WO2022139680A1

WO2022139680A1 - Sars-cov-2 binding molecules and uses thereof

Info

Publication number: WO2022139680A1
Application number: PCT/SG2021/050801
Authority: WO
Inventors: Taichi Kuramochi; Nagisa KAGEJI; Siok Wan GAN; Cheng-I Wang; Bei Wang; Wen-Hsin Lee
Original assignee: Chugai Seiyaku Kabushiki Kaisha; Agency For Science, Technology And Research
Priority date: 2020-12-21
Filing date: 2021-12-17
Publication date: 2022-06-30
Also published as: TW202241942A

Abstract

The present disclosure provides affinity matured SARS-CoV-2 antibodies with improved physiochemical property derived from a parent antibody that binds to the RBD domain of the SARS-CoV-2 spike protein. Said antibodies can also bind to SARS-CoV-2 protein muteins and have increased solubility. It further discloses a pharmaceutical composition comprising said antibodies and the use of said antibodies for treating and preventing SARS-CoV-2 infection, and for diagnosis of SARS-CoV-2.

Description

SARS-COV-2 BINDING MOLECULES AND USES THEREOF

[Technical Field]

The present disclosure relates to SARS-CoV-2 binding molecules and methods of using the same.

[Background Art]

Antibodies are potent therapeutics as both the Fab and Fc parts of the antibody can be harnessed to neutralize a target. After an antibody binds to its target via the Fab region, the Fc region can recruit molecules such as complement molecules or Fc receptors to further activate the immune system. The target will then be eliminated by mechanisms such as complementdependent cytotoxicity (CDC), antibody -dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

Complement-dependent cytotoxicity (CDC) is mediated by the "classical" complement pathway, which is a cascade of enzymatic reactions involving complement proteins Cl through C9. Activation of the classical pathway is first triggered when complement Clq binds to the antibody Fc. Complement protein Clq is a large protein complex consisting of six globular heads and a collagen-like tail, and each globular head is able to interact with an antibody Fc. As the affinity of an individual globular head in Clq for an antibody Fc is weak, Clq binds only weakly to monomeric IgG, and does not activate the classical pathway. This weak affinity is essential for homeostasis as the concentrations of Clq and antibodies in blood are high. However, when a target is densely coated with antibodies, Clq is able to engage multiple Fes and bind with high avidity, and thus activating the complement pathway. Activation of the classical pathway cascade leads to deposition of proteins such as complement C4b and C3b onto the surface of the target. C4b and C3b mark the target for phagocytic uptake by cells that express complement receptors such as CR1 through CR4 and CRIg. From C3b, the classical pathway cascade also progresses further and results in the deposition of C5b, C6, C7, C8 and C9 proteins, which assemble to become a pore-forming C5b-9 membrane- attack complex (MAC). The formation of MAC on the target surface disrupts the membrane integrity and ultimately leads to target lysis. Antibody-mediated CDC activity has been long known for its ability to mediate the killing of bacteria, and was discovered in 1895 by Jules Bordet. More recently, antibody mediated CDC activity has also been described to mediate the clearance of several different types of viruses (Front Microbiol. 2017; 8: 1117).

Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are mediated by interactions between the antibody Fc and cells expressing Fc gamma receptors. In ADCC, effector cytotoxic cells such as natural killer cells recognize antibody-bound targets and release lytic enzymes to destroy the target. In ADCP, phagocytes such as macrophages, monocytes, and neutrophils take up the antibody opsonized targets and clear them from the circulation. Although ADCP is an important process for protective immunity, some pathogens exploit the ability of ADCP to enhance their infectivity in a process known as antibody -dependent enhancement (ADE). ADE occurs when antibodies enhance pathogen entry into host cells via Fc gamma receptors. This occurs when antibody titres are insufficient to neutralize the pathogen, or when the antibodies against the pathogen are non-neutralizing in nature. To avoid therapeutically administered antibodies from having ADE function, mutations are introduced into the Fc region to reduce or silence Fc gamma receptor binding functions. Alternatively, Fc subclasses with naturally weak Fc gamma receptor binding function such as IgG2 and IgG4 are used.

The ability of sub-neutralizing antibodies to promote disease via ADE in humans has been well documented for Dengue viruses. Although there is no definitive evidence for ADE in human coronavirus diseases in vivo at the present moment, there is in vitro evidence that ADE can occur. Yip et. al. (Yip et. al. Virol. J. 11, 82 (2014)) showed that human macrophages can be infected by SARS-CoV as a result of IgG-mediated ADE, and that this infection route requires binding and signaling through FcyRII receptors. Similarly, Wan et. al. (Wan et. al. J. Virol. 94, e02015-19 (2020)) showed that an antibody against the receptor binding domain (RBD) of the Middle East respiratory syndrome (MERS) coronavirus spike could enhance the viral entry.

Some antibodies binding to SARS-CoV-2 have been reported (Hansen et. al. Science. Vol. 369, Issue 6506, pp. 1010-1014). [Introduction]

However, it remains a challenge to design a SARS-CoV-2-binding molecule with sufficient binding affinity to and/or physicochemical property suitable for treating or preventing or reducing an incidence of viral infections, e.g., coronavirus infections, and furthermore, to design a SARS-Cov-2-binding molecule having substantially decreased Fc gamma receptor binding and maintained or increased complement Clq-binding activity. Generating such a molecule is one of potential strategy to reduce the risk of antibodydependent enhancement (ADE) in coronavirus infections, caused by the virus entry into host cells via Fc gamma receptors, while having enough clearance of the virus of interest. The risk of ADE may arise from antibodies against coronavirus strains endemic in human populations, such as HKU1, OC43, NL63 and 229E. Cross-reactive antibodies that weakly bind to SARS- CoV-2 may have the potential to mediate ADE.

Thus, there is a need to provide alternative SARS-CoV-2 binding molecules, e.g., SARS-CoV-2 binding molecules having favourable properties, and related methods.

[Summary]

The present disclosure provides the following:

[1] An isolated antibody that binds to SARS-CoV-2, wherein the antibody comprises:

(i) HVR-H1 comprising the amino acid sequence XiYEMN, wherein Xi is L, I or S (SEQ ID NO: 109),

(ii) HVR-H2 comprising the amino acid sequence VISYXiGSNKYYADSVKG, wherein Xi is E or D (SEQ ID NO: 110),

(iii) HVR-H3 comprising the amino acid sequence LITMX1RGX2X3X4, wherein Xi is T or V, X₂ is P or A, X₃ is D or Q, X₄ is Y or G (SEQ ID NO: 111),

(iv) HVR-L1 comprising the amino acid sequence RASQX1IX2X3YLN, wherein Xi is S or E, X₂ is S or E, X₃ is S or D (SEQ ID NO: 112),

(v) HVR-L2 comprising the amino acid sequence AAX1X2LQX3, wherein Xi is S or E, X₂ is S or E, X₃ is I or G (SEQ ID NO: 113), and

(vi) HVR-L3 comprising the amino acid sequence QXiSYNLPRT, wherein Xi is E or Q (SEQ ID NO: 114). [2] The antibody of [1], wherein the antibody comprises:

(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61, 64, 67, 70, 73 or 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, 65, 68, 71, 74 or 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:79, 82, 85, 88, 91, 94, 97, 100, 103 or 106, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 86, 89, 92, 95, 98, 101, 104 or 107, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108.

[3] An isolated antibody that binds to SARS-CoV-2, wherein the antibody comprises:

(a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 88, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 90;

(b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:91, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93;

(c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 87;

(d) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:94, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 95, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 96;

(e) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 88, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 90; (f) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:91, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93;

(g) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 87;

(h) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:94, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 95, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 96;

(i) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 88, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 90;

(j) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:91, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93;

(k) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 87;

(l) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:94, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 95, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 96;

(m) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 98, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 99;

(n) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 64, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 65, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(o) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(p) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83 (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(q) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(r) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 100, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 102; (s) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 98, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 99;

(t) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 103, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 104, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 105;

(u) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 107, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 108;

(v) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:79, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81;

(w) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:79, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81; or

(x) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 64, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 65, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 100, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 102. [4] The antibody of any one of [1] to [3], which is a monoclonal antibody.

[5] The antibody of any one of [1] to [4], which is a human, humanized, or chimeric antibody.

[6] The antibody of any one of [1] to [5], comprising (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2 to 7; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 42 and 44 to 52; or (c) the VH sequence as in (a) and the VL sequence as in

(b).

[7] The antibody of [6], comprising a VH sequence of any one of SEQ ID NOs: 2 to 7.

[8] The antibody of [6] or [7], comprising a VL sequence of any one of SEQ ID NOs: 42 and 44 to 52.

[9] An isolated antibody that binds to SARS-CoV-2, wherein the antibody comprises:

(a) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 46,

(b) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 47,

(c) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 45,

(d) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 48,

(e) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 46,

(f) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 47,

(g) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 45,

(h) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 48,

(i) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 46,

(j) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 47,

(k) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 45,

(l) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 48,

(m) a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 49,

(n) a VH sequence of SEQ ID NO: 3 and a VL sequence of SEQ ID NO: 44,

(o) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 44,

(p) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 44,

(q) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 44,

(r) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 50,

(s) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 49,

(t) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 51,

(u) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 52,

(v) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 42, (w) a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 42, or

(x) a VH sequence of SEQ ID NO: 3 and a VL sequence of SEQ ID NO: 50,

[10] The antibody of any one of [1] to [9], wherein the antibody further comprises a variant Fc region comprising at least one amino acid alteration to a parent Fc region, wherein, when compared to the parent Fc region, the variant Fc region has a substantially decreased Fc gamma R-binding activity and has a maintained or increased Clq-binding activity.

[11] The antibody of [10], wherein the variant Fc region comprises Ala at position 234 according to EU numbering and Ala at position 235 according to EU numbering.

[12] The antibody of [10] or [11], wherein the variant Fc region comprises amino acid alterations at positions of any one of the following (a)-(c):

(a) positions 267, 268, and 324;

(b) positions 236, 267, 268, 324, and 332; and

(c) positions 326 and 333, wherein the positions are according to EU numbering.

[13] The antibody of [12], wherein the variant Fc region comprises an amino acid(s) selected from the group consisting of (a) to (g) below:

(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, wherein the positions are according to EU numbering.

[14] The antibody of any one of [10] to [13], wherein the variant Fc region comprises an amino acid(s) selected from the group consisting of (a) to (f) below:

(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, and Thr at position 436

(d) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440;

(e) Leu at position 428, and Ala at position 434; and (f) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440; wherein the positions are according to EU numbering.

[15] A pharmaceutical composition comprising the antibody of any one of [1] to [14], and a pharmaceutically acceptable carrier.

[16] The antibody of any one of [1] to [14] or the pharmaceutical composition of [15] for use in treatment and/or prevention of a SARS-CoV-2 infection.

[17] A method for treating a SARS-CoV-2 infection, comprising administering to an individual having the SARS-CoV-2 infection an effective amount of the antibody of any one of [1] to [14] or the pharmaceutical composition of [15].

[18] Use of the antibody of any one of [1] to [14] or the pharmaceutical composition of [15] for the manufacture of a medicament for treatment and/or prevention of a SARS-CoV-2 infection.

[19] Use of the antibody of any one of [1] to [14] or the pharmaceutical composition of [15] for treatment and/or prevention of a SARS-CoV-2 infection.

[20] An isolated nucleic acid encoding the antibody of any one of [1] to [14].

[21] A host cell or vector comprising the nucleic acid of [20].

[22] A method of producing the antibody of any one of [1] to [14], comprising culturing the host cell of [21].

[23] An isolated antibody produced by the method of [22].

[24] A kit comprising the composition of [15], and a package insert comprising instructions for administering to a subject to treat or prevent a SARS-CoV-2 infection.

[25] A method of detecting the presence of SARS-CoV-2 in a sample, the method comprising: contacting the sample with the antibody of any one of [1] to [14] under conditions permissive for binding of the antibody to SARS-CoV-2; detecting whether a complex is formed between the antibody and SARS-CoV-2; wherein the formation of a complex is indicative of the presence of SARS-CoV-2 in the sample.

[Brief Description of Drawings]

[FIG.l] FIG. 1 illustrates plasma concentration-time profiles of 5A6CCS1-SG1095ACT3 and 5A6CCS1-SG1095 in human FcRn transgenic mice administrated with IVIG after intravenous administration. [FIG.2] FIG. 2 illustrates a relative binding of RBD mutants to RBD wildtype for 5A6 and 5A6CCS1.

[FIG.3-1] FIG. 3-1 illustrates neutralization of SARS-CoV-2 pseudovirus by parental 5A6 and engineered 5A6CCS1 antibodies.

[FIG.3-2] FIG. 3-2 illustrates neutralization of SARS-CoV-2 pseudovirus by parental 5A6 and engineered 5A6CCS1 antibodies.

[FIG.4] FIG. 4 illustrates lung viral titer of hamsters infected with live SARS-CoV-2 virus after treatment with 5A6CCS1 antibody.

[FIG.5] FIG. 5 illustrates neutralization of SARS-CoV-2 pseudoviruses of various VOCs and VOIs (Beta, Gamma, Kappa, Delta and Epsilon) by parental 5A6 and engineered 5A6CCS1 antibodies.

[Description of Embodiments]

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

(M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:

Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

The definitions and detailed description below are provided to facilitate understanding of the present disclosure illustrated herein.

I. DEFINITIONS

Alteration of Amino Acids

Amino acid alteration means any of substitution, deletion, addition, and insertion, or a combination thereof. In the present disclosure, amino acid alteration may be rephrased as amino acid mutation or amino acid modification. For amino acid alteration in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR may be appropriately employed. Furthermore, several known methods may also be employed as amino acid alteration methods for substitution to non-natural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). For example, it is suitable to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA which has a non-natural amino acid bound to a complementary amber suppressor tRNA of one of the stop codons, the UAG codon (amber codon).

In the present specification, the meaning of the term “and/or” when describing the site of amino acid alteration includes every combination where “and” and “or” are suitably combined. Specifically, for example, “the amino acids at positions 33, 55, and/or 96 are substituted” includes the following variation of amino acid alterations: amino acid(s) at (a) position 33, (b) position 55, (c) position 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) positions 33, 55, and 96.

Furthermore, herein, as an expression showing alteration of amino acids, an expression that shows, at the left and the right of a number indicating a specific position, one-letter or three-letter codes for amino acids before and after alteration, respectively, may be used appropriately. For example, the alteration NIOObL or AsnlOObLeu used when substituting an amino acid contained in an antibody variable region indicates substitution of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number shows the amino acid position according to Kabat numbering, the one-letter or three-letter amino-acid code written before the number (at the left of the number) shows the amino acid before substitution, and the one-letter or three-letter amino-acid code written after the number (at the right of the number) shows the amino acid after substitution. Similarly, the alteration P238D or Pro238Asp used when substituting an amino acid of the Fc region contained in an antibody constant region indicates substitution of Pro at position 238 (according to EU numbering) with Asp. That is, the number shows the amino acid position according to EU numbering, the one-letter or three- letter amino-acid code written before the number (at the left of the number) shows the amino acid before substitution, and the one-letter or three-letter amino-acid code written after the number (at the right of the number) shows the amino acid after substitution.

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule or an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antigen-binding molecule and antigen, or antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd (KD)). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following. The structure of the antigen-binding domain of an antigen-binding molecule/antibody that binds to the epitope is called paratope. The paratope stably binds to the epitope through a hydrogen bond, electrostatic force, van der Waals’ forces, a hydrophobic bond, or the like acting between the epitope and the paratope. This binding force between the epitope and the paratope is called “affinity” (see also above). The total binding force when a plurality of antigen binding domains bind to a plurality of antigens is called “avidity”. The affinity works synergistically when, for example, an antibody comprising a plurality of antigen binding domains (i.e., a polyvalent or a multivalent antibody) bind to a plurality of epitopes, and the avidity may be higher than the affinity.

An “affinity matured” antigen-binding molecule/antibody refers to an antigen-binding molecule/antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antigen-binding molecule/ parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antigen-binding molecule/antibody for antigen.

The terms “anti-SARS-CoV-2 antigen-binding molecule”, “anti-SARS-CoV-2 antibody”, “an antigen-binding molecule that binds to SARS-CoV-2” and “an antibody that binds to SARS-CoV-2” refer to an antigen-binding molecule/antibody that is capable of binding SARS-CoV-2 with sufficient affinity such that the antigen-binding molecule/antibody is useful as a diagnostic and/or therapeutic and/or prevention agent in targeting SARS-CoV-2. In one embodiment, the extent of binding of an anti-SARS-CoV-2 antigen-binding molecule or an anti-SARS-CoV-2 antibody to an unrelated, non-SARS-CoV-2 protein is less than about 10% of the binding of the antigen-binding molecule/antibody to SARS-CoV-2 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antigen-binding molecule/antibody that binds to SARS-CoV-2 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. 10’⁸ 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-SARS-CoV-2 antigen-binding molecule or an anti-SARS-CoV-2 antibody binds to an epitope of SARS-CoV-2 that is conserved among SARS-CoV-2 from different species.

Antigen-binding molecules binding to SARS-CoV-2 The term “antigen-binding molecule”, as used herein, refers to any molecule that comprises an antigen-binding site, antigen-bindng moiety, or any molecule that has binding activity to an antigen, and may further refer to molecules such as a peptide or protein having a length of about five amino acids or more. The peptide and protein are not limited to those derived from a living organism, and for example, they may be a polypeptide produced from an artificially designed sequence. They may also be any naturally-occurring polypeptide, synthetic polypeptide, recombinant polypeptide, and such. Scaffold molecules comprising a known stable conformational structure such as alpha/beta barrel as scaffold, and in which part of the molecule is made into an antigen-binding site, is also one embodiment of the antigenbinding molecule described herein.

In one aspect, the antigen-binding molecure described herein can be an antigen-binding molecule binding to SARS-CoV-2 (also referred to herein as a “SARS-CoV-2-binding molecule” or “SARS-CoV-2 binding molecule”).

In certain embodiments, the SARS-CoV2-binding molcule of the present disclosure is generally an antibody. In certain embodiments, the SARS-CoV-2-binding molecule of the present disclosure is “single chain Fv (scFv)”, “single chain antibody”, “Fv”, “single chain Fv 2 (scFv2)”, “Fab”, “F(ab’)2”, VHH, VL, VH, single domain antibody, or any antibody fragment.

In certain embodiments, the SARS-CoV2-binding molcule of the present disclosure binds to SARS-CoV-2 spike (S) protein. In some embodiments, the SARS-CoV2-binding molcule of the present disclosure binds to SARS-CoV-2 spike (S) protein receptor binding domain (RBD).

The term "antibody" herein 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 or antigenbinding fragments so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" and “antigen-binding fragment” refer 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 and antigen-binding 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); VHH; VL; VH; single domain antibody; multispecific antibodies formed from antibody fragments/ antigen-binding fragments and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region (HVR) of an antibody (e.g. an isolated complementarity determining region (CDR)). In some embodiments, antibody fragments or antigen binding fragments may retain at least about one, at least about two, at least about three, at least about four, at least about five or at least about six of the HVR/CDR regions/sequences of the antibody.

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

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

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG₂, IgGs, IgG₄, IgAi, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

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, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

“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: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. Dosages and administration of agent, e.g., a pharmaceutical formulation, of the present disclosure may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. See, for example, Mordenti and Rescigno, (1992) Pharmaceutical Research. 9:17-25; Morenti et al., (1991) Pharmaceutical Research. 8:1351- 1359; and Mordenti and Chappell, "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al. (eds) (Pergamon Press: NY, 1989), pp. 42-96. An effective amount of the active agent of the present disclosure to be employed will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the subject. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VE): FR1-H1(E1)-FR2-H2(E2)-FR3-H3(L3)-FR4.

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

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody -encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences 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 the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

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, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs: three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 3 l-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

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

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

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

An “individual” or “subject” 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 embodiments, the individual or subject is a human. The term “subject” includes patients and non-patients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition, such as a SARS-CoV-2 infection, while “non-patients” refer to individuals not suffering and are likely to not suffer from the medical condition. “Non-patients” include healthy individuals, non-diseased individuals and/or an individual free from the medical condition.

An "isolated" antibody is one which has been separated from a component of its natural environment. The term “isolated” does not necessarily mean the exclusion of artificial or synthetic mixtures with other components, or the presence of impurities, for example, due to incomplete purification. In particular, isolated antibodies are also meant to include those that are chemically synthesised or engineered. In various embodiments, an isolated antibody is obtained by removing or purifying it from its natural environment, by selection from an antibody source as a phage display library or a B-cell repertoire, by chemical synthesis and/or through use of antibody engineering techniques such as modification of sequences and/or structures of a parental antibody. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

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 ordinarily 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.

“Isolated nucleic acid encoding an anti-SARS-CoV-2 antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies composing the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

"Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

“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 with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the 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 in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU5 10087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B . It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term "pharmaceutical formulation" (also referred to herein as “pharemaceutical composition”) refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “SARS-CoV-2,” as used herein, refers to any native SARS-CoV-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length” unprocessed SARS- CoV-2 as well as any form of SARS-CoV-2 that results from processing in the cell. The term also encompasses naturally occurring variants of SARS-CoV-2, e.g., splice variants or allelic variants. An antigen-binding molecule of the disclosure may bind to any variants of SARS- CoV-2 proteins. The amino acid sequence of an exemplary SARS-CoV-2 is shown in NCBI Reference Sequence: NC_045512.2 (Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu- 1, complete genome). The amino acid sequence of an exemplary spike protein (surface glycoprotein) of SARS-CoV-2 is shown in NCBI Reference Sequence: YP_009724390.1. Examples of naturally occurring variants of SARS-CoV-2 include, but are not limited to, Alpha (e.g., B.l.1.7), Beta (e.g., B.1.351, B.1.351.2, B.1.351.3), Delta (e.g., B.1.617.2, AY.l, AY.2, AY.3), Epsilon (e.g., B.1.427 and B.1.429), Gamma (e.g., P.l, P.1.1, P.1.2), Kappa (e.g., B.1.617.1) and Lambda (e.g., C.37) variants. A variant of SARS-CoV-2 may share at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, at least about 99.91%, at least about 99.92%, at least about 99.93%, at least about 99.94%, at least about 99.95%, at least about 99.96%, at least about 99.97%, at least about 99.98% or at least about 99.99% sequence identity with the sequence shown in NCBI Reference Sequence: NC_045512.2 over its entire length. The disease caused by the coronavirus which was first reported in 2019 is called Coronavirus Disease 2019 (COVID-19).

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, reducing an incidence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the disclosure are used to delay development of a disease or to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to 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 framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6^th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating 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 operatively linked. Such vectors are referred to herein as "expression vectors." The expression vector can be introduced into a host cell by a method using a virus, an electroporation method, or the like, but the introduction of an expression vector is not limited to in vitro introduction, and it is also possible to directly introduce a vector into a body.

The terms "coupled" or "connected" as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

The term "and/or", e.g., "X and/or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of +/- 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value.

Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible subranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1.00% to 5.00% and also 1.0% to 5.0% and all their intermediate values (such as 1.01%, 1.02% . . . 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth/breadth of a range.

Additionally, when describing some embodiments, the disclosure may have disclosed a method and/or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features/characteristics discussed herein, one or more of these features/characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

II. COMPOSITIONS AND METHODS

In one aspect, the disclosure is based, in part, on SARS-CoV-2-binding molecules and uses thereof. In one aspect, the disclosure is based, in part, on anti-SARS-CoV-2 antibodies and uses thereof. Antibodies of the disclosure are useful, e.g., for the diagnosis or treatment of coronavirus infetions, especially for Coronavirus Disease 2019 (COVID- 19). Exemplary, nonlimiting embodiments of SARS-CoV-2-binding molecules, SARS-CoV-2 antibodies and uses are disclosed hereinafter.

A. Exemplary Anti-SARS-CoV-2 Antibodies

In one aspect, the disclosure provides isolated antibodies that bind to SARS-CoV-2 and/or SARS-CoV-2 spike protein and/or SARS-CoV-2 spike protein receptor binding domain (RBD).

In certain embodiments, an anti-SARS-CoV-2 antibody of the present disclosure blocks binding of SARS-CoV-2 to a host cell receptor. In certain embodiments, an anti-SARS-CoV-2 antibody of the present disclosure inhibits SARS-CoV-2 entry into a host cell. In further embodiments, an anti-SARS-CoV-2 antibody of the present disclosure inhibits the interaction between the Receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2).

In certain embodiments, an anti-SARS-CoV-2 antibody of the present disclosure binds to the spike protein of SARS-CoV-2. In further embodiments, an anti-SARS-CoV-2 antibody of the present disclosure binds to the SI domain of the spike protein of SARS-CoV-2. In further embodiments, an anti-SARS-CoV-2 antibody of the present disclosure binds to the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2.

In ceratin embodiments, an anti-SARS-CoV-2 antibody of the present disclosure exerts Complement dependent cytotoxicity (CDC) against a target virus, e.g., SARS-CoV-2. In ceratin embodiments, an anti-SARS-CoV-2 antibody of the present disclosure exerts the lysis of a target virus, e.g., SARS-CoV2, (virolysis) or reduction of the virus’ ability to infect cells by complement.

In certain embodiments, an anti-SARS-CoV-2 antibody of the present disclosure suppresses antibody-dependent enhancement (ADE) observed with conventional anti-SARS- CoV-2 antibodies.

In certain embodiments, an anti-SARS-CoV-2 antibody of the present disclosure binds to and/or neutralizes the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. In some embodiments, an anti-SARS-CoV-2 antibody of the present disclosure binds to and/or neutralizes one or more variants having a mutation in the RBD of the spike protein of SARS- CoV-2. For example, spike protein variants have been mentioned in a preliminary report (preprint) by Nelson-Sathi et al. (“Mutational landscape and in silico structure models of SARS-CoV-2 Spike Receptor Binding Domain reveal key molecular determinants for virushost interaction”; Cold Spring Harbor Laboratory, bioRxiv (the preprint server for biology); https://doi.or /10.1101/2020.05.02.Q71811). RBD variants (mutants) of SARS-CoV-2 spike protein may include, but are not limited to, mutations at amino acid positions P322, T323, P33O, F338, V341, A344, R346, A348, N354, S359, V367, N370, P384, A411, Q414, K417, A435, N439, K444, G446, L452, Y453, L455, 1468, E471, A475, G476, S477, T478, P479, N481, G482, V483, E484, G485, F486, N487, F490, Q493, S494, N501, Y508, E516, H519, A520, P521, and A522 from SARS-CoV-2 S protein of the Wuhan strain (Ref Seq: YP_009724390.1). Such mutants may include, but are not limited to: P322A, T323I, P33OS, F338L, V341I, A344S, R346K, A348S, N354D, N354S, S359N, V367F, N370S, P384L, P384S, A411S, Q414K, Q414R, K417N, A435S, N439K, K444R, G446V, L452R, Y453F, L455F, I468T, I468V, E471Q, A475V, G476S, S477G, S477I, S477N, T478I, P479S, N481D, G482S, V483A, V483F, V483I, E484D, E484K, E484Q, G485S, F486S, N487R, F490L, F490S, Q493L, S494P, N501Y, Y508H, E516Q, H519Q, A520S, A520V, P521R, P521S, A522S, and A522V. The above mutants and mutations may be used alone or in combination. The mutants and mutations may be combined with any amino acid modifications (such as addition, insertion, deletion, and substitution) within or outside the RBD. Examples of such combinations include, but are not limited to, K417N/E484K/D614G (herein called South Africa triple mutation) and del69-70/dell44- 145/N501Y/A570D/D614G/P681H/T716US982A/D1118H (herein called UK variant).

An anti-SARS-CoV-2 antibody of the present disclosure may bind to and/or neutralize any spike protein mutants, including the above mutants, with any amino acid mutations such as substitutions. In some embodiments, an anti-SARS-CoV-2 antibody of the present disclosure binds to and/or neutralizes SARS-CoV-2 S protein RBD mutants such as V483A and F490S. In some embodiments, an anti-SARS-CoV-2 antibody of the present disclosure binds to and/or neutralizes SARS-CoV-2 S protein mutants with a non-RBD mutation such D614G.

An anti-SARS-CoV-2 antibody of the present disclosure may have favourable physicochemical properties. In some embodiments, an anti-SARS-CoV-2 antibody of the present disclosure exhibits high solubility. In some embodiments, an anti-SARS-CoV-2 antibody of the present disclosure is substantially soluble when concentrated to about 5 mg/mL, e.g., a little or no visible particles were observed by visual inspection at the concentration.

An anti-SARS-CoV-2 antibody of the present disclosure may have favourable pharmacokinetic properties. In some embodiments, an anti-SARS-CoV-2 antibody of the present disclosure exhibits favourable exposure, clearance and/or half-life properties. In some embodiments, the exposure is from about 100 to about 800 pg/mL, or from about 200 to 600 pg/mL after 28 days administration. In some embodiments, the clearance is from about 0.5 to about 15 mL/day/kg or from about 2 to about 10 mL/day/kg. In some embodiments, the halflife is from about 1 to about 30 days, from about 5 to about 20 days or from about 8 to about 18 days.

In one aspect, the disclosure provides an anti-SARS-CoV-2 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: 61, 64, 67, 70, 73 or 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, 65, 68, 71, 74 or 77; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79, 82, 85, 88, 91, 94, 97, 100, 103 or 106; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 86, 89, 92, 95, 98, 101, 104 or 107; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108.

In one aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61, 64, 67, 70, 73 or 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, 65, 68, 71, 74 or 77; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, 65, 68, 71, 74 or 77. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61, 64, 67, 70, 73 or 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, 65, 68, 71, 74 or 77; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78.

In another aspect, the disclosure provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79, 82, 85, 88, 91, 94, 97, 100, 103 or 106; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 86, 89, 92, 95, 98, 101, 104 or 107; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79, 82, 85, 88, 91, 94, 97, 100, 103 or 106; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 86, 89, 92, 95, 98, 101, 104 or 107; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108. In another aspect, an antibody of the disclosure comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61, 64, 67, 70, 73 or 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, 65, 68, 71, 74 or 77, and (iii) HVR- H3 comprising an amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79, 82, 85, 88, 91, 94, 97, 100, 103 or 106, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 86, 89, 92, 95, 98, 101, 104 or 107, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108.

In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 88; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 90. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 91; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 93. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 87. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 94; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 95; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 96. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 88; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 90. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 91; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 93. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 87. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 94; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 95; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 96. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 88; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 90. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 91; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 93. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 87. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 94; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 95; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 96. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 97; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 98; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 99. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 64; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 65; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 84. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 84. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 84. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 84. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 100; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 102. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 97; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 98; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 99. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 103; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 104; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 105. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 106; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 107; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 108. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 81. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 81. In another aspect, the disclosure provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 64; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 65; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 100; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101; and (f) HVR- L3 comprising an amino acid sequence selected from SEQ ID NO: 102.

In another aspect, the disclosure provides an antibody that binds to SARS-CoV-2, wherein the antibody comprises at least one, two, three, four, five, or six HVRs selected from

(i) HVR-H1 comprising the amino acid sequence XiYEMN, wherein Xi is L, I or S (SEQ ID NO: 109), (ii) HVR-H2 comprising the amino acid sequence VISYXiGSNKYYADSVKG, wherein Xi is E or D (SEQ ID NO: 110), (iii) HVR-H3 comprising the amino acid sequence LITMX1RGX2X3X4, wherein Xi is T or V, X₂ is P or A, X₃ is D or Q, X₄ is Y or G (SEQ ID NO: 111), (iv) HVR-L1 comprising the amino acid sequence RASQX1IX2X3YLN, wherein Xi is S or E, X₂ is S or E, X3 is S or D (SEQ ID NO: 112), (v) HVR-L2 comprising the amino acid sequence AAX1X2LQX3, wherein Xi is S or E, X2 is S or E, X3 is I or G (SEQ ID NO: 113), and (vi) HVR-L3 comprising the amino acid sequence QXiSYNLPRT, wherein Xi is E or Q (SEQ ID NO: 114).

In another aspect, the disclosure provides an antibody that binds to SARS-CoV-2, wherein the antibody comprises:

(i) HVR-H1 comprising the amino acid sequence XiYEMN, wherein Xi is S, A, I, L, M, P, V, H, F, W or Y (SEQ ID NO: 115), (ii) HVR-H2 comprising the amino acid sequence VISYX1X2SNKX3YADSVKG, wherein XI is D, S, T or E, X₂ is G or A, X₃ is Y or F (SEQ ID NO: 116),

(iii) HVR-H3 comprising the amino acid sequence LITMX1RGX2X3X4, wherein Xi is V or T, X₂ is E, A, P,V, G, H, K or R, X₃ is D or Q, X₄ is Y, P, G or R (SEQ ID NO: 117),

(i) HVR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO: 118),

(ii) HVR-L2 comprising the amino acid sequence AASSLQXi, wherein Xi is S, I, L, M, G, or F (SEQ ID NO: 119), and

(iii) HVR-L3 comprising the amino acid sequence QXiSYNLPRT, wherein Xi is Q, N or E (SEQ ID NO: 120); provided that the antibody is not an antibody comprising HVR-H1, HVR-H2 and HVR-H3 comprised in H-chain variable region of SEQ ID NO: 1, and HVR-L1, HVR-L2 and HVR-L3 comprised in L-chain variable region of SEQ ID NO: 43.

In any of the above embodiments, an anti-SARS-CoV-2 antibody can be humanized or human. In one embodiment, an anti-SARS-CoV-2 antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework. In some embodiments, an isolated anti-SARS-CoV-2 antibody of the present disclosure further comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO: 121, 122, 123, or 124, FR2 comprising the amino acid sequence of SEQ ID NO: 125, FR3 comprising the amino acid sequence of SEQ ID NO: 126, FR4 comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, an isolated anti-SARS-CoV-2 antibody of the present disclosure further comprises a light chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO: 128, FR2 comprising the amino acid sequence of SEQ ID NO: 129, FR3 comprising the amino acid sequence of SEQ ID NO: 130, FR4 comprising the amino acid sequence of SEQ ID NO: 131.

In another aspect, an anti-SARS-CoV-2 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 to 7. 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 relative to the reference sequence, but an anti-SARS-CoV-2 antibody comprising that sequence retains the ability to bind to SARS-CoV-2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2 to 7. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-SARS-CoV-2 antibody comprises the VH sequence in SEQ ID NO: 2 to 7, including post-translational modifications of that sequence. In certain embodiments, the antibody mentioned above is not an antibody comprising HVR-H1, HVR-H2 and HVR-H3 comprised in H-chain variable region of SEQ ID NO: 1. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 61, 64, 67, 70, 73 or 76, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 62, 65, 68, 71, 74 or 77, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, 66, 69, 72, 75 or 78. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N- terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

In another aspect, an anti- SARS-CoV-2 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42, 44 to 52. 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-PRO antibody comprising that sequence retains the ability to bind to SARS-CoV-2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 42, 44 to 52. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-SARS-CoV-2 antibody comprises the VL sequence in SEQ ID NO: 42, 44 to 52, including post-translational modifications of that sequence. In certain embodiments, the antibody mentioned above is not an antibody comprising HVR-L1, HVR-L2 and HVR-L3 comprised in L-chain variable region of SEQ ID NO: 43. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 79, 82, 85, 88, 91, 94, 97, 100, 103 or 106; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 86, 89, 92, 95, 98, 101, 104 or 107; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90, 93, 96, 99, 102, 105 or 108. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N- terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation. In another aspect, an anti-SARS-CoV-2 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 SEQ ID NO: 2 to 7 and SEQ ID NO: 42, 44 to 52, respectively, including post- translational modifications of those sequences. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

In another aspect, an anti-SARS-CoV-2 antibody is provided, wherein the antibody that binds to SARS-CoV-2, wherein the antibody comprises: (a) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 46, (b) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 47, (c) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 45, (d) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 48, (e) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 46, (f) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 47, (g) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 45, (h) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 48, (i) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 46, (j) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 47, (k) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 45, (1) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 48, (m) a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 49, (n) a VH sequence of SEQ ID NO: 3 and a VL sequence of SEQ ID NO: 44, (o) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 44, (p) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 44, (q) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 44, (r) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 50, (s) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 49, (t) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 51, (u) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 52, (v) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 42, (w) a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 42, or (x) a VH sequence of SEQ ID NO: 3 and a VL sequence of SEQ ID NO: 50, including post-translational modifications of those sequences. Post-translational modifications include but are not limited to a modification of glutamine or glutamate in N-terminal of heavy chain or light chain to pyroglutamic acid by pyroglutamylation In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 4; (b) a CH sequence comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 44; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequence comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 45; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60.

In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 46; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 47; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 48; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 6; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 46; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 6; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 47; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 6; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 45; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 6; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 48; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 7; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 46; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 7; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 47; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 7; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 45; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 7; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 48; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 2; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 49; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 3; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 44; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 7; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 44; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 6; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 44; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 4; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 50; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 49; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 51; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 52; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 5; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 42; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 2; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 42; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60. In another aspect, the disclosure provides an antibody comprising (a) a VH sequence comprising the amino acid sequence of SEQ ID NO: 3; (b) a CH sequnce comprising the amino acid sequence of SEQ ID NO: 40 or 41; c) a VL sequence comprising the amino acid sequence of SEQ ID NO: 50; and d) a CL sequence comprising the amino acid sequence of SEQ ID NO: 60.

In a further aspect, the disclosure provides an antibody that binds to the same epitope as an anti-SARS-CoV-2 antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as any antibodies described above. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of SARS-CoV-2, especially for SARS-CoV-2 spike protein receptor binding domain (RBD). In certain embodiments, an antibody is provided that binds to the same epitope as any one of antibodies described above, whrein the antibody comprises a variant Fc region comprising at least one amino acid alteration in a parent Fc region, wherein the variant Fc region has a substantially decreased Fc gamma R-binding activity and has a maintained or increased Clq- binding activity when compared to the parent Fc region.

In one aspect, the disclosure provids a SARS-CoV-2-binding molecule comprising: (i) an antigen-binding moiety which specifically binds to a SARS-CoV-2, and (ii) a variant Fc region comprising at least one amino acid alteration relative to a parent Fc region, wherein the variant Fc region has a substantially decreased Fc gamma R-binding activity and has a maintained or increased Clq-binding activity when compared to the parent Fc region, and wherein the antigen-binding molecule specifically binds to the same epitope as any one of antibodies described above.

In a further aspect of the disclosure, an anti-SARS-CoV-2 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, an anti-SARS-CoV-2 antibody is an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment. In another embodiment, the antibody can be a full length antibody, e.g., an intact antibody (e.g., a human IgGl, IgG2, IgG3, or IgG4) or other antibody class or isotype as defined herein.

In a further aspect, an anti-SARS-CoV-2 antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:

1. Antibody Affinity

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. 10’⁸ 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 affinity matured antibody provided herein has improved affinity (or lower Kd) and/or neutralizing power/potentcy for SARS-CoV-2 as compared to its parent antibody (e.g., an antibody having a H-chain variable region set forth in SEQ ID NO: 1 and a L-chain variable region set forth in SEQ ID NO: 43). In certain embodiments, an affinity matured antibody provided herein has an affinity for SARS-CoV-2 that is least about 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold or about 1000 fold higher than that of its parent antibody.

In certain embodiments, an antibody provided herein exhibits similar binding/binding affinity for different variants of SARS-CoV-2. In certain embodiments, an antibody provided herein exhibits similar binding/binding affinity for at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16 or at least about 17 variants of SARS-CoV-2. In certain embodiments, an antibody provided herein exhibits similar binding to RBD wildtype and RBD mutants. For example, antibody provided herein may show more than 0.5 relative binding to RBD wildtype for RBD mutants. In certain embodiments, an antibody provided herein exhibits similar binding to RBD wildtype for at least at about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15 or at least about 16 RBD mutants. In certain embodiments, an antibody provided herein is capable of neutralizing various Variants of Concern (VOCs) and/or Variants of Interest (VOIs). In certain embodiments, an antibody provided herein is capable of neutralizing the Beta, Gamma, Kappa, Delta and/or Epsilon variants of SARS-CoV-2.

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, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody- coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER (registered trademark) multi-well plates (Thermo Scientific) are coated overnight with 5 microgram (micro g)/ml of a capturing anti-Fab antibody (Cappel Fabs) 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 [¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment 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, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, 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 (registered trademark)) in PBS. When the plates have dried, 150 micro 1/well of scintillant (MICROSCINT-20 ™; Packard) is 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 maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE (registered trademark) surface plasmon resonance assay. For example, an assay using a BIACORE (registered trademark)-2000 or a BIACORE(registered trademark)-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25 degrees C with immobilized antigen CM5 chips at -10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with A-ethyl-TW- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and A-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 micro g/ml (~0.2 micro M) before injection at a flow rate of 5 micro 1/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25 degrees C at a flow rate of approximately 25 micro 1/min. Association rates (k_on) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/k_on. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M ¹ s ¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25 degrees C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or a 8000- series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment.

Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, VHH, VL, VH, single domain antibody 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, e.g., Pluckthiin, 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 U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; 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).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., 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 a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

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

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

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

272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

4. Human Antibodies

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

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB (registered trademark) technology; U.S. Patent No. 7,041,870 describing K-M MOUSE (registered trademark) technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE (registered trademark) technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

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

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-Derived Antibodies

Antibodies of the disclosure may 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 phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., 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, e.g., in the McCafferty et al., Nature 348:552- 554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as singlechain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

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

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecific antibody, e.g. 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 SARS-CoV-2 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of SARS-CoV-2. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express SARS-CoV-2. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multi- specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547- 1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006/0025576A1). The antibody or fragment herein also includes a “Dual Acting Fab” or “DAF” comprising an antigen binding site that binds to SARS-CoV-2 as well as another, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

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 may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. 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, e.g., antigen-binding. a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[TABLE 1]

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(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.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may, for example, be outside of antigen contacting residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a 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 insertional variants of the antibody molecule include the fusion of an enzyme (e.g. for ADEPT) or a polypeptide which increases the plasma half-life of the antibody to the N- or C-terminus of the antibody. b) Glycosylation variants

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

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the disclosure may be made in order to create antibody variants with certain improved properties.

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 antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 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 Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about +/- 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose- deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; 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 Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-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 attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). c) Fc region variants

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 (also referred to herein as a “variant Fc region”). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

Clq

"Clq" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. Clq together with two serine proteases, Clr and Cis, forms the complex Cl, the first component of the complement dependent cytotoxicity (CDC) pathway. Human Clq can be purchased commercially from, e. g. from Quidel, San Diego, CA.

"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 (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with an Fc region variant) and increased or decreased Clq binding capability are described, e.g., in US Patent No. 6,194,551 B l and WO 1999/51642. See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000). “Complement dependent cytotoxicity” or “CDC” herein may further refer to the lysis of a target virus (virolysis) or reduction of the virus’ ability to infect cells by complement. Methods for assessing complement dependent lysis or complement dependent reduction of virus infectivity are widely known in the art, such as the use of heat inactivated serum or serum depleted of complement components. Examples of complement dependent virolysis or inactivation are detailed in Springer Semin Immunopathol. 1983; 6(4): 327-347.

"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: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. The phrase "substantially decreased", "substantially increased", or "substantially different", as used herein, refers to a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill 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 said values (e.g., Kd values).

The term "Fc region" or “Fc domain” herein 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, the term “Fc region” or “Fc domain” comprises a fragment consisting of a hinge or a portion thereof and CH2 and CH3 domains in an antibody molecule. The Fc region of IgG class means, but is not limited to, a region from, for example, cysteine 226 (EU numbering (also referred to as EU index herein)) to the C terminus or proline 230 (EU numbering) to the C terminus. 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, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The Fc region can be preferably obtained by the partial digestion of, for example, an IgGl, IgG2, IgG3, or IgG4 monoclonal antibody with a proteolytic enzyme such as pepsin followed by the re-elution of a fraction adsorbed on a protein A column or a protein G column. Such a proteolytic enzyme is not particularly limited as long as the enzyme is capable of digesting a whole antibody to restrictively form Fab or F(ab')2 under appropriately set reaction conditions (e.g., pH) of the enzyme. Examples thereof can include pepsin and papain.

An Fc region derived from, for example, naturally occurring (wild type) IgG can be used as the "Fc region" of an antigen-binding molecule. In this context, the naturally occurring IgG means a polypeptide that contains an amino acid sequence identical to that of IgG found in nature and belongs to a class of an antibody substantially encoded by an immunoglobulin gamma gene. The naturally occurring human IgG means, for example, naturally occurring human IgGl, naturally occurring human IgG2, naturally occurring human IgG3, or naturally occurring human IgG4. The naturally occurring IgG also includes variants or the like spontaneously derived therefrom. A plurality of allotype sequences based on gene polymorphism are described as the constant regions of human IgGl, human IgG2, human IgG3, and human IgG4 antibodies in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, any of which can be used in the present disclosure. Particularly, the sequence of human IgGl may have DEL or EEM as an amino acid sequence of EU numbering positions 356 to 358.

Fc receptor

The term "Fc receptor" or “FcR” refers to 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 which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc gamma RI, Fc gamma RII, and Fc gamma RIII subclasses, including allelic variants and alternatively spliced forms of those receptors. Fc gamma RII receptors include Fc gamma RIIA (an "activating receptor") and Fc gamma RUB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor Fc gamma RIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. Inhibiting receptor Fc gamma RUB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25- 34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

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 homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., 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.).

Binding to human FcRn in vivo and plasma half-life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with an Fc region variant are administered. WO 2000/42072 (Presta) describes antibody variants with increased or decreased binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

Fc gamma receptor

Fc gamma receptor refers to a receptor capable of binding to the Fc domain of monoclonal IgGl, IgG2, IgG3, or IgG4 antibodies, and includes all members belonging to the family of proteins substantially encoded by an Fc gamma receptor gene. In human, the family includes Fc gamma RI (CD64) including isoforms Fc gamma Ria, Fc gamma Rib and Fc gamma RIc; Fc gamma RII (CD32) including isoforms Fc gamma Rlla (including allotype H131 and R131), Fc gamma Rllb (including Fc gamma RIIb-1 and Fc gamma RIIb-2), and Fc gamma Rile; and Fc gamma RIII (CD16) including isoform Fc gamma Rllla (including allotype V158 and F158) and Fc gamma Rlllb (including allotype Fc gamma RIIIb-NAl and Fc gamma RIIIb-NA2); as well as all unidentified human Fc gamma receptors, Fc gamma receptor isoforms, and allotypes thereof. However, Fc gamma receptor is not limited to these examples. Without being limited thereto, Fc gamma receptor includes those derived from humans, mice, rats, rabbits, and monkeys. Fc gamma receptor may be derived from any organisms. Mouse Fc gamma receptor includes, without being limited to, Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD16), and Fc gamma RIII-2 (CD16-2), as well as all unidentified mouse Fc gamma receptors, Fc gamma receptor isoforms, and allotypes thereof. Such preferred Fc gamma receptors include, for example, human Fc gamma RI (CD64), Fc gamma RIIA (CD32), Fc gamma RIIB (CD32), Fc gamma RIIIA (CD16), and/or Fc gamma RIIIB (CD 16). The polynucleotide sequence and amino acid sequence of Fc gamma RI are shown in RefSeq accession number NM_000566.3 and RefSeq accession number NP_000557.1, respectively; the polynucleotide sequence and amino acid sequence of Fc gamma RIIA are shown in RefSeq accession number BC020823.1 and RefSeq accession number AAH20823.1, respectively; the polynucleotide sequence and amino acid sequence of Fc gamma RIIB are shown in RefSeq accession number BC 146678.1 and RefSeq accession number AAI46679.1, respectively; the polynucleotide sequence and amino acid sequence of Fc gamma RIIIA are shown in RefSeq accession number BC033678.1 and RefSeq accession number AAH33678.1, respectively; and the polynucleotide sequence and amino acid sequence of Fc gamma RIIIB are shown in RefSeq accession number BC 128562.1 and RefSeq accession number AAI28563.1, respectively. Whether an Fc gamma receptor has binding activity to the Fc domain of a monoclonal IgGl, IgG2, IgG3, or IgG4 antibody can be assessed by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR)-based BIACORE method, and others (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010), in addition to the above-described FACS and ELISA formats.

Meanwhile, “Fc ligand” or “effector ligand” refers to a molecule and preferably a polypeptide that binds to an antibody Fc domain, forming an Fc/Fc ligand complex. The molecule may be derived from any organisms. The binding of an Fc ligand to Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fc gamma receptor, Fc alpha receptor, Fc beta receptor, FcRn, Clq, and C3, mannan-binding lectin, mannose receptor, Staphylococcus Protein A, Staphylococcus Protein G, and viral Fc gamma receptors. The Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), which are a family of Fc receptors homologous to Fc gamma receptor. The Fc ligands also include unidentified molecules that bind to Fc.

Fc gamma receptor-binding activity

The impaired binding activity of Fc domain to any of the Fc gamma receptors Fc gamma RI, Fc gamma RIIA, Fc gamma RUB, Fc gamma RIIIA, and/or Fc gamma RIIIB can be assessed by using the above-described FACS and ELISA formats as well as ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and surface plasmon resonance (SPR)-based BIACORE method (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

ALPHA screen is performed by the ALPHA technology based on the principle described below using two types of beads: donor and acceptor beads. A luminescent signal is detected only when molecules linked to the donor beads interact biologically with molecules linked to the acceptor beads and when the two beads are located in close proximity. Excited by laser beam, the photosensitizer in a donor bead converts oxygen around the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor beads and reaches the acceptor beads located in close proximity, a chemiluminescent reaction within the acceptor beads is induced. This reaction ultimately results in light emission. If molecules linked to the donor beads do not interact with molecules linked to the acceptor beads, the singlet oxygen produced by donor beads do not reach the acceptor beads and chemiluminescent reaction does not occur. For example, a biotin-labeled antigen-binding molecule or antibody is immobilized to the donor beads and glutathione S-transferase (GST)-tagged Fc gamma receptor is immobilized to the acceptor beads. In the absence of an antigen-binding molecule or antibody comprising a competitive mutant Fc domain, Fc gamma receptor interacts with an antigenbinding molecule or antibody comprising a wild-type Fc domain, inducing a signal of 520 to 620 nm as a result. The antigen-binding molecule or antibody having a non-tagged mutant Fc domain competes with the antigen-binding molecule or antibody comprising a wild-type Fc domain for the interaction with Fc gamma receptor. The relative binding affinity can be determined by quantifying the reduction of fluorescence as a result of competition. Methods for biotinylating the antigen-binding molecules or antibodies such as antibodies using Sulfo- NHS-biotin or the like are known. Appropriate methods for adding the GST tag to an Fc gamma receptor include methods that involve fusing polypeptides encoding Fc gamma receptor and GST in-frame, expressing the fused gene using cells introduced with a vector carrying the gene, and then purifying using a glutathione column. The induced signal can be preferably analyzed, for example, by fitting to a one- site competition model based on nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

One of the substances for observing their interaction is immobilized as a ligand onto the gold thin layer of a sensor chip. When light is shed on the rear surface of the sensor chip so that total reflection occurs at the interface between the gold thin layer and glass, the intensity of reflected light is partially reduced at a certain site (SPR signal). The other substance for observing their interaction is injected as an analyte onto the surface of the sensor chip. The mass of immobilized ligand molecule increases when the analyte binds to the ligand. This alters the refraction index of solvent on the surface of the sensor chip. The change in refraction index causes a positional shift of SPR signal (conversely, the dissociation shifts the signal back to the original position). In the Biacore system, the amount of shift described above (i.e., the change of mass on the sensor chip surface) is plotted on the vertical axis, and thus the change of mass over time is shown as measured data (sensorgram). Kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the curve of sensorgram, and affinity (KD) is determined from the ratio between these two constants. Inhibition assay is preferably used in the BIACORE methods. Examples of such inhibition assay are described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

Fc region variants (or variant Fc regions/Fc domain variants/variant Fc domains) In one aspect, the Fc region of the present disclosure comprises a Fc region variant comprising at least one amino acid alteration (or mutation or modification, including substitution) relative to a parent Fc region. Such an Fc region variant may also be called “variant Fc region”, “Fc domain variant”, or “variant Fc domain” in this disclosure.

In certain embodiments, one or more amino acid alterations (mutations or modifications, including amino acid substitutions, deletions, and insertions) may be introduced into an Fc region of an antibody (a parent Fc region), thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions. For instance, the heavy chain constant region of human IgGl, human IgG2, human IgG3, and human IgG4 are shown in SEQ ID NOs: 132 to 135, respectively. For instance, the Fc region of human IgGl, human IgG2, human IgG3, and human IgG4 are shown as a partial sequence of SEQ ID NOs: 132 to 135.

In certain embodiments, the present disclosure contemplates an antigen-binding molecule that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet 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 has Fc gamma R binding (hence likely having ADCC activity) and/or FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc gamma RIII only, whereas 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 to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 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, ACT1TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95: 652-656 (1998). Clq binding assays may also be carried out to confirm whether the antibody is able to bind Clq and hence has CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Known methods for assessing complement dependent lysis or complement dependent reduction of virus infectivity, such as the use of heat inactivated serum or serum depleted of complement components, may also be used to assess Clq binding/complement activation. FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., IntT. Immunol. 18( 12): 1759- 1769 (2006)).

In certain embodiments, the Fc region variants (or variant Fc region (s)) comprise 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 herein as a "parent" Fc region). The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with a parent Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

A "parent Fc region" as used herein refers to an Fc region prior to the introduction of an amino acid alteration(s) described herein. Preferred examples of the parent Fc region include Fc regions derived from native antibodies. Antibodies include, for example, IgA (IgAl, IgA2), IgD, IgE, IgG (IgGl, IgG2, IgG3, IgG4), and IgM, or such. Antibodies may be derived from human or monkey (e.g., cynomolgus, rhesus macaque, marmoset, chimpanzee, or baboon). Native antibodies may also include naturally-occurring mutations. A plurality of allotype sequences of IgGs due to genetic polymorphism are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, and any of them may be used in the present disclosure. In particular, for human IgGl, the amino acid sequence at positions 356 to 358 (EU numbering) may be either DEL or EEM. Preferred examples of the parent Fc region include Fc regions derived from a heavy chain constant region of human IgGl (SEQ ID NO: 132), human IgG2 (SEQ ID NO: 133), human IgG3 (SEQ ID NO: 134), and human IgG4 (SEQ ID NO: 135). Another preferred example of the parent Fc region is an Fc region derived from a heavy chain constant region SGI (SEQ ID NO: 136). Another preferred example of the parent Fc region is an Fc region derived from a heavy chain constant region SG182 (SEQ ID NO: 137). Furthermore, the parent Fc region may be an Fc region produced by adding an amino acid alteration(s) other than the amino acid alteration(s) described herein to an Fc region derived from a native antibody.

In certain embodiments, the variant Fc region of the present disclosure has a substantially decreased Fc gamma receptor-binding activity compared to the parent Fc region. In certain embodiments, the variant Fc region of the present disclosure has a maintained (does not have a substantially decreased) Clq-binding activity or increased Clq-binding activity compared to the parent Fc region. In certain embodiments, Fc gamma receptor is human Fc gamma receptor, monkey Fc gamma receptor (e.g., cynomolgus, rhesus macaque, marmoset, chimpanzee, or baboon Fc gamma receptor), or mouse Fc gamma receptor.

In a variant Fc region (or an antigen-binding molecule comprising the variant Fc region) of the present disclosure having a substantially decreased binding activity for one or more human Fc gamma receptors, typically, one or more amino acid mutations are present in the Fc region. In certain embodiments, the variant Fc region described herein exhibits reduced binding affinity to a Fc gamma receptor, as compared to a native IgGl Fc region. Herein, human Fc gamma receptors (Fc gamma Rs) include, but are not limited to Fc gamma Ria, Fc gamma Rlla (including allelic variants 167H and 167R), Fc gamma RUb, Fc gamma Rllla (including allelic variants 158F and 158V), and Fc gamma RIHb (including allelic variants NA1 and NA2). In a further aspect, a variant Fc region of the present disclosure has a substantially decreased binding activity for human Fc gamma Ria, Fc gamma Rlla (including allelic variants 167H and 167R), Fc gamma RUb, Fc gamma Rllla (including allelic variants 158F and 158V), and Fc gamma RIHb (including allelic variants NA1 and NA2), as compared to a parent Fc region. In one aspect, a variant Fc region of the present disclosure has a substantially decreased binding activity for one or more mouse Fc gamma Rs including, but not limited to Fc gamma RI, Fc gamma Rllb, Fc gamma RIII, and Fc gamma RIV, as compared to a parent Fc region. In a further aspect, a variant Fc region of the present disclosure has a substantially decreased binding activity for mouse Fc gamma RI, Fc gamma Rllb, Fc gamma RIII, and Fc gamma RIV, as compared to a parent Fc region.

"Fc gamma receptors" (herein, referred to as Fc gamma receptors, Fc gamma R or FcgR) refers to receptors that may bind to the Fc region of IgGl, IgG2, IgG3, and IgG4 monoclonal antibodies, and practically means any member of the family of proteins encoded by the Fc gamma receptor genes. In humans, this family includes Fc gamma RI (CD64) including isoforms Fc gamma Ria, Fc gamma Rib, and Fc gamma RIc; Fc gamma RII (CD32) including isoforms Fc gamma Rlla (including allotypes H131 (type H) and R131 (type R)), Fc gamma Rllb (including Fc gamma RIIb-1 and Fc gamma RIIb-2), and Fc gamma Rile; and Fc gamma RIII (CD16) including isoforms Fc gamma Rllla (including allotypes V158 and F158), and Fc gamma Rlllb (including allotypes Fc gamma RIIIb-NAl and Fc gamma RIIIb-NA2), and any human Fc gamma Rs, Fc gamma R isoforms or allotypes yet to be discovered, but is not limited thereto. Fc gamma Rllbl and Fc gamma RIIb2 have been reported as splicing variants of human Fc gamma Rllb. In addition, a splicing variant named Fc gamma RIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human Fc gamma Rllb includes all splicing variants registered in NCBI, which are NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human Fc gamma Rllb includes every previously -reported genetic polymorphism, as well as Fc gamma Rllb (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.

In Fc gamma Rlla, there are two allotypes, one where the amino acid at position 167 of Fc gamma Rlla 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)).

The Fc gamma R includes human, mouse, rat, rabbit, and monkey -derived Fc gamma Rs but is not limited thereto, and may be derived from any organism. Mouse Fc gamma Rs include Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD 16), and Fc gamma RIV (CD16-2), and any mouse Fc gamma Rs, or Fc gamma R isoforms, but are not limited thereto.

The amino acid sequence of human Fc gamma Ria is set forth in SEQ ID NO: 138; the amino acid sequence of human Fc gamma Rlla (167H) is set forth in SEQ ID NO: 139; the amino acid sequence of human Fc gamma Rlla (167R) is set forth in SEQ ID NO: 140; the amino acid sequence of human Fc gamma Rllb is set forth in SEQ ID NO: 141; the amino acid sequence of human Fc gamma Rllla (158F) is set forth in SEQ ID NO: 142; the amino acid sequence of human Fc gamma Rllla (158V) is set forth in SEQ ID NO: 143; the amino acid sequence of human Fc gamma Rlllb (NA1) is set forth in SEQ ID NO: 144; and the amino acid sequence of human Fc gamma Rlllb (NA2) is set forth in SEQ ID NO: 145.

The amino acid sequence of mouse Fc gamma RI is set forth in SEQ ID NO: 146; the amino acid sequence of mouse Fc gamma Rllb is set forth in SEQ ID NO: 147; the amino acid sequence of mouse Fc gamma RIII is set forth in SEQ ID NO: 148; and the amino acid sequence of mouse Fc gamma RIV is set forth in SEQ ID NO: 149.

In one aspect, a variant Fc region of the present disclosure (or an antigen-binding molecule comprising said variant Fc region) has a substantially decreased Fc gamma R-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 Fc gamma R-binding activity for the parent Fc region (or an antigen-binding molecule comprising said parent Fc region). In one aspect, a variant Fc region of the present disclosure has a substantially decreased Fc gamma R-binding activity, which means that the ratio of [the difference in the RU values of sensorgrams that changed before and after interaction of Fc gamma R with the variant Fc region]/[the difference in the RU values of sensorgrams that changed before and after capturing Fc gamma R 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 than 0.002, or less than 0.001. In one embodiment, the variant Fc region (or the antigen -binding molecule comprising said variant Fc region) does not substantially bind to an Fc gamma receptor. In one aspect, a variant Fc region of the present disclosure has a maintained (does not have a substantially decreased) Clq-binding activity or increased Clq-binding activity. “Maintained” or “not substantially decreased” Clq-binding activity means that the difference of Clq-binding activities between a variant Fc region and a parent Fc region of the present disclosure 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 Clq-binding activity for the parent Fc region. In the case that a variant Fc region has an “increased” Clq-binding activity, the difference of Clq-binding activities between a variant Fc region and a parent Fc region of the present disclosure may be more than 50% and the variant Fc region of the present disclosure may have a Clq-binding activity that is 100% or more, 150% or more, 200% or more, 400% or more, 800% or more, or 1600% or more as a function of the Clq-binding activity for the parent Fc region. The comparison may be made at any concentration of antigen-binding molecule, but it is preferred that the comparison is made in the presence of a high concentration of antigen-binding molecule, which allows the antigenbinding molecule comprising a variant Fc region or a parent Fc region (control) to assemble into hexamers. Binding activity of an antigen-binding molecule to Clq can be evaluated using conventional Clq binding assay (e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402) or by using a CDC assay (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Known methods for assessing complement dependent lysis or complement dependent reduction of virus infectivity, such as the use of heat inactivated serum or serum depleted of complement components, may also be used to assess Clq binding.

Binding between hexameric antigen-binding molecules and Clq can be evaluated using known methods, such as ELISA-based methods, surface plasmon resonance (SPR)-based methods, etc. (see, e.g., Biologicals 2019 Sep;61:76-79). Such assay may be carried out particularly under conditions that allow an antigen-binding molecule comprising a variant Fc region or a parent Fc region (control) to assemble into hexamers.

For example, to determine the binding activity of a polypeptide containing a variant Fc region towards Clq, a Clq binding ELISA may be performed. Briefly, assay plates may be coated overnight at 4 degrees C with a polypeptide containing a variant Fc region or a polypeptide containing a parent Fc region (control) in coating buffer. The plates may then be washed and blocked. Following washing, an aliquot of human Clq may be added to each well and incubated for 2 hours at room temperature. Following a further wash, 100 microliters of a sheep anti-complement Clq peroxidase conjugated antibody may be added to each well and incubated for 1 hour at room temperature. The plate may again be washed with wash buffer and 100 microliters of substrate buffer containing OPD (o-phenylenediamine dihydrochloride (Sigma)) may be added to each well. The oxidation reaction, observed by the appearance of a yellow color, may be allowed to proceed for 30 minutes and stopped by the addition of 100 microliters of 4.5 N H2SO4. The absorbance may then read at (492-405) nm. The binding activity of an Fc region for Clq can be determined by a method described in WO2018/052375.

For another example, binding activity of an antigen-binding molecule to Clq can be evaluated using a CDC assay, as the occurrence of target lysis by CDC indicates the occurrence of the binding of Clq to an antibody Fc which triggers the classical complement pathway. CDC assay described in Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004) may suitably be used. For example, Clq binding may be assayed as detailed in the Examples of the present disclosure. Briefly, cells stably transfected to overexpress an antigen are suspended at a suitable concentration and seeded onto an assay plate. A suitable concentration of human serum is added to each well. Antibodies are diluted over a suitable range and added to each well. After mixing the components well, the plate is placed in an incubator and incubated at 37 degrees C with 5% CO2 for about 1 hour. The cells are washed with buffer and stained with a viability dye for example 7AAD, and analyzed by flow cytometry to determine the percentage of cells lysed by antibody-mediated CDC.

In one aspect, the present disclosure provides an antigen-binding molecule comprising a variant Fc region with a substantially decreased ADCC activity. In one aspect, the present disclosure provides an antigen-binding molecule comprising a variant Fc region with a maintained (without a substantially decreased) CDC activity or increased CDC activity. In one aspect, the present disclosure provides an antigen-binding molecule comprising a variant Fc region with a substantially decreased ADCC activity and a maintained (without a substantially decreased) CDC activity or increased CDC activity.

In one aspect, a variant Fc region of the present disclosure confers to an antigenbinding molecule comprising the variant Fc region 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 antigen-binding molecule comprising the parent Fc region.

In one aspect, a variant Fc region of the present disclosure confers to an antigenbinding molecule comprising the variant Fc region a maintained (i.e., not substantially decreased) CDC activity or an increased CDC activity. “Maintained” or “not substantially decreased” CDC activity means that the difference of CDC activities between the antigenbinding molecule comprising the variant Fc region and the antigen-binding molecule comprising the parent Fc region is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%. In one aspect, a variant Fc region of the present disclosure confers to an antigen-binding molecule comprising the variant Fc region increased CDC activity that is more than 100%, more than 200%, more than 400%, more than 800%, or more than 1600% for the antigen -binding molecule comprising the parent Fc region, in which the CDC activity is determined as the concentration of the antibody required to achieve 50% of the maximum complementdependent lysis of a target cell.

In further aspects, the variant Fc region of the present disclosure comprises at least one amino acid alteration of at least one position selected from the group consisting of: 234, 235, 236, 267, 268, 324, 326, 332, and 333, according to EU numbering (see, e.g., WO2018/052375).

In one aspect, the variant Fc region with a substantially decreased Fc gamma receptorbinding activity and a maintained (without a substantially decreased) or increased Clq-binding activity comprises Ala at position 234, Ala at position 235 and at least one amino acid alteration of at least one position selected from the group consisting of: 236, 267, 268, 324, 326, 332, and 333, according to EU numbering.

In one aspect, the variant Fc region with a substantially decreased Fc gamma receptorbinding activity and a maintained (without a substantially decreased) or increased Clq-binding activity comprises Ala at position 234, Ala at position 235 and further amino acid alterations of any one of the following (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. In a further aspect, the variant Fc region with a substantially decreased Fc gamma receptor-binding activity and maintained (without a substantially decreased) or increased Clq- 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 236; (e) Glu at position 332; (f) Ala, Asp, Glu, Met, or Trp at position 326; and (g) Ser at position 333, according to EU numbering.

In one aspect, the variant Fc region with a substantially decreased Fc gamma receptorbinding activity and maintained (without a substantially decreased) or increased Clq-binding activity comprises amino acids of: 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 a substantially decreased Fc gamma receptorbinding activity and maintained (without a substantially decreased) or increased Clq-binding activity comprises amino acids of: 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 a substantially decreased Fc gamma receptorbinding activity and maintained (without a substantially decreased) or increased Clq-binding activity comprises amino acids of: 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 a substantially decreased Fc gamma receptorbinding activity and maintained (without a substantially decreased) or increased Clq-binding activity comprises amino acids of: 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 a substantially decreased Fc gamma receptorbinding activity and maintained (without a substantially decreased) or increased Clq-binding activity comprises amino acids of: Ala at position 234, Ala at position 235, Trp at position 326, and Ser at position 333, according to EU numbering. In one aspect, the variant Fc region of the present disclosure has an increased FcRn binding activity under acidic pH, compared to the parent Fc region.

In one aspect, it is preferable that a variant Fc region of the present disclosure does not have a substantially increased FcRn binding activity, especially at pH7.4, compared to the parent Fc region.

"FcRn" is structurally similar to polypeptides of 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 beta or light chain (beta 2 microglobulin) complexed with a transmembrane alpha or heavy chain. Like MHC, the alpha chain of FcRn contains three extracellular domains (alphal, alpha2, and alpha3), and its short cytoplasmic domain tethers them to the cell surface. The alphal and alpha2 domains interact with the FcRn-binding domain of the antibody Fc region. The polynucleotide and amino acid sequences of human FcRn may be derived, for example, from the precursors shown in NM_004107.4 and NP_004098.1 (containing the signal sequence), respectively.

The amino acid sequence of human FcRn (alpha chain) is set forth in SEQ ID NO: 150; and the amino acid sequence of human beta2 microglobulin is set forth in SEQ ID NO: 151.

In one aspect, it is preferable that a variant Fc region of the present disclosure does not have a substantially increased FcRn binding activity, especially at pH7.4, that is less than 1000 fold, less than 500 fold, less than 200 fold, less than 100 fold, less than 90 fold, less than 80 fold, less than 70 fold, less than 60 fold, less than 50 fold, less than 40 fold, less than 30 fold, less than 20 fold, less than 10 fold, less than 5 fold, less than 3 fold, or less than 2 fold compared to the FcRn binding activity for the parent Fc region. In one aspect, a variant Fc region of the present disclosure does not have a substantially increased FcRn binding activity, especially at pH7.4, which means that the ratio of [the difference in the RU values of sensorgrams that changed before and after interaction of FcRn with the variant Fc region]/[the difference in the RU values of sensorgrams that changed before and after capturing 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. In another aspect, the variant Fc region of the present disclosure can further comprise at least one amino acid alteration of at least one position selected from the group consisting of: 428, 434, 436, 438, and 440, according to EU numbering.

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

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

In another aspect, the variant Fc region of the present disclosure comprises any of the amino acid alterations, singly or in combination, described in Table 2 below (see also, e.g., WO2018/052375). In another aspect, the variant Fc region of the present disclosure comprises at least any one of the amino acid alterations described in Table 2.

[Table 2]

In one aspect, the antigen-binding molecule of the present disclosure has a substantially decreased Fc gamma R-binding activity. In one aspect, the antigen-binding molecule of the present disclosure has a maintained (not substantially decreased) or increased Clq-binding activity.

In one aspect, the antigen-binding molecule of the present disclosure has a substantially decreased Fc gamma R-binding activity and has a maintained (not substantially decreased) or increased Clq-binding activity.

In one aspect, the present disclosure provides an antigen-binding molecule comprising the Fc region which is:

(i) an Fc region which exhibits reduced binding affinity to human Fc gamma receptor, as compared to a parent Fc region (which may be a parent native human IgGl Fc region), wherein the Fc region variant comprises (fl) or (f2) below:

(fl) Ala at position 234 and Ala at position 235; (f2) Ala at position 234, Ala at position 235, and Ala at position 297; wherein the amino acid positions are numbered according to EU index.

(i) an Fc region which exhibits reduced binding affinity to human Fc gamma receptor, as compared to a parent Fc region (which may be a parent native human IgGl Fc region), wherein the Fc region further exhibits maintained (not substantially decreased) or increased Clq-binding activity as compared to the parent Fc region (which may be the parent native human IgGl Fc region), wherein the Fc region variant comprises (fl) or (f2) below:

(fl) Ala at position 234 and Ala at position 235; (f2) Ala at position 234, Ala at position 235, and Ala at position 297; and wherein the Fc region variant further comprises amino acids selected from the group consisting of (f3) to (f9) below:

(f3) Glu at position 267; (f4) Phe at position 268; (f5) Thr at position 324; (f6) Ala at position 236; (f7) Glu at position 332; (f8) Ala, Asp, Glu, Met, or Trp at position 326; and

(f9) Ser at position 333; wherein the amino acid positions are numbered according to EU index.

(i) an Fc region which exhibits reduced binding affinity to human Fc gamma receptor, as compared to a parent Fc region (which may be a parent native human IgGl Fc region), wherein the Fc region further exhibits maintained (not substantially decreased) or increased Clq-binding activity as compared to the parent Fc region (which may be the parent native human IgGl Fc region), and wherein the Fc region further exhibits stronger FcRn binding affinity to human FcRn under acidic condition, as compared to the parent Fc region.

(i) an Fc region which exhibits reduced binding affinity to human Fc gamma receptor, as compared to a parent Fc region (which may be a parent native human IgGl Fc region), wherein the Fc region further exhibits maintained (not substantially decreased) or increased Clq-binding activity as compared to the parent Fc region (which may be the parent native human IgGl Fc region), wherein the Fc region further exhibits stronger FcRn binding affinity to human FcRn under acidic condition, as compared to the parent Fc region, wherein the Fc region variant comprises, in addition to (fl) or (f2) above and amino acids selected from the group consisting of (f3) to (f9) above, Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and/or Glu at position 440, and wherein the amino acid positions are numbered according to EU index. In one aspect, the present disclosure provides an antigen-binding molecule comprising the Fc region which is:

(i) an Fc region which exhibits reduced binding affinity to human Fc gamma receptor, as compared to a parent Fc region (which may be a parent native human IgGl Fc region), wherein the Fc region further exhibits maintained (not substantially decreased) or increased Clq-binding activity as compared to the parent Fc region (which may be the parent native human IgGl Fc region), wherein the Fc region further exhibits stronger FcRn binding affinity to human FcRn under acidic condition, as compared to the parent Fc region, wherein the Fc region variant comprises, in addition to (fl) or (f2) above and amino acids selected from the group consisting of (f3) to (f9) above, Leu at position 428, Ala at position 434, and/or Thr at position 436, and wherein the amino acid positions are numbered according to EU index.

In addition, amino acid alterations performed for other purpose(s) can be combined in a variant Fc region described herein.

For example, in addition to amino acid alterations at positions 234 and 235, an amino acid substitution at a position selected from the group of E233, N297, P331, and P329 may be introduced to reduce the binding affinity of an Fc region to an Fc gamma receptor. In one embodiment, the variant Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment the variant Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297, and P331. In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In particular embodiments, the Fc region comprises amino acid substitutions at positions P329, L234, and L235. In more particular embodiments the Fc region comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the Fc region is an IgGl Fc region, particularly a human IgGl Fc region. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fc gamma receptor (as well as complement) binding of a human IgGl Fc region, as described in PCT publication no. WO 2012/130831. WO 2012/130831 also describes methods of preparing such mutant Fc regions and methods for determining its properties such as Fc receptor binding or effector functions. In certain embodiments, N-glycosylation of the Fc region has been eliminated. In one such embodiment, the Fc region comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D).

In a particular embodiment, the variant Fc region exhibiting reduced binding affinity to an Fc receptor, as compared to a native IgGl Fc domain, is a human IgGl Fc region comprising the amino acid substitutions L234A, L235A, and N297A.

For example, amino acid substitutions that improve FcRn-binding activity (Hinton et al., J. Immunol. 176(l):346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514- 23524 (2006); Petkova et al., Inti. Immunol. 18(12): 1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159 (2010); WO 2006/019447; WO 2006/053301; and WO 2009/086320), and amino acid substitutions for improving antibody heterogeneity or stability (WO 2009/041613) 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 for repeated binding to a plurality of antigen molecules, which are described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, can be combined with a variant Fc region described herein. Alternatively, with the objective of conferring binding ability to other antigens, the amino acid alterations disclosed in EP1752471 and EP1772465 may be combined in CH3 of a variant Fc region described herein. Alternatively, with the objective of increasing plasma retention, amino acid alterations that decrease the pl of the constant region (WO 2012/016227) may be combined in a variant Fc region described herein. Alternatively, with the objective of promoting uptake into cells, amino acid alterations that increase the pl of the constant region (WO 2014/145159) may be combined in a variant Fc region described herein. Alternatively, with the objective of promoting elimination of a target molecule from plasma, amino acid alterations that increase the pl of the constant region (WO2016/125495 and WO2016/098357) may be combined in a variant Fc region described herein.

Amino acid alterations of enhancing human FcRn-binding activity under acidic pH can also be combined in a variant Fc region described herein. Specifically, such alterations may include, for example, substitution of Leu for Met at position 428 and substitution of Ser for Asn at position 434, according to EU numbering (Nat Biotechnol, 2010, 28: 157-159); substitution of Ala for Asn at position 434 (Drug Metab Dispos, 2010 Apr; 38(4): 600-605); substitution of Tyr for Met at position 252, substitution of Thr for Ser at position 254 and substitution of Glu for Thr at position 256 (J Biol Chem, 2006, 281: 23514-23524); substitution of Gin for Thr at position 250 and substitution of Leu for Met at position 428 (J Immunol, 2006, 176(1): 346-356); substitution of His for Asn at position 434 (Clin Pharmacol Ther, 2011, 89(2): 283-290), and alterations described in W02010/106180, WO2010/045193, W02009/058492, W02008/022152, W02006/050166, W02006/053301, W02006/031370, W02005/123780, W02005/047327, W02005/037867, W02004/035752, W02002/060919, or such. In another embodiment, such alterations may include, for example, at least one alteration 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. Those alterations may further include substitution of Arg for Gin at position 438 and/or substitution of Glu for Ser at position 440 (WO2016/125495).

In one embodiment, the antigen-binding molecule of the present disclosure comprising the variant Fc regions with modified effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

In one embodiment, the antigen-binding molecule of the present disclosure comprising the variant Fc regions may have altered (e.g., increased or decreased) binding to FcRs as described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region with one or more amino acid substitutions which alter ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., either increased or decreased) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, WO2011/091078, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

In one embodiment, the antigen-binding molecule of the present disclosure 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 US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).

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

In another embodiment, an antigen-binding molecule may comprise an Fc region variant of the present disclosure described herein below in detail.

In one embodiment described herein, the Fc region of the antigen binding molecule of the present disclosure is an IgG Fc region. In a particular embodiment, the Fc region is an IgGl Fc region. In a further particular embodiment, the Fc region is a human IgGl Fc region.

In one non-limiting embodiment, the antigen-binding molecule of the present disclosure specifically binds to a virus which can cause the risk of Antibody-Dependent Enhancement (ADE) when the antigen-binding molecule is administered.

In the present disclosure, amino acid alteration means any of substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present disclosure, amino acid alteration may be rephrased as amino acid mutation.

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

The number of amino acid alterations introduced into an Fc region is not limited. In certain embodiments, it can 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.

Furthermore, an antigen-binding molecule comprising a variant Fc region of the present disclosure may be chemically modified with various molecules such as polyethylene glycol (PEG) and cytotoxic substances. Methods for such chemical modification of a polypeptide are established in the art.

The antigen-binding molecule of the present disclosure comprising an Fc region variant has a substantially decreased Fc gamma receptor-binding activity, and/or has a maintained (does not have a substantially decreased) Clq-binding activity or increased Clq- binding activity, and/or has an increased FcRn binding activity under acidic pH, and/or does not have a substantially increased FcRn binding activity at neutral pH, when compared to an antigen-binding molecule comprising the parent Fc region.

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541. e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n- vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, 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 may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

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

The antigen binding molecules of the present disclosure herein can be combined with a variety of existing technologies. As a non-limiting embodiment of such a combination of technologies, the generation of cells that have expressed a chimeric antigen receptor (CAR) utilizing the antigen binding molecule of the present disclosure is exemplified. Cells herein include, for example, T cells, gamma delta T cells, NK cells, NKT cells, cytokine-induced killer (CIK) cells, and macrophages (Int J Mol Sci. (2019) 20(11), 2839, Nat Rev Drug Discov. (2020) 19(5), 308). One of the non-limiting methods for the generation of a T cell expressing a CAR (CAR-T) involves, for example, a method in which a CAR comprising antigen binding molecules of the present disclosure that specifically bind to SARS-CoV-2, the transmembrane domains of the TCR, and the intracellular signal domains of costimulatory molecules such as CD28 to enhance T-cell activation is introduced into an effector cell such as a T cell by genetic modification techniques.

As non-limiting examples of techniques that can be combined with antigen binding molecules of the present disclosure herein, the generation of T-cell redirecting antigen binding molecules utilizing the antigen binding molecules of the present disclosure is exemplified (Nature (1985) 314 (6012), 628-31, Int J Cancer (1988) 41 (4), 609-15, Proc Natl Acad Sci USA (1986) 83 (5), 1453-7). The T-cell redirecting antigen binding molecule is a bispecific antigen-binding molecule comprising a binding domain for any one of the subunits forming a T-cell receptor (TCR) complex on T-cells, in particular a binding domain for CD3 epsilon chain among CD3 and a binding domain for the target antigen bound by the antigen binding molecules of the present disclosure.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-SARS-CoV-2 antibody 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 a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes 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 that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp2/0 cell). In one embodiment, a method of making an anti-SARS-CoV-2 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 expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an anti-SARS-CoV-2 antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody -encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

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

Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., 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, e.g., in Mather et al., Annals N.Y. 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 DHFR’ CHO 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, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

C. Assays

Anti-SARS-CoV-2 antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

I. Binding assays and other assays

In one aspect, an antibody of the disclosure is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.

In another aspect, competition assays may be used to identify an antibody that competes for binding to SARS-CoV-2 with any anti-SARS-CoV-2 antibody described herein. In certain embodiments, when such a competing antibody is present in excess, it blocks (e.g., reduces) the binding of a reference antibody to SARS-CoV-2 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an anti-SARS-CoV-2 antibody described herein (e.g., an epitope within a fragment of SARS-CoV-2, especially for SARS-CoV-2 spike protein receptor binding domain (RBD)). 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).

In an exemplary competition assay, immobilized SARS-CoV-2 is incubated in a solution comprising a first labeled antibody that binds to SARS-CoV-2 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to SARS-CoV-2. The second antibody may be present in a hybridoma supernatant. As a control, immobilized SARS-CoV-2 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 SARS-CoV-2, excess unbound antibody is removed, and the amount of label associated with immobilized SARS-CoV-2 is measured. If the amount of label associated with immobilized SARS-CoV-2 is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to SARS-CoV-2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity assays

In one aspect, assays are provided for identifying anti-SARS-CoV-2 antibodies having biological activity. Biological activity may include, e.g., blocking binding of SARS-CoV-2 to a host cell receptor, inhibiting SARS-CoV-2 entry into a host cell, inhibiting and/or preventing and/or reducing an incidence of SARS-CoV-2 infection of a host cell, complement-mediated opsonization and/or inactivation of the SARS-CoV-2 virus. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the disclosure is tested for such biological activity. Depending on the assay, cell and/or tissue culture may be required. A cell may be examined using any of a number of different physiologic assays. Alternatively or additionally, molecular analysis may be performed, including, but not limited to, western blotting to monitor protein expression and/or test for protein-protein interactions; mass spectrometry to monitor other chemical modifications; etc. In some embodiments, such methods utilize an animal host. For example, animal hosts suitable for the disclosure can 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 with, infected with, or otherwise exposed to virus prior to or concurrent with administration of a test antibody. Naive 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 is 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 efficacy of the test antibody in blocking virus binding and/or infectivity in the animal host.

In certain embodiments, an antibody of the disclosure is tested for its biological activity in blocking the binding interaction between the SARS-CoV-2 spike protein and a host cell receptor including, but not limited to, ACE2. The binding interaction between the spike protein and the host cell receptor can be performed by ELISA or BIACORE®.

In certain embodiments, an antibody of the disclosure is tested for its biological activity in a SARS-CoV-2 spike protein pseudotyped lentivirus neutralization assay. This method is described in detail by Crawford et. al. Viruses. 2020 May; 12(5). Briefly, antibodies are preincubated with SARS-CoV-2 spike protein pseudotyped lentiviruses and thereafter added to target cells which express a SARS-CoV2 viral entry receptor such as ACE2. The pseudoviruses are engineered to carry a reporter gene such as luciferase or a fluorescent protein which is expressed upon successful entry and infection of the target cells. The neutralizing activity of the antibody is determined by its ability to inhibit pseudovirus infection of the target cell.

In certain embodiments, an antibody of the disclosure is tested for its biological activity in inhibiting killing of target cells by live SARS-CoV-2 virus. Similar to the pseudotyped lentivirus assay, antibodies are first pre-incubated with live SARS-CoV-2 virus and thereafter added to the target cell. In this assay, the target cells not only express the SARS-CoV2 viral entry receptor for infection but are also sensitive to infection and are killed by the virus. One such candidate target cells is the Vero E6 cells. The ability of the antibody to inhibit viral infection of the target cell is assayed using a cell viability assay to enumerate the percentage of live cells remaining after exposure to the virus. Alternatively, the target cells can be fixed shortly after infection and stained for the presence viral antigens to quantitate the number of viral infection foci. This method will allow the ability of the antibodies to block early viral entry to be assessed.

In certain embodiments, the biological activity of antibodies is tested for its ability to prevent or reduce infection in animals which are permissive for SARS-CoV-2 infection, such as, but not limited to, transgenic mice, hamsters and non-human primates. As the SARS-CoV-2 spike protein does not bind well to mouse ACE2, transgenic mice overexpressing human ACE2, such as the K18-hACE2 mice can be used. Hamsters are a suitable small animal model for infection as they are susceptible to infection with SARS-CoV-2 virus and lung lesions are similar to that observed in COVID-19 patients. The antibody can either be administered to an animal either before or after infection with SARS-CoV-2 virus. The ability of the antibody to prevent or reduce infection of an animal can be determined by reduction in weight loss, reduction in lung viral titre measured by qPCR, reduction of cytokine production measured by ELISA or qPCR, and/or changes in lung histopathology.

D. Immunoconjugates

The disclosure also provides immunoconjugates comprising an anti-SARS-CoV-2 antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

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. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B l); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative 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. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Leters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

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, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody as described herein 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 spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine- 123 again, iodine- 131, indium-i l l, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionuclide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase- sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

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

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-SARS-CoV-2 antibodies provided herein is useful for detecting the presence of SARS-CoV-2 in a sample. The sample may be obtained from any source. For example, the sample may be a biological sample, a pharmaceutical sample, an environmental sample, a food sample etc. In some embodiments, the sample comprises a biological sample. The term “detecting” 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, ocular lens fluid, nasal fluid, anterior nares fluid, nasal mid-turbinate fluid, pharyngeal fluid, nasopharyngeal fluid, oropharyngeal fluid, mucus or processed fractions thereof.

In one embodiment, an anti-SARS-CoV-2 antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of SARS-CoV-2 in a sample is provided. In certain embodiments, the method comprises contacting the sample with an anti-SARS-CoV-2 antibody as described herein under conditions permissive for binding of the anti-SARS-CoV-2 antibody to SARS-CoV-2, and detecting whether a complex is formed between the anti-SARS-CoV-2 antibody and SARS-CoV-2. In various embodiments, the formation of a complex is indicative of the presence of SARS-CoV-2 in the sample. In various embodiments, the absence of a formation of a complex is indicative of the absence of SARS-CoV-2 in the sample. Such methods may be an in vitro or in vivo method. In one embodiment, an anti-SARS-CoV-2 antibody is used to select subjects eligible for therapy with an anti-SARS-CoV-2 antibody, e.g. where SARS-CoV-2 is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of the disclosure include SARS-CoV-2 infection and diseases and/or symptoms caused by or associated with SARS- CoV-2 infection.

In certain embodiments, labeled anti-SARS-CoV-2 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, lucerif erases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6- phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations (also referrd to herein as “Pharmaceutical composition”) of an anti-SARS-CoV-2 antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic 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 (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble 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 US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-SARS-CoV-2 antibodies provided herein may be used in therapeutic methods.

In one aspect, an anti-SARS-CoV-2 antibody for use as in therapy is provided. In one aspect, an anti-SARS-CoV-2 antibody for use as a medicament is provided. In further aspects, an anti-SARS-CoV-2 antibody for use in treating and/or preventing and/or reducing an incidence of SARS-CoV-2 infection is provided. In certain embodiments, an anti-SARS-CoV- 2 antibody for use in a method of treatment and/or prevention and/or reduction of an incidence of SARS-CoV-2 infection is provided. In certain embodiments, the disclosure provides an anti-SARS-CoV-2 antibody for use in a method of treating and/or preventing and/or reducing an incidence of SARS-CoV-2 infection in an individual having SARS-CoV-2 infection comprising administering to the individual an effective amount of the anti-SARS-CoV-2 antibody.

In further embodiments, the disclosure provides an anti-SARS-CoV-2 antibody for use in blocking binding of the receptor-binding domain of SARS-CoV-2 to and/or SARS-CoV-2 entry into a host cell. In certain embodiments, the disclosure provides an anti-SARS-CoV-2 antibody for use in a method of blocking binding of the receptor-binding domain of SARS- CoV-2 to and/or SARS-CoV-2 entry into a host cell in an individual comprising administering to the individual an effective of the anti- SARS-CoV-2 antibody to block binding of the receptor-binding domain of SARS-CoV-2 to and/or SARS-CoV-2 entry into a host cell. An "individual" according to any of the above embodiments is preferably a human.

In further embodiments, the disclosure provides an anti-SARS-CoV-2 antibody for use in exerting complement dependent cytotoxicity (CDC) against a target virus, e.g., SARS-CoV- 2. In further embodiments, the disclosure provides an anti-SARS-CoV-2 antibody for use in exerting the lysis of a target virus, e.g. SARS-CoV-2, (virolysis) or reduction of the virus ability to infect cells by complement. In further embodiments, the disclosure provides an anti- SARS-CoV-2 antibody for use in suppressing antibody-dependent enhancement (ADE) observed with conventional anti-SARS-CoV-2 antibodies.

In a further aspect, the disclosure provides for the use of an anti-SARS-CoV-2 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment and/or prevention and/or reduction of an incidence of SARS-CoV-2 infection. In a further embodiment, the medicament is for use in a method of treating and/or preventing and/or reducing an incidence of SARS-CoV-2 infection comprising administering to an individual having SARS-CoV-2 infection an effective amount of the medicament. In a further embodiment, the medicament is for blocking binding of the receptor-binding domain of SARS-CoV-2 to and/or SARS-CoV-2 entry into into a host cell. In a further embodiment, the medicament is for use in a method of blocking binding of the receptor-binding domain of SARS-CoV-2 to and/or SARS-CoV-2 entry into a host cell in an individual comprising administering to the individual an amount effective of the medicament to block binding of SARS-CoV-2 protein to and/or SARS-CoV-2 entry into a host cell. An "individual" according to any of the above embodiments may be a human.

In a further embodiment, the medicament is for use in exerting complement dependent cytotoxicity (CDC) against a target virus, e.g., SARS-CoV-2. In a further embodiment, the medicament is for use in exerting the lysis of a target virus, e.g. SARS-CoV-2, (virolysis) or reduction of the virus ability to infect cells by complement. In a further embodiment, the medicament is for use in suppressing antibody -dependent enhancement (ADE) observed with conventional anti-SARS-CoV-2 antibodies.

In a further aspect, the disclosure provides a method for treating and/or preventing and/or reducing an incidence of a SARS-CoV-2 infection. In one embodiment, the method comprises administering to an individual having such SARS-CoV-2 infection an effective amount of an anti-SARS-CoV-2 antibody. An “individual” according to any of the above embodiments may be a human. In some embodiments, the disclosure provides use of an anti- SARS-CoV-2 antibody for treating and/or preventing and/or reducing an incidence of a SARS- CoV-2 infection. In a further aspect, the disclosure provides a method for blocking binding of the receptor-binding domain of SARS-CoV-2 to and/or SARS-CoV-2 entry into into a host cell in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-SARS-CoV-2 antibody to block binding of the receptor-binding domain of SARS-CoV-2 to and/or SARS-CoV-2 entry into into a host cell. In some embodiments, the disclosure provides use of an anti-SARS-CoV-2 antibody for blocking binding of the receptor-binding domain of SARS-CoV-2 to and/or SARS-CoV-2 entry into into a host cell in an individual. In one embodiment, an “individual” is a human.

In further embodiments, the disclosure provides a method for exerting complement dependent cytotoxicity (CDC) against a target virus, e.g., SARS-CoV-2. In one embodiment, the method comprises administering to the individual an effective amount of an anti-SARS- CoV-2 antibody to exert complement dependent cytotoxicity (CDC) against a target virus, e.g., SARS-CoV-2. In some embodiments, the disclosure provides use of an anti-SARS-CoV-2 antibody for exerting complement dependent cytotoxicity (CDC) against a target virus, e.g., SARS-CoV-2.

In further embodiments, the disclosure provides a method for exerting the lysis of a target virus, e.g. SARS-CoV-2, (virolysis) or reduction of the virus ability to infect cells by complement. In one embodiment, the method comprises administering to the individual an effective amount of an anti-SARS-CoV-2 antibody to exert the lysis of a target virus, e.g. SARS-CoV-2, (virolysis) or reduction of the virus ability to infect cells by complement. In some embodiments, the disclosure provides use of an anti-SARS-CoV-2 antibody for exerting the lysis of a target virus, e.g. SARS-CoV-2, (virolysis) or reduction of the virus ability to infect cells by complement.

In further embodiments, the disclosure provides a method for suppressing antibodydependent enhancement (ADE) observed with conventional anti-SARS-CoV-2 antibodies. In one embodiment, the method comprises administering to the individual an effective amount of an anti-SARS-CoV-2 antibody to suppress antibody -dependent enhancement (ADE) observed with conventional anti-SARS-CoV-2 antibodies. In some embodiments, the disclosure provides use of an anti-SARS-CoV-2 antibody for suppressing antibody-dependent enhancement (ADE) observed with conventional anti-SARS-CoV-2 antibodies. In a further aspect, the disclosure provides pharmaceutical formulations comprising any of the anti-SARS-CoV-2 antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-SARS-CoV-2 antibodies provided herein and a pharmaceutically acceptable carrier.

In a further aspect, the pharmaceutical formulation is for treatment and/or prevention and/or reduction of an incidence of SARS-CoV-2 infection. In a further embodiment, the pharmaceutical formulation is for blocking binding of the receptor-binding domain of SARS- CoV-2 to and/or SARS-CoV-2 entry into a host cell. In one embodiment, the pharmaceutical formulation is administered to an individual having SARS-CoV-2 infection. An "individual" according to any of the above embodiments is preferably a human. In certain embodiments, SARS-CoV-2 infection may include diseases and/or symptoms caused by or associated with SARS-CoV-2 infection such as acute respiratory distress, pneumonia, dyspnea, fever, rhinitis, nasal congestion, loss of smell, fatigue, diarrhea etc.

In one embodiment, anti-SARS-CoV-2 antibodies that comprise a variant Fc region of the present disclosure can suppress antibody-dependent enhancement (ADE) observed with conventional anti-SARS-CoV-2 antibodies. ADE is a phenomenon where a virus bound to an antibody is phagocytosed via activating Fc gamma Rs so that infection of the virus to a cell is enhanced. Fc modifications that reduce interaction with activating Fc gamma Rs could 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 of dengue infection in vivo (Cell Host Microbe (2010) 8, 271-283).

Such modifications, however, reduce the other effector immune functions mediated by antibodies, such as ADCC and CDC. Especially, CDC can be expected to play an important role in inhibiting ADE, therefore complement component Clq binding of Fc regions should not be reduced for therapeutic efficacy. Furthermore, antibody half-life can be extended by engineering Fc regions that change binding affinity to its salvage receptor, FcRn, which might lead to prophylactic use of antibodies for protecting viral infection.

An antibody of the disclosure can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time -points, bolus administration, and pulse infusion are contemplated herein.

Antibodies of the disclosure would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated (e.g., into the form of a pharmaceutical composition) with one or more agents currently used to prevent or treat or reduce an incidence of the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically /clinically determined to be appropriate.

For the prevention or treatment or reducing an incidence of disease, the appropriate dosage of an antibody of the disclosure will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 micro g/kg to 15 mg/kg (e.g. O.lmg/kg-lOmg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 micro g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 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, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. An exemplary dosing regimen comprises administering [[add exemplary dosing regimen, if known, e.g., “an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the antibody”]]. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the disclosure in place of or in addition to an anti- SARS-CoV-2 antibody.

The antigen binding molecule of the present disclosure herein can be administered by a method in which a nucleic acid encoding the antigen binding molecule of the present disclosure is administered or incorporated into a body using a vector or the like, and the antigen binding molecule is directly expressed in a body, but may be administered without using a vector. As vectors, viral vectors, plasmid vectors are exemplified, and even adenoviral vectors are exemplified. The nucleic acid encoding the antigen binding molecule of the present disclosure may be administered directly to the body or directly to the body by electroporation. For example, the antigen binding molecule of the present disclosure may be administered by a method in which mRNA encoding the antigen binding molecule of the present disclosure may be subjected to chemical modifications to enhance the stability of mRNA in vivo, and mRNA may be administered directly to humans and the antigen binding molecule of the present disclosure may be expressed in vivo (see EP2101823B, WO20 13/ 120629). Also, B cells introduced with the antigen binding molecule of the present disclosure may be administered (Sci Immunol. (2019), 4(35), eaax0644). Bacteria introduced with the antigen binding molecule of the present disclosure may also be administered (Nature Reviews Cancer (2018) 18, 727-743).

As a non-limiting example of techniques that can be combined with antigen binding molecules of the present disclosure herein, the generation of T cells secreting T cell redirecting antigen binding molecules utilizing the antigen binding molecules of the present disclosure is exemplified (Trends Immunol.(2019) 40(3) 243-257). One of the non-limiting techniques is the introduction of bispecific antigen binding molecules, comprising a binding domain for any one of the subunits forming a T-cell receptor (TCR) complex on T cells, in particular a binding domain for CD3 epsilon chain among CD3 and a binding domain for the target antigen bound by antigen binding molecules of the present disclosure, into an effector cell such as a T cell by genetic modification techniques.

H. Articles of Manufacture

In another aspect of the disclosure, an article of manufacture containing materials useful for the treatment, prevention, reduction of an incidence of and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label on or a 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 which is by itself or combined with another composition effective for treating, preventing, reducing an incidence of, and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of the disclosure. The label or package insert indicates that the composition is used for treating or preventing or reducing an incidence of the condition of choice. The article of manufacture in this embodiment of the disclosure may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture may include an immunoconjugate of the disclosure in place of or in addition to an anti-SARS-CoV-2 antibody.

III. EXAMPLES

Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. The following are examples of methods and compositions of the disclosure. It is understood that various other embodiments may be practiced, given the general description provided above. It should also be appreciated that other modifications within the purview of the skilled person in the art may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

EXAMPLE 1.1: Identification of mutations to improve binding affinity

Amino acid residues in a variable region are numbered according to Kabat (Kabat et al., Sequence of proteins of immunological interest, 5^th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Amino acid residues in a heavy constant region are numbered according to "EU numbering system". (Kabat et al., Sequence of proteins of immunological interest, 5^th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991))

An anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody, named as 5A6 (H-chain and L-chain variable regions are set forth in SEQ ID NO: 1 and SEQ ID NO: 43, respectively), was isolated from a human Fab library using phase display technology as a lead antibody. 5A6 blocks RBD of the viral spike protein from binding to the host receptor angiotensin converting enzyme 2 receptor (ACE2). Variable regions of the heavy chain and the light chain of 5A6 are named as 5A6H.GS and 5A6L.GS, respectively. The polynucleotides encoding variable regions of the heavy and light chain were synthesised, and were cloned into expression vectors containing a polynucleotide encoding the heavy chain constant region SGI sequence or the light chain constant region SKI sequence, respectively. Recombinant antibodies were expressed transiently using the Expi293 Expression System (Thermo Fisher), according to the manufacturer’s instructions. It is called a parent antibody or wild type and referred as 5A6-SG1 or 5A6H.GS-SG1/5A6L.GS-SK1. Recombinant antibodies were purified with protein A (GE Healthcare) from supernatant of transient expression and the formulation of the purified antibody was either D-PBS or His buffer (20mM Histidine, 150mM NaCl, pH6.0). Size exclusion chromatography was further conducted to remove high molecular weight and/or low molecular weight component, if necessary. To identify mutations which improve affinity, more than 1000 variants were generated from the parent antibody (5A6-SG1 or 5A6H.GS-SG1/5A6L.GS-SK1). These variants had each position in the CDRs substituted with 18 other amino acids, excluding the original amino acid and Cysteine. Some positions in the frameworks were also substituted. Binding affinities of clone 5A6 and variants were measured at 25 °C or 37°C using Biacore 8K instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized onto all flow cells of a CM4 sensor chip using amine coupling kit (GE Healthcare). Antibodies were captured onto the antihuman Fc sensor surfaces to around 200RU capture level. Recombinant SARS-CoV-2 S protein RBD were injected at two concentrations, 12.5nM & 50nM . All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. Assay temperature was set at at 25°C . Sensor surface was regenerated each cycle with 3M MgCh . Binding affinities were determined by processing and fitting the data to 1:1 binding model using Biacore Insight Evaluation Software, version 3.0.12.15655 (GE Healthcare).

Variants with improved affinity were selected and kinetics of the selected variants are listed in Table 3.

[Table 3]

EXAMPLE 1.2: Generation of an affinity maturated antibody

The frameworks of 5A6, derived from the human Fab library, are not human germline framework sequences. The frameworks of 5A6 for the heavy chain and the light chain were replaced with human germline frameworks sequences. After assessing several human germline sequences, 5A602H and 5A605L were generated as the variable regions of the heavy chain and the light chain, respectively. The selected human germline frameworks are listed in Table 4.

[Table 4]

Mutations identified in Table 3 were combined, and this combination generated several variants with increased affinity against the SARS-CoV-2 S protein RBD. These mutations in CDRs and/or frameworks were introduced into 5A602H and 5A605L, and finally one variant was selected. The name of the selected variant of variable regions are named as 5A6AM1, consisting of 5A602H0835 (SEQ ID NO: 2) and 5A605L0494 (SEQ ID NO: 42) as variable regions of the heavy chain and the light chain, respectively.

EXAMPLE 2.1: Further engineering to improve various properties of 5A6AM1 antibody

When the solution of 5A6AM1-SG1 was concentrated, visible/insoluble particles were observed. Visible/invisible insoluble particles in protein formulations are considered to indicate low solubility of the protein of interest. Low solubility of therapeutic protein may limit the concentration in formulation and make drug development difficult especially when the drug needs to be high concentration in formulation/solution.

Various assays have been reported to evaluate antibody characteristics, such as antibody self-interaction, cross-interaction, melting temperature, hydrophobicity, aggregation species, low molecular species, monomer contents, non-specific binding. (PNAS January 31, 2017 114 (5) 944-949., PLoS ONE 8(2): e57479., MAbs. 2020 Jan-Dec; 12(1): 1683432.). 5A6AM1-SG1 was assessed in several assays and found to be unfavorable physicochemical properties compared with a negative control antibody, Antibody A, as shown in Table 6.

To overcome these challenges, further engineering was carried out by introducing mutations into 5A6AM1-SG1. After several rounds of the combinations, several variants with improved physicochemical properties were successfully generated as shown in Table 6. To compare the solubility of each antibody, the visual inspection assessment was carried out. All the variants were concentrated to 5 mg/mL using Amicon Ultra 0.5mL filters (Merck) and transferred to deactivated clear glass inserts (Waters). Inspectors checked samples in the vials and scored the degree of visible particles (VP) using the scoring system shown below in Table 5. The visual inspection score was calculated as the average from at least three inspectors, as shown in Table 6. All the variants showed lesser visible particles in the solution compared to 5A6AM1-SG1. In addition to reduced visible particles, several variants showed improved binding affinity against SARS-CoV-2 S protein RBD domain compared to 5A6AM1.

[Table 5]

[Table 6]

EXAMPLE 2.2: Characterization of engineering antibodies

Several variants were selected and produced with the human or human modified heavy constant regions. The names of antibodies and sequences are shown in Table 7.

[Table 7]

Binding affinity of clone 5A6 and variants towards recombinant SARS-CoV-2 S protein RBD and mutants (V483A, and F490S) were measured using Biacore 8K instrument (GE Healthcare). Protein A/G was immobilized onto all flow cells of a CM4 sensor chip using amine coupling kit (GE Healthcare). Antibodies were captured onto the protein A/G sensor surfaces to around 200RU capture level. Recombinant SARS-CoV-2 S protein RBD and mutants (V483A, and F490S) were injected at different concentrations (6.25nM - lOOnM, twofold serial dilution). All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. Assay temperature was set at 37 °C. Sensor surface was regenerated each cycle with 10 mM Glycine-HCl, pH 1.5. Binding affinity were determined by processing and fitting the data to 1:1 binding model using Biacore Insight Evaluation Software, version 3.0.12.15655 (GE Healthcare). Binding affinities of clone 5A6 and variants binding to trimeric SARS-CoV-2 S protein were determined at 37°C using Biacore T200 instrument (GE Healthcare). Anti-histidine antibody (GE Healthcare) was immobilized onto all flow cells of a CM5 sensor chip using amine coupling kit (GE Healthcare). Trimeric SARS-CoV-2 S protein (D614G) with a polyhistidine tag at the C- terminus and trimeric SARS-CoV-2 S protein with a polyhistidine tag at the C-terminus was captured onto the anti-histidine sensor surfaces between 200-300RU capture level. Then clone 5A6 and variants were injected at different concentration (6.25nM - lOOnM, two-fold serial dilution). All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. Sensor surface was regenerated each cycle with lOmM Gly-HCl pH 1.5. Binding kinetic and affinity was determined by processing and fitting the data to 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). Binding kinetics parameters for the clone 5A6 and affinity improved variants binding to SARS-CoV-2 S protein RBD and mutants (V483A, and F490S), trimeric SARS- CoV-2 S protein and trimeric SARS-CoV-2 (D614G) are shown in Table 8. Affinity maturation of clone 5A6 obtained more than 1000 fold affinity improvement for binding to SARS-CoV-2 S protein RBD and recovery of binding to SARS-CoV-2 S protein RBD mutants (V483A, and F490S). Clone 5A6 also showed binding improvement to trimeric SARS-CoV-2 S protein and trimeric SARS-CoV-2 (D614G) after affinity maturation.

[Table 8]

Notes: n.d. KD cannot be determined due to low binding response.

# kinetic constant kd is approaching the limit that can be measured by the instrument

* Biphasic binding, KD cannot be uniquely determined a Antibody was captured on a protein AG sensor surface and different concentration of specific antigen were injected . b Antigen was captured on an anti-His tag sensor surface and different concentration of each antibody were injected.

Size-exclusion chromatography (SEC) analysis was performed to measure high molecular weight species (HWM) and low molecular weight species (LWM). Each of the selected antibodies were transiently expressed twice and purified individually, and SEC analysis was carried out. The averages of HWM, Main, and LWM peaks were calculated as shown in Table 9. Engineered antibodies showed lesser amount of HMW compared to the parent antibody.

[Table 9]

Average of N=2

The full-length amino acid sequences of the antibody variable regions are shown in Table 10.

[Table 10] Full length amino acid sequence of variable region of antibody

EXAMPLE 3: Evaluation for PK profile in human FcRn transgenic mice

Pharmacokinetic profiles in human FcRn transgenic mice were evaluated. The results are shown in FIG. 1. FIG. 1 illustrates plasma concentration-time profiles of 5A6CCS1-SG1095ACT3 and 5A6CCS1-SG1095 (which are listed in Table 7) in human FcRn transgenic mice administrated with IVIG after intravenous administration. Antibody concentration was quantified by ECLIA with SARS-CoV-2 S Protein RBD from Aero Biosystems coated on plate and SULFO tag- labelled anti-human IgG which is recognized specific mutation of administrated antibody as a detection antibody.

5A6CCS1-SG1095ACT3 showed higher concentration than 5A6CCS1-SG1095 after 28 days administration. As shown in Table 11, AUC was 564 day*pg/mL in 5A6CCS1-SG1095ACT3 and 245 day*qg/mL in 5A6CCS1-SG1095. Half-life was 16.7 days in 5A6CCS1- SG1095ACT3 and 10.8 days in 5A6CCS1-SG1095. Clearance was 3.57 mL/day/kg in 5A6CCS1-SG1095ACT3 and 8.25 mL/day/kg in 5A6CCS1-SG1095. 5A6CCS1- SG1095ACT3 showed higher exposure, longer half-life, and lower clearance than 5A6CCS1- SG1095.

[Table 11] CL T1/2

mL/day/kg day

5A6CCS1-SG1095ACT3 564 3.57 16.7

5A6CCS1-SG1095 245 8.25 10.8

Table 11 shows area under the curve (AUC), half-life (T1/2) and total clearance of 5A6CCS1- SG1095ACT3 and 5A6CCS1-SG1095 in human FcRn transgenic mice administrated with IVIG after intravenous administration.

EXAMPLE 4.1: Biacore binding analysis of 5A6CCS1 to the RBD mutants

A list of RBD mutants reported in circulation was tested for binding to 5A6CCS 1 using Biacore 8K instrument (GE Healthcare). 5A6 and 5A6CCS1 were captured onto the protein A/G sensor surfaces to around 300RU capture level. Recombinant SARS-CoV-2 S protein RBD and mutants (K417N, N439K, K444R, G446V, Y453F, N481D, G482S, V483A, E484K, E484Q, G485S, F486S, N487R, F490S, N501Y, and triple RBD mutation K417N, E484K, N501Y) were injected at 100 nM concentrations. Binding responses of 5A6 and 5A6CCS1 to recombinant RBD wildtype or mutants were normalized to capture level.

FIG. 2 illustrates a relative binding of RBD mutants to RBD wildtype for 5A6 and 5A6CCS1. 5A6 showed less than 0.5 relative binding to RBD wildtype for RBD mutants V483A, E484K, F486S, F490S, and triple RBD mutation K417N, E484K, N501Y. 5A6CCS1 showed similar binding to RBD wildtype for all of the mutants, although the binding to E484K and the triple RBD mutation K417N, E484K, N501Y was slightly lower, while the relative binding to RBD wildtype was above 0.5.

EXAMPLE 4-2: Antibody neutralization assay with SARS-CoV-2 Spike glycoprotein pseudovirus

METHODS

Antibody neutralization assay with SARS-CoV-2 Spike glycoprotein pseudovirus CHO-ACE2 cells were seeded at a density of 3.2 x 10⁴ cells in 100 pL of complete medium without Geneticin in 96-well Flat Clear Bottom Black Polystyrene TC-treated Microplates (Corning, #3904). Serially diluted IgGs were incubated in a 96-well flat-bottom cell culture plate (Costar, #3596) with an equal volume of pseudovirus (6 ng of p24) at the final volume of 50pL at 37 degrees Celsius (deg C) for one hour, and the mixture was added to the monolayer of pre-seeded CHO ACE2 cells in duplicate. After one hour of pseudovirus infection at 37 deg C, 150p 1 of culture medium was added to each well and the cells were further incubated for another 48 hours. Upon removal of culture medium, cells were washed twice with sterile PBS, and then lysed in 20 pL of lx Passive lysis buffer (Promega, E1941) with gentle shaking at 400 rpm at 37 deg C for 30 minutes. Luciferase activity was then assessed using the Luciferase Assay System (Promega, E1510) on a Promega GloMax Luminometer. The relative luciferase units (RLU) were converted to percent neutralization and plotted with a nonlinear regression curve fit using PRISM (Graphpad).

To evaluate the neutralization potential of antibodies, pseudoviruses were generated expressing wildtype or mutant SARS-CoV-2 spike glycoprotein tagged with a luciferase reporter. Neutralization of pseuodovirus was evaluated using CHO cells stably expressing human ACE2 (CHO-ACE2) as targets. The following mutant pseudoviruses with single mutation were generated: K417N, V483A, E484K, N501Y, and D614G. In addition, pseudoviruses carrying multiple mutations were also generated: K417N/E484K/D614G (hereafter called South Africa triple mutation) and del69-70/dell44- 145/N501Y/A570D/D614G/P681H/T716FS982A/D1118H (hereafter called UK variant).

FIG. 3 illustrates the neutralization of SARS-CoV-2 pseudovirus by parental 5A6 and engineered 5A6CCS1 antibodies. As shown in FIG. 3, parental 5A6 antibody was not able to neutralize V483A and E484K single mutant pseudoviruses, and also the South Africa triple mutant which contains E484K mutation. In contrast, affinity improved 5A6CCS1 was able to neutralize V483A, E484K, and South Africa triple mutant viruses.

EXAMPLE 4-3: Antibody treatment of Golden Syrian Hamsters infected with SARS- CoV-2

METHODS

Antibody treatment of Golden Syrian Hamsters infected with SARS-CoV-2

Female Golden Syrian Hamsters (Janvier labs, France), 6-8 weeks old, were anesthetized and infected intranasally with 70pL of virus suspension containing 1 x 10⁵ pfu of SARS-CoV-2 (D614G) virus, strain Slovakia/SK-BMC5/2020 (European Virus Archive global). Six hours after infection, hamsters received an intraperitoneal injection of antibodies at the indicated dose. The left lungs were placed in RNAlater overnight at 4 deg C then stored at -80 deg C until RNA extraction for quantification of viral load by qRT-PCR. The superior, middle, post caval and inferior right lobes were snap frozen in liquid nitrogen, then stored at -80 deg C until processing for infectious particle detection (TCID50).

Viral load determination in lungs by qRT-PCR

Extraction of viral RNA was performed using QIAamp viral RNA mini kit (Qiagen), and RT- PCR was performed using SuperScript III One-Step qRT-PCR Kitl (Life Technologies, #11732). SARS-CoV-2 RNA was quantitated using IP2 and IP4 primers which detect the RNA dependent RNA polymerase gene (RdRp) and ORFlab gene detection primers. The sequence of forward, reverse primers and control probe are provided in Table 12.

[Table 12] Primers used for detection of SARS-CoV-2 RNA

RdRp gene / nCoV_IP nCoV_IP2-12669Fw ATGAGCTTAGTCCTGTTG nCoV_IP2-12759Rv CTCCCTTTGTTGTGTTGT nCo V_IP2- 12696b Probe(+) AGATGTCTTGTGCTGCCGGTA [5']Hex [3']BHQ-1

RdRp gene / nCoV_IP4 nCoV_IP4-14059Fw GGTAACTGGTATGATTTCG nCoV_IP4-14146Rv CTGGTCAAGGTTAATATAGG nCo V_IP4- 14084Probe(+) TCATACAAACCACGCCAGG [5']Fam [3']BHQ-1

ORFlab gene / nCoV

ORFlab_Fw CCGCAAGGTTCTTCTTCGTAAG

ORFlab_Rv TGCTATGTTTAGTGTTCCAGTTTTC ORFlab_probe AAGGATCAGTGCCAAGCTCGTCGCC [5']Hex [3'] BHQ-1

FIG. 4 illustrates lung viral titre of hamsters infected with live SARS-CoV-2 virus after treatment with 5A6CCS1 antibody.

To confirm if the engineered 5A6CCS1 antibody had neutralizing potency against live SARS- CoV-2 virus, the efficacy of the antibody was tested in a hamster model of SARS-CoV-2 infection. Hamsters were intranasally infected with 1 x 10⁵ pfu of SARS-CoV-2 (D614G) virus, strain Slovakia/SK-BMC5/2020, and 6 hours later, 5A6CCS1 antibody was administered intraperitoneally. For control antibody, IC17-hIgGl which binds to an irrelevant antigen (KLH) was administered. The hamsters were sacrificed 4 days later, and lungs were harvested to determine the viral load. As shown in FIG. 4, 5A6CCS 1 significantly reduced lung viral load as determined by qRT-PCR of the SARS-CoV-2 RNA-dependent RNA polymerase gene and the ORFlab gene in a dose dependent manner.

EXAMPLE 5: Neutralization of SARS-CoV-2 pseudo viruses of various Variants of Concern (VOCs) and Variants of Interest (VOIs) by parental 5A6 and engineered 5A6CCS1 antibodies.

FIG. 5 illustrates neutralization of SARS-CoV-2 pseudoviruses of various VOCs and VOIs (Beta, Gamma, Kappa, Delta and Epsilon) by parental 5A6 and engineered 5A6CCS 1 antibodies.

As shown in FIG. 5, parental 5A6 antibody was not able to neutralize the Beta (B.1.351) or Gamma (P.l) variants which both contain E484K mutation, or Kappa (B.1.617.1) variant which contains E484Q mutation. In contrast, affinity improved 5A6CCS 1 was able to neutralize these three mutant pseudoviruses. Moreover, affinity improved 5A6CCS1 also showed enhanced potency in neutralizing Delta (B.1.617.2) variant and Epsilon (B.1.429) variant pseudoviruses.

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

The present disclosure provides affinity matured SARS-CoV-2-binding molecules with improved physicochemical property, SARS-CoV-2-binding molecules that can increase the clearance of the coronavirus of interest while reducing the risk of ADE, methods for producing the antigen-binding molecules, pharmaceutical compositions comprising such SARS-CoV-2- binding molecule as an active ingredient, and therapeutic methods using the pharmaceutical compositions.

As demonstrated in the examples, to identify mutations that improve affinity to SARS-CoV-2, variants were generated from the parent antibody 5A6 (which is an anti-SARS-CoV-2 spike (S) protein receptor binding domain (RBD) antibody). Furthermore, to reduce the insolubility of the antibody, further engineering was carried out by introducing mutations that improve physicochemical properties. The resulting variants showed useful characteristics such as enhanced affinity to SARS-CoV-2 S protein RBD, trimeric SARS-CoV-2 S protein and several SARS-CoV-2 S protein mutants, as well as increased solubility.

Claims

[CLAIMS] WHAT IS CLAIMED IS:

1. An isolated antibody that binds to SARS-CoV-2, wherein the antibody comprises:

(vi) HVR-L3 comprising the amino acid sequence QXiSYNLPRT, wherein Xi is E or Q (SEQ ID NO: 114).

2. The antibody of claim 1, wherein the antibody comprises:

3. An isolated antibody that binds to SARS-CoV-2, wherein the antibody comprises:

(a) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:88, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 90;

(b) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:91, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93; (c) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:85, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 87;

(e) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:88, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 90;

(f) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:91, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93;

(g) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:85, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 87;

(i) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:88, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 89, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 90;

(k) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:85, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 87;

(n) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 64, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 65, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(o) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 76, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 78, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(p) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, (iii) HVR-H3 comprising the

115 amino acid sequence of SEQ ID NO: 75, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83 (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(q) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84;

(r) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 100, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 102;

(s) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:97, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 98, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 99;

(v) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 71, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72, (iv) HVR-L1 comprising the amino acid

116 sequence of SEQ ID NO:79, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81;

(x) (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 64, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 65, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 100, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 101, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 102.

4. The antibody of any one of claims 1 to 3, comprising (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2 to 7; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 42 and 44 to 52; or (c) the VH sequence as in (a) and the VL sequence as in (b).

5. The antibody of claim 4, comprising a VH sequence of any one of SEQ ID NOs: 2 to 7.

6. The antibody of claim 4 or 5, comprising a VL sequence of any one of SEQ ID NOs: 42 and 44 to 52.

7. An isolated antibody that binds to SARS-CoV-2, wherein the antibody comprises:

(a) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 46,

(b) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 47,

(c) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 45,

(d) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 48,

(e) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 46,

(f) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 47,

(g) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 45,

(h) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 48,

(i) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 46,

(j) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 47,

(k) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 45,

(l) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 48,

117 (m) a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 49,

(n) a VH sequence of SEQ ID NO: 3 and a VL sequence of SEQ ID NO: 44,

(o) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 44,

(p) a VH sequence of SEQ ID NO: 6 and a VL sequence of SEQ ID NO: 44,

(q) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 44,

(r) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 50,

(s) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 49,

(t) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 51,

(u) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 52,

(v) a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 42,

(w) a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 42, or

(x) a VH sequence of SEQ ID NO: 3 and a VL sequence of SEQ ID NO: 50,

8. The antibody of any one of claims 1 to 7, wherein the antibody further comprises a variant Fc region comprising at least one amino acid alteration to a parent Fc region, wherein, when compared to the parent Fc region, the variant Fc region has a substantially decreased Fc gamma R-binding activity and has a maintained or increased Clq-binding activity.

9. The antibody of claim 8, wherein the variant Fc region comprises Ala at position 234 according to EU numbering and Ala at position 235 according to EU numbering.

10. The antibody of claim 8 or 9, wherein the variant Fc region comprises amino acid alterations at positions of any one of the following (a)-(c):

(a) positions 267, 268, and 324;

(b) positions 236, 267, 268, 324, and 332; and

(c) positions 326 and 333, wherein the positions are according to EU numbering.

11. The antibody of claim 10, wherein the variant Fc region comprises an amino acid(s) selected from the group consisting of (a) to (g) below:

(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, wherein the positions are according to EU numbering.

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12. The antibody of any one of claims 8 to 11, wherein the variant Fc region comprises an amino acid(s) selected from the group consisting of (a) to (f) below:

(a) Ala at position 434;

(c) Leu at position 428, Ala at position 434, and Thr at position 436

(d) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position

438, and Glu at position 440;

(e) Leu at position 428, and Ala at position 434; and

(f) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440; wherein the positions are according to EU numbering.

13. A pharmaceutical composition comprising the antibody of any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

14. The antibody of any one of claims 1 to 12 or the pharmaceutical composition of claim 13 for use in treatment and/or prevention of a SARS-CoV-2 infection.

15. A method for treating a SARS-CoV-2 infection, comprising administering to an individual having the SARS-CoV-2 infection an effective amount of the antibody of any one of claims 1 to 12 or the pharmaceutical composition of claim 13.

16. Use of the antibody of any one of claims 1 to 12 or the pharmaceutical composition of claim 13 in the manufacture of a medicament for treatment and/or prevention of a SARS-CoV- 2 infection.

17. An isolated nucleic acid encoding the antibody of any one of claims 1 to 12.

18. A host cell or vector comprising the nucleic acid of claim 17.

19. A method of producing the antibody of any one of 1 to 12, comprising culturing the host cell of claim 18.

20. A method of detecting the presence of SARS-CoV-2 in a sample, the method comprising: contacting the sample with the antibody of any one of 1 to 12 under conditions permissive for binding of the antibody to SARS-CoV-2; detecting whether a complex is formed between the antibody and SARS-CoV-2; wherein the formation of a complex is indicative of the presence of SARS-CoV-2 in the sample.

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