in vitro or in vivo method. In one embodiment, an anti-DENV antibody is used to select individuals who can be treated with an anti-DENV antibody, for example, where DENV or DENV E protein is a biomarker for patient selection.

Examples of disorders that can be diagnosed using the antibody of the invention include, but are not limited to, DENV infection and diseases and/or symptoms caused by or associated with DENV infection, such as dengue fever, dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS).

One embodiment of the invention is an anti-ZIKV antibody as provided herein for use in a diagnostic or detection method. Another aspect of the invention relates to a method for detecting the presence of ZIKV in a biological sample. In some embodiments, the method comprises contacting a biological sample with an anti-ZIKV antibody as provided herein under conditions that allow the anti-ZIKV antibody to bind to ZIKV and detecting the formation of a complex between the anti-ZIKV antibody and ZIKV. Said method may be an in vitro or in vivo method. Another aspect of the invention relates to a method for detecting the presence of ZIKV in a biological sample. In some embodiments, the method comprises contacting a biological sample with an anti-ZIKV antibody as provided herein under conditions that allow the anti-ZIKV antibody to bind to ZIKV and detecting the formation of a complex between the anti-ZIKV antibody and ZIKV. Said method may be an in vitro or in vivo method. In one embodiment, an anti-ZIKV antibody is used to select individuals who can be treated with an anti-ZIKV antibody, for example, when ZIKV is a biomarker for patient selection.

Examples of disorders that can be diagnosed using the antibody of the invention include, but are not limited to, ZIKV infection and diseases and/or symptoms caused by or associated with ZIKV infection, such as fever, rash, headache, joint pain, red eyes and muscle pain.

Some embodiments of the invention are labeled antibodies to DENV. Some embodiments of the invention are labeled anti-ZIKV antibodies. Labels include, but are not limited to, tags or moieties that can be detected directly (such as fluorescent, chromophore, electron dense, chemiluminescent, and radioactive tags), as well as moieties such as enzymes or ligands that can be detected indirectly, e.g. enzymatic reaction or molecular interaction.

Examples of labels include, but are not limited to, the radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ³¹ I, fluorophores such as rare earth metal chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, e.g. firefly luciferase and bacterial luciferase (US No. 4737456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g. glucose oxidase, galactose oxidase and glucose 6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase cross-linked with an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin tags, bacteriophage tags, stable free radicals and etc.

In one embodiment, an antibody containing an Fc region variant of the invention can be used as an affinity purification agent. In this process, the antibody variant is immobilized on a solid phase, such as Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody variant is contacted with a sample containing the antigen to be purified, and the support is then washed with a suitable solvent that should remove substantially all of the materials in the sample other than the antigen to be purified, which is bound to the immobilized antibody variant. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, which should separate the antigen from the antibody variant.

The antibody variant can also be used in diagnostic assays, for example, to detect the expression of an antigen of interest in specific cells, tissues or serum.

The antibody variant can be used to perform any known assays, such as competition binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, 1987, pp. 147-158, CRC Press, Inc.).

E. Pharmaceutical compositions

Pharmaceutical compositions of the anti-DENV antibody provided herein are prepared by mixing said antibody, having the required degree of purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed. edited by Osol A., 1980) in in the form of lyophilized compositions or aqueous solutions.

Pharmaceutical compositions of the anti-ZIKV antibody provided herein are prepared by mixing said antibody, having the required degree of purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed. edited by Osol A., 1980) in in the form of lyophilized compositions or aqueous solutions.

Pharmaceutical compositions of a polypeptide containing a variant Fc region provided herein are prepared by mixing said polypeptide having the required degree of purity with one or more optional pharmaceutically acceptable carriers in the form of lyophilized compositions or aqueous solutions.

Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used and include, but are not limited to: buffers such as phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and meta-cresol); low molecular weight polypeptides (containing less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming agents against ions such as sodium; metal-containing complexes (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). As used herein, examples of pharmaceutically acceptable carriers also include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble hyaluronidase glycoproteins PH-20, such as rHuPH20 ( ^HYLENEX® , Baxter International, Inc.). Some examples of sHASEGP and methods for their use, including rHuPH20, are described in US Patent Publications Nos. 2005/0260186 and 2006/0104968. In one aspect of the invention, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Examples of lyophilized antibody compositions are described in US No. 6,267,958. Aqueous antibody compositions include those described in US No. 6171586 and WO 2006/044908, the latter compositions including histidine acetate buffer.

The composition according to the invention may also contain more than one active substance if necessary for the particular indication being treated, preferably substances with complementary activities that do not adversely affect each other. For example, the use of an additional antiviral agent may be required, such as, but not limited to, interferons (e.g., interferon α-2b, interferon-γ, etc.), anti-DENV monoclonal antibodies, anti-DENV polyclonal antibodies, RNA inhibitors polymerases, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, short interfering RNAs, short hairpin RNAs, microRNAs, RNA aptamers, ribozymes and combinations thereof. Such active ingredients must be present in combination in quantities effective to achieve the intended purpose.

The active substances can also be included in microcapsules, for example those obtained using coacervation or interfacial polymerization methods, for example in hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug administration systems (for example in liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in a macroemulsion. Such methods are described in Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980.

Slow-release preparations can be prepared. Suitable examples of sustained release formulations are semi-permeable matrices of solid hydrophobic polymers comprising the antibody or conjugate, such matrices being shaped articles such as films or microcapsules.

Compositions intended for use in vivo generally must be sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

G. Therapeutic methods and compositions

Any of the antibodies to DENV presented herein can be used in therapeutic methods.

One object of the invention is an anti-DENV antibody, as provided herein, for use as a drug. Other aspects of the invention include an anti-DENV antibody provided herein for use in the treatment of DENV infection. Some embodiments of the invention provide an anti-DENV antibody for use in a method of treatment. Some embodiments of the invention provide an anti-DENV antibody for use in a method of treating an individual having a DENV infection, which includes administering to the individual an effective amount of an anti-DENV antibody. In one of these embodiments, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In other embodiments, the anti-DENV antibody is for use in blocking DENV E protein binding to a host cell and/or DENV entry into a host cell. In some embodiments, an anti-DENV antibody is provided for use in a method of blocking binding of DENV E protein to a host cell and/or entry of DENV into a host cell in an individual, which comprises administering to the individual an effective amount of an anti-DENV antibody to block binding of E protein. DENV protein with the host cell and/or entry of DENV into the host cell. "Individual" in the context of any of the above embodiments of the invention is preferably a human.

Another object of the invention is the use of an antibody to DENV for the preparation or production of a medicinal product. In one embodiment, the medicament is for treating a DENV infection. In another embodiment, the drug is used in a method of treating a DENV infection, which includes administering to an individual having a DENV infection an effective amount of the drug. In one of these embodiments, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In a further embodiment, the drug is designed to block the binding of DENV E protein to a host cell and/or the entry of DENV into the host cell. In another embodiment, the drug is for use in a method of blocking binding of DENV E protein to a host cell and/or entry of DENV into a host cell, comprising administering to an individual an effective amount of the drug to block binding of DENV E protein to a host cell. host and/or entry of DENV into the host cell. "Individual" in the context of any of the above embodiments of the invention may be a human.

Another object of the invention is a method for treating DENV infection. In one embodiment, the method includes administering to an individual having a DENV infection an effective amount of an anti-DENV antibody. In one embodiment, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent described below. "Individual" in the context of any of the above embodiments of the invention may be a human.

Another aspect of the invention is a method of blocking the binding of DENV E protein to a host cell and/or entry of DENV into a host cell in an individual. In one embodiment, the method comprises administering to an individual an effective amount of an anti-DENV antibody to block binding of DENV E protein to a host cell and/or entry of DENV into the host cell. In one embodiment of the invention, the “individual” is a human.

Another object of the invention is pharmaceutical compositions containing any of the antibodies to DENV presented in the present description, which are intended, for example, for use in any of the above therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the anti-DENV antibodies described herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition contains any of the anti-DENV antibodies described herein and at least one additional therapeutic agent, such as those described below.

Another object of the invention is a pharmaceutical composition intended for the treatment of infection caused by DENV. In a further embodiment, the pharmaceutical composition is designed to block the binding of DENV E protein to a host cell and/or the entry of DENV into a host cell. In one embodiment of the invention, the pharmaceutical composition is administered to an individual having a DENV infection. "Individual" in the context of any of the above embodiments of the invention is preferably a human.

In some embodiments, DENV infection may include diseases and/or symptoms caused by or associated with DENV infection, such as dengue fever, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS).

One aspect of the invention is methods of treating or preventing infection caused by the Zika virus, including the administration of an antibody that binds to ZIKV. One object of the invention is methods for inhibiting transmission of the Zika virus from a pregnant mother to her fetus. One object of the invention is methods of preventing congenital syndrome caused by the Zika virus (eg, mental retardation, microcephaly, etc.), including the administration of an antibody that binds to ZIKV. One aspect of the invention is methods of inhibiting the reduction of fetal weight (eg, whole body weight of the child, head weight, etc.), including the administration of an antibody that binds to ZIKV.

In one example, the term “congenital Zika virus syndrome” refers to a special pattern of birth defects unique to fetuses and infants infected with the Zika virus before birth. As will be apparent to one of ordinary skill in the art, individuals with congenital Zika virus syndrome may have syndromes such as, but not limited to, intellectual disability, microcephaly, severe microcephaly in which there is partial destruction of the skull, tissue loss brain with a specific pattern of brain damage, including subcortical calcification, damage to the posterior part of the eye, including macular scars and patchy retinal pigmentation, congenital contractures such as clubfoot or arthrogryposis, hypertension limiting body movement soon after birth, etc. It is likely that administration of the antibodies provided herein can prevent at least one, or at least two, or at least three, or at least four, or all symptoms of the congenital syndrome caused by the Zika virus. For example, an antibody provided herein prevents Zika virus infection of a fetus or infant before birth, thereby inhibiting the decrease in fetal weight (eg, infant's total body weight, head weight, etc.).

Any of the anti-ZIKV antibodies provided herein can be used in therapeutic methods.

One object of the invention is an anti-ZIKV antibody, as described herein, for use as a drug. Other aspects of the invention include an anti-ZIKV antibody as provided herein for use in the treatment of ZIKV infection. Some embodiments of the invention provide an anti-ZIKV antibody for use in a method of treatment. Some embodiments of the invention provide an anti-ZIKV antibody for use in a method of treating an individual having a ZIKV infection, which includes administering to the individual an effective amount of an anti-ZIKV antibody. In one of these embodiments, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In other embodiments, the anti-ZIKV antibody is for use in blocking the binding of ZIKV to a host cell and/or the entry of ZIKV into a host cell. In some embodiments, the anti-ZIKV antibody is for use in a method of blocking ZIKV binding to a host cell and/or entry of ZIKV into a host cell in an individual, comprising administering to the individual an effective amount of an anti-ZIKV antibody to block ZIKV binding to a host cell and /or entry of ZIKV into the host cell. "Individual" in the context of any of the above embodiments of the invention is preferably a human.

Another object of the invention is the use of an antibody to ZIKV for the preparation or production of a medicinal product. In one embodiment, the medicament is for treating a ZIKV infection. In another embodiment, the drug is used in a method of treating a ZIKV infection, comprising administering to an individual who has a ZIKV infection an effective amount of the drug. In one of these embodiments, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In a further embodiment, the drug is designed to block the binding of ZIKV to a host cell and/or the entry of ZIKV into a host cell. In another embodiment, the drug is for use in a method of blocking ZIKV binding to a host cell and/or entry of ZIKV into a host cell, which comprises administering to an individual an effective amount of a drug to block ZIKV binding to a host cell and/or entry into a host cell. ZIKV into the host cell. "Individual" in the context of any of the above embodiments of the invention may be a human.

Another object of the invention is a method for treating a ZIKV infection. In one embodiment, the method includes administering to an individual having a ZIKV infection an effective amount of an anti-ZIKV antibody. In one of these embodiments, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent described below. "Individual" in the context of any of the above embodiments of the invention may be a human.

Another aspect of the invention is a method of blocking the binding of ZIKV to a host cell and/or entry of ZIKV into a host cell in an individual. In one embodiment, the method comprises administering to an individual an effective amount of an anti-ZIKV antibody to block ZIKV binding to a host cell and/or entry of ZIKV into the host cell. In one embodiment of the invention, the “individual” is a human.

Another object of the invention is pharmaceutical compositions containing any of the antibodies to ZIKV presented in the present description, which are intended, for example, for use in any of the above therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the anti-ZIKV antibodies described herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition contains any of the anti-ZIKV antibodies described herein and at least one additional therapeutic agent, such as those described below.

Another object of the invention is a pharmaceutical composition intended for the treatment of infection caused by ZIKV. In a further embodiment of the invention, the pharmaceutical composition is designed to block the binding of ZIKV to a host cell and/or the entry of ZIKV into a host cell. In one embodiment of the invention, the pharmaceutical composition is administered to an individual having a ZIKV infection. "Individual" in the context of any of the above embodiments of the invention is preferably a human.

In some embodiments, a ZIKV infection may include illnesses and/or symptoms caused by or associated with ZIKV infection, such as fever, rash, headache, joint pain, red eyes, and muscle pain.

Any of the polypeptides containing a variant Fc region presented herein can be used in therapeutic methods.

One object of the invention is a polypeptide containing a variant of the Fc region, intended for use as a drug. In some embodiments, the polypeptide comprising an Fc region variant is for use in a method of treatment. In some embodiments, a polypeptide containing an Fc region variant is for use in a method of treating an individual having a disorder, which includes administering to the individual an effective amount of a polypeptide containing an Fc region variant. In one of these embodiments, the method also includes administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment of the invention, the disorder is a viral infection. In one embodiment of the invention, the “individual” is a human.

Another object of the invention is the use of a polypeptide containing a variant of the Fc region for the preparation or production of a medicinal product. In one embodiment of the invention, the medicament is for treating a disorder. In some aspects of the invention, the polypeptide is an antibody. In some aspects of the invention, the polypeptide is an Fc fusion protein. In another embodiment of the invention, the drug is for use in a method of treating a disorder, comprising administering to an individual who has the disorder to be treated an effective amount of the drug. In one of these embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment of the invention, the disorder is a viral infection. In one embodiment of the invention, the “individual” is a human.

Another object of the invention is a method for treating a disorder. In one embodiment, the method comprises administering to an individual who has the disorder an effective amount of a polypeptide comprising an Fc region variant. In one embodiment, the method further includes administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment of the invention, the disorder is a viral infection. In one embodiment of the invention, the “individual” is a human.

Another object of the invention is pharmaceutical compositions containing a polypeptide that contains a variant of the Fc region presented herein, which are intended for use in a therapeutic method, such as any of the therapeutic methods presented herein. In one embodiment of the invention, the pharmaceutical composition contains a polypeptide that contains a variant of the Fc region presented in the present description, and a pharmaceutically acceptable carrier. In another embodiment of the invention, the pharmaceutical composition contains a polypeptide that contains a variant of the Fc region presented in the present description, and at least one additional therapeutic agent.

According to a further aspect of the invention, the pharmaceutical composition is for treating a disorder. In one embodiment of the invention, the pharmaceutical composition is administered to an individual who has a disorder. In one embodiment of the invention, the disorder is a viral infection. In one embodiment of the invention, the “individual” is a human.

Antiviral antibodies that contain the Fc region variant of the present invention can suppress antibody-dependent enhancement (ADE) that occurs with canonical antiviral antibodies. ADE is a phenomenon in which phagocytosis of antibody-bound virus occurs through activating FcγRs, resulting in increased viral infection of the cell. Fc modifications that reduce interactions through activating FcγRs may reduce the risk of ADE. Mutations at positions 234 and 235, resulting in the substitution of leucine for alanine, resulting in the formation of LALA mutants, have been found to reduce the risk of ADE during dengue virus infection in vivo (Cell Host Microbe 8, 2010, pp. 271-283). However, these modifications reduce other antibody-mediated immune effector functions such as ADCC and CDC. First of all, CDC can be expected to play an important role in the inhibition of ADE, so for therapeutic efficacy, the binding of Fc regions to the complement component C1q should not be reduced. In addition, the half-life of an antibody can be extended by engineering Fc regions with altered binding affinity to their salvage receptor, FcRn, which may allow prophylactic use of antibodies to protect against viral infection.

The virus is preferably selected from adenovirus, astrovirus, hepadnavirus, herpesvirus, papovirus, poxyvirus, arenavirus, bunyavirus, calcivirus, coronavirus, filovirus, flavivirus, orthomyxovirus, paramycovirus, picornavirus, reovirus, retrovirus, rhabdovirus or togavirus.

In preferred embodiments, the adenovirus includes, but is not limited to, human adenovirus. In preferred embodiments, the astrovirus includes, but is not limited to, a mammalian astrovirus. In preferred embodiments, the hepadnavirus includes, but is not limited to, hepatitis B virus. In preferred embodiments, the herpes virus includes, but is not limited to, herpes simplex virus type I, herpes simplex virus type 2, human cytomegalovirus, Epstein virus Barr, varicella-zoster virus, roseolovirus and Kaposi's sarcoma-associated herpesvirus. In preferred embodiments, 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, vaccinia virus, monkeypox virus, humanpox virus, pseudocowpox virus, papular stomatitis virus, Tanapox virus, Yaba monkey tumor virus, and molluscum contagiosum. In preferred embodiments, the arenavirus includes, but is not limited to, lymphocytic choriomeningitis virus, Lassa virus, Machupo virus, and Yunin virus. In preferred embodiments, bunyavirus includes, but is not limited to, hantavirus, nairovirus, orthobunyavirus, and phlebovirus. In preferred embodiments, calcivirus includes, but is not limited to, vesivirus, norovirus such as Norfolk virus, and sapovirus. In preferred embodiments, the coronavirus includes, but is not limited to, human coronavirus (the etiologic agent 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, the flavivirus includes, but is not limited to, yellow fever virus, West Nile virus, dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4), hepatitis C virus, tick-borne encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Kiasanur forest disease virus and Powassan encephalitis virus. In preferred embodiments, the orthomyxovirus includes, but is not limited to, influenza virus type A, influenza virus type B, and influenza virus type C. In preferred embodiments, the paramyxovirus includes, but is not limited to, parainfluenza virus, rubella (mumps) virus ), morbillivirus (measles virus), pneumovirus such as human respiratory syncytial virus and subacute sclerosing panencephalitis virus. In preferred embodiments, picornavirus includes, but is not limited to, poliovirus, rhinovirus, Coxsackie A virus, Coxsackie B virus, hepatitis A virus, echovirus, and enterovirus. In preferred embodiments, the reovirus includes, but is not limited to, Colorado tick fever virus and rotavirus. In preferred embodiments, the retrovirus includes, but is not limited to, a lentivirus such as human immunodeficiency virus and human T-lymphotropic virus (HTLV). In preferred embodiments, rhabdovirus includes, but is not limited to, lyssavirus such as rabies virus, vesicular stomatitis virus, and infectious hematopoietic necrosis virus. In preferred embodiments, togavirus includes, but is not limited to, alphavirus such as Ross River virus, O'Nyong-Nyong virus, Sindbis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus and Western equine encephalitis virus, and rubella virus.

Another aspect of the invention is methods of preparing a medicament or pharmaceutical composition comprising admixing any of the anti-DENV antibodies provided herein with a pharmaceutically acceptable carrier, for example, for use in any of the above therapeutic methods. In one embodiment of the invention, methods of preparing a drug or pharmaceutical composition further comprise adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

Another aspect of the invention is methods of preparing a medicament or pharmaceutical composition comprising admixing any of the anti-ZIKV antibodies provided herein with a pharmaceutically acceptable carrier, for example, for use in any of the above therapeutic methods. In one embodiment of the invention, methods of preparing a drug or pharmaceutical composition further comprise adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

Antibodies proposed in the invention can be used either individually or in combination with other agents used in therapy. For example, an antibody of the invention may be used in conjunction with at least one additional therapeutic agent. In some embodiments, 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, short interfering RNAs, short hairpin RNAs, microRNAs, RNA aptamers, ribozymes and combinations thereof.

Another aspect of the invention is methods of preparing a medicament or pharmaceutical composition comprising mixing any of the Fc region variant-containing polypeptides described herein with a pharmaceutically acceptable carrier, for example, for use in any of the above therapeutic methods. In one embodiment of the invention, methods of preparing a drug or pharmaceutical composition further comprise adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

The Fc region variant containing polypeptides of the invention can be used either alone or in combination with other therapeutic agents. For example, a polypeptide comprising an Fc region variant of the invention may be used in conjunction with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antiviral agent such as, but not limited to, interferons (e.g., interferon α-2b, interferon-γ, etc.), antiviral monoclonal antibodies, antiviral polyclonal antibodies, inhibitors RNA polymerases, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, short interfering RNAs, short hairpin RNAs, microRNAs, RNA aptamers, ribozymes and combinations thereof.

Such combination therapies as noted above include coadministration (where two or more therapeutic agents are included in the same or different compositions) and separate administration, in which case administration of an antibody or polypeptide containing an Fc region variant of the invention , can be performed before, simultaneously and/or after the administration of an additional therapeutic agent or agents. In one embodiment, administration of the DENV antibody and administration of the additional therapeutic agent can be accomplished within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days relative to each other. In another embodiment, administration of the Fc region variant-containing polypeptide and administration of the additional therapeutic agent can be accomplished within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5 or 6 days relative to each other.

An antibody or polypeptide containing an Fc region variant of the invention (and any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary and intranasal and, if necessary, used for local treatment, introduction into the lesion. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be carried out by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending, among other things, on whether the treatment is short-term or chronic. Various dosing regimens can be used, including, but not limited to, single administration or multiple administrations at different times, bolus administration, and pulsatile infusion.

Antibodies or polypeptides containing a variant Fc region of the invention can be formulated, dosed and administered in accordance with good clinical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the schedule of administration and other factors known to medical practitioners. The agent may, but is not required to, be included in the composition in combination with one or more other agents currently used for the prevention and treatment of the disorder in question. The effective amount of these other agents depends on the amount of agent present in the composition, the type of disorder or treatment, and the other factors noted above. They are generally administered in the same doses and by the same routes of administration as specified herein, or in amounts ranging from 1 to 99% of the dose specified herein, or in any dose and by any means. pathways that have been empirically/clinically assessed to be suitable.

For the prevention or treatment of a disease, the appropriate dose of an antibody or polypeptide comprising an Fc region variant of the invention (whether used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, type of polypeptide containing the Fc region variant, severity and course of disease, whether the antibody or polypeptide containing the Fc region variant is used for preventive or therapeutic purposes, prior therapy, patient medical history and response to the antibody or polypeptide containing the Fc region variant , as well as the instructions of the attending physician. An antibody or polypeptide containing an Fc region variant may be administered to a patient once or as a series of treatments. Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.1-10 mg/kg) of an antibody or polypeptide containing an Fc region variant may represent an initial potential dose to be administered to a patient. for example, by one or more separate treatments or by continuous infusion. One example of typical daily doses would be from about 1 μg/kg to 100 mg/kg or more depending on the factors noted above. When repeated administrations are used over several days or a longer period of time, depending on the condition, treatment should generally be continued until suppression of disease symptoms is achieved. One example dose of an antibody or polypeptide containing an Fc region variant is a dose of from about 0.05 to about 10 mg/kg. Thus, the patient may be administered one or more of the following doses: approximately 0.5, 2.0, 4.0, or 10 mg/kg (or any combination thereof). These doses can be administered in divided doses, for example, every week or every three weeks (for example, such that the patient receives from about two to about twenty, for example about six doses of the antibody or polypeptide containing an Fc region variant). An initial higher loading dose may be used followed by one or more lower doses. Using generally accepted methods and assays, the effect of this therapy can be easily monitored.

It is apparent that any of the above compositions or therapeutic methods can be administered using the immunoconjugate of the invention instead of or in addition to an anti-DENV antibody or an anti-ZIKV antibody. It is apparent that any of the above compositions or therapeutic methods can be administered using the immunoconjugate of the invention instead of or in addition to the Fc region variant-containing polypeptide provided herein.

H. Products

Another object of the invention is a product that contains products used for the treatment, prevention and/or diagnosis of the above disorders. The product consists of a container and a label or package insert placed on or inserted into the container. Acceptable containers include, for example, jars, vials, syringes, intravenous (IV) bags, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective for treating, preventing and/or diagnosing a condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial equipped with a stopper that can be pierced using a hypodermic needle). At least one active substance in the composition is an antibody or polypeptide containing a variant of the Fc region proposed/proposed in the invention. The label or package insert states that the composition is used to treat the selected condition. In addition, the product may include (a) a first container containing a composition, where the composition contains an antibody or polypeptide containing an Fc region variant as provided by the invention; and (b) a second container containing the composition, wherein the composition contains an additional cytotoxic or other therapeutic agent. According to this embodiment, the product may include a package insert that contains information that the compositions can be used to treat a particular condition. In an alternative or additional embodiment, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BSI), phosphate buffered saline, Ringer's solution, and dextrose solution. In addition, it may include other products required from a commercial and consumer point of view, in particular other buffers, diluents, filters, needles and syringes.

As should be apparent, any of the above articles may include an immunoconjugate of the invention instead of or in addition to an anti-DENV antibody or an anti-ZIKV antibody. As will be apparent, any of the above articles may include an immunoconjugate of the invention instead of or in addition to an Fc region variant-containing polypeptide.

Examples

Below are examples of methods and compositions proposed in the invention. As will be apparent, various other embodiments of the invention may be practiced in light of the general description of the invention presented above.

Example 1: Preparation of antigens and antibodies

Expression and purification of recombinant soluble E protein from DENV-1, DENV-2, DENV-3 and DENV-4

Recombinant soluble E proteins (0.8E-His) from DENV-1, DENV-2, DENV-3 and DENV-4 with a carboxy-terminal 8× histidine tag (SEQ ID NO: 65-68, respectively) were transiently expressed using a cell line FreeStyle293-F or Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). By expressing prM0.8E-His from DENV-1, DENV-2, DENV-3 and DENV-4 (SEQ ID NOs: 61-64, respectively), prM0.8E-His was expressed as an individual polypeptide in cells, which was then processed intracellularly , splitting between prM and 0.8E-His. As a result, 0.8E-His was secreted into the cell culture medium. Conditioned media containing 0.8E-His was applied to a column packed with immobilized metal affinity chromatography (IMAC) resin loaded with nickel or cobalt, followed by imidazole elution. Fractions containing 0.8E-His were pooled and applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing 0.8E-His were pooled and stored at -80°C.

Expression and purification of recombinant human FcγRs

Extracellular domains of human FcγRs were prepared as follows. First, the FcγR extracellular domain gene was synthesized using a method well known to those skilled in the art. Simultaneously, the sequence of each FcγR was obtained based on information registered in NCBI. In particular, the basis for the creation of FcγRIa was the NCBI sequence with access code NM_000566 (version no. NM_000566.3), the basis for the creation of FcγRIIa was the NCBI sequence with access code NM_001136219 (version no. NM_001136219.1), the basis for the creation of FcγRIIb was the NCBI sequence , having the NCBI accession code NM_004001 (version no. NM_004001.3), the basis for the creation of FcγRIIIa was the NCBI sequence, having the NCBI accession code NM_001127593 (version no. NM_001127593.1), and the basis for the creation of FcγRIIIb was the NCBI sequence, having the accession code NM_000570 ( version no. NM_000570.3), and a His tag was attached to the C terminus of each FcγR construct. In addition, polymorphisms are known to exist for FcγRIIa, FcγRIIIa and FcγRIIIb, and polymorphism sites were derived from the data described in Warmerdam et al. (J Exp Med 172, 1990, pp. 19-25) for FcγRIIa; in Wu et al. (J Clin Invest 100, 1997, pp. 1059-1070) for FcγRIIIa; and Ory et al. (J Clin Invest 84, 1989, pp. 1688-1691) for FcγRIIIb.

Expression vectors were created by inserting the resulting gene fragments into expression vectors for animal cells. The created expression vectors were briefly introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cancer cells to express proteins of interest. Liquids obtained by filtration through a 0.22 μm pore size filter of culture supernatants, which were obtained from the culture medium of the above cells subjected to short-term introduction, were purified using, in general, the following four steps: (I) cation exchange column chromatography (SP Sepharose FF ); (II) anti-His tag affinity column chromatography (HisTrap HP); (III) column gel filtration (Superdex 200); and (IV) sterilization by filtration. Anion exchange column chromatography using Q Sepharose FF was used to purify FcγRI in step (I). The absorbance at 280 nm of the purified proteins was measured using a spectrophotometer. Based on the measured values, the concentration of the purified proteins was determined using the extinction coefficient calculated by a method such as PACE (Protein Science 4, 1995, pp. 2411-2423).

Expression and purification of recombinant mouse FcγRs

Extracellular domains of mouse FcγRs (mFcγRs) were prepared as follows. First, the FcγR extracellular domain gene was synthesized using a method well known to those skilled in the art. To carry out this synthesis, the sequence of each FcγR was obtained based on information registered in NCBI. In particular, mFcγRI was obtained based on the reference sequence registered in NCBI: NP_034316.1; mFcγPIIb was obtained based on the reference sequence registered in NCBI: NP_034317.1; mFcγRIII was obtained based on the reference sequence registered in NCBI: NP_034318.2; and mFcγRIV were obtained based on the NCBI-registered reference sequence: NP_653142.2. Each of these sequences was tagged at the C-terminus with a His tag.

Each resulting gene fragment was inserted into vectors for expression in animal cells to obtain expression vectors. FreeStyle 293 cells (Invitrogen) derived from human embryonic kidney cancer cells were transiently transfected with the resulting expression vectors to express the protein of interest. The resulting culture supernatant was isolated and then passed through a 0.22 μm pore size filter to obtain the culture supernatant. The resulting culture supernatant was purified typically using the following four steps: (I) ion exchange column chromatography; (II) anti-His tag affinity column chromatography (HisTrap HP); (III) column gel filtration (Superdex 200); and (IV) sterilization by filtration. 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 a solvent in step (III) or later stages, and D-PBS(-) containing 0.1 M arginine was used for mFcγRIII. Absorbance at 280 nm of purified proteins was measured using a spectrophotometer. Based on the measured values, the concentration of the purified proteins was determined using the extinction coefficient calculated by a method such as PACE (Protein Science 4, 1995, pp. 2411-2423).

Expression and purification of recombinant human FcRn

FcRn is a heterodimer of FcRn consisting of an alpha chain and beta2-microglobulin. Oligo-DNA primers were derived from the published sequence of the human FcRn gene (J Exp Med, 180, 1994, pp. 2377-2381). A DNA fragment encoding the complete gene was obtained by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template, and primers were prepared. Using the resulting DNA fragment as a template, the DNA fragment encoding the extracellular domain containing the signal region (Met1-Leu290) was amplified by PCR and inserted into an expression vector for mammalian cells. Similarly, oligo-DNA primers were prepared based on the published sequence of the human beta2-microglobulin gene (Proc Natl Acad Sci USA 99, 2002, cc. 16899-16903). A DNA fragment encoding the complete gene was obtained by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template, and primers were prepared. Using the resulting DNA fragment as a template, the DNA fragment encoding the complete protein containing the signal region (Met1-Met119) was amplified by PCR and inserted into a mammalian cell expression vector.

Soluble human FcRn was expressed using the following procedure. Plasmids designed to express human FcRn alpha chain (SEQ ID NO: 81) and beta2-microglobulin (SEQ ID NO: 82) were introduced into cells of the human embryonic kidney cancer cell line HEK293H (Invitrogen) using the lipofection method. using PEI (polyethylimine) (Polyscience). The resulting culture supernatant was collected and FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences), followed by further purification using HiTrap Q HP (GE Healthcare) (J Immunol 169, 2002, pp. 5171-5180).

Expression and purification of recombinant antibodies

Recombinant antibodies were expressed transiently using either the FreeStyle293-F cell line or the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Purification from the conditioned medium expressing antibodies was carried out by a conventional method using protein A. If necessary, additional gel filtration was performed.

Expression and purification of recombinant soluble human CD154

The human CD154 gene was synthesized based on published protein sequence data (NP_000065.1). A DNA fragment encoding a soluble form of human CD154 (shCD154) with a FLAG tag was obtained by PCR using the synthesized DNA as a template, and the resulting DNA fragments were inserted into an expression vector for mammalian cells, thereby obtaining the expression vector FLAG-shCD154 (SEQ ID NO: 106). The nucleotide sequences of the resulting expression vectors were determined using conventional methodologies known to those skilled in the art. FLAG-shCD154 was expressed using the FreeStyle 293 cell line (Invitrogen) according to the manufacturer's protocol. After transfection, cells were grown for the required period of time, after which conditioned media was collected. The conditioned medium was subjected to cation exchange chromatography using 25 mM MES (pH 6.0), and FLAG-shCD154 was eluted using a continuous gradient of 25 mM MES, 1 M NaCl (pH 6.0). Peak fractions were pooled, concentrated using an AmiconUltra Ultracel device and subjected to gel filtration using phosphate buffered saline (Wako). Peak fractions were again pooled and concentrated using AmiconUltra Ultracel, then sterilized by filtration using a 0.22 μm PVDF membrane filter. To determine the concentration of purified FLAG-shCD154, absorbance was measured at 280 nm using a spectrophotometer. Protein concentrations were calculated from the measured values using the absorption coefficient calculated by the method described in Protein Science 4, 1995, pp. 2411-2423.

Example 2: Generation of Antibody Variants with Improved Affinity for DENV E Protein

Genes encoding VH (3CH, SEQ ID NO: 1) and VL (3CL, SEQ ID NO: 7) antibodies to DENV E protein 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 in the same expression vector. In the present description, the antibody is designated as DG_3CH-SG182/3CL-SK1 or as 3C.

The number of mutations and their combinations was assessed to identify mutations and combinations that increase the binding properties of 3C. Multiple mutations were then introduced into the variable regions to increase binding affinity to the E protein. Thus, optimized VH variants were created, such as 3CH912 (SEQ ID NIO: 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 variants such as 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). Genes encoding VH were combined with the human IgG1 CH gene (either SG182, SEQ ID NO: 46; SG1095, SEQ ID NO: 54; or SG1106, SEQ ID NO: 59), and genes encoding VL were combined with the human CL (SK1, SEQ ID NO: 60). Each of the constructs was cloned into an expression vector. The amino acid sequence data of the antibody variants are summarized in Table 2. One of the variants, DG_3CH1047-SG182/3CL658-SK1, is also referred to herein as 3Cam, and the other variant, DG_3CH1047-SG182/3CL-SK1, is also referred to herein as 3Cam2 .

Antibodies were expressed in HEK293 cells cotransfected with a mixture of heavy and light chain expression vectors and purified using protein A.

The affinity of antibodies to DENV E protein binding to DENV-1, DENV-2, DENV-3 and DENV-4 E protein was determined at 25°C using a Biacore T200 device (GE Healthcare). Anti-histidine antibody (GE Healthcare) was immobilized onto all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in PBS, pH 7.4, containing 20 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20 and 0.005% NaN ₃ . The E protein of DENV-1, DENV-2, DENV-3 and DENV-4 with a C-terminal His tag was captured on flow cell 2 or 3, and flow cell 1 was the reference flow cell E protein immobilization level had to correspond to 200 resonance units (RU). Antibodies to DENV E protein were injected at a concentration of 250 nM onto the entire sensory surface for 180 s, followed by dissociation for 300 s. The sensory surface was regenerated after each cycle using 10 mM Gly-HCl, pH 1.5. Binding affinity was determined by processing and fitting the data to a 1:1 binding model using BIACORE® T200 Evaluation software, version 2.0 (GE Healthcare).

Tables 3a and 3b present data on the affinity (K _d ) of antibodies to the DENV E protein that bind to the E protein DENV-1 (indicated in the table as DV1), DENV-2 (indicated in the table as DV2), DENV-3 ( designated in table DV3) and DENV-4 (designated in table DV4). Each 3C variant demonstrated increased binding affinity to all four DENV serotypes compared to parental 3C. As an example, in FIG. Figure 1 shows sensorgrams of the parent antibody 3C (DG_3CH-SG182/3CL-SK1) and one of the variants of the 3Cam antibody (DG_3CH1047-SG182/3CL658-SK1). In addition, in FIG. Figure 12 shows sensorgrams of the parent antibody 3C (DG_3CH-SG182/3CL-SK1) and one of the variants of the 3Cam2 antibody (DG_3CH1047-SG182/3CL-SK1).

Example 3: Creation of CH variants to improve antibody performance

Multiple mutations were introduced into the human IgG1 CH heavy chain constant region (SG182, SEQ ID NO: 46), resulting in the 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). Genes encoding CH variants were combined with VH 3C (3CH, SEQ ID NO: 1), one of the 3C variants (3CH1047, SEQ ID NO: 6) or with an antibody to CD154 (SLAPH0336a, SEQ ID NO: 88). The anti-CD154 VL gene (SLAPL0336a, SEQ ID NO: 89) was combined with the human CL gene (SK1, SEQ ID NO: 60). Each of the constructs was cloned into an expression vector. Details regarding CH variants are summarized in Table 4. The variable region of the anti-CD154 antibody SLAPH0336a/SLAPL0366a, which can form a large immune complex in the presence of trimeric CD154 antigen, was used to assess the binding avidity of CH variants to each Fc receptor.

Antibodies were expressed in HEK293 cells cotransfected with a mixture of heavy and light chain expression vectors and purified using protein A.

Example 4: Binding Affinities of Antibody Having Fc Variants to Complement C1q

Human C1q binding assay

Anti-CD154 Fc-variant antibodies (antibodies produced in our laboratory, which were generated using the method described in Example 3) were distributed onto Nunc-ImmunoPlate MaxiSorp plates (Nalge Nunc International) and allowed to stand overnight at 4°C. After washing with PBS-T, the plate was blocked with TBST containing 0.5% BSA and 1× Block Ace: blocking reagent (DS Pharma) for 2 hours at room temperature. After washing the plate, human C1q (Calbiochem) was spread onto the plate and allowed to stand for 1 hour at room temperature. The plate was washed and HRP-labeled anti-human C1q antibody (Bio Rad) was added, allowed to react for 1 hour at room temperature, and washed. TMB substrate (Invitrogen) was then added. The signal was measured using a plate reader at 450 nm (working wavelength) and 570 nm (reference wavelength). The binding affinity of an antibody having a wild-type (WT) Fc region to human C1q was reduced by the introduction of LALA mutations into the Fc region, and the reduced binding affinity to human C1q was restored by the introduction of additional KWES, EFT, or EFT+AE mutations in addition to LALA (Fig. .2). The LALA+KMES mutations slightly increased binding affinity, while the LALA+KWES, LALA+KAES, and LALA+KEES mutants bound C1q with affinities comparable to those of the WT (Fig. 3). The binding characteristics of the LALA or LALA+KAES mutants described above were not altered by the additional introduction of AC3 or ACT5 mutations (Fig. 4).

Mouse C1q binding assay

Anti-CD154 Fc-variant antibodies (antibodies produced in our laboratory, which were generated using the method described in Example 3) were distributed onto Nunc-ImmunoPlate MaxiSorp plates (Nalge Nunc International) and allowed to stand overnight at 4°C. After washing with PBS-T, the plate was blocked with TBST containing 0.5% BSA and 1× Block Ace: blocking reagent (DS Pharma) for 7 hours at 4°C. After washing the plate, 10% mouse plasma (Innovative Research) was spread on the plate and allowed to stand overnight at 4°C. The plate was washed and biotinylated anti-mouse C1q antibody (Hycult Biotech) was added, allowed to react for 1 hour at room temperature, and washed. Streptavidin-HRP (Pierce) was added, allowed to react for 1 hour at room temperature, and washed. Subsequently, ABTS substrate for HRP (KPL) used in ELISA was added. The signal was measured using a plate reader at a length of 405 nm. The binding affinity of an antibody having a wild-type (WT) Fc region to mouse C1q was reduced by the introduction of LALA mutations into the Fc region, and the reduced binding affinity to mouse C1q was restored by the introduction of an additional KAES mutation in addition to LALA. The binding characteristics of the LALA or LALA+KAES mutants described above were not altered by the introduction of additional ACT3 or ACT5 mutations (Fig. 5).

Example 5: Biacore analysis of binding of Fc variants to FcγR and FcRn

The binding of Fc variants to human or murine FcγR and human FcRn at pH 7.4 was determined at 25°C using a Biacore T200 device (GE Healthcare). All antibodies and FcγR or FcRn were prepared in PBS-R, pH 7.4, containing 50 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20 and 0.005% NaN ₃ . For the FcγR binding assay, anti-histidine antibody (GE Healthcare) was immobilized with anti-histidine antibody onto all flow cells of the CM4 sensor chip using an amine coupling kit (GE Healthcare). Each of the FcγRs was immobilized on flow cell 2, 3, or 4, and flow cell 1 was the reference flow cell. FcγR immobilization levels were required to correspond to 400 resonance units (RU). All antibodies were injected at a concentration of 100 nM into all flow cells. The immune complex was prepared by mixing antibody and trimeric CD154 in a 1:1 molar ratio and incubated at room temperature for 1 hour. The sensor surface was regenerated after each cycle using 10 mM glycine-HCl, pH 1.5.

For the FcRn binding assay, a biotinylated capture reagent (Biotin CAPture reagent (GE Healthcare)) was immobilized onto both flow cells 1 and 2 of the CAP sensor chip using the Biotin CAPture kit (GE Healthcare). Biotinylated FcRn was immobilized on flow cell 2, and flow cell 1 was a reference flow cell. FcRn immobilization levels were required to correspond to 400 RU. All antibodies were injected at a concentration of 100 nM into flow cells 1 and 2. Immune complexes were prepared by mixing the antibody and trimeric CD154 in a 1:1 molar ratio and incubated at room temperature for 1 hour. The sensor surface was regenerated after each cycle using 8 M guanidine- HCl, 1M NaOH (3:1 v/v).

Binding levels were standardized against the level of immobilization of the corresponding FcγR or FcRn. Binding of the antibody alone or the immune complex (antibody and trimeric CD154 antigen) to human or murine FcγR and human FcRn was monitored based on binding responses. The immune complex was used to evaluate the resulting increased avidity effect of binding to FcγR or FcRn. The results for human FcγR1a, FcγR2a 167H and 167R, FcγR2b, FcγR3a 158F and 158V, FcγR3b NA1 and NA2 are presented in Fig. 6(a)-(h). The results for mouse FcγR1, FcγR2b, FcγR3, FcγR4 are presented in Fig. 7 (a)-(d). Binding of the wild-type Fc region (designated WT IgG) was significantly reduced when the LALA, LALA+KAES, or LALA+KWES mutations were introduced into the Fc region for each of the FcγRs studied. This trend was roughly maintained in assays using antibody alone (denoted Ab alone) and immune complex (denoted CD154 IC). Binding of KAES or KWES mutants was not significantly reduced compared with WT IgG for most FcγRs studied. Results for human FcRn are presented in FIG. 8. Binding of the wild-type Fc region (designated hIgG1) to human FcRn was not noticeably altered even after the introduction of the LALA or LALA+KAES mutations into the Fc region. Binding increased slightly after additional introduction of ACT3 or ACT5 mutations, but still remained relatively low (Fig. 8).

Example 6: In Vivo Efficacy of Anti-DENV Antibodies against DENV Infection

6-8 week old AG129 mice were infected intraperitoneally using ¹⁰⁶ plaque forming units (PFU) with DENV-2 strain D2Y98P. After 48 hours, mice were treated with 1-30 μg of antibody in P1 buffer. The antibody was injected intravenously through the retroorbital route. After 24 hours, i.e. 72 h after initial infection, blood samples were collected. Previous studies (Zust et al., J Virol 88, 2014, pp. 7276-7285; Tan et al., PLOS Negl Trop Dis 4, 2010, e672) found that the peak of viremia after D2Y98P infection was reached between 3-4 days after infection. Viral RNA was extracted from the plasma of each mouse and subjected to qPCR and compared to a DENV-2 standard of known infectivity using a plaque assay. Treatment with both antibodies 3C and 3Cam significantly reduced viremia compared with PBS control in this mouse model, and the effectiveness of both antibodies was comparable. An antibody containing the LALA+KAES mutation in the Fc region was more effective than an antibody containing only the LALA mutation. This was the case for both antibodies 3C and 3Cam (Fig. 9). This result suggests that the restoration of C1q-binding activity as a result of the addition of the KAES mutation to the LALA mutation is involved in the antiviral effectiveness of the antibodies.

6-8 week old AG129 mice were infected intraperitoneally using 10 ⁶ -10 ⁷ plaque forming units (PFU) with DENV virus (DENV-1 strain 08K3126, DENV-2 strain D2Y98P, DENV-3 strain VN32/96, DENV-4 strain TVP360 ). After 48 hours, mice were treated with 1-30 μg of antibody in P1 buffer. The antibody was injected intravenously through the retroorbital route. After 24 hours, i.e. 72 h after initial infection, blood samples were collected. Previous studies (Zust et al., J Virol 88, 2014, cc. 7276-7285; Tan et al., PLOS Negl Trop Dis 4, 2010, e672) found that peak viremia after D2Y98P infection was reached between 3-4 days after infection. Viral RNA was extracted from the plasma of each mouse and subjected to qPCR and compared to a DENV standard of known infectivity using a focus assay. Both 3C and 3Cam2 antibodies with the same Fc variant (LALA+KAES) significantly reduced viremia compared to P1 buffer control in this mouse model, and 3Cam2 reduced viremia to a greater extent than 3C antibody (Fig. 10). .

Antibody 3Cam2, having the LALA+KAES mutation in the F region, had greater efficacy compared to the antibody having only the LALA mutation, when administered at a higher dose of the antibody (Fig. 11). This result suggests that the restoration of C1q-binding activity as a result of the addition of the KAES mutation to the LALA mutation is involved in the antiviral effectiveness of the antibodies.

Although the invention has been described above for better understanding by providing certain details for purposes of illustration and examples, the description and examples should not be construed as limiting the scope of the invention. The description of all patent and scientific literature cited herein is expressly incorporated herein by reference in its entirety.

Example 7: In vitro testing of cross-reactivity with Zika virus of dengue virus-specific antibodies 3C and 3Cam2

7.1. FRNT analysis

To evaluate the neutralization ability of DG_3CH1047-SG1106/3CL_SK1 (or 3Cam2-LALA+KAES+ACT5) against Zika virus, a focus reduction neutralization (FRNT) assay (virus neutralization reaction) was performed. As a result, it was found that the 3Cam2-LALA+KAES+ACT5 antibody had a more pronounced neutralizing ability against the Zika virus compared to DG_3CH-SG1106/3CL_SK1 (or 3C-LALA+KAES+ACT5) (Fig. 13). The average neutralizing capacity or EC ₅₀ value for Zika virus is presented in Table 5.

Below are the sequences of the antibodies studied:

3Cam2:

DG_3СН1047 (SEQ ID NO: 6)-SG182 (SEQ ID NO: 46)/3CL (SEQ ID NO: 7)_ SK1 (SEQ ID NO: 60)

3Cam2-LALA+KAES:

DG_3CH1047 (SEQ ID NO: 6)-SG1095 (SEQ ID NO: 54)/3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO: 60)

3Cam2-LALA+KAES+ACT5:

DG_3CH1047 (SEQ ID NO: 6)-SG1106 (SEQ ID NO: 59)/3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO: 60)

3C:

DG_3CH (SEQ ID NO: 1)-SG182 (SEQ ID NO: 46)/3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO: 60)

3C-LALA:

DG_3CH (SEQ ID NO: 1)-SG192 (SEQ ID NO: 47)/3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO: 60)

3C-LALA+KAES+ACT5:

DG_3CH (SEQ ID NO: 1)-SG1106 (SEQ ID NO: 59)/3CL (SEQ ID NO: 7)_SK1 (SEQ ID NO: 60)

7.2. ADE analysis for Zika virus

To address the question of whether the 3Cam2-LALA+KAES+ACT5 antibody could abolish not only the enhancement of DENV infection, but also the enhancement of Zika virus infection compared to the wild-type Fc antibody, an ADE assay was performed using K562 cells (Fig. 14). The same methodology as for DENV was used. Enhancement was detected with the antibody with the wild-type Fc version DG_3CH1047-SG182/3CL_SK1 (or 3Cam2), but not with its Fc variants DG_3CH1047-SG1095/3CL_SK1 (or 3Cam2-LALA+KAES) and 3Cam2-LALA+KAES+ACT5 (Fig. 14A). The experiment, repeated using a new batch of 3Cam2 At, confirmed the reversal of enhancement with the Fc variant antibody.

Overall, the data demonstrated that the LALA mutation abolished the enhancement of both DENV and Zika virus infection, and that additional KAES and ACT5 mutations did not reduce the effect of LALA.

7.3. In vitro methods

7.3.1. Virus

Zika virus (Asian strain, registration number KF993678) was obtained from the Environmental Health Institute, Singapore. The virus was grown in C6/36 cells (ATCC) and quantified using the following focusing assay: 20-24 hours before infection, 1 × 10 ⁵ BHK-21 cells were seeded per well of 24-well plates and incubated overnight at 37°C. C, 5% CO ₂ . The virus-containing supernatant was serially diluted in a ratio from 1:100 to 1:100,000 and incubated in a volume of 500 μl on cell monolayers at 37°C, 5% CO ₂ for 2-3 hours. At the end of the incubation period, 0. 5 ml 0.8% methylcellulose in RPMI. The plates were incubated at 37°C, 5% CO ₂ for days.

To stain infected cells, the culture medium with methylcellulose was removed and the cells were fixed with 3.7% formaldehyde for 30 min. After formaldehyde was removed, the plates were washed with tap water and the cells were permeabilized with 1% Triton X-100 in 1× PBS. After a second washing step, 4G2 antibody diluted in l%FBS/1×PBS was used to stain infected cells, followed by incubation with polyclonal goat anti-mouse antibody-HRP diluted 1:2000 in 1% FBS/PBS. Trueblue Peroxidase Substrate (KPL) was used for the color reaction. Foci were counted manually in each well and the initial concentration was calculated based on the average counts for all wells in which counts were countable.

7.3.2. FRNT analysis

20-24 hours before infection, 1×10 ⁵ BHK-21 cells were seeded into a well of 24-well plates and incubated overnight at 37°C, 5% CO ₂ . Antibody dilutions were prepared in 96-well sterile U-bottom plates in RPMI medium and a constant amount of virus was added. This mixture was incubated for 30 min at 37°C, 5% CO ₂ before adding to the cells. After overnight incubation, the medium from the 24-well plates was removed, replacing 450 μl of fresh medium with 5% FBS/RPMI, and 50 μl of the virus/antibody mixture was added to the wells and incubated at 37°C, 5% CO ₂ for 2-3 h.

At the end of the incubation period, each well was layered with 0.5 ml of 0.8% methylcellulose in RPMI. The plates were incubated at 37°C, 5% CO ₂ for days.

To stain infected cells, the culture medium with methylcellulose was removed and the cells were fixed with 3.7% formaldehyde for 30 min. After formaldehyde was removed, the plates were washed with tap water and the cells were permeabilized with 1% Triton X-100 in 1× PBS. After a second wash step, 4G2 antibody diluted in 1% FBS/1×PBS was used to stain infected cells, followed by incubation with polyclonal goat anti-mouse antibody-HRP diluted 1:2000 in 1% FBS/PBS. Trueblue Peroxidase Substrate (KPL) was used for the color reaction. Foci were counted manually in each well and EC ₅₀ values were calculated using Prism (three-parameter curve fitting) software.

7.3.3. ADE analysis using K562 cells

^6x104 k562 cells/well were seeded in RPMI/10%FCS medium in 96-well round-bottom tissue culture plates and cultured overnight. Serial dilutions of the antibody were prepared in other round-bottom plates and a constant amount of virus was added. The mixture was incubated for 30 min at 37°C, after which 50 μl was added to K562 cells. A multiplicity of infection (MOI) of 1 was used. After overnight incubation, the medium was removed and the antibody/virus mixtures were added. After incubation for 90 min at 37°C, RPMI, 10% FCS was added for an additional 2.5 days of incubation. The cells were then fixed with Cytofix/Cytoperm solution (Becton Dickinson) and then stained with the anti-E protein antibody 4G2-Alexa647 and the anti-NS1-Alexa488 antibody. Samples were analyzed using a FACS Verse flow cytometer with a 96-well HTS device (BD).

Example 8: In vivo testing of the protective ability of dengue virus-specific antibodies 3C and 3Cam2 against Zika virus infection

8.1. Efficacy of Antibody Therapeutic Treatment

Since AG129 mice are highly susceptible to the Zika virus and develop infection even earlier than after infection with DENV, the IFNAR model was chosen instead of this mouse strain to study the Zika virus.

IFNAR mice are deficient in type I IFN receptor (IFNAR) but have normal expression of type II IFN receptor or IFN gamma receptor (IFNGR).

The dose of the virus is 10 ⁴ PFU/mouse, selected on the basis of a well-known publication (Cell Host Microbe. 19(5), 2016, cc. 720-730. doi: 10.1016/j.chom.2016.03.010). The strain of Zika virus used was isolated in Canada from a traveler returning from Thailand. The strain belongs to the Asian line. Mice were treated with the antibody at a dose of 100 μg.

Mice treated with 3Cam2-LALA+KAES+ACT5 2 days before Zika virus infection showed significantly lower viremia at day 3 and day 5 post-infection compared with control mice treated with P1 buffer. Antibody 3Cam2-LALA+KAES+ACT5 reduced viremia more effectively than 3C-LALA+KAES+ACT5 (Fig. 15). Survival analysis demonstrated that in both antibody-treated groups the animals survived until at least day 50 (end of the experiment), in contrast to the P1-treated group in which all mice died 15 days after infection (Fig. 16).

8.2. In vivo methods

8.2.1. Preparation of Antibody for In Vivo Treatment

The antibody was concentrated to several milligrams/ml in P1 buffer (20 mM His, 150 mM Arg-Asp, pH 6.0) and diluted in P1 buffer to the required concentration immediately before in vivo treatment.

8.2.2. Mice

AG129 or IFNAR mice were used in in vivo experiments according to the indicated method. AG129 mice were purchased from B&K Universal, UK. IFNAR mice (B6.129S2-Ifnar1tm1 Agt/Mmjax) were purchased from The Jackson Laboratory (USA).

8.2.3. Therapeutic treatment

Mice were infected intraperitoneally (ip) using 10 ⁴ PFU/mouse. At 48 h postinfection, mice were anesthetized with isoflurane for intravenous (iv) injection of antibody. Abs were diluted in P1 buffer and injected via the retroorbital route in a volume of 100 μl.

Example 9: In vitro efficacy of 3Cam2-LALA+KAES+ACT5 against Zika virus

9.1 Linking

The binding of human antibodies DG_3CH-SG182/3CL_SK1 (or 3C), DG_3CH-SG192/3CL_SK1 (or 3C-LALA) and 3Cam2-LALA+KAES+ACT5 to Zika virus (ZIKV) was assessed.

In general, the method was as follows: MAbs were diluted and then incubated for 1 h at 37°C on purified ZIKV-coated ELISA plates. An HRP-conjugated anti-human IgG secondary antibody was used to detect the binding of 3C, 3C-LALA, and 3Cam2-LALA+KAES+ACT5 to ZIKV virions. TMB substrate was used for detection. After the reaction stopped, absorbance was measured using duplicate readings.

Antibody 3Cam2-LALA+KAES+ACT5 bound to ZIKV similarly to antibodies 3C and 3C-LALA (Fig. 17).

9.2 Neutralization

The ability of the antibody to inhibit ZIKV infection of cells was also studied. ZIKV was mixed using an MOI of 10 with diluted human MAb and incubated for 2 h at 37°C with gentle mixing. Virus/MAb mixtures were then added to HEK 293T cells seeded in 96-well plates. After culture for 2 days at 37°C, the level of cell infection was measured by detection of ZIKV-specific antigen using flow cytometry. All reads were performed in triplicate, and neutralizing capacity was calculated relative to cells infected with ZIKV in the absence of antibodies.

All antibodies, i.e. 3Cam2-LALA+KAES+ACT5, 3C and 3C-LALA had similar abilities to neutralize ZIKV infection of HEK cells (Fig. 18).

Example 10: In vivo efficacy of 3Cam2-LALA+KAES+ACT5 against Zika virus: maternal-to-fetal transmission

Further, in vivo experiments studied the ability of antibodies to inhibit transmission of the virus from mother to offspring.

Pregnant IFNαR knockdown mice were inoculated with ZIKV 10.5 days after mating. Antibodies were administered on days −1, 0, and 3 postinfection. On day 16.5 after mating, pregnant mice were selected and fetuses were removed. The weight of the fetus was determined, including the weight of the whole body and the weight of the head; viral load was measured in amniotic fluid, placenta, fetal brain and liver.

Fetuses from treated mothers were significantly heavier than untreated mouse fetuses, suggesting protection against the congenital developmental failure found in untreated ZIKV-infected animals (Fig. 19). The antibodies significantly inhibited transmission of the virus from the pregnant mother to the fetus (Fig. 20).

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LIST OF SEQUENCES

<110> CHUGAI SEYAKU KABUSHIKI REPENT

<120> ANTIBODIES TO DENGUE VIRUS WITH CROSS-REACTIVITY

WITH ZIKA VIRUS, AND WAYS OF THEIR APPLICATION

<130> 34246SG6/MBR(JKN)/ndh

<160> 109

<170> Patent In 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

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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

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Gln Val Gln Leu Val Gln Ser Gly Pro Asp Val Glu Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn

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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

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<213> Artificial Sequence

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<223>VH

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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

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<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

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<211> 132

<212>PRT

<213> Artificial Sequence

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<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

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Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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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

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<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

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<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

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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

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Ser Asn Tyr Ile His

15

<210> 12

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<223> HVR-H1

<400> 12

Ser Tyr Tyr Met His

15

<210> 13

<211> 17

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<400> 13

Val Ile Asn Pro Arg Gly Gly Ser Thr Ala Ser Ala Gln Lys Phe Gln

1 5 10 15

Gly

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<400> 14

Val Ile Asn Pro Arg Gly Gly Ser Arg Ala Ser Ala Gln Lys Phe Gln

1 5 10 15

Gly

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<400> 15

Val Ile Asn Pro Arg Gly Gly Ser Arg Arg Ser Ala Gln Lys Phe Gln

1 5 10 15

Gly

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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

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<212>PRT

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<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

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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

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<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

15

<210> 25

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> HVR-L2

<400> 25

Asp Ala Ser Asn Leu Lys Phe

15

<210> 26

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> HVR-L2

<400> 26

Asp Ala Ser Glu Leu Lys Thr

15

<210> 27

<211> 9

<212>PRT

<213> Homo sapiens

<400> 27

Gln Gln Phe Asp Asp Leu Pro Ile Thr

15

<210> 28

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> HVR-L3

<400> 28

Gln Gln Phe Asp Ala Leu Pro Ile Thr

15

<210> 29

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> HVR-L3

<400> 29

Gln Gln Phe Ser Ala Leu Pro Ile Thr

15

<210> 30

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> HVR-L3

<400> 30

Gln Gln Phe Glu Asp Leu Pro Ile Thr

15

<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

15

<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

15

<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

15

<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

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> 53

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 53

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 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> 54

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223>CH

<400> 54

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

Ala 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> 55

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 55

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

Asp 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> 56

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223>CH

<400> 56

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

Glu 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> 57

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223>CH

<400> 57

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

Met 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> 58

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 58

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

Ala 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 Leu His Glu Ala Leu His Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 59

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223>CH

<400> 59

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

Ala 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 Leu His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 60

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223>CL

<400> 60

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 61

<211> 585

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 61

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Phe His Leu Thr Thr Arg Gly Gly Glu Pro His Met Ile Val Ser Lys

20 25 30

Gln Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ser Ala Gly Val Asn

35 40 45

Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Leu Cys Glu Asp Thr

50 55 60

Met Thr Tyr Lys Cys Pro Arg Ile Thr Glu Ala Glu Pro Asp Asp Val

65 70 75 80

Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val Thr Tyr Gly Thr Cys

85 90 95

Ser Gln Thr Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala Leu Ala

100 105 110

Pro His Val Gly Leu Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser

115 120 125

Ser Glu Gly Ala Trp Lys Gln Ile Gln Lys Val Glu Thr Trp Ala Leu

130 135 140

Arg His Pro Gly Phe Thr Val Ile Ala Leu Phe Leu Ala His Ala Ile

145 150 155 160

Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe Ile Leu Leu Met Leu

165 170 175

Val Thr Pro Ser Met Ala Met Arg Cys Val Gly Ile Gly Asn Arg Asp

180 185 190

Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val Leu Glu

195 200 205

His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp

210 215 220

Ile Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val Leu Arg Lys

225 230 235 240

Leu Cys Ile Glu Ala Lys Ile Ser Asn Thr Thr Thr Asp Ser Arg Cys

245 250 255

Pro Thr Gln Gly Glu Ala Thr Leu Val Glu Glu Gln Asp Ala Asn Phe

260 265 270

Val Cys Arg Arg Thr Val Val Asp Arg Gly Trp Gly Asn Gly Cys Gly

275 280 285

Leu Phe Gly Lys Gly Ser Leu Leu Thr Cys Ala Lys Phe Lys Cys Val

290 295 300

Thr Lys Leu Glu Gly Lys Ile Val Gln Tyr Glu Asn Leu Lys Tyr Ser

305 310 315 320

Val Ile Val Thr Val His Thr Gly Asp Gln His Gln Val Gly Asn Glu

325 330 335

Thr Thr Glu His Gly Thr Ile Ala Thr Ile Thr Pro Gln Ala Pro Thr

340 345 350

Ser Glu Ile Gln Leu Thr Asp Tyr Gly Thr Leu Thr Leu Asp Cys Ser

355 360 365

Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Thr Met Lys

370 375 380

Glu Lys Ser Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu Pro Leu

385 390 395 400

Pro Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn Arg Gln

405 410 415

Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gln Glu Val

420 425 430

Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly

435 440 445

Ala Thr Glu Ile Gln Thr Ser Gly Thr Thr Thr Ile Phe Ala Gly His

450 455 460

Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr Leu Lys Gly Met Ser

465 470 475 480

Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val Ala Glu

485 490 495

Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly Thr Asp

500 505 510

Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly Val Thr

515 520 525

Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp Lys Glu

530 535 540

Lys Pro Val Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr Ile

545 550 555 560

Ile Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe Lys Lys

565 570 575

Gly His His His His His His His

580 585

<210> 62

<211> 585

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 62

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val Ser Arg

20 25 30

Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Asp Gly Val Asn

35 40 45

Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu Asp Thr

50 55 60

Ile Thr Tyr Lys Cys Pro Phe Leu Arg Gln Asn Glu Pro Glu Asp Ile

65 70 75 80

Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys

85 90 95

Thr Thr Thr Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala Leu Val

100 105 110

Pro His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser

115 120 125

Ser Glu Gly Ala Trp Lys His Ala Gln Arg Ile Glu Thr Trp Ile Leu

130 135 140

Arg His Pro Gly Phe Thr Ile Met Ala Ala Ile Leu Ala Tyr Thr Ile

145 150 155 160

Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu Thr Ala

165 170 175

Val Ala Pro Ser Met Thr Met Arg Cys Ile Gly Ile Ser Asn Arg Asp

180 185 190

Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val Leu Glu

195 200 205

His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp

210 215 220

Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys

225 230 235 240

Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Asp Ser Arg Cys

245 250 255

Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys Arg Phe

260 265 270

Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly Cys Gly

275 280 285

Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Thr Cys Lys

290 295 300

Lys Asn Met Lys Gly Lys Val Val Gln Pro Glu Asn Leu Glu Tyr Thr

305 310 315 320

Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly Asn Asp

325 330 335

Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser Ser Ile

340 345 350

Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu Cys Ser

355 360 365

Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln Met Glu

370 375 380

Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu Pro Leu

385 390 395 400

Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser Asn Trp Ile Gln Lys

405 410 415

Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln Asp Val

420 425 430

Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly

435 440 445

Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr Gly His

450 455 460

Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly Met Ser

465 470 475 480

Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile Ala Glu

485 490 495

Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly Asp Gly

500 505 510

Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys Arg His

515 520 525

Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu Lys Asp

530 535 540

Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile

545 550 555 560

Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys

565 570 575

Gly His His His His His His His

580 585

<210> 63

<211> 583

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 63

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Phe His Leu Thr Ser Arg Asp Gly Glu Pro Arg Met Ile Val Gly Lys

20 25 30

Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ala Ser Gly Ile Asn

35 40 45

Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Asp Asp Thr

50 55 60

Val Thr Tyr Lys Cys Pro His Ile Thr Glu Val Glu Pro Glu Asp Ile

65 70 75 80

Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys

85 90 95

Asn Gln Ala Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala Leu Ala

100 105 110

Pro His Val Gly Met Gly Leu Asp Thr Arg Thr Gln Thr Trp Met Ser

115 120 125

Ala Glu Gly Ala Trp Arg Gln Val Glu Lys Val Glu Thr Trp Ala Leu

130 135 140

Arg His Pro Gly Phe Thr Ile Leu Ala Leu Phe Leu Ala His Tyr Ile

145 150 155 160

Gly Thr Ser Leu Thr Gln Lys Val Val Ile Phe Ile Leu Leu Met Leu

165 170 175

Val Thr Pro Ser Met Thr Met Arg Cys Val Gly Val Gly Asn Arg Asp

180 185 190

Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val Leu Glu

195 200 205

His Gly Gly Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp

210 215 220

Ile Glu Leu Gln Lys Thr Glu Ala Thr Gln Leu Ala Thr Leu Arg Lys

225 230 235 240

Leu Cys Ile Glu Gly Lys Ile Thr Asn Ile Thr Thr Asp Ser Arg Cys

245 250 255

Pro Thr Gln Gly Glu Ala Ile Leu Pro Glu Glu Gln Asp Gln Asn Tyr

260 265 270

Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp Gly Asn Gly Cys Gly

275 280 285

Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys Phe Gln Cys Leu

290 295 300

Glu Ser Ile Glu Gly Lys Val Val Gln His Glu Asn Leu Lys Tyr Thr

305 310 315 320

Val Ile Ile Thr Val His Thr Gly Asp Gln His Gln Val Gly Asn Glu

325 330 335

Thr Gln Gly Val Thr Ala Glu Ile Thr Ser Gln Ala Ser Thr Ala Glu

340 345 350

Ala Ile Leu Pro Glu Tyr Gly Thr Leu Gly Leu Glu Cys Ser Pro Arg

355 360 365

Thr Gly Leu Asp Phe Asn Glu Met Ile Leu Leu Thr Met Lys Asn Lys

370 375 380

Ala Trp Met Val His Arg Gln Trp Phe Phe Asp Leu Pro Leu Pro Trp

385 390 395 400

Thr Ser Gly Ala Thr Thr Lys Thr Pro Thr Trp Asn Arg Lys Glu Leu

405 410 415

Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys Gln Glu Val Val Val

420 425 430

Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr

435 440 445

Glu Ile Gln Thr Ser Gly Gly Thr Ser Ile Phe Ala Gly His Leu Lys

450 455 460

Cys Arg Leu Lys Met Asp Lys Leu Lys Leu Lys Gly Met Ser Tyr Ala

465 470 475 480

Met Cys Leu Asn Thr Phe Val Leu Lys Lys Glu Val Ser Glu Thr Gln

485 490 495

His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu Asp Ala Pro

500 505 510

Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala His Asn

515 520 525

Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu Glu Pro

530 535 540

Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn Ile Val Ile

545 550 555 560

Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Arg Lys Gly His

565 570 575

His His His His His His

580

<210> 64

<211> 585

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 64

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Phe Ser Leu Ser Thr Arg Asp Gly Glu Pro Leu Met Ile Val Ala Lys

20 25 30

His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly Ile Asn

35 40 45

Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Glu Asp Thr

50 55 60

Val Thr Tyr Lys Cys Pro Leu Leu Val Asn Thr Glu Pro Glu Asp Ile

65 70 75 80

Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Met Tyr Gly Thr Cys

85 90 95

Thr Gln Ser Gly Glu Arg Arg Arg Glu Lys Arg Ser Val Ala Leu Thr

100 105 110

Pro His Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp Met Ser

115 120 125

Ser Glu Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp Ile Leu

130 135 140

Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr Met Ile

145 150 155 160

Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val Leu Met Met Leu

165 170 175

Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val Gly Asn Arg Asp

180 185 190

Phe Val Glu Gly Val Ser Gly Gly Ala Trp Val Asp Leu Val Leu Glu

195 200 205

His Gly Gly Cys Val Thr Thr Met Ala Gln Gly Lys Pro Thr Leu Asp

210 215 220

Phe Glu Leu Thr Lys Thr Thr Ala Lys Glu Val Ala Leu Leu Arg Thr

225 230 235 240

Tyr Cys Ile Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr Arg Cys

245 250 255

Pro Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln Gln Tyr

260 265 270

Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly Cys Gly

275 280 285

Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser Cys Ser

290 295 300

Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu Asn Leu Glu Tyr Thr

305 310 315 320

Val Val Val Thr Val His Asn Gly Asp Thr His Ala Val Gly Asn Asp

325 330 335

Thr Ser Asn His Gly Val Thr Ala Met Ile Thr Pro Arg Ser Pro Ser

340 345 350

Val Glu Val Lys Leu Pro Asp Tyr Gly Glu Leu Thr Leu Asp Cys Glu

355 360 365

Pro Arg Ser Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys Met Lys

370 375 380

Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu Pro Leu

385 390 395 400

Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn Tyr Lys

405 410 415

Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg Gln Asp Val

420 425 430

Thr Val Leu Gly Ser Gln Glu Gly Ala Met His Ser Ala Leu Ala Gly

435 440 445

Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His Met Phe Ala Gly His

450 455 460

Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly Met Ser

465 470 475 480

Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met Ala Glu

485 490 495

Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala Gly

500 505 510

Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys Glu Lys

515 520 525

Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn Thr Asn

530 535 540

Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile

545 550 555 560

Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg Lys

565 570 575

Gly His His His His His His His

580 585

<210> 65

<211> 403

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 65

Met Arg Cys Val Gly Ile Gly Asn Arg Asp Phe Val Glu Gly Leu Ser

1 5 10 15

Gly Ala Thr Trp Val Asp Val Val Leu Glu His Gly Ser Cys Val Thr

20 25 30

Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Ile Glu Leu Leu Lys Thr

35 40 45

Glu Val Thr Asn Pro Ala Val Leu Arg Lys Leu Cys Ile Glu Ala Lys

50 55 60

Ile Ser Asn Thr Thr Thr Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala

65 70 75 80

Thr Leu Val Glu Glu Gln Asp Ala Asn Phe Val Cys Arg Arg Thr Val

85 90 95

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser

100 105 110

Leu Leu Thr Cys Ala Lys Phe Lys Cys Val Thr Lys Leu Glu Gly Lys

115 120 125

Ile Val Gln Tyr Glu Asn Leu Lys Tyr Ser Val Ile Val Thr Val His

130 135 140

Thr Gly Asp Gln His Gln Val Gly Asn Glu Thr Thr Glu His Gly Thr

145 150 155 160

Ile Ala Thr Ile Thr Pro Gln Ala Pro Thr Ser Glu Ile Gln Leu Thr

165 170 175

Asp Tyr Gly Thr Leu Thr Leu Asp Cys Ser Pro Arg Thr Gly Leu Asp

180 185 190

Phe Asn Glu Met Val Leu Leu Thr Met Lys Glu Lys Ser Trp Leu Val

195 200 205

His Lys Gln Trp Phe Leu Asp Leu Pro Leu Pro Trp Thr Ser Gly Ala

210 215 220

Ser Thr Ser Gln Glu Thr Trp Asn Arg Gln Asp Leu Leu Val Thr Phe

225 230 235 240

Lys Thr Ala His Ala Lys Lys Gln Glu Val Val Val Leu Gly Ser Gln

245 250 255

Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Thr

260 265 270

Ser Gly Thr Thr Thr Ile Phe Ala Gly His Leu Lys Cys Arg Leu Lys

275 280 285

Met Asp Lys Leu Thr Leu Lys Gly Met Ser Tyr Val Met Cys Thr Gly

290 295 300

Ser Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln His Gly Thr Val

305 310 315 320

Leu Val Gln Val Lys Tyr Glu Gly Thr Asp Ala Pro Cys Lys Ile Pro

325 330 335

Phe Ser Thr Gln Asp Glu Lys Gly Val Thr Gln Asn Gly Arg Leu Ile

340 345 350

Thr Ala Asn Pro Ile Val Thr Asp Lys Glu Lys Pro Val Asn Ile Glu

355 360 365

Thr Glu Pro Pro Phe Gly Glu Ser Tyr Ile Ile Val Gly Ala Gly Glu

370 375 380

Lys Ala Leu Lys Leu Ser Trp Phe Lys Lys Gly His His His His

385 390 395 400

His His His

<210> 66

<211> 403

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 66

Met Arg Cys Ile Gly Ile Ser Asn Arg Asp Phe Val Glu Gly Val Ser

1 5 10 15

Gly Gly Ser Trp Val Asp Ile Val Leu Glu His Gly Ser Cys Val Thr

20 25 30

Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Phe Glu Leu Ile Lys Thr

35 40 45

Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys Tyr Cys Ile Glu Ala Lys

50 55 60

Leu Thr Asn Thr Thr Thr Asp Ser Arg Cys Pro Thr Gln Gly Glu Pro

65 70 75 80

Ser Leu Asn Glu Glu Gln Asp Lys Arg Phe Val Cys Lys His Ser Met

85 90 95

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Gly

100 105 110

Ile Val Thr Cys Ala Met Phe Thr Cys Lys Lys Asn Met Lys Gly Lys

115 120 125

Val Val Gln Pro Glu Asn Leu Glu Tyr Thr Ile Val Ile Thr Pro His

130 135 140

Ser Gly Glu Glu His Ala Val Gly Asn Asp Thr Gly Lys His Gly Lys

145 150 155 160

Glu Ile Lys Ile Thr Pro Gln Ser Ser Ile Thr Glu Ala Glu Leu Thr

165 170 175

Gly Tyr Gly Thr Val Thr Met Glu Cys Ser Pro Arg Thr Gly Leu Asp

180 185 190

Phe Asn Glu Met Val Leu Leu Gln Met Glu Asn Lys Ala Trp Leu Val

195 200 205

His Arg Gln Trp Phe Leu Asp Leu Pro Leu Pro Trp Leu Pro Gly Ala

210 215 220

Asp Thr Gln Gly Ser Asn Trp Ile Gln Lys Glu Thr Leu Val Thr Phe

225 230 235 240

Lys Asn Pro His Ala Lys Lys Gln Asp Val Val Val Leu Gly Ser Gln

245 250 255

Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Met

260 265 270

Ser Ser Gly Asn Leu Leu Phe Thr Gly His Leu Lys Cys Arg Leu Arg

275 280 285

Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly

290 295 300

Lys Phe Lys Val Val Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile

305 310 315 320

Val Ile Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro

325 330 335

Phe Glu Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile

340 345 350

Thr Val Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu

355 360 365

Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro

370 375 380

Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys Gly His His His His His

385 390 395 400

His His His

<210> 67

<211> 401

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 67

Met Arg Cys Val Gly Val Gly Asn Arg Asp Phe Val Glu Gly Leu Ser

1 5 10 15

Gly Ala Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr

20 25 30

Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Ile Glu Leu Gln Lys Thr

35 40 45

Glu Ala Thr Gln Leu Ala Thr Leu Arg Lys Leu Cys Ile Glu Gly Lys

50 55 60

Ile Thr Asn Ile Thr Thr Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala

65 70 75 80

Ile Leu Pro Glu Glu Gln Asp Gln Asn Tyr Val Cys Lys His Thr Tyr

85 90 95

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser

100 105 110

Leu Val Thr Cys Ala Lys Phe Gln Cys Leu Glu Ser Ile Glu Gly Lys

115 120 125

Val Val Gln His Glu Asn Leu Lys Tyr Thr Val Ile Ile Thr Val His

130 135 140

Thr Gly Asp Gln His Gln Val Gly Asn Glu Thr Gln Gly Val Thr Ala

145 150 155 160

Glu Ile Thr Ser Gln Ala Ser Thr Ala Glu Ala Ile Leu Pro Glu Tyr

165 170 175

Gly Thr Leu Gly Leu Glu Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn

180 185 190

Glu Met Ile Leu Leu Thr Met Lys Asn Lys Ala Trp Met Val His Arg

195 200 205

Gln Trp Phe Phe Asp Leu Pro Leu Pro Trp Thr Ser Gly Ala Thr Thr

210 215 220

Lys Thr Pro Thr Trp Asn Arg Lys Glu Leu Leu Val Thr Phe Lys Asn

225 230 235 240

Ala His Ala Lys Lys Gln Glu Val Val Val Leu Gly Ser Gln Glu Gly

245 250 255

Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Thr Ser Gly

260 265 270

Gly Thr Ser Ile Phe Ala Gly His Leu Lys Cys Arg Leu Lys Met Asp

275 280 285

Lys Leu Lys Leu Lys Gly Met Ser Tyr Ala Met Cys Leu Asn Thr Phe

290 295 300

Val Leu Lys Lys Glu Val Ser Glu Thr Gln His Gly Thr Ile Leu Ile

305 310 315 320

Lys Val Glu Tyr Lys Gly Glu Asp Ala Pro Cys Lys Ile Pro Phe Ser

325 330 335

Thr Glu Asp Gly Gln Gly Lys Ala His Asn Gly Arg Leu Ile Thr Ala

340 345 350

Asn Pro Val Val Thr Lys Lys Glu Glu Pro Val Asn Ile Glu Ala Glu

355 360 365

Pro Pro Phe Gly Glu Ser Asn Ile Val Ile Gly Ile Gly Asp Lys Ala

370 375 380

Leu Lys Ile Asn Trp Tyr Arg Lys Gly His His His His His His

385 390 395 400

His

<210> 68

<211> 403

<212>PRT

<213> Artificial Sequence

<220>

<223> DENV

<400> 68

Met Arg Cys Val Gly Val Gly Asn Arg Asp Phe Val Glu Gly Val Ser

1 5 10 15

Gly Gly Ala Trp Val Asp Leu Val Leu Glu His Gly Gly Cys Val Thr

20 25 30

Thr Met Ala Gln Gly Lys Pro Thr Leu Asp Phe Glu Leu Thr Lys Thr

35 40 45

Thr Ala Lys Glu Val Ala Leu Leu Arg Thr Tyr Cys Ile Glu Ala Ser

50 55 60

Ile Ser Asn Ile Thr Thr Ala Thr Arg Cys Pro Thr Gln Gly Glu Pro

65 70 75 80

Tyr Leu Lys Glu Glu Gln Asp Gln Gln Tyr Ile Cys Arg Arg Asp Val

85 90 95

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Gly

100 105 110

Val Val Thr Cys Ala Lys Phe Ser Cys Ser Gly Lys Ile Thr Gly Asn

115 120 125

Leu Val Gln Ile Glu Asn Leu Glu Tyr Thr Val Val Val Thr Val His

130 135 140

Asn Gly Asp Thr His Ala Val Gly Asn Asp Thr Ser Asn His Gly Val

145 150 155 160

Thr Ala Met Ile Thr Pro Arg Ser Pro Ser Val Glu Val Lys Leu Pro

165 170 175

Asp Tyr Gly Glu Leu Thr Leu Asp Cys Glu Pro Arg Ser Gly Ile Asp

180 185 190

Phe Asn Glu Met Ile Leu Met Lys Met Lys Lys Lys Thr Trp Leu Val

195 200 205

His Lys Gln Trp Phe Leu Asp Leu Pro Leu Pro Trp Thr Ala Gly Ala

210 215 220

Asp Thr Ser Glu Val His Trp Asn Tyr Lys Glu Arg Met Val Thr Phe

225 230 235 240

Lys Val Pro His Ala Lys Arg Gln Asp Val Thr Val Leu Gly Ser Gln

245 250 255

Glu Gly Ala Met His Ser Ala Leu Ala Gly Ala Thr Glu Val Asp Ser

260 265 270

Gly Asp Gly Asn His Met Phe Ala Gly His Leu Lys Cys Lys Val Arg

275 280 285

Met Glu Lys Leu Arg Ile Lys Gly Met Ser Tyr Thr Met Cys Ser Gly

290 295 300

Lys Phe Ser Ile Asp Lys Glu Met Ala Glu Thr Gln His Gly Thr Thr

305 310 315 320

Val Val Lys Val Lys Tyr Glu Gly Ala Gly Ala Pro Cys Lys Val Pro

325 330 335

Ile Glu Ile Arg Asp Val Asn Lys Glu Lys Val Val Gly Arg Ile Ile

340 345 350

Ser Ser Thr Pro Leu Ala Glu Asn Thr Asn Ser Val Thr Asn Ile Glu

355 360 365

Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Asn

370 375 380

Ser Ala Leu Thr Leu His Trp Phe Arg Lys Gly His His His His

385 390 395 400

His His His

<210> 69

<211> 374

<212>PRT

<213> Homo sapiens

<400> 69

Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln

1 5 10 15

Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser

20 25 30

Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu

35 40 45

Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln

50 55 60

Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser

65 70 75 80

Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile

85 90 95

Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg

100 105 110

Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys

115 120 125

Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe

130 135 140

Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile

145 150 155 160

Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr

165 170 175

Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro

180 185 190

Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val

195 200 205

Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln

210 215 220

Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn

225 230 235 240

Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly

245 250 255

Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg

260 265 270

Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro

275 280 285

Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu

290 295 300

Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys

305 310 315 320

Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys

325 330 335

Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys

340 345 350

Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys

355 360 365

Glu Pro Gln Gly Ala Thr

370

<210> 70

<211> 316

<212>PRT

<213> Homo sapiens

<400> 70

Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu

1 5 10 15

Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp

20 25 30

Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp

35 40 45

Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala

50 55 60

Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu

65 70 75 80

Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn

85 90 95

Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp

100 105 110

Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro

115 120 125

His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser

130 135 140

Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys

145 150 155 160

Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala

165 170 175

Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr

180 185 190

Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser

195 200 205

Met Gly Ser Ser Ser Pro Met Gly Val Ile Val Ala Val Val Ile Ala

210 215 220

Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys

225 230 235 240

Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala

245 250 255

Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln

260 265 270

Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met

275 280 285

Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu

290 295 300

Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn

305 310 315

<210> 71

<211> 316

<212>PRT

<213> Homo sapiens

<400> 71

Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu

1 5 10 15

Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp

20 25 30

Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp

35 40 45

Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala

50 55 60

Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu

65 70 75 80

Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn

85 90 95

Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp

100 105 110

Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro

115 120 125

His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser

130 135 140

Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys

145 150 155 160

Ser Gln Lys Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala

165 170 175

Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr

180 185 190

Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser

195 200 205

Met Gly Ser Ser Ser Pro Met Gly Val Ile Val Ala Val Val Ile Ala

210 215 220

Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys

225 230 235 240

Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala

245 250 255

Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln

260 265 270

Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met

275 280 285

Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu

290 295 300

Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn

305 310 315

<210> 72

<211> 291

<212>PRT

<213> Homo sapiens

<400> 72

Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp

1 5 10 15

Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr

20 25 30

Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro

35 40 45

Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu

50 55 60

Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp

65 70 75 80

Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln

85 90 95

Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr

100 105 110

Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val

115 120 125

Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu

130 135 140

Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu

145 150 155 160

Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg

165 170 175

Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly

180 185 190

Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys

195 200 205

Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile

210 215 220

Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala

225 230 235 240

Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Pro

245 250 255

Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn Thr Ile Thr

260 265 270

Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln

275 280 285

Asn Arg Ile

290

<210> 73

<211> 254

<212>PRT

<213> Homo sapiens

<400> 73

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ser Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp

225 230 235 240

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys

245 250

<210> 74

<211> 254

<212>PRT

<213> Homo sapiens

<400> 74

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ser Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp

225 230 235 240

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys

245 250

<210> 75

<211> 233

<212>PRT

<213> Homo sapiens

<400> 75

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Asn Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Val Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile

225 230

<210> 76

<211> 233

<212>PRT

<213> Homo sapiens

<400> 76

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile

225 230

<210> 77

<211> 404

<212>PRT

<213> Mus musculus

<400> 77

Met Ile Leu Thr Ser Phe Gly Asp Asp Met Trp Leu Leu Thr Thr Leu

1 5 10 15

Leu Leu Trp Val Pro Val Gly Gly Glu Val Val Asn Ala Thr Lys Ala

20 25 30

Val Ile Thr Leu Gln Pro Pro Trp Val Ser Ile Phe Gln Lys Glu Asn

35 40 45

Val Thr Leu Trp Cys Glu Gly Pro His Leu Pro Gly Asp Ser Ser Thr

50 55 60

Gln Trp Phe Ile Asn Gly Thr Ala Val Gln Ile Ser Thr Pro Ser Tyr

65 70 75 80

Ser Ile Pro Glu Ala Ser Phe Gln Asp Ser Gly Glu Tyr Arg Cys Gln

85 90 95

Ile Gly Ser Ser Met Pro Ser Asp Pro Val Gln Leu Gln Ile His Asn

100 105 110

Asp Trp Leu Leu Leu Gln Ala Ser Arg Arg Val Leu Thr Glu Gly Glu

115 120 125

Pro Leu Ala Leu Arg Cys His Gly Trp Lys Asn Lys Leu Val Tyr Asn

130 135 140

Val Val Phe Tyr Arg Asn Gly Lys Ser Phe Gln Phe Ser Ser Asp Ser

145 150 155 160

Glu Val Ala Ile Leu Lys Thr Asn Leu Ser His Ser Gly Ile Tyr His

165 170 175

Cys Ser Gly Thr Gly Arg His Arg Tyr Thr Ser Ala Gly Val Ser Ile

180 185 190

Thr Val Lys Glu Leu Phe Thr Thr Pro Val Leu Arg Ala Ser Val Ser

195 200 205

Ser Pro Phe Pro Glu Gly Ser Leu Val Thr Leu Asn Cys Glu Thr Asn

210 215 220

Leu Leu Leu Gln Arg Pro Gly Leu Gln Leu His Phe Ser Phe Tyr Val

225 230 235 240

Gly Ser Lys Ile Leu Glu Tyr Arg Asn Thr Ser Ser Glu Tyr His Ile

245 250 255

Ala Arg Ala Glu Arg Glu Asp Ala Gly Phe Tyr Trp Cys Glu Val Ala

260 265 270

Thr Glu Asp Ser Ser Val Leu Lys Arg Ser Pro Glu Leu Glu Leu Gln

275 280 285

Val Leu Gly Pro Gln Ser Ser Ala Pro Val Trp Phe His Ile Leu Phe

290 295 300

Tyr Leu Ser Val Gly Ile Met Phe Ser Leu Asn Thr Val Leu Tyr Val

305 310 315 320

Lys Ile His Arg Leu Gln Arg Glu Lys Lys Tyr Asn Leu Glu Val Pro

325 330 335

Leu Val Ser Glu Gln Gly Lys Lys Ala Asn Ser Phe Gln Gln Val Arg

340 345 350

Ser Asp Gly Val Tyr Glu Glu Val Thr Ala Thr Ala Ser Gln Thr Thr

355 360 365

Pro Lys Glu Ala Pro Asp Gly Pro Arg Ser Ser Val Gly Asp Cys Gly

370 375 380

Pro Glu Gln Pro Glu Pro Leu Pro Pro Ser Asp Ser Thr Gly Ala Gln

385 390 395 400

Thr Ser Gln Ser

<210> 78

<211> 293

<212>PRT

<213> Mus musculus

<400> 78

Met Gly Ile Leu Pro Phe Leu Leu Ile Pro Met Glu Ser Asn Trp Thr

1 5 10 15

Val His Val Phe Ser Arg Thr Leu Cys His Met Leu Leu Trp Thr Ala

20 25 30

Val Leu Asn Leu Ala Ala Gly Thr His Asp Leu Pro Lys Ala Val Val

35 40 45

Lys Leu Glu Pro Pro Trp Ile Gln Val Leu Lys Glu Asp Thr Val Thr

50 55 60

Leu Thr Cys Glu Gly Thr His Asn Pro Gly Asn Ser Ser Thr Gln Trp

65 70 75 80

Phe His Asn Gly Arg Ser Ile Arg Ser Gln Val Gln Ala Ser Tyr Thr

85 90 95

Phe Lys Ala Thr Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Met Glu

100 105 110

Gln Thr Arg Leu Ser Asp Pro Val Asp Leu Gly Val Ile Ser Asp Trp

115 120 125

Leu Leu Leu Gln Thr Pro Gln Leu Val Phe Leu Glu Gly Glu Thr Ile

130 135 140

Thr Leu Arg Cys His Ser Trp Arg Asn Lys Leu Leu Asn Arg Ile Ser

145 150 155 160

Phe Phe His Asn Glu Lys Ser Val Arg Tyr His His Tyr Ser Ser Asn

165 170 175

Phe Ser Ile Pro Lys Ala Asn His Ser His Ser Gly Asp Tyr Tyr Cys

180 185 190

Lys Gly Ser Leu Gly Arg Thr Leu His Gln Ser Lys Pro Val Thr Ile

195 200 205

Thr Val Gln Gly Pro Lys Ser Ser Arg Ser Leu Pro Val Leu Thr Ile

210 215 220

Val Ala Ala Val Thr Gly Ile Ala Val Ala Ala Ile Val Ile Ile Leu

225 230 235 240

Val Ser Leu Val Tyr Leu Lys Lys Lys Gln Val Pro Asp Asn Pro Pro

245 250 255

Asp Leu Glu Glu Ala Ala Lys Thr Glu Ala Glu Asn Thr Ile Thr Tyr

260 265 270

Ser Leu Leu Lys His Pro Glu Ala Leu Asp Glu Glu Thr Glu His Asp

275 280 285

Tyr Gln Asn His Ile

290

<210> 79

<211> 267

<212>PRT

<213> Mus musculus

<400> 79

Met Thr Leu Asp Thr Gln Met Phe Gln Asn Ala His Ser Gly Ser Gln

1 5 10 15

Trp Leu Leu Pro Pro Leu Thr Ile Leu Leu Leu Phe Ala Phe Ala Asp

20 25 30

Arg Gln Ser Ala Ala Leu Pro Lys Ala Val Val Lys Leu Asp Pro Pro

35 40 45

Trp Ile Gln Val Leu Lys Glu Asp Met Val Thr Leu Met Cys Glu Gly

50 55 60

Thr His Asn Pro Gly Asn Ser Ser Thr Gln Trp Phe His Asn Trp Ser

65 70 75 80

Ser Ile Arg Ser Gln Val Gln Ser Ser Tyr Thr Phe Lys Ala Thr Val

85 90 95

Asn Asp Ser Gly Glu Tyr Arg Cys Gln Met Glu Gln Thr Arg Leu Ser

100 105 110

Asp Pro Val Asp Leu Gly Val Ile Ser Asp Trp Leu Leu Leu Gln Thr

115 120 125

Pro Gln Arg Val Phe Leu Glu Gly Glu Thr Ile Thr Leu Arg Cys His

130 135 140

Ser Trp Arg Asn Lys Leu Leu Asn Arg Ile Ser Phe Phe His Asn Glu

145 150 155 160

Lys Ser Val Arg Tyr His His Tyr Lys Ser Asn Phe Ser Ile Pro Lys

165 170 175

Ala Asn His Ser His Ser Gly Asp Tyr Tyr Cys Lys Gly Ser Leu Gly

180 185 190

Ser Thr Gln His Gln Ser Lys Pro Val Thr Ile Thr Val Gln Asp Pro

195 200 205

Ala Thr Thr Ser Ser Ile Ser Leu Val Trp Tyr His Thr Ala Phe Ser

210 215 220

Leu Val Met Cys Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Tyr

225 230 235 240

Val Arg Arg Asn Leu Gln Thr Pro Arg Asp Tyr Trp Arg Lys Ser Leu

245 250 255

Ser Ile Arg Lys His Gln Ala Pro Gln Asp Lys

260 265

<210> 80

<211> 249

<212>PRT

<213> Mus musculus

<400> 80

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Val Leu Thr Ala Phe Ser

1 5 10 15

Gly Ile Gln Ala Gly Leu Gln Lys Ala Val Val Asn Leu Asp Pro Lys

20 25 30

Trp Val Arg Val Leu Glu Glu Asp Ser Val Thr Leu Arg Cys Gln Gly

35 40 45

Thr Phe Ser Pro Glu Asp Asn Ser Ile Lys Trp Phe His Asn Glu Ser

50 55 60

Leu Ile Pro His Gln Asp Ala Asn Tyr Val Ile Gln Ser Ala Arg Val

65 70 75 80

Lys Asp Ser Gly Met Tyr Arg Cys Gln Thr Ala Leu Ser Thr Ile Ser

85 90 95

Asp Pro Val Gln Leu Glu Val His Met Gly Trp Leu Leu Leu Gln Thr

100 105 110

Thr Lys Trp Leu Phe Gln Glu Gly Asp Pro Ile His Leu Arg Cys His

115 120 125

Ser Trp Gln Asn Arg Pro Val Arg Lys Val Thr Tyr Leu Gln Asn Gly

130 135 140

Lys Gly Lys Lys Tyr Phe His Glu Asn Ser Glu Leu Leu Ile Pro Lys

145 150 155 160

Ala Thr His Asn Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Ile Gly

165 170 175

His Asn Asn Lys Ser Ser Ala Ser Phe Arg Ile Ser Leu Gly Asp Pro

180 185 190

Gly Ser Pro Ser Met Phe Pro Pro Trp His Gln Ile Thr Phe Cys Leu

195 200 205

Leu Ile Gly Leu Leu Phe Ala Ile Asp Thr Val Leu Tyr Phe Ser Val

210 215 220

Arg Arg Gly Leu Gln Ser Pro Val Ala Asp Tyr Glu Glu Pro Lys Ile

225 230 235 240

Gln Trp Ser Lys Glu Pro Gln Asp Lys

245

<210> 81

<211> 365

<212>PRT

<213> Homo sapiens

<400> 81

Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe

1 5 10 15

Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr

20 25 30

His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp

35 40 45

Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu

50 55 60

Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val

65 70 75 80

Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys

85 90 95

Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr

100 105 110

Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val

115 120 125

Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp

130 135 140

Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile

145 150 155 160

Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr

165 170 175

Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg

180 185 190

Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys

195 200 205

Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe

210 215 220

Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu

225 230 235 240

Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser

245 250 255

Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His

260 265 270

Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val

275 280 285

Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val

290 295 300

Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu

305 310 315 320

Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg

325 330 335

Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp

340 345 350

Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala

355 360 365

<210> 82

<211> 119

<212>PRT

<213> Homo sapiens

<400> 82

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

20 25 30

His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

35 40 45

Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

50 55 60

Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

65 70 75 80

Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

85 90 95

Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

100 105 110

Val Lys Trp Asp Arg Asp Met

115

<210> 83

<211> 330

<212>PRT

<213> Homo sapiens

<400> 83

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

Lys 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 Asp Glu

225 230 235 240

Leu 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 Gly Lys

325 330

<210> 84

<211> 326

<212>PRT

<213> Homo sapiens

<400> 84

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser 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 Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 85

<211> 377

<212>PRT

<213> Homo sapiens

<400> 85

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

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 Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105 110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

115 120 125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135 140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155 160

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

165 170 175

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

180 185 190

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

195 200 205

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

210 215 220

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

225 230 235 240

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

245 250 255

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

260 265 270

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

275 280 285

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

290 295 300

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

305 310 315 320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330 335

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

340 345 350

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

355 360 365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375

<210> 86

<211> 327

<212>PRT

<213> Homo sapiens

<400> 86

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser 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 Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 87

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 87

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

Lys 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> 88

<211> 119

<212>PRT

<213> Artificial Sequence

<220>

<223>VH

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His

20 25 30

Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ile Ile Ser Gly Gly Gly Ser Tyr Thr Ser Tyr Pro Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu His

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Arg Glu Ser Asn Gly Lys Pro Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 89

<211> 111

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 89

Asp Ile Glu Leu Thr Gln Ser Pro Thr Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Ala

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 90

<211> 132

<212>PRT

<213> Artificial Sequence

<220>

<223>VH

<400> 90

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 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> 91

<211> 132

<212>PRT

<213> Artificial Sequence

<220>

<223>VH

<400> 91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 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 Val 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> 92

<211> 132

<212>PRT

<213> Artificial Sequence

<220>

<223>VH

<400> 92

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 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 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 Val 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> 93

<211> 132

<212>PRT

<213> Artificial Sequence

<220>

<223>VH

<400> 93

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 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> 94

<211> 132

<212>PRT

<213> Artificial Sequence

<220>

<223>VH

<400> 94

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 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 Val 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> 95

<211> 132

<212>PRT

<213> Artificial Sequence

<220>

<223>VH

<400> 95

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 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 Val 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> 96

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 96

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 Glu Asp Leu Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Gln Ile Lys

100 105

<210> 97

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 97

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 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> 98

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 98

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile 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 Leu 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> 99

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 99

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile 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 Asn Leu Lys Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu 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> 100

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223>VL

<400> 100

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile 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 Leu 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> 101

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> HVR-H1

<400> 101

Ser Tyr Tyr Ile His

15

<210> 102

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223>FR-H1

<400> 102

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 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> 103

<211> 32

<212>PRT

<213> Artificial Sequence

<220>

<223>FR-H3

<400> 103

Arg Val 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> 104

<211> 23

<212>PRT

<213> Artificial Sequence

<220>

<223>FR-L1

<400> 104

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210> 105

<211> 32

<212>PRT

<213> Artificial Sequence

<220>

<223>FR-L3

<400> 105

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys

20 25 30

<210> 106

<211> 157

<212>PRT

<213> Artificial Sequence

<220>

<223>CD154

<400> 106

Asp Tyr Lys Asp Asp Asp Asp Lys Met Gln Lys Gly Asp Gln Asn Pro

1 5 10 15

Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser

20 25 30

Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu

35 40 45

Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu

50 55 60

Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser

65 70 75 80

Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg

85 90 95

Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys

100 105 110

Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln

115 120 125

Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser

130 135 140

His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu

145 150 155

<210> 107

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 107

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

Ala 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> 108

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223>CH

<400> 108

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 Leu His Glu Ala Leu His Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 109

<211> 328

<212>PRT

<213> Artificial Sequence

<220>

<223>CH

<400> 109

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 Leu His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<---

Claims (2)

1. Use of an antibody for the production of medicinal products for the treatment or prevention of infection caused by the Zika virus, where the antibody contains a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 7, wherein the antibody also contains the amino acid sequence of SEQ ID NO: 59 . 2. The use of claim 1, wherein the antibody comprises a VH sequence as shown in SEQ ID NO: 6, with a human IgG1 CH sequence as shown in SEQ ID NO: 59, and a VL sequence as shown in SEQ ID NO: 7, with the human CL sequence as shown in SEQ ID NO: 60.

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