Variant hinge and molecules comprising same

Abstract

Provided herein are variant hinge sequences, Fc variants comprising variant hinge sequences, and one-armed antibodies comprising variant hinge sequences.

Description

VARIANT HINGE AND MOLECULES COMPRISING SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of US provisional application no. 63 / 633,504, filed April 12, 2025, the disclosure of which is hereby incorporated by reference herein.FIELD

[0002] The present disclosure relates to variant hinge sequences, Fc variants comprising variant hinge sequences, and one-armed antibodies comprising variant hinge sequences.REFERENCE TO SEQUENCE LISTING

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification as a WIPO Standard ST.26 formatted XML file with file name “09402- 102WO1.xml”, a creation date of April 10, 2024, and a size of 616,508 bytes.BACKGROUND

[0004] Antibodies are employed in a wide range of applications, including as therapeutics for various diseases, including cancers, immune disorders, and infectious diseases. Typically, such antibodies are bivalent, binding two antigens at two antigen binding sites. In addition to this traditional format, alternate antibody formats having varying symmetries, valencies, Fc amendments, and other customizations have been developed to better serve differing systems and / or uses. Despite the availability of available antibody formats, there still exist applications for which a new antibody format would provide a best solution.SUMMARY

[0005] The present disclosure relates to variant hinge sequences, Fc variants comprising variant hinge sequences, and one-armed antibodies comprising variant hinge sequences. This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0006] Provided herein are variant hinge regions, comprising a first amino acid chain of a hinge region of a reference IgG 1 comprising amino acids 216-230 of the reference IgG 1 and a second amino acid chain of the hinge region of the reference lgG1 consisting of amino acids 223-230 of the reference IgG 1 according to the Eu numbering system.

[0007] Also provided herein are variant hinge regions, comprising a first amino acid chain of a hinge region of a reference lgG1 comprising amino acids 216-230 of the reference lgG1 and a second amino acid chain of the hinge region of the reference lgG1 comprising aminoacids 223-230 of the reference lgG1, comprising deletion of amino acids 216-222 of the second amino acid chain, according to the Eu numbering system.

[0008] Also provided herein are variant hinge regions, comprising a first amino acid chain of a hinge region of a reference lgG1 and a second amino acid chain of the hinge region of the reference IgG 1 , and comprising one or more amino acid substitutions selected from the group consisting of: C226A, C226S, C226G, C229A, C229S, and C229G according to the Eu numbering system.

[0009] Also provided herein are variant hinge regions comprising a first amino acid chain of a hinge region of a reference lgG1 and a second amino acid chain of the hinge region of the reference lgG1 , and comprising the amino acid substitutions T223A and T225A according to the Eu numbering system.

[0010] Also provided herein are variant hinge regions, comprising a first amino chain of a hinge region of a reference IgG 1 and a second amino acid chain of the hinge region of the reference lgG1 , and comprising deletion of K222 through T225 of the reference lgG1 according to the Eu numbering system.

[0011] In some embodiments, the hinge region of the reference IgG 1 has a sequence of SEQ ID NO: 8.

[0012] Also provided herein are Fc variant comprising the variant hinge region provided herein. Provided Fc variants herein comprise variant hinge regions comprising at least one of (i) a first amino acid chain of a hinge region of a reference IgG 1 comprising amino acids 216-230 of the reference IgG 1 and a second amino acid chain of the hinge region of the reference IgG 1 consisting of amino acids 223-230 of the reference IgG 1 according to the Eu numbering system, (ii) a first amino acid chain of a hinge region of a reference lgG1 comprising amino acids 216-230 of the reference lgG1 and a second amino acid chain of the hinge region of the reference lgG1 comprising amino acids 223-230 of the reference lgG1 , comprising deletion of amino acids 216-222 of the second amino acid chain, according to the Eu numbering system, (iii) a first amino acid chain of a hinge region of a reference lgG1 and a second amino acid chain of the hinge region of the reference lgG1 , and comprising one or more amino acid substitutions selected from the group consisting of: C226A, C226S, C226G, C229A, C229S, and C229G according to the Eu numbering system, (iv) a first amino acid chain of a hinge region of a reference IgG 1 and a second amino acid chain of the hinge region of the reference lgG1 , and comprising the amino acid substitutions T223A and T225A according to the Eu numbering system, and (v) a first amino chain of a hinge region of a reference IgG 1 and a second amino acid chain of the hinge region of the reference lgG1 , and comprising deletion of K222 through T225 of the reference IgG 1 according to the Eu numbering system.

[0013] In some embodiments, the Fc variant has a first heavy chain comprising the first amino acid chain of the variant hinge region and a second heavy chain comprising the second amino acid chain of the variant hinge region. In some embodiments, the first heavy chain comprises a hinge sequence, CH1 , CH2, and CH3. In some embodiments, the Fc variant of any of claims 5-8, further comprising one or more amino acid substitutions selected from the group consisting of: C226A, C226S, C226G, C229A, C229S, and C229G according to the Eu numbering system.

[0014] In some embodiments, the first chain of the variant hinge region comprises (i) C226A, (ii) C226S, (iii) C229A, (iv) C229S, (v) C226A and C229A, (vi) C226A and C229S, (vii) C226S and C229A, or (viii) C226S and C229S.

[0015] In some Fc variants, the second heavy chain is a shortened heavy chain. In some Fc variants, the second heavy chain lacks a CH1 segment. In some Fc variants, the second heavy chain comprises CH2 and CH3.

[0016] In some embodiments, the second chain of the variant hinge region comprises (i) C226A, (ii) C226S, (iii) C229A, (iv) C229S, (v) C226A and C229A, (vi) C226A and C229S, (vii) C226S and C229A, or (viii) C226S and C229S.

[0017] In some Fc variants, the first heavy chain comprises amino acids 216-446 (residues of hinge, CH2, CH3) of an IgG 1 Fc region. In some Fc variants, the second heavy chain comprises amino acids 223-446 (residues of CH2 and CH3) of an lgG1 Fc region.

[0018] In some embodiments, the second heavy chain further comprises amino acids 216- 230 (residues of hinge) of an IgG Fc region.

[0019] In some embodiments, the Fc is of an lgG1 . In further embodiments, the IgG 1 is a human lgG1. In some embodiments, the IgG is a human lgG1 allotype. In some embodiments, the human IgG 1 allotype comprises the first heavy chain sequence SEQ ID NO:1 and the second heavy chain sequence. In some embodiments, the Fc comprises CH1 , CH2, or CH3 of an IgG, an IgM, an IgD, an IgA, or an IgE.

[0020] In some embodiments, a variant hinge region, Fc variant, or one-armed antibody comprises an amino acid substitution of Thr residues in the variant hinge region. In some embodiments, the first chain of the variant hinge region and the second chain of the variant hinge region comprise T225A and T223A.

[0021] In some embodiments, the first chain of the variant hinge region comprises the deletion of amino acids 222 through 225 and the second chain of the variant hinge region comprises deletion of amino acids 223 through 225.

[0022] In some embodiments, the Fc variant comprises a light chain constant region (CL) . In some embodiments, the CL sequence is associated with CH1 of the first heavy chain. In some embodiments, the CL sequence is of SEQ ID NO: 7.

[0023] In some embodiments, the Fc variant comprises an effectorless mutation. In some embodiments, the effectorless mutation is in the CH2 domain. In some embodiments, the effectorless mutation is selected from the group consisting of LALA, LALAPG, LALAGA, and LAGA. In some embodiments, the effectorless mutation is LALAGA.

[0024] In some embodiments, the Fc variant comprises a knob-in-hole structure. In some embodiments, the knob-in-hole structure comprises (i) T366S, L368A, and Y407V amino acid substitutions on the first heavy chain, and (ii) a T366W amino acid substitution on the second heavy chain. In some embodiments, the knob in hole structure comprises mutations in the CH3 domain.

[0025] In some embodiments, the Fc variant further comprises an N297G amino acid substitution

[0026] In some embodiments, the Fc variant is an Fc variant of an Fc fusion.

[0027] Also provided herein are antibodies comprising a variant hinge region or an Fc variant provided herein. In some embodiments, the antibody is a one-armed antibody. In some embodiments, the antibody is an antigen binding fragment of an antibody, a bispecific antibody, a fusion of an antibody or antigen binding fragment thereof and another protein, a fusion of a fragment of a first antibody, or a fragment of a second antibody.

[0028] Also provided herein are isolated nucleic acid sequences encoding hinge regions, Fc variants, and antibodies provided herein.

[0029] Also provided herein are isolated cells comprising the nucleic acids provided herein.

[0030] Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of an antibody provided herein and an excipient.

[0031] In some Fc variants, the first heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-169 and 495-548, and the second heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 170-563. In some Fc variants, the first heavy chain and second heavy chain are selected from the first heavy chain and second heavy chain pairs provided in Table 12. In some Fc variants, the first heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-169 and 495-548, and the second heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 170-563.

[0032] In some antibodies herein, the first heavy chain and second heavy chain are selected from the first heavy chain and second heavy chain pairs provided in Table 12.

[0033] Also provided herein are Fc variants and one-armed antibodies, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein HC1 comprises: a CH1 domain, a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 , comprising the amino acid substitutions C226A, T223A and T225A, a CH2 domain comprising a LALAGA mutation, and a CH3 comprising a holemutation; and HC2 comprises: a hinge comprising an amino acid chain consisting of amino acids 236-230 of a reference lgG1 , comprising the amino acid substitution C226A, a CH2 domain comprising a L235A mutation, and a CH3 comprising a knob mutation.

[0034] Also provided herein are Fc variants and one-armed antibodies, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein HC1 comprises: a CH1 domain, a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 , comprising the amino acid substitutions C226A, C229A, T223A and T225A, a CH2 domain comprising a LALAGA mutation, and a CH3 comprising a hole mutation; and HC2 comprises: no hinge, a CH2 domain comprising deletion of A231 through L234 and a LALAGA mutation, and a CH3 domain comprising a knob mutation.

[0035] Also provided herein are Fc variants and one-armed antibodies, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein HC1 comprises: a CH1 domain, a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 comprising the amino acid substitution C226A and a deletion of K222 through T225, a CH2 domain comprising a LALAGA mutation, and a CH3 comprising a hole mutation; and HC2 comprises: a hinge comprising an amino acid chain consisting of amino acids 236-230 of a reference IgG 1 , comprising the amino acid substitution C226A, a CH2 domain comprising a LALAGA mutation, and a CH3 comprising a knob mutation.

[0036] An Fc variant or one-armed antibody, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein HC1 comprises: a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 comprising the amino acid substitutions C226A and C229A and a deletion of K222 through T225, a CH2 domain comprising a LALAGA mutation, and a CH3 comprising a hole mutation; and HC2 comprises: no hinge, a CH2 domain comprising deletion of A231 through L234 and a LALAGA mutation, and a CH3 domain comprising a knob mutation, and wherein HC1 and HC2 comprise a C226A amino acid substitution and a deletion of K222 through T225, a LALAGA effectorless mutation

[0037] Also provided herein are Fc variants and one-armed antibodies comprising: an HC1 having an amino acid sequence of SEQ ID NO: 153 and an HC2 having an amino acid sequence of SEQ ID NO: 378; an HC1 having an amino acid sequence of SEQ ID NO: 155 and an HC2 having an amino acid sequence of SEQ ID NO: 554; an HC1 having an amino acid sequence of SEQ ID NO: 162 and an HC2 having an amino acid sequence of SEQ ID NO: 378; or an HC1 having an amino acid sequence of SEQ ID NO: 164 and an HC2 having an amino acid sequence of 554.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0039] FIG. 1 depicts a representative SDS-PAGE gel having bands corresponding to the heavy chain (HC), light chain (LC) and heterogeneity of Fc (Fc).

[0040] FIG. 2 depicts reduced SDS-PAGE gels comprising antibody having received deglycosidase treatment, which appears to reduce heterogeneity.

[0041] FIG. 3A, FIG. 3B, and FIG. 3C depict representative LC-MS analysis plots. FIG. 3A depicts a plot showing Intact mass peaks of the xh2DQJ ‘Intact hinge, N297G’ (PUR4914) showing Fc-HC-LC plus a 948.5 Da, which matches with mass of one of the most common O-glycans with the composition of 1 Gal, IGalNac, and 2 Sialic acids. FIG. 3B and FIG. 3C depict a representative LC-MS analysis plot (PUR4914 shown) split into two panels. FIG. 3B panel includes mass peaks less than 35000, and FIG. 3C panel includes mass peaks from 35000 to 57000 showing the HC peak. As shown in FIG. 3B panel, the PUR4914 sample treated with PNGase F followed by reduction with DDT, has peaks indicating the presence of O-glycosylation on the Fc chain (Fc+947.9Da).

[0042] FIG. 4A and FIG. 4B depict LC / MS analysis plots for an original length hinge variant (FIG. 4A) of a representative one-armed antibody, and a shorter hinge variant (FIG. 4B), wherein clipping of the antibody can be seen in the original length variant but not in the shorter length variant.

[0043] FIG. 5A and FIG. 5B depict SEC-MALS results comprising SEC chromatograms with percentage composition labeled, together with molar masses provided by MALS listed in the table inset into each panel. FIG. 5A panels represent (top to bottom): a one armed antibody with a short hinge lacking a second amino acid chain in the hinge, a one armed antibody with a hinge lacking a second amino acid chain in the hinge. FIG. 5B panels represent (top to bottom): a one armed antibody with a short hinge comprising a truncated second amino acid chain in the hinge, and a one armed antibody with a hinge comprising a truncated second amino acid chain in the hinge.DETAILED DESCRIPTION OF THE INVENTION

[0044] For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody” includes more than one antibody, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection withthe recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.”

[0045] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0046] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011 , and now available in journal format online as Current Protocols in Molecular Biology, Vols. 00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”).

[0047] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For purposes ofinterpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.

[0049] Throughout this specification, amino acid residues in amino acid sequences are numbered according to the Eu numbering convention (unless otherwise noted).

[0050] Variant hinge regions and molecules comprising same

[0051] Provided herein are hinge regions that are variant hinge regions of an lgG1 hinge region (i.e., the hinge region of a reference lgG1). Variant hinge regions can comprise modifications relative to the reference IgG 1 hinge region, which can yield improvements in one or more properties of the antibodies. For example, select improvements are discussed herein including reduction in heterogeneity, reduction in o-glycosylation, or other improvements of an Fc region or one-armed antibody compared with an Fc region or one- armed antibody not having said improvements. Variant hinge regions provided herein can also comprise other improvements not discussed.

[0052] A variant hinge region can comprise a first amino acid chain of a hinge region of a reference lgG1 and a second amino acid chain of the hinge region of the reference IgG 1 . In some embodiments, the hinge region of the reference lgG1 can have a sequence of SEQ ID NO: 8, though other reference sequences are envisioned. In some embodiments, a reference sequence can be one of SEQ ID NOs: 8-61 , or another sequence of an IgG hinge. A variant hinge region can comprise one or more modifications to one or more of the first and second amino acid chains, such as modifications described herein. In some embodiments, the first amino acid chain of the variant hinge region has a sequence of one of SEQ ID NOS.: 8-34. In some embodiments, the second amino acid chain of the variant hinge region has a sequence of one of SEQ ID NOS.: 9-61. In some embodiments, first and second amino acid chains provided hereinbelow of variant hinge regions can be selected from a pair of amino acid chains in Table 9.

[0053] Also provided herein are Fc variants, wherein an Fc variant is a variant of an Fc domain. An “Fc domain” refers to the tail region of an antibody or antigen binding fragment thereof. While an Fc domain can be of an IgG, IgA, IgD, IgM, or IgE isotype, Fc variants provided herein are typically of an IgG isotype.

[0054] An Fc variant herein can comprise a first heavy chain (HC1) and a second heavy chain (HC2). The first heavy chain of an Fc variant can comprise the first amino acid chain of a variant hinge region provided herein, and the second heavy chain of the Fc variant can comprise the second amino acid chain of the variant hinge region.

[0055] An Fc variant can lack an N terminal constant heavy chain domain or fragment thereof. In some embodiments, the Fc variant can lack a CH1 domain (or a fragment thereof). Notably, the second heavy chain of an Fc variant can lack a CH1 domain. Typically, the CH1 domain of the first heavy chain of the Fc variant is present, and is appended to theN terminus of the first amino acid chain of the variant hinge region. Typically, the CH1 can have a sequence of an IgG CH1 ; a CH1 herein can in some embodiments have a sequence of SEQ ID NO.: 1.

[0056] A first or second heavy chain of an Fc variant can comprise a constant heavy chain or fragment thereof appended to the C terminus of the first or second amino acid chain of the variant hinge region. Such an Fc variant can comprise a CH2 appended to the C terminus of the variant hinge region, and can further comprise a CH3 appended to the C terminus of the CH2. In some such cases, the Fc variant can be of an IgG (e.g., an lgG1). A CH1 or CH2 can have a sequence of a reference IgG (e.g., SEQ ID NOS.: 2 and 4, respectively) or can have one or more modifications such as those provided hereinbelow (for example but not limited to SEQ ID NOS: 3, 5, or 6).

[0057] An Fc variant herein can further comprise a light chain constant region (LC) that associates with the CH1 (e.g., of the first heavy chain). Typically, the LC domain can have a sequence of an IgG LC; an LC herein can in some embodiments have a sequence of SEQ ID NO.: 7.

[0058] A CH1 , CH2, and / or CH3 domain of an Fc variant herein can be of an immunoglobulin (Ig), such as an IgG, an IgM, an IgD, an IgA, or an IgE. In some cases, an Fc variant herein can be of an immunoglobulin (Ig), such as an IgG, an IgM, an IgD, an IgA, or an IgE. In some embodiments, an Fc variant can be of an IgG that in an IgG 1 , an lgG2, an lgG3, or an lgG4. Notably, an Fc variant can be of an IgG 1 .

[0059] An Fc variant can be of a human Ig allotype, such as a human allotype of an IgG (e.g., IgG 1 , lgG2, lgG3, or lgG4), an IgM, and IgD, an IgA, or an IgE. Notably, an Fc variant can be of a human lgG1 allotype. For example, a human lgG1 allotype can comprise the first heavy chain sequence of the Fc region of a human lgG1 , (e.g., SEQ ID NO: 1) and the second heavy chain sequence of the Fc region of a human IgG 1 (e.g., SEQ ID NO: 2). In some embodiments, an Fc variant can be of an allotype that is not human.

[0060] A Fc variant herein can be a variant of an IgG Fc region, such as an IgG 1 Fc region. The first heavy chain of such an Fc variant can comprise amino acids 216-446 of an IgG Fc region (e.g., and IgG 1 Fc region), which can correspond to the residues of the hinge, CH2, and CH3 of the first heavy chain of the Fc variant. The second heavy chain of such an Fc variant can comprise amino acids 231-446 of an IgG Fc region (e.g., and IgG 1 Fc region), which can correspond to the residues of the CH2, and CH3 of the second heavy chain of the Fc variant. In some embodiments, the second heavy chain of an Fc variant comprising amino acids 231-446 of an IgG 1 Fc region can further comprise amino acids 223-230 of an IgG Fc region (e.g., an lgG1 Fc region), which can correspond to residues of the hinge of the second heavy chain of the Fc variant.

[0061] The first heavy chain of an Fc variant herein can comprise a first amino acid chain of a variant hinge region provided herein and an additional constant heavy chain domain or fragment thereof. In some embodiments, for example, the Fc variant can comprise a first amino acid chain of a variant hinge region and CH1, and can further comprise CH2. In some embodiments, the Fc variant can comprise a first amino acid chain of a variant hinge region and CH2 and can further comprise CH3. In some embodiments, the Fc variant can comprise a first amino acid chain of a variant hinge region, CH1 , CH2, and CH3. Typically, a CH1 can be attached by a peptide bond to the N terminus of first amino acid chain of a variant hinge region, which can be attached by a peptide bond to the N terminus of a CH2, which can be attached by a peptide bond to the N terminus of a CH3. In some cases, the Fc variant can be of an IgG (e.g., an lgG1 ). In some embodiments, an Fc variant can comprise a first heavy chain of one of SEQ ID NOS.: 62-169 and a second heavy chain of one of SEQ ID NOS.: 170-386. In some embodiments, first and second heavy chains provided hereinbelow of Fc variants can be selected from a pair of amino acid chains in Table 12.

[0062] An Fc domain provided herein can be of a one-arm antibody. A one arm antibody can be an antibody comprising an Fc variant provided herein, one variable heavy chain domain (VH), and one variable light chain domain (VL). The one arm antibody can comprise a first heavy chain and a second heavy chain, wherein the first heavy chain of the one arm antibody can comprise the first heavy chain of the Fc variant and the second heavy chain of the one arm antibody can comprise the second heavy chain of the Fc variant. The VH can be attached by a peptide bond to the N terminus of the first heavy chain of the one arm antibody, for example to the N terminus of a CH1 , and the VL can be attached by a peptide bond to the N terminus of a CL associated with the first heavy chain of the one arm antibody. The VH and VL can be associated with each other to provide an antigen binding domain. Typically, the second heavy chain of a one arm antibody will not comprise a VH or VL. Further, the second heavy chain of a one arm antibody may not comprise a CH1 or a CL. Generally, one arm antibodies provided herein can be devoid of a second antigen binding domain typically present in a standard bivalent antibody format. However, embodiments wherein an additional antigen binding domain or other domain(s) is fused with an Fc variant provided herein are contemplated (e.g., Fc fusion proteins or similar).

[0063] In some embodiments, a one-armed antibody can be produced, for example in a manner similar to that of an IgG, including but not limited to a hybridoma method, recombinant DNA methods, phage- display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other exemplary methods for making monoclonal antibodies being described herein.

[0064] In some embodiments, an Fc variant or one-armed antibody can be humanized. A “Humanized” Fc variant or one-armed antibody can be engineered to have its CDRs derivedfrom a non-human donor immunoglobulin , the remaining immunoglobulin parts of the molecule being derived from one or more human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. A suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of a donor antibody. The acceptor antibody can be heterologous to the donor antibody, and in some embodiments can be a human antibody. The donor antibody can contribute the amino acid sequences of its variable region, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner. A donor can provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and / or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating heavy and / or light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.

[0065] A humanized Fc variant herein can comprise one or both of a humanized first heavy chain and a humanized second heavy chain. A humanized first heavy chain can comprise one, two, three, or all of a humanized CH1 domain, a humanized first hinge region, a humanized CH2 domain, and a humanized CH3 domain. Further, a humanized Fc variant comprising a CL1 domain can comprise a humanized CL1 domain.

[0066] A heavy chain of a humanized Fc variant may be humanized for example using the human germline sequence selected from IGHV3-48, IGHV7-4-1 and IGHV1-2. A humanized CL1 domain may be humanized for example using a human germline sequence selected from IGKV1-33, IGKV3-11 and IGKV1-16.

[0067] In some embodiments, a variant hinge region, Fc variant, or one-armed antibody provided herein can be human. A human antibody can be obtained using one or more of a variety of methods, for example using yeast-based libraries or transgenic animals (e.g., mice) that are capable of producing repertoires of human antibodies. Yeast presenting human antibodies on their surface that bind to an antigen of interest can be selected using FACS (Fluorescence-Activated Cell Sorting) based methods or by capture on beads using labeled antigens. Transgenic animals that have been modified to express human immunoglobulin genes can be immunized with an antigen of interest and antigen-specific human antibodies isolated using B-cell sorting techniques. Human antibodies produced using these techniques can then be characterized for properties such as affinity, developability and selectivity.

[0068] A one-armed antibody can provide a benefit over another antibody format. For example, a target of an antibody may be agonized upon dimerization. In some such cases, a standard antibody with two binding sites can bind to a target molecule at each of two binding sites. This can bring the two target molecules in close proximity, which can promote dimerization and thus agonistic activity. This agonistic activity can hinder a desired activity (e.g., antagonistic) of an antibody. Such agonistic activity can be ameliorated by use of an antibody that has only one binding site available to bind the target. For example, a one- armed antibody can bind to one target molecule at one binding site, thereby reducing a propensity for dimerization, yielding a reduction in agonistic activity.

[0069] Agonism of a target molecule (e.g., upon dimerization) by binding to a standard antibody (i.e., having two binding sites in close proximity) can be reduced when the target molecule is instead bound to a one-armed antibody, such as provided herein. This can occur when the one-armed antibody binds to the same epitope on the target molecule, or when the one-armed antibody binds to a different epitope on the target molecule. Agonism of a target molecule bound to a one-armed antibody can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower than agonism of the target molecule bound to a standard antibody having affinity for the same epitope. In some cases, agonism of a target molecule bound to a one-armed antibody can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower than agonism of the target molecule bound to a standard antibody having affinity for a different epitope. In some cases, agonism of a target molecule bound to a one-armed antibody can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% lower than agonism of the target molecule bound to a standard antibody having affinity for a an epitope that overlaps with the epitope of the one-armed antibody, e.g., by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0070] An Fc region provided herein, a molecule comprising an Fc region provided herein such as a one-armed antibody, can be isolated. An “isolated” molecule can be removed from an environment, such as an environment in which it can be found in nature, or an environment in which it is otherwise generated. For example, such a molecule may be purified away from substances with which it would normally exist in nature. In some such cases, the molecule can be at least about 90% pure, at least about 95% pure, at least about 99% pure, or about 100% pure.

[0071] In some embodiments, a variant hinge region, Fc variant, or one-armed antibody provided herein can be produced using recombinant methods and materials known in the art of antibody production. In some embodiments, the present disclosure provides an isolated nucleic acid encoding a variant hinge region, Fc variant, or one-armed antibody herein. The nucleic acid can encode an amino acid sequence of the first heavy chain, an amino acidsequence of the second heavy chain, and / or an amino acid sequence of a light chain of the variant hinge region, Fc variant, or one-armed antibody. In some embodiments, one or more vectors (e.g., expression vectors) comprising nucleic acid sequences encoding a variant hinge region, Fc variant, or one-armed antibody herein are provided. In some embodiments, a host cell comprising nucleic acid sequences encoding an anti-CD200R1 antibody of the present disclosure are provided. In some embodiments, the host cell is transformed with a vector comprising a nucleic acid that encodes an amino acid sequence herein. In some embodiments, the host cell is transformed with a vector comprising a first nucleic acid that encodes an amino acid sequence of the first heavy chain and a second nucleic acid that encodes an amino acid sequence of the second heavy chain of a variant hinge region, Fc variant, or one-armed antibody herein. In some embodiments, the host cell is transformed with a first vector comprising a nucleic acid that encodes an amino acid sequence of the first heavy chain of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence of the second heavy chain of a variant hinge region, Fc variant, or one-armed antibody herein. In further embodiments, the host cell is transformed with a third vector comprising a nucleic acid sequence that encodes an amino acid sequence of a light chain of a Fc variant or one-armed antibody herein.

[0072] Also provided herein are methods of producing variant hinge regions, Fc variants, and one-armed antibodies. Methods can comprise contacting a cell, tissue or organism with an isolated nucleic acid, polynucleotide, or vector encoding a variant hinge region, Fc variant, or one-armed antibody herein. In some embodiments, a cell of such methods can be other than E.coli, such as a eukaryotic cell. Such methods can further comprise culturing and / or growing said cell, tissue, or organism. In some embodiments, methods can further comprise inducing expression of the variant hinge region, Fc variant, or one-armed antibody. In some embodiments, methods can comprise isolating said variant hinge region, Fc variant, or one-armed antibody.

[0073] Modifications of variant hinge regions and molecules comprising same

[0074] In some embodiments, one or more modifications of an Fc variant or one-armed antibody provided herein or a composition thereof can provide optimization of one or more properties. For example, modifications can contribute to optimization of properties that can facilitate or enable production, such as the percentage of Fc variant or one-armed antibody in a composition produced that is a monomer, the stability of the Fc variant or one-armed antibody (e.g., the thermal stability of the Fc region), or the reduce heterogeneity in the Fc variant or one-armed antibody in a composition. Some modifications can remove liabilities (e.g., clipping) that can arise during production of an Fc variant or one-armed antibody. Further, additional modifications can be employed to ensure correct pairing of the first heavy chain and the second heavy chain of an Fc variant or one-armed antibody.

[0075] Compositions comprising an Fc variant or one-armed antibody provided herein can include the Fc variant or one-armed antibody as a monomer (i.e., existing by itself and not aggregated with another Fc variant, one-armed antibody, or other molecule). It can be beneficial for monomer content of such a composition to be improved (e.g., increased) by modifications of an Fc variant or one-armed antibody. Monomer content can be determined as the percentage of monomer of an Fc region or one-armed antibody provided herein present in a composition comprising the Fc region or one-armed antibody.

[0076] In some embodiments, one or more modifications such as provided herein can improve (e.g., increase) monomer content of a composition. Modifications that can improve monomer content can include modification of disulfide bonds in the variant hinge region and / or effectorless mutations (e.g., as provided herein) of the Fc variant. In some cases, one or more other modifications herein can improve monomer content.

[0077] In some embodiments, one or more modifications provided herein can increase monomer content by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% compared to the monomer content a composition comprising an Fc variant or one-armed antibody wherein the one or more modifications is absent. In some embodiments, the percentage of monomer of a composition comprising an Fc variant or one-armed antibody provided herein can be at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In select embodiments, the percentage of monomer of a composition comprising an Fc variant or one-armed antibody provided herein can be at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0078] An Fc variant or one-armed antibody provided herein can comprise one or more modifications that can be beneficial to the thermal stability of the molecule. Thermal stability can be described by the temperature at which the molecule starts to unfold (e.g., Ton). Typically, an Fc variant or one-armed antibody with a higher Tonhas better thermostability. Thermal stability of the Fc variant or one-armed antibody can be affected for example by bonds and / or other interactions between amino acid chains of the Fc variant or one-armed antibody, and modifications thereof.

[0079] In some embodiments one or more modifications such as provided herein can improve thermal stability (i.e., increase Ton) in a composition. Modifications that can improve thermal stability can include modifications in the Fc region such as effector attenuating or effectorless mutations, other modifications to the Fc region, modifications to the hinge such as those a mutation(s) that eliminate one or more disulfide bonds, or other modifications to the hinge region.

[0080] In some embodiments, one or more modifications provided herein can increase Tonof an Fc variant or one-armed antibody provided herein by at least 5%, at least about 10%, at least about 15%, or at least about 20% compared to the Tonof an Fc region or one-armed antibody without the one or more modifications.

[0081] In some embodiments, an Fc variant or one-armed antibody provided herein can have a Tonthat is at least about 54°C, at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, or at least about 60°C. In some embodiments, an Fc variant or one-armed antibody provided herein can have a Tonthat is between about 54°C and about 61°C, between about 55°C and about 61°C, between about 56°C and about 61°C, between about 57°C and about 61°C, between about 58°C and about 61 °C, between about 59°C and about 61 °C, between about 60°C and about 61°C, between about 54°C and about 60°C, between about 55°C and about 60°C, between about 56°C and about 60°C, between about 57°C and about 60°C, between about 58°C and about 60°C, between about 59°C and about 60°C, between about 54°C and about 59°C, between about 55°C and about 59°C, between about 56°C and about 59°C, between about 56°C and about 59°C, between about 57°C and about 59, between about 58°C and about 59°C, between about 54°C and about 58°C, between about 55°C and about 58°C, between about 56°C and about 58°C, between about 57°C and about 58°C, between about 54°C and about 57°C, between about 55°C and about 57°C, between about 56°C and about 57°C, between about 54°C and about 56°C, between about 55°C and about 56°C, or between about 54°C and about 55°C.

[0082] Heterogeneity can be a description or measure of a mixture of components in a composition. Compositions comprising an Fc variant or one-armed antibody provided herein can include the Fc variant or one-armed antibody (i.e., a first amino acid chain and second amino acid chain associated together as provided herein). Such compositions can also comprise additional components, such as a homodimer(s) (i.e., two first amino acid chains associated together or two second amino acid chains associated together), single amino acid chains, a glycosylated variant of an Fc variants or one-armed antibody, or another unintended Fc or antibody fragments or amino acid molecules. Any one or combination of such components present in a composition can contribute to heterogeneity of that composition. In some cases, heterogeneity can arise, for example, from homodimerization of a heavy chain or Fc, disulfide mispairing, O-glycosylation, N-terminal clipping or alternative processing of the Fc chain, proteolysis, or other causes. It may be beneficial to reduce or eliminate heterogeneity from one or more sources. In some cases, heterogeneity from one source, a combination of sources, or all sources can be reduced to a threshold amount or essentially eliminated.

[0083] In some embodiments one or more modifications such as provided herein can improve (i.e. , reduce) heterogeneity in a composition. Modifications that can improve heterogeneity can include but are not limited to asymmetric formats (i.e., knob-in-hole mutations), modifications which reduce or prevent o-glycosylation, modifications which reduce or prevent truncation, modifications that can reduce or prevent disulfide mispairing, modifications that can reduce or prevent alternative processing of an amino acid chain, or modification that can reduce or prevent proteolysis, for example as provided herein.

[0084] One or more modification(s) can reduce heterogeneity as determined by SDS-PAGE analysis of the composition. For example, bands in such an SDS-PAGE that are present due to heterogeneity can be reduced in a composition comprising a Fc variant or one-arm antibody comprising one or more modification(s) herein, compared with a composition where the one or more modification(s) are absent. In some embodiments, bands in such an SDS- PAGE can be reduced by at least 10%, at least 25%, at least 50%, at least 75%, or at least 90%. In some embodiments, one or more bands in such an SDS-PAGE can be eliminated. In further embodiments, only bands corresponding to the amino acid chains of the Fc variant or one-arm antibody are present in the SDS-PAGE. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the protein product within the optimized composition can be an Fc variant or one-armed antibody as described, herein. In some embodiments, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of protein product within the optimized composition can be another amino acid product, e.g., as described above.

[0085] In some embodiments one or more modifications such as provided herein can improve (i.e., reduce) heterogeneity in a composition. Modifications that can improve heterogeneity can include but are not limited to asymmetric formats (i.e., knob-in-hole mutations), modifications which reduce or prevent o-glycosylation, modifications which reduce or prevent truncation, modifications that can reduce or prevent disulfide mispairing, modifications that can reduce or prevent alternative processing of an amino acid chain, or modification that can reduce or prevent proteolysis, for example as provided herein. One or more modification(s) can reduce heterogeneity as determined by SDS-PAGE analysis of the composition. For example, bands in such an SDS-PAGE that are present due to heterogeneity can be reduced in a composition comprising a Fc variant or one-arm antibody comprising one or more modification(s) herein, compared with a composition where the one or more modification(s) are absent. In some embodiments, bands in such an SDS-PAGE can be reduced by at least 10%, at least 25%, at least 50%, at least 75%, or at least 90%. In some embodiments, one or more bands in such an SDS-PAGE can be eliminated. In further embodiments, only bands corresponding to the amino acid chains of the Fc variant or one- arm antibody are present in the SDS-PAGE. In some embodiments, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% of the protein product within the optimized composition can be an Fc variant or one-armed antibody as described, herein. In some embodiments, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of protein product within the optimized composition can be another amino acid product, e.g., as described above. In some embodiments one or more modifications such as provided herein can improve (i.e., reduce) heterogeneity in a composition. Modifications that can improve heterogeneity can include but are not limited to asymmetric formats (i.e., knob-in- hole mutations), modifications which reduce or prevent o-glycosylation, modifications which reduce or prevent truncation, modifications that can reduce or prevent disulfide mispairing, modifications that can reduce or prevent alternative processing of an amino acid chain, or modification that can reduce or prevent proteolysis, for example as provided herein. One or more modification(s) can reduce heterogeneity as determined by SDS-PAGE analysis of the composition. For example, bands in such an SDS-PAGE that are present due to heterogeneity can be reduced in a composition comprising a Fc variant or one-arm antibody comprising one or more modification(s) herein, compared with a composition where the one or more modification(s) are absent. In some embodiments, bands in such an SDS-PAGE can be reduced by at least 10%, at least 25%, at least 50%, at least 75%, or at least 90%. In some embodiments, one or more bands in such an SDS-PAGE can be eliminated. In further embodiments, only bands corresponding to the amino acid chains of the Fc variant or one- arm antibody are present in the SDS-PAGE. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the protein product within the optimized composition can be an Fc variant or one-armed antibody as described, herein. In some embodiments, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of protein product within the optimized composition can be another amino acid product, e.g., as described above.

[0086] Modifications to an Fc variant or one-armed antibody herein can enhance ADCC. Fc engineering can be used to enhance ADCC. For example (with reference to lgG1), F243L / R292P / Y300LA / 305I / P396L; S239D / I332E; and S298A / E333A / K334A increase FcyRllla binding; S239D / I332E / A330L increases FcyRllla binding and decreases FcyRllb binding; G236A / S239D / I332E improves binding to FcyRlla, improves the FcyRlla / FcyRllb binding ratio (activating / inhibitory ratio), and enhances phagocytosis of antibody-coated target cells by macrophages. Fc variants or one-armed antibodies provided herein can comprise a modified Fc region comprising amino acid substitutions selected from S239D / A330L / I332E, G236A / S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L or F243L / R292P / Y300L / V305I / P396L, An asymmetric Fc in which one heavy chain contains L234Y / L235Q / G236W / S239M / H268D / D270E / S298A mutations and D270E / K326D / A330M / K334E in the opposing heavy chain, can increase affinity for FcyRlllaF158 (a lower-affinity allele) and FcyRllla V158 (a higher-affinity allele) with no increased binding affinity to inhibitory FcyRllb.

[0087] Modifications to an Fc variant or one-armed antibody herein can enhance antibody dependent cellular phagocytosis (ADCP). Fc engineering can be used to enhance ADCP. For example (with reference to IgG 1), a G236A / S239D / I332E modification can increase FcyRlla binding and increase FcyRllla binding.

[0088] Modifications to an Fc variant or one-armed antibody herein can enhance coengagement. Fc engineering can be used to increase co-engagement with FcRs. For example (with reference to IgG 1), S267E / L328F increases FcyRllb binding; N325S / L328F increases FcyRlla binding and decreases FcyRllla binding.

[0089] An antigen binding protein described herein may comprise a heavy chain constant region with an altered glycosylation profile, such that the antigen binding protein has an enhanced effector function, e.g., enhanced ADCC, enhanced CDC, or both enhanced ADCC and CDC. Examples of suitable methodologies to produce antigen binding proteins with an altered glycosylation profile are described in W02003011878, W02006014679 and EP1229125, all of which can be applied to the antigen binding proteins herein described.

[0090] Fc variants and / or one-armed antibodies herein with reduced effector function can exhibit reduced ADCC and / or CDC. Such molecules can comprise a naturally disabled constant region of an lgG2 or lgG4 isotype or a mutated IgG 1 constant region. Examples of suitable modifications are described for example in EP0307434. One example comprises substitution with alanine at positions 235 and 237 (EU index numbering), i.e., L235A and G237A (commonly referred to as “LAGA” mutations). Another example comprises substitution with alanine at positions 234 and 235 (EU index numbering), i.e., L234A and L235A (commonly referred to as “LALA” mutations). Another example comprises substitution with alanine at positions 234, 235, and 237 (EU index numbering), i.e., L234A, L235A and G237A (commonly referred to as “LALAGA” mutations). Further examples, described in EP2691417 and US8969526, comprise P329G or P329R, in combination with the LALA mutations (EU index numbering) for IgG 1 Fes and P329G or P329R in combination with S228P and L235E for lgG4 Fes (EU index numbering).

[0091] Other mutations that decrease effector function include L234F / L235E / P331S; a chimeric antibody created using the CH1 and hinge region from human lgG2 and the CH2 and CH3 regions from human lgG4; lgG2m4, based on the lgG2 isotype with four key amino acid residue changes derived from lgG4 (H268Q, V309L, A330S and P331S); lgG2a that contains V234A / G237A / P238S / H268AA / 309L / A330S / P331S substitutions to eliminate affinity for Fey receptors and C1q complement protein; lgG2m4 (H268Q / V309L / A330S / P331S, changes to lgG4); lgG4 (S228P / L234A / L235A); hulgG1 L234A / L235A (AA); hulgG4 S228P / L234A / L235A; lgG1s(L234A / L235A / G237A / P238S / H268A / A330S / P331 S); lgG4s1 (S228P / F234A / L235A / G237A / P238S); and lgG4s2 (S228P / F234A / L235A / DG236 / G237A / P238S, wherein D denotes a deletion).

[0092] Additional alterations and mutations to decrease effector function include: (with reference to IgG 1 unless otherwise noted): aglycosylated N297A or N297Q or N297G; L235E; lgG4:F234A / L235A; and chimeric lgG2 / lgG4. lgG2: H268QA / 309L / A330S / P331S, and lgG2: V234A / G237A / P238S / H268AA / 309L / A330S / P331S can reduce FcyR and C1q binding (US8961967).

[0093] Correct pairing of the first heavy chain and the second heavy chain of an Fc variant or one-armed antibody herein can be essential to production thereof. Notably, incorrect pairing contributes to heterogeneity in a composition comprising a Fc variant or one-armed antibody herein; thus increasing correct pairing can reduce heterogeneity in such composition. In some cases, without optimization, incorrect pairing or mis-matching of the heavy chains can result in a reduction in efficacy, tolerance, potency, or another property of the Fc variant, one-armed antibody, or composition thereof. In some embodiments, correct pairing can be accomplished by employing an asymmetric Fc format.

[0094] Asymmetric formats retain as closely as possible the native architecture of natural antibodies by forcing correct HL chain pairing and / or promoting H chain heterodimerization during the co-expression of three (if common heavy or light chains are used) or four polypeptide chains e.g., Triomab, asymmetric reengineering technology immunoglobulin (ART-lg), CrossMab, Biclonics common light chain, ZW1 common light chain, DuoBody and knobs into holes, DuetMab, KA body, Xmab, YBODY, HET-mAb, HET-Fab, DART-Fc, SEEDbody, mouse / rat chimeric IgG.

[0095] In some embodiments, an Fc variant or one-armed antibody provided herein can comprise a knob-in-hole feature (e.g., mutation of Fc). A knob-in-hole feature can favor heterodimerization of the Fc variant or one-armed antibody compared with an Fc or one- armed antibody without the knob-in-hole feature (i.e., correct association of the first amino acid chain and the second amino acid chain). For example, a knob-in-hole feature can comprise a mutation at the CH3 domain of the Fc variant or one-armed antibody.

[0096] One contemplated configuration of a knob-in-hole feature comprises a T366W mutation (the “knob” feature) on the first amino acid chain of an Fc variant or one-armed antibody and T366S, L368A, and Y407V mutations (the “hole” feature) on the second amino acid chain of the Fc variant or one-armed antibody. A configuration wherein the knob is on the second amino acid chain and the hole is on the first amino acid chain, though either or both configurations can be effective in reducing heterogeneity as described herein, e.g., by promoting correct pairing of the first amino acid chain and the second amino acid chain of the Fc variant or one-armed antibody.

[0097] Other Fc architectures promoting correct heterodimer formation are also envisioned, for example but not limited to those described in Ha J-H, Kim J-E and Kim Y-S (2016) Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins. Front. Immunol. 7:394. doi: 10.3389 / fimmu.2016.00394, which is incorporated herein by reference.

[0098] Glycosylation of a variant hinge region, Fc variant, or one-armed antibody herein can affect heterogeneity of a composition comprising said Fc variant or one-armed antibody.This can be glycosylation other than N-linked glycosylation, including but not limited to cases wherein the Fc variants or one-armed antibodies in the composition comprise N297G mutations. O-glycosylation in compositions comprising an Fc variant or one-armed antibody herein can contribute to the heterogeneity of the composition, which can be determined for example by treating the one-armed antibodies with deglycosidase and visualizing the composition via reducing SDS-PAGE.

[0099] Heterogeneity due to O-glycosylation can be optimized (i.e., reduced or eliminated) by removal of o-glycosylation in the variant hinge region, Fc variant, or one-armed antibody herein. Removal of o-glycosylation can be accomplished by mutation of threonine (T) and / or serine (S) residues in the hinge region to alanine (A) residues. For example, a variant hinge region, Fc variant, or one-armed antibody herein can comprise one of T223A or T225A, according to the Eu numbering system. In some such cases, the variant hinge region, Fc variant, or one-armed antibody can comprise both T223A and T225A, according to the Eu numbering system. In embodiments wherein a cysteine (C) residue is modified (e.g., as provided herein), said cysteine residue can be replaced with an alanine (A) residue rather than a serine (S) residue to reduce O-glycosylation in the hinge (i.e., C226A instead of C226S, and / or C229A instead of C229S).

[0100] A variant hinge region provided herein or an Fc variant provided herein can comprise an amino acid substitution in which Thr residues are removed from the variant hinge region. Notably, Thr residues can be substituted with Ala (e.g., T223A and T225A). While such substitution of Thr residues can be made in one of the first chain of the variant hinge region and the second chain of the variant hinge region, in some embodiments a best result can be accomplished by substitution of Thr residues in both the first and second chains of the variant hinge region. In some embodiments, the first chain of the variant hinge region and the second chain of the variant hinge region can comprise T225A and T223A.

[0101] Heterogeneity of a composition comprising a variant hinge region, an Fc variant or one-armed antibody herein can arise by clipping, alternative processing, or proteolysis of one or both of the amino acid chains of the variant hinge region, Fc variant or one-armed antibody. Such clipping, processing, or proteolysis can result in truncation (e.g., of an N- terminal portion) of the variant hinge region, Fc variant or one-armed antibody. In somecases, heterogeneity of a composition can arise via clipping, alternative processing, or proteolysis of one or both of the amino acid chains of the variant hinge region, Fc variant or one-armed antibody of the composition.

[0102] In some embodiments, clipping, alternative processing, or proteolysis of a variant hinge region, Fc variant, or one-armed antibody herein, can be reduced or eliminated, e.g., by removal of one or more amino acids involved in said clipping, alternative processing, or proteolysis. Such a modification can comprise deletion of amino acids 222 through 225 of the variant hinge region, Fc variant, or one-armed antibody herein. In some embodiments, truncation of the hinge region can comprise removal of amino acids K222 through T225 of the hinge region of the first heavy chain constant region (e.g., the amino acids “KTHT”) and removal of amino acids T223 through T225 of the hinge region of the second heavy chain constant region (e.g., the amino acids “THT”). Such truncation can eliminate one or more restriction sites or other modifiable sites in the variant hinge region, thus reducing clipping, alternative processing, and / or proteolysis.

[0103] Heterogeneity due to clipping, alternative processing, or proteolysis can be optimized (i.e. , reduced or eliminated) by modification in the region of the clipping, alternative processing, or proteolysis. In some embodiments, this can be accomplished by a modification of the variant hinge region, Fc variant, or one-armed antibody. In some such cases, a variant hinge region, Fc variant or one-armed antibody herein can comprise an intentionally truncated hinge region. Truncation of a hinge region can, for example, ameliorate undesired clipping, alternative processing, or proteolysis within the hinge region. Clipping can occur for example in the presence of an alanine residue (e.g., C226A) in the hinge region. Such clipping can be improved (i.e., reduced or eliminated) by pairing the alanine residue (e.g., C226A in one amino acid chain of the variant hinge region, Fc variant, or one-armed antibody) with a truncation of the hinge region in the other amino acid chain of the variant hinge region, Fc variant, or one-armed antibody.

[0104] Fc regions provided herein can comprise further asymmetric architecture, which can improve heterogeneity during production of a molecule comprising such an Fc region.

[0105] Asymmetric formats retain as closely as possible the native architecture of natural antibodies by forcing correct HL chain pairing and / or promoting H chain heterodimerization during the co-expression of three (if common heavy or light chains are used) or four polypeptide chains e.g. Triomab, asymmetric reengineering technology immunoglobulin (ART-lg), CrossMab, Biclonics common light chain, ZW1 common light chain, DuoBody and knobs into holes (KIH), DuetMab, KA body, Xmab, YBODY, HET-mAb, HET-Fab, DART-Fc, SEEDbody, mouse / rat chimeric IgG.

[0106] Notably, Fc regions provided herein can comprise knob-hole, which can comprise knob (T366W) and hole (T366S, L368A, and Y407V) amino acid substitutions in the secondand first heavy chains, respectively. Also envisioned are knob-knob, hole-knob, and holehole configurations. However, in some embodiments, different configurations can tend to form homodimers (i.e., two first heavy chains or two second heavy chains) more than other configurations. In some embodiments, knob-hole configuration can yield fewer homodimers than knob-knob, hole-knob, and / or hole-hole configurations, or configurations comprising neither a knob nor a hole amino acid substitution.

[0107] A variant hinge region can comprise a truncated second amino acid chain of the reference IgG 1. In some embodiments, the first amino acid chain of the variant hinge region can comprise amino acids 216-230 of the first amino acid chain of the hinge region of the reference lgG1 and the second amino acid chain of the variant hinge region can comprise amino acids 223-230 of the hinge region of the second amino acid chain of the reference IgG 1 , according to the Eu numbering system. In some embodiments, the second amino acid chain of the hinge region of the reference lgG1 consists of amino acids 223-230 of the reference IgG 1 according to the Eu numbering system (i.e., a truncated second amino acid chain). In some embodiments, the first amino acid chain of the variant hinge region has a sequence of one of SEQ ID NOS.: 8-34. In some embodiments, the second amino acid chain of the variant hinge region has a sequence of one of SEQ ID NOS.: 35-61. An amino acid chain of a variant hinge region can, but need not, further comprise one or more additional modifications, such as described herein.

[0108] In some embodiments, the second heavy chain of an Fc variant can be a truncated heavy chain. A truncated heavy chain can comprise a truncated second amino acid chain of a variant hinge region provided herein. A truncated heavy chain can comprise one or more other modifications of a variant hinge region, such as those provided herein. For example, the second heavy chain of an Fc variant can comprise a second amino acid chain of a variant hinge region consisting of amino acids 223-230 of the hinge region of a reference IgG 1 . In some such embodiments, the variant hinge region of an Fc variant can comprise a sequence of one of SEQ ID NOS.: 8-34 and the second amino acid chain of the variant hinge region of an Fc variant can comprise a sequence of one of SEQ ID NOS.: 35-61 . Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9. In some embodiments, an Fc variant comprising a truncated second amino acid chain of a variant hinge region can have a first heavy chain having a sequence of one of SEQ ID NOS.: 62-169 or 495-548, and a second heavy chain having a sequence of one of SEQ ID NOS.: 197-223, 251-277, 305-331 , 359-385, 413-439, or 467-493. Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0109] The first and second amino acid chains of a variant hinge region associate together to yield said variant hinge region. In some embodiments, there can be two disulfide bridges joining the two amino acid chains of the variant hinge region. However, some variant hingeregions may comprise one or no disulfide bridges (achieved, for example, through deletion or substitution of one or more residues involved in the disulfide bridge formation, such as described herein).

[0110] Disulfide bonds within a variant hinge region, Fc variant, or one-armed antibody herein occur between amino acid residues comprising a thiol group (e.g., cysteine). In some embodiments, modifications that can contribute to the optimization of monomer production and / or thermal stability Some embodiments can comprise modification to the disulfide bond configuration of a variant hinge region, Fc variant, or one-armed antibody herein. In some such cases, monomer content and / or thermal stability can be improved. An unmodified hinge region can comprise two disulfide bonds: a first at C226 and a second at C229. In variant hinge regions, Fc regions, and one-armed antibodies provided herein, one or both of these disulfide bonds can be removed. Removal of a disulfide bond can result from modification of the amino acid sequence, for example by mutation of one or both cysteine residues participating in the disulfide bond. In some cases, this can be accomplished via a mutation of cysteine to serine or alanine (e.g., C226S, C226A, C229S, or C229A). In some cases, it may be preferred to remove the disulfide bond at C226 while retaining the disulfide bond at C229. This can be accomplished by a C226S or a C226A mutation in one or both amino acid chains of a variant hinge region, Fc variant, or one-armed antibody herein. Introduction of a C226S mutation, a C226A mutation, or both a C226S and a C226A mutation in both amino acid chains of a variant hinge region, Fc variant, or one-armed antibody herein can yield improved thermal stability compared with a Fc region having both disulfide bonds intact.

[0111] In some embodiments, a variant hinge region, Fc variant, or one-armed antibody herein can be engineered to comprise a single disulfide bond. In some such cases, the variant hinge region, cysteinylation of the Fc variant, or one-armed antibody can be reduced or eliminated, which can improve heterogeneity of a composition thereof.

[0112] A variant hinge region provided herein can comprise one or amino acid substitutions of residues involved in the formation of one or more disulfide bridges, which can result in a variant wherein the one or more disulfide bridge is absent. In some embodiments, disulfide bridge formation can occur at cysteine residues present in a hinge region, wherein there is a cysteine residue at that location on each of the two amino acid chains of the hinge region. Modification of such cysteine residues to another residue at the site of a disulfide bond on one or both amino acid chains of the hinge can prevent the formation of the disulfide bond. In selected embodiments, the same modification of one or more cysteine residues is present in both the first and second amino acid chains of the variant hinge region. Such cysteine residues can be substituted with, for example, alanine, serine, or glycine. For example, a variant hinge region can comprise one or more amino acid substitutions selected from thegroup consisting of C226A, C226S, C226G, C229A, C229S, and C229G. In some cases, a variant hinge region can comprise one or more amino acid substitutions selected from the group consisting of C226A, C226S, C229A, C229S. A variant hinge region comprising a substitution of one or more residues involved in disulfide bond formation can have a first amino acid chain having a sequence of one of: SEQ ID NOS.: 9-16, 18-25, 27-34, and can have a second amino acid chain having a sequence of one of SEQ ID NOS.: 9-16, 18-25, 27-34, 36-43, 45-52, or 54-61. In some embodiments, the first amino acid chain of the variant hinge region can comprise (i) C226A (e.g., having a sequence of one of SEQ ID NOS.: 9, 11 , 15, 18, 20, 24, 27, 29, or 33), (ii) C226S (e.g., having a sequence of one of SEQ ID NOS.: 12, 14, 16, 21 , 23, 25, 30, 32, or 34), (Hi) C229A (e.g., having a sequence of one of SEQ ID NOS.: 10, 11 , 16, 19, 20, 25, 28, 29, or 34), (iv) C229S (e.g., having a sequence of one of SEQ ID NOS.: 13, 14, 15, 22, 23, 24, 31 , 32, or 33), (v) C226A and C229A (e.g., having a sequence of one of SEQ ID NOS.: 11 , 20, or 29), (vi) C226A and C229S (e.g., having a sequence of one of SEQ ID NOS.: 15, 24, or 33), (vii) C226S and C229A (e.g., having a sequence of one of SEQ ID NOS.: 16, 25, or 34), or (viii) C226S and C229S (e.g., having a sequence of one of SEQ ID NOS.: 14, 23, or 32) according to the Eu numbering system. In some embodiments, the second amino acid chain of the variant hinge region can comprise (i) C226A (e.g., having a sequence of one of SEQ ID NOS.: 9, 11 , 15, 18, 20, 24, 27, 29, 33, 36, 38, 42, 45, 47, 51 , 54, 56, or 60), (ii) C226S (e.g., having a sequence of one of SEQ ID NOS.: 12, 14, 16, 21 , 23, 25, 30, 32, 34, 39, 41 , 43, 48, 50, 52, 57, 59, or 61), (iii) C229A (e.g., having a sequence of one of SEQ ID NOS.: 10, 11 , 16, 19, 20, 25, 28, 29, 34, 37, 38, 43, 46, 47, 52, 55, 56, or 61), (iv) C229S (e.g., having a sequence of one of SEQ ID NOS.: 13, 14, 15, 22, 23, 24, 31 , 32, 33, 40, 41 , 42, 49, 50, 51 , 58, 59, or 60), (v) C226A and C229A (e.g., having a sequence of one of SEQ ID NOS.: 11 , 20, 29, 38, 47, or 56), (vi) C226A and C229S (e.g., having a sequence of one of SEQ ID NOS.: 15, 24, 33, 42, 51 , or 60), (vii) C226S and C229A (e.g., having a sequence of one of SEQ ID NOS.: 16, 25, 34, 43, 52, or 61), or (viii) C226S and C229S (e.g., having a sequence of one of SEQ ID NOS.: 14, 23, 32, 41 , 50, or 59) according to the Eu numbering system. In some embodiments, both the first and the second amino acid chains of the variant hinge region can comprise one or more such amino acid substitutions, for example selected from the group consisting of C226A, C226S, C226G, C229A, C229S, and C229G or selected from the group consisting of C226A, C226S, C229A, and C229S. In some embodiments, both the first and the second amino acid chains of the variant hinge region can comprise the same one or more amino acid substitutions, for example for example selected from the group consisting of C226A, C226S, C226G, C229A, C229S, and C229G or selected from the group consisting of C226A, C226S, C229A, and C229S. Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9.

[0113] The first heavy chain, the second heavy chain, or both of an Fc variant can comprise an amino acid substitution of one or more residues involved in disulfide bridge formation in the variant hinge region, such as described above. Such an Fc variant can comprise a first heavy chain having a sequence of one of SEQ ID NOS.: 63-70, 72-79, 81-88, 90-97, 99-106, 108-115, 117-124, 126-133, 135-142, 144-151 , 153-160, 162-169, 496-503, 505-512, 514- 521 , 523-530, 532-539, or 541-548, and a second heavy chain having a sequence of one of SEQ ID NOS.: 171-178, 180-187, 189-196, 198-205, 207-214, 216-223, 225-232, 234-241 , 243-250, 252-259, 261-268, 270-277, 279-286, 288-295, 297-304, 306-313, 315-322, 324- 331 , 333-340, 342-349, 351-358, 360-367, 369-376, 378-385, 387-394, 396-403, 405-412, 414-421 , 423-430, 432-439, 441-448, 450-457, 459-466, 468-475, 477-484, 486-493.

[0114] For example, a variant hinge region of an Fc variant can comprise one or more amino acid substitutions selected from the group consisting of C226A, C226S, C226G, C229A, C229S, and C229G. In some cases, a variant hinge region of an Fc variant can comprise one or more amino acid substitutions selected from the group consisting of C226A, C226S, C229A, C229S. For example, the first amino acid chain of the variant hinge region of an Fc variant herein can comprise (i) C226A (e.g., having a sequence of one of SEQ ID NOS.: 9, 11 , 15, 18, 20, 24, 27, 29, or 33), (ii) C226S (e.g., having a sequence of one of SEQ ID NOS.: 12, 14, 16, 21 , 23, 25, 30, 32, or 34), (ill) C229A (e.g., having a sequence of one of SEQ ID NOS.: 10, 11 , 16, 19, 20, 25, 28, 29, or 34), (iv) C229S (e.g., having a sequence of one of SEQ ID NOS.: 13, 14, 15, 22, 23, 24, 31 , 32, or 33), (v) C226A and C229A (e.g., having a sequence of one of SEQ ID NOS.: 11 , 20, or 29), (vi) C226A andC229S (e.g., having a sequence of one of SEQ ID NOS.: 15, 24, or 33), (vii) C226S andC229A (e.g., having a sequence of one of SEQ ID NOS.: 16, 25, or 34), or (viii) C226S andC229S (e.g., having a sequence of one of SEQ ID NOS.: 14, 23, or 32) according to the Eu numbering system, and the second amino acid chain of the variant hinge region of an Fc variant herein can comprise (i) C226A (e.g., having a sequence of one of SEQ ID NOS.: 9, 11 , 15, 18, 20, 24, 27, 29, 33, 36, 38, 42, 45, 47, 51 , 54, 56, or 60), (ii) C226S (e.g., having a sequence of one of SEQ ID NOS.: 12, 14, 16, 21 , 23, 25, 30, 32, 34, 39, 41 , 43, 48, 50, 52, 57, 59, or 61), (Hi) C229A (e.g., having a sequence of one of SEQ ID NOS.: 10, 11 , 16, 19, 20, 25, 28, 29, 34, 37, 38, 43, 46, 47, 52, 55, 56, or 61), (iv) C229S (e.g., having a sequence of one of SEQ ID NOS.: 13, 14, 15, 22, 23, 24, 31 , 32, 33, 40, 41 , 42, 49, 50, 51 , 58, 59, or 60), (v) C226A and C229A (e.g., having a sequence of one of SEQ ID NOS.: 11 , 20, 29, 38, 47, or 56), (vi) C226A and C229S (e.g., having a sequence of one of SEQ ID NOS.: 15, 24, 33, 42, 51 , or 60), (vii) C226S and C229A (e.g., having a sequence of one of SEQ ID NOS.: 16, 25, 34, 43, 52, or 61), or (viii) C226S and C229S (e.g., having a sequence of one of SEQ ID NOS.: 14, 23, 32, 41 , 50, or 59) according to the Eu numbering system. In some embodiments, both the first and the second amino acid chains of the variant hinge region ofan Fc variant can comprise one or more such amino acid substitutions, and in some embodiments, both the first and second amino acid chains of the variant hinge region of an Fc variant can comprise the same one or more such amino acid substitutions. Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9.

[0115] An Fc variant comprising an amino acid substitution of one or more residues involved in disulfide bridge formation in the variant hinge region can comprise a first heavy chain comprising (i) C226A (e.g., having a sequence of one of SEQ ID NOS.: 63, 65, 69, 72, 74, 78, 81 , 83, 87, 90, 92, 96, 99, 101 , 105, 108, 110, 114, 117, 119, 123, 126, 128, 132, 135,137, 141 , 144, 146, 150, 153, 155, 159, 162, 164, 168, 496, 498, 502, 505, 507, 511 , 514,516, 520, 523, 525, 529, 532, 534, 538, 541 , 543, or 547), (II) C226S (e.g., having a sequence of one of SEQ ID NOS.: 66, 68, 70, 75, 77, 79, 84, 86, 88, 93, 95, 97, 102, 104, 106, 111 , 113, 115, 120, 122, 124, 129, 131 , 133, 138, 140, 142, 147, 149, 151 , 156, 158,160, 165, 167, 169, 499, 501 , 503, 508, 510, 512, 517, 519, 521 , 526, 528, 530, 535, 537,539, 544, 546, or 548), (iii) C229A (e.g., having a sequence of one of SEQ ID NOS.: 64, 65, 70, 73, 74, 79, 82, 83, 88, 91 , 92, 97, 100, 101 , 106, 109, 110, 115, 118, 119, 124, 127, 128, 133, 136, 137, 142, 145, 146, 151, 154, 155, 160, 163, 164, 169, 497, 498, 503, 506, 507,512, 515, 516, 521 , 524, 525, 530, 533, 534, 539, 542, 543, or 548), (iv) C229S (e.g., having a sequence of one of SEQ ID NOS.: 67, 68, 69, 76, 77, 78, 85, 86, 87, 94, 95, 96, 103, 104, 105, 112, 113, 114, 121 , 122, 123, 130, 131 , 132, 129, 140, 141 , 148, 149, 150, 157, 158,159, 166, 167, 168, 500, 501 , 502, 509, 510, 511 , 518, 519, 520, 527, 528, 529, 536, 537,538, 545, 546, or 457), (v) C226A and C229A (e.g., having a sequence of one of SEQ ID NOS.: 65, 74, 83, 92, 101 , 110, 119, 128, 137, 146, 155, 164, 498, 507, 516, 525, 534, or 543), (vi) C226A and C229S (e.g., having a sequence of one of SEQ ID NOS.: 69, 78, 87, 96, 105, 114, 123, 132, 141 , 150, 159, 168, 502, 511 , 520, 529, 538, or 547), (vii) C226S and C229A (e.g., having a sequence of one of SEQ ID NOS.: 70, 79, 88, 97, 106, 115, 124, 133, 142, 151 , 160, 169, 503, 512, 521 , 530, 539, or 548), or (vii) C226S and C229S (e.g., having a sequence of one of SEQ ID NOS.: 68, 77, 86, 95, 104, 113, 122, 131 , 140, 149, 158, 167, 501 , 510, 519, 528, 537, or 546) according to the Eu numbering system. An Fc variant comprising an amino acid substitution of one or more residues involved in disulfide bridge formation in the variant hinge region can comprise a second heavy chain comprising (i) C226A (e.g., having a sequence of one of SEQ ID NOS.: 171 , 173, 177, 180, 182, 186, 189, 191 , 195, 198, 200, 204, 207, 209, 213, 216, 218, 222, 225, 227, 231 , 234, 236, 240,243, 245, 249, 252, 254, 258, 261 , 263, 267, 270, 272, 276, 279, 281 , 285, 288, 290, 294,297, 299, 303, 306, 308, 312, 315, 317, 321 , 324, 326, 330, 333, 335, 339, 342, 344, 348,351 , 353, 357, 360, 362, 366, 369, 371 , 375, 378, 380, 384, 387, 389, 393, 396, 398, 402,405, 407, 411 , 414, 416, 420, 423, 425, 429, 432, 434, 438, 442, 443, 447, 450, 452, 456, 459, 451 , 465, 468, 470, 474, 477, 479, 483, 486, 488, or 492), (ii) C226S (e.g., having asequence of one of SEQ ID NOS.: 171 , 173, 177, 180, 182, 186, 189, 191 , 195, 198, 200, 204, 207, 209, 213, 216, 218, 222, 225, 227, 231 , 234, 236, 240, 243, 245, 249, 252, 254, 258, 261 , 263, 267, 270, 272, 276, 279, 281 , 285, 288, 290, 294, 297, 299, 303, 306, 308,312, 315, 317, 321 , 324, 326, 330, 333, 335, 339, 342, 344, 348, 351 , 353, 357, 360, 362,366, 369, 371 , 375, 378, 380, 384, 387, 389, 393, 396, 398, 402, 405, 407, 411 , 414, 416,420, 423, 425, 429, 432, 434, 438, 442, 443, 447, 450, 452, 456, 459, 451 , 465, 468, 470,474, 477, 479, 483, 486, 488, or 492), (iii) C229A (e.g., having a sequence of one of SEQ ID NOS.: 172, 173, 178, 181 , 182, 187, 190, 191 , 196, 199, 200, 205, 208, 209, 214, 217, 218, 223, 226, 227, 232, 235, 236, 241, 244, 245, 250, 253, 254, 259, 262, 263, 268, 271 , 272,277, 280, 281 , 286, 289, 290, 295, 298, 299, 304, 307, 308, 313, 316, 317, 322, 325, 326,331 , 334, 335, 340, 343, 344, 349, 352, 353, 358, 361 , 362, 367, 370, 371 , 376, 379, 380,385, 388, 389, 394, 397, 398, 403, 406, 407, 412, 415, 416, 421 , 424, 425, 430, 433, 434,439, 442, 443, 448, 451 , 452, 457, 460, 461 , 466, 469, 470, 475, 478, 479, 484, 487, 488, or 493), (iv) C229S (e.g., having a sequence of one of SEQ ID NOS.: 175, 176, 177, 184, 185, 186, 193, 194, 195, 202, 203, 204, 211 , 212, 213, 220, 221 , 222, 229, 230, 231 , 238, 239,240, 247, 248, 249, 256, 257, 258, 265, 266, 267, 274, 275, 276, 283, 284, 285, 292, 293,294, 301 , 302, 303, 310, 311 , 312, 319, 320, 321 , 328, 329, 330, 337, 338, 339, 346, 347,348, 355, 356, 357, 364, 365, ,366, 373, 374, 375, 382, 383, 384, 391 , 392, 393, 400, 401 ,402, 409, 410, 411 , 418, 419, 420, 427, 428, 429, 436, 437, 438, 445, 446, 447, 454, 455,456, 463, 464, 465, 472, 473, 474, 481 , 482, 483, 490, 491 , or 492), (v) C226A and C229A (e.g., having a sequence of one of SEQ ID NOS.: 173, 182, 191 , 200, 209, 218, 227, 236, 245, 254, 263, 272, 281 , 290, 299, 208, 217, 326, 335, 344, 353, 362, 371 , 380, 389, 398, 407, 416, 425, 434, 443, 452, 461 , 470, 479, or 488), (vi) C226A and C229S (e.g., having a sequence of one of SEQ ID NOS.: 173, 182, 191 , 200, 209, 218, 227, 236, 245, 254, 263, 272, 281 , 290, 299, 208, 217, 326, 335, 344, 353, 362, 371 , 380, 389, 398, 407, 416, 425,434, 443, 452, 461 , 470, 479, or 488), (vii) C226S and C229A (e.g., having a sequence of one of SEQ ID NOS.: 178, 187, 196, 205, 214, 223, 232, 241 , 250, 259, 268, 277, 286, 295, 304, 313, 322, 331 , 340, 349, 358, 367, 376, 385, 394, 403, 412, 421 , 430, 439, 448, 457,466, 475, 484, or 493), or (vii) C226S and C229S (e.g., having a sequence of one of SEQ IDNOS.: 176, 185, 194, 203, 212, 221, 230, 239, 248, 257, 266, 275, 284, 293, 302, 311 , 320, 329, 338, 347, 356, 365, 374, 383, 392, 401 , 410, 419, 428, 437, 446, 455, 464, 473, 482, or 491) according to the Eu numbering system. In some embodiments, both the first heavy chain and the heavy chain of an Fc variant can comprise one or more such amino acid substitutions, and in some embodiments, both the first heavy chain and the heavy chain of an Fc variant can comprise the same one or more such amino acid substitutions. Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0116] In some embodiments, a variant hinge region provided herein can comprise a modification that can modify remove one or more Thr residues, for example such that heterogeneity of a composition can be improved. Such modifications can, in some cases, result in improved monomer content. Such modifications can comprise, for example, substitution of amino acids of one or more restriction sites, or deletion of amino acids of one or more of the restriction sites.

[0117] In some embodiments, the first and second amino chains of a variant hinge region can comprise substitution of one or more threonine residues, for example, one or both of the amino acid substitutions T223A and T225A according to the Eu numbering system on both the first and second amino acid chains of the variant hinge region. In some cases, a variant hinge region comprising an amino acid sequence comprising both T223A and T225A can be preferred. A first amino acid chain of variant hinge region can comprise a sequence of one of SEQ ID NOS.: 17-25, and a second amino acid chain of a variant hinge region can comprise a sequence of one of SEQ ID NOS.: 17-25 or 44-52. Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 12.

[0118] An Fc variant herein can comprise a variant hinge region comprising substitution of one or more threonine residues, for example, one or both of the amino acid substitutions T223A and T225A according to the Eu numbering system on both the first and second amino acid chains; In some cases, a variant hinge region having both T223A and T225A substitutions can be preferred. Such Fc variant can comprise a variant hinge region comprising a first amino acid chain comprising a sequence of one of SEQ ID NOS.: 17-25, and a second amino acid chain comprising a sequence of one of SEQ ID NOS.: 17-25 or 44- 52. Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9. In some embodiments, an Fc variant comprising T223A and T225A substitutions can comprise a first heavy chain sequence of one of SEQ ID NOS.: 71-79, 98- 106, 125-133, 152-160, 504-512, or 531-539, and a second heavy chain sequence of one of SEQ ID NOS.: 179-187, 206-214, 233-241 , 260-268, 287-295, 314-322, 341-349, 368-376, 396-403, 422-430, 449-457, or 476-484. Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0119] In some embodiments, a variant hinge region can comprise a deletion of one or more amino acids within the first and second amino acid chains of the variant hinge region. Such deletion can for example result in deletion of a restriction site, such that the restriction site is no longer recognized by a corresponding restriction enzyme. For example, a variant hinge region can comprise deletion of K222 through T225 of the hinge region of the reference lgG1. In some such embodiments, the first amino acid chain of the variant hinge region can have a sequence of one of SEQ ID NOS.: 26-34, and the second amino acid chain of the variant hinge region can have a sequence of one of SEQ ID NOS.: 26-34 and 53-61. Somesuch pairs of first and second amino acid chains of variant hinge regions are provided in Table 9.

[0120] An Fc variant herein can comprise a variant hinge region comprising deletion of one or more amino acids within the first and second amino acid chains of the variant hinge region, for example deletion of K222 through T225 of the hinge region of the reference IgG 1 . Such an Fc variant can comprise a variant hinge region comprising a first amino acid sequence of one of SEQ ID NOS.: 26-34 and a second amino acid chain of one of SEQ ID NOS.: 26-34 and 53-61 . Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9. In some embodiments, an Fc variant comprising a variant hinge region comprising a deletion of one or more amino acids (e.g., deletion of K222 through T225) can comprise a first heavy chain of one of SEQ ID NOS.: 179-187, 206-214, 233-241 , 260-268, 287-295, 314-322, 341-349, 368-376, 396-403, 422-430, 449-457, or 476-484, and a second heavy chain of one of SEQ ID NOS.: 188-196, 215-223, 242-250, 269-277, 296-304, 323-331 , 350-358, 377-385, 404-412, 431-439, 458-466, or 485-493. Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0121] A variant hinge region or hinge region of an Fc variant or one-armed antibody herein can comprise no second amino acid chain of the hinge. In some embodiments, a variant hinge region or hinge region of an Fc variant or one-armed antibody herein can comprise deletion of each amino acid in the second chain of the variant hinge region; such a variant hinge region or hinge region of an Fc variant or one-armed antibody herein may comprise only the first amino acid chain of the variant hinge region.

[0122] An Fc variant lacking a second amino acid chain of a hinge (e.g., a variant hinge region) as provided herein can comprise a first amino acid chain of a hinge region of one of SEQ ID NOS.: 8-34. An Fc variant lacking a second amino acid chain of a hinge as provided herein can comprise a first heavy chain having a sequence of one of SEQ ID NOS.: 62-169 or 495-548, and a second heavy chain having a sequence of one of SEQ ID NOS.: 224, 278, 332, 386, 440, 494, or 549-563. Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0123] A CH2 domain, for example of an Fc variant or one-armed antibody provided herein can be a truncated CH2 domain. In some embodiments, a truncated CH2 domain can modify heterogeneity of a composition comprising the Fc variant or one-armed antibody, for example by changing the amount of clipping of the Fc variant or one-armed antibody. In some embodiments, an Fc variant or one-armed antibody can have less clipping than an otherwise same Fc variant or one-armed antibody not having a truncated CH2 domain. In some embodiments, a composition comprising an Fc variant or one-armed antibody can have better heterogeneity than an otherwise same composition comprising an otherwise same Fc variant or one-armed antibody.

[0124] A truncated CH2 domain can comprise deletion of one or more N terminal amino acids. For example, one, two, three, four, five, or more amino acids can be deleted from the N terminal end of the CH2 domain. In some embodiments, the first three (e.g., A231 through E233) or the first four (A231 through L234) amino acids can be deleted. A truncated CH2 domain can further comprise a knob or hole mutation, for example as provided herein.

[0125] Atruncated CH2 domain can comprise effectorless mutations, such as those provided herein. For example, a truncated CH2 domain can comprise one, two, or three of the amino acid substitutions L234A, L235A and G237A (i.e., LALAGA). In some embodiments, a truncated CH2 domain can comprise all of LALAGA effectorless amino acid substitutions. In some embodiments, a truncated CH2 domain can comprise deletion of A231 through E288 and one or two of L234A, L235A and G237A effectorless amino acid substitutions. In some embodiments, a truncated CH2 domain can comprise deletion of A231 through E288 and each of LALAGA effectorless amino acid substitutions. In some embodiments, a truncated CH2 domain can comprise deletion of A231 through L234 and the amino acid substitution L235A. In some embodiments, a truncated CH2 domain can comprise deletion of A231 through L234 and the amino acid substitutions G237A. In some embodiments, a truncated CH2 domain can comprise deletion of A231 through L234 and both of the amino acid substitutions L235A and G237A. In some embodiments, a truncated CH2 domain comprising one set of effectorless modifications can yield less clipping in an Fc variant or one-armed antibody than a truncated CH2 domain comprising a second set of effectorless modifications.

[0126] An Fc variant having a truncated CH2 domain as provided herein can comprise a first heavy chain having a sequence of one of SEQ ID NOS.: 62-169, and a second heavy chain having a sequence of one of SEQ ID NOS.: 549-563. Some such pairs of first and second heavy chains of Fc variants are provided in Table 12

[0127] In some embodiments, a variant hinge region can have both a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference IgG 1) and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein. Such a variant hinge region can have a first amino acid chain having a sequence of one of SEQ ID NOS.: 9-16 or 18-25 and a second amino acid chain having a sequence of one of SEQ ID NOS.: 36-43, 45-52, or 54-61. Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9.

[0128] In some embodiments, an Fc variant can comprise a variant hinge region comprising a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference IgG 1 ) and an amino acid substitution of one or more residues involved in disulfide bond formation, such as provided herein. Such an Fc variant can comprise a first heavy chain having a sequence of one of SEQ ID NOS.: 63-70, 72-79, 81-88, 90-97, 99-106, 108-115, 117-124, 126-133, 135-142, 144-151 , 153-160, 162-169, 496-503, 505-512, 514-521 , 523-530, 532-539, or 541-548, and a second heavy chain having a sequence of one of SEQ ID NOS.: 216-223, 270-277, 324-331 , 378-385, 432-439, or 486-493. Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0129] In some embodiments, a variant hinge region can comprise (i) substitution of threonine residues (e.g., amino acid substitutions T223A and T225A) and a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference lgG1) (e.g., having a first amino acid chain of one of SEQ ID NOS. :17-25 and a second amino acid chain of one of SEQ ID NOS.: 44-52); (ii) substitution of threonine residues (e.g., amino acid substitutions T223A and T225A) and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first amino acid chain of one of SEQ ID NOS.: 18-25 and a second amino acid chain of one of SEQ ID NOS.: 18-25 or 45-52); or (iii) substitution of threonine residues (e.g., amino acid substitutions T223A and T225A), a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference lgG1), and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first amino acid chain of one of SEQ ID NOS.: 18-25 and a second amino acid chain of one of SEQ ID NOS.: 45-52). Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9.

[0130] In some embodiments, an Fc variant can comprise a variant hinge region comprising (i) substitution of threonine residues (e.g., amino acid substitutions T223A and T225A) and a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference IgG 1 ) (e.g., having a first heavy chain of one of SEQ ID NOS.: 71-79, 98-106, 125-133, 152- 160, 504-512, or 531-539 and a second heavy chain of one of SEQ ID NOS.: 206-214, 260- 268, 314-322, 368-376, 422-430, or 476-484); (ii) substitution of threonine residues (e.g., amino acid substitutions T223A and T225A) and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first heavy chain of one of SEQ ID NOS.: 72-79, 99-106, 126-133, 153-160, 505-512, or 532-539 and a second heavy chain of one of SEQ ID NOS.: 180-187, 207-214, 234-241 , 261-268, 288-295, 315-322, 342-349, 369-376, 396-403, 423-430, 450-457, 477- 484); or (iii) substitution of threonine residues (e.g., amino acid substitutions T223A and T225A), a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference lgG1), and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first heavy chain of one of SEQ ID NOS.: 72-79, 99-106, 126-133, 153-160, 505-512, or 532-539 and a second heavy chain of one of SEQ ID NOS.: 207-214, 261-268, 315-322, 369-376, 423-430,or 477-484). Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0131] In some embodiments, a variant hinge region can comprise (i) a deletion of one or amino acids (e.g., deletion of K222 through T225) and a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference lgG1) (e.g., having a first amino acid chain of one of SEQ ID NOS.: 26-34 and a second amino acid chain of one of SEQ ID NOS.: 53-61); (ii) a deletion of one or amino acids (e.g., deletion of K222 through T225) and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first amino acid chain of one of SEQ ID NOS.: 27-34 and a second amino acid chain of one of SEQ ID NOS.: 27-37 or 57- 61); or (iii) a deletion of one or amino acids (e.g., deletion of K222 through T225), a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference lgG1), and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first amino acid chain of one of SEQ ID NOS.: 27-34 and a second amino acid chain of one of SEQ ID NOS.: 57- 61). Some such pairs of first and second amino acid chains of variant hinge regions are provided in Table 9.

[0132] In some embodiments, an Fc variant can comprise a variant hinge region comprising (i) a deletion of one or amino acids (e.g., deletion of K222 through T225) and a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference lgG1) (e.g., having a first heavy chain of one of SEQ ID NOS.: 80-88, 107-115, 134-142, 161-169, 513-521 , or 540-548 and a second heavy chain of one of SEQ ID NOS.: 215-223, 269-277, 323-331 , 377-385, 431-439, or 485-493); (ii) a deletion of one or amino acids (e.g., deletion of K222 through T225) and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first heavy chain of one of SEQ ID NOS.: 81-88, 108-115, 135-142 162-169, 519-526, or 546- 553 and a second heavy chain of one of SEQ ID NOS.: 189-196, 216-223, 243-250, 270- 277, 297-304, 324-331 , 351-358, 378-385, 405-412, 432-439, 459-466, or 486-493); or (iii) a deletion of one or amino acids (e.g., deletion of K222 through T225), a truncated second amino acid chain (e.g., consisting of amino acids 223-230 of the reference lgG1), and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein (e.g., having a first amino acid chain of one of SEQ ID NOS.: 81-88, 108-115, 135-142 162-169, 519-526, or 546-553 and a second heavy chain of one of SEQ ID NOS.: 216-223, 270-277, 324-331 , 378-385, 432-439, or 486-493). Some such pairs of first and second heavy chains of Fc variants are provided in Table 12.

[0133] In some embodiments, an Fc variant can comprise a variant hinge region comprising (i) a deletion of one or more amino acids (e.g., deletion of K222 through T225) in the firstamino acid chain and a lack of a second amino acid chain (e.g., having a first heavy chain of one of SEQ ID NOS.: 216-223, 270-277, 324-331 , 378-385, 432-439, or 486-493 and a second heavy chain of one of SEQ ID NOS.: 224, 278, 332, 386, 440, 494, or 549-563); (ii) an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein in the first amino acid chain and a lack of a second amino acid chain (e.g., having a first heavy chain of one of SEQ ID NOS.: 63-70, 72- 79, 81-88, 90-97, 99-106, 108-115, 117-124, 126-133, 135-142, 144-151 , 153-160, 162-169, 496-503, 505-512, 514-521 , 523-530, 532-539, or 541-548 and a second heavy chain of one of SEQ ID NOS.: 224, 278, 332, 386, 440, 494, or 549-563); (Hi) substitution of threonine residues (e.g., amino acid substitutions T223A and T225A) in the first amino acid chain and a lack of a second amino acid chain (e.g., having a first heavy chain of one of SEQ ID NOS.: 71-79, 98-106, 125-133, 152-160, 504-512, or 531-539 and a second heavy chain of one of SEQ ID NOS.: 224, 278, 332, 386, 440, 494, or 549-563); (iv) a deletion of one or a deletion of one or amino acids (e.g., deletion of K222 through T225) and an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein in the first amino acid chain and a lack of a second amino acid chain (e.g., having a first heavy chain of one of SEQ ID NOS.: 81-88, 108-115, 135-142 162-169, 514- 521 , or 541-548 and a second heavy chain of one of SEQ ID NOS.: 224, 278, 332, 386, 440, 494, or 549-563); or (v) an amino acid substitution of one or more residues involved in disulfide bridge formation, such as those substitutions provided herein and substitution of threonine residues (e.g., amino acid substitutions T223A and T225A) in the first amino acid chain and a lack of a second amino acid chain (e.g., having a first heavy chain of one of SEQ ID NOS.: 72-79, 99-106, 126-133, 153-160, 505-512, or 532-539 and a second heavy chain of one of SEQ ID NOS.: 224, 278, 332, 386, 440, 494, or 549-563).

[0134] Conservative variants of variant hinge regions, Fc variants and one-armed antibodies provided herein are contemplated. A conservative variant can comprise one or more conservative amino acid substitution(s) and / or substitution(s) based on common side-chain class or properties are well-known in the art and can be used in the embodiments of the present disclosure. For example, a conservative substitution can comprise a substitution of a member of a class of side chain to another member of the same class of side chain, as in Table 1 below. The present disclosure also contemplates variants based on nonconservative amino acid substitutions in which a member of one amino acid side chain class is exchanged for an amino acid from another class.

[0135] Table 1: Conservative Substitutions

[0136] In some embodiments, a variant hinge region, Fc variant or one-armed antibody can comprise a sequence that is 100% identical to a sequence provided in herein (e.g., in Table 7, Table 8, Table 10, or Table 11). In some embodiments, a variant hinge region, Fc variant, or one-armed antibody can comprise a sequence that is at least 80%, at least 85% at least 90%, at least 95%, or at least 99% identical to a sequence provided herein (e.g., in Table 7, Table 8, Table 10, or Table 11). In some such embodiments, a variant hinge region, Fc variant, or one-armed antibody can comprise one or more conservative substitutions in the amino acid sequence. In some such embodiments, each amino acid substitution of an amino acid sequence can be a conservative substitution.

[0137] For example, contemplated herein are variant hinge regions comprising a conservatively substituted amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to a sequence of SEQ ID NO: 8-61 . Also contemplated herein are Fc variants comprising a conservatively substituted HC1 amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a sequence of SEQ ID NO: 62-169 or 495-548 and a conservatively substituted HC2 amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a sequence of SEQ ID NO: 170-349 or 549-563. Also contemplated herein are one-armed antibodies comprising Fc variants comprising a conservatively substituted HC1 amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a sequence of SEQ ID NO: 62-169 or 495-548 and a conservatively substituted HC2 amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a sequence of SEQ ID NO: 170-349 or 549-563. Typically, a conservative substitution will not be of a residue that is specifically modified as described herein.EXAMPLES

[0138] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodimentand feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.

[0139] Example 1: Existing one-arm antibody format utilized in Onartuzumab poses challenges for manufacturing with mammalian cell lines

[0140] Methods

[0141] Typical antibodies and antigen binding fragments thereof are bivalent. Such antibodies can bind well to a target antigen, but can yield undesired activity in some biological settings. For example, an antagonist antibody can exert agonist activity if signaling is activated by proximity of antigen at each of the two binding sites of the bivalent antibody or antigen binding fragment thereof.

[0142] A one-armed (OA) antibody or antigen binding fragment thereof comprising only one antigen binding arm can engage an antigen monovalently. Although there can be several formats available, it can be desirable to engineer said one-armed antibody or antigen binding fragment thereof with a full length crystallizable fragment (Fc) domain. This can allow for FcRn mediated antibody recycling and a sufficiently long half-life.

[0143] To produce such a one-armed antibody in a mammalian cell expression system (e.g., to enable post-translational addition of an N-linked glycan at Asn297), a one-armed antibody was expressed transiently in an Expi293 cell line. This one-armed antibody comprised an N297G mutation to disrupt glycosylation and a knob-in-hole modification in CH3 (referred to herein as “intact hinge, N297G). To probe the effects of homodimerization of HC or Fc, disulfide mispairing in the HR and / or other domains, or other properties on heterogeneity, additional one-armed antibody constructs were produced.

[0144] To generate one-arm antibodies transiently in mammalian cells, 30ml_ of Expi293F cells were transfected with 30 ug of total DNA containing HC, LC, and Fc DNA at a ratio of 1 :4:1 , following a standard protocol by Thermo Fisher. The cultures were harvested by centrifugation at seven days post transfection, and the filtered supernatant was batch-bound to pre-equilibrated POROS MabSelect resin for affinity purification. The loaded resin was washed with 20 CV of 1X PBS with 500 mM NaCI, followed by 5 CV of 1X PBS. The antibody was eluted with 3 CV of Elution Buffer (0.1 M acetic acid, 0.15 M NaCI) and neutralized immediately with 1 M MOPS using 1 / 6 the volume of eluate. The neutralized antibody was injected onto HiLoad Superdex 16 / 600 200 pg at a flow rate of 1.0 mL / min using 2X PBS, pH 6.5 as the running buffer. Fractions containing the OA (one-armed antibody) were pooled.

[0145] Two “intact hinge, N297G” one-armed antibodies against different antigens (xh2DQJ and xhP006Hy1a.36) as well as an intact hinge variant comprising a C226S mutation (“C226S, N297G) and an intact hinge variant comprising a C229S mutation (C229S N297G), as well as an intact hinge variant comprising C226S and C229S mutations (“no-hinge,N297G) were generated. One-arm antibody variants of “no-hinge, N297G” with additional modifications (i.e., LALA, LALAPG, LALAGA, AND LAGA) which can suppress immune effector function were also generated using these methods.

[0146] SDS-PAGE analysis of the eight samples in a reducing environment was run using a standard protocol as provided in Table 2 below.

[0147] Table 2: SDS-PAGE analysis of one-armed constructs

[0148] Melting temperatures of the constructs were determined by differential scanning fluorimetry using a Nanotemper Prometheus instrument. The antibodies were subjected to temperature ramps from 20°C to 95°C at a rate of rc / min, and the changes in intrinsic fluorescence were monitored to determine melting temperature.

[0149] Results

[0150] A representative SDS-PAGE gel having bands corresponding to the heavy chain (HC), light chain (LC) and heterogeneity of Fc (Fc) is depicted in FIG. 1 .

[0151] Table 3 shows the percentage of potentially monomeric entities (%monomer) and temperature parameters measured for nine one-armed antibodies generated as described above. Tonis the temperature at which molecules start to unfold (or melt). Taggis the temperature at which molecules start to aggregate. Tm_i, Tm-2, and Tm_3(melting temperature) are each midpoint temperatures of a distinct unfolding event of a domain, which can be descriptive of thermostability. An IgG can have three melting temperatures, since it comprises three domains, each with different thermal properties. Tm_i describes the melting temperature (midpoint) of the CH2 domain or a combination of CH2 and Fab. Tm_2 describes the melting temperature of the Fab or a combination of Fab and CH2. Tm 3describes the melting temperature of the CH3 domain or a combination of Fab and CH3. Tonis used to compare the stability of these variants.

[0152] Table 3: Percent monomer and melting temperatures of one-armed constructs

[0153] The “Intact hinge, N297G” one-armed antibody constructs generated from mammalian cells displayed low monomer content (between 82% and 87%) and low thermostability, (Tonbetween 52°C - 53°C). Further, heterogeneity of these constructs was observed in reduced SDS-PAGE analysis (FIG. 1).

[0154] N297G “no hinge” constructs (comprising no disulfide bond in the hinge region) appeared more stable with higher Ton than “intact hinge” (one or two disulfide bonds in the hinge region, including C226S and C229S) constructs. The hinge_C229S and intact hinge constructs had worse percent monomer content than ‘no hinge’. Among the two single disulfide bond variants, hinge_C226S had a superior percent monomer content.

[0155] All non-N297G effector function attenuating mutations (in “no-hinge” background) except for FES resulted in significantly better thermal stability than N297G with Tonat 58°C- 60°C. Among the effector function-attenuating mutations, LALAGA displayed superior percent monomer content.

[0156] Example 2: O-glycosylation of the hinge contributes to heterogeneity

[0157] Wherein antibodies comprise N297G mutation (eliminating N-linked glycosylation), heterogeneity of a composition comprising one-armed antibodies can arise from glycosylation other than N-linked glycosylation. One-armed antibodies were treated with deglycosidase to remove O-linked glycan(s), and assessed using SDS-PAGE. For LC-MS analysis, In addition, antibodies with N297G or LALAGA mutations were treated with PNGase to remove N-linked glycan(s) and analyzed using LC-MS. to identify O-linked glycan(s).

[0158] Materials and methods

[0159] Deqlycosylation for SDS-PAGE assay

[0160] 20 ug of SEC-purified one-armed antibody was diluted to a concentration of 1 .0 mg / mL with 1XPBS, pH6.5, to which 2.5 pL of Deglycosylation Mix Buffer 2 (NEB, Cat#: P6044S) was added. The reaction mixture was incubated at 75°C for 10 minutes, and then cooled to 12°C. 2.5 pL of Protein Deglycosylation Mix II was then added to the reaction and mixed gently by pipetting. The mixture was incubated at 25°C for 30 minutes, and then at 37°C for 1 hour. The samples were analyzed on a reduced SDS-PAGE gel by loading 4pL of the reaction mixture per lane, and the SDS-PAGE was performed according to Invitrogen's NuPAGE instructions.

[0161] Deqlycosylation and reduction sample preparation for LC-MS analysis

[0162] 3pL PNGase F was used for 10ug antibody deglycosylation to remove N-glycans under non-denaturing conditions at 37C overnight. The deglycosylated samples were further reduced to break disulfide bonds in 5mM dithiothreitol (DTT) and 25mM ammonium bicarbonate at 56C for 30min.

[0163] 1-1.5 ug samples were analyzed using a PLRP-S 1000A, 2.1 x 50 mm, 8 pm (Agilent, P / N: PL1912-1802) column on a Thermo Fisher Ultimate 3000 high-performance liquid chromatography (HPLC) coupled with Thermo Fisher Q Exactive Mass Spectrometer (MS). For intact mass analysis, samples in 1xPBS, pH6.5 were directly injected and analyzed by LC-MS. For reduced mass analysis, samples after deglycosylation and reduction were injected and analyzed by LC-MS.

[0164] LC-MS data acquisition and analysis

[0165] Mobile phases are MS-grade water + 0.1% formic acid (A) and MS-grade acetonitrile + 0.1% formic acid (B). Below are HPLC instrumental method parameters: Method duration: 28min. Flow rate: 0.3 mL / mL. Flow gradient: Begin with 10% B. From 3.01 min to 18min, % B was increased linearly to 80%, followed by keeping constant for 5. OOmin, returning in 0.01 Omin to the initial condition, and equilibrating for 4.99min afterwards. The curve for all steps is 5. Column temperature was set at 60°C. Below are mass spec instrumental method parameters: Chromatographic peak width 15s. Full MS-SIM:run time 3-17.98min. Polarity positive. In-source CID 15.0 eV. Full MS-SIM: microscans 7. Resolution 17,500. AGC target 1e6. Maximum IT 200ms. Scan range 500-3000 m / z. Data was processed using PMI-Byos, intact mass workflow.

[0166] Results

[0167] Deglycosidase treatment appears to eliminate much of the heterogeneity as shown in the reduced SDS-PAGE gels (FIG. 2). U and T are samples that were untreated or treated with a Deglycosidase mix, respectively, as described in the Method section above. Samples, PUR4914, PUR4916, PUR4918, and PUR4920, were further analyzed by mass spectrometry.

[0168] Intact MS analysis of “Intact hinge, N297G” xh2DQJ (PUR4914) before and after PNGase indicates the presence of O-glycosylation in PUR4914 (FIGS. 3A, 3B, and 3C) Mutation of Threonine and Serine residues (including C226S) in the hinge region to Alanine eliminates the O-glycosylation site in the hinge in these samples, as shown in Table 4.

[0169] Table 4: LC-MS analysis of one-armed antibody constructs.

[0170] Example 3: Engineering approach to eliminate heterogeneity resulting from N- terminal clipping or alternative processing of the Fc chain and potential proteolysis within the hinge region while maintaining thermostability, binding function and limiting o-glycosylation

[0171] Methods

[0172] The sequences encoding the heavy chain, Fc-only chain, and light chains for anti- CD200R1 in the various hin ge configurations were individually cloned into a pRK mammalian expression vector that used a CMV promoter. Vectors contain an N-terminal IgHV secretory signal peptide. Recombinant one-armed antibodies were expressed using the Expi293 expression system according to standard protocols. The antibodies were harvested after 7 days of expression. Media supernatant was collected after centrifugation at 300 x g for 10 minutes followed by a second centrifugation at 12,857 x g for 15 minutes and subsequently filtered with a 0.22-micron filter. Recombinant antibodies were purified from clarified media using POROS MabSelect resin affinity chromatography on a benchtop gravity column. The eluate was concentrated and loaded onto a Superdex 200 pg 16 / 600 column using an AKTA FPLC system and eluted into 2x phosphate buffered saline (PBS) pH 6.5. The major peak fractions were pooled, concentrated, and filtered using a 0.22-micron syringe filter.

[0173] To determine thermostability of hinge variants of anti-hCD200R1 antibodies, DSF measurements were performed using a Prometheus Nanotemper. Antibodies were normalized to 1 mg / mL in 2X PBS pH 6.5 and subjected to temperature increases from 20°C to 95°C at 1 °C / minute. Intrinsic fluorescence was monitored and fit to determine the melting temperatures.

[0174] To determine the binding affinity of hinge variants of anti-hCD200R1 antibodies, SPR measurements were performed using a Biacore 8K+ instrument at 25 °C. Biotinylated CD200R1 (0.5 pg / mL) in HBS-P+ buffer (0.01 M 4-[2-hydroxyethyl]-1- piperazineethanesulfonic acid [HEPES], pH 7.4; 0.15 M NaCI, 0.005% Surfactant P20) was captured onto a CAPture chip using a flow rate of 30 pL / min. Flow cell 1 (FC1) was left blank and kept as a reference. Next, 3-fold serial dilutions of antibodies in 1X HBS-P+ buffer ranging from 0.27 to 200 nM were injected at a flow rate of 30 pL / min. Each sensorgram was recorded one time and subjected to reference and buffer subtraction prior to analysis. Analysis was performed using Biacore 8K+ Evaluation Software (version 1.1.1.7442). Association rates (ka) and dissociation rates (kd) were calculated using a one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the ratio of the dissociation and association rates (kd / ka).

[0175] Deqlycosylation and reduction sample preparation for LC-MS analysis

[0176] 3 L PNGase F was used for 10ug antibody deglycosylation to remove N-glycans under non-denaturing conditions at 37C overnight. The deglycosylated samples were further reduced to break disulfide bonds in 5mM dithiothreitol and 25mM ammonium bicarbonate at 56C for 30min.

[0177] LC-MS data acquisition and analysis

[0178] 1-1.5 ug samples were analyzed using a PLRP-S 1000A, 2.1 x 50 mm, 8 pm (Agilent, P / N: PL1912-1802) column on a Thermo Fisher Ultimate 3000 high-performance liquid chromatography (HPLC) coupled with Thermo Fisher Q Exactive Mass Spectrometer (MS). For intact mass analysis, samples in 1xPBS, pH6.5 were directly injected and analyzed by LC-MS. For reduced mass analysis, samples after deglycosylation and reduction were injected and analyzed by LC-MS.

[0179] Mobile phases are MS-grade water + 0.1% formic acid (A) and MS-grade acetonitrile + 0.1% formic acid (B). Below are HPLC instrumental method parameters: Method duration: 28min. Flow rate: 0.3 mL / mL. Flow gradient: Begin with 10% B. From 3.01 min to 18min, % B was increased linearly to 80%, followed by keeping constant for 5. OOmin, returning in 0.01 Omin to the initial condition, and equilibrating for 4.99min afterwards. The curve for all steps is 5. Column temperature was set at 60°C. Below are mass spec instrumental method parameters: Chromatographic peak width 15s. Full MS-SIM: run time 3-17.98min. Polarity positive. In-source CID 15.0 eV. Full MS-SIM: microscans 7. Resolution 17,500. AGC target 1e6. Maximum IT 200ms. Scan range 500-3000 m / z. Data was processed using PMI-Byos, intact mass workflow.

[0180] Results

[0181] With mutation in the hinge for the removal of o-linked glycosylation, we further confirm the product quality and stability and we examine the detailed product heterogeneity using LC-MS. Antibodies in “Intact hinge”, “Intact hinge_C226A” or “No hinge” one-arm formats with either LALAGA or N297G for Fc effector suppression were examined. Firstly, their monomer content (%) after protein A affinity column purification and their thermostability after AKTA-SEC 2nd step purification is consistent with what was observed as One-arm antibodies without the mutation for removal of O-glycan (Table 4). Secondly, LC-MS showed that O-glycan was not present. Thirdly, LC-MS also showed that Fc chain can be present in different N-term sequences from potential enzymatic processing to different extents for each one-arm antibody. In Table 4, the N terminus of the Fc chain is shown for each one-arm antibody and the ones that are showing clipping has ain between the amino acid residues where clipping may have happened based on the mass identified. The extent of clipping is estimated from the relative peak size of non-clipped peak vs clipped peak and noted under the column “Fc clipping %.” An example of LC-MS identifying the clipping is in FIG. 4A.

[0182] To reduce the heterogeneity from alternative clipping or processing at the N terminus of the Fc chain, we designed a shorter hinge for “Intact hinge_C226A” for the Fc chain and have the N terminus starting from APPCPAPEAA instead of AHAAPPCPAPEAA and the one arm antibodies with this Fc chain do not show clipping anymore. For “No hinge” antibodies, mutating the N-terminus of the Fc chain to AGGPSVFLEPP resulted in removal of this clipping issue for all one arm “No hinge” antibodies (Table 5). Binding kinetics for select hinge variants as determined by Biacore CAPture are provided in Table 6.

[0183] Table 5: Clipping in one-armed antibody constructs

[0184] Table 6: Binding kinetics of one-armed antibodies

[0185] Example 4: Hinge_C226A preserves the stability that can withhold the low pH incubation after Protein A column elution

[0186] Methods

[0187] Samples in four OA formats in the arginine succinate buffer, pH 5.5, at 100 mg / mL were adjusted to pH~3.5 by adding protein A elution buffer (0.1 M Acetic Acid, 0.15 M NaCI, pH = 3). The sample concentration was diluted to 2.5 mg / mL after pH adjustment. The samples were incubated at room temperature for 4.5 hours to assess low pH stability Samples were exchanged back to arginine succinate buffer, pH 5.5, using Amicon Ultra 0.5 mL 10k Centrifugal Filters (Millipore Sigma). Size exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) was performed to evaluate the low pH stability of the OA molecules in four formats.

[0188] SEC-MALS was carried out on a Waters XBridge BEH SEC column (200A, 3.5um, 7.8*300 mm, PN: 186007640) on a Thermo Scientific Ultimate 3000 coupled with Wyatt Technology miniDAWN TREOS and Optilab T-rEX. The running buffer is 2xPBS, diluted from 10xPBS (Teknova). Buffer was filtered through a 0.2um PES membrane (Thermo Scientific). Below are the parameters in instrumental software (Chromeleon 7) : 40min isocratic method, 0.5mL / min flow rate, and UV wavelength 280 nm. Data was processed using Chameleon 7. Peaks were auto-integrated by the software and examined by the user.

[0189] Results

[0190] SEC-MALS results (SEC chromatograms were shown with percentage composition labeled, together with molar masses provided by MALS listed in the table inset in each figure) (FIG. 5A and FIG. 5B)

[0191] Example 5: Extending the hinge engineering to Fc fusion protein design

[0192] Methods

[0193] The gene for human LTBR (UniProt ID: P36941) encompassing residues A32-M225 was attached to the N-terminus of the sequences encoding the Fc variants that differ in their hinge sequences (Table 1), and were codon-optimized for human cell expression, synthesized, and cloned into a pRK vector downstream of and in frame with a mouse Ig HC signal sequence for strong secretion.

[0194] Each construct was expressed using 100 mL of Expi293F, following Thermo Fisher’s protocol. The harvested and filtered conditioned medium was put through an affinity purification by batch-binding to MabSelectSuRe resin for 1 hour at 4°C, followed by gravityflow purification. The load resin was washed with 40CV of 1XPBS, pH7.2, and the protein was eluted with 2x10 CV of Elution Buffer (0.1 M Gly-HCI, pH3.5, 150mM NaCI) and neutralized with 1 M TRIS, pH7.5 using 1 / 1 Oth the eluate volume. The eluate was concentrated using a 10K MWCO concentrator to 1mL and was injected onto the HiLoad Superdex 200 pg SEC column. The fractions containing the non-aggregated protein of interest were pooled, and the proteins were further characterized by differential scanning fluorimetry to determine their thermal stabilities.

[0195] Results

[0196] In contrast with one-armed antibodies, the purified LTBR-Fc proteins did not exhibit obvious heterogeneity on SDS-PAGE gel. Samples with a single disulfide bond, SS and short hinge SS, had higher thermal stability than without any disulfide bonds. Samples with shortened hinge, shortSS and short hinge noSS, had significantly lower product quality than those with normal length hinge.

[0197] Sequence Tables

[0198] Amino acid sequences of Fc regions, portions thereof, and other polypeptides described herein are provided in Table 7 below:

[0199] Table 7: Sequences of amino acid chains of selected Fc components

[0200] Amino acid sequences of amino acid chains of variant hinge regions described herein are provided in Table 8 below:

[0201] Table 8: sequences of amino acid chains of variant hinge regions

[0202] Exemplary combinations of first and second amino acid chain sequences that can yield variant hinge regions described herein are provided in Table 9 below:

[0203] Table 9: Exemplary combinations of first and second amino acid chains yielding variant hinge regions

[0204] Table 10: sequences of HC1 amino acid chains of Fc variants

[0205] Table 11 : sequences of HC2 amino acid chains of Fc variants

[0206] Table 12: Exemplary combinations of HC1 and HC2 yielding Fc variants

[0207] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the following claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the following claims.

Claims

CLAIMS1. A variant hinge region, comprising a first amino acid chain of a hinge region of a reference IgG 1 comprising amino acids 216-230 of the reference IgG 1 and a second amino acid chain of the hinge region of the reference lgG1 consisting of amino acids 223-230 of the reference IgG 1 according to the Eu numbering system.

2. A variant hinge region, comprising a first amino acid chain of a hinge region of a reference IgG 1 comprising amino acids 216-230 of the reference IgG 1 and a second amino acid chain of the hinge region of the reference lgG1 comprising amino acids 223- 230 of the reference lgG1 , comprising deletion of amino acids 216-222 of the second amino acid chain, according to the Eu numbering system.

3. A variant hinge region, comprising a first amino acid chain of a hinge region of a reference lgG1 and a second amino acid chain of the hinge region of the reference lgG1 , and comprising one or more amino acid substitutions selected from the group consisting of: C226A, C226S, C226G, C229A, C229S, and C229G, according to the Eu numbering system.

4. A variant hinge region comprising a first amino acid chain of a hinge region of a reference lgG1 and a second amino acid chain of the hinge region of the reference lgG1 , and comprising the amino acid substitutions T223A and T225A according to the Eu numbering system.

5. A variant hinge region, comprising a first amino chain of a hinge region of a reference lgG1 and a second amino acid chain of the hinge region of the reference IgG 1 , and comprising deletion of K222 through T225 of the reference I gG 1 according to the Eu numbering system.

6. The variant hinge region of any of claims 1-5, wherein the hinge region of the reference lgG1 has a sequence of SEQ ID NO: 8.

7. An Fc variant comprising the variant hinge region of any of claims 1 -6.

8. The Fc variant of claim 7, having a first heavy chain comprising the first amino acid chain of the variant hinge region and a second heavy chain comprising the second amino acid chain of the variant hinge region.

9. The Fc variant of any of claims 5-7, wherein the first heavy chain comprises a hinge sequence, CH1 , CH2, and CH3.

10. The variant hinge region of any of claims 1 , 2, or 4-7, or the Fc variant of any of claims 5- 8, further comprising one or more amino acid substitutions selected from the group consisting of: C226A, C226S, C226G, C229A, C229S, and C229G according to the Eu numbering system.

11. The variant hinge region of any of claims 1-7 or claim 10, or the Fc variant of any of claims 7-10, wherein the first chain of the variant hinge region comprises (i) C226A, (ii) C226S, (iii) C229A, (iv) C229S, (v) C226A and C229A, (vi) C226A and C229S, (vii) C226S and C229A, or (viii) C226S and C229S.

12. The Fc variant of any of claims 7-11 , wherein the second heavy chain is a shortened heavy chain.

13. The Fc variant of any of claims 7-12, wherein the second heavy chain lacks a CH1 segment.

14. The Fc variant of any of claims 7-13, wherein the second heavy chain comprises CH2 and CH3.

15. The variant hinge region of any of claims 1-7 or claims 10-11, or the Fc variant of any of claims 7-14, wherein the second chain of the variant hinge region comprises (i) C226A, (ii) C226S, (iii) C229A, (iv) C229S, (v) C226A and C229A, (vi) C226A and C229S, (vii) C226S and C229A, or (viii) C226S and C229S.

16. The Fc variant of any of claims 7-15, wherein the first heavy chain comprises amino acids 216-446 (residues of hinge, CH2, CH3) of an lgG1 Fc region.

17. The Fc variant of any of claims 7-16 wherein the second heavy chain comprises amino acids 223-446 (residues of CH2 and CH3) of an IgG 1 Fc region.

18. The Fc variant of any of claims 7-17 or claims 13-16, wherein the second heavy chain further comprises amino acids 216-230 (residues of hinge) of an IgG Fc region.

19. The Fc variant of any of claims 7-18, wherein the Fc is of an IgG 1 .

20. The Fc variant of any of claims 7-19, wherein the IgG 1 is a human IgG 1 .21 . The Fc variant of any of claims 7-20, wherein the IgG is a human IgG 1 allotype.

22. The Fc variant of claim 7-21 , wherein the human IgG 1 allotype comprises the first heavy chain sequence SEQ ID NO:1 and the second heavy chain sequence.

23. The Fc variant of any of claims 7-22, wherein the Fc comprises CH1 , CH2, or CH3 of an IgG, an IgM, an IgD, an IgA, or an IgE.

24. The variant hinge region of any of claims 1-7 or claims 10-11 , claim 15, or the Fc variant of any of claims 7-24, comprising an amino acid substitution of Thr residues in the variant hinge region.

25. The variant hinge region of any of claims 1-7, claims 10-11 , claim 15, or the Fc variant of any of claims 5-23, wherein the first chain of the variant hinge region and the second chain of the variant hinge region comprise T225A and T223A.

26. The variant hinge region of any of claims 1-7, claims 10-11 , claim 15, or claim 25, or the Fc variant of any of claims 5-23 or claim 24, wherein the first chain of the variant hinge region comprises the deletion of amino acids 222 through 225 and the second chain of the variant hinge region comprises deletion of amino acids 223 through 225.

27. The Fc variant of any of claims 5-26, further comprising a light chain constant region (CL) .

28. The Fc variant of claim 28, wherein the CL sequence is associated with CH1 of the first heavy chain.

29. The Fc variant of claim 28 or claim 29, wherein the CL sequence is of SEQ ID NO: 7.

30. The Fc variant of any of claims 5-30, further comprising an effectorless mutation.31 . The Fc variant of any previous claim, wherein the effectorless mutation is in the CH2 domain.

32. The Fc variant of claim 31 , wherein the effectorless mutation is selected from the group consisting of LALA, LALAPG, LALAGA, and LAGA.

33. The Fc variant of claim 31 or claim 32, wherein the effectorless mutation is LALAGA.

34. The Fc variant of any of claims 5-33, further comprising a knob-in-hole structure.

35. The Fc variant of claim 34, wherein the knob-in-hole structure comprises (i) T366S, L368A, and Y407V amino acid substitutions on the first heavy chain, and (ii) a T366W amino acid substitution on the second heavy chain.

36. The Fc variant of claim 34 or claim 35, wherein the knob in hole structure comprises mutations in the CH3 domain.

37. The Fc variant of any of claims 7-36, further comprising an N297G amino acid substitution38. Fc variant of any of claims 7-37, wherein the Fc variant is an Fc variant of an Fc fusion.

39. An antibody comprising variant hinge region of any of claims 1-6, claims 10-11 , or claim 15, or an Fc variant of any of claims 7-38.

40. The antibody of claim 39, wherein the antibody is a one-armed antibody.41 . The antibody of claim 39 or claim 40, wherein the antibody is an antigen binding fragment of an antibody, a bispecific antibody, a fusion of an antibody or antigen binding fragment thereof and another protein, a fusion of a fragment of a first antibody, or a fragment of a second antibody.

42. An isolated nucleic acid sequence encoding a hinge region of any of claims 1 -6, claims 10-11 , or claim 15, an Fc variant of any of claims 7-38, or an antibody of any of claims 39-41.

43. An isolated cell comprising the nucleic acid of claim 43.

44. A pharmaceutical composition comprising a therapeutically effective amount of an antibody of any of claims 39-41 and an excipient.

45. The Fc variant of any of claims 7-38, wherein the first heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-169 and 495-548, and the second heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 170-563.

46. The Fc variant of any of claims 7-38, wherein the first heavy chain and second heavy chain are selected from the first heavy chain and second heavy chain pairs provided in Table 12.

47. The antibody of any of claims 39-41 , wherein the first heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-169 and 495-548, and the second heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 170-563.

48. The antibody of any of claims 39-41 , wherein the first heavy chain and second heavy chain are selected from the first heavy chain and second heavy chain pairs provided in Table 12.

49. An Fc variant or one-armed antibody, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein a. HC1 comprises: i. a CH1 domain, ii. a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 , comprising the amino acid substitutions C226A, T223A and T225A, iii. a CH2 domain comprising a LALAGA mutation, and iv. a CH3 comprising a hole mutation; and b. HC2 comprises: i. a hinge comprising an amino acid chain consisting of amino acids 236- 230 of a reference IgG 1 , comprising the amino acid substitution C226A, ii. a CH2 domain comprising a L235A mutation, and iii. a CH3 comprising a knob mutation.

50. An Fc variant or one-armed antibody, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein a. HC1 comprises: i. a CH1 domain, ii. a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 , comprising the amino acid substitutions C226A, C229A, T223A and T225A, iii. a CH2 domain comprising a LALAGA mutation, and iv. a CH3 comprising a hole mutation; and b. HC2 comprises: i. no hinge, ii. a CH2 domain comprising deletion of A231 through L234 and a LALAGA mutation, and iii. a CH3 domain comprising a knob mutation.51 . An Fc variant or one-armed antibody, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein a. HC1 comprises: i. a CH1 domain, ii. a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 comprising the amino acid substitution C226A and a deletion of K222 through T225,iii. a CH2 domain comprising a LALAGA mutation, and iv. a CH3 comprising a hole mutation; and b. HC2 comprises: i. a hinge comprising an amino acid chain consisting of amino acids 236- 230 of a reference IgG 1 , comprising the amino acid substitution C226A, ii. a CH2 domain comprising a LALAGA mutation, and iii. a CH3 comprising a knob mutation.

52. An Fc variant or one-armed antibody, comprising: a first heavy chain (HC1) and a second heavy chain (HC2), wherein a. HC1 comprises: i. a hinge comprising an amino acid chain comprising amino acids 216-230 of a hinge region of a reference IgG 1 comprising the amino acid substitutions C226A and C229A and a deletion of K222 through T225, ii. a CH2 domain comprising a LALAGA mutation, and iii. a CH3 comprising a hole mutation; and b. HC2 comprises: i. no hinge, ii. a CH2 domain comprising deletion of A231 through L234 and a LALAGA mutation, and iii. a CH3 domain comprising a knob mutation. and wherein HC1 and HC2 comprise a C226A amino acid substitution and a deletion of K222 through T225, a LALAGA effectorless mutation53. An Fc variant or one-armed antibody comprising: a. an HC1 having an amino acid sequence of SEQ ID NO: 153 and an HC2 having an amino acid sequence of SEQ ID NO: 378; b. an HC1 having an amino acid sequence of SEQ ID NO: 155 and an HC2 having an amino acid sequence of SEQ ID NO: 554; c. an HC1 having an amino acid sequence of SEQ ID NO: 162 and an HC2 having an amino acid sequence of SEQ ID NO: 378; or d. an HC1 having an amino acid sequence of SEQ ID NO: 164 and an HC2 having an amino acid sequence of 554.

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