POLYPEPTIDE, PHARMACEUTICAL FORMULATION AND PROCESS FOR PREPARING AN ANTIBODY

Description

1 / 153 POLYPEPTIDE, PHARMACEUTICAL FORMULATION AND PROCESS FOR PREPARING AN ANTIBODY Divided from BR112019004770-0, filed on September 15, 2017. FIELD OF THE INVENTION

[001] The present invention relates to dengue virus antibodies and methods of their use. The present invention also relates to polypeptides containing variant Fc regions and methods of their use. BACKGROUND

[002] Dengue fever is the most common arthropod-borne viral disease in the world. The virus that causes dengue (or referred to here as DENV) can be divided into four different infectious serotypes such as DENV-1, DENV-2, DENV-3, and DENV-4. Symptoms of dengue infection include fever, muscle pain, headache, low platelet and white blood cell counts, coagulopathy, hemorrhage, and vascular effusion that can lead to dengue shock syndrome. When a person is exposed to the dengue virus after a previous dengue infection, antiviral antibodies can increase the uptake of the virus into host cells, and the patient is at higher risk of developing a severe form of dengue. Severe forms of dengue can, however, also occur during a first infection.

[003] Although it is the most common arthropod-borne viral disease, to date, there is no drug available for the treatment of dengue. Approaches to dengue as a disease have been primarily focused on preventing infection and / or treating symptoms.

[004] Thus, there is a need to provide agents capable of neutralizing and / or binding to at least one dengue serotype. Recently, several groups have reported on antibodies to Petition 870260064115, dated 06 / 29 / 2026, p. 10 / 485 2 / 153 anti-DENV neutralization (see, for example, WO2012 / 082073, WO2013 / 089647, WO2013 / 151764, WO2013 / 173348, WO2014 / 025546, WO2015 / 123362, WO2015 / 122995 and WO2016 / 012800). However, antibodies with superior therapeutic properties are still needed.

[005] Vaccines and antibody-based therapeutic products are currently under development to prevent and treat viral infection. However, antibody-based treatments are not without risks. One such risk is increased antibody dependence (ADE), which occurs when non-neutralizing antiviral antibodies facilitate viral entry into host cells, leading to the possibility of increased infection in cells (Expert Rev Anti Infect Ther (2013) 11, 1147-1157). The most common mechanism for ADE is the interaction of the virus-antibody complex through the Fc portion of the antibody with Fc receptors (FcRs) on the cell surface. A normally mild viral infection can be enhanced by ADE to become a fatal disease. An anti-DENV antibody with mutations in the Fc region that prevent binding to FcR has been reported to have failed to enhance DENV infection (WO2010 / 043977).However, there is still a need for antibody-based therapeutic agents without increasing the risk of antibody-dependent enhancement of infection. SUMMARY.

[006] The invention provides anti-DENV antibodies, polypeptides containing variant Fc regions, and methods of using the same.

[007] In some embodiments, an anti-DENV antibody isolated from the present invention binds to the DENV E protein. In some embodiments, an anti-DENV antibody of the invention comprises: (a) (i) HVR-H3 comprising the amino acid sequence GGX1ALFYDSYTTPX2DX3GSWWFDP, where X1 is R or E, X2 is R or F, X3 is G, D or L (SEQ ID NO: 42), (ii) HVR-L3 comprising the Petition 870260064115, dated 06 / 29 / 2026, p. 11 / 485 3 / 153 amino acid sequence QQFX1X2LPIT, where Xi is D, S or E, X2 is D or A (SEQ ID NO: 45), and (iii) HVR-H2 comprising amino acid sequence VINPRGGSX1X2SAQKFQG, where X1 is T or R, X2 is A or R (SEQ ID NO: 41); (b) (i) HVR-H1 comprising the amino acid sequence SX1YX2H, where X1 is N or Y, X2 is I or M (SEQ ID NO: 40), (ii) HVR-H2 comprising the amino acid sequence VINPRGGSX1X2SAQKFQG, where X1 is T or R, X2 is A or R (SEQ ID NO: 41), and (iii) HVR-H3 comprising the amino acid sequence GGX1ALFYDSYTTPX2DX3GSWWFDP, where X1 is R or E, X2 is R or F, X3 is G, D or L (SEQ ID NO: 42); (c) (i) HVR-H1 comprising amino acid sequence SX1YX2H, where X1 is N or Y, X2 is I or M (SEQ ID NO: 40), (ii) HVR-H2 comprising amino acid sequence VINPRGGSX1X2SAQKFQG, where X1 is T or R, X2 is A or R (SEQ ID NO: 41), (iii) HVR-H3 comprising amino acid sequence GGX1ALFYDSYTTPX2DX3GSWWFDP, where X1 is R or E, X2 is R or F, X3 is G, D or L (SEQ ID NO: 42), (iv) HVR-L1 comprising amino acid sequence QASQX1IRX2YLN, where X1 is D or E, X2 is K or Q (SEQ ID NO: 43); (v) HVR-L2 comprising the amino acid sequence DASX1LKX2, where X1 is N or E, X2 is T or F (SEQ ID NO: 44); and (vi) HVR-L3 comprising the amino acid sequence QQFX1X2LPIT, where X1 is D, S, or E, X2 is D or A (SEQ ID NO: 45); or (d) (i) HVR-L1 comprising the amino acid sequence QASQX1IRX2YLN, where X1 is D or E, X2 is K or Q (SEQ ID NO: 43); (ii) HVR-L2 comprising the amino acid sequence DASX1LKX2, where X1 is N or E, X2 is T or F (SEQ ID NO: 44);and (iii) HVR-L3 comprising the amino acid sequence QQFX1X2LPIT, where X1 is D, S, or E, X2 is D or A (SEQ ID NO: 45). In some embodiments, the; Petition 870260064115, dated 06 / 29 / 2026, p. 12 / 485 4 / 153 antibody of the invention is not an antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[008] In some embodiments, an anti-DENV antibody isolated from the invention comprises: (a) (i) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (ii) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10, and (iii) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (b) (i) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (ii) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6, and (iii) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (c) (i) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (ii) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (iii) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (iv) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 8 to 10; (v) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 8 to 10; and (vi) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10; (d) (i) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 8 to 10; (ii) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 8 to 10; and (iii) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10; or (e) (i) HVR-H1 of the VH sequence of any of the SEQ Petition 870260064115, dated 06 / 29 / 2026, p. 13 / 485 5 / 153 (i) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (ii) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (iii) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 7; (v) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 7; and (vi) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 7.

[009] In some embodiments, the antibody of the invention is not an antibody (i) HVR-H1 of the VH sequence of SEQ ID NO: 1, (ii) HVR-H2 of the VH sequence of SEQ ID NO: 1, (iii) HVR-H3 of the VH sequence of SEQ ID NO: 1, (iv) HVR-L1 of the VL sequence of SEQ ID NO: 7, (v) HVRL2 of the VL sequence of SEQ ID NO: 7, and (vi) HVR-L3 of the VL sequence of SEQ ID NO: 7.

[010] In some embodiments, an anti-DENV antibody isolated from the present invention further comprises a variable domain heavy chain structure FR1 comprising the amino acid sequence of SEQ ID NO: 31, FR2 comprising the amino acid sequence of SEQ ID NO: 32, FR3 comprising 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, an anti-DENV antibody isolated from the present invention further comprises a variable domain light chain structure FR1 comprising the amino acid sequence of SEQ ID NO: 36, FR2 comprising the amino acid sequence of SEQ ID NO: 37, FR3 comprising the amino acid sequence of SEQ ID NO: 38, FR4 comprising the amino acid sequence of SEQ ID NO: 39.

[011] In some embodiments, an anti-DENV antibody isolated from the present invention comprises (a) a VH sequence having at least 95% sequence identity with the amino acid sequence of any of the SEQ ID NOs: 2 to 6; (b) a VL sequence having at least 95% sequence identity with the Petition 870260064115, dated 06 / 29 / 2026, p. 14 / 485 6 / 153 amino acid sequence of any of the SEQ ID NOs: 8 to 10; (c) a VH sequence of any of the SEQ ID NOs: 2 to 6 and a VL sequence of any of the SEQ ID NOs: 8 to 10; or (d) a VH sequence of any of the SEQ ID NOs: 2 to 6 and a VL sequence of any of the SEQ ID NOs: 7.

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

[013] In some embodiments, an Fc region of an anti-DENV antibody of the present invention comprises Ala at position 234 and Ala at position 235 according to EU numbering. In some embodiments, an Fc region of an anti-DENV antibody of the present invention may be selected from the variant Fc regions described herein.

[014] The invention also provides isolated nucleic acids encoding an anti-DENV antibody of the present invention. The invention also provides host cells comprising a nucleic acid of the present invention. The invention also provides a method for producing an antibody comprising culturing a host cell of the present invention such that the antibody is produced.

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

[016] The anti-DENV antibodies of the present invention can be used as a medicament. In some embodiments, the Petition 870260064115, dated 06 / 29 / 2026, p. 15 / 485 7 / 153 anti-DENV antibodies of the present invention can be used for the treatment of DENV infection.

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

[018] The invention also provides a method of treating an individual who has a DENV infection. In some embodiments, the method comprises administering to the individual an effective amount of an anti-DENV antibody of the present invention. In some embodiments, the method further comprises administering to the individual an additional therapeutic agent, for example, as described below.

[019] In some embodiments, a variant Fc region of the present invention comprises at least one amino acid change in an original Fc region. In other embodiments, the variant Fc region has substantially decreased FcyR binding activity when compared with the original Fc region. In other embodiments, the variant Fc region does not have substantially decreased C1q binding activity when compared with the original Fc region.

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

[021] In some embodiments, a variant Fc region of the present invention comprises amino acids selected from the group consisting of: (a) Glu at position 267, (b) Phe at position 268, (c) Petition 870260064115, dated 06 / 29 / 2026, page 16 / 485 8 / 153 Thr in position 324, (d) Ala in position 236, (e) Glu in position 332, (f) Ala, Asp, Glu, Met or Trp in position 326 and (g) Ser in position 333; according to EU numbering.

[022] In some embodiments, a variant Fc region of the present invention further comprises 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.

[023] In some embodiments, a variant Fc region of the present invention comprises any of the amino acid changes, individually or in combination, as described in Table 4. In some embodiments, an Fc region of origin described in the present invention is derived from human IgG1. The invention provides a polypeptide comprising the amino acid sequence of any of the SEQ ID NOs: 51 to 59.

[024] In some embodiments, a polypeptide comprising a variant Fc region of the present invention is an antibody. In other embodiments, the antibody is a viral antibody. In other embodiments, the antibody comprises a variable region derived from an anti-DENV antibody described herein.

[025] In other forms, the antibody comprises: (a) (i) HVR-H3 comprising amino acid sequence SEQ ID NO: 16, (ii) HVR-L3 comprising amino acid sequence SEQ ID NO: 27, and (iii) HVR-H2 comprising amino acid sequence SEQ ID NO: 13; (b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the sequence of Petition 870260064115, dated 06 / 29 / 2026, p. 17 / 485 9 / 153 amino acid of SEQ ID NO: 13, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[026] In other forms, the antibody comprises: (a) (i) HVR-H3 of the VH sequence of SEQ ID NO: 6, (ii) HVRL3 of the VL sequence of SEQ ID NO: 10, and (iii) HVR-H2 of the VH sequence of SEQ ID NO: 6; (b) (i) HVR-H1 of VH sequence of SEQ ID NO: 6, (ii) HVRH2 of VH sequence of SEQ ID NO: 6, and (iii) HVR-H3 of VH sequence of SEQ ID NO: 6; (c) (i) HVR-H1 of VH sequence of SEQ ID NO: 6, (ii) HVRH2 of VH sequence of SEQ ID NO: 6, (iii) HVR-H3 of VH sequence of SEQ ID NO: 6, (iv) HVR-L1 of VL sequence of SEQ ID NO: 10; (v) HVR-L2 of the VL sequence of SEQ ID NO: 10; and (vi) HVR-L3 of sequence VL of SEQ ID NO: 10; (d) (i) HVR-L1 of sequence VL of SEQ ID NO: 10; (ii) HVRL2 of the VL sequence of SEQ ID NO: 10; and (iii) HVR-L3 of sequence VL of SEQ ID NO: 10; or (e) (i) HVR-H1 of sequence VH of SEQ ID NO: 6, (ii) HVR Petition 870260064115, of 29 / 06 / 2026, p. 18 / 485 10 / 153 H2 of VH sequence of SEQ ID NO: 6, (iii) HVR-H3 of VH sequence of SEQ ID NO: 6, (iv) HVR-L1 of VL sequence of SEQ ID NO: 7; (v) HVR-L2 of the VL sequence of SEQ ID NO: 7; and (vi) HVR-L3 of sequence VL of SEQ ID NO: 7.

[027] The invention also provides isolated nucleic acids encoding a polypeptide comprising a variant Fc region of the present invention. The invention also provides host cells comprising a nucleic acid of the present invention. The invention also provides a method of producing a polypeptide comprising a variant Fc region comprising culturing a host of the present invention such that the polypeptide is produced.

[028] The invention also provides a pharmaceutical formulation comprising a polypeptide comprising an Fc variant region of the present invention and a pharmaceutically acceptable carrier.

[029] The polypeptides comprising the variant Fc regions of the present invention may be for use as a medicament. In some embodiments, the polypeptides comprising the variant Fc regions of the present invention may be for use in the treatment of a viral infection.

[030] Polypeptides comprising variant Fc regions of the present invention can be used in the manufacture of a medicament. In some embodiments, the medicament is for the treatment of a viral infection.

[031] The invention also provides a method of treating an individual who has a viral infection. In some embodiments, the method comprises administering to the individual an effective amount of a polypeptide comprising an Fc variant region of the present invention. Petition 870260064115, dated 06 / 29 / 2026, page 19 / 485 11 / 153

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

[033] In one embodiment, the present invention relates to a process for preparing an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, the process comprising the steps of: (a) combine a selected VH variant sequence from the group consisting of: 3CH1047 (SEQ ID NO: 6) and 3CH1049 (SEQ ID NO: 95) with a selected human lgG1 CH sequence from the group consisting of: SG182 (SEQ ID NO: 46), SG1095 (SEQ ID NO: 54) and SG1106 (SEQ ID NO: 59); (b) combine a selected VL variant sequence from the group consisting of 3CL (SEQ ID NO: 7) and 3CL633 (SEQ ID NO: 98), with a human SK1 CL sequence (SEQ ID NO: 60); (c) clone each of the combinations into an expression vector; (d) express the resulting expression vectors in the co-transfected cells (host cells); and (e) purify the antibody resulting from step (d). BRIEF DESCRIPTION OF THE FIGURES

[034] Figures 1 (A) to (D) illustrate the BIACORE® sensograms of anti-DENV 3C and 3C antibodies for DENV-1 protein E (A), DENV-2 protein E (B), DENV-3 protein E (C) and protein Petition 870260064115, dated 06 / 29 / 2026, p. 20 / 485 12 / 153 And of DENV-4 (D), as described in Example 2.

[035] Figure 2 illustrates the binding affinities of antibodies with different Fc variants towards human C1q, as described in Example 4. Binding activities were measured by ELISA. The Fc variants tested are: WT, LALA + KWES, LALA + EFT + AE, LALA + EFT and LALA.

[036] Figure 3 illustrates the binding affinities of antibodies with different Fc variants towards human C1q, as described in Example 4. Binding activities were measured by ELISA. The Fc variants tested are: WT, LALA, LALA + KWES, LALA + KAES, LALA + KDES, LALA + KEES and LALA + KMES.

[037] Figure 4 illustrates the binding affinities of antibodies with different Fc variants toward human C1q, as described in Example 4. Binding activities were measured by ELISA. The Fc variants tested are: WT, LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + ACT3 + KAES and LALA + ACT5 + KAES.

[038] Figure 5 illustrates antibody binding affinities with different Fc variants toward mouse C1q, as described in Example 4. Binding activities were measured by ELISA. The Fc variants tested are: WT, LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + ACT3 + KAES and LALA + ACT5 + KAES.

[039] Figures 6(a) to (h) illustrate the Biacore analysis for Fc variants that bind to human FcyRs, as described in Example 5. The Fc variants tested are: WT (described as WT IgG in the figure), 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. These Fc variants were tested for binding to FcyRs including: (a) human FcyR1a, (b) human FcyR2a allelic variant 167H, (c) human FcyR2a allelic variant 167R, (d) human FcyR2b, (e) variant Petition 870260064115, dated 06 / 29 / 2026, p. 21 / 485 13 / 153 allelic variant of human FcYR3a 158F, (f) allelic variant of human FcYR3a 158V, (g) allelic variant of human FcYR3b NA1 and (h) allelic variant of human FcγR3b NA2. Each Fc variant was evaluated both as an antibody alone with the Fc variant (described simply as Ab) and as an immune complex formed with the antibody and a trimeric CD154 antigen (described as CD154 IC).

[040] Figures 7 (a) to (d) illustrate the Biacore analysis with respect to mouse Fc variants that bind to FcyRs, as described in Example 5. The Fc variants tested are: WT (described as WT IgG in the figure), 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. These Fc variants were tested for binding to FcyRs including: (a) mouse FcyRI, (b) mouse FcYR2b, (c) mouse FcyR3 and (d) mouse FcyR4. Each Fc variant was evaluated both as an antibody alone containing the Fc variant (described simply as Ab) and as an immune complex formed with the antibody and a trimeric CD154 antigen (described as CD154 IC).

[041] Figure 8 illustrates the Biacore analysis with respect to Fc variants that bind to human FcRn, as described in Example 5. The Fc variants tested are: WT (described as hIgG1 in the figure), LALA, LALA + ACT3, LALA + ACT5, LALA + KAES, LALA + KAES + ACT3, and LALA + KAES + ACT5. Each Fc variant was evaluated both as an antibody alone with the Fc variant (described simply as Ab) and as an immune complex formed with the antibody and a trimeric CD154 antigen (described as CD154 IC).

[042] Figure 9 illustrates viremia on day 3 after DENV-2 virus infection in AG129 mice, as described in Example 6. Anti-DENV 3C and 3Cam antibodies in combination with the Fc variants of WT (described as hIgG1), LALA and LALA + KAES, or PBS as a Petition 870260064115, dated 06 / 29 / 2026, page 22 / 485 14 / 153 negative controls were administered on day 2 after viral infection.

[043] Figure 12 (A) to (D) illustrates the BIACORE® sensograms of anti-DENV 3C and 3Cam2 antibodies against DENV-1 protein E (A), DENV-2 protein E (B), DENV-3 protein E (C), and DENV-4 protein E (D), as described in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

[044] The techniques and procedures described or referred to in this invention are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely used methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al., eds., (2003)); the Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)); Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RIFreshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers,. Petição 870260064115, de 29 / 06 / 2026, pág. 23 / 485 15 / 153 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); e Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993). I. DEFINIÇÕES

[045] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Reactions, Mechanisms and Advanced Organic Structure, 4th ed., John Wiley & Sons (New York, NY 1992), provide a skill in the art with a general guide to many of the terms used in the present application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[046] For the purposes of interpreting this descriptive report, the following definitions will apply and, where appropriate, terms used in the singular also include the plural and vice versa. It should be understood that the terminology used here is for the purpose of describing only particular modalities, and is not intended to be limiting. In the event that any definition shown below conflicts with any document incorporated herein by reference, the definition shown below shall prevail.

[047] A human receptor structure for the purposes of this invention is a structure comprising the amino acid sequence Petition 870260064115, dated 06 / 29 / 2026, p. 24 / 485 16 / 153 of a variable light chain (VL) domain structure or a variable heavy chain (VH) domain structure derived from a human immunoglobulin structure or a human consensus structure, as defined below. A human receptor structure derived from a human immunoglobulin structure or a human consensus structure may comprise its same amino acid sequence, or it may contain amino acid sequence changes. 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 receptor structure is sequence-identical to the human immunoglobulin structure sequence or human consensus structure sequence of the VL.

[048] Affinity refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, binding affinity refers to intrinsic binding affinity reflecting a 1:1 interaction between the members of a binding partner (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Illustrative and exemplary specific embodiments for measuring binding affinity are described in what follows.

[049] An affinity-matured antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to an antibody of origin that does not have such alterations, such alterations resulting in an improvement in the antibody's affinity for the antigen. Petition 870260064115, dated 06 / 29 / 2026, page 25 / 485 17 / 153

[050] The terms anti-DENV antibody and a DENV-binding antibody refer to an antibody that is capable of binding to DENV with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting DENV. The antibody can bind to the DENV E protein. The terms anti-DENV E protein antibody and DENV E protein-binding antibody refer to an antibody that is capable of binding to the DENV E protein with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting DENV. In one embodiment, the extent of binding of an anti-DENV E protein antibody to an unrelated protein other than the DENV E protein is less than about 10% of the antibody binding to the DENV E protein as measured, for example, by radioimmunoassay (RIA).In certain embodiments, antibody that binds to DENV and / or DENV E protein has a dissociation constant (Kd) of 1 microM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-DENV antibody binds to a DENV epitope that is conserved among DENV of different serotypes. In certain embodiments, an anti-DENV E protein antibody binds to a DENV E protein epitope that is conserved among DENV E proteins of different serotypes.

[051] The term antibody in this invention is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided they exhibit the desired antigen-binding activity. Petition 870260064115, dated 06 / 29 / 2026, page 26 / 485 18 / 153

[052] Antibody-dependent cell-mediated cytotoxicity or ADCC refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to an antigen-carrying target cell and subsequently neutralize the target cell with cytotoxins. The primary cells for ADCC mediation, NK cells, express only Fc gamma RIII, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Imunol 9: 457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), can be performed.Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that reported in Clynes et al. PNAS (USA) 95:652656 (1998).

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

[054] An antibody that binds to the same epitope as a reference antibody refers to an antibody that blocks binding. Petition 870260064115, dated 06 / 29 / 2026, p. 27 / 485 19 / 153 of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen in a competition assay. An exemplary competition assay is provided here.

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

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

[057] The class of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different immunoglobulin classes are called alpha, delta, epsilon, gamma, and mu, respectively.

[058] 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 appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Variants of Petition 870260064115, dated 06 / 29 / 2026, page 28 / 485 20 / 153 polypeptides with altered Fc region amino acid sequences (polypeptides with an Fc variant region) and increased or decreased C1q binding capacity are described, for example, in US Patent No. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[059] The term cytotoxic agent as used herein refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211At, 1311, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 212Pb and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, periwinkle alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth-inhibiting agents; enzymes and their fragments such as nucleolytic enzymes; antibiotics; Toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or their variants; and the various antitumor or anticancer agents disclosed below.

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

[061] An effective quantity of an agent, for example, a pharmaceutical formulation, refers to an effective amount, in dosages and for periods of time necessary, to obtain the Petition 870260064115, dated 06 / 29 / 2026, p. 29 / 485 21 / 153 desired therapeutic or prophylactic outcome.

[062] The term epitope includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen, and includes specific amino acids that directly come into contact with the antibody. Epitope determinants may include chemically surfactant clusters of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural features and / or specific charge characteristics. In general, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[063] Fc receptor or FcR describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcyRII receptors include FcyRIIA (an activating receptor) and FcyRIIB (an inhibiting receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibiting motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15: 203-234 (1997). FcRs that are reviewed, for example, in Ravetch and Kinet, Annu. Rev.)Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. Petition 870260064115, of 29 / 06 / 2026, p. 30 / 485 22 / 153 126:330-41 (1995). Other FcRs, including those to be identified in the future, are included by the term FcR in this invention.

[064] The term Fc receptor or FcR also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).The in vivo binding to human FcRn and serum half-life of high-affinity FcRn-binding polypeptides can be analyzed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with a variant Fc region are administered. WO 2000 / 42072 (Presta) describes antibody variants with improved or impaired binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[065] The term Fc region in this invention is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region 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 otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also called the EU index. Petition 870260064115, dated 06 / 29 / 2026, page 31 / 485 23 / 153 as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[066] The term antibody comprising the Fc region refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447) or glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or through recombinant engineering of the nucleic acid encoding the antibody. Consequently, a composition comprising an antibody having an Fc region according to this invention may comprise an antibody with G446-K447, with G446 and without K447, with all G446-K447 removed, or a mixture of three types of antibodies described above.

[067] Structure or FR refers to variable domain residuals other than hypervariable region (HVR) residuals. A variable domain FR usually consists of four FR domains: FR1, FR2, FR3, and FR4. Consequently, HVR and FR sequences usually appear in the following sequence in VH (or VL): FR1H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

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

[069] A functional Fc region has an effector function of a native sequence Fc region. Exemplary effector functions include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a domain. Petition 870260064115, dated 06 / 29 / 2026, p. 32 / 485 24 / 153 binding (e.g., a variable antibody domain) and can be evaluated using various assays as described, for example, in the definitions in this invention.

[070] The terms host cell, host cell line, co-transfected cell, and host cell culture are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and its derived progeny without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a source cell, but may contain mutations. Mutant progeny that possesses the same biological function or activity when screened or selected in the originally transformed cell is included here. In certain embodiments, a host cell or co-transfected cell is CHO-DXB11, CHOK1, or CHO-DG44.Such a host cell or co-transfected cell may be a taurine expression carrier, obtained by introducing the DNA-encoding taurine carrier (WO 2007 / 119774).

[071] A human antibody is one that has an amino acid sequence that corresponds to that of an antibody produced by a human being or human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-coding sequences. This definition of a human antibody specifically excludes a humanized antibody that comprises non-human antigen-binding residues.

[072] A human consensus structure is a structure that represents the amino acid residues that occur most commonly in a selection of VL or VH structure sequences of immunoglobulin Petition 870260064115, dated 06 / 29 / 2026, page 33 / 485 25 / 153 human. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the sequence subgroup is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[073] A humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A humanized form of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.

[074] The term hypervariable region or HVR as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence (complementarity-determining regions or CDRs) and / or form structurally defined loops (hypervariable loops) and / or contain antigen-contact residues (antigen contacts). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs in this invention include: (a) hypervariable loops that occur in residuals of Petition 870260064115, dated 06 / 29 / 2026, page 34 / 485 26 / 153 amino acids 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2) and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). (b) CDRs that occur at amino acid residues 24 to 34 (L1), 50 to 56 (L2), 89 to 97 (L3), 31 to 35b (H1), 50 to 65 (H2) and 95 to 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts that occur at amino acid residues 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2) and 93 to 101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b) and / or (c), including amino acid residues from HVR 46 to 56 (L2), 47 to 56 (L2), 48 to 56 (L2), 49 to 56 (L2), 26 to 35 (H1), 26 to 35b (H1), 49 to 65 (H2), 93 to 102 (H3) and 94 to 102 (H3).

[075] Unless otherwise indicated, HVR residuals and other residuals in the variable domain (e.g., FR residuals) are numbered here in accordance with Kabat et al., supra.

[076] An immunoconjugate is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

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

[078] An isolated antibody is one that has been separated from a component of its natural environment. In some embodiments, a Petition 870260064115, dated 06 / 29 / 2026, page 35 / 485 27 / 153 antibody is purified to more than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion-exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[079] An isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that commonly contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[080] Isolated nucleic acid encoding an antibody refers to one or more nucleic acid molecules that encode the heavy and light chains of the antibody (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[081] The term monoclonal antibody as used here refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or mutations that arise during the production of a monoclonal antibody preparation, such variants generally being present in small quantities. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in an antibody preparation Petition 870260064115, dated 06 / 29 / 2026, page 36 / 485 28 / 153 monoclonal is directed against a single determinant on an antigen. Thus, the monoclonal modifier indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be produced by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage presentation methods, and methods using transgenic animals containing all or part of the human immunoglobulin sites, such methods and other exemplary methods for the production of monoclonal antibodies being described herein.

[082] A pure antibody refers to an antibody that is not conjugated with a heterologous component (e.g., a cytotoxic component) or radioactive labeling. Pure antibodies may be present in a pharmaceutical formulation.

[083] Native antibodies refer to naturally occurring immunoglobulin molecules with variable structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-linked. From the N- to the C-terminal, each heavy chain has a variable region (VH), also called a variable heavy domain or a variable heavy chain domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N- to the C-terminal, each light chain has a variable region (VL), also called a variable light domain or variable light chain domain, followed by a constant light domain (CL). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and Petition 870260064115, dated 06 / 29 / 2026, page 37 / 485 29 / 153 lambda (λ), based on the amino acid sequence of its constant domain.

[084] A native sequence Fc region comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Human native sequence Fc regions include a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; a native sequence human IgG4 Fc region, as well as their naturally occurring variants.

[085] The term package insert is used to refer to the instructions usually included in commercial packaging of therapeutic products, which contain information on indications, use, dosage, administration, combination therapy, contraindications and / or warnings regarding the use of such therapeutic products.

[086] Percent amino acid sequence identity (%) with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, the Megalign software (DNASTAR), or GENTYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for sequence alignment, including Petition 870260064115, dated 06 / 29 / 2026, page 38 / 485 30 / 153 any algorithms necessary to obtain maximum alignment with respect to the total length of the sequences being compared.

[087] The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code was deposited with the user documentation at the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, including UNIX V4.0D digital. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In situations where ALIGN-2 is used for amino acid sequence comparisons, the amino acid sequence identity % of a given amino acid sequence A in, with, or against a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A that possesses or comprises a certain amino acid sequence identity % in, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y

[088] where X is the number of amino acid residues scored as identical matches by the ALIGN-2 sequence alignment program with the alignment program for A and B, and where Y is the total number of amino acid residues in B. It will be observed that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity of A with B will not be equal to the amino acid sequence identity of B with A. Unless specifically Petition 870260064115, dated 06 / 29 / 2026, p. 39 / 485 31 / 153 mentioned otherwise, all amino acid sequence identity values ​​% used herein are obtained as described in the immediately preceding paragraph, using the ALIGN-2 computer program.

[089] The term pharmaceutical formulation refers to a preparation that is in such a form that allows the biological activity of an ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to an individual to whom the formulation would be administered.

[090] A pharmaceutically acceptable vehicle refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to an individual. A pharmaceutically acceptable vehicle includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[091] The term DENV E protein, as used herein, refers to any DENV E protein native to any DENV serotype, including DENV-1, DENV-2, DENV-3, and DENV-4, unless otherwise indicated. The DENV genome encodes three structural proteins (capsid (C), pre-membrane / membrane (prM / M), and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The E protein, a glycoprotein 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 have revealed three distinct beta-barrel domains connected to the viral membrane by a helical rod anchoring and two antiparallel transmembrane domains. Domain III (EDIII) adopts an immunoglobulin-like fold and has been suggested to play a critical role in receptor interactions.Domain II (EDII) is an elongated domain composed of two long finger-like structures and contains a fusion loop of 13. Petition 870260064115, dated 06 / 29 / 2026, page 40 / 485 The core domain (domain I; EDI) is a nine-stranded β-barrel that is connected to EDIII and EDII by one and four flexible connectors, respectively. E proteins are important for viral assembly, receptor binding, entry, viral fusion, and possibly immune evasion during the virus life cycle and are thus required to adopt various distinct conformations and arrangements on the virus particle. The term encompasses the unprocessed full-length DENV E protein as well as any form of DENV E protein resulting from processing within the cell. The term also encompasses naturally occurring variants of DENV E protein, for example, serotype variants or quasi-species variants. The core domain (domain I; EDI) is a nine-stranded β-barrel that is connected to EDIII and EDII by one and four flexible connectors, respectively. E proteins are important for viral assembly, receptor binding, entry, viral fusion, and possibly immune evasion during the virus life cycle and, thus, dynamic proteins are needed to adopt various distinct conformations and arrangements on the virus particle.

[092] The phrase substantially decreased, substantially increased or substantially different, as used herein, refers to a sufficiently high degree of difference between two numerical values ​​(usually one associated with a molecule and the other associated with a reference / comparator molecule) such that a person skilled in the art would consider the difference between the two values ​​to be of statistical significance within the context of the biological characteristic measured by the said values ​​(e.g., Kd values).

[093] As used in this invention, treatment (and its grammatical variations such as treat or treating) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of occurrence or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention Petition 870260064115, dated 06 / 29 / 2026, p. 41 / 485 33 / 153 metastasis, decreased disease progression rate, improved or mitigated disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to slow the development of a disease or to slow the progression of a disease.

[094] The term variable region or variable domain refers to the domain of an antibody heavy or light chain that is involved in antibody binding to the antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved structure regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to the antigen to select a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[095] A variant Fc region comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared with a native sequence Fc region or the Fc region of a source polypeptide, for example, from about one to about ten amino acid substitutions and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the source polypeptide. Petition 870260064115, dated 06 / 29 / 2026, page 42 / 485 34 / 153 origin. The variant Fc region in this invention will preferably contain at least about 80% homology with a native sequence Fc region and / or with an Fc region of a polypeptide of origin, and more preferably at least around 90% homology from this, more preferably also at least around 95% homology.

[096] The term vector, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are attached in an operable manner. Such vectors are referred to herein as expression vectors. II. COMPOSITIONS AND METHODS

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

[098] In one aspect, the invention is based, in part, on polypeptides comprising variant Fc regions and their use. In certain embodiments, polypeptides comprising variant Fc regions with substantially decreased FcyR binding activity are provided. In certain embodiments, polypeptides comprising variant Fc regions without substantially decreased C1q binding activity are provided. In particular embodiments, the polypeptides of the invention are antibodies. Polypeptides comprising variant Fc regions of the invention are useful, for example, for the diagnosis or treatment of viral infection. Petition 870260064115, dated 06 / 29 / 2026, p. 43 / 485 35 / 153

[099] A. Anti-DENV Antibodies Samples and Polypeptides comprising variant Fc regions

[0100] In one aspect, the invention provides isolated antibodies that bind to DENV and / or DENV E protein. In certain embodiments, an anti-DENV antibody blocks the binding of DENV and / or DENV E protein to a host cell. In certain embodiments, an anti-DENV antibody inhibits the entry of DENV into a host cell. In certain embodiments, an anti-DENV antibody binds to a whole DENV particle. In other embodiments, the antibody binds to a whole DENV particle better than to a monomeric DENV E protein. In certain embodiments, an 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 certain embodiments, the anti-DENV antibody binds to and / or neutralizes the DENV E protein derived 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. A serotype refers to the distinct variations within a species of virus.

[0101] In one aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of any of the SEQ ID NOs: 11 to 12; (b) HVR-H2 comprising the amino acid sequence of any of the SEQ ID NOs: 13 to 15; (c) HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20; (d) HVR-L1 comprising the amino acid sequence of any of the SEQ ID NOs: 21 to 23; (e) HVR-L2 comprising the sequence of Petition 870260064115, dated 06 / 29 / 2026, p. 44 / 485 36 / 153 amino acid from any of the SEQ ID NOs: 24 to 26; and (f) HVR-L3 comprising the amino acid sequence from any of the SEQ ID NOs: 27 to 30.

[0102] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0103] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0104] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: Petition 870260064115, dated 06 / 29 / 2026, p. 45 / 485 37 / 153 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0105] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VH sequences selected from (a) HVR-H1 comprising the amino acid sequence of any of the SEQ ID NOs: 11 to 12; (b) HVR-H2 comprising the amino acid sequence of any of the SEQ ID NOs: 13 to 15; and (c) HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20 and HVR-L3 comprising the amino acid sequence of any of the SEQ ID NOs: 27 to 30.In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20, HVR-L3 comprising the amino acid sequence of any of the SEQ ID NOs: 27 to 30, and HVR-H2 comprising the amino acid sequence of any of the SEQ ID NOs: 13 to 15. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of any of the SEQ ID NOs: 11 to 12; (b) HVR-H2 comprising the amino acid sequence of any of the SEQ ID NOs: 13 to 15; and (c) HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20.

[0106] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VH sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 Petition 870260064115, dated 06 / 29 / 2026, p. 46 / 485 38 / 153 comprising the amino acid sequence of SEQ ID NO: 41; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42.

[0107] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VH sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In yet another embodiment, the Petition 870260064115, dated 06 / 29 / 2026, p. 47 / 485 39 / 153 antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20.

[0108] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VL sequences selected from (a) HVR-L1 comprising the amino acid sequence of any of the SEQ ID NOs: 21 to 23; (b) HVR-L2 comprising the amino acid sequence of any of the SEQ ID NOs: 24 to 26; and (c) HVR-L3 comprising the amino acid sequence of any of the SEQ ID NOs: 27 to 30. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of any of the SEQ ID NOs: 21-23; (b) HVR-L2 comprising the amino acid sequence of any of the SEQ ID NOs: 24 to 26; and (c) HVR-L3 comprising the amino acid sequence of any of the SEQ ID NOs: 27 to 30.

[0109] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VL sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2 Petition 870260064115, dated 06 / 29 / 2026, p. 48 / 485 40 / 153 comprising the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0110] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VL sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0111] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 comprising the amino acid sequence of any of the SEQ ID NOs: 11 to 12, (ii) HVR-H2 comprising the amino acid sequence of any of the SEQ ID NOs: 13 to 15, and (iii) HVR-H3 comprising an amino acid sequence of any of the SEQ ID NOs: 16 to 20; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 comprising the amino acid sequence of any of the SEQ ID NOs: 21 to 23, (ii) HVR-L2 comprising the amino acid sequence of any of the SEQ Petition 870260064115, dated 06 / 29 / 2026, p. 49 / 485 41 / 153 ID NOs: 24 to 26, and (iii) HVR-L3 comprising the amino acid sequence of any of the SEQ ID NOs: 27 to 30.

[0112] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 42; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0113] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 20; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0114] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) Petition 870260064115, dated 06 / 29 / 2026, p. 50 / 485 42 / 153 (a) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0115] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of any of the SEQ ID NOs: 11 to 12; (b) HVR-H2 comprising the amino acid sequence of any of the SEQ ID NOs: 13 to 15; (c) HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20; (d) HVR-L1 comprising the amino acid sequence of any of the SEQ ID NOs: 21 to 23; (e) HVR-L2 comprising the amino acid sequence of any of the SEQ ID NOs: 24 to 26; and (f) HVR-L3 comprising an amino acid sequence of any of the SEQ ID NOs: 27 to 30.

[0116] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 45.

[0117] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence Petition 870260064115, dated 06 / 29 / 2026, p. 51 / 485 43 / 153 of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 30.

[0118] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 27.

[0119] In certain embodiments, any one or more amino acids of an anti-DENV antibody as provided above are substituted at the following HVR positions: (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), at positions 4 and 7; and (f) in HVR-L3 (SEQ ID NO: 27), at positions 4 and 5.

[0120] In certain embodiments, one or more amino acid substitutions of an anti-DENV antibody are conservative substitutions, as provided herein. In certain embodiments, any one or more of the following substitutions may be produced 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 Petition 870260064115, dated 06 / 29 / 2026, p. 52 / 485 44 / 153 ID NO: 16), R3E; R14F; G16D or L; (d) in HVR-L1 (SEQ ID NO: 21), D5E; K8Q; (e) in HVR-L2 (SEQ ID NO: 24), N4E; T7F; and (f) in HVR-L3 (SEQ ID NO: 27), D4S or E; D5A.

[0121] All possible combinations of the above substitutions are included by the consensus sequence of SEQ ID NOs: 40, 41, 42, 43, 44, and 45 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVRL3, respectively.

[0122] In another embodiment, an anti-DENV antibody of the invention is not an antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24 and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0123] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (b) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (c) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (d) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 8 to 10; (e) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 8 to 10; and (f) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10.

[0124] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (b) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (c) HVR-H3 of the VH sequence of any Petition 870260064115, dated 06 / 29 / 2026, p. 53 / 485 45 / 153 one of the SEQ ID NOs: 2 to 6; (d) HVR-L1 of the VL sequence of any of the SEQ ID NO: 7; (e) HVR-L2 of the VL sequence of any of the SEQ ID NO: 7; and (f) HVR-L3 of the VL sequence of any of the SEQ ID NO: 7.

[0125] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 of the VH sequence of SEQ ID NO: 6; (b) HVR-H2 of the VH sequence of SEQ ID NO: 6; (c) HVR-H3 of the VH sequence of SEQ ID NO: 6; (d) HVR-L1 of the VL sequence of SEQ ID NO: 10; (e) HVR-L2 of the VL sequence of SEQ ID NO: 10; and (f) HVRL3 of the VL sequence of SEQ ID NO: 10.

[0126] In another aspect, the invention provides an anti-DENV antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 of the VH sequence of SEQ ID NO: 6; (b) HVR-H2 of the VH sequence of SEQ ID NO: 6; (c) HVR-H3 of the VH sequence of SEQ ID NO: 6; (d) HVR-L1 of the VL sequence of SEQ ID NO: 7; (e) HVR-L2 of the VL sequence of SEQ ID NO: 7; and (f) HVR-L3 of the VL sequence of SEQ ID NO: 7.

[0127] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VH sequences selected from (a) HVR-H1 of the VH sequence from any of the SEQ ID NOs: 2 to 6; (b) HVR-H2 of the VH sequence from any of the SEQ ID NOs: 2 to 6; (c) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6. In one embodiment, the antibody comprises HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6. In another embodiment, the antibody comprises HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6 and HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10. In another embodiment, the antibody comprises HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6 and HVR-L3 of the sequence Petition 870260064115, dated 06 / 29 / 2026, p. 54 / 485 46 / 153 VL of any of the SEQ ID NOs: 7. In one embodiment, the antibody comprises HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6, HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10, and HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6. In another embodiment, the antibody comprises HVRH3 of the VH sequence of any of the SEQ ID NOs: 2 to 6, HVR-L3 of the VL sequence of any of the SEQ ID NOs: 7, and HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6. In yet another embodiment, the antibody comprises (a) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (b) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6; and (c) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6.

[0128] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VH sequences selected from (a) HVR-H1 of the VH sequence from SEQ ID NO: 6; (b) HVR-H2 of the VH sequence from SEQ ID NO: 6; (c) HVR-H3 of the VH sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises HVR-H3 of the VH sequence of SEQ ID NO: 6. In another embodiment, the antibody comprises HVR-H3 of the VH sequence of SEQ ID NO: 6 and HVR-L3 of the VL sequence of SEQ ID NO: 10. In another embodiment, the antibody comprises HVR-H3 of the VH sequence of SEQ ID NO: 6 and HVR-L3 of the VL sequence of SEQ ID NO: 7. In another embodiment, the antibody comprises HVR-H3 of the VH sequence of SEQ ID NO: 6, HVR-L3 of the VL sequence of SEQ ID NO: 10, and HVRH2 of the VH sequence of SEQ ID NO: 6. In another embodiment, the antibody comprises HVR-H3 of the VH sequence of SEQ ID NO: 6, HVR-L3 of the VL sequence of SEQ ID NO: 7, and HVR-H2 of sequence VH of SEQ ID NO: 6.In another embodiment, the antibody comprises (a) HVR-H1 of the VH sequence from SEQ ID NO: 6; (b) HVRH2 of the VH sequence from SEQ ID NO: 6; and (c) HVR-H3 of the VH sequence. Petition 870260064115, dated 06 / 29 / 2026, p. 55 / 485 47 / 153 of SEQ ID NO: 6.

[0129] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VL sequences selected from (a) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 8 to 10; (b) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 8 to 10; and (c) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10. In one embodiment, the antibody comprises (a) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 8 to 10; (b) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 8 to 10; and (c) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10.

[0130] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three HVR VL sequences selected from (a) HVR-L1 of the VL sequence of SEQ ID NO: 10; (b) HVR-L2 of the VL sequence of SEQ ID NO: 10; and (c) HVR-L3 of the VL sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises (a) HVR-L1 of the VL sequence of SEQ ID NO: 10; (b) HVR-L2 of the VL sequence of SEQ ID NO: 10; and (c) HVR-L3 of the VL sequence of SEQ ID NO: 10.

[0131] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (ii) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6, and (iii) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 8 to 10, (ii) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 8 to 10, and (iii) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10. Petition 870260064115, dated 06 / 29 / 2026, p. 56 / 485 48 / 153

[0132] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6, (ii) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6, and (iii) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 7, (ii) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 7, and (iii) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 7.

[0133] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 of the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 of the VH sequence of SEQ ID NO: 6, and (iii) HVR-H3 of the VH sequence of SEQ ID NO: 6; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 of the VL sequence of SEQ ID NO: 10, (ii) HVR-L2 of the VL sequence of SEQ ID NO: 10, and (iii) HVR-L3 of the VL sequence of SEQ ID NO: 10.

[0134] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 of the VH sequence of SEQ ID NO: 6, (ii) HVR-H2 of the VH sequence of SEQ ID NO: 6, and (iii) HVR-H3 of the VH sequence of SEQ ID NO: 6; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 of the VL sequence of SEQ ID NO: 7, (ii) HVR-L2 of the VL sequence of SEQ ID NO: 7, and (iii) HVR-L3 of the VL sequence of SEQ ID NO: 7.

[0135] In another aspect, the invention provides an antibody that Petition 870260064115, dated 06 / 29 / 2026, p. 57 / 485 49 / 153 comprises (a) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (b) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (c) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (d) HVR-L1 of the VL sequence of any of the SEQ ID NOs: 8 to 10; (e) HVR-L2 of the VL sequence of any of the SEQ ID NOs: 8 to 10; and (f) HVR-L3 of the VL sequence of any of the SEQ ID NOs: 8 to 10.

[0136] In another aspect, the invention provides an antibody comprising (a) HVR-H1 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (b) HVR-H2 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (c) HVR-H3 of the VH sequence of any of the SEQ ID NOs: 2 to 6; (d) HVR-L1 of the VL sequence of any of the SEQ ID NO: 7; (e) HVR-L2 of the VL sequence of any of the SEQ ID NO: 7; and (f) HVR-L3 of the VL sequence of any of the SEQ ID NO: 7.

[0137] In another aspect, the invention provides an antibody comprising (a) HVR-H1 of the VH sequence of SEQ ID NO: 6; (b) HVR-H2 of the VH sequence of SEQ ID NO: 6; (c) HVR-H3 of the VH sequence of SEQ ID NO: 6; (d) HVR-L1 of the VL sequence of SEQ ID NO: 10; (e) HVR-L2 of the VL sequence of SEQ ID NO: 10; and (f) HVR-L3 of the VL sequence of SEQ ID NO: 10.

[0138] In another aspect, the invention provides an antibody comprising (a) HVR-H1 of the VH sequence of SEQ ID NO: 6; (b) HVR-H2 of the VH sequence of SEQ ID NO: 6; (c) HVR-H3 of the VH sequence of SEQ ID NO: 6; (d) HVR-L1 of the VL sequence of SEQ ID NO: 7; (e) HVR-L2 of the VL sequence of SEQ ID NO: 7; and (f) HVR-L3 of the VL sequence of SEQ ID NO: 7.

[0139] In another embodiment, an anti-DENV antibody of the invention is not an antibody comprising (i) HVR-H1 of the VH sequence of SEQ ID NO: 1, (ii) HVR-H2 of the VH sequence of SEQ ID NO: 1, (iii) HVR-H3 of the VH sequence of SEQ ID NO: 1, (iv) HVR-L1 of Petition 870260064115, dated 06 / 29 / 2026, p. 58 / 485 50 / 153 sequence VL of SEQ ID NO: 7, (v) HVR-L2 of sequence VL of SEQ ID NO: 7, and (vi) HVR-L3 of sequence VL of SEQ ID NO: 7.

[0140] In any of the above embodiments, an anti-DENV antibody can be humanized. In one embodiment, an anti-DENV antibody comprises HVRs as in any of the above embodiments, and further comprises a human receptor framework, for example, a human immunoglobulin framework or a human consensus framework. In another embodiment, an anti-DENV antibody comprises HVRs as in any of the above embodiments, and further comprises a VH or VL comprising an FR sequence. In yet another embodiment, the anti-DENV antibody comprises the following variable heavy chain and / or light chain FR sequences: For the variable heavy chain domain, FR1 comprises the amino acid sequence of SEQ ID NO: 31, FR2 comprises the amino acid sequence of SEQ ID NO: 32, FR3 comprises the amino acid sequence of SEQ ID NO: 33 or 34, FR4 comprises the amino acid sequence of SEQ ID NO: 35.For the variable light chain domain, FR1 comprises the amino acid sequence of SEQ ID NO: 36, FR2 comprises the amino acid sequence of SEQ ID NO: 37, FR3 comprises the amino acid sequence of SEQ ID NO: 38, and FR4 comprises the amino acid sequence of SEQ ID NO: 39.

[0141] In another aspect, an anti-DENV antibody comprises a variable heavy chain (VH) domain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of any of the SEQ ID NOs: 2 to 6. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions in Petition 870260064115, dated 06 / 29 / 2026, p. 59 / 485 51 / 153 relative to the reference sequence, but an anti-DENV antibody comprising this sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in any of the SEQ ID NOs: 2 to 6. In certain embodiments, substitutions, insertions or deletions occur in regions of the HVRs (i.e., in the FRs). Optionally, the anti-DENV antibody comprises the VH sequence in any of the SEQ ID NOs: 2 to 6, including post-translational modifications of this sequence. In a particular embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of any of the SEQ ID NOs: 11 to 12, (b) HVR-H2 comprising the amino acid sequence of any of the SEQ ID NOs: 13 to 15, and (c) HVR-H3 comprising the amino acid sequence of any of the SEQ ID NOs: 16 to 20.Post-translational modifications include, but are not limited to, a modification of glutamine or glutamate at the N-terminal heavy chain or light chain into pyroglutamic acid via pyroglutamylation.

[0142] In another aspect, an anti-DENV antibody comprises a variable heavy chain (VH) domain sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 6. In certain embodiments, a VH sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-DENV antibody comprising this sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids were substituted, inserted, and / or eliminated in SEQ ID NO: 6. In certain embodiments, the Petition 870260064115, dated 06 / 29 / 2026, p. 60 / 485 52 / 153 Substitutions, insertions, or deletions occur in the outer regions of the HVRs (i.e., in the FRs). Optionally, the anti-DENV antibody comprises the VH sequence at SEQ ID NO: 6, including post-translational modifications of this sequence. In a particular embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence at SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence at SEQ ID NO: 15, and (c) HVR-H3 comprising the amino acid sequence at SEQ ID NO: 20. Post-translational modifications include, but are not limited to, a glutamine or glutamate modification at the N-terminal of the heavy chain or light chain to pyroglutamic acid via pyroglutamylation.

[0143] In another aspect, an anti-DENV antibody is provided, wherein the antibody comprises a variable light chain (VL) domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of any of the SEQ ID NOs: 8 to 10. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-DENV antibody comprising this sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids were substituted, inserted, and / or deleted in any of the SEQ ID NOs: 8 to 10. In certain embodiments, substitutions, insertions, or deletions occur in the outer regions of the HVRs (i.e., in the FRs).Optionally, the anti-DENV antibody comprises the VL sequence in any of the SEQ ID NOs: 8 to 10, including post-translational modifications of this sequence. In one particular embodiment, the VL comprises one, two, or three selected HVRs. Petition 870260064115, dated 06 / 29 / 2026, p. 61 / 485 53 / 153 (a) HVR-L1 comprising the amino acid sequence of any of the SEQ ID NOs: 21 to 23; (b) HVR-L2 comprising the amino acid sequence of any of the SEQ ID NOs: 24 to 26; and (c) HVR-L3 comprising the amino acid sequence of any of the SEQ ID NOs: 27 to 30. Post-translational modifications include, but are not limited to, a modification of glutamine or glutamate at the N-terminal heavy chain or light chain to pyroglutamic acid via pyroglutamylation.

[0144] In another aspect, an anti-DENV antibody is provided, wherein the antibody comprises a variable light chain (VL) domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-DENV antibody comprising this sequence retains the ability to bind to DENV. In certain embodiments, a total of 1 to 10 amino acids were substituted, inserted, and / or deleted in SEQ ID NO: 10. In certain embodiments, substitutions, insertions, or deletions occur in the outer regions of the HVRs (i.e., in the FRs).Optionally, the antiDENV antibody comprises the VL sequence at SEQ ID NO: 10, including post-translational modifications of this sequence. In a particular embodiment, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence at SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence at SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence at SEQ ID NO: 30. Post-translational modifications include, but are not limited to, a glutamine or glutamate modification at the N-terminus. Petition 870260064115, dated 06 / 29 / 2026, p. 62 / 485 54 / 153 of heavy chain or light chain in pyroglutamic acid through pyroglutamylation.

[0145] In another aspect, an anti-DENV antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in any of the SEQ ID NOs: 2 to 6 and any of the SEQ ID NOs: 8 to 10, respectively, including post-translational modifications of these sequences. In another embodiment, the antibody comprises the VH and VL sequences in any of the SEQ ID NOs: 2 to 6 and SEQ ID NO: 7, respectively, including post-translational modifications of these sequences. Post-translational modifications include, but are not limited to, a modification of glutamine or glutamate at the N-terminal heavy chain or light chain to pyroglutamic acid via pyroglutamylation.

[0146] In another aspect, an anti-DENV antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences at SEQ ID NO: 6 and SEQ ID NO: 10, respectively, including post-translational modifications of these sequences. In another embodiment, the antibody comprises the VH and VL sequences at SEQ ID NO: 6 and SEQ ID NO: 7, respectively, including post-translational modifications of these sequences. Post-translational modifications include, but are not limited to, a modification of glutamine or glutamate at the N-terminal heavy chain or light chain to pyroglutamic acid via pyroglutamylation.

[0147] In another aspect, the invention provides an antibody that binds to the same epitope as an anti-DENV antibody provided herein. Petition 870260064115, dated 06 / 29 / 2026, p. 63 / 485 55 / 153 In certain embodiments, an antibody is provided that binds to an epitope comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all of the amino acids selected from the group consisting of G100, W101, K122, I162, S274, K310, W391, and F392 in the DENV-2 E protein. In certain embodiments, an antibody is provided which binds to an epitope comprising at least one, at least two, or all of the amino acids selected from the group consisting of K122, I162, and S274 in the DENV-2 E protein. In certain embodiments, an antibody is provided that binds to an epitope that still comprises at least one of the amino acids selected from the group consisting of G100, W101, K310, W391, and F392, whereas the epitope comprises at least one, at least two, or all of the amino acids selected from the group consisting of K122, I162, and S274.

[0148] In another aspect of the invention, an anti-DENV antibody according to any of the above embodiments is a monoclonal antibody, which includes a chimeric, humanized or human antibody. In one embodiment, an anti-DENV antibody is an antibody fragment, for example, an Fv, Fab, Fab', scFv diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, for example, an intact IgG1 antibody or another antibody class or isotype as defined herein.

[0149] In another aspect, an anti-DENV antibody of the invention may have a modification that suppresses antibody binding to Fc gamma receptors (FcγR). Without wishing to be limited by theory, a modification that suppresses antibody binding to Fc gamma receptors may be advantageous because decreased binding to FcγR may prevent the ADE (antibody-dependent enhancement) phenomenon of infection, which is believed to be primarily mediated by interaction with FcγR. In one embodiment, an Fc region of a Petition 870260064115, dated 06 / 29 / 2026, p. 64 / 485 56 / 153 anti-DENV antibody of the invention comprises Ala at position 234 and Ala at position 235 according to EU numbering.

[0150] In another aspect, an anti-DENV antibody according to any of the above embodiments may incorporate any of the features, individually or in combination, as described in Sections 1 to 7 below. 1. Antibody Affinity

[0151] In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of 1 micro M or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M).

[0152] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, the binding affinity of the Fab solution for the antigen is measured by equilibrizing Fab with a minimum concentration of labeled antigen (125I) in the presence of a titration series of unlabeled antigen, then capturing the bound antigen with an anti-Fab antibody-coated plate (see, for example, Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish assay conditions, MICROTITER (registered trademark) multi-reservoir plates (Thermo Scientific) are coated overnight with 5 µg / mL of an anti-Fab capture antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 degrees C).In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM of [125I] antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent). Petition 870260064115, dated 06 / 29 / 2026, page 65 / 485 57 / 153 with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may continue for a longer period (e.g., around 65 hours) to ensure equilibrium is reached. After this, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20 (trademark)) in PBS. Once the plates have dried, 150 micro 1 / reservoir of scintillating agent (MICROSCINT-20™; Packard) are added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of the maximum binding are chosen for use in competitive binding assays.

[0153] According to another embodiment, Kd is measured using a BIACORE (trademark) surface plasmon resonance assay. For example, an assay using a BIACORE (trademark)-2000 or a BIACORE (trademark)-3000 (BIAcore, Inc, Piscataway, NJ) is run at 25 degrees C with the antigen-immobilized CM5 chips in ~10 response units (RU). In one embodiment, the carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) hydrochloride according to the vendor's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, at 5 pg / mL (-0.2 pM) before injection at a flow rate of 5 pL / minute to achieve approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups.For kinetic measurements, doubled serial dilutions of Fab (0.78 nM to 500 nM) are injected into PBS with surfactant. Petition 870260064115, dated 06 / 29 / 2026, p. 66 / 485 58 / 153 polysorbate 20 at 0.05% (TWEEN-20™) (PBST) at 25°C at a flow rate of approximately 25 μL / min. Association rates (kon) and dissociation rates (koff) are calculated using an individualized Langmuir simple binding model (BIACORE (registered trademark) Evaluation Software version 3.2) through simultaneous fitting of association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999).If the association rate exceeds 106M-1s-1 by the surface plasmon resonance assay above, then the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25 degrees C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing antigen concentrations as measured in a spectrometer, such as a spectrophotometer equipped with a stop flow (Aviv Instruments) or an SLM-AMINCO™ 8000 series spectrophotometer (ThermoSpectronic) with a shake cuvette. 2. Antibody Fragments

[0154] In certain embodiments, an antibody supplied here is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab% Fv and scFv) fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and US Patent Nos. Petition 870260064115, dated 06 / 29 / 2026, p. 67 / 485 59 / 153 epitope residues binding to the recovery receptor and having an increased in vivo half-life, see US Patent No. 5,849,046.

[0155] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0156] Single-domain antibodies are antibody fragments comprising all or part of the variable heavy-chain domain or all or part of the variable light-chain domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).

[0157] Antibody fragments can be produced by various techniques, including but not limited to proteolytic digestion of an intact antibody, as well as production via recombinant host cells (e.g., E. coli or phage), as described in this invention. 3. Chimeric and Humanized Antibodies

[0158] In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a class-switched antibody in which the class or subclass has been altered from the source antibody. Chimeric antibodies include their Petition 870260064115, dated 06 / 29 / 2026, p. 68 / 485 60 / 153 antigen-binding fragments.

[0159] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the non-human antibody of origin. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, for example, CDRs, (or parts thereof) are derived from a non-human antibody, and the FRs (or parts thereof) are derived from human antibody sequences. A humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve the antibody's specificity or affinity.

[0160] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. EUA 86: 10029-10033 (1989); US Patents Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (which describes reemergence); Dall'Acqua et al., Methods 36:43-60 (2005) (which describes FR rearrangement); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (describing the guided selection approach for FR rearrangement).

[0161] Regions of human structure that can be used for humanization include, but are not limited to: regions Petition 870260064115, dated 06 / 29 / 2026, p. 69 / 485 61 / 153 selected structural regions using the best-fit method (see, for example, Sims et al. J. Immunol. 151:2296 (1993)); structural regions derived from the consensus sequence of human antibodies from a particular subgroup of variable light or heavy chain regions (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); mature human (somatically modified) structural regions or human germline structural regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and structural regions derived from selection from RF libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)). 4. Human Antibodies

[0162] In certain embodiments, an antibody provided here is a human antibody. Human antibodies can be produced using various techniques known in the specialty. Human antibodies are described in general in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0163] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with variable human regions in response to an antigenic challenge. Such animals typically contain all or part of the human immunoglobulin sites, which replace endogenous immunoglobulin sites, or which are present extrachromosically or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin sites have generally been inactivated. For a review of methods for obtaining human antibodies from Petition 870260064115, dated 06 / 29 / 2026, page 70 / 485 62 / 153 transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patents Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB technology (trademark); U.S. Patent No. 7,041,870 describing KM MOUSE technology (trademark); and U.S. Patent Application Publication No. 2007 / 0061900 describing VELOCIMOUSE technology (trademark). The human variable regions of intact antibodies generated by such animals can be further modified, for example, by combining them with a different human constant region.

[0164] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Techniques and Applications of Monoclonal Antibody Production, pp. 5163 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies generated by means of human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of human IgM monoclonal antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas).Human hybridoma technology (Triome technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Petition 870260064115, dated 06 / 29 / 2026, page 71 / 485 63 / 153

[0165] Human antibodies can also be generated by isolating variable domain sequences from selected Fv clones from human-derived phage presentation libraries. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. 5. Library-Derived Antibodies

[0166] The antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating display libraries in phages and selecting such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Academic. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol.Methods 284(12): 119-132(2004).

[0167] In certain phage presentation methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for the antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The Petition 870260064115, dated 06 / 29 / 2026, page 72 / 485 64 / 153 phages typically present antibody fragments, such as single-chain Fv fragments (scFv) or Fab fragments. Immunized source libraries provide high-affinity antibodies for the immunogen without the requirement of hybridoma construction.

[0168] Alternatively, the untested repertoire can be cloned (e.g., from humans) to provide a single source of antibodies for a wide range of non-self-antigens and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, exempt libraries can also be produced synthetically by cloning non-rearranged V-gene segments from stem cells, and using PCR primers containing random sequences to encode the highly variable CDR3 regions and perform the rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing libraries in human antibody phages include, for example: US Patent No. 5,750,373, and US Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 00236002.

[0169] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments in this invention. 6. Multispecific Antibodies

[0170] In certain embodiments, an antibody provided here is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for DENV protein E and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two Petition 870260064115, dated 06 / 29 / 2026, page 73 / 485 65 / 153 different epitopes of DENV protein E. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing DENV protein E. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0171] Techniques for the production of multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole design (see, for example, US Patent No. 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic steering effects for the production of Fc-heterodimeric antibody molecules (WO 2009 / 089004A1); Crosslinking of two or more antibodies or fragments (see, for example, US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); the use of diabody technology for the production of bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv dimers (scFv) (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0172] Antibodies designed with three or more functional antigen-binding sites, including Octopus antibodies, are also included in this invention (see, for example, US 2006 / 0025576A1).

[0173] The antibody or fragment in this invention also includes a Dual-Acting Fab or DAF comprising a binding site. Petition 870260064115, dated 06 / 29 / 2026, p. 74 / 485 66 / 153 to the antigen that binds to DENV E protein as well as another different antigen (see, for example, US 2008 / 0069820). 7. Antibody Variants

[0174] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequences. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, for example, antigen binding. a) Replacement, Insertion and Deletion Variants

[0175] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of preferred substitutions. More substantial alterations are provided in Table 1 under the heading of exemplary substitutions and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products selected for a desired activity, e.g., retained / enhanced antigen binding, decreased immunogenicity, or enhanced ADCC or CDC. [TABLE 1] Petition 870260064115, dated 06 / 29 / 2026, p. 75 / 485 67 / 153 Original Residue Exemplary Substitutions Preferred Substitutions Wing (A) Vai; Leu; llê Vai Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gene Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg ΙΙθ (D Leu; Vai; Met; Ala; Phe; Norleucina Leu Leu (L) Norleucina; lie; Vai; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; llê Pro Phe(F) Trp; Leu; Ser(S) Thr Thr (T) Ser Ser Trp (W) Phe Thr Phe Leu;

[0176] Amino acids can be grouped according to the following common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0177] Non-conservative substitutions will result Petition 870260064115, dated 06 / 29 / 2026, page 76 / 485 68 / 153 necessarily in the exchange of a member from one of these classes to another class.

[0178] One type of surrogate variant involves the substitution of one or more residues in the hypervariable region of a source antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the source antibody and / or will have substantially retained certain biological properties of the source antibody. An exemplary surrogate variant is an affinity-ripened antibody, which can be conveniently generated, for example, using phage-presentation-based affinity maturation techniques such as those described in this invention. In summary, one or more HVR residues are mutated and the variant antibodies presented on phages are screened for particular biological activity (e.g., binding affinity).

[0179] Alterations (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such alterations can be made at HVR access points, i.e., residues encoded by codons that mutate at high frequency during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that come into contact with the antigen, with the resulting VH or VL variant being tested for binding affinity. Affinity maturation through the construction and re-selection of secondary libraries has been described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some affinity maturation modalities, diversity is Petition 870260064115, dated 06 / 29 / 2026, p. 77 / 485 69 / 153 introduced into selected variable genes for maturation by any of a variety of methods (e.g., error-prone PCR, chain rearrangement, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, in which multiple HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine screening mutagenesis or modeling. CDR-H3 and CDRL3 in particular are frequently targeted.

[0180] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as granted herein) that do not substantially reduce binding affinity may be created in HVRs. Such changes may, for example, be outside the antigen-contact residues in HVRs. In certain embodiments of the VH and VL variant sequences given above, each HVR is unchanged, or contains no more than one, two, or three amino acid substitutions.

[0181] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called alanine screening mutagenesis as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a target residue or group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Petition 870260064115, dated 06 / 29 / 2026, page 78 / 485 70 / 153 alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Other substitutions can be introduced at amino acid sites demonstrating functional sensitivity to the initial substitutions. Alternatively or additionally, a crystal structure of an antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and nearby residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine if they contain the desired properties.

[0182] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions that vary in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue.Other variants of antibody molecule insertion include the fusion of an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody to the N- or C-terminal of the antibody. b) Glycosylation Variants

[0183] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0184] Where the antibody comprises an Fc region, the carbohydrate attached to it may be altered. Native antibodies produced by mammalian cells typically comprise a biantennary branched oligosaccharide that is usually linked by an N-linkage to the Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. Petition 870260064115, dated 06 / 29 / 2026, page 79 / 485 71 / 153 TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, for example, mannose, N-Acetylglucosamine (GlcNAc), galactose and sialic acid, as well as a fucose linked to a GlcNAc in the stem of the biantenna oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain enhanced properties.

[0185] In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid, and higher mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located around position 297 in the Fc region (EU numbering of Fc region residues); However, Asn297 can also be located around + / 3 amino acids upstream or downstream of position 297, that is, between positions 294 and 300, due to minor sequence variations in antibodies.Such fucosylation variants may have an improved ADCC function. See, for example, US Patent Publications Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to fucose-deficient or defucosylated antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO. Petition 870260064115, dated 06 / 29 / 2026, page 80 / 485 72 / 153 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient CHO Lec13 cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially at Example 11), and neutralized cell lines, such as alpha-1,6-fucosyltransferase, FUT8, gene-neutralized CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0186] Antibody variants are also provided with sectioned oligosaccharides, for example, where a biantennary oligosaccharide linked to the Fc region of the antibody is sectioned by GlcNAc.These antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jan-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 3087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). c) Variants of the Fc region

[0187] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a sequence of the human Fc region (by Petition 870260064115, dated 06 / 29 / 2026, page 81 / 485 73 / 153 example, a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0188] In certain embodiments, the invention contemplates an antibody variant that possesses some, but not all, effector functions, making it a desirable candidate for applications where the antibody's in vivo half-life is important, but certain effector functions (such as ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to measure CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to confirm whether the antibody possesses Fc gamma R binding (consequently likely having ADCC activity) and / or FcRn binding capacity. Primary cells for ADCC mediation, NK cells, express only Fc gamma RIII, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in US Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, the ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and the CytoTox 96 non-radioactive cytotoxicity assay (trademark) (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or. Petition 870260064115, dated 06 / 29 / 2026, p. 82 / 485 74 / 153 Additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). C1q binding assays can also be performed to confirm whether the antibody is capable of binding to C1q and therefore possesses CDC activity. See, for example, C1q and C3c binding ELISA 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 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0189] Antibodies with modified effector function include those with substitution of one or more of the residues in the Fc region 238, 265, 269, 270, 297, 327 and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297 and 327, including the so-called Fc mutant DANA with substitution of residues 265 and 297 for alanine (US Patent No. 7,332,581).

[0190] Certain antibody variants with altered binding to FcRs are described. (See, for example, US Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0191] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that alter ADCC, for example, substitutions at positions 298, 333 and / or 334 of the Fc region (EU numbering of residues).

[0192] In some modalities, the changes are made in the region Petition 870260064115, dated 06 / 29 / 2026, p. 83 / 485 75 / 153 Fc resulting in altered (i.e., increased or decreased) C1q binding and / or Complement-Dependent Cytotoxicity (CDC), for example, as described in US Patent No. 6,194,551, WO 99 / 51642, WO 2011 / 091078, and Idusogie et al. J. Immunol. 164: 41784184 (2000).

[0193] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that increase the binding of the Fc region to FcRn. Such Fc variants include those with substitutions in one or more of the residues in the Fc region: 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 the residue in the Fc region 434 (US Patent No. 7,371,826).

[0194] See also Duncan & Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94 / 29351 with respect to other examples of variants of the Fc region.

[0195] In another embodiment, an antibody may comprise a variant Fc region of the present invention described in more detail below. d) Cysteine-based antibody variants

[0196] In certain embodiments, it may be desirable to create engineered cysteine ​​antibodies, for example, tioMAbs, in which one or more residues of an antibody are replaced with cysteine ​​residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thus positioned at sites Petition 870260064115, dated 06 / 29 / 2026, page 84 / 485 Accessible 76 / 153 of the antibody can be used to conjugate the antibody to other components, such as drug components or drug-connector components, to create an immunoconjugate, as further described in this invention. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain. The designed cysteine ​​antibodies can be generated as described, for example, in US Patent No. 7,521,541. e) Antibody Derivatives

[0197] In certain embodiments, an antibody provided herein may be further modified to contain additional non-protein components that are known in the art and readily available. Suitable components for antibody derivation include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers linked to the antibody can vary, and if more than one polymer is linked, they can be the same or different. In general, the number and / or type of polymers used for derivation can be determined based on considerations including, but not limited to. Petition 870260064115, dated 06 / 29 / 2026, p. 85 / 485 77 / 153 limited to these, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0198] In another embodiment, conjugates of an antibody and a non-protein component that can be selectively heated through exposure to radiation are provided. In one embodiment, the non-protein component is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells but that heat the non-protein component to a temperature at which cells near the non-protein component of the antibody are killed.

[0199] In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity. In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region without substantially decreased C1q binding activity.In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the variant Fc region comprises at least one amino acid residue alteration (e.g., substitution) compared to the corresponding sequence in the Fc region of a native or reference variant sequence (sometimes collectively referred to in this invention as the source Fc region). In certain embodiments, the variant Fc region of the invention has substantially decreased FcyR binding activity compared to the source Fc region. In certain embodiments, a. Petition 870260064115, dated 06 / 29 / 2026, page 86 / 485 78 / 153 The variant Fc region of the invention does not have substantially decreased C1q binding activity compared to the original Fc region. In certain embodiments, the FcyR is a human FcyR, a monkey FcyR (e.g., cynomolgus, rhesus monkey, marmoset, chimpanzee, or baboon FcyR), or a mouse FcyR.

[0200] In one aspect, a variant Fc region of the invention has substantially decreased binding activity for one or more human FcyRs including, but not limited to, FcYRIa, FcYRIIa (including allelic variants 167H and 167R), FcYRIIb, FcYRIIIa (including allelic variants 158F and 158V) and FcYRIIIb (including allelic variants NA1 and NA2), compared with an original Fc region. In another aspect, a variant Fc region of the invention has substantially decreased binding activity for human FcYRIa, FcYRIIa (including allelic variants 167H and 167R), FcYRIIb, FcYRIIIa (including allelic variants 158F and 158V), and FcYRIIIb (including allelic variants NA1 and NA2), compared to a source Fc region.

[0201] In one aspect, a variant Fc region of the invention has substantially decreased binding activity for one or more mouse FcYRs including, but not limited to, FcYRI, FcYRIIb, FcYRIII, and FcYRIV, compared to a source Fc region. In a further aspect, a variant Fc region of the invention has substantially decreased binding activity for mouse FcYRI, FcYRIIb, FcYRIII, and FcYRIV, compared to a source Fc region.

[0202] Fcy receptors (hereinafter referred to as Fcy receptors, FcyR or FcgR) refers to receptors that can bind to the Fc region of IgG1, IgG2, IgG3 and IgG4 monoclonal antibodies, and practically means any element of the protein family encoded by the Fcy receptor genes. In humans, this family includes FcyRI (CD64) Petition 870260064115, dated 06 / 29 / 2026, p. 87 / 485 79 / 153 including the FcYRIa, FcYRIb, and fcYRIc isoforms; FcyRII (CD32) including the FcYRIIa isoforms (including H131 (H type) and R131 (R type) allotypes), FcyRIIb (including FcyRIIb-' and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD16) including the fcyRIIIa isoforms (including V158 and F158 allotypes) and FcyRIIIb isoforms (including FcyRiiib-NA1 and FcyRiiib-NA2 allotypes), and any human FcyRs, FcyR isoforms, or allotypes yet to be discovered, but not limited to these. FcyRIIb1 and FcyRIIb2 have been reported as junctional variants of human FcyRIIb. In addition, a junction variant called FcyRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). Besides these junction variants, human FcyRIIb includes all junction variants registered with the NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992.3. In addition, human FcyRIIb includes every previously reported genetic polymorphism, as well as FcyRIIb (Arthritis Rheum. 48:3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46:1242-1254 (2002)), and every genetic polymorphism that will be reported in the future.

[0203] In FcyRIIa, there are two allotypes, one where the amino acid at position 167 of FcyRIIa is histidine (type H) and the other where the amino acid at position 167 is substituted with arginine (type R) (Warrmerdam, J. Exp. Med. 172:19-25 (1990)).

[0204] FcyR includes FcyRs from humans, mice, rats, rabbits, and monkeys, but is not limited to them, and can be derived from any organism. Mouse FcyRs include FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIV (CD16-2), and any mouse FcyRs or FcyR isoforms, but are not limited to these.

[0205] The amino acid sequence of human FcyRIa is shown in SEQ ID NO: 69; the amino acid sequence of human FcyRIIa (167H) is shown in SEQ ID NO: 70; the amino acid sequence of human FcyRIIa (167R) is shown in SEQ ID NO: Petition 870260064115, dated 06 / 29 / 2026, p. 88 / 485 80 / 153 71; the amino acid sequence of human FcYRIIb is shown in SEQ ID NO: 72; the amino acid sequence of human FcγRIIIa (158F) is shown in SEQ ID NO: 73; the amino acid sequence of human FcγRIIIa (158V) is shown in SEQ ID NO: 74; the amino acid sequence of human FcγRIIIb (NA1) is shown in SEQ ID NO: 75; and the amino acid sequence of human FcγRIIIb (NA2) is shown in SEQ ID NO: 76.

[0206] The amino acid sequence of mouse FcyRI is shown in SEQ ID NO: 77; the amino acid sequence of mouse FcγRIIb is shown in SEQ ID NO: 78; the amino acid sequence of mouse FcyRIII is shown in SEQ ID NO: 79; and the amino acid sequence of mouse FcyRIV is shown in SEQ ID NO: 80.

[0207] In one aspect, a variant Fc region of the invention has a substantially decreased FcyR binding activity that is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% as a function of the FcyR binding activity for the original Fc region.In one aspect, a variant Fc region of the invention has substantially decreased FcyR binding activity, meaning that the ratio of [the difference in RU values ​​of the sensorgrams that changed before and after the interaction of FcyR with the variant Fc region] / [the difference in RU values ​​of the sensorgrams that changed before and after the capture of FcyR to the 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. Petition 870260064115, dated 06 / 29 / 2026, p. 89 / 485 81 / 153 that is 0.002 or less than 0.001.

[0208] In one aspect, a variant Fc region of the invention does not have substantially decreased C1q binding activity, meaning that the difference in C1q binding activities between a variant Fc region and an original 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% as a function of the C1q binding activity for the original Fc region.

[0209] In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region with substantially decreased ADCC activity. In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region without substantially decreased CDC activity. In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region with substantially decreased ADCC activity and without substantially decreased CDC activity.

[0210] In one aspect, a variant Fc region of the invention has a substantially decreased ADCC activity that is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% as a function of the ADCC activity for the source Fc region.

[0211] In one aspect, a variant Fc region of the invention does not have substantially diminished CDC activity, meaning that the difference in CDC activities between a variant Fc region and an original Fc region of the invention is less than 50%, less than 45%, less than 40%, less than 35%, less than Petition 870260064115, dated 06 / 29 / 2026, p. 90 / 485 82 / 153 that is 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% depending on CDC activity for the Fc region of origin.

[0212] In one aspect, the invention provides an isolated polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity, compared to a polypeptide comprising an original Fc region. In other aspects, the polypeptide of the invention comprises 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.

[0213] In one aspect, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises Ala at position 234, Ala at position 235 and at least one amino acid change from at least one position selected from the group consisting of: 236, 267, 268, 324, 326, 332 and 333, according to EU numbering.

[0214] In one aspect, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises Ala at position 234, Ala at position 235 and other amino acid changes from any of the following from (a) to (c): (a) positions 267, 268 and 324; (b) headings 236, 267, 268, 324 and 332; and (c) headings 326 and 333, according to the EU numbering system.

[0215] In another aspect, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises 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 Petition 870260064115, dated 06 / 29 / 2026, page 91 / 485 83 / 153 236; (e) Glu in position 332; (f) Ala, Asp, Glu, Met or Trp in position 326; and (g) Ser in position 333, according to EU numbering.

[0216] In one aspect, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises 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, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises 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, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises 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, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises 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, the variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity comprises amino acids: Ala at position 234, Ala at position 235, Trp at position 326, and Ser at position 333, according to EU numbering.

[0217] In another aspect, the variant Fc region of the invention may further comprise at least one amino acid change of at least one position selected from the group consisting of: 428, 434, 436, 438 and 440, according to EU numbering. Petition 870260064115, dated 06 / 29 / 2026, page 92 / 485 84 / 153

[0218] In another aspect, the variant Fc region may also 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 which describes a connection between amino acid changes and FcRn binding activity of a variant Fc region).

[0219] In another aspect, the variant Fc region of the invention comprises 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, the variant Fc region of the invention comprises 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.

[0220] In one aspect, it is preferable that a variant Fc region of the invention does not have substantially increased FcRn binding activity, especially at pH 7.4, compared to the original Fc region.

[0221] FcRn is structurally similar to the polypeptides of the major histocompatibility complex (MHC) class I, and exhibits 22% to 29% sequence identity with MHC class I molecules. FcRn is expressed as a heterodimer consisting of a soluble or light β chain (β2-microglobulin) complexed with a transmembrane or heavy α chain. Like MHC, the α chain of FcRn contains three extracellular domains (α1, α2, and α3), and its short cytoplasmic domain attaches to the cell surface. The α1 and α2 domains Petition 870260064115, dated 06 / 29 / 2026, page 93 / 485 85 / 153 interact with the FcRn binding domain of the antibody Fc region. The polynucleotide and amino acid sequences of human FcRn can be derived, for example, from the precursors shown in NM_004107.4 and NP_004098.1 (containing the signal sequence), respectively.

[0222] 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.

[0223] In one aspect, it is preferable that a variant Fc region of the invention does not have a substantially increased FcRn binding activity, especially at pH 7.4, that is less than 1000 times, less than 500 times, 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 times, less than 10 times, less than 5 times, less than 3 times or less than 2 times compared to the FcRn binding activity for the original Fc region.In one aspect, a variant Fc region of the invention does not possess a substantially increased FcRn binding activity, especially at pH 7.4, meaning that the ratio of [the difference in RU values ​​of the sensorgrams that changed before and after the interaction of FcRn with the variant Fc region] / [the difference in RU values ​​of the sensorgrams that changed before and after the capture of FcRn to the 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.

[0224] In another aspect, the variant Fc region of the invention comprises any of the amino acid changes, individually Petition 870260064115, dated 06 / 29 / 2026, p. 94 / 485 86 / 153 or in combination, as described in Table 4. In another aspect, the variant Fc region of the invention comprises at least any of the amino acid changes described in Table 4. In another aspect, the invention provides a polypeptide comprising the amino acid sequence of any of the SEQ ID NOs: 51 to 59.

[0225] In some embodiments, a polypeptide comprising a variant Fc region of the present invention is a constant region of antibody heavy chain. In some embodiments, a polypeptide comprising a variant Fc region of the present invention further comprises an antigen-binding domain. In another embodiment, the polypeptide is an antibody heavy chain. In another embodiment, the polypeptide is an antibody. In certain embodiments, an antibody is a chimeric antibody, or a humanized antibody. The origin of an antibody is not particularly limited, but examples include a human antibody, a mouse antibody, a rat antibody, and a rabbit antibody. In another embodiment, the polypeptide is an Fc fusion protein.

[0226] Two or more polypeptides comprising a variant Fc region described in this invention may be included in a molecule, wherein two polypeptides comprising variant Fc regions are associated, much like an antibody. The type of antibody is not limited, and IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) and IgM, or similar, may be used.

[0227] In some embodiments, a polypeptide comprising a variant Fc region of the present invention is an antibody. In other embodiments, a polypeptide comprising a variant Fc region of the present invention is a viral antibody.

[0228] In other embodiments, a polypeptide comprising a variant Fc region of the present invention comprises a variable antibody region comprising: Petition 870260064115, dated 06 / 29 / 2026, page 95 / 485 87 / 153 (a) (i) HVR-H3 comprising amino acid sequence SEQ ID NO: 16, (ii) HVR-L3 comprising amino acid sequence SEQ ID NO: 27, and (iii) HVR-H2 comprising amino acid sequence SEQ ID NO: 13; (b) HVR-H1 comprising the amino acid sequence with SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence with SEQ ID NO: 13, and (iii) HVR-H3 comprising the amino acid sequence with SEQ ID NO: 16; (c) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27; or (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0229] In other embodiments, a polypeptide comprising a variant Fc region of the present invention comprises a variable antibody region comprising: (a) HVR-H3 from the VH sequence of SEQ ID NO: 6, (ii) HVRL3 from the VL sequence of SEQ ID NO: 10, and (iii) HVR-H2 from the VH sequence of SEQ ID NO: 6; (b) HVR-H1 of the VH sequence with SEQ ID NO: 6, (ii) HVRH2 of the VH sequence with SEQ ID NO: 6, and (iii) HVR-H3 of the VH sequence with SEQ ID NO: 6; (c) HVR-H1 of sequence VH of SEQ ID NO: 6, (ii) HVR Petition 870260064115, dated 06 / 29 / 2026, p. 96 / 485 88 / 153 H2 of the VH sequence of SEQ ID NO: 6, (iii) HVR-H3 of the VH sequence of SEQ ID NO: 6, (iv) HVR-L1 of the VL sequence of SEQ ID NO: 10; (v) HVR-L2 of the VL sequence of SEQ ID NO: 10; and (vi) HVR-L3 of the VL sequence of SEQ ID NO: 10; (d) HVR-L1 of the VL sequence of SEQ ID NO: 10; (ii) HVRL2 of the VL sequence of SEQ ID NO: 10; and (iii) HVR-L3 of the VL sequence of SEQ ID NO: 10; or (e) HVR-H1 of the VH sequence of SEQ ID NO: 6, (ii) HVRH2 of the VH sequence of SEQ ID NO: 6, (iii) HVR-H3 of the VH sequence of SEQ ID NO: 6, (iv) HVR-L1 of the VL sequence of SEQ ID NO: 7; (v) HVR-L2 of the VL sequence of SEQ ID NO: 7; and (vi) HVR-L3 of the VL sequence of SEQ ID NO: 7.

[0230] In other embodiments, a polypeptide comprising 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 provides an antibody comprising a VH region comprising the amino acid sequence of SEQ ID NO: 1 and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 54 in the heavy chain, and a VL region comprising the amino acid sequence of SEQ ID NO: 7 in the light chain. In other embodiments, the invention provides an antibody comprising a VH region comprising the amino acid sequence of SEQ ID NO: 1 and a variant Fc region comprising the amino acid sequence of SEQ ID NO: 58 in the heavy chain, and a VL region comprising the amino acid sequence of SEQ ID NO: 7 in the light chain.In other embodiments, the invention provides an antibody comprising the VH region comprising the amino acid sequence of SEQ ID NO: 1 and an Fc variant region comprising the amino acid sequence of SEQ ID NO: 59 in the heavy chain, and the VL region which... Petition 870260064115, dated 06 / 29 / 2026, page 97 / 485 89 / 153 comprises the amino acid sequence of SEQ ID NO: 7 in the light chain.

[0231] The invention also provides an anti-DENV antibody described in this invention, further comprising a polypeptide comprising a variant Fc region of the present invention. In some embodiments, an anti-DENV antibody of the invention comprises the 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 the VL region comprising the amino acid sequence of SEQ ID NO: 10 in the light chain. In some embodiments, an anti-DENV antibody of the invention comprises the 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 the VL region comprising the amino acid sequence of SEQ ID NO: 10 in the light chain.In some embodiments, an anti-DENV antibody of the invention comprises the 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 the VL region comprising the amino acid sequence of SEQ ID NO: 10 in the light chain.

[0232] In some embodiments, an anti-DENV antibody of the invention comprises the 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 the VL region comprising the amino acid sequence of SEQ ID NO: 7 in the light chain.

[0233] An Fc origin region as used here refers to an Fc region prior to the introduction of an amino acid change described herein. Preferred examples of the Fc origin region include Petition 870260064115, dated 06 / 29 / 2026, p. 98 / 485 90 / 153 Fc regions derived from native antibodies. Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM, or similar. Antibodies may be derived from humans or monkeys (e.g., cynomolgus, rhesus monkey, marmoset, chimpanzee, or baboon). Native antibodies may also include naturally occurring mutations. A plurality of IgG allotype sequences due to genetic polymorphism is described in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, and any of these may be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU numbering) may be DEL or EEM. Preferred examples of the Fc region of origin include Fc regions derived from a constant heavy chain 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 the originating Fc region is an Fc region derived from an SG1 heavy chain constant region (SEQ ID NO: 87). Another preferred example of the originating Fc region is an Fc region derived from an SG182 heavy chain constant region (SEQ ID NO: 46). Furthermore, the originating Fc region may be an Fc region produced by adding an amino acid change other than the amino acid change described here to an Fc region derived from a native antibody.

[0234] Furthermore, amino acid changes performed for other purposes can be combined in a variant Fc region described in this invention. For example, amino acid substitutions that improve FcRn binding activity (Hinton et al., J. Immunol. 176(1):346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33):2351423524 (2006); Petkova et al., Intl. Immunol. 18(12):1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159 (2010); WO 2006 / 019447; Petition 870260064115, dated 06 / 29 / 2026, p. 99 / 485 91 / 153 amino acid substitutions to improve antibody heterogeneity or stability (WO 2009 / 041613) may be added. Alternatively, polypeptides with the property of promoting antigen clearance, which are described in WO 2011 / 122011, WO 2012 / 132067, WO 2013 / 046704 or WO 2013 / 180201, polypeptides with the property of specific binding to a target tissue, which are described in WO 2013 / 180200, polypeptides with the property of repeated binding to a plurality of antigen molecules, which are described in WO 2009 / 125825, WO 2012 / 073992 or WO 2013 / 047752, may be combined with a variant Fc region described in this invention. Alternatively, in order to confer the ability to bind to other antigens, the amino acid changes disclosed in EP1752471 and EP1772465 can be combined in CH3 of a variant Fc region described herein.Alternatively, with the aim of increasing plasma retention, amino acid changes that decrease the pI of the constant region (WO 2012 / 016227) can be combined in a variant Fc region described herein. Alternatively, with the aim of promoting absorption into cells, amino acid changes that increase the pI of the constant region (WO 2014 / 145159) can be combined in a variant Fc region described in this invention. Alternatively, with the aim of promoting the elimination of a target molecule from the plasma, amino acid changes that increase the pI of the constant region (WO 2016 / 125495 and WO 2016 / 098357) can be combined in a variant Fc region described herein.

[0235] Amino acid alterations to increase the binding activity to human FcRn under acidic pH can also be combined in a variant Fc region described in this invention. Specifically, such alterations may include, for example, the substitution of Leu for Met at position 428 and the substitution of Ser for Asn at position 434, of Petition 870260064115, dated 06 / 29 / 2026, page 100 / 485 92 / 153 according to EU numbering (Nat Biotechnol, 2010, 28: 157-159); the substitution of Ala for Asn at position 434 (Drug Metab Dispos, 2010 Apr; 38(4): 600-605); the substitution of Tyr for Met at position 252, the substitution of Thr for Ser at position 254 and the substitution of Glu for Thr at position 256 (J Biol Chem, 2006, 281: 23514-23524); the substitution of Gin for Thr at position 250 and the substitution of Leu for Met at position 428 (J Immunol, 2006, 176(1): 346-356); the substitution of His for Asn at position 434 (Clin Pharmacol Ther, 2011,89(2): 283290), 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 similar.In another embodiment, such changes may include, for example, at least one change selected from the group consisting of substitution of Leu for Met at position 428, substitution of Ala for Asn at position 434 and substitution of Thr for Tyr at position 436. These changes may further include substitution of Arg for Gin at position 438 and / or substitution of Glu for Ser at position 440 (WO 2016 / 125495).

[0236] In the present invention, amino acid change means any substitution, deletion, addition, insertion and modification, or a combination thereof. In the present invention, amino acid change may be reformulated as amino acid mutation.

[0237] Amino acid alterations are produced by several methods known to those skilled in the art. Such methods include the site-directed mutagenesis method (HashimotoGotoh et al., Gene 152: 271-275 (1995); Zoller, Meth. Enzymol. 100: 468500 (1983); Kramer et al., Nucleic Acids Res. 12: 9441-9456 (1984)); Kramer and Fritz, Enzymol. Methods 154: 350-367 (1987); and Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), the mutation method Petition 870260064115, dated 06 / 29 / 2026, p. 101 / 485 93 / 153 of PCR and the cassette mutation method, but are not limited to these.

[0238] The number of amino acid changes introduced in an Fc region is not limited. In certain 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.

[0239] Furthermore, a polypeptide comprising a variant Fc region of the present invention can be chemically modified with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Methods for such chemical modification of a polypeptide are set out in the art.

[0240] In some embodiments, a polypeptide comprising a variant Fc region of the present invention is an antibody or an Fc fusion protein comprising a domain that can bind to any antigen. Examples of antigens that can be bound by such antibodies and Fc fusion proteins include, but are not limited to, ligands (cytokines, chemokines, and the like), receptors, cancer antigens, viral antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes partially containing immunoglobulins. B. Recombinant Methods and Compositions

[0241] In one example, it refers to a process for preparing an antibody as described herein, wherein the process comprises the steps of: (a) combining a variant VH sequence as described herein with a human lgG1 CH sequence as described in this invention; (b) combine a variant VL sequence as described here, with a human SK1 CL sequence; Petition 870260064115, dated 06 / 29 / 2026, p. 102 / 485 94 / 153 (c) clone each of the combinations into an expression vector; (d) express the resulting expression vectors in co-transfected cells (host cell); and (e) purify the antibody resulting from step (d). In certain examples, a host cell is CHO-DXB11, CHO-K1, or CHODG44. Such a host cell may be a taurine expression carrier cell, obtained by introducing the DNA-encoding taurine carrier (WO 2007 / 119774). Vectors that can be used for the manufacture of such antibodies are known in the art. Generally, antibodies can be produced using recombinant methods and compositions, for example, as described in US Patent No. 4,816,567. In one embodiment, the isolated nucleic acid encoding an anti-DENV antibody described in this invention is provided. In another embodiment, the isolated nucleic acid encoding a polypeptide comprising a variant Fc region or an Fc region of origin described herein is provided.Such nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In such an embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector. Petition 870260064115, dated 06 / 29 / 2026, page 103 / 485 95 / 153 comprising a nucleic acid encoding an amino acid sequence comprising the antibody's VH. In one embodiment, the host cell is eukaryotic, for example, Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp2 / 0 cells). In one embodiment, a method for producing an anti-DENV antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).In another embodiment, a method for producing a polypeptide comprising a variant Fc region or a source Fc region is provided, wherein the method comprises culturing a host cell comprising nucleic acids encoding a polypeptide such as an antibody, Fc region or variant Fc region, as provided above, under conditions suitable for polypeptide expression, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

[0242] For recombinant production of an anti-DENV antibody, the nucleic acid encoding an antibody, for example, as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. For recombinant production of an Fc region, the nucleic acid encoding an Fc region is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional procedures (e.g., through the use of oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

[0243] The host cells suitable for cloning or Petition 870260064115, dated 06 / 29 / 2026, page 104 / 485 96 / 153 Expression of antibody-encoding vectors includes prokaryotic or eukaryotic cells described in this invention. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0244] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been humanized, resulting in the production of an antibody with a partially or completely human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0245] Suitable host cells for glycosylated antibody expression are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified, which can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0246] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (which describe the PLANTIBODIES™ technology for the production of antibodies in plants). Petition 870260064115, dated 06 / 29 / 2026, page 105 / 485 97 / 153 transgenic).

[0247] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described, for example, in Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO DHFR- cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0248] Polyclonal antibodies are preferably increased in animals by means of multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, for example, hemocyanin from limpet burrow. Petition 870260064115, dated 06 / 29 / 2026, page 106 / 485 98 / 153 lock, serum albumin, bovine thyroglobulin or soy trypsin inhibitor using a bifunctional or derivative agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R1N=C=NR, where R and R1 are different alkyl groups.

[0249] Animals (usually non-human mammals) are immunized against the antigen, immunogenic conjugates, or derivatives by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is analyzed for antibody titer. The animals are boosted until the titer stabilizes. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and / or through a different crosslinking reagent. Conjugates can also be produced in recombinant cell culture as protein fusions.Similarly, aggregating agents such as alum are appropriately used to enhance the immune response.

[0250] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerizations, amidations) that may be present in small quantities. Thus, the monoclonal modifier indicates the character of the antibody as Petition 870260064115, dated 06 / 29 / 2026, page 107 / 485 99 / 153 not being a mixture of discrete antibodies.

[0251] For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to extract lymphocytes that produce or are capable of producing antibodies that bind specifically to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0252] The immunization agent will typically include the antigenic protein or a fusion variant thereof. In general, peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0253] Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are usually employed. The hybridoma cells thus prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused myeloma cells of origin. For example, if the myeloma cells of origin lack the enzyme hypoxanthine guanine phosphorobosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium). Petition 870260064115, dated 06 / 29 / 2026, page 108 / 485 100 / 153 which are substances that inhibit the growth of HGPRT-deficient cells.

[0254] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, murine myeloma cell lines are preferred, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 cells (and their derivatives, e.g., X63Ag8-653) available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al. J. Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0255] The culture medium in which hybridoma cells are growing is analyzed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the specialty. For example, binding affinity can be determined by Scatchard analysis of Munson, Anal. Biochem. 107(1):220-239 (1980).

[0256] After hybridoma cells are identified as producing antibodies of the desired specificity, affinity, and / or activity, clones can be subcloned by limiting the Petition 870260064115, dated 06 / 29 / 2026, page 109 / 485 101 / 153 dilution procedures and cultured by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM medium or RPMI-1640. In addition, hybridoma cells can be cultured in vivo as tumors in a mammal.

[0257] Monoclonal antibodies secreted by subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0258] An Fc region can be obtained by re-eluting the adsorbed fraction onto the protein A column after partial digestion of IgG1, IgG2, IgG3, IgG4 or similar monoclonal antibodies using a protease such as pepsin. The protease is not particularly limited as long as it can digest a full-length antibody so that Fab and F(ab')2 are produced in a restrictive manner by appropriately adjusting the enzymatic reaction conditions such as pH, and examples include pepsin and papain.

[0259] Furthermore, the present invention provides a method for producing a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity compared to a polypeptide comprising an original Fc region, comprising introducing at least one amino acid change in the original Fc region. In some aspects, the polypeptide produced is an antibody. In certain embodiments, an antibody is a chimeric antibody, or a humanized antibody. In some aspects, the polypeptide produced is an Fc fusion protein.

[0260] In one aspect, at least one amino acid is changed in the above-mentioned method for producing a polypeptide that Petition 870260064115, dated 06 / 29 / 2026, page 110 / 485 102 / 153 comprises a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity, from at least one position selected from the group consisting of: 234, 235, 236, 267, 268, 324, 326, 332 and 333, according to EU numbering.

[0261] In another aspect, two amino acids are altered in the above-mentioned method to produce a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity, at positions 234 and 235.

[0262] In another aspect, the amino acids are altered in the method mentioned above to produce a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity, the alterations comprising: (a) two amino acid alterations at positions 234 and 235, and (b) at least one amino acid alteration at at least one position selected from the group consisting of: 236, 267, 268, 324, 326, 332 and 333, according to EU numbering.

[0263] In another aspect, the amino acids are altered in the above-mentioned method to produce a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity, the alterations comprising: (a) two amino acid alterations at positions 234 and 235, and (b) at least one amino acid alteration at any of the following (i) to (iii): (i) positions 267, 268 and 324; (ii) positions 236, 267, 268, 324 and 332; and (iii) positions 326 and 333, in accordance with EU numbering.

[0264] In another aspect, an amino acid change in the method mentioned above for the production of a polypeptide that Petition 870260064115, dated 06 / 29 / 2026, p. 111 / 485 103 / 153 comprises a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity, selected at each position of 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; (f) Ala at position 236; (g) Glu at position 332; (h) Ala, Asp, Glu, Met, Trp at position 326; and (l) Ser at position 333, according to EU numbering.

[0265] In another aspect, the amino acid changes in the above-mentioned method for producing a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased 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 another aspect, the amino acid changes in the above-mentioned method for producing a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity and without substantially decreased C1q binding activity are: Ala at position 234, Ala at position 235, Asp at position 326, and Ser at position 333; according to EU numbering.In a further aspect, the amino acid changes in the method mentioned above for the production of a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity, and without substantially decreased 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 another aspect, the amino acid changes in the method mentioned above for the production of a polypeptide comprising a variant Fc region with substantially decreased FcyR binding activity, and without... Petition 870260064115, dated 06 / 29 / 2026, page 112 / 485 104 / 153 with substantially decreased C1q binding activity, are: Ala at position 234, Ala at position 235, Met at position 326 and Ser at position 333; according to EU numbering. In a further aspect, the amino acid changes in the method mentioned above for the production of a polypeptide comprising an Fc variant region with substantially decreased FcyR binding activity, and without substantially decreased 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.

[0266] In another aspect, at least one amino acid is further altered in the method mentioned above, from at least one position selected from the group consisting of: 428, 434, 436, 438 and 440, according to the EU numbering.

[0267] In another aspect, an amino acid change in the above-mentioned method is further selected from the following (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, in accordance with EU numbering (see also WO 2016 / 125495 which describes a connection between amino acid changes and FcRn binding activity of a variant Fc region).

[0268] In an additional aspect, the amino acid changes in the above-mentioned method 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 another aspect, the amino acid changes in the aforementioned method 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. Petition 870260064115, dated 06 / 29 / 2026, page 113 / 485 105 / 153 in position 440, according to the EU numbering system.

[0269] In one respect, it is preferable that a variant Fc region of the invention does not have substantially increased FcRn binding activity, especially at pH 7.4, compared to the original Fc region.

[0270] In another aspect, the amino acid changes in the production methods mentioned above are selected from any single change, combination of single changes, or combination changes described in Table 4.

[0271] Polypeptides comprising a variant Fc region produced by any of the above-mentioned methods or other methods known in the art are included in the present invention. C. Tests

[0272] The anti-DENV antibodies provided here can be identified, screened, or characterized for their physicochemical properties and / or biological activities by various assays known in the art.

[0273] The variant Fc regions provided here can be identified, screened, or characterized for their physicochemical properties and / or biological activities through various assays known in the art. 1. Bond tests and other tests

[0274] In one aspect, an antibody of the invention is tested for its antigen-binding activity, for example, by known methods such as ELISA, Western blot, etc. In another aspect, a polypeptide comprising a variant Fc region of the invention is tested for its antigen-binding activity, for example, by known methods such as ELISA, Western blot, etc.

[0275] In another aspect, competition tests can be Petition 870260064115, dated 06 / 29 / 2026, p. 114 / 485 106 / 153 used to identify an antibody that competes for binding to DENV and / or DENV protein E with any anti-DENV antibody described in this invention. In certain embodiments, when such a competitive antibody is present in excess, it blocks (e.g., reduces) the binding of a reference antibody to DENV and / or DENV protein E 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 certain embodiments, such a competitive antibody binds to the same epitope (e.g., a linear or conformational epitope) that is bound by an anti-DENV antibody described herein. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) Epitope Mapping Protocols, in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0276] In an exemplary competition assay, immobilized DENV or DENV protein E is incubated in a solution comprising a first labeled antibody that binds to DENV and / or DENV protein E and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to DENV or DENV protein E. The second antibody may be present in a hybridoma supernatant. As a control, immobilized DENV or DENV protein E is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to DENV or DENV protein E, the excess unbound antibody is removed, and the amount of labeling associated with the immobilized DENV or DENV protein E is measured.If the amount of labeling associated with DENV or immobilized DENV protein E is substantially reduced in the test sample. Petition 870260064115, dated 06 / 29 / 2026, p. 115 / 485 107 / 153 in relation to the control sample, so this indicates that the second antibody is competing with the first antibody with respect to binding to DENV or DENV E protein. See Harlow and Lane (1988) Antibodies: A Laboratory Handbook ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0277] Assays for determining the binding activity of a polypeptide containing a variant Fc region toward one or more members of the FcR family are described herein or otherwise known in the art. Such binding assays include, but are not limited to, BIACORE® analysis, which utilizes the surface plasmon resonance (SPR) phenomenon, Amplified Luminescent Proximity Homogeneous Assay (ALPHA) screening, ELISA, and fluorescence-activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103(11): 4005-4010).

[0278] In one embodiment, BIACORE® analysis can be used to evaluate whether the binding activity of a polypeptide comprising a variant Fc region is enhanced, maintained, or diminished with respect to a particular FcR family member. For example, by observing whether there is a decrease or increase in the dissociation constant (Kd) value obtained from sensorgram analysis, where several FcRs are subjected to interaction as an analyte with polypeptides comprising a variant Fc region immobilized or captured on the sensor chip using known methods and reagents such as Protein A, Protein L, Protein A / G, Protein G, anti-lambda chain antibodies, anti-kappa chain antibodies, antigenic peptides, antigenic proteins.Changes in binding activity can also be determined by comparing changes in the resonance unit (RU) value on the sensorgram before and after one or more types of FcRs are subjected to interaction as analytes with the captured polypeptides. Petition 870260064115, dated 06 / 29 / 2026, page 116 / 485 108 / 153 comprising the variant Fc region. Alternatively, the FcR can be immobilized or captured on sensor chips, and the polypeptides comprising the variant Fc region are used as an analyte.

[0279] In BIACORE® analysis, one of the substances (the ligand) under observation for an interaction is immobilized on a thin gold film on a sensor chip, and by shining light from the reverse side of the sensor chip, so that total reflection occurs at the interface between the thin gold film and the glass, a portion of the reduced reflection intensity is formed in part of the reflected light (SPR signal). When the other of the substances (the analyte) under observation for an interaction is allowed to flow over the surface of the sensor chip and 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. The position of the SPR signal shifts as a result of this change in refractive index (conversely, the position of the signal returns when this bond dissociates).The BIACORE® system indicates the aforementioned displacement quantity, or more specifically the time variable of mass, by plotting the mass change on the sensor chip surface on the ordinate as measurement data (sensorgram). The amount of analyte bound to the ligand trapped on the sensor chip surface is determined from the sensorgram. Kinetic parameters such as the association rate constants (ka) and dissociation rate constants (kd) are determined from the sensorgram curves, and the dissociation constants (Kd) are determined from the ratio of these constants. In the BIACORE® method, a method for measuring inhibition is preferably used. An example of a method for measuring inhibition is described in Lazar et al., Proc. Natl. Acad. Sci. USA 103(11):4005-4010 (2006).

[0280] ALPHA screening is performed using ALPHA technology. Petition 870260064115, dated 06 / 29 / 2026, p. 117 / 485 109 / 153 which uses two blood cells, one donor and one acceptor, based on the following principles. Luminescent signals are detected only when molecules bound to the donor blood cells physically interact with molecules bound to the acceptor blood cells, and the two blood cells are in close proximity to each other. The laser-excited photosensitizer in the donor blood cells converts ambient oxygen into excited singlet oxygen. The singlet oxygen is dispersed around the donor blood cells, and when it reaches the adjacent acceptor blood cells, the chemiluminescent reaction is induced in the blood cells, and light is finally emitted. When molecules bound to the donor blood cells do not interact with molecules bound to the acceptor blood cells, the chemiluminescent reaction does not occur because the singlet oxygen produced by the donor blood cells does not reach the acceptor blood cells.

[0281] For example, a biotinylated polypeptide complex is bound to donor cells, and the Fc receptor labeled with glutathione S-transferase (GST) is bound to acceptor cells. In the absence of a competing polypeptide complex comprising a variant Fc region, the polypeptide complex comprising an original Fc region interacts with the Fc receptor and produces signals from 520 to 620 nm. The polypeptide complex comprising an unlabeled variant Fc region competes with the polypeptide complex comprising an original Fc region for interaction with the Fc receptor. The relative binding activities can be determined by quantifying the decrease in fluorescence observed as a result of the competition. The biotinylation of polypeptide complexes such as antibodies using sulfo-NHS-biotin and the like is well known.The method of expressing the Fc receptor and GST in a cell carrying a fusion gene is produced by fusing a polynucleotide encoding the Fc receptor into a structure with a... Petition 870260064115, dated 06 / 29 / 2026, p. 118 / 485 The 110 / 153 polynucleotide encoding GST in an expression vector, and performing purification using a glutathione column, is appropriately adopted as a method for labeling an Fc receptor with GST. The signals obtained are preferably analyzed, for example, by fitting them with a single-site competition model using nonlinear regression analysis using software such as GRAPPAD PRISM (GraphPad, San Diego).

[0282] A variant Fc region with decreased FcR binding activity refers to an Fc region that binds to FcR with essentially weaker binding activity than a source Fc region when assays are performed using substantially the same amount of a corresponding source Fc region and a variant Fc region. Furthermore, a variant Fc region with increased FcR binding activity refers to an Fc region that binds to FcR with essentially stronger binding activity than a corresponding source Fc region when assays are performed using substantially the same amount of a source Fc region and a variant Fc region.A variant Fc region with maintained FcR binding activity refers to an Fc region that binds to FcR with binding activity equivalent to, or essentially not different from, that of a source Fc region when assays are performed using substantially the same amount of the corresponding source Fc region and the polypeptide containing the variant Fc region.

[0283] If the binding activities of an Fc region towards multiple FcRs have been increased or decreased, this can be determined from the increase or decrease in the amount of binding of the various FcRs to the Fc region, which were determined according to the measurement method mentioned above. Here, the amount of binding of the various FcRs to the Fc region can be evaluated as a value obtained by division. Petition 870260064115, dated 06 / 29 / 2026, page 119 / 485 111 / 153 of the difference in RU values ​​of sensorgrams that were altered before and after the interaction of several FcRs as the analyte with the Fc region, by the difference in RU values ​​of sensorgrams that were altered before and after the capture of the Fc regions to the sensor chips. The binding activity of an Fc region to an FcyR or FcRn can be determined by a method described in Example 5 of this invention.

[0284] In the present invention, a substantially decreased FcyR binding activity preferably means, for example, that the binding activity of a variant Fc region to an FcyR is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2% or less than 0.1% as a function of the FcyR binding activity of the source Fc region.Similarly, preference means, for example, that the ratio of [the difference in RU values ​​of the sensorgrams that changed before and after the interaction of FcyR with the variant Fc region] / [the difference in RU values ​​of the sensorgrams that changed before and after the capture of FcyR by the 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.

[0285] In the present invention, not a substantially increased FcRn binding activity, especially at pH 7.4, preferably means, for example, that the binding activity of a variant Fc region to an FcRn is less than 1000 times, less than 500 times, less than 200 times, less than 100 times. Petition 870260064115, dated 06 / 29 / 2026, p. 120 / 485 112 / 153 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 times, less than 10 times, less than 5 times, less than 3 times, or less than 2 times compared to the FcRn binding activity of the Fc origin region. Also preferably means, for example, that the ratio of [the difference in RU values ​​of the sensorgrams that were altered before and after the interaction of FcRn with the variant Fc region] / [the difference in RU values ​​of the sensorgrams that were altered before and after the capture of FcRn on the 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.

[0286] To determine the binding activity of a polypeptide containing a variant Fc region toward C1q, a C1q binding ELISA can be performed. In summary, assay plates can be coated overnight at 4°C with a polypeptide containing a variant Fc region or a polypeptide containing an original Fc region (control) in coating buffer. The plates can then be washed and blocked. After washing, an aliquot of human C1q can be added to each reservoir and incubated for 2 hours at room temperature. After another wash, 100 μL of a sheep complement C1q peroxidase-conjugated antibody can be added to each reservoir and incubated for 1 hour at room temperature. The plate can again be washed with washing buffer and 100 μL of substrate buffer containing OPD (o-phenylenediamine dichloride (Sigma)) can be added to each reservoir. The oxidation reaction, Petition 870260064115, dated 06 / 29 / 2026, p. 121 / 485 113 / 153 observed by the appearance of a yellow color, can be allowed to proceed for 30 minutes and stopped by the addition of 100 μL of 4.5 N H2SO4. The absorbance can then be read at (492-405) nm. The binding activity of an Fc region to C1q can be determined by a method described in Example 4 of this invention.

[0287] In one aspect, the difference in C1q binding activity between a variant Fc region and an original 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% as a function of the C1q binding activity by the original Fc region. 2. Activity trials

[0288] In one aspect, assays are provided for the identification of anti-DENV antibodies having biological activity. Biological activity may include, for example, blocking the binding of DENV E protein to a host cell, inhibiting DENV entry into a host cell, inhibiting and / or preventing DENV infection of a host cell, etc. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0289] In certain embodiments, an antibody of the invention is tested with respect to such biological activity. In certain embodiments, the plaque reduction neutralization test (PRNT) assay can be used to measure the activity or neutralizing potency of a test antibody. In some embodiments, an animal host can be used to measure anti-DENV activity in vivo.

[0290] In certain modalities, cells can be analyzed directly with respect to the binding between DENV and a test antibody. Immunohistochemistry techniques, confocal techniques, and / or other techniques for evaluating binding are well known to those skilled in the art. Several cell lines can be used. Petition 870260064115, dated 06 / 29 / 2026, page 122 / 485 114 / 153 for such screening assays, including cells specifically designed for this purpose. Examples of cells used in screening assays include mammalian cells, fungal cells, bacterial cells, or viral cells. A cell may be a stimulated cell, such as a cell stimulated with a growth factor. A person skilled in the art should understand that the invention disclosed herein contemplates a wide variety of assays for measuring the ability of a test antibody to bind to DENV.

[0291] Depending on the assay, cell and / or tissue culture may be required. A cell can be examined using any of several different physiological assays. Alternatively or additionally, molecular analysis can be performed, including, but not limited to, western blotting to monitor expression and / or protein testing for protein-protein interactions; mass spectrometry to monitor other chemical modifications; etc.

[0292] In some embodiments, such methods utilize an animal host. For example, suitable animal hosts for the invention may be any mammalian hosts, including primates, ferrets, cats, dogs, cows, horses, and rodents such as mice, hamsters, rabbits, and rats. In some embodiments, the animal host is inoculated, infected, or otherwise exposed to the virus before or simultaneously with the administration of a test antibody. Unexperimented and / or inoculated animals may be used for any of a variety of studies. For example, such animal models may be used for virus transmission studies as known in the art. A test antibody may be administered to a suitable animal host before, during, or after virus transmission studies in order to determine the effectiveness of the test antibody in blocking virus binding and / or the possibility of Petition 870260064115, dated 06 / 29 / 2026, page 123 / 485 115 / 153 contamination in the animal host.

[0293] In one aspect, assays are provided for the identification of polypeptides comprising variant Fc regions having biological activity. Biological activity may include, for example, ADCC activity and CDC activity. Polypeptides comprising variant Fc regions having biological activity in vivo and / or in vitro are also provided.

[0294] In certain embodiments, a polypeptide comprising a variant Fc region of the invention is tested with respect to such biological activity. In certain aspects, a polypeptide comprising a variant Fc region of the T gene of the invention modulates an effector function compared to the polypeptide comprising an original Fc region. In a certain aspect, this modulation is a modulation of ADCC and / or CDC.

[0295] In vitro and / or in vivo cytotoxicity assays can be conducted to confirm CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody has FcyR binding (therefore likely having ADCC activity) and retains FcRn binding capacity. Primary cells for ADCC mediation, only NK cells express FcyRIII, while monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to evaluate the ADCC activity of KT, a molecule of interest, are described in US Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, methods of. Petition 870260064115, dated 06 / 29 / 2026, p. 124 / 485 116 / 153 Non-radioactive assays can be employed (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). C1q binding assays can also be performed to confirm whether the antibody binds to C1q and consequently possesses CDC activity. See, for example, C1q and C3c binding ELISA 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 202:163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)). D. Immunoconjugates

[0296] In some embodiments, the invention also provides immunoconjugates comprising an anti-DENV antibody conjugated herein to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (for example, protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes. In some embodiments, the invention also provides immunoconjugates comprising a Petition 870260064115, dated 06 / 29 / 2026, p. 125 / 485 117 / 153 polypeptide comprising an Fc variant region conjugated herein to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

[0297] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including, but not limited to, a maytansinoid (see U.S. Patents Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin, such as the monomethylauristatin DE and DF (MMAE and MMAF) medicinal components (see U.S. Patents Nos. 5,635,483 and 5,780,588 and 7,498,298); a dolastatin; a calicheamicin or derivatives thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. 16:717-721 (2005); Nagy et al., Proc. Natl.USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and US Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel,thesistaxel and ortataxel; a trichothecene; and CC1065.

[0298] In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, active non-binding fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), chain Petition 870260064115, dated 06 / 29 / 2026, page 126 / 485 118 / 153 ricin A, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolacca americana proteins (PAPI, FPA and PAP-S), Moordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

[0299] In another embodiment, an immunoconjugate comprises an antibody as described in this invention conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 212Pb and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example, Tc-99m or 123I, or a fusiform label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0300] Antibody-cytotoxic agent conjugates can be produced 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), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniobenzoyl)ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be Petition 870260064115, dated 06 / 29 / 2026, page 127 / 485 119 / 153 prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminopentaacetic acid (MX-DTPA) is an exemplary chelating agent for radionuclide-antibody conjugation. See WO 94 / 11026. The ligand may be a cleavable ligand that facilitates the release of a cytotoxic drug into the cell. For example, an acid-unstable ligand, peptidase-sensitive ligand, photolabile ligand, dimethyl ligand, or disulfide-containing ligand (Chari et al., Cancer Res. 52: 127-131 (1992); US Patent No. 5,208,020) may be used.

[0301] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with crosslinking reagents including, but not limited to, BMPs, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfoMBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) that are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0302] E. Diagnostic and Detection Methods and Compositions

[0303] In certain embodiments, any of the anti-DENV antibodies provided herein is useful for detecting the presence of DENV and / or DENV E protein in a biological sample. The term detect as used herein encompasses quantitative or qualitative detection. In certain embodiments, a 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 gland secretion, lymph, urine, sweat, lacrimal fluid, gastric fluid, synovial fluid, peritoneal fluid, lens fluid, or mucus.

[0304] In one embodiment, an anti-DENV antibody for use in a diagnostic or detection method is provided. In another Petition 870260064115, dated 06 / 29 / 2026, p. 128 / 485 In aspect 120 / 153, a method for detecting the presence of DENV in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-DENV antibody as described herein under conditions permissive for binding of the anti-DENV antibody to DENV, and detecting whether a complex is formed between the anti-DENV antibody and DENV. Such a method may be an in vitro or in vivo method. In a further aspect, a method for detecting the presence of DENV protein E in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-DENV antibody as described herein under conditions permissive for binding of the anti-DENV antibody to DENV protein E, and detecting whether a complex is formed between the anti-DENV antibody and DENV protein E. Such a method may be an in vitro or in vivo method.In one embodiment, an anti-DENV antibody is used to select individuals eligible for therapy with an anti-DENV antibody, for example, where DENV or DENV E protein is a biomarker for patient selection.

[0305] Exemplary disorders that can be diagnosed using an antibody of the invention include DENV infection and diseases and / or symptoms caused by or associated with DENV infection such as dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).

[0306] In certain embodiments, labeled anti-DENV antibodies are provided. Markers include, but are not limited to, markers or components that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent and radioactive markers), as well as components, such as enzymes or ligands, that are detected indirectly, for example, through an enzymatic reaction or a molecular interaction. Exemplary markers include, but are not limited to, the Petition 870260064115, dated 06 / 29 / 2026, p. 129 / 485 121 / 153 radioisotopes 32P, 14C, 125I, 3H and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, for example, firefly luciferase and bacterial luciferase (US Patent No. 4,737,556), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those coupled with an enzyme employing peroxide hydrogen to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, fusiform markers, bacteriophage markers, stable free radicals, and the like.

[0307] In one embodiment, an antibody comprising a variant Fc region 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 then the support is washed with a suitable solvent that will remove substantially all material in the sample except the antigen to be purified, which is bound to the immobilized antibody variant. Finally, the support is washed with another suitable solvent, such as a glycine buffer, pH 5.0, which will release the antigen from the antibody variant.

[0308] The antibody variant can also be useful in diagnostic tests, for example, for detecting the expression of an antigen of interest in specific cells, tissues or serum.

[0309] The antibody variant can be used in any known assay method, such as competitive binding assays, Petition 870260064115, dated 06 / 29 / 2026, p. 130 / 485 122 / 153 direct and indirect intercalated assays and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, (1987) pp. 147-158, CRC Press, Inc. F. Pharmaceutical Formulations

[0310] Pharmaceutical formulations of an anti-DENV antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable vehicles (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.

[0311] Pharmaceutical formulations of a polypeptide comprising a variant Fc region as described in this invention are prepared by mixing such polypeptide having the desired degree of purity with one or more optional pharmaceutically acceptable carriers in the form of lyophilized formulations or aqueous solutions.

[0312] Pharmaceutically acceptable vehicles are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or Petition 870260064115, dated 06 / 29 / 2026, page 131 / 485 123 / 153 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable vehicles in this invention further include interstitial drug dispersing agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, soluble human PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0313] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 04408, the latter formulations including a histidine-acetate buffer.

[0314] The formulation in this invention may also contain more than one active ingredient as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an antiviral agent, such as, but not limited to, interferons (e.g., interferon α2b, interferon-γ, etc.), anti-DENV monoclonal antibodies, anti-DENV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, small interfering RNAs, small hairpin RNAs, microRNAs, RNA aptamers, ribozymes and their Petition 870260064115, dated 06 / 29 / 2026, page 132 / 485 124 / 153 combinations. These active ingredients are appropriately present in combination with amounts that are effective for the intended purpose.

[0315] Active ingredients can be captured in microcapsules prepared, for example, by means of coacervation techniques or by means of interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0316] Controlled-release preparations can be manufactured. Suitable examples of controlled-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices of which are in the form of molded articles, for example, films or microcapsules.

[0317] The formulations to be used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes. G. Therapeutic Methods and Compositions

[0318] Any of the anti-DENV antibodies provided in this invention can be used in therapeutic methods.

[0319] In one aspect, an anti-DENV antibody for use as a medicament is provided. In other aspects, an anti-DENV antibody is provided for use in the treatment of DENV infection. In certain embodiments, an anti-DENV antibody for use in a treatment method is provided. In certain embodiments, the invention provides an anti-DENV antibody for use in a treatment method of a Petition 870260064115, dated 06 / 29 / 2026, p. 133 / 485 125 / 153 individual having DENV infection comprising administering to the individual an effective amount of anti-DENV antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In other embodiments, the invention provides an anti-DENV antibody for use in blocking DENV protein E binding and / or DENV entry into a host cell. In certain embodiments, the invention provides an anti-DENV antibody for use in a method of blocking DENV protein E binding and / or DENV entry into a host cell in an individual, comprising administering to the individual an effective amount of anti-DENV antibody to block DENV protein E binding and / or DENV entry into a host cell. An individual according to any of the above embodiments is preferably a human being.

[0320] In another aspect, the invention provides the use of an anti-DENV antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of DENV infection. In another embodiment, the medicament is for use in a method of treating DENV infection comprising administering to an individual having DENV infection an effective amount of the medicament. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In another embodiment, the medicament is for blocking the binding of DENV protein E and / or the entry of DENV into a host cell. In another embodiment, the medicament is for use in a method of blocking the binding of DENV protein E and / or the entry of DENV into a host cell in an individual comprising administering to the individual a Petition 870260064115, dated 06 / 29 / 2026, p. 134 / 485 126 / 153 effective amount of the drug to block DENV protein E binding and / or DENV entry into a host cell. An individual according to either of the above embodiments may be a human being.

[0321] In a further aspect, the invention provides a method for treating a DENV infection. In one embodiment, the method comprises administering to an individual having such a DENV infection an effective amount of an anti-DENV antibody. In another such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An individual according to any of the above embodiments may be a human being.

[0322] In another aspect, the invention provides a method for blocking DENV protein E binding and / or DENV entry into a host cell in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-DENV antibody to block DENV protein E binding and / or DENV entry into a host cell. In one embodiment, an individual is a human being.

[0323] In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-DENV antibodies provided herein, for example, for use in any of the therapeutic methods described above. In one embodiment, a pharmaceutical formulation comprises any of the anti-DENV antibodies provided in this invention and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-DENV antibodies provided herein and at least one additional therapeutic agent, for example, as described below.

[0324] In a further aspect, the pharmaceutical formulation is for the treatment of DENV infection. In another embodiment, the Petition 870260064115, dated 06 / 29 / 2026, page 135 / 485 The pharmaceutical formulation 127 / 153 is intended to block the binding of DENV E protein and / or the entry of DENV into a host cell. In one embodiment, the pharmaceutical formulation is administered to an individual having DENV infection. An individual according to either of the above embodiments is preferably a human being.

[0325] In certain forms, 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).

[0326] Any of the polypeptides comprising a variant Fc region provided herein may be used in therapeutic methods.

[0327] In one aspect, a polypeptide comprising a variant Fc region for use as a medicament is provided. In certain embodiments, a polypeptide comprising a variant Fc region is provided for use in a treatment method. In certain embodiments, the invention provides a polypeptide comprising a variant Fc region for use in a treatment method for an individual having a disorder comprising administering to the individual an effective amount of the polypeptide comprising a variant Fc region. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the disorder is a viral infection. In one embodiment, the individual is a human being.

[0328] In another aspect, the invention provides the use of a polypeptide comprising a variant Fc region in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a disorder. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In another embodiment, the medicament is for use in a Petition 870260064115, dated 06 / 29 / 2026, page 136 / 485 128 / 153 A method of treating a disorder comprising administering to an individual having the disorder to be treated an effective amount of the drug. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the disorder is a viral infection. In one embodiment, the individual is a human being.

[0329] In another aspect, the invention provides a method for treating a disorder. In one embodiment, the method comprises administering to an individual who has such a disorder an effective amount of a polypeptide comprising a variant Fc region. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In one embodiment, the disorder is a viral infection. In one embodiment, the individual is a human being.

[0330] In a further aspect, the invention provides pharmaceutical formulations comprising a polypeptide comprising a variant Fc region provided herein, for use in a therapeutic method such as any of the therapeutic methods described herein. In one embodiment, a pharmaceutical formulation comprises a polypeptide comprising a variant Fc region provided in this invention and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises a polypeptide comprising a variant Fc region granted herein and at least one additional therapeutic agent.

[0331] In another aspect, the pharmaceutical formulation is for the treatment of a disorder. In one embodiment, the pharmaceutical formulation is administered to an individual having a disorder. In one embodiment, the disorder is a viral infection. In one embodiment, the individual is a human being.

[0332] Antivirus antibodies comprising an Fc region Petition 870260064115, dated 06 / 29 / 2026, p. 137 / 485 A variant of the present invention, 129 / 153, can suppress antibody-dependent enhancement (ADE) observed with conventional antiviral antibodies. ADE is a phenomenon where a virus bound to an antibody undergoes phagocytosis through the activation of FcyRs, such that the virus's infection of a cell is enhanced. Fc modifications that reduce interaction with FcyRs can alleviate the risk of ADE. Mutations at positions 234 and 235 from leucine to alanine to form LALA mutants have been shown to reduce the risk of ADE from dengue infection in vivo (Cell Host Microbe (2010) 8, 271-283). However, such modifications reduce other antibody-mediated effector immune functions, such as ADCC and CDC. In particular, CDC is expected to play an important role in inhibiting ADE; therefore, the binding of the complement component C1q to the Fc regions should not be reduced for therapeutic efficacy.Furthermore, the antibody half-life can be extended by designing Fc regions that alter the binding affinity with their salvage receptor, FcRn, which may lead to the prophylactic use of antibodies for protection against viral infection.

[0333] The virus is preferably selected from an adenovirus, an astrovirus, a hepadnavirus, a herpesvirus, a papovavirus, a poxvirus, an arenavirus, a bunyvirus, a calcivirus, a coronavirus, a filovirus, a flavivirus, an orthomyxovirus, a paramyxovirus, a picornavirus, a reovirus, a retrovirus, a rhabdovirus or a togavirus.

[0334] In preferred embodiments, adenovirus includes, but is not limited to, a human adenovirus. In preferred embodiments, astrovirus includes, but is not limited to, a mammastrovirus. In preferred embodiments, hepadnavirus includes, but is not limited to, hepatitis B virus. In preferred embodiments, herpesvirus includes, but is not limited to, a herpes simplex virus type I, a virus Petition 870260064115, dated 06 / 29 / 2026, p. 138 / 485 130 / 153 of herpes simplex type 2, a human cytomegalovirus, an Epstein-Barr virus, a varicella-zoster virus, a roseolovirus, and a herpesvirus associated with Kaposi's sarcoma. In preferred embodiments, papovavirus includes, but is not limited to, human papillomavirus and a human polyomavirus. In preferred embodiments, poxvirus includes, but is not limited to, a smallpox virus, a vaccinia virus, a cowpox virus, a monkeypox virus, a smallpox virus, a bovine pseudopox virus, papular stomatitis virus, tanapox virus, yaba monkey tumor virus, and molluscum contagiosum virus. In preferred embodiments, arenavirus includes, but is not limited to, lymphocytic choriomeningitis virus, a lasso virus, a machupo virus, and a junin 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, a vesivirus, a norovirus such as Norwalk virus, and a sapovirus. In preferred embodiments, coronavirus includes, but is not limited to, a human coronavirus (the causative agent of severe acute respiratory syndrome (SARS)). In preferred embodiments, filovirus includes, but is not limited to, an Ebola virus and a Marburg virus. In preferred forms, flaviviruses include, but are not limited to, a yellow fever virus, a West Nile virus, dengue viruses (DENV-1, DENV-2, DENV-3, and DENV-4), a hepatitis C virus, a tick-borne encephalitis virus, a Japanese encephalitis virus, a Murray Valley encephalitis virus, a St. Louis encephalitis virus, a Russian spring-summer encephalitis virus, an Omsk hemorrhagic fever virus, a bovine viral diarrhea virus, a Kyasanus Forest disease virus, and a Powssan encephalitis virus.In preferred forms, orthomyxovirus includes, but is not limited to, influenza A virus, influenza B virus, and influenza B virus. Petition 870260064115, dated 06 / 29 / 2026, p. 139 / 485 131 / 153 influenza type C. In preferred embodiments, paramyxovirus includes, but is not limited to, a parainfluenza virus, a rubella (mumps) virus, a morbillivirus (measles), a pneumovirus, such as a human respiratory syncytial virus, and a subacute sclerosing panencephalitis virus. In preferred embodiments, picornavirus includes, but is not limited to, a poliovirus, a rhinovirus, a coxsackievirus A, a coxsackievirus B, a hepatitis A virus, an echovirus, and an enterovirus. In preferred embodiments, reovirus includes, but is not limited to, a Colorado tick fever virus and a rotavirus. In preferred embodiments, retrovirus includes, but is not limited to, a lentivirus, such as a human immunodeficiency virus and a human T-lymphotropic virus (HTLV).In preferred embodiments, rhabdovirus includes, but is not limited to, a lyssavirus, such as rabies virus, vesicular stomatitis virus, and infectious hematopoietic necrosis virus. In preferred embodiments, togavirus includes, but is not limited to, an 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 a rubella virus.

[0335] In another aspect, the invention provides methods for the preparation of a medicament or a pharmaceutical formulation, comprising mixing any of the anti-DENV antibodies provided herein with a pharmaceutically acceptable vehicle, for example, for use in any of the therapeutic methods described above. In one embodiment, the methods for the preparation of a medicament or a pharmaceutical formulation further comprise the addition of at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0336] The antibodies of the invention can be used Petition 870260064115, dated 06 / 29 / 2026, p. 140 / 485 132 / 153 alone or in combination with other agents in a therapy. For example, an antibody of the invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is an antiviral agent, such as, but not limited to, interferons (e.g., interferon α-2b, interferon-γ, etc.), anti-DENV monoclonal antibodies, anti-DENV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, small interfering RNAs, small hairpin RNAs, microRNAs, RNA aptamers, ribozymes, and combinations thereof.

[0337] In a further aspect, the invention provides methods for the preparation of a medicament or a pharmaceutical formulation comprising mixing any of the polypeptides comprising a variant Fc region provided herein, with a pharmaceutically acceptable carrier, for example, for use in any of the therapeutic methods above. In one embodiment, the methods for the preparation of a medicament or a pharmaceutical formulation further comprise the addition of at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0338] Polypeptides comprising a variant Fc region of the invention may be used alone or in combination with other agents in therapy. For example, a polypeptide comprising a variant Fc region of the invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, an 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, RNA polymerase inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, Petition 870260064115, dated 06 / 29 / 2026, page 141 / 485 133 / 153 antisense compounds, small interfering RNAs, small hairpin RNAs, microRNAs, RNA aptamers, ribozymes, and combinations thereof.

[0339] The aforementioned combination therapies encompass combined administration (where two or more therapeutic agents are included in the same formulations or separately), and separate administration, in which case the administration of the antibody or polypeptide comprising a variant Fc region of the invention may occur before, simultaneously with, and / or after the administration of the additional therapeutic agent or agents. In one embodiment, the administration of the anti-DENV antibody and the administration of an additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.In another embodiment, the administration of the polypeptide comprising the variant Fc region and the administration of an additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0340] An antibody or a polypeptide comprising an Fc variant region of the invention (and any additional therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. Dosing may be by any suitable route, for example, by injections such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic. Various dosing schedules including, but not limited to, single or multiple administrations. Petition 870260064115, dated 06 / 29 / 2026, page 142 / 485 134 / 153 on various occasions, cake administration and pulse infusion are contemplated in this invention.

[0341] Antibodies or polypeptides comprising an Fc region variant of the invention would be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the specific disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of application of the agent, the method of administration, the administration schedule, and other factors known to medical professionals. The agent need not be, but is optionally formulated with, one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of agent present in the formulation, the type of disorder or treatment, and other factors discussed above.These are generally used at the same dosages and by the routes of administration as described herein, or at around 1 to 99% of the dosages described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.

[0342] For the prevention or treatment of disease, the appropriate dosage of an antibody or polypeptide comprising a variant Fc region of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the type of polypeptide comprising the variant Fc region, the severity and course of the disease, whether the antibody or polypeptide comprising the variant Fc region is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody or polypeptide comprising the variant Fc region, and the discretion of the attending physician. The antibody or polypeptide Petition 870260064115, dated 06 / 29 / 2026, page 143 / 485 135 / 153 comprising an Fc variant region is appropriately administered to the patient at once or in a series of treatments. Depending on the type and severity of the disease, approximately 1 µg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody or polypeptide comprising an Fc variant region may be a candidate initial dosage for administration to the patient, either, for example, through one or more separate administrations or by continuous infusion. A typical daily dosage may range from approximately 1 µg / kg to 100 mg / kg or more, depending on the aforementioned factors. For repeated administrations over several days or more, depending on the condition, treatment would generally be sustained until a desired suppression of disease symptoms occurs. An exemplary dosage of the antibody or polypeptide comprising the Fc variant region would be in the range of approximately 0.05 mg / kg to approximately 10 mg / kg.Thus, one or more doses around 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (for example, in such a way that the patient receives from about two to about twenty, or for example, around six doses of the antibody or polypeptide comprising the variant Fc region). A higher initial loading dose, followed by one or more lower doses, may be administered. The progress of this therapy is easily monitored by conventional techniques and examinations.

[0343] It is understood that any of the above formulations or therapeutic methods may be performed using an immunoconjugate of the invention in place of or in addition to an anti-DENV antibody.It is also understood that any of the above formulations or therapeutic methods may be performed using an immunoconjugate of the invention in place of or in addition to a polypeptide comprising a... Petition 870260064115, dated 06 / 29 / 2026, page 144 / 485 136 / 153 region Fc variant granted here. H. Manufacturing Articles

[0344] In another aspect of the invention, a manufactured article containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The manufactured article comprises a container and a label or package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is isolated or combined with another composition effective for the treatment, prevention and / or diagnosis of the condition and may have a sterile access orifice (for example, the container may be an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle). At least one active ingredient in the composition is an antibody or a polypeptide comprising an Fc variant region of the invention.The label or package insert indicates that the composition is used for the treatment of the chosen condition. Furthermore, the manufactured article may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody or a polypeptide comprising an Fc variant region of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises another cytotoxic or otherwise therapeutic agent. The manufactured article of this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the manufactured article may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water. Petition 870260064115, dated 06 / 29 / 2026, page 145 / 485 137 / 153 for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0345] It is understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-DENV antibody. It is probably understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to a polypeptide comprising a variant Fc region. III. EXAMPLES

[0346] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided 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.

[0347] Recombinant soluble E proteins (0,8E-His) from DENV-1, DENV-2, DENV-3, and DENV-4 with a terminal 8x histidine carboxy marker (SEQ ID NOs: 65 to 68, respectively) were transiently expressed using the FreeStyle293-F cell line or Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Through prM0,8E-His expression from DENV-1, DENV-2, DENV-3, and DENV-4 (SEQ ID NOs: 61 to 64, respectively), prM0,8E-His was expressed as a single polypeptide in the cells, which is then processed intracellularly to be cleaved between prM and 0,8E-His. As a result, 0,8E-His was secreted into the cell culture medium. Conditioned medium containing 0.8E-His was applied to a column packed with an immobilized metal affinity chromatography (IMAC) resin loaded with nickel or cobalt, followed Petition 870260064115, dated 06 / 29 / 2026, p. 146 / 485 138 / 153 by elution with imidazole. The fractions containing 0.8E-His were pooled and applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). The fractions containing 0.8E-His were pooled and stored at -80 °C. Expression and purification of recombinant human FcyRs

[0348] Extracellular domains of human FcyRs were prepared by the following method. First, an FcyR extracellular domain gene was synthesized by a method well known to those skilled in the art. At that point, the sequence of each FcyR was produced based on information registered in the NCBI. Specifically, FcYRIa was produced based on the NCBI Accession sequence No. NM_000566 (Version No. NM_000566.3), FcyRIIa was produced based on the NCBI Accession sequence No. NM_001136219 (Version No. NM_001136219.1), FcyRIIb was produced based on the NCBI Accession sequence No. NM_004001 (Version No. NM_004001.3), FcyRIIIa was produced based on the NCBI Accession sequence No. NM_001127593 (Version No. NM_001127593.1), and FcyRIIIb was produced based on the NCBI Accession sequence No. NM_000570 (Version No. NM_000570.3), and a His mark was attached to it. C-terminal of each FcyR construct.Furthermore, the presence of polymorphism is known for FcyRIIa, FcyRIIIa, and FcyRIIIb, and the polymorphic sites were produced by reference to Warmerdam et al. (J Exp Med (1990) 172, 19-25) for FcyRIIa; Wu et al. (J Clin Invest (1997) 100, 1059-1070) for FcyRIIIa; and Ory et al. (J Clin Invest (1989) 84, 1688-1691) for FcyRIIIb.

[0349] Expression vectors were constructed by inserting the obtained gene fragments into animal cell expression vectors. The constructed expression vectors were transiently introduced into FreeStyle293 cells derived from cancer cells. Petition 870260064115, dated 06 / 29 / 2026, p. 147 / 485 139 / 153 human embryonic renal cell (Invitrogen) to express the proteins of interest. The liquids prepared by filtration through a 0.22 μm filter of the culture supernatants obtained from the culture medium of the above cells subjected to transient introduction were purified, in principle, by the following four steps: (i) cation exchange column chromatography (SP Sepharose FF); (ii) His-tag affinity column chromatography (HisTrap HP); (iii) gel filtration column chromatography (Superdex 200); and (iv) sterile filtration. To purify FcyRI, anion exchange column chromatography with Q sepharose FF was used for step (i). The absorbance of the purified protein was measured at 280 nm using a spectrophotometer. Based on the measured values, the concentrations of purified proteins were calculated using the extinction coefficient determined by a method such as PACE (Protein Science (1995) 4, 2411-2423). Expression and purification of recombinant mouse FcyRs

[0350] The extracellular domain of mouse FcyRs (mFcyRs) was prepared using the following method: first, the FcyR extracellular domain gene was synthesized by a method generally known to those skilled in the art. For this synthesis, the sequence of each FcyR was prepared based on information registered in the NCBI. Specifically, mFcyRI was prepared based on the NCBI Reference Sequence: NP_034316.1; mFcyRIIb was prepared based on the NCBI Reference Sequence: NP_034317.1; mFcyRIII was prepared based on the NCBI Reference Sequence: NP_034318.2; and mFcyRIV was prepared based on the NCBI Reference Sequence: NP_653142.2. Each of these sequences was joined in a C-terminal fashion with a His tag.

[0351] Each gene fragment obtained was inserted into the vectors to Petition 870260064115, dated 06 / 29 / 2026, pp. 148 / 485140 / 153 expression in animal cells to prepare expression vectors. The prepared expression vectors were transiently transferred to FreeStyle 293 cells derived from human embryonic renal cancer cells (Invitrogen) to express the protein of interest. The culture supernatant obtained was recovered and then passed through a 0.22 μm filter to obtain a culture supernatant. The culture supernatant obtained was purified, as a rule, by the following four steps: (i) ion-exchange column chromatography, (ii) affinity column chromatography for the His label (HisTrap HP), (iii) gel filtration column chromatography (Superdex200) and (iv) sterile filtration. The ion exchange column chromatography of step (i) was performed using Q Sepharose HP for mFcyRI, SP Sepharose FF for mFcyRIIb and mFcyRIV, and SP Sepharose HP for mFcyRIII.D-PBS(-) was used as a solvent in step (iii) or later, while D-PBS(-) containing 0.1 M arginine was used for mFcγRIII. Absorbance was measured for each purified protein at 280 nm using a spectrophotometer, and the concentration of the purified protein was calculated using an extinction coefficient calculated from the value obtained by a method such as PACE (Protein Science (1995) 4, 2411-2423). Expression and purification of recombinant human FcRn

[0352] FcRn is a heterodimer of FcRn alpha and beta2-microglobulin chains. Oligo-DNA primers were prepared based on the published human FcRn gene sequence (J Exp Med (1994) 180, 2377-2381). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers. Using the obtained DNA fragment as a template, a DNA fragment encoding the extracellular domain containing the signal region (Met1-Leu290) was amplified by PCR, and Petition 870260064115, dated 06 / 29 / 2026, page 149 / 485 141 / 153 inserted into a mammalian cell expression vector. Similarly, oligo-DNA primers were prepared based on the published human beta2-microglobulin gene sequence (Proc Natl Acad Sci USA (2002) 99, 16899-16903). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the primers prepared. Using the obtained DNA fragment as a template, a DNA fragment encoding the total protein containing a signal region (Met1-Met119) was amplified by PCR and inserted into a mammalian cell expression vector.

[0353] Soluble human FcRn was expressed by the following procedure. Plasmids constructed for the expression of human FcRn alpha chain (SEQ ID NO: 81) and beta2-microglobulin (SEQ ID NO: 82) were introduced into human embryonic renal cancer-derived cell line HEK293H (Invitrogen) by lipofection using PEI (Polyscience). The resulting culture supernatant was collected and the FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences), followed by further purification using HiTrap Q HP (GE Healthcare) (J Immunol (2002) 169, 5171-5180). Expression and purification of recombinant antibodies

[0354] Recombinant antibodies were transiently expressed using a FreeStyle293-F cell line or an Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Purification of the conditioned antibody-expressing medium was performed using the conventional method with protein A. Gel filtration was also conducted if necessary. Expression and purification of recombinant soluble human CD154

[0355] The human CD154 gene was synthesized based on the published protein sequence (NP_000065.1). A fragment of Petition 870260064115, dated 06 / 29 / 2026, page 150 / 485 142 / 153 DNA encoding the soluble form of human CD154 (shCD154) with a FLAG tag was prepared by PCR, using the synthesized DNA as a template, and the resulting DNA fragments were inserted into a mammalian cell expression vector, thus producing the FLAG-shCD154 expression vector (SEQ ID NO: 106). The nucleotide sequences of the obtained 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) as described by the manufacturer's protocol. After transfection, the cells were cultured for an appropriate time before the conditioned medium was harvested. The conditioned medium was subjected to cation exchange chromatography using 25 mM MES (pH 6.0), and FLAG-shCD154 was eluted with a continuous gradient of 25 mM MES, 1M NaCl (pH 6.0).The maximum fractions were pooled, concentrated using AmiconUltra Ultracel, and subjected to gel filtration chromatography using phosphate-buffered saline (Wako). The maximum fractions were again pooled and concentrated using AmiconUltra Ultracel, then sterilized by filtration with a 0.22 micrometer 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 determined values ​​using an absorbance coefficient calculated by the method described in Protein Science (1995) 4: 2411-2423. Example 2: Generation of antibody variants with improved affinity to the DENV E protein.

[0356] The genes encoding VH (3CH, SEQ ID NO: 1) and VL (3CL, SEQ ID NO: 7) of an anti-DENV E protein antibody were synthesized and combined with a human IgG1 CH (SG182, Petition 870260064115, dated 06 / 29 / 2026, p. 151 / 485 143 / 153 A human CL (SK1, SEQ ID NO: 46) and a human CL (SK1, SEQ ID NO: 60), respectively, were developed and both constructs were cloned into a single expression vector. The antibody is referred to here as DG_3CHSG182 / 3CL-SK1, or as 3C.

[0357] Several mutations and their combinations were examined to identify mutations and combinations that improved the 3C binding properties. Multiple mutations were then introduced into the variable regions to increase the binding affinity with the E protein. Optimized VH variants, 3CH912 (SEQ ID NO: 2), 3CH953 (SEQ ID NO: 3), 3CH954 (SEQ ID NO: 4), 3CH955 (SEQ ID NO: 5), 3CH1047 (SEQ ID NO: 6), 3CH987 (SEQ ID NO: 90), 3CH989 (SEQ ID NO: 91), 3CH992 (SEQ ID NO: 92), 3CH1000 (SEQ ID NO: 93), 3CH1046 (SEQ ID NO: 94), 3CH1049 (SEQ ID NO: 95), and optimized VL variants, 3CL499 (SEQ ID NO: 8), 3CL563 (SEQ ID NO: 9), 3CL658 (SEQ ID NO: 10), 3CL012 (SEQ ID NO: 96), 3CL119 (SEQ ID NO: 97), 3CL633 (SEQ ID NO: 98), 3CL666 (SEQ ID NO: 99), 3CL668 (SEQ ID NO: 100), were thus generated.The genes encoding VH were combined with human IgG1 CH (any one from SG182, SEQ ID NO: 46; SG1095, SEQ ID NO: 54; or SG1106, SEQ ID NO: 59), and the genes encoding VL were combined with a human CL (SKI, SEQ ID NO: 60). Each was cloned into an expression vector. The amino acid sequences of the antibody variants are summarized in Table 2. One of the variants, DG_3CH1047-SG182 / 3CL658-SK1, is also referred to here as 3Cam, and another variant, DG_3CH1047-SG182 / 3CL-SK1, is referred to here as 3Cam2.

[0358] Antibodies were expressed in HEK293 cells cotransfected with a mixture of heavy and light chain expression vectors, and were purified using protein A. Petition 870260064115, dated 06 / 29 / 2026, p. 152 / 485 144 / 153 Table 2 3C amino acid sequences and 3C variants Anticorpo SEQ ID NO: VH VL HVR-H1 HVR-H2 HVR-H3 HVR-L1 HVR-L2 HVR-L3 CH CL DG 3CH-SG182 / 3CL-SK1 1 7 11 13 16 21 24 27 46 60 DG 3CH912-SG182 / 3CL-SK1 2 7 11 14 17 21 24 27 46 60 DG 3CH953-SG182 / 3CL-SK1 3 7 11 15 17 21 24 27 46 60 DG 3CH954-SG182 / 3CL-SK1 4 7 11 15 18 21 24 27 46 60 DG 3CH955-SG182 / 3CL-SK1 5 7 11 15 19 21 24 27 46 60 DG 3CH-SG182 / 3CL499-SK1 1 8 11 13 16 21 25 28 46 60 DG 3CH-SG182 / 3CL563-SK1 1 9 11 13 16 22 25 29 46 60 DG 3CH953-SG182 / 3CL499-SK1 3 8 11 15 17 21 25 28 46 60 DG 3CH953-SG182 / 3CL563-SK1 3 9 11 15 17 22 25 29 46 60 DG 3CH955-SG182 / 3CL499-SK1 5 8 11 15 19 21 25 28 46 60 DG 3CH955-SG182 / 3CL563-SK1 5 9 11 15 19 22 25 29 46 60 OG_3CH1047-SG182 / 3CL658-SK1 6 10 12 15 20 23 26 30 46 60 DG 3CH100O-SG182 / 3CL-SK1 93 7 12 15 17 21 24 27 46 60 DG 3CH1047-SG182 / 3CL-SK1 6 7 12 15 20 21 24 27 46 60 DG 3CH953-SG1095 / 3CL-SK1 3 7 11 15 17 21 24 27 54 60 DG 3CH100O-SG1095 / 3CL-SK1 93 7 12 15 17 21 24 27 54 60 DG 3CH1047-SG1095 / 3CL-SK1 6 7 12 15 20 21 24 27 54 60 DG3CH953-SG1106 / 3CL-SK1 3 7 11 15 17 21 24 27 59 60 DG 3CH100O-SG1106 / 3CL-SK1 93 7 12 15 17 21 24 27 59 60 DG 3CH1047-SG1106 / 3CL-SK1 6 7 12 15 20 21 24 27 59 60 DG 3CH953-SG1095 / 3CL012-SK1 3 96 11 15 17 21 24 30 54 60 DG 3CH100O-SG1095 / 3CL012-SK1 93 96 12 15 17 21 24 30 54 60 DG 3CH1047-SG1095 / 3CL012-SK1 6 96 12 15 20 21 24 30 54 60 DG 3CH953-SG1106 / 3CL012-SK1 3 96 11 15 17 21 24 30 59 60 DG_3CH1000-SG1106 / 3CL012-SK1 93 96 12 15 17 21 24 30 59 60 DG 3CH1047-SG1106 / 3CL012-SK1 6 96 12 15 20 21 24 30 59 60 DG_3CH953-SG1O95 / 3CL119-SK1 3 97 11 15 17 23 24 27 54 60 DG 3CH1OOO-SG1095 / 3CL119-SK1 93 97 12 15 17 23 24 27 54 60 DG 3CH1047-SG1095 / 3CL119-SK1 6 97 12 15 20 23 24 27 54 60 DG_3CH953-SG1106 / 3CL119-SKl 3 97 11 15 17 23 24 27 59 60 DG 3CH1OOO-SG11O6 / 3CL119-SK1 93 97 12 15 17 23 24 27 59 60 DG.3CH1047-SG1106 / 3CL119-SK1 6 97 12 15 20 23 24 27 59 60 DG 3CH1049-SG182 / 3CL-SK1 95 7 12 15 20 21 24 27 46 60 DG 3CH1046-SG182 / 3CL-SK1 94 7 12 15 17 21 24 27 46 60 DG_3CH989-SG182 / 3CL-SK1 91 7 11 15 1721 24 27 46 60 DG 3CH992-SG182 / 3CL-SK1 92 7 101 15 17 21 24 27 46 60 DG 3CH987-SG182 / 3CL-SK1 90 7 101 15 17 21 24 27 46 60 DG.3CH1049-SG182 / 3CL012-SK1 95 96 12 15 20 21 24 30 46 60 DG 3CH1046-SG182 / 3CL012-SK1 94 96 12 15 17 21 24 30 46 60 DG 3CH989-SG182 / 3CL012-SK1 91 96 11 15 17 21 24 30 46 60 DG 3CH992-SG182 / 3CL012-SK1 92 96 101 15 17 21 24 30 46 60 DG 3CH987-SG182 / 3CL012-SK1 90 96 101 15 17 21 24 30 46 60 DG_3CH1049-SG182 / 3CL119-SK1 95 97 12 15 20 23 24 27 46 60 DG 3CH1046-SG182 / 3CL119-SK1 94 97 12 15 17 23 24 27 46 60 DG 3CH989-SG182 / 3CL119-SK1 91 97 11 15 17 23 24 27 46 60 DG_3CH992-SG182 / 3CL119-SK1 92 97 101 15 17 23 24 27 46 60 DG 3CH987-SG182 / 3CL119-SK1 90 97 101 15 17 23 24 27 46 60 DG_3CH1049-SG182 / 3CL668-SKl 95 100 12 15 20 21 24 30 46 60 DG 3CH1046-SG182 / 3CL668-SK1 94 100 12 15 17 21 24 30 46 60 DG 3CH989-SG182 / 3CL668-SK1 91 100 11 15 17 21 24 30 46 60 DG.3CH1049-SG182 / 3CL666-SK1 95 99 12 15 20 23 24 27 46 60 DG 3CH1046-SG182 / 3CL666-SK1 94 99 12 15 17 23 24 27 46 60 DG3CH989-SG182 / 3CL666-SK1 91 99 11 15 17 23 24 27 46 60 DG_3CH1049-SG182 / 3CL633-SKl 95 98 12 15 20 21 24 27 46 60 DG 3CH1046-SG182 / 3CL633-SK1 94 98 12 15 17 21 24 27 46 60 DG 3CH989-SG182 / 3CL633-SK1 91 98 11 15 17 21 24 27 46 60 DG 3CH953-SG182 / 3CL668-SK1 3 100 11 15 17 21 24 30 46 60 DG 3CH1000-SG182 / 3CL668-SK1 93 100 12 15 17 21 24 30 46 60 DG.3CH953-SG182 / 3CL666-SK1 3 99 11 15 17 23 24 27 46 60 DG 3CH1000-SG182 / 3CL666-SK1 93 99 12 15 17 23 24 27 46 60 06 3CH953-SG182 / 3CL633-SK1 3 98 11 15 17 21 24 27 46 60 DG 3CH1OOO-SG182 / 3CL633-SK1 93 98 12 15 17 21 24 27 46 60 DG 3CH1047-SG182 / 3CL666-SK1 6 99 12 15 20 23 24 27 46 60 DG 3CH992-SG182 / 3CL666-SK1 92 99 101 15 17 23 24 27 46 60 DG 3CH1047-SG182 / 3CL633-SK1 6 98 12 15 20 21 24 27 46 60 OG.3CH992-SG182 / 3CL633-SK1 92 98 101 15 17 21 24 27 46 60

[0359] This anti-virus protects against DENV. Petition: 870260064115, on 06 / 29 / 2026, page. 153 / 485 The 145 / 153 binding to DENV-1, DENV-2, DENV-3, and DENV-4 E protein was determined at 25°C using a Biacore T200 instrument (GE Healthcare). Antihistidine antibody (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in PBS, pH 7.4, containing 20 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaNsa. The E protein of DENV-1, DENV-2, DENV-3, and DENV-4 with the His C-terminal marker was captured on flow cell 2 or 3, with flow cell 1 as the reference flow cell. The capture level for the E protein was measured at 200 resonance units (RU). Anti-DENV E protein antibodies were injected at 250 nM over the entire sensor surface for 180 seconds, followed by 300 seconds of dissociation. The sensor surface was regenerated after each cycle using 10 mM Gly-HCl pH 1.5.Linkage affinity was determined by processing and fitting the data with the 1:1 linkage model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).

[0360] Tables 3a and 3b show the affinity (Kd) of anti-DENV E protein antibodies that bind to the E protein of DENV-1 (described as DV1 in the Table), DENV-2 (described as DV2 in the Table), DENV3 (described as DV3 in the Table), and DENV-4 (described as DV4 in the Table). Each 3C variant showed increased binding affinity against all four DENV serotypes, compared to the original 3C. As examples, the sensorgrams of the antibody of origin 3C (DG_3CH-SG182 / 3CL-SK1) and one of the variant antibody 3Cam (DG_3CH1047-SG182 / 3CL658-SK1) are shown in Figure 1. Similarly, the sensorgrams of the antibody of origin 3C (DG_3CHSG182 / 3CL-SK1) and one of the variant antibodies 3Cam2 (DG_3CH1047-SG182 / 3CL-SK1) are shown in Figure 12. Petition 870260064115, dated 06 / 29 / 2026, page 154 / 485 146 / 153 Tabela 3a Valores Kd de 3C e variantes 3C contra quatro sorotipos do DENV Variante Fv KD (M) DV1 DV2 DV3 DV4 DG_3CH-SG182 / 3CL-SK1 3.21E-08 1.40E-08 6.07E-09 1.69E-08 DG_3CH912-SG182 / 3CL-SK1 1.85E-09 8.11E-10 2.44E-10 8.78E-09 DG_3CH953-SG182 / 3CL-SK1 1.41E-09 7.76E-10 1.36E-10 6.20E-09 DG_3CH954-SG182 / 3CL-SK1 8.87E-10 1.22E-10 1.24E-10 4.35E-09 DG_3CH955-SG182 / 3CL-SK1 1.13E-09 7.94E-10 2.05E-10 7.80E-09 DG_3CH-SG182 / 3CL499-SK1 1.11E-08 4.73E-09 4.03E-09 8.35E-09 DG_3CH-SG182 / 3CL563-SK1 1.34E-08 4.43E-09 3.52E-09 1.18E-08 DG_3CH953-SG182 / 3CL499-SK1 1.46E-09 9.05E-10 2.11E-10 5.99E-09 DG_3CH953-SG182 / 3CL563-SK1 1.88E-09 9.22E-10 2.64E-10 8.21E-09 DG_3CH955-SG182 / 3CL499-SK1 1.26E-09 1.03E-09 3.31E-10 8.26E-09 DG_3CH955-SG182 / 3CL563-SK1 1.91E-09 1.04E-09 2.90E-10 1.22E-08 DG_3CH1047-SG182 / 3CL658-SKl 3.51E-09 7.65E-10 3.82E-12* 9.11E-09 DG_3CH1047-SG182 / 3CL-SKl 2.14E-09 8.43E-10 2.31E-13* 5.61E-09 Note: * strong binders, slow off rate <1E-05, KD cannot be uniquely determined. Note: * Strong binders, slow dissociation index < 1 E-05, KD cannot be specifically determined. Petition 870260064115, dated 06 / 29 / 2026, page 155 / 485 147 / 153 Table 3b Kd values ​​of 3C and 3C variants against DENV1 serotypes and DENV3 Variante Fv KD (M) DV1 DV3 DG_3CH-SG182 / 3CL-SK1 2.25E-08 6.35E-09 DG_3CH 1047-SG182 / 3C L658-SK1 2.98E-09 5.01E-12* DG_3CH953-SG182 / 3CL-SK1 1.80E-09 9.85E-13* DG_3CH 1000-SG182 / 3C L-SK1 1.79E-09 3.49E-12* DG_3CH 1047-SG182 / 3C L-SK1 2.06E-09 7.22E-11 * DG_3CH 953-SG1095 / 3C L-SK1 1.79E-09 7.03E-12* DG_3CH 1000-SG1095 / 3CL-SK1 1.80E-09 1.68E-11 * DG_3CH 1047-SG1095 / 3CL-SK1 2.09E-09 2.93E-12* DG_3CH953-SG 1106 / 3C L-SK1 1.85E-09 7.08E-12* DG_3CH1000-SG1106 / 3CL-SK1 1.84E-09 2.99E-13* DG_3CH1047-SG1106 / 3CL-SK1 2.12E-09 4.71E-12* DG_3CH953-SG1095 / 3CL012-SK1 1.86E-09 5.53E-12* DG_3CH 1000-SG1095 / 3CL012-SK1 1.83E-09 9.59E-13* DG_3CH 1047-SG1095 / 3CL012-SK1 2.21E-09 3.17E-12* DG_3CH 953-SG 1106 / 3C L012-SK1 1.89E-09 1.06E-11 * DG_3CH1000-SG1106 / 3CL012-SK1 1.91E-09 1.01E-13* DG_3CH1047-SG1106 / 3CL012-SK1 2.18E-09 2.36E-12* DG_3CH953-SG1095 / 3CL119-SK1 1.99E-09 7.18E-12* DG_3CH 1000-SG1095 / 3CL119-SK1 1.92E-09 4.28E-14* DG_3CH 1047-SG1095 / 3CL119-SK1 2.23E-09 3.79E-12* DG_3CH953-SG1106 / 3CL119-SK1 1.96E-09 2.17E-12* DG_3CH 1000-SG1106 / 3CL119-SK1 1.91E-09 1.82E-12* DG_3CH 1047-SG1106 / 3CL119-SK1 2.29E-09 4.36E-12* DG_3CH 1049-SG182 / 3C L-SK1 2.14E-09 2.39E-12* DG_3CH 1046-SG182 / 3C L-SK1 1.91E-09 2.13E-13* DG_3CH989-SG182 / 3CL-SK1 1.84E-09 4.48E-13* DG_3CH992-SG182 / 3CL-SK1 2.02E-09 4.03E-12* DG_3CH987-SG182 / 3CL-SK1 1.82E-09 9.86E-13* DG_3CH 1049-SG182 / 30 L012-SK1 2.26E-09 1.05E-12* DG_3CH 1046-SG182 / 3CL012-SK1 1.92E-09 1.45E-13* DG_3CH989-SG182 / 3CL012-SK1 1.52E-09 9.67E-14* DG_3CH992-SG182 / 3CL012-SK1 1.72E-09 1.07E-12* DG_3CH987-SG182 / 3CL012-SK1 142E-09 2.67E-12* DG_3CH1049-SG182 / 3CL119-SK1 1.72E-09 5.09E-12* DG_3CH1046-SG182 / 3CL119-SK1 1.53E-09 2.73E-12* DG_3CH989-SG182 / 3CL119-SK1 1.53E-09 6.83E-12* DG_3CH992-SG182 / 3CL119-SK1 1.87E-09 5.15E-13* DG_3CH987-SG182 / 3CL119-SK1 1.53E-09 7.15E-13* DG_3CH 1049-SG 182 / 3C L668-SK1 1.70E-09 1.55E-12* DG_3CH 1046-SG182 / 3C L668-SK1 1.56E-09 5.59E-13* DG_3CH989-SG182 / 3CL668-SK1 1.50E-09 3.03E-12* DG_3CH 1049-SG 182 / 3C L666-SK1 1.72E-09 2.95E-12* DG_3CH 1046-SG182 / 3C L666-SK1 1.52E-09 4.70E-13* DG_3CH989-SG182 / 3CL666-SK1 1.50E-09 1.96E-12* DG_3CH 1049-SG 182 / 3C L633-SK1 1.73E-09 4.10E-12* DG_3CH 1046-SG182 / 3C L633-SK1 1.64E-09 2.01E-13* DG_3CH989-SG182 / 3CL633-SK1 1.54E-09 2.37E-12* DG_3CH953-SG182 / 3CL668-SK1 1.68E-09 2.36E-12* DG_3CH 1000-SG182 / 3C L668-SK1 1.68E-09 8.00E-13* DG_3CH953-SG182 / 3CL666-SK1 1 69E-09 5.48E-12* DG_3CH 1000-SG182 / 3C L666-SK1 1.66E-09 2.50E-12* DG_3CH953-SG182 / 3CL633-SK1 1.65E-09 4.96E-12* DG_3CH 1000-SG182 / 3C L633-SK1 1.77E-09 4.25E-12* DG_3CH 1047-SG182 / 3C L666-SK1 9.82E-10 4.20E-11 * DG_3CH992-SG182 / 3CL666-SK1 1.39E-09 8.02E-13* DG_3CH 1047-SG182 / 3C L633-SK1 9.56E-10 4.37E-13* DG_3CH992-SG182 / 3CL633-SK1 1.27E-09 2.95E-12*. Note: * strong binders, slow off rate <1E-O5, KD cannot be uniquely determined. Petição 870260064115, de 29 / 06 / 2026, pág. 156 / 485 148 / 153

[0361] Note: * strong binders, slow dissociation index < 1E-05, KD cannot be specifically determined. Example 3: Generation of CH antibody variants for improved properties

[0362] Multiple mutations have been introduced into a constant region of the human IgG1 CH heavy chain (SG182, SEQ ID NO: 46), and as a result, 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: The genes encoding the CH variants (3CH, SEQ ID NO: 1), one of the 3C variants (3CH1047, SEQ ID NO: 6), or an anti-CD154 antibody (SLAPH0336a, SEQ ID NO: 88) were generated. The VL gene of the anti-CD154 antibody (SLAPL0336a, SEQ ID NO: 89) was combined with a human CL (SKI, SEQ ID NO: 60). Each of these was cloned into an expression vector.The details of the CH variants are summarized in Table 4. The variable region of an anti-CD154 antibody SLAPH0336a / SLAPL0366a, which can form a large immune complex in the presence of the trimeric CD154 antigen, was used to evaluate the avidity binding of the CH variants to each Fc receptor.

[0363] Antibodies were expressed in HEK293 cells cotransfected with a mixture of heavy and light chain expression vectors, and were purified by protein A. Petition 870260064115, dated 06 / 29 / 2026, page 157 / 485 149 / 153 Table 4 Amino acid sequences of variant Fc regions Nome da variante ID mutações SEQID NO: WT SG182 - 46 LAIA SG192 L234A, L235A 47 KWES SG1085 K326W, E333S 48 EFT + AE SG1086 G236A, S267E, H268F, S324T, I332E 49 EFT SG1087 S267E, H268F, S324T 50 LAIA + KWES SG1088 L234A, L235A, K326W, E333S 51 LAIA + EFT + AE SG1089 L234A, L235A, G236A, S267E, H268F, S324T, I332E 52 LAIA + EFT SG1090 L234A, L235A, S267E, H268F, S324T 53 LALA + KAES SG1095 L234A, L235A, K326A, E333S 54 LALA+ KDES SG1096 L234A, L235A, K326D, E333S 55 LALA+ KEES SG1097 L234A, L235A, K326E, E333S 56 LALA + KMES SG1098 L234A, L235A, K326M, E333S 57 LALA + ACT3 + KAES SG1105 L234A, L235A, K326A, E333S, M428L, N434A, Y436T, Q438R, S440E 58 LAIA + ACT5 + KAES SGI106 L234A, L235A, K326A, E333S, M428L, N434A, Q438R, S440E 59 KAES SGI109 K326A, E333S 107 LALA +ACTS SG1044 L234A, L235A, M428L, N434A, Y436T, Q438R, S440E 108 LALA + ACTS SG1045 L234A, L235A, M4281, N434A, Q438R, S440E 109 Example 4: Binding affinities of an antibody with Fc variants to complement C1q Human C1q binding assay

[0364] Anti-CD154 antibodies with Fc variants (internal antibodies generated using the method described in Example 3) were distributed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left to stand overnight at 4°C. After washing with PBST, the plate was blocked with PBST containing 0.5% BSA and 1x Block Ace: Blocking Reagent (DS Pharma) for 2 hours at room temperature. After washing the plate, human C1q (Calbiochem) was dispensed onto the plate and left to stand for 1 hour at room temperature. The plate was washed, and HRP-labeled anti-human C1q antibody (Bio-Rad) was added to react for 1 hour at room temperature, and washing was performed. Subsequently, TMB Substrate (Invitrogen) was added. The signal was measured by a plate reader at a wavelength of 450 nm (test wavelength) and 570 nm (reference wavelength).The binding affinity of the antibody having a wild-type (WT) Fc region with human C1q was decreased by... Petition 870260064115, dated 06 / 29 / 2026, page 158 / 485 150 / 153 introduction of LALA mutations in the Fc region, and the decreased binding affinity with human C1q was recovered by the additional introduction of KWES, EFT, or EFT + AE in addition to the LALA mutations (Figure 2). LALA + KMES mutations slightly increased the binding affinity, while LALA + KWES, LALA + KAES, and LALA + KEES mutants bound to C1q with affinity comparable to WT (Figure 3). The binding property of the LALA or LALA + KAES mutants described above was not affected by the additional introduction of ACT3 or ACT5 mutations (Figure 4). Mouse C1q binding assay

[0365] Anti-CD154 antibodies with Fc variants (internal antibodies generated using the method described in Example 3) were distributed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and left to stand overnight at 4°C. After washing with PBST, the plate was blocked with PBST containing 0.5% BSA and 1x Block Ace: Blocking Reagent (DS Pharma) for 7 hours at 4°C. After washing the plate, 10% mouse plasma (Innovative Research) was dispensed onto the plate and left to stand overnight at 4°C. The plate was washed, and biotinylated anti-mouse C1q antibody (Hycult Biotech) was added to react for 1 hour at room temperature, and washing was performed. Streptavidin-HRP (Pierce) was added to react for 1 hour at room temperature, and washing was performed. Subsequently, ABTS ELISA HRP Substrate (KPL) was added. The signal was measured by a plate reader at a wavelength of 405 nm.The binding affinity of the antibody having a wild-type (WT) Fc region with mouse C1q was decreased by the introduction of LALA mutations in the Fc region, and the decreased binding affinity with mouse C1q was recovered through the additional introduction of KAES beyond the LALA mutations. The binding property of the mutants. Petition 870260064115, dated 06 / 29 / 2026, p. 159 / 485 151 / 153 LALA or LALA + KAES described above was not affected by the additional introduction of ACT3 or ACT5 mutations (Figure 5). Example 5: Biacore analysis for Fc variants that bind to FcyRs and FcRn

[0366] The binding of Fc variants to human or mouse FcyRs and human FcRn at pH 7.4 was determined at 25°C using the Biacore T200 instrument (GE Healthcare). All antibodies and FcyRs or FcRn were prepared in PBS-P pH 7.4 containing 50 mM Na-Phosphate, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. For the FcyR binding assay, anti-Histidine antibody (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using the amine coupling kit (GE Healthcare). Each of the FcyRs was captured in flow cell 2, 3, or 4 by the anti-Histidine antibody, with flow cell 1 as the reference flow cell. FcyR capture levels were targeted at 400 resonance units (RU). All antibodies were injected at 100 nM onto all flow cells.The immune complex was prepared by mixing a 1:1 molar ratio of antibody and trimeric CD154, and incubated at room temperature for one hour. The sensor surface was regenerated after each cycle using 10 mM Glycine-HCl, pH 1.5.

[0367] For the FcRn binding assay, Biotin CAPture Reagent (GE Healthcare) was immobilized on both flow cells 1 and 2 of a sensor chip using the Biotin CAPture kit (GE Healthcare). Biotinylated FcRn was captured in flow cell 2 with flow cell 1 as a reference flow cell. FcRn capture levels were targeted at 400 RU. All antibodies were injected at 100 nM onto flow cells 1 and 2. Immune complexes were prepared by mixing a 1:1 molar ratio of antibody and trimeric CD154, and incubated at room temperature for a Petition 870260064115, dated 06 / 29 / 2026, page 160 / 485 152 / 153 hour. The sensor surface was regenerated after each cycle using 8M Guanidine-HCl, 1M NaOH (3:1 vol / vol).

[0368] Binding levels were normalized with the capture level of corresponding FcyRs or FcRn. Binding of a single antibody or an immune complex (an antibody and a trimeric CD154 antigen) towards human or mouse FcyRs and human FcRn was monitored based on the binding response. The immune complex was used to assess enhanced binding towards FcyRs or FcRn through avidity effects. Results for human FcYR1a, FcYR2a 167H and 167R, FcYR2b, FcYR3a 158F and 158V, FcYR3b NA1 and NA2 are shown in Figures 6(a) to (h). Results for mouse FcyRI, FcYR2b, FcyR3, FcyR4 are shown in Figures 7(a) to (d). The binding of a wild-type Fc region (described as WT IgG) was significantly decreased by introducing LALA, LALA+KAES, or LALA+KWES mutations into the Fc region for each of the FcyRs tested.The trend was approximately the same between assays using the antibody alone (described as Ab alone) and the immune complex (described as CD154 IC). Binding of KAES or KWES mutants was not greatly reduced compared to that of WT IgG for most FcyRs tested. Results for human FcRn are shown in Figure 8. Binding of a wild-type Fc region (described as hIgG1) to human FcRn did not appear to be affected, even after the introduction of LALA or LALA+KAES mutations in the Fc region. Binding was slightly improved by the additional introduction of ACT3 or ACT5 mutations, but still remained comparatively low (Figure 8). Example 6: In vivo efficacy of anti-DENV antibodies against DENV infection.

[0369] AG129 mice aged 6 to 8 weeks were Petition 870260064115, dated 06 / 29 / 2026, p. 161 / 485 153 / 153 mice were infected intraperitoneally with 10⁶ plaque-forming units (pfu) of the DENV-2 D2Y98P strain. 48 hours later, the mice were treated with 25 pg of antibody in PBS. The antibody was injected intravenously via the retro-orbital route. A further 24 hours later, i.e., 72 hours after the initial infection, blood was collected. In previous studies (Zust et al, J Virol (2014) 88, 72767285; Tan et al, PLOS Negl Trop Dis (2010) 4, e672), it was established that peak viremia after infection with D2Y98P is reached between day 3 and 4 post-infection. Viral RNA was extracted from the plasma of each mouse and a quantitative PCR was performed and compared against a DENV-2 standard with known contamination potential in a plaque assay. Both 3C and 3Cam antibodies greatly reduced viremia compared to the PBS control in this mouse model, and the efficacy of both antibodies was comparable.The antibody with the LALA + KAES mutation in the Fc region showed stronger efficacy compared to the antibody with only the LALA mutation. This was the case for both 3C and 3Cam antibodies (Figure 9). This result indicates that the recovery of C1q binding activity by adding the KAES mutation to the LALA mutation contributes to the antiviral efficacy of the antibodies.

[0370] Although the preceding invention has been described in some detail by way of illustration and example for the purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. Disclosures of all patents and scientific literature cited herein are expressly incorporated in their entirety by reference. Petition 870260064115, dated 06 / 29 / 2026, page 162 / 485

Claims

1 / 2 CLAIMS 1. Polypeptide, characterized in that it comprises a variant Fc region comprising at least one amino acid change in an original Fc region, wherein the variant Fc region has substantially decreased FcyR binding activity and does not have substantially decreased C1q binding activity when compared with the original Fc region.

2. Polypeptide, according to claim 1, characterized in that the variant Fc region comprises Ala at position 234, Ala at position 235 and additional amino acid changes at any of the following positions (a)-(c): (a) positions 267, 268 and 324; (b) positions 236, 267, 268, 324 and 332; and (c) positions 326 and 333; according to EU numbering. 3.Polypeptide according to claim 2, characterized in that the variant Fc region comprises 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.

4. Polypeptide according to any one of claims 1 to 3, characterized in that the variant Fc region further comprises amino acids selected from the group consisting of: Petition 870260064115, dated 29 / 06 / 2026, p. 163 / 485. 2 / 2 (a) Wing in position 434; (b) Wing in position 434, Thr in position 436, Arg in position 438 and Glu in position 440; (c) Leu in position 428, Wing in position 434, Thr in position 436, Arg in position 438 and Glu in position 440; and (d) Leu in position 428, Wing in position 434, Arg in position 438 and Glu in position 440; according to EU numbering.

5. Polypeptide, according to any one of claims 1 to 4, characterized in that it comprises the amino acid sequence of any one of the SEQ ID NOS: 51 to 59.

6. Polypeptide, according to any one of claims 1 to 5, characterized in that the polypeptide is an antibody.

7. Pharmaceutical formulation, characterized in that it comprises the polypeptide, as defined in any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

8. Process for preparing an antibody, characterized in that the process comprises the steps of: (a) combining a VH sequence with a human IgG Fc variant sequence of any of the SEQ ID NOs: 51-59; (b) combining a VL sequence; (c) cloning each of the combinations into an expression vector; (d) expressing the resulting expression vectors in co-transfected cells; and (e) purifying the antibody resulting from step (d). Petition 870260064115, dated 06 / 29 / 2026, p. 164 / 485

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