Antibodies containing modified heavy chain constant regions

Abstract

Provided herein are heavy chain constant regions (referred to as "modified heavy chain constant regions") or functionally equivalent fragments thereof that enhance the biological properties of an antibody compared to the unmodified form of the same antibody. An example of a modified heavy chain constant region comprises an IgG2 hinge and three constant domains (i.e., CH1, CH2, and CH3 domains), wherein one or more of the constant region domains are of a non-IgG2 isotype (e.g., IgG1, IgG3, or IgG4). The heavy chain constant region can comprise a wild-type human IgG domain sequence or a variant of the same sequence. Also provided herein are methods for enhancing certain biological properties, such as internalization, agonism, and antagonism, of antibodies comprising a non-IgG2 hinge, comprising replacing the non-IgG2 hinge of the antibody with an IgG2 hinge.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos. 62 / 511,178, filed May 25, 2017, and 62 / 599,221, filed December 15, 2017. The contents of all patents, patent applications, and references cited herein are hereby incorporated by reference in their entirety. [Background technology]

[0002] background Antibody therapeutics are one of the fastest growing areas in the treatment of diseases such as cancer and immune disorders. Nevertheless, efficient antigen targeting by therapeutic antibodies remains a major challenge in medicine. Therefore, antibody engineering has become a major focus of interest in the pharmaceutical industry. This has led to the emergence of many novel engineered antibodies, including antibody fragments, antibody-drug conjugates (ADCs), antibodies with modified effector domains, and bispecific antibodies.

[0003] Antibodies exert their therapeutic potential through a variety of mechanisms. Antibodies directly inhibit or activate target antigens, thereby regulating cell signaling. Antibodies can inhibit the binding of ligands to receptors. Antibodies can also induce or inhibit immune responses (e.g., as costimulators of T cell activation), for example, by activating a subject's immune system to fight infection or cancer.

[0004] Furthermore, antibody-mediated internalization of cell surface receptors / antigens is recognized as a major mechanism of action for therapeutic antibodies. In this case, the antibody removes the target from the cell surface, triggering its internalization within the cell and preventing its function. Indeed, one of the pioneering examples of antibody therapeutics is trastuzumab for the treatment of breast cancer. Trastuzumab targets the ErbB2 receptor, triggering receptor / antibody internalization and thus blocking EGFR signaling. However, antibodies do not always exhibit efficient internalization capabilities, and therefore there is a continuing need for antibodies with improved internalization functions. Therefore, methods to improve the internalization of existing therapeutic antibodies are highly desirable. Summary of the Invention

[0005] summary The present invention provides heavy chain constant regions (referred to as "modified heavy chain constant regions"), or functionally equivalent fragments thereof, that enhance or modify the biological properties of an antibody compared to the unmodified form of the same antibody. For example, antibodies comprising modified constant regions exhibit increased internalization and / or agonist or antagonist activity. Thus, the antibodies of the present invention are optimized versions of the original, unmodified antibody. In certain embodiments, the heavy chain comprises a modified constant region that contains one or more mutations or modifications compared to the wild-type heavy chain constant region. In certain embodiments, the modified heavy chain constant region comprises an IgG2 hinge and three constant domains (i.e., CH1, CH2, and CH3 domains), wherein one or more of the constant region domains are of a non-IgG2 human isotype (e.g., IgG1, IgG3, or IgG4) or a functionally equivalent fragment thereof. The modified constant region can include one or more (e.g., 1 to 10 or more) amino acid substitutions or deletions in the hinge or CH1, CH2, CH3 domains relative to the corresponding wild-type amino acid sequence or a variant thereof, e.g., the amino acid sequence of the hinge and / or each constant domain is at least about 80%, 85%, 90%, 95% or more (i.e., 96%, 97%, 98%, 99% or 100%) identical to the corresponding wild-type amino acid sequence.

[0006] In certain embodiments, the modified heavy chain constant region comprises a wild-type human IgG2 hinge or an amino acid sequence at least 95% identical to the amino acid sequence of a wild-type human IgG2 hinge. The hinge may further comprise additional modifications, for example, to reduce disulfide bond formation. In certain embodiments, the hinge comprises the amino acid substitution C219S compared to a wild-type human IgG2 hinge. In certain embodiments, the hinge comprises the amino acid sequence set forth in any of SEQ ID NOs: 8, 21-23, 126-132, and 134-147, or one of these sequences with 1 to 3 amino acids inserted between the CVE and CPP.

[0007] In certain embodiments, the modified heavy chain constant region comprises an IgG2 CH1 domain, eg, a wild-type human IgG2 CH1 domain or an amino acid sequence that is at least 95% identical to the amino acid sequence of the wild-type human IgG2 CH1 domain (SEQ ID NO: 7).

[0008] In some embodiments, the modified heavy chain constant region comprises an IgG1 CH2 domain, e.g., a wild-type human IgG1 CH2 domain or an amino acid sequence at least 95% identical to the amino acid sequence of the wild-type human IgG1 CH2 domain. The CH2 domain may further comprise additional modifications (e.g., to reduce or eliminate effector function). In some embodiments, the CH2 domain comprises amino acid substitutions A330S and P331S compared to wild-type full-length human IgG1 CH2. In some embodiments, the CH2 domain comprises SEQ ID NO: 24.

[0009] In certain embodiments, the modified heavy chain constant region comprises an IgG1 CH3 domain, e.g., a wild-type human IgG1 CH3 domain or an amino acid sequence at least 95% identical to the amino acid sequence of the wild-type human IgG1 CH3 domain. The CH3 domain may further comprise additional modifications to confer a particular allotype. In certain embodiments, the CH3 domain comprises amino acid residue E at position 356 and amino acid M at position 358 (the "f" allotype), compared to wild-type full-length human IgG1 of another allotype (e.g., the "fa" allotype, which has D and L at positions 356 and 358, respectively). In certain embodiments, the CH3 domain comprises SEQ ID NO: 5.

[0010] In certain embodiments, the antibody comprises a modified heavy chain constant region, wherein (a) the CH1 domain is a wild-type human IgG2 CH1 domain or a wild-type IgG1 CH1 domain, with or without additional modifications, (b) the hinge is a wild-type IgG2 hinge, with or without a C219S substitution, (c) the CH2 domain is a wild-type human IgG1 CH2 domain or a wild-type IgG2 CH2 domain, with or without additional modifications, and (d) the CH3 domain is a wild-type human IgG1 CH3 domain or a wild-type human IgG2 CH3 domain (e.g., of allotype f or fa), with or without the amino acid E at position 356 and the amino acid M at position 358. In certain embodiments, the modified heavy chain constant region comprises an amino acid sequence described herein, e.g., as set forth in any of SEQ ID NOs: 26-37 and 78-93.

[0011] The antibodies of the invention (i.e., antibodies with modified constant regions) may be fully human or humanized and may further exhibit one or more enhanced or altered characteristics compared to the same antibody lacking the modified heavy chain constant region. These characteristics may include increased or altered cellular internalization, agonist activity, formation of large cross-linked complexes, ADCC, receptor-mediated signaling, antagonist activity, immunomodulatory activity, and anti-tumor activity; or the introduction of novel properties, e.g., agonist activity.

[0012] Also provided are bispecific molecules and immunoconjugates comprising the modified constant regions of the invention, as well as compositions comprising the antibody, bispecific, or immunoconjugate and an acceptable pharmaceutical carrier. Such compositions may also include one or more additional therapeutic agents, such as agents that stimulate the immune system, such as a checkpoint inhibitor, a costimulatory molecule, an anti-CD39 antibody, or an anti-A2AR antibody.

[0013] Methods for producing antibodies comprising modified heavy chain constant regions are also provided. Some methods provided herein include methods for increasing cellular internalization of an antibody and methods for increasing the agonistic activity of an antibody compared to the same antibody comprising a hinge of a non-IgG2 isotype. Such methods include providing an antibody having a hinge that is not an IgG2 hinge and replacing the hinge with an IgG2 hinge (e.g., a hinge that is a wild-type human IgG2 hinge, a hinge having an amino acid sequence at least 95% identical to the amino acid sequence of a wild-type human IgG2 hinge, or a hinge that has been modified to reduce disulfide bond formation, e.g., a hinge containing the amino acid substitution C219S). In certain embodiments, the internalization of the antibody is enhanced or increased by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more, and the T 1 / 2 In certain embodiments, agonist activity is increased or enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more, as defined by increased cytokine release or increased proliferation of effector T cells; decreased T regulatory cell activity if binding to Tregs reduces Treg function; or increased Treg depletion.

[0014] In certain embodiments, the method further comprises replacing at least one of the CH1, CH2, or CH3 domains with a CH1, CH2, or CH3 domain of a different isotype. Such replacements include, for example, (a) replacing the CH1 domain with an IgG1 CH1 domain or an IgG2 CH1 domain; (b) replacing the CH2 domain with an IgG1 CH2 domain or an IgG2 CH2 domain; and / or (b) replacing the CH3 domain with an IgG1 CH3 domain or an IgG2 CH3 domain, wherein the replaced domain has a wild-type sequence or at least 95% identity to the wild-type sequence. In certain embodiments, the CH1 domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the CH2 domain is modified to reduce or eliminate effector function, for example, the CH2 domain comprises the amino acid substitutions A330S and P331S (SEQ ID NO: 24). In certain embodiments, the CH3 domain comprises amino acid residue E at position 356 and amino acid M at position 358 (SEQ ID NO: 5, allotype "f"), and in certain embodiments, the CH3 domain comprises allotype "fa."

[0015] The methods provided herein include methods of treating a subject by administering an antibody, bispecific molecule, or immunoconjugate comprising a modified heavy chain constant region. One or more additional therapeutic agents may also be co-administered, e.g., a therapeutic agent that stimulates the immune system, such as a checkpoint inhibitor, a costimulatory molecule, or the like.

[0016] Provided herein are antibodies comprising a modified heavy chain constant region comprising, in N-terminal to C-terminal order, a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, wherein (a) the CH1 domain comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs by up to 5 amino acids from or is at least 95% identical to SEQ ID NO: 7, wherein at least one of C131, R133, E137, S138, or R217 is not substituted or deleted; (b) the hinge comprises the amino acid sequence of SEQ ID NO: 8, 21-23, 126-132, or or 134-147, or a sequence that includes 1-3 amino acids inserted between CVE and CPP, or a sequence that differs by up to 5 amino acids, wherein the hinge does not include a simultaneous substitution or deletion of both C219 and C220; (c) the antibody has at least one enhanced property or a newly introduced property compared to the same antibody comprising an IgG1 hinge and CH1 domain; and (d) the modified heavy chain constant region is not a wild-type IgG2 constant region or an IgG2 constant region containing C219S and / or C220S. The hinge may comprise the amino acid sequence ERKXCVECPPCPAP (SEQ ID NO: 129) or ERKCXVECPPCPAP (SEQ ID NO: 130), where X is any amino acid except cysteine. For example, the hinge may comprise the amino acid sequence ERKSCVECPPCPAP (SEQ ID NO: 131) or ERKCSVECPPCPAP (SEQ ID NO: 132). In some embodiments, at least one or all of amino acid residues P233, V234, A235, and G237 are deleted or substituted with other amino acid residues, for example, the corresponding amino acid in the IgG1 hinge. In some embodiments, none of amino acid residues R133, E137, S138, and R217, or none of C131, R133, E137, S138, and R217, are substituted or deleted. In some embodiments, N192 and / or F193 are substituted with other amino acids. The antibody may comprise a CH2 domain that is at least 95% identical to that of wild-type IgG1. The antibody may comprise a CH3 domain that is at least 95% identical to that of wild-type IgG1.In certain embodiments, the CH2 and / or CH3 domain is not a wild-type IgG1 CH2 and / or CH3 domain, and the antibody has an effector function that is more potent than that of wild-type IgG1. In certain embodiments, the CH2 and / or CH3 domain is not a wild-type IgG1 CH2 and / or CH3 domain, and the antibody has an effector function that is less potent than that of wild-type IgG1. In certain embodiments, the antibody comprises a CH2 domain and / or CH1 domain that is at least 95% identical to that of wild-type IgG1 or IgG4. In certain embodiments, the antibody has at least one enhanced property or newly introduced property that is agonistic activity selected from agonistic activity, antibody-mediated receptor internalization, ADCC, receptor-mediated signaling, antagonistic activity, immunomodulatory activity, or anti-tumor activity.

[0017] In some embodiments, the antibody comprises a modified heavy chain constant region, wherein (a) the CH1 domain is a wild-type human IgG2 CH1 domain; (b) the hinge comprises any of SEQ ID NOs: 8, 21-23, 126-132, or 134-147, or a sequence comprising 1 to 3 amino acids inserted between the CVE and the CPP; (c) the CH2 domain is a wild-type human IgG1 CH2 domain or a modified CH2 domain that confers enhanced or reduced effector function to the antibody; and (d) the CH3 domain is a wild-type human IgG1 CH3 domain or a modified CH3 domain that confers enhanced or reduced effector function to the antibody. The modified heavy chain constant domain can comprise an amino acid sequence set forth in any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262, or an amino acid sequence at least 95% identical to one or more of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262. For Fc-containing heavy chains having an amino acid sequence at least 95% identical to any of these sequences, it is preferred that specific amino acid mutations made in these sequences to modulate biological activity remain unchanged.

[0018] In one embodiment, the antibody comprises a modified heavy chain constant region, wherein the heavy chain constant region has the sequence [ka] or a CH1 domain and hinge comprising an amino acid sequence that differs by up to 10 amino acids from SEQ ID NO: 133 or is at least 90% identical to SEQ ID NO: 133, wherein (i) at least one of C131, R133, E137, S138, and R217 is not substituted with another amino acid or deleted; (ii) C219 and C220 may be substituted with another amino acid or deleted, but not both C219 and C220; (iii) 1 to 3 amino acids may be inserted between CVE and CPP in the hinge; (iv) the hinge optionally contains an additional amino acid at the C-terminus, e.g., G; (v) one or more of the amino acids P233, V234, A235, and G237 may be substituted with another amino acid (e.g., the corresponding amino acid from IgG1) or deleted; (vi) the CH2 and CH3 domains are selected from wild-type or modified IgG1, IgG2, IgG3, or IgG4. (vii) the modified heavy chain constant region is not a wild-type IgG2 heavy chain constant region or a wild-type IgG2 heavy chain constant domain having C219S or C220S; and (viii) the antibody has at least one enhanced or newly introduced property compared to the same antibody comprising an IgG1 hinge and CH1 domain. In some embodiments, the antibody has at least one enhanced or newly introduced property selected from agonist activity, antibody-mediated receptor internalization, ADCC, receptor-mediated signal transduction, antagonist activity, immunomodulatory activity, or antitumor activity. In some embodiments, none of the amino acids C131; R133; E137; S138; or R217 is substituted with another amino acid or deleted. In some embodiments, N192 and / or F193 are not substituted or are N192 and / or F193, respectively. In certain embodiments, C219 is C219S, C220 is C220S, and P233 to G237 are substituted or deleted; V234 to G237 are substituted or deleted; A235 to G237 are substituted or deleted; G237 is substituted or deleted; P233 is substituted or deleted; P233 to V234 are substituted or deleted; or P233 to A235 are substituted or deleted.The antibody may have effector function or may have effector function removed. The antibody may comprise a wild-type or modified IgG1 CH2 domain and / or a wild-type or modified IgG1 CH3 domain.

[0019] In one embodiment, the antibody comprises a modified heavy chain constant region, wherein the heavy chain constant region has the sequence [ka] Alternatively, the antibody may comprise an amino acid sequence that differs by up to 10 amino acids from SEQ ID NO:7 or is at least 90% identical to SEQ ID NO:7, wherein (i) at least one of C131, R133, E137, S138, and R217 is not substituted or deleted; (ii) the modified heavy chain constant region is not a wild-type IgG2 heavy chain constant region or a wild-type IgG2 heavy constant domain having C219S or C220S; and (iii) the antibody has at least one enhanced or newly introduced property compared to the same antibody comprising an IgG1 hinge and CH1 domain. The antibody may have at least one enhanced or newly introduced property selected from agonist activity, antibody-mediated receptor internalization, ADCC, receptor-mediated signal transduction, antagonist activity, immunomodulatory activity, or anti-tumor activity. In some embodiments, none of the amino acids C131, R133, E137, and S138 is substituted with another amino acid or deleted. In some embodiments, N192 and / or F193 are not substituted or are N192 and / or F193, respectively. The antibody may have effector function or may have effector function removed. The antibody may comprise a wild-type or modified IgG1 CH2 domain and / or a wild-type or modified IgG1 CH3 domain.

[0020] The antibody may comprise a modified heavy chain constant region, wherein the heavy chain constant region has the sequence [ka] or an amino acid sequence that differs by a maximum of 5 amino acids from SEQ ID NO: 8, wherein (i) C219 and C220 may be substituted with other amino acids or deleted, but not both C219 and C220; (ii) one or more of amino acids P233, V234, A235, and G237 may be substituted or deleted; (iii) 1 to 3 amino acids may be inserted between the CVE and CPP of the hinge; (iv) the hinge optionally comprises an additional amino acid, e.g., G, at the C-terminus; (v) the CH2 and CH3 domains may be wild-type or modified IgG1, IgG2, IgG3, or IgG4 CH2 and CH3 domains; (vi) the modified heavy chain constant region is not a wild-type IgG2 heavy chain constant region or a wild-type IgG2 heavy constant domain having C219S or C220S; and (vii) the antibody has at least one enhanced property or a de novo introduced property compared to the same antibody comprising an IgG1 hinge and CH1 domain. The antibody may have at least one enhanced or newly introduced property selected from agonist activity, antibody-mediated receptor internalization, ADCC, receptor-mediated signal transduction, antagonist activity, immunomodulatory activity, or antitumor activity, which property is agonist activity. In some embodiments, C219 is C219S, C220 is C220S, and P233 to G237 are substituted or deleted; V234 to G237 are substituted or deleted; A235 to G237 are substituted or deleted; G237 is substituted or deleted; P233 is substituted or deleted; P233 to V234 are substituted or deleted; or P233 to A235 are substituted or deleted. The antibody may have effector function or may have ablated effector function. The antibody may comprise a wild-type or modified IgG1 CH2 domain and / or a wild-type or modified IgG1 CH3 domain.

[0021] Also provided are antibodies comprising a modified heavy chain constant region, wherein the heavy chain constant region comprises an IgG1 or IgG2 hinge, and the hinge lacks 1 to 7 amino acids, and the antibody has at least one enhanced or newly introduced property compared to the same antibody comprising an IgG1 hinge and CH1 domain. The antibody may have at least one enhanced or newly introduced property selected from agonist activity, antibody-mediated receptor internalization, ADCC, receptor-mediated signal transduction, antagonist activity, immunomodulatory activity, or antitumor activity. The hinge may be an IgG2 hinge lacking 1 to 4 amino acids, e.g., amino acids C219, C220, V222, and E224. The hinge may be an IgG1 hinge lacking amino acids 219, C220, D221, K222, T223, H224, and T225. The antibody may comprise a wild-type or modified IgG2 CH1 domain; a wild-type or modified IgG1 CH1 domain and an IgG1, IgG2 or IgG4 CH2 domain and an IgG1, IgG2 or IgG4 CH3 domain.

[0022] The antibody having a modified heavy chain constant region may be a human or humanized antibody, or an antigen-binding portion thereof. In some embodiments, the antibody specifically binds to an antigen involved in immune regulation. The antibody may be an agonist of a costimulatory receptor or an antagonist of an inhibitory receptor. For example, the antibody may bind to a costimulatory receptor selected from the group consisting of B7-1, B7-2, CD28, 4-1BB, GITR, OX40, ICOS, CD70, CD27, CD40, DR3, or CD28H, or may bind to an inhibitory receptor selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4. The antigen may be an antigen that needs to be internalized, for example, CD73. The antigen may be CD39.

[0023] In certain embodiments, the antibody comprising a modified heavy chain constant region specifically binds to a costimulatory receptor, e.g., GITR, OX40, 4-1BB, CD28, ICOS, CD40, CD27, or any other TNFR superfamily member, and comprises a modified heavy chain constant region selected from the group consisting of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262. In certain embodiments, the antibody exhibits enhanced or altered agonist activity relative to an antibody having the same variable region and light chain but comprising an IgG1 heavy chain constant region.

[0024] In certain embodiments, the antibody comprising a modified heavy chain constant region specifically binds to a cell surface molecule, e.g., CD73, induces antibody-mediated internalization of the cell surface molecule, and comprises a modified heavy chain constant region selected from the group consisting of SEQ ID NOs: 26-37, 54-56, 78-125, and 152-232. In certain embodiments, the antibody has enhanced or altered internalization properties compared to an antibody having the same variable region and light chain but comprising an IgG1 heavy chain constant region. The anti-CD73 antibody may also bind to an Fc having any amino acid sequence selected from the group consisting of SEQ ID NOs: 234-245 and 247-262.

[0025] In some embodiments, the antibody comprising a modified heavy chain constant region specifically binds to an inhibitory receptor, e.g., CTLA-4, PD-1, LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and comprises a modified heavy chain constant region selected from the group consisting of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262. In some embodiments, the antibody exhibits more potent or altered antagonist activity, or a novel activity is introduced, compared to the same antibody having an IgG1 heavy chain constant region. In some embodiments, the Fc comprises one or more mutations to modulate, e.g., reduce, effector function.

[0026] In certain embodiments, an antibody comprising a modified heavy chain constant region specifically binds to a cell surface molecule and induces intracellular signaling, wherein the antibody comprises a modified heavy chain constant region selected from the group consisting of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262. In certain embodiments, the intracellular signaling mediates agonist activity, antagonist activity, internalization of a cell surface molecule, or ADCC. In certain embodiments, the antibody induces stronger intracellular signaling than an antibody having the same variable region and light chain but comprising an IgG1 heavy chain constant region.

[0027] In certain embodiments, an antibody comprising a modified heavy chain constant region specifically binds to a cell surface molecule and induces the formation of a high molecular weight antibody-cell surface molecule complex, wherein the antibody comprises a modified heavy chain constant region selected from the group consisting of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262. In certain embodiments, the antibody induces the formation of a higher molecular weight complex than an antibody having the same variable region and light chain but comprising an IgG1 heavy chain constant region.

[0028] In certain embodiments, an antibody comprising a modified heavy chain constant region specifically binds to a cell surface molecule and induces clustering or oligomerization of the cell surface molecule, wherein the antibody comprises a modified heavy chain constant region selected from the group consisting of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262. In certain embodiments, the antibody induces greater clustering or oligomerization of cell surface molecules compared to an antibody having the same variable region and light chain but comprising an IgG1 heavy chain constant region.

[0029] Also provided herein are bispecific molecules comprising an antibody comprising a modified heavy chain constant region linked to a molecule having a second binding specificity. Also provided herein are immunoconjugates comprising an antibody comprising a modified heavy chain constant region linked to a second agent. Also provided are compositions comprising the antibodies, bispecifics, or immunoconjugates described herein and a carrier. The compositions may include one or more additional therapeutic agents, e.g., a therapeutic agent that stimulates the immune system, e.g., an antagonist of a checkpoint inhibitor or a costimulatory receptor.

[0030] Also provided herein is a method for producing an antibody comprising a modified heavy chain constant region comprising, in N-terminal to C-terminal order, a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, the method comprising the steps of: (a) providing an antibody comprising a hinge and / or CH1 domain that is not an IgG2 hinge and / or IgG2 CH1 domain; and (b) replacing the hinge and / or CH1 domain with an IgG2 hinge and / or IgG2 CH1 domain, respectively. Also provided herein is a method for increasing antibody internalization by cells, the method comprising: (a) providing an antibody comprising a hinge and / or CH1 domain that is not an IgG2 hinge and / or IgG2 CH1 domain; and (b) replacing the hinge and / or CH1 domain with an IgG2 hinge and / or IgG2 CH1 domain, respectively. Antibody internalization can be increased compared to the internalization of the same antibody comprising a hinge of a non-IgG2 isotype, for example, an antibody comprising an IgG1 constant region. Also provided is a method for increasing the agonistic activity of an antibody, comprising: (a) providing an antibody comprising a hinge and / or CH1 domain that is not an IgG2 hinge and / or IgG2 CH1 domain; and (b) replacing the hinge and / or CH1 domain with an IgG2 hinge and / or IgG2 CH1 domain, respectively. The agonistic activity may be increased compared to the agonistic activity of the same antibody comprising a hinge of a non-IgG2 isotype, e.g., an antibody comprising an IgG1 constant region. The IgG2 hinge may be a wild-type human IgG2 hinge or may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of a wild-type human IgG2 hinge, e.g., a sequence shown in Table 4. The method may include replacing at least one of the CH1, CH2, or CH3 domains with a CH1, CH2, or CH3 domain, respectively, of a different isotype. The method may comprise the steps of: (a) replacing the CH1 domain with an IgG2 CH1 domain; (b) replacing the CH2 domain with an IgG1 CH2 domain; and / or (b) replacing the CH3 domain with an IgG1 CH3 domain.The method may include the steps of (a) replacing the CH1 domain with a wild-type human IgG2 CH1 domain or a domain at least 95% identical thereto; (b) replacing the CH2 domain with a wild-type human IgG1 CH2 domain or a domain at least 95% identical thereto; and / or (b) replacing the CH3 domain with a wild-type human IgG1 CH3 domain or a domain at least 95% identical thereto. The method may include the steps of replacing the heavy chain constant region with a modified heavy chain constant region comprising any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262, or a region at least 95% identical to SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262 (or introducing amino acid mutations of these sequences into the Fc). The hinge may be modified to reduce or alter disulfide bond formation. The hinge may contain the amino acid substitution C219S. The hinge may contain the amino acid sequence set forth in any of SEQ ID NOS: 8, 21-23, 126-132, or 134-147, or a sequence containing 1 to 3 amino acids inserted between the CVE and CPP. The CH1 domain may contain the amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 7). The CH2 domain may be modified to reduce or eliminate effector function. The CH2 domain may contain the amino acid substitutions A330S and P331S. The CH2 domain can comprise the amino acid sequence PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 4). The CH2 domain can comprise the amino acid substitutions A330S and P331S. The CH3 domain can comprise the amino acid sequence: GQPREPQVYTLPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) may include:

[0031] Also provided herein are modified heavy chain constant regions that have reduced or undetectable binding to one or more FcγRs (e.g., CD16, CD32, CD64). Such modified heavy chain constant regions can have one to five, one to three, one to two, or a single mutation (e.g., substitution) relative to the wild-type heavy chain constant region.

[0032] Also provided are antibodies or antigen-binding portions thereof, e.g., human or humanized antibodies, described herein, e.g., produced by the methods described above. Methods of treating a subject, e.g., a subject with cancer, with any of the antibodies described herein are also encompassed herein. The methods may include administering one or more additional therapeutic agents, e.g., a therapeutic agent that stimulates the immune system. For example, the therapeutic agent may target a checkpoint inhibitor or a costimulatory molecule. The methods may include administering a composition, bispecific molecule, or immunoconjugate described herein. [Brief explanation of the drawings]

[0033] [Figure 1A]Figure 1A shows the kinetics of antibody-mediated internalization of CD73 in H2228 cells (non-small cell lung cancer cell line) by the following antibodies: 11F11, 4C3, 6D11, CD73.3-IgG1.1f with 4C3Vk1 light chain ("3-Vh-hHC-IgG1.1f / 4C3Vk1"), 11F11 The CD73.4-IgG2CS ("4-Vh-hHC-IgG2-C219S / 11F11-Vk2"), CD73.10-IgG2CS ("CD73.10-Vh-hHC-IgG2-C219S"), CD73.10-IgG2CS-IgG1.1f ("CD73.10-Vh-hHC-IgG2-C219S-IgG1.1f"), and CD73.10-IgG1.1f ("CD73.10-Vh-hHC-IgG1.1f") antibodies have a Vk2 light chain. The 11F11 (which is of the IgG2 isotype), CD73.4-IgG2CS, CD73.10-IgG2CS, and CD73.10-IgG2CS-IgG1.1f antibodies are internalized more rapidly and to a greater extent than the other tested antibodies, which are of the IgG1 isotype.

[0034] [Figure 1B] FIG. 1B shows the kinetics of antibody-mediated CD73 internalization for the same antibodies shown in FIG. 1A in HCC15 cells (a non-small cell lung cancer cell line), showing similar results to those obtained with H2228 cells.

[0035] [Figure 1C] Figure 1C shows the kinetics of antibody-mediated CD73 internalization in Calu6 cells using the same antibodies shown in Figures 1A and 1B as well as CD73.11-IgG2CS ("11-Vh-hVC-IgG2-C219S"), showing similar results to those obtained with H2228 and HCC15 cells.

[0036] [Figure 1D] Figure 1D shows the kinetics of antibody-mediated CD73 internalization for the same antibodies shown in Figure 1C in NCI-2030 cells (a non-small cell lung cancer cell line), showing similar results to those obtained in H2228, HCC15, and Calu6 cells.

[0037] [Figure 1E] FIG. 1E shows the kinetics of antibody-mediated CD73 internalization of the indicated antibodies in Calu6 cells as measured by flow cytometry.

[0038] [Figure 1F] Figure IF shows the kinetics of antibody-mediated CD73 internalization of the indicated antibodies in NCI-H292 cells (a mucoepidermoid lung carcinoma cell line) as measured by flow cytometry, where the antibodies were not washed off after initial incubation of the cells with the antibodies.

[0039] [Figure 1G] FIG. 1G shows the percentage of CD73 internalized in Calu6 cells treated with the indicated antibodies and shows antibody-mediated CD73 internalization over time for the indicated antibodies in Calu6 cells.

[0040] [Figure 1H] FIG. 1H shows the percentage of CD73 internalized in NCI-H292 cells treated with the indicated antibodies over time, showing antibody-mediated CD73 internalization over time for the indicated antibodies in NCI-H292 cells.

[0041] [Figure 1I] Figure 1I shows the percentage of CD73 internalized in SNU-C1 cells (a colon cancer cell line) treated with the indicated antibodies over time, demonstrating antibody-mediated CD73 internalization over time for the indicated antibodies in SNU-C1 cells.

[0042] [Figure 1J] Figure 1J shows the percentage of CD73 internalized in NCI-H1437 cells (non-small cell lung cancer cell line) treated with the indicated antibodies over time, demonstrating antibody-mediated CD73 internalization over time for the indicated antibodies in NCI-H1437 cells.

[0043] [Figure 2]FIG. 2 shows the binding kinetics of the indicated anti-human GITR antibodies to anti-CD3 (plate-coated) and CD28-activated human CD4 T cells and their corresponding graphically derived EC50 values.

[0044] [Figure 3] Figures 3A, 3B, and 3C show the secretion of IFN-γ and IL-2 from donor CD4 T cells stimulated with soluble anti-human GITR antibodies having various heavy chain constant regions. Figure 3A shows IFN-γ secretion from donor CD4 T cells stimulated with OKT3-expressing CHO cells and various concentrations of anti-human GITR antibodies having an IgG2-IgG1 constant region. Figure 3B shows IL-2 secretion from donor CD4 T cells stimulated with OKT3-expressing CHO cells and various concentrations of an IgG1 heavy chain constant domain or an IgG2-IgG1 hybrid heavy chain constant domain. Figure 3C shows IL-2 secretion from donor CD4 T cells stimulated with OKT3-expressing CHO cells and various concentrations of an effector-less version (IgG1.1) of the antibody in Figures 3A and B.

[0045] [Figure 4] Figure 4 shows IL-2 secretion from 3A9-hGITR cells cultured on anti-CD3 monoclonal antibody-coated plates in the presence of increasing amounts of the indicated anti-human GITR antibodies: hybridoma anti-GITR (IgG2) and its recombinant derivatives as chimeras with IgG1f, IgG1.1 (effectorless), or IgG2 hinge.

[0046] [Figure 5] Figures 5A, 5B, 5C, and 5D show the effect of an IgG2 hinge on antibody / antigen complex size. Figures 5A, 5B, and 5C show SEC chromatogram, DLS, and MALS data for complexes of hCD73-his and antibody CD73.4 containing various constant regions. Figure 5D shows a schematic model of the hCD73-his / mAb complex derived from the MALS-determined masses in Figure 5C.

[0047] [Figure 6]FIG. 6 shows the SEC-MALS data of the CD73 / mAb complex.

[0048] [Figure 7] FIG. 7 shows DLS data for CD73 / mAb complexes.

[0049] [Figure 8A] FIG. 8A shows the percentage of CD73 internalized in Calu6 cells treated with the indicated antibodies over time, demonstrating antibody-mediated CD73 internalization over time for the indicated antibodies in Calu6 cells.

[0050] [Figure 8B] FIG. 8B shows the percentage of CD73 internalized in NCI-H292 cells treated with the indicated antibodies over time, demonstrating antibody-mediated CD73 internalization over time for the indicated antibodies in NCI-H292 cells.

[0051] [Figure 8C] FIG. 8C shows the levels of CD73 on the surface of Calu6 cells treated with 5 μg / ml of the indicated antibodies for 0, 5, 15, or 30 minutes.

[0052] [Figure 9] FIG. 9 shows the levels of IL-2 secreted by CD4+ T cells co-cultured with CHO-OKT3 cells in the presence of anti-GITR antibodies with the indicated constant regions.

[0053] [Figure 10] Figure 10 shows the percentage of antibody-mediated CD73 internalization at 1 hour, 4 hours, or 21 hours after addition of each of the indicated antibodies. Bars for each antibody are shown at 21 hours (left), 4 hours (middle), and 1 hour (right).

[0054] [Figure 11A] FIG. 11A shows an overlay of SEC chromatogram data for 1:1 molar complexes of hCD73-his and 16 different CD73.4 antibodies containing various constant region sequences.

[0055] [Figure 11B] FIG. 11B is an expansion of the chromatogram data from 11 to 19.5 minutes of the chromatogram in FIG. 10A, showing four distinct eluting species.

[0056] [Figure 11C] Figure 11C shows the percentage of UV signal area for peak 2 in Figure 11B plotted for 16 different antibody / CD73-his complexes. The data are sorted from left to right in order of increasing peak area.

[0057] [Figure 12] Figure 12 shows antibody binding to anti-his Fab-captured FcγR-his protein. Binding responses are plotted as a percentage of the theoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. Bars for each antibody are shown in the order provided in the legend below the slide.

[0058] [Figure 13] Figure 13 shows antibody binding to anti-his Fab-captured FcgR-his protein. Binding responses are plotted as a percentage of the theoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. Bars for each antibody are shown in the order provided in the legend below the slide.

[0059] [Figure 14A] Figure 14A shows antibody binding to anti-his Fab-captured FcγR-his protein. Binding responses are plotted as a percentage of the theoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. Bars for each antibody are shown in the order provided in the legend below the slide.

[0060] [Figure 14B]Figure 14B shows antibody binding to anti-his Fab-captured FcγR-his protein. Binding responses are plotted as a percentage of the theoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. Bars for each antibody are shown in the order provided in the legend below the slide.

[0061] [Figure 15] FIG. 15 shows a time course analysis of the internalization of anti-GITR antibodies.

[0062] [Figure 16A] FIG. 16A shows colocalization analysis of GITR and early endosome marker EEA2 at time 0.

[0063] [Figure 16B] FIG. 16B shows colocalization analysis of GITR and the early endosome marker EEA2 at 30 and 120 minutes.

[0064] [Figure 16C] FIG. 16C shows the results of quantification of the endosomal colocalization shown in FIGS. 16A and 16B plotted as the ratio of colocalized pixel intensity to total staining.

[0065] [Figure 17A] FIG. 17A shows NFkB signaling activation in CD8+ T cells treated with the indicated anti-GITR antibodies.

[0066] [Figure 17B] FIG. 17B shows NFkB signaling activation in CD4+ T cells treated with the indicated anti-GITR antibodies.

[0067] [Figure 18] FIG. 18 shows p38 activation in CD4+ T cells treated with the indicated anti-GITR antibodies.

[0068] [Figure 19]FIG. 19 shows the disulfide bond arrangement in an IgG2 antibody having conformations A, B, or A / B.

[0069] [Figure 20A] FIG. 20A shows the levels of IL-2 secreted by CD4+ T cells cocultured with CHO-OKT3 cells in the presence of various concentrations of anti-GITR antibodies with the indicated constant regions.

[0070] [Figure 20B] Figure 20B shows the levels of IL-2 secreted by CD4+ T cells cocultured with CHO-OKT3 cells in the presence of 5 μg / ml of anti-GITR antibodies with the indicated constant regions (same experiment as in Figure 20A).

[0071] [Figure 20C] FIG. 20C shows the levels of IL-2 secreted by CD4+ T cells cocultured with CHO-OKT3 cells in the presence of 1.25 μg / ml of anti-GITR antibodies with the indicated constant regions (same experiment as in FIG. 20A).

[0072] [Figure 20D] Figure 20D shows the levels of IL-2 secreted by CD4+ T cells cocultured with CHO-OKT3 cells in the presence of 0.313 μg / ml of anti-GITR antibodies with the indicated constant regions (same experiment as in Figure 20A).

[0073] [Figure 21] FIG. 21 shows the amino acid sequence of part of hIgG1f, where the underlined sequence is reproduced below and indicates the position of mutations in the hIgG1, hIgG1.1f, hIgG1.3f and hIgG1-P238K amino acid sequences relative to wild-type IgG1.

[0074] [Figure 22]Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, 22I, 22J, 22K and 22L show a comparison of the dissociation rates of antibody Y1238 in various Fc region contexts from the indicated Fc receptors based on sensorgram data.

[0075] [Figure 23] Figures 23A, 23B, 23C, 23D, 23E and 23F show the charge profile of the dAb-Fc molecule characterized by icIEF. DETAILED DESCRIPTION OF THE INVENTION

[0076] Detailed Description In certain embodiments, the present invention is based, at least in part, on the discovery that the following properties of an antibody are enhanced or modified when the antibody comprises an IgG2 hinge, compared to the same antibody comprising a non-IgG2 hinge (or compared to an antibody comprising the same IgG1 constant region): (i) internalization; (ii) agonist function; (iii) receptor-mediated intracellular signaling; (iv) ADCC; and (v) antibody / antigen complex weight. Furthermore, these enhanced or modified antibody characteristics are further enhanced or modified when the antibody comprises an IgG2 CH1 domain in addition to the IgG2 hinge. It has also been observed that antibodies having an IgG2 CH1 domain but not an IgG2 hinge have enhanced or modified activity compared to the same antibody having an IgG1 CH1 domain. While not intending to be limited to a particular mechanism of action, the enhancing effect of the IgG2 hinge has been found to correlate with an increased size of the antibody / antigen complex. When an antibody has an IgG2 hinge, the enhanced size of the antibody / antigen complex may result from the greater rigidity of the IgG2 hinge compared to other isotypes. Furthermore, it has been shown that certain regions or amino acid residues in the IgG2 hinge and CH1 domain may be modified, while others preferably are not, in order to retain enhanced or altered activity.

[0077] As further described herein, these modified heavy chain constant regions that confer enhanced or modified activity to the antibody (or antigen-binding region thereof) may have effector function. Thus, it has been demonstrated that antibodies can be generated that have the advantageous properties conferred by the IgG2 hinge and / or CH1 domain and also have effector function.

[0078] The present invention is also based, at least in part, on the discovery that deleting a portion of the hinge of an IgG1 or IgG2 antibody results in an antibody with enhanced or altered properties relative to antibodies having an IgG1 constant region.

[0079] Also described herein are modified heavy chain regions having mutations that reduce ADCC and / or CDC effector function, e.g., P238 mutations, e.g., P238K, and in certain embodiments, one or more such mutations are combined with mutations that enhance (i) internalization; (ii) agonist function; (iii) receptor-mediated intracellular signaling; (iv) ADCC; and / or (v) antibody / antigen complex weight.

[0080] Accordingly, provided herein are (i) antibodies having modified heavy chain constant regions that confer enhanced or altered properties on the antigen-binding region of the antibody, and methods for using the same, and (ii) methods for enhancing or altering certain biological properties, such as internalization, agonism, and antagonism, of antibodies comprising a non-IgG2 hinge and / or CH1 domain, wherein the method comprises replacing the non-IgG2 hinge and / or CH1 domain of the antibody with an IgG2 hinge and / or IgG2 CH1 domain or a portion thereof.

[0081] Provided herein are "modified heavy chain constant regions" that enhance certain biological properties of antibodies, e.g., antibodies having a non-IgG2 hinge and / or a non-IgG2 CH1 domain, relative to the same antibody with a different constant region. An example of a modified heavy chain constant region comprises an IgG2 hinge, CH1 domain, CH2 domain, and CH3 domain, where at least one of these constant domains is not of the IgG2 isotype but can be, for example, IgG1, IgG3, or IgG4. In certain embodiments, the modified heavy chain constant region comprises an IgG2 hinge and an IgG1 CH2 and CH3 domain. In certain embodiments, the modified heavy chain constant region comprises an IgG2 CH1 domain and an IgG2 hinge. In certain embodiments, the modified heavy chain constant region comprises an IgG2 CH1 domain, an IgG2 hinge, an IgG1 CH2 domain, and an IgG1 CH3 domain. The modified heavy chain constant region may have effector function similar to that of wild-type IgG1, or may be engineered to have reduced or enhanced effector function relative to that of wild-type IgG. The modified heavy chain constant region may comprise a wild-type CH1, hinge, CH2 and / or CH3 domain or a variant thereof, e.g., a CH1, hinge, CH2 and / or CH3 domain having one or more amino acid substitutions, deletions or additions compared to the corresponding wild-type domains and / or having an amino acid sequence that is at least 90% or more identical to the corresponding wild-type sequence.

[0082] Also provided are antibodies and fusion proteins comprising the IgG1.3 heavy chain constant region. The antibody comprising the IgG1.3 heavy chain constant region can be an antagonist or agonist antibody, such as an antagonist antibody against a checkpoint inhibitor or an agonist antibody against a checkpoint stimulator.

[0083] definition In order that this description may be more readily understood, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0084] As used herein, the term "antibody" can include whole antibodies and any antigen-binding fragments thereof (e.g., an antigen-binding fragment comprising the hinge, an antigen-binding fragment comprising the hinge and CH1 domain, an antigen-binding fragment comprising the hinge and CH2 domain, or an antigen-binding fragment comprising a portion of the hinge, CH2 domain, and CH3 domain) or single chains. In certain embodiments, an "antibody" includes a protein, e.g., a glycoprotein, or an antigen-binding portion thereof, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of a hinge, CH1 domain, CH2 domain, and CH3 domain. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (herein abbreviated as V L The light chain constant region consists of one domain, CL. H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L Each antibody consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0085] Immunoglobulins may be of any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In certain embodiments, the antibodies described herein are of the human IgG1 or IgG2 subtype. Immunoglobulins, such as human IgG1, exist in several allotypes, which differ from each other by at most a few amino acids. "Antibody" can include, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies; and single-chain antibodies.

[0086] In some embodiments, the heavy chain of the antibody comprises a C-terminal lysine; a C-terminal glycine (the C-terminal lysine is deleted) or lacks GK or lacks K. When referring to antibodies described herein that comprise modified heavy chain constant regions, the antibodies can comprise the provided sequences with or lacking a C-terminal GK or K.

[0087] Amino acid numbering is according to the EU index in Kabat (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD) and Figures 3c-3f of US Patent Application Publication 2008 / 0248028.

[0088] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. An antigen-binding portion of an antibody may be a "hinge-containing antigen-binding portion." The antigen-binding function of an antibody has been shown to be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody described herein include: (i) V L , V H(ii) a Fab fragment, which is a monovalent fragment consisting of the CL and CH1 domains; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region; H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody; L and V H Fv fragment consisting of domains, (v) V H dAb fragments comprise (vi) isolated complementarity-determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, optionally linked by a synthetic linker. Additionally, the two domains of the Fv fragment, V, L and V H is encoded by a different gene, but V L and V H The regions can be linked using recombinant methods by synthetic linkers, which allow them to be produced as a single protein chain that pairs to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0089] The "CDRs" of a variable domain are amino acid residues within the hypervariable regions, as defined by Kabat, Chothia, a combination of Kabat and Chothia, AbM, contact and / or conformational definitions, or any other method of CDR determination known in the art. Antibody CDRs can be identified as the hypervariable regions originally defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington, DC). CDR locations can also be identified as the structural loop structures originally described by Chothia et al. (See, e.g., Chothia et al., 1989, Nature 342:877-883). Other approaches to CDR identification include the "AbM definition," which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contacts as presented in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. In another approach, referred to herein as the "conformational definition" of CDRs, CDR positions may be defined as residues that make an enthalpic contribution to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but nevertheless overlap with at least a portion of the Kabat CDRs, but may be shortened or extended according to predictions or experimental findings that certain residues, groups of residues, or entire CDRs do not significantly affect antigen binding. As used herein, a CDR is a CDR defined by any approach known in the art, including a combination of approaches. The methods used herein may use CDRs defined by any of these approaches.For any embodiment comprising more than one CDR, the CDRs may be defined by any of the Kabat, Chothia, extended, AbM, contact and / or conformational definitions.

[0090] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by the heavy chain constant domain genes. The full-length amino acid sequences of each wild-type human IgG constant region (including all domains, i.e., CH1 domain, hinge domain, CH2 domain, and CH3 domain) are listed in the online UniProt database, for example, as P01857 (IgG1), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4), or their different allotypes (SEQ ID NOs: 1, 6, 11, and 16, respectively). As used herein, a domain of a heavy chain constant region, e.g., a hinge, is considered to be of an "IgG1 isotype," an "IgG2 isotype," an "IgG3 isotype," or an "IgG4 isotype" if the domain contains the amino acid sequence of a corresponding domain of each isotype or a variant thereof (having a higher homology to the corresponding domain of each isotype than to other isotypes).

[0091] "Allotype" refers to naturally occurring variants within a particular isotype group, which variants differ by a few amino acids (see, e.g., Jefferies et al. (2009) mAbs 1:1). The antibodies described herein can be of any allotype.

[0092] A "wild-type" protein or a portion thereof is a version of a protein that exists in nature. The amino acid sequence of a wild-type protein, such as a heavy chain constant region, is the amino acid sequence of a protein that exists in nature. Due to allotypic differences, there may be more than one amino acid sequence for a wild-type protein. For example, there are several allotypes of the naturally occurring human IGg1 heavy chain constant region (see, for example, Jeffries et al. (2009) mAbs 1:1).

[0093] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. Thus, the Fc region of an antibody of isotype IgG includes the heavy chain constant region of the antibody except for the first constant region immunoglobulin domain (CH1). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the C1 domain in each of the antibody's two heavy chains. H2 and C H3 IgM and IgE Fc regions contain three heavy chain constant domains (C HFor IgG, the Fc region includes the immunoglobulin domains consisting of the hinge, CH2, and CH3. For purposes of the present invention, the Fc region is defined as beginning at amino acid 216 and ending at amino acid 447, where the numbering is according to the EU index in Kabat (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD) and Figures 3c-3f of U.S. Patent Application Publication No. 2008 / 0248028. The Fc can be a native (or naturally occurring or wild-type) Fc, including any allotypic variant or variant Fc (e.g., a non-naturally occurring Fc), containing, for example, 1, 2, 3, 4, 5, 1-5, 1-10, or 5-10 or more amino acid mutations, e.g., substitutions, additions, or deletions. For example, a variant Fc comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a wild-type Fc. A modified or mutated Fc may have enhanced or reduced effector function and / or half-life. The CH2 and CH3 regions are the primary sites of effector function and FcRn binding. Fc may refer to this region in isolation or in the context of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0094] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event resulting therefrom. Examples of "effector function" include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cellular phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).

[0095] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor. The various properties of human FcγRs are summarized in Table 1. Most innate effector cell types coexpress one or more activating FcγRs and an inhibitory FcγRIIB in mice and humans, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not express an inhibitory FcγRIIB. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a with respect to binding activating Fc receptors.

[0096] [Table 1]

[0097] "Hinge," "hinge domain," or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that connects the CH1 domain to the CH2 domain, and includes the upper, middle, and lower portions of the hinge (Roux et al. J. Immunol. 1998 161:4083). The hinge provides varying levels of flexibility between the binding and effector regions of the antibody and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, the hinge begins at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., 1998 J Immunol 161:4083). The sequences of wild-type IgG1, IgG2, IgG3, and IgG4 hinges are shown in Table 2.

[0098] [Table 2] * C-terminal amino acid sequence of the CH1 domain.

[0099] The term "hinge" includes wild-type hinges (e.g., those shown in Table 3) and variants thereof (e.g., non-naturally occurring hinges or modified hinges). For example, the term "IgG2 hinge" includes wild-type IgG2 hinges shown in Table 3 and variants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, or additions. An example of an IgG2 hinge variant is an IgG2 hinge in which 1, 2, 3, or all four cysteines (C219, C220, C226, and C229) have been changed to other amino acids. In certain embodiments, the IgG2 hinge contains a C219X or C220X substitution, where X is any amino acid other than cysteine. The IgG2 hinge may contain substitutions that, alone or in combination with one or more substitutions in other regions of the heavy or light chain, result in an antibody comprising a hinge that adopts an A or B configuration (see, e.g., Allen et al. (2009) Biochemistry 48:3755). In certain embodiments, the hinge is a hybrid hinge comprising sequences from at least two isotypes. For example, the hinge may comprise an upper, middle, or lower hinge from one isotype, with the remainder of the hinge being from one or more other isotypes. For example, the hinge may be an IgG2 / IgG1 hinge, e.g., comprising an IgG2 upper and middle hinge and an IgG1 lower hinge. The hinge may have effector function or may have effector function removed. For example, the lower hinge of wild-type IgG1 provides effector function.

[0100] A "non-IgG2" hinge refers to a hinge that is not of the IgG2 isotype.

[0101] The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge in the heavy chain constant domain. As used herein, the CH1 domain begins at A118 and ends at V215. The term "CH1 domain" includes wild-type CH1 domains (e.g., having SEQ ID NO: 2 for IgG1 and SEQ ID NO: 7 for IgG2; Table 3) and variants thereof (e.g., non-naturally occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wild-type CH1 domains and variants thereof with 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, or additions. Examples of CH1 domains include CH1 domains with mutations that modify the biological activity of the antibody, such as ADCC, CDC, or half-life. Modifications of CH1 domains that affect the biological activity of the antibody are provided herein.

[0102] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain in the heavy chain constant domain. As used herein, a CH2 domain begins at P238 and ends at K340. The term "CH2 domain" includes wild-type CH2 domains (e.g., having SEQ ID NO: 4 for IgG1; Table 3) and variants thereof (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type CH2 domains and variants thereof with 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, or additions. Exemplary CH2 domains include CH2 domains with mutations that modify antibody biological activities, such as ADCC, CDC, or half-life. In one embodiment, the CH2 domain contains the substitutions A330S / P331S, which reduce effector function. Modifications of CH2 domains that affect other antibody biological activities are provided herein.

[0103] The term "CH3 domain" refers to the heavy chain constant region C-terminal to the CH2 domain. As used herein, a CH3 domain begins at G341 and ends at K447. The term "CH3 domain" includes wild-type CH3 domains (e.g., having SEQ ID NO: 5 for IgG1; Table 3) and variants thereof (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type CH3 domains and variants thereof with 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, or additions. Exemplary CH3 domains include CH3 domains with mutations that modify the biological activity of the antibody, such as ADCC, CDC, or half-life. Modifications of CH3 domains that affect the biological activity of the antibody are provided herein.

[0104] [Table 3]

[0105] As used herein, the term "monoclonal antibody" refers to an antibody or composition of antibodies in which the entire antibody exhibits a single binding specificity and affinity for a particular epitope. Generally, such monoclonal antibodies are derived from a single cell or antibody-encoding nucleic acid and propagated without intentionally introducing any sequence alterations. Thus, the term "human monoclonal antibody" refers to a monoclonal antibody having variable and optional constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas obtained by fusing B cells from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome containing human heavy chain and light chain transgenes) with immortalized cells.

[0106] As used herein, the term "recombinant human antibody" includes fully human antibodies prepared, expressed, engineered, or isolated by recombinant means, e.g., (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, engineered, or isolated by any other means, including substitution of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies utilize specific human germline immunoglobulin sequences encoded by germline genes, but include variable and constant regions, including subsequent rearrangements and mutations that occur, e.g., during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region comprises an antigen-binding domain encoded by various genes that are rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region changes in further response to antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid sequences encoding light and heavy chain immunoglobulin polypeptides in response to an antigen may not be identical to the original germline sequences, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0107] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0108] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids from a human immunoglobulin. In some embodiments of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not eliminate the antibody's ability to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0109] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0110] "Bispecific" or "bifunctional antibody" refers to an artificial hybrid antibody having two different heavy / light chain pairs, resulting in two antigen-binding sites with different antigen specificities. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0111] The terms "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0112] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to antigen "x" is substantially free of antibodies that specifically bind other antigens than "x"). However, an isolated antibody that specifically binds to an epitope of antigen "x" may have cross-reactivity with other antigen "x" proteins from different species.

[0113] As used herein, an "agonist antibody" refers to an antibody that is an agonist of a costimulatory receptor, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40, OX40L, CD70, or CD27, DR3, or CD28H protein, e.g., an antibody that can boost a subject's immune system (or immune response) by stimulating the activity of the protein and subsequent stimulation of immune cells, e.g., T cells. In certain embodiments, an agonist antibody is an antibody that enhances the activity of an inhibitory receptor, e.g., CTLA-4, PD-1, PD-L1, PD-L2, or LAG-3, TIM-3, Galectin-9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, CD73, PD1H, LAIR1, TIM-1, or TIM-4, thereby blocking the immune response.

[0114] As used herein, an "antagonist antibody" refers to an antibody that is an antagonist of an inhibitory signal in an immune cell, e.g., a T cell, e.g., an antibody that can block or block CTLA-4, PD-1, PD-L1, PD-L2, or proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, CD73, PD1H, LAIR1, TIM-1, or TIM-4, thereby stimulating an immune response. In certain embodiments, an antagonist antibody refers to an antibody that blocks the activity of a stimulatory receptor, e.g., B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40, OX40L, CD70 or CD27, DR3 or CD28H, thereby inhibiting an immune response.

[0115] Both agonist and antagonist antibodies result in either amplification of antigen-specific T cell responses or inhibition of antigen-specific T cell responses (immune checkpoint regulators).

[0116] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., GITR) to which an immunoglobulin or antibody specifically binds. Epitopes within a protein antigen can be formed by both contiguous amino acids (usually linear epitopes) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are generally, but not always, retained by exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost by treatment with denaturing solvents. Epitopes generally contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblot and immunoprecipitation assays, in which overlapping or adjacent peptides are tested for reactivity with a given antibody. Methods for determining the spatial structure of epitopes include techniques described in the literature and herein, such as x-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0117] As used herein, the term "naturally occurring" with respect to an object refers to the fact that the object exists in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and exists in an organism (including a virus) that has not been intentionally modified in a laboratory is naturally occurring.

[0118] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the chain length. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" may include one or more polypeptides.

[0119] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0120] Also provided are "conservative sequence modifications" of the sequences set forth herein, including, for example, conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into SEQ ID NOS: 1-74 by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0121] In some embodiments, amino acid sequence modifications to the heavy chain constant region or a domain thereof do not modify or eliminate certain properties of the heavy chain constant region. These properties include, for example, hinge rigidity or stiffness and antibody agonist or antagonist activity. In some embodiments, amino acid sequence modifications to the heavy chain constant region or a domain thereof modify or eliminate certain properties of the heavy chain constant region.

[0122] Methods for identifying conservative amino acid substitutions that do and do not abolish antibody and / or constant region properties are well known in the art, for example, as described in the Examples section herein.

[0123] With respect to nucleic acids, the term "homology" indicates that two nucleic acids or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, usually at least about 90% to 95% or more, and preferably at least about 98% to 99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to a complementary strand.

[0124] With respect to polypeptides, the term "substantial homology" indicates that two polypeptides or designated sequences, when optimally aligned and compared, are identical in at least about 80% of their amino acids, usually at least about 90% to 95% or more, and preferably at least about 98% to 99.5% of their amino acids, with appropriate amino acid insertions or deletions.

[0125] The percent identity between two sequences is a function of the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), and optimal alignment is determined by taking into account the number of gaps and the length of each gap that needed to be introduced to optimally align the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, non-limiting examples of which are set forth below.

[0126] The percent identity between two nucleotide sequences can be determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0127] The nucleic acid and protein sequences described herein can further be used as "query sequences" to conduct searches against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program at a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program at a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0128] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. An antigen can be a full-length or mature protein or a fragment thereof.

[0129] An "immune response" is a biological response in a vertebrate to foreign agents, which response protects the organism from these agents and the diseases they cause. An immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (antibodies, cytokines, and complement) produced by any of these cells or the liver, which result in the selective targeting, binding, damaging, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues within the vertebrate. An immune response can result, for example, in the activation or inhibition of T cells, e.g., effector T cells or Th cells, e.g., CD4+ or CD8+ T cells, or T reg This includes cell inhibition.

[0130] "Immunomodulator" or "immunoregulator" refers to a factor, e.g., a component of a signal transduction pathway, that may be involved in the regulation, control, or modification of an immune response. "Modulation," "regulation," or "modulation" of an immune response refers to any alteration of cells of the immune system or the activity of such cells (e.g., effector T cells). Such modulation includes stimulation or suppression of the immune system, which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In preferred embodiments, the immunomodulator is located on the surface of T cells. An "immunomodulatory target" or "immunoregulatory target" is an immunomodulator that is the target of binding of a substance, factor, moiety, compound, or molecule, the activity of which is altered by said binding. Immunomodulatory targets include, for example, cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0131] "Immunotherapy" refers to the treatment of a subject with a disease or at risk of recurrence or recurring disease by methods that involve inducing, enhancing, suppressing or otherwise modifying the immune response.

[0132] "Immunostimulatory therapy" or "immunostimulatory therapy" refers to increasing (inducing or enhancing) the immune response in a subject, for example, to treat cancer.

[0133] "Enhancing an endogenous immune response" means increasing the efficacy or potency of the immune response present in a subject. This increase in efficacy and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0134] "T Effector" ("T eff ") cells refer to T cells with cytolytic activity (e.g., CD4+ and CD8+ T cells) and T helper (Th) cells, which secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (T reg cells) are not included.

[0135] As used herein, the term "linkage" refers to the association of two or more molecules. Linkage may be covalent or non-covalent. Linkage may also be genetic (i.e., recombinant fusion). Such linkage may be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0136] As used herein, "administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject, using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein can be administered by topical, epithelial, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times and / or over one or more extended periods of time.

[0137] As used herein, the term "T cell-mediated response" refers to the response of effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0138] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response elicited by cytotoxic T cells. CTL responses are primarily CD8 + It is mediated by T cells.

[0139] As used herein, the terms "inhibition" or "blocking" (e.g., referring to the inhibition / blocking of a ligand to its receptor or subsequent intracellular response) are used interchangeably and include partial and complete inhibition / blocking. In certain embodiments, the antibody inhibits binding by at least about 50%, e.g., at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, e.g., as determined further herein.

[0140] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that can invade nearby tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0141] As used herein, the terms "treat" and "treatment" refer to any type of intervention or procedure or administration of an active agent to a subject for the purpose of ameliorating, alleviating, ameliorating, arresting, or delaying or preventing the progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease. Prevention refers to intervention in a subject who does not have a disease to prevent the disease from occurring or to minimize its effects if it does occur.

[0142] "Hematopoietic malignancies" refers to lymphomas, leukemias, myelomas, or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Examples of lymphomas include both B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include both Hodgkin's lymphoma and most non-Hodgkin's lymphomas. Non-limiting examples of B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlap with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis. Non-limiting examples of T-cell lymphomas include extranodal T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma. Hematological tumors also include leukemias, including, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia. Hematological tumors further include myelomas, such as, but not limited to, multiple myeloma and smoldering multiple myeloma. Other hematological and / or B-cell or T-cell-related cancers are encompassed by the term hematopoietic tumors.

[0143] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with other therapeutic agents, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to disease morbidity. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug is an amount that, when administered alone or in combination with other therapeutic agents to a subject at risk of developing or experiencing disease recurrence, prevents the onset or recurrence of disease. The ability of a therapeutic or prophylactic agent to promote disease regression or prevent the onset or recurrence of disease can be evaluated using a variety of methods known to skilled practitioners, such as assaying the activity of a drug in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0144] For example, an anti-cancer drug is a drug that delays cancer progression or promotes cancer regression in a subject. In a preferred embodiment, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that the administration of an effective amount of a drug alone or in combination with an anti-neoplastic agent results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptom intervals, prevention of functional impairment or physical disability due to disease, or other improvement of disease symptoms in a patient. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to an acceptably low level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal levels resulting from drug administration.

[0145] As an example for tumor treatment, a therapeutically effective amount or dosage of a drug preferably inhibits cell proliferation or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated subjects. In the most preferred embodiment, a therapeutically effective amount or dosage of a drug completely inhibits cell proliferation or tumor growth, i.e., preferably inhibits cell proliferation or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of a composition can be assessed by testing the ability of a compound to inhibit cell proliferation, and such inhibition can be measured in vitro by assays known to those skilled in the art. In other preferred embodiments described herein, tumor regression can be observed and can last for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.

[0146] The terms "patient" and "subject" refer to any human or non-human animal receiving prophylactic or therapeutic treatment. For example, the methods and compositions described herein can be used to treat subjects with cancer. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0147] Various aspects described herein are described in further detail in the following subsections.

[0148] I. Modified heavy chain constant region Described herein are "modified heavy chain constant regions" that, when present in an antibody, enhance or alter a certain biological property or characteristic of the antibody relative to the same antibody without the modified heavy chain constant region, such as an antibody comprising a non-IgG2 hinge, e.g., an IgG1 antibody. The enhanced or altered biological property of the antibody is: (a) Increased or altered internalization by cells; (b) increased or altered agonist activity; (c) increased or altered antagonist or blocking activity; (d) enhanced ADCC; (d) the emergence of novel properties; (e) increased or altered signal transduction; (f) formation of large antibody / antigen cross-linked complexes; (g) increased clustering or oligomerization of target cell surface molecules; (h) increased stimulation or enhancement of the immune response; and / or (i) increased inhibition of the immune response Includes.

[0149] Also provided herein are antibodies comprising a heavy chain comprising one or more amino acid mutations that modify effector function, e.g., reduce effector function.

[0150] In certain embodiments, an antibody comprising a modified heavy chain constant region mediates antibody-dependent receptor (or ligand or surface molecule) internalization more efficiently after the antibody binds to its target on the cell membrane than the same antibody not comprising a modified heavy chain constant region, e.g., comprising an IgG1 heavy chain, e.g., the antibody internalizes the target or surface molecule (e.g., receptor or ligand) at a faster rate and / or to a greater extent into the cell and / or is internalized itself. The rate and extent of antibody internalization can be determined, e.g., as shown in the Examples. The rate of internalization can be determined, e.g., by measuring the T of internalization, e.g., as shown in the Examples. 1 / 2 may be enhanced or increased by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more, as measured by T 1 / 2 leads to at least a 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or greater decrease in T 1 / 2 Instead of having a modified heavy chain constant region, the internalization rate is increased, thereby increasing the T 1 / 2 is reduced to 5 min (i.e., a two-fold increase in the internalization rate or T 1 / 2 ). "T 1 / 2 " is defined as the time at which half of maximal internalization is achieved, measured from the time the antibody is added to the cells. In one embodiment, T 1 / 2is reduced by at least 10 minutes, 30 minutes, or 1 hour. The maximum level of internalization can be the level of internalization at a plateau, representing internalization plotted against antibody concentration or time. The modified heavy chain constant region can increase the maximum level of antibody internalization by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more. Another method for comparing the internalization efficacy of various antibodies, such as the same antibody with and without a modified heavy chain constant region, is to compare the internalization levels at a certain antibody concentration (e.g., 100 nM) and / or a certain time (e.g., 2 minutes, 5 minutes, 10 minutes, or 30 minutes). Comparing internalization levels can also be used to measure the EC of internalization. 50 Internalization may also be measured by comparing the levels of internalization of an antibody to a (control) antibody, such as an antibody described herein, e.g., 11F11 or CD73.4-IgG2CS-IgG1, and expressed as a percentage of the value obtained with the (control) antibody. The degree of internalization may be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more when compared by any of these methods.

[0151] In some embodiments, an antibody comprising a modified heavy chain constant region has stronger agonistic activity than the same antibody that does not comprise a modified heavy chain constant region, for example, an IgG1 heavy chain. In some embodiments, the enhanced agonistic activity enhances the stimulatory activity of a target molecule, such as GITR, or other molecules that stimulate or co-stimulate an immune response, for example, T cell activity. In some embodiments, the enhanced agonistic activity enhances the inhibitory activity of a target molecule (e.g., a checkpoint inhibitor) that inhibits an immune response, for example, T cell activity. The enhanced agonistic activity of an antibody to regulate T cell activity can be determined, for example, by measuring the level of IFN-γ or IL-2 secretion from T cells contacted with the antibody, as shown in the Examples. The agonist activity of an antibody that binds to a stimulatory target can be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more, as defined by increased cytokine release or increased effector T cell proliferation; decreased T regulatory cell activity if binding to Tregs reduces Treg function; or increased Treg depletion. For example, the amount of IFN-γ or IL-2 secreted by T cells stimulated with an antibody that binds to a stimulatory target containing a modified heavy chain constant region is at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more higher than T cells stimulated with the same antibody without the modified heavy chain constant region. The agonist activity of an antibody that binds to an inhibitory target can be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more, as defined by decreased cytokine release or decreased effector T cell proliferation; increased T regulatory cell activity; or decreased Treg depletion. For example, the amount of IFN-γ or IL-2 secreted by T cells stimulated with an antibody that binds to an inhibitory target containing a modified heavy chain constant region may be at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more less than T cells stimulated with the same antibody that does not contain a modified heavy chain constant region.

[0152] In some embodiments, an antibody comprising a modified heavy chain constant region has more potent antagonist or blocking activity than the same antibody without the modified heavy chain constant region, e.g., comprising an IgG1 heavy chain. The enhanced antagonist activity of an antibody can be determined, for example, by measuring cytokine release and / or proliferation under conditions that include conditions of T cell activation. Antagonist activity can be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

[0153] In some embodiments, an antibody comprising a modified heavy chain constant region has enhanced ADCC activity compared to the same antibody not comprising the modified heavy chain constant region, e.g., comprising an IgG1 heavy chain. Enhanced ADCC can be determined by methods known in the art. ADCC can be enhanced by at least 10%, 30%, 50%, 2-fold, 5-fold, or more.

[0154] In some embodiments, antibodies comprising a modified heavy chain constant region have the ability to form larger antibody / antigen cross-linked complexes compared to the same antibody that does not comprise a modified heavy chain constant region, for example, an IgG1 heavy chain. The ability to form complexes can be determined, for example, as described in the Examples. Antibody / antigen complexes formed with antibodies comprising a modified heavy chain constant region can be at least 50%, 2-fold, 3-fold, 5-fold, or 10-fold larger than complexes formed with the same antibody that does not comprise a modified heavy chain constant region. In some embodiments, complexes of at least 2,000 kDa, 3,000 kDa, 5,000 kDa, 10,000 kDa, 50,000 kDa, or 100,000 kDa are formed with antibodies having a modified heavy chain constant region.

[0155] In some embodiments, an antibody comprising a modified heavy chain constant region induces greater clustering or oligomerization of target molecules on the cell surface than the same antibody that does not comprise a modified heavy chain constant region, e.g., comprises an IgG1 heavy chain. The extent of clustering and oligomerization can be determined, for example, by measuring the size of the antibody / antigen complex.

[0156] In certain embodiments, an antibody comprising a modified heavy chain constant region transduces a higher level or a different type of signaling or transduction compared to the same antibody without the modified heavy chain constant region, e.g., comprising an IgG1 heavy chain. Signaling can be monitored by determining the activation level of one or more proteins in a signaling pathway. In certain embodiments, signaling is determined, e.g., by measuring the activity (or phosphorylation) of a signaling protein, e.g., NFkB or p38, as described in the Examples. Signaling induced by an antibody comprising a modified heavy chain constant region can be at least 10%, 20%, 50%, 2-fold, 5-fold, or more higher or lower than signaling induced by the same antibody without the modified heavy chain constant region. For example, signaling induced by an antibody comprising a modified heavy chain constant region that binds to a stimulatory molecule (e.g., GITR) can be at least 10% enhanced than that obtained with the same antibody having an IgG1 heavy chain. For example, the EC 50 may be reduced by at least 50%, 2-fold, 5-fold or more.

[0157] In some embodiments, an antibody comprising a modified heavy chain constant region has an increased ability to stimulate or enhance an immune response or the immune system compared to the same antibody not comprising a modified heavy chain constant region, for example, an IgG1 heavy chain. The increased ability to stimulate an immune response or the immune system may be due to increased agonistic activity of T cell costimulatory receptors and / or increased antagonistic activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system may be due to increased EC in assays measuring immune responses, for example, cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly by detecting CD107a or granzymes), and assays measuring changes in proliferation. 50 Or may be reflected in a fold increase in the maximum level of activity. The ability to stimulate an immune response or immune system activity may be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more.

[0158] In some embodiments, an antibody comprising a modified heavy chain constant region has increased anti-proliferative or anti-tumor activity compared to the same antibody not comprising a modified heavy chain constant region, e.g., comprising an IgG1 heavy chain. The enhanced anti-tumor activity of an antibody can be determined, for example, by tumor growth in an animal treated with the antibody. The anti-tumor activity can be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more. The anti-tumor activity can be measured, for example, as a reduced tumor burden, manifested, for example, by reduced tumor growth kinetics and complete tumor regression.

[0159] In some embodiments, an antibody comprising a modified heavy chain constant region has an increased immune response or ability to inhibit or suppress the immune system compared to the same antibody not comprising a modified heavy chain constant region, e.g., an IgG1 heavy chain. The increased immune response or ability to inhibit or suppress the immune system may be due to increased antagonist activity of T cell costimulatory receptors and / or increased agonist activity of inhibitory receptors. The increased ability to stimulate the immune response or immune system may be measured by an increased EC200 / EC200 / EC200 in assays that measure immune responses, such as cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly by detecting CD107a or granzymes), and assays that measure changes in proliferation. 50 Or may be reflected in the fold increase in the maximum level of activity. The ability to inhibit or suppress an immune response or immune system activity may be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more.

[0160] In some embodiments, the modified heavy chain constant region or a portion thereof, e.g., a hinge, is more rigid than other heavy chain constant regions, e.g., IgG1, IgG2, IgG3, and / or IgG4 heavy chain constant regions. For example, the modified heavy chain constant region is a non-naturally occurring heavy chain constant region having a more rigid portion, e.g., a hinge, than a naturally occurring heavy chain constant region or its hinge. The rigidity of a heavy chain constant region or a portion thereof, e.g., a hinge, can be determined, for example, by computer modeling, electron microscopy, spectroscopy such as nuclear magnetic resonance (NMR), X-ray crystallography (B factor), or ultracentrifugal sedimentation velocity (AUC) to measure or compare the radius of gyration of an antibody containing the hinge. Alternatively, the rigidity of a heavy chain constant region or a portion thereof can be determined, for example, by measuring the size of an antibody / antigen complex, as further described herein.

[0161] It is understood that an antibody comprising a modified heavy chain constant region and having enhanced functional properties as determined by methods known in the art and described herein will be associated with a statistically significant difference in a specific activity compared to that seen with the same antibody but having a different heavy chain constant region.

[0162] In certain embodiments, the modified heavy chain constant region comprises a hinge and CH1, CH2, and CH3 domains of an IgG2 isotype ("IgG2 hinge"). In certain embodiments, the modified heavy chain constant region comprises an IgG2 hinge and CH1, CH2, and CH3 domains, wherein at least one of the CH1, CH2, and CH3 domains is not of the IgG2 isotype. In certain embodiments, the modified heavy chain constant region comprises an IgG2 hinge and CH1, CH2, and CH3 domains, wherein the heavy chain constant domain is not a wild-type IgG2 constant region or is not an IgG2 constant region having a mutation at amino acid 219 or 220. The IgG2 hinge can be a wild-type IgG2 hinge, e.g., a wild-type human IgG2 hinge (e.g., having SEQ ID NO: 8), or a variant thereof, provided that the IgG2 hinge confers the ability of the antibody to have enhanced activity relative to the same antibody comprising a non-IgG2 hinge or comprising an IgG1 heavy chain. In certain embodiments, an IgG2 hinge variant retains a rigidity or stiffness similar to that of a wild-type IgG2 hinge. The rigidity of a hinge can be determined, for example, by computer modeling, electron microscopy, spectroscopy such as nuclear magnetic resonance (NMR), X-ray crystallography (B factor), or area under ultracentrifugation (AUC) to measure or compare the radius of gyration of antibodies comprising the hinge. A hinge has a similar or higher rigidity than that of another hinge if the value obtained by one of the above tests for the hinge differs by less than 5%, 10%, 25%, 50%, 75%, or 100% from the value for the same antibody having a different hinge, e.g., an IgG1 hinge. Those skilled in the art can determine from the tests whether a hinge has a rigidity at least similar to that of another hinge by interpreting the test results.

[0163] An example of a human IgG2 hinge variant is an IgG2 hinge comprising substitutions of four cysteine ​​residues (i.e., C219, C220, C226, and C229) with one or more other amino acids. Cysteines may be replaced with serine. An example of an IgG2 hinge is a human IgG2 hinge comprising a C219X mutation or a C220X mutation, where X is any amino acid other than cysteine. In certain embodiments, the IgG2 hinge does not comprise both a C219X substitution and a C220X substitution. In certain embodiments, the IgG2 hinge comprises C219S or C220S, but not both C219S and C22S. Other IgG2 hinge variants that may be used are human IgG2 hinges comprising C220, C226, and / or C229 substitutions, for example, a C220S, C226S, or C229S mutation (which may be combined with the C219S mutation). An IgG2 hinge can also be an IgG2 hinge in which part of the hinge is of another isotype (i.e., a chimeric or hybrid hinge), but where the rigidity of the chimeric hinge is at least similar to that of a wild-type IgG2 hinge. For example, the IgG2 hinge can be an IgG2 hinge in which the lower hinge (defined in Table 2) is of the IgG1 isotype, e.g., a wild-type IgG1 lower hinge.

[0164] A "hybrid" or "chimeric" hinge is said to be of a particular isotype if more than half of the consecutive amino acids of the hinge are from that isotype. For example, a hinge having an IgG2 upper and middle hinge and an IgG1 lower hinge is considered an IgG2 hybrid hinge.

[0165] In certain embodiments, the antibody comprises a modified heavy chain constant region comprising an IgG2 hinge comprising one of the sequences shown in Table 4, for example, amino acid sequences 8, 21, 22, 23, 126-129, and 134-147 of the following: In certain embodiments, the hinge comprises SEQ ID NO: 8, 21, 126, 134 or 135, wherein one, two, three or all four amino acids of P233, V234, A235 and G23 (corresponding to the C-terminal four amino acids "PVAG" (SEQ ID NO: 148)) are deleted or substituted with other amino acids, e.g., the C-terminal amino acids of an IgG1 hinge (ELLG (SEQ ID NO: 149) or ELLGG (SEQ ID NO: 150). In certain embodiments, the hinge comprises SEQ ID NO: 8, 21, 126, 134 or 135, wherein V234, A235 and G237 are deleted or substituted with other amino acids. In certain embodiments, the hinge comprises SEQ ID NO: 8, 21, 126, 134 or 135, wherein A235 and G237 are deleted or substituted with other amino acids. In certain embodiments, the hinge comprises SEQ ID NO: 8, 21, 126, 134, or 135, wherein G237 is deleted or substituted with another amino acid. In certain embodiments, the hinge comprises SEQ ID NO: 8, 21, 126, 134, or 135, wherein V234 and A235 are deleted or substituted with another amino acid. Substitution of PVAG (SEQ ID NO: 143) of IgG2 with the corresponding amino acids of an IgG1 hinge, i.e., (ELLG (SEQ ID NO: 144) or ELLGG (SEQ ID NO: 145)), to obtain a hybrid hinge having SEQ ID NO: 22 or 138, or a variant thereof (see, e.g., Table 4), provides a hinge with the advantages of an IgG2 hinge and the effector function of an IgG1 hinge.

[0166] In certain embodiments, the modified heavy chain constant region comprises a hinge consisting of or consisting essentially of one of the sequences in Table 4, e.g., SEQ ID NOs: 8, 21, 22, 23, 127-132 and 134-141, and in certain embodiments, no additional hinge amino acid residues.

[0167] [Table 4] X is any amino acid other than cysteine.

[0168] In some embodiments, the modified heavy chain constant region comprises an IgG2 hinge as shown in Table 4, in which 1 to 5, 1 to 3, 1 to 2, or 1 amino acid is inserted between amino acid residues CVE and CPP. In some embodiments, THT or GGG is inserted. In some embodiments, 1, 1 to 2, or 1 to 3 amino acids can be inserted between the hinge and the CH2 domain. For example, an additional glycine can be inserted between the hinge and the CH2 domain.

[0169] In certain embodiments, the modified heavy chain constant region is an IgG1 or IgG2 constant region, wherein the hinge comprises a deletion of 1 to 10 amino acids. As shown in the Examples, an IgG1 antibody lacking amino acid residues SCDKTHT (S219, C220, D221, K222, T223, H224, and T225; SEQ ID NO: 151) contributed more efficiently to antibody-mediated CD73 internalization than the same antibody having a wild-type IgG1 constant region. Similarly, in the case of an IgG2 antibody, an IgG2 antibody lacking amino acid residues CCVE (C219, C220, V222, and E224; SEQ ID NO: 152) contributed more efficiently to antibody-mediated CD73 internalization than the same antibody having a wild-type IgG1 constant region. Thus, provided herein are modified heavy chain constant regions in which the hinge comprises a deletion of 1, 2, 3, 4, 5, 6, or 7 amino acid residues selected from residues 219, C220, D221, K222, T223, H224, and T225 for IgG1 antibodies, and residues C219, C220, V222, and E224 for IgG2 antibodies.

[0170] In certain embodiments, the modified heavy chain constant region comprises a CH1 domain that is a wild-type CH1 domain of an IgG1 or IgG2 isotype ("IgG1 CH1 domain" or "IgG2 CH1 domain," respectively). CH1 domains of isotypes IgG3 and IgG4 ("IgG3 CH1 domain" and "IgG2 CH1 domain," respectively) can also be used. The CH1 domain can also be a variant of the wild-type CH1 domain, for example, a variant of the wild-type IgG1, IgG2, IgG3, or IgG4 CH1 domain. Examples of CH1 domain variants include A114C, C131S, and / or T173C. The CH1 domain, for example, the IgG2 CH1 domain, can comprise the substitution C131S, which confers the B-form (or conformation) to an IgG2 antibody or an antibody having an IgG2 CH1 and hinge.

[0171] In some embodiments, the modified heavy chain constant region comprises a CH1 domain that is of the IgG2 isotype. In some embodiments, the CH1 domain comprises, for example, the amino acid sequence [ka] In one embodiment, the CH1 domain is a variant of SEQ ID NO: 7, comprising 1 to 10, 1 to 5, 1 to 2, or 1 amino acid substitution or deletion relative to SEQ ID NO: 7. As further described in the Examples, it has been shown herein that an IgG2 CH1 domain or variant thereof confers enhanced properties to an antibody relative to an IgG1 antibody, and further enhanced properties when the antibody also comprises an IgG2 hinge. In one embodiment, the IgG2 CH1 variant does not comprise an amino acid substitution or deletion at one or more of the following amino acid residues: C131, R133, E137, and S138, which are shown in bold and underlined in SEQ ID NO: 7 above. For example, the modified heavy chain constant region may comprise an IgG2 CH1 domain in which none of R133, E137, and S138 are deleted or substituted with other amino acids, and none of C131, R133, E137, and S138 are deleted or substituted with other amino acids. In one embodiment, C131 is replaced with another amino acid, e.g., C131S, and this substitution induces the antibody to adopt conformation B. Both conformation A and conformation B antibodies with modified heavy chain constant regions are shown herein to have enhanced activity compared to the same antibodies with an IgG1 constant region.

[0172] In certain embodiments, N192 and / or F193 (residues shown as italics and underlined in SEQ ID NO: 7 above) are substituted with other amino acids, e.g., the corresponding amino acids in IgG1, i.e., N192S and / or F193L.

[0173] In certain embodiments, one or more amino acid residues in the IgG2 CH1 domain are substituted with the corresponding amino acid residue in IgG4. For example, N192 may be N192S; F193 may be F193L; C131 may be C131K; and / or T214 may be T214R.

[0174] The antibody may comprise a modified heavy chain constant region comprising an IgG2 CH1 domain or variant thereof and an IgG2 hinge or variant thereof. The hinge and CH1 domain may be in combination with any IgG2 hinge and IgG2 CH1 domain described herein. In one embodiment, the IgG2 CH1 and hinge have the following amino acid sequence: [ka] or an amino acid sequence that differs therefrom by at most 1 to 10 amino acids. Amino acid variants are as described above for the hinge and CH1 domains.

[0175] In certain embodiments, the antibody comprises at least an IgG2 hinge and optionally also an IgG2 CH1 domain or fragment, or a derivative of the hinge and / or CH1 domain, and the antibody is in (conformational) Form A (see, e.g., Allen et al. (2009) Biochemistry 48:3755). In certain embodiments, the antibody comprises at least an IgG2 hinge and optionally also an IgG2 CH1 domain or fragment, or a derivative of the hinge and / or CH1 domain, and the antibody is in Form B (see, e.g., Allen et al. (2009) Biochemistry 48:3755).

[0176] In certain embodiments, the modified heavy chain constant region comprises a CH2 domain that is a wild-type CH2 domain of an IgG1, IgG2, IgG3, or IgG4 isotype ("IgG1 CH2 domain," "IgG2 CH2 domain," "IgG3 CH2 domain," or "IgG4 CH2 domain," respectively). The CH2 domain can also be a variant of the wild-type CH2 domain, for example, a variant of the wild-type IgG1, IgG2, IgG3, or IgG4 CH2 domain. Examples of CH2 domain variants include variants that modulate the biological activity of the Fc region of an antibody, such as ADCC or CDC, or that modulate the half-life or stability of the antibody. In certain embodiments, the CH2 domain is a human IgG1 CH2 domain with the A330S and / or P331S mutation, wherein the CH2 domain has reduced effector function compared to the same CH2 mutation without the mutation. The CH2 domain may have enhanced effector function. The CH2 domain may contain one or more of the following mutations: SE (S267E), SELF (S267E / L328F), SDIE (S239D / I332E), SEFF, GASDALIE (G236A / S239D / A330L / I332E) and / or one or more mutations at the following amino acids: E233, L235, G237, P238, H268, P271, L328, A330, and K322. It should be noted that some of these mutations are actually part of the hinge, rather than the CH2 domain as defined herein. Other mutations are further set forth elsewhere herein.

[0177] In certain embodiments, the modified heavy chain constant region comprises a CH3 domain that is a wild-type CH3 domain of an IgG1, IgG2, IgG3, or IgG4 isotype ("IgG1 CH3 domain," "IgG2 CH3 domain," "IgG3 CH3 domain," or "IgG4 CH3 domain," respectively). The CH3 domain can also be a variant of the wild-type CH3 domain, e.g., a variant of the wild-type IgG1, IgG2, IgG3, or IgG4 CH3 domain. Examples of CH3 domain variants include variants that modulate a biological activity of the Fc region of an antibody, such as ADCC or CDC, or that modulate the half-life or stability of the antibody.

[0178] Generally, a variant of a CH1, hinge, CH2 or CH3 domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations and / or up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations or 1-10 or 1-5 mutations in the corresponding wild-type domain (CH1, hinge, CH2 or CH3 domain, respectively) or may comprise an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, provided that the heavy chain constant region containing the particular variant retains the required biological activity.

[0179] Table 5 shows examples of human heavy chain constant regions containing human CH1, hinge, CH2, and / or CH3 domains, where each domain is a wild-type domain or a variant thereof that provides the heavy chain constant region with the desired biological activity. Blank cells in Table 5 indicate that the domain is present or absent, and if present, may be of any isotype, e.g., IgG1, IgG2, IgG3, or IgG4. For example, an antibody containing heavy chain constant region 1 in Table 5 is an antibody containing a heavy chain constant region that includes at least an IgG2 hinge and may also include CH1, CH2, and / or CH3 domains, where the CH1, CH2, and / or CH3 domains, if present, are of the IgG1, IgG2, IgG3, or IgG4 isotype. As another example for understanding Table 5, an antibody containing heavy chain constant region 8 is an antibody containing a heavy chain constant region that includes an IgG1 CH1 domain and an IgG2 hinge, an IgG1 CH2 domain, and may or may not also include a CH3 domain, and when present, may be of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0180] [Table 5] * Modified heavy chain constant region

[0181] In certain embodiments, antibodies comprising a heavy chain constant region shown in Table 5 exhibit enhanced biological activity compared to the same antibody comprising a heavy chain constant region that does not include the particular heavy chain constant region or the same antibody comprising an IgG1 constant region.

[0182] In certain embodiments, a method for improving the biological activity of an antibody comprising a non-IgG2 hinge and / or a non-IgG2 CH1 domain comprises providing an antibody comprising a non-IgG2 hinge and / or a non-IgG2 CH1 domain, and replacing the non-IgG2 hinge and non-IgG2 CH1 domain with an IgG2 hinge and an IgG2 CH1 domain, respectively. A method for improving the biological activity of an antibody that does not comprise a modified heavy chain constant region can comprise providing an antibody that does not comprise a modified heavy chain constant region, and replacing the heavy chain constant region with a modified heavy chain constant region.

[0183] Examples of modified heavy chain constant regions are provided in Table 6, indicating the affiliation of each of the domains.

[0184] [Table 6]

[0185] In certain embodiments, the antibody comprises an IgG2 hinge comprising any of SEQ ID NOs: 8, 21, 22, 23, 126-132, 134-136, and 137, or a variant thereof, such as: (i) which differs from any of SEQ ID NOs: 8, 21, 22, 23, 126-132, 134-136, and 137 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) which differs from any of SEQ ID NOs: 8, 21, 22, 23, 126-132, 134-136, and 137 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; or (iii) which differs from any of SEQ ID NOs: 8, 21, 22, 23, 126-132, 134-136, and 137 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acids. and (iv) a modified heavy chain constant region comprising an IgG2 hinge comprising an amino acid sequence that differs by amino acid substitution, addition, or deletion, and / or an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 8, 21, 22, 23, 126-132, 134-136, or 137, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative; and wherein the modified heavy chain constant region has enhanced biological activity relative to other heavy chain constant regions, for example, heavy chain constant regions comprising a non-IgG2 hinge or the same modified heavy chain constant region comprising a non-IgG2 hinge.

[0186] In certain embodiments, the hinge comprises a sequence that is a variant of any of SEQ ID NOs: 8, 21, 22, 23, 126-132, 134-136, and 137, wherein R217 (the second amino acid in the wild-type IgG2 hinge (SEQ ID NO: 8)) is not deleted or substituted with another amino acid. In certain embodiments in which the hinge is a variant of any of SEQ ID NOs: 8, 21, 22, 23, 126-132, 134-136, and 137, the hinge has a rigidity similar to that of wild-type IgG2.

[0187] In certain embodiments, the antibody comprises an IgG1 CH1 domain comprising SEQ ID NO: 2 or an IgG2 CH1 domain comprising SEQ ID NO: 7, or a modified heavy chain constant region comprising a variant of SEQ ID NO: 2 or 7, wherein the variant (i) differs from SEQ ID NO: 2 or 7 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 2 or 7 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 2 or 7 by 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and / or (iv) differs from SEQ ID NO: and (iv) a modified heavy chain constant region that comprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to Nos. 2 or 7, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative; and wherein the modified heavy chain constant region has enhanced biological activity relative to other heavy chain constant regions, for example, a heavy chain constant region comprising a non-IgG2 hinge or the same modified heavy chain constant region comprising a non-IgG2 hinge.

[0188] In certain embodiments, the antibody comprises a modified heavy chain constant region comprising an IgG1 CH2 domain comprising SEQ ID NO: 4 or 24 or a variant of SEQ ID NO: 4 or 24, wherein the variant (i) differs from SEQ ID NO: 4 or 24 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 4 or 24 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 4 or 24 by 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and / or (iv) differs from SEQ ID NO: 4 or 24 by at least one amino acid substitution, addition, or deletion. and (iv) a heavy chain constant region that comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain constant region of (i) to (iv), wherein the amino acid substitutions in any of (i) to (iv) may be conservative or non-conservative; and wherein the modified heavy chain constant region has enhanced biological activity relative to other heavy chain constant regions, for example, a heavy chain constant region comprising a non-IgG2 hinge or the same modified heavy chain constant region comprising a non-IgG2 hinge.

[0189] In one embodiment, the antibody is an IgG1 comprising SEQ ID NO: 5 or a variant of SEQ ID NO: 5. The variants include a modified heavy chain constant region comprising a CH3 domain, which variants (i) differ from SEQ ID NO: 5 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differ from SEQ ID NO: 5 by at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differ from SEQ ID NO: 5 by 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and / or (iv) comprise an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative; and wherein the modified heavy chain constant region has enhanced biological activity compared to other heavy chain constant regions, for example, heavy chain constant regions comprising a non-IgG2 hinge or the same modified heavy chain constant region comprising a non-IgG2 hinge.

[0190] The modified heavy chain constant region can also contain a combination of the CH1, hinge, CH2 and CH3 domains described above.

[0191] In some embodiments, the antibody comprises a modified heavy chain constant region described herein or a variant of a modified heavy chain constant region described herein, wherein the variant (i) differs from a modified heavy chain constant region described herein by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, additions, or deletions; (ii) differs from a modified heavy chain constant region described herein by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; or (iii) differs from a modified heavy chain constant region described herein by 1 to 5, 1 to 3, 1 to 2, 2 to 5, 3 to 5, 1 to 10, or 5 to 10 amino acid substitutions, additions, or deletions. and (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a modified heavy chain constant region described herein, wherein in any of (i) through (iv), the amino acid substitutions can be conservative or non-conservative; and wherein the modified heavy chain constant region has enhanced biological activity relative to other heavy chain constant regions, e.g., heavy chain constant regions comprising a non-IgG2 hinge or the same modified heavy chain constant region comprising a non-IgG2 hinge.

[0192] In certain embodiments, the antibody comprises a modified heavy chain constant region comprising any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262, or a variant of any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262, wherein the variant is selected from the group consisting of: (i) a heavy chain constant region comprising any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262; (ii) differs from any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, additions, or deletions; (iii) differs from any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; and / or (iv) differs from any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262 by 1-5, 1-3, 1-2, 2-5, 3-5, 1-10, or 5-10 amino acid substitutions, additions, or deletions; and / or (iv) differs from any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262 by at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more identical to any of SEQ ID NOs: 26-37, 54-56, 78-125, 152-232, 234-245, and 247-262. comprises an amino acid sequence that is 99% identical, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative; and wherein the modified heavy chain constant region has enhanced biological activity (and / or reduced effector function) relative to other heavy chain constant regions, for example, a heavy chain constant region comprising a non-IgG2 hinge or the same modified heavy chain constant region comprising a non-IgG2 hinge.

[0193] The modified heavy chain constant region may have (i) a similar, reduced, or increased effector function (e.g., binding to FcγR) compared to the wild-type heavy chain constant region, and or (ii) a similar, reduced, or increased half-life (or binding to the FcRn receptor) compared to the wild-type heavy chain constant region.

[0194] In some embodiments, the antibody (or antigen-binding fragment thereof) comprises a modified heavy chain constant region comprising SEQ ID NO: 198 or a portion thereof comprising P238K, or any variant of SEQ ID NO: 198 or a portion thereof, wherein the variant (i) differs from SEQ ID NO: 198 or a portion thereof comprising P238K by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 198 or a portion thereof comprising P238K by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 198 or a portion thereof comprising P238K by 1-5, 1-3, 1-2, 2-5, 3-5, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43, 1-44, 1-45, 1-46, 1-47, 1- and (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 198, or a portion thereof, including P238K, wherein the amino acid substitutions can be conservative or non-conservative; and wherein the modified heavy chain constant region has reduced effector function, e.g., undetectable binding to low affinity FcγRs (e.g., CD32a, CD32b and CD16a), and optionally undetectable binding to high affinity FcγR (CD64), as determined by the assays described herein.

[0195] In certain embodiments, the IgG1 Fc comprising the P238K mutation (e.g., comprising SEQ ID NO: 198 or a portion thereof) contains no other mutations compared to a wild-type IgG1 Fc, e.g., one described herein. In certain embodiments, the IgG1 Fc comprising the P238K mutation (e.g., comprising SEQ ID NO: 198 or a portion thereof) contains 1 to 5 amino acid changes in addition to P238K compared to a wild-type human IgG1 Fc, e.g., comprises SEQ ID NO: 198 or a portion thereof and 1 to 5 amino acid changes compared to SEQ ID NO: 198 or a portion thereof, with the proviso that the IgG1 Fc has reduced effector function.

[0196] In certain embodiments, the IgG1 Fc comprising the P238K mutation does not comprise any other mutations that reduce effector function. In certain embodiments, the IgG1 Fc comprising the P238K mutation comprises 1 to 5 mutations that reduce effector function.

[0197] In certain embodiments, the IgG Fc comprising the P238K mutation also comprises an L235E mutation and / or a K322A mutation, and in certain embodiments may not comprise additional Fc mutations that modulate Fc effector function, e.g., no mutations at P330, P331, or the lower hinge, e.g., amino acids 234 and 236-237. The IgG may be IgG1 or IgG2.

[0198] In one embodiment, the antibody comprises a heavy chain constant region comprising an IgG2 constant domain or at least the hinge thereof, wherein the IgG2 constant domain or the hinge thereof comprises a mutation selected from the group consisting of P238A, P238K, L235A, K322A and optionally a C219 and / or C220 mutation, e.g., C219S and / or C220S.

[0199] In one embodiment, the antibody comprises a heavy chain constant region comprising an IgG1 constant domain comprising one or more of L234A, L235E, and G237A. As used herein, "IgG1.3" refers to an IgG1 heavy chain comprising L234A, L235E, and G237A (see, e.g., SEQ ID NO: 248). An IgG1 constant region comprising these three mutations may also comprise additional mutations, such as those described herein. Exemplary sequences comprising the L234A, L235E, and G237A mutations and additional mutations are provided herein in the Sequence Listing. IgG1.3 Fc provides antibodies with significantly reduced effector functions, such as ADCC and CDC. In one embodiment, the Fc comprises the IgG1.3 mutation and an additional mutation, such as P238K.

[0200] In one embodiment, the antibody comprises an IgG1.3 heavy chain constant region, which does not contain any other mutations that modulate effector function in addition to L234A, L235E, and G237A. In one embodiment, the antibody comprises an IgG1.3 heavy chain constant region, which does not contain any other mutations in addition to L234A, L235E, and G237A.

[0201] The heavy chain constant regions are provided in the Sequence Listing. In some embodiments, the antibody comprises one of the heavy chain constant regions shown in the table, wherein the constant region does not contain any mutations in addition to the sequence shown in the table. In certain embodiments, the antibody comprises one of the heavy chain constant regions set forth in the Tables, wherein the constant region (i) differs from a sequence in the Sequence Listing by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from a sequence in the Sequence Listing by at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from a sequence in the Sequence Listing by 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence in the Sequence Listing, wherein in any of (i)-(iv), the amino acid substitutions can be conservative or non-conservative; and wherein the biological activity of the constant region is not significantly altered by these mutations.

[0202] The heavy chain constant region may contain a combination of mutations that confer the biological activity of each individual mutation to an antibody comprising the heavy chain region. For example, one or more mutations that enhance agonist activity, large cell surface complex formation, or antibody internalization may be combined with one or more mutations that regulate effector function. Examples of constant chain sequences containing combinations of mutations that confer various biological functions are shown in the sequence listing.

[0203] II. Antibodies with modified heavy chain constant regions and their target antigens Modified heavy chain constant regions can be used in a wide range of antibodies, including antibodies that require internalization (e.g., antibody drug conjugates (ADCs) and anti-CD73 antibodies), agonistic activity (e.g., antibodies effective in modulating an immune response, e.g., stimulating T cell activation, e.g., agonistic anti-GITR antibodies), antagonistic activity (e.g., antibodies that inhibit or block proteins that inhibit an immune response, e.g., T cell activation, e.g., antagonist PD-1 antibodies), effector function, e.g., ADCC and CDC, or reduced effector function, signal transduction, or anti-tumor activity. For example, internalization of a cell surface inhibitory receptor can limit the ability to interact with that receptor and reduce cellular function.

[0204] In some embodiments, the antibody comprising the modified heavy chain constant domain is an antibody (e.g., an antibody specific for a cell surface receptor) that requires internalization for activity when bound to the cell surface, for example, by triggering receptor-mediated endocytosis. Such antibodies can be used as vehicles for targeted delivery of drugs, toxins, enzymes, or DNA for therapeutic applications, and therefore, increasing the internalization properties of these antibodies is desirable. An example of an antibody that can benefit from effective internalization is an antibody-drug conjugate. Various assays for measuring the internalization properties of antibodies are known in the art and are described herein. These assays utilize, for example, a wide range of dyes for antibody labeling, which can be used in wash- or quench-based assays to monitor internalization. Antibody internalization can also be monitored in no-wash assays that rely on fluorescent labels.

[0205] In some embodiments, antibodies comprising modified heavy chain constant domains are antibodies that require internalization of the antigen to which they bind, e.g., a cell surface molecule such as a receptor or ligand, for activity. Thus, antibodies against cell surface proteins that require downregulation for biological (e.g., therapeutic) activity can use the modified heavy chain constant regions described herein.

[0206] In one embodiment, the antibody comprising the modified heavy chain constant domain binds to a cell surface molecule, e.g., an immune cell, e.g., a T cell, a Teff cell, a Th1 cell, a Th2 cell, a CD4+ T cell, a CD8+ T cell, a T reg The antibodies agonize or antagonize the biological activity of cell surface molecules of immune cells, dendritic cells, macrophages, monocytes, Langerhans cells, NK cells, myeloid-derived suppressor cells, B cells, or any other immune cell. The cell surface molecules can be stimulatory, for example, costimulatory molecules (e.g., GITR, OX40, CD137, CD40, ICOS, and other TNFR family members), and antibodies can further stimulate activity (agonist antibodies) or inhibit activity (antagonist antibodies). The cell surface molecules can be inhibitory molecules (e.g., CTLA-4, PD-1, PD-L1, LAG-3, TIM-3), and antibodies can further stimulate activity (agonist antibodies) or inhibit activity (antagonist antibodies).

[0207] In some embodiments, the antibody comprising the modified heavy chain constant domain is an agonist antibody of a stimulatory (or costimulatory) molecule (e.g., an anti-GITR antibody), which boosts the subject's immune system, for example, by inducing IL-2 and / or IFN-γ secretion from T cells. Other agonist antibodies have the potential to control cancer in the absence of T cell immunity, and have been shown to activate APCs, promote anti-tumor T cell responses, and / or foster cytotoxic bone marrow cells. Agonist antibodies of stimulatory molecules differ from antagonist antibodies of inhibitory molecules, which block negative immune checkpoints, such as anti-CTLA-4 or anti-PD-1. Agonist activity, such as T cell proliferation, can be measured using a variety of methods known in the art.

[0208] In some embodiments, the antibody comprising the modified heavy chain constant domain is an antagonist antibody of a checkpoint inhibitor that boosts a subject's immune response by blocking or inhibiting negative immune checkpoints, such as anti-CTLA-4 or anti-PD-1 antibodies, for example, by targeting inhibitory receptors expressed on activated T cells. Antagonist activity, such as inhibition of T cell proliferation, can be measured using a variety of methods known in the art.

[0209] In some embodiments, the antibody is, for example, an agonist of (i) a costimulatory receptor or an antagonist of (ii) an inhibitory signal in a T cell, both of which can result in an enhanced immune response, for example, an antigen-specific T cell response (immune checkpoint regulator). In some embodiments, the antibody is, for example, an antagonist of (i) a costimulatory receptor or an agonist of (ii) an inhibitory signal in a T cell. Costimulatory and co-inhibitory molecules are members of the immunoglobulin superfamily (IgSF), and antibodies with modified heavy chain constant regions can bind to any of these. One important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6, and antibodies with modified heavy chain constant regions can bind to any of these. Another family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors are the TNF family of molecules that bind to cognate TNF receptor (TNFR) family members, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, and TWEAKR / Fn1. 4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, including lymphotoxin α / TNFβ, TNFR2, TNFα, LTα, LTβ, LTβR, lymphotoxin α1β2, FAS, FASL (CD178), DR3 (TNFRSF25), RELT, DR6, TROY, NGFR (e.g., Tansey (2009) Drug Discovery Today 00:1).Thus, the antibodies described herein can bind to these surface molecules and can be, for example, (i) agonists or antagonists (or inhibitors or blockers) of proteins of the IgSF family or the B7 family or the TNFR family that inhibit T cell activation or antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF; "immunosuppressive cytokines") and / or (ii) agonists or antagonists of stimulatory receptors of the IgSF family, the B7 family, or the TNF family or cytokines that stimulate T cell activation, for modulation, e.g., stimulation, of the immune response, e.g., for the treatment of a proliferative disease such as cancer.

[0210] Thus, an antibody with a modified heavy chain constant domain may be used as one of the following agents: (1) an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3, or CD28H; or (2) Antagonists (inhibitors or blockers) of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as the above-mentioned CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, and any of the following proteins: TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, CD73, PD1H, LAIR1, TIM-1, TIM-4, and CD39.

[0211] Other antibodies include antagonists of inhibitory receptors of NK cells and agonists of activating receptors of NK cells, such as KIR, TIGIT, NKG2A.

[0212] In general, antibodies that may benefit from modified heavy chain constant regions include, for example, agonistic antibodies that ligate positive costimulatory receptors, blocking antibodies that attenuate signaling through inhibitory receptors, antagonistic antibodies, and antibodies that systemically increase the frequency of anti-tumor T cells, antibodies that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor binding (e.g., PD-L1 / PD-1 interaction), inhibiting or depleting Tregs (e.g., anti-CD25 monoclonal antibodies), inhibiting metabolic enzymes such as IDO, or reversing / preventing T cell anergy or exhaustion), and antibodies that induce innate immune activation and / or inflammation at the tumor site. Increased internalization of inhibitory receptors can translate into reduced levels of potential inhibitors.

[0213] In some embodiments, the antibody comprising the modified heavy chain constant region is conjugated with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC), which requires internalization for its activity. In ADCs, the antibody functions as a targeting agent to direct the ADC to target cells that express the antigen, such as an antigen on cancer cells. In this case, the antigen may be a tumor-associated antigen, i.e., one that is specifically expressed or overexpressed on cancer cells. Once there, the drug is released at or near the target cell and acts as a therapeutic agent. For a review of the mechanism of action and use of ADCs in cancer treatment, see Schrama et al., Nature Rev. Drug Disc. 2006, 5, 147.

[0214] For cancer treatment, the therapeutic agent or drug of the ADC is preferably a cytotoxic drug that causes the death of the targeted cancer cells. Cytotoxic drugs that can be used in ADCs include the following types of compounds and their analogs and derivatives: (a) enediynes, such as calicheamicin (see, e.g., Lee et al., J. Am. Chem. Soc. 1987, 109, 3464 and 3466) and uncialamicin (see, e.g., Davies et al., WO2007 / 038868A2(2007) and Chowdari et al., US8,709,431B2(2012)); (b) tubulysin (see, e.g., Domling et al., US7,778,814B2(2010); Cheng et al., US8,394,922B2(2013); and Cong et al., US2014 / 0227295A1); (c) CC-1065 and duocarmycin (see, e.g., Boger, US6,5458,530 B1 (2003); Sufi et al., US8,461,117B2 (2013); and Zhang et al., US2012 / 0301490A1 (2012)); (d) epothilones (see, e.g., Vite et al., US2007 / 0275904A1 (2007) and USRE42930E (2011)); (e) auristatins (see, e.g., Senter et al., US Pat. No. 6,844,869B2 (2005) and Doronina et al., US Pat. No. 7,498,298B2 (2009)); (f) pyrrolobenzodiazepine (PBD) dimers (see, e.g., Howard et al., US2013 / 0059800A1(2013); US2013 / 0028919A1(2013); and WO2013 / 041606A1 (2013)); and (g) Maytansinoids, such as DM1 and DM4 (see, for example, Chari et al., US Pat. No. 5,208,020 (1993) and Amphlett et al., US Pat. No. 7,374,762B2 (2008)).

[0215] In ADCs, the antibody and therapeutic agent may be conjugated via a linker, e.g., a cleavable linker such as a peptidyl, disulfide, or hydrazone linker. For example, the linker may be a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Patents 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO02 / 096910; WO07 / 038658; WO07 / 051081; WO07 / 059404; WO08 / 083312; and WO08 / 103693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295; the disclosures of which are incorporated herein by reference.

[0216] Examples of targets of ADCs that can be enhanced with modified heavy chain constant regions include B7H4 (Korman et al., US2009 / 0074660A1); CD19 (Rao-Naik et al., US2009 / 0074660A1); CD22 (King et al., US2010 / 0143368A1); CD30 (Keler et al., US7,387,776B2 (2008)); CD70 (Terrett et al., US8,124,738B2); CTLA-4 (Korman et al., US6,984,720B1 (2006)); PD-1 (Korman et al., US8,008,449B2 (2011)); PSMA (Huang et al., US2009 / 0297438A1 and Cardarelli et al., US7,875,278B2); PTK7 (Terrett et al., US2010 / 0034826A1); glypican-3 (Terrett et al., US2010 / 0209432(A1)); RG1 (Harkins et al., US7,335,748B2(2008)); mesothelin (Terrett et al., US8,268,970B2(2012)); and CD44 (Xu et al., US2010 / 0092484A1).

[0217] The modified heavy chain constant domain may be part of an antibody for use in immunological diseases other than oncology, such as rheumatoid arthritis, lupus, etc.

[0218] The modified heavy chain constant domain can be fused to a non-antibody molecule (or antibody variant) or fragment thereof, and can be fused to any polypeptide required for the presence of Fc. The modified heavy chain constant domain can be fused to an antigen-binding fragment of an antibody, as further defined herein (e.g., defined portion).

[0219] In some embodiments, a heavy chain constant domain or portion thereof containing the P238K mutation, lacking certain effector functions, can be fused to a polypeptide, e.g., the heavy chain portion of an antigen-binding fragment of an antibody. As further described herein, the Fc of an IgG, e.g., IgG1, containing the P238K mutation and comprising, for example, the amino acid sequence set forth in SEQ ID NO: 198, can be fused to the heavy chain variable domain of an antibody, wherein the antibody binds to any target, e.g., a target protein described herein (e.g., CD40 or CD40L). The IgG1 Fc containing the P238K mutation (e.g., P238K IgG1 fa having the amino acid sequence of SEQ ID NO: 198, or in the case of IgG1, having allotype f) can be used with any antibody or any antigen-binding fragment thereof for which effector function, particularly binding to FcγR CD32a, CD32b, and CD16a, is undesirable. In addition to P238K, the heavy chain constant region may contain one or two additional mutations, e.g., substitutions, that reduce binding to FcγR CD64, or P238K may be used in the context of an IgG2 hinge, e.g., an IgG2 hinge comprising C219S, as further described herein.

[0220] III. Methods for Modifying the Biological Activity of Antibodies Provided herein are methods for enhancing the biological activities of an antibody, such as one or more of the following biological activities: (a) Increased or altered internalization by cells; (b) increased or altered agonist activity; (c) increased or altered antagonist or blocking activity; (d) enhanced or reduced ADCC; (d) the emergence of novel properties; (e) increased or altered signal transduction; (f) formation of large antibody / antigen cross-linked complexes; (g) increased clustering or oligomerization of target cell surface molecules; (h) increased stimulation or enhancement of the immune response; and / or (i) Increased inhibition of immune responses.

[0221] Methods for enhancing the biological activity of an antibody can include replacing the heavy chain constant region or portion thereof, such as the hinge and / or CH1 domain, with a modified heavy chain constant region or portion thereof, such as an IgG2 hinge and / or IgG2 CH1 domain.

[0222] In certain embodiments, a method for improving the biological activity of an antibody comprises: (i) preparing an antibody that does not contain a modified heavy chain constant region described herein; and (ii) replacing the heavy chain constant region of the antibody with a modified heavy chain constant region or portion thereof that enhances the biological activity of the antibody. In certain embodiments, a method for improving the biological activity of an antibody comprises: (i) preparing an antibody that contains a non-IgG2 hinge (e.g., an IgG1 hinge, an IgG3 hinge, or an IgG4 hinge); and (ii) replacing the non-IgG2 hinge of the antibody with an IgG2 hinge. In certain embodiments, a method for improving the biological activity of an antibody comprises: (i) preparing an antibody that contains a non-enhanced IgG2 hinge; and (ii) replacing the non-enhanced IgG2 hinge of the antibody with an IgG2 hinge. A "non-enhanced IgG2 hinge" is a variant IgG2 hinge that differs from an IgG2 hinge in such a way that it no longer has the properties required for enhanced antibody biological activity, e.g., a variant hinge that no longer has the rigidity of a wild-type IgG2 hinge.

[0223] An example of a method for enhancing the biological activity of an antibody includes (i) preparing an antibody comprising a non-IgG2 hinge or a non-enhanced IgG2 hinge, and (ii) replacing the hinge with a hinge comprising SEQ ID NO: 8, 21, 22, 23, 126-132, 134-136, or 137, or a variant thereof, such as a variant described herein. A method for enhancing the biological activity of an antibody can also include (i) preparing an antibody comprising a heavy chain constant region that is not a modified heavy chain constant region, and (ii) replacing the heavy chain constant region with a modified heavy chain constant region. The replacement of the heavy chain constant region can include replacing the CH1, hinge, CH2, and / or CH3 domains. For example, the heavy chain constant region can be modified by replacing the hinge with an IgG2 hinge or a variant thereof and / or replacing the CH1 domain with an IgG1 or IgG2 CH1 domain or a variant thereof. In one embodiment, the hinge is replaced with an IgG2 hinge, and the CH2 domain is replaced with an IgG1 CH2 domain. In certain embodiments, the hinge is replaced with an IgG2 hinge, and the CH3 domain is replaced with an IgG1 CH3 domain. In certain embodiments, the hinge is replaced with an IgG2 hinge, the CH1 is replaced with an IgG2 hinge, the CH2 domain is replaced with an IgG1 CH2 domain, and the CH3 domain is replaced with an IgG1 CH3 domain. In certain embodiments, the heavy chain constant region is replaced with modified heavy chain regions 1-27 shown in Table 5 above, or a heavy chain constant region shown in Table 6 or described herein.

[0224] Also provided herein is a method for enhancing the biological activity of an IgG1 or IgG2 antibody, comprising deleting 1 to 10 amino acids from the hinge of the IgG1 or IgG2 antibody, respectively. For example, one or more of amino acids 219, C22, D221, K222, T223, H224, and T225 can be deleted. In one embodiment, amino acids 219, C22, D221, K222, T223, H224, and T225 are all deleted.

[0225] Further provided herein are methods for producing and providing effectorless antibodies or antigen-binding fragments thereof, e.g., by converting P238, e.g., P238K, to eliminate or reduce the effector function of the antibody.

[0226] In some embodiments, for example, replacement of an antibody's heavy chain constant region to modify biological activity does not result in a decrease or significant decrease in its binding activity to its target antigen. As described in the Examples, replacement of the heavy chain constant regions of anti-GITR and anti-CD73 antibodies did not significantly change their affinity for human GITR and human CD73 antigens, respectively.

[0227] When referring to the replacement of a domain of a particular isotype with the same domain of a different isotype or a domain containing a mutation, e.g., the P238 mutation, it is understood that the domains need not be literally replaced, but rather, it may only be necessary to change the amino acids that differ between the two isotypes.

[0228] Standard assays for assessing the binding ability of antibodies to various antigens are known in the art and further described herein, and include, for example, ELISA, Western blot, and RIA. Suitable assays are detailed in the Examples. The binding kinetics (e.g., binding affinity) of an antibody can also be measured by BIACORE. (登録商標) This can be assessed by standard assays known in the art, such as by SPR analysis. Assays to assess the properties of antibodies with modified constant regions (e.g., ligand binding, T cell proliferation, cytokine production) are further detailed below and in the Examples.

[0229] Examples of antibodies that can be modified as described herein include, for example, antibodies for treating cancer, e.g., Yervoy TM(ipilimumab) or tremelimumab (against CTLA-4), galiximab (against B7.1), BMS-936558 (against PD-1), CT-011 (against PD-1), MK-3475 (against PD-1), AMP224 (against B7DC), BMS-936559 (against B7-H1), MPDL3280A (against B7-H1), MEDI-570 (against ICOS), AMG557 (against B7H2), MGA271 (against B7H3), IMP321 (against LAG-3), BMS-663513 (against CD137), PF-05082566 (against CD137), CDX-1127 (against CD27), anti-OX40 (Providence Health Services), huMAbOX40L (against OX40L), atacicept (against TACI), CP-870893 (against CD40), lucatumumab (against CD40), dacetuzumab (against CD40), muromonab-CD3 (against CD3), and ipilimumab (against CTLA-4).

[0230] Other antibodies that can be modified as described herein include PD-1 and PD-L1 antagonist antibodies. Examples of anti-PD-1 antibodies that can be modified as described herein include nivolumab (BMS-936558); antibodies comprising one of the CDRs or variable regions of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4, and 4A11, described in WO2006 / 121168; MK-3475 (lambrolizumab), described in WO2012 / 145493; AMP-514, described in WO2012 / 145493; CT-011 (pidilizumab; formerly CT-AcTibody or BAT; see, e.g., Rosenblatt et al. (2011) J. Immunotherapy 34:409); WO2009 / 014708, WO03 / 099196, WO2009 / 114335, WO2011 / 066389, WO2011 / 161699, WO2012 / 145493, WO2013 / 173223, U.S. Patents 7,635,757 and 8,217,149 and U.S. Patent Publication 2009 / 0317368.

[0231] Further antibodies that may be modified include anti-PD-L1 antibodies, such as BMS-936559 (referred to as 12A4 in WO 2007 / 005874 and US Patent 7,943,743); the CDRs or sequences of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, as described in PCT Publication WO 2007 / 005874 and US Patent 7,943,743. or antibodies comprising the variable regions thereof; MEDI4736 (also known as anti-B7-H1); MPDL3280A (also known as RG7446); any of the anti-PD-L1 antibodies disclosed in WO2013 / 173223, WO2011 / 066389, WO2012 / 145493, U.S. Patents 7,635,757 and 8,217,149, and U.S. Publication No. 2009 / 145493.

[0232] Other antibodies that can be modified include anti-CTLA-4 antibodies, such as Yervoy TM (ipilimumab or antibody 10D1, described in PCT Publication WO01 / 14424); tremelimumab (formerly ticilimumab, CP-675,206); the following publications: WO98 / 42752; WO00 / 37504; U.S. Patent 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304; and any of the anti-CTLA-4 antibodies disclosed in WO2013 / 173223.

[0233] Other antibodies that can be modified include anti-LAG-3 antibodies, such as BMS-986016; IMP731 described in US2011 / 007023; and IMP-321.

[0234] Other antibodies that can be modified include anti-GITR agonist antibodies, such as the anti-GITR antibody 6C8 or humanized versions thereof described in WO2006 / 105021; the antibodies described in WO2011 / 028683; and the antibodies described in JP 2008-278814.

[0235] Antibodies targeting other antigens, including those described elsewhere herein, may also be modified. For example, antibodies that require anti-Her2 internalization, such as trastuzumab (Herceptin), may be modified as described herein.

[0236] IV. Further Heavy Chain Constant Domain Modifications In addition to the modifications described herein to antibodies for enhanced biological activity or reduced effector function, additional mutations can be made, e.g., to the CH1, hinge, CH2, or CH3 domains, e.g., to further reduce antibody effector function, FcγR binding, and / or stability. For example, any of the modifications described herein, e.g., below, can be combined with a P238, e.g., P238K, mutation, such as in an IgG1 or IgG1-IgG2 hybrid Fc or portion thereof.

[0237] Fc and modified Fc The antibodies described herein generally can comprise an Fc region containing one or more modifications to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cellular cytotoxicity. For example, modifications can be made to the Fc region to produce Fc variants with (a) increased or decreased antibody-dependent cellular cytotoxicity (ADCC), (b) increased or decreased complement-dependent cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors, relative to the parent Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications may be particularly desirable. For example, a variant Fc region can contain two, three, four, five, etc., substitutions, e.g., at specific Fc region positions identified herein. Examples of Fc sequence variants are described herein and in U.S. Patents 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publications WO00 / 42072; WO01 / 58957; WO04 / 016750; WO04 / 029207; WO04 / 035752; WO04 / 074455; WO04 / 099249; WO04 / 063351; WO05 / 070963; WO05 / 040217, WO05 / 092925, and WO06 / 020114.

[0238] Reduced effector function ADCC activity can be reduced by modifying the Fc region. In some embodiments, sites that affect binding to Fc receptors, preferably sites other than the salvage receptor binding site, can be removed (e.g., by mutation). In other embodiments, the Fc region can be modified to remove the ADCC site. ADCC sites are known in the art; for example, see Sarmay et al. (1992) Molec. Immunol. 29 (5): 633-9 for the ADCC site in IgG1. In some embodiments, the G236R and L328R variants of human IgG1 effectively eliminate FcγR binding. Horton et al. (2011) J. Immunol. 186:4223 and Chu et al. (2008) Mol. Immunol. 45:3926. In other embodiments, an Fc with reduced FcγR binding contains amino acid substitutions L234A, L235E, and G237A. Gross et al. (2001) Immunity 15:289.

[0239] CDC activity can also be reduced by Fc region modifications. Mutations at positions D270, K322, P329, and P331, particularly the alanine mutations D270A, K322A, P329A, and P331A, significantly reduce the ability of the corresponding antibody to bind to C1q and activate complement. Idusogie et al. (2000) J. Immunol. 164:4178; WO99 / 51642. Modifications at position 331 of IgG1 (e.g., P331S) have been shown to reduce complement fixation. Tao et al. (1993) J. Exp. Med. 178:661 and Canfield & Morrison (1991) J. Exp. Med. 173:1483. In another example, one or more amino acid residues within amino acid positions 231-239 are altered, thereby reducing the antibody's ability to fix complement. WO94 / 29351.

[0240] In one embodiment, the Fc with reduced complement fixation has the amino acid substitutions A330S and P331S. Gross et al. (2001) Immunity 15:289.

[0241] For uses where effector function is to be avoided entirely, e.g., when antigen binding alone is sufficient for the desired therapeutic benefit and effector function only results in (or increases the risk of) undesirable side effects, IgG4 antibodies can be used, or antibodies or fragments lacking the Fc region or a substantial portion can be devised, or the Fc can be mutated to eliminate glycosylation entirely (e.g., N297A). Alternatively, human IgG2 (C H 1 domain and hinge region) and human IgG4 (C H 2 and C H Hybrid constructs of the Fc domain (IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG1, IgG1, IgG1a, IgG1b, IgG1c, IgG1d, IgG1e, IgG1f ...

[0242] In other embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to reduce the overall effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be replaced with a different amino acid residue such that the antibody has reduced affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is modified can be, for example, an Fc receptor (residues 234, 235, 236, 237, 297) or the complement C1 component (residues 297, 318, 320, 322). U.S. Patents 5,624,821 and 5,648,260, both to Winter et al.

[0243] WO 88 / 007089 proposed modifications in the IgG Fc region to reduce binding to FcγRI (234A; 235E; 236A; G237A) to decrease ADCC or to block binding to complement component C1q (E318A or V / K320A and K322A / Q) to eliminate CDC. See also Duncan & Winter (1988) Nature 332:563; Chappel et al. (1991) Proc. Nat'l Acad. Sci. (USA) 88:9036; and Sondermann et al. (2000) Nature 406:267 (describing the effect of these mutations on FcγRII binding).

[0244] Fc modifications that reduce effector function also include substitutions, insertions and deletions at positions 234, 235, 236, 237, 267, 269, 325 and 328, such as 234G, 235G, 236R, 237K, 267R, 269R, 325L and 328R. An Fc variant may include 236R / 328R. Other modifications for reduced FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P and 234V. These and other modifications are reviewed in Strohl (2009) Current Opinion in Biotechnology 20:685-691. Mutation of one or more IgG residues at positions 233-236 and 327-331, such as E233P, L234V, L235A, optionally G236Δ, A327G, A330S, and P331S in IgG1; E233P, F234V, L235A, optionally G236Δ in IgG4; and A330S and P331S in IgG2, can reduce effector function (both ADCC and complement activation) while maintaining neonatal FcR binding (maintaining half-life). See Armour et al. (1999) Eur. J. Immunol. 29:2613; WO99 / 58572. Other mutations that reduce effector function include L234A and L235A in IgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A in IgG2 (Cole et al. (1997) J. Immunol. 159:3613; see also U.S. Patent 5,834,597); and S228P and L235E in IgG4 (Reddy et al. (2000) J. Immunol. 164:1925). Another combination of mutations to reduce effector function in human IgG1 includes L234F, L235E, and P331S. Oganesyan et al. (2008) Acta Crystallogr. D. Biol. Crystallogr. 64:700.See generally Labrijn et al. (2008) Curr. Op. Immunol. 20:479. Additional mutations found to reduce effector function in Fc(IgG1) fusion proteins (abatacept) are C226S, C229S, and P238S (EU residue numbering). Davis et al. (2007) J. Immunol. 34:2204.

[0245] Other Fc variants with reduced ADCC and / or CDC are described in Glaesner et al. (2010) Diabetes Metab. Res. Rev. 26:287 (F234A and L235A to reduce ADCC and ADCP in IgG4); Hutchins et al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:11980 (F234A, G237A, and E318A in IgG4); An et al. (2009) MAbs 1:572 and US Patent Publication 2007 / 0148167 (H268Q, V309L, A330S, and P331S in IgG2); McEarchern et al. (2007) Blood 109:1185 (C226S, C229S, E233P, L234V, L235A in IgG1); Vafa et al. (2014) Methods 65:114 (V234V, G237A, P238S, H268A, V309L, A330S, P331S in IgG2).

[0246] In some embodiments, the Fc is selected to have essentially no effector function, i.e., reduced FcγR binding and reduced complement fixation. An example of an effector-less Fc, such as an IgG1 Fc, contains the following five mutations: L234A, L235E, G237A, A330S, and P331S. Gross et al. (2001) Immunity 15:289. These five substitutions can be combined with N297A to similarly eliminate glycosylation.

[0247] Enhanced effector function Alternatively, ADCC activity can be increased by Fc region modification. With regard to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2, and IgG1 constant domains, rather than IgG2 or IgG4, should be selected for use with drugs when ADCC is desired. Alternatively, the Fc region can be modified at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, The modifications may be made by modifying one or more amino acids at 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. See WO2012 / 142515; see also WO00 / 42072. Examples of substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Examples of variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. For example, a human IgG1Fc containing the G236A variant, optionally combined with I332E, has been shown to increase the FcγIIA / FcγIIB binding affinity ratio by approximately 15-fold. Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010) mAbs 2:181.Other modifications for enhancing FcyR and complement interactions include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl (2009) Current Opinion in Biotechnology 20:685-691. In particular, both ADCC and CDC can be enhanced by altering the E333 position of IgG1, e.g., E333A. Shields et al. (2001) J. Biol. Chem. 276:6591. P247I and A339D / Q mutations for enhancing effector function in IgG1 are disclosed in WO2006 / 020114, and D280H, K290S±S298D / V are disclosed in WO2004 / 074455. K326A / W and E333A / S variants have been shown to increase effector function in human IgG1, and E333S in IgG2. Idusogie et al. (2001) J. Immunol. 166:2571.

[0248] In particular, the binding sites of human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described. Shields et al. (2001) J. Biol. Chem. 276:6591-6604. Specific mutations at positions 256, 290, 298, 333, 334, and 339, including the combination mutants T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, showed improved binding to FcγRIII (with enhanced FcγRIIIa binding and ADCC activity). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with the S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys. Lazar et al. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005; Awan et al. (2010) Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res. 317:1278. Triple mutations in antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, and the S239D / I332E variant showed increased ability to deplete B cells in monkeys. Lazar et al. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005. Furthermore, IgG1 mutants containing L235V, F243L, R292P, Y300L, V305I, and P396L mutations have been identified that showed enhanced binding to FcγRIIIa and concomitant enhanced ADCC activity in transgenic mice expressing human FcγRIIIa in B cell malignancies and breast cancer models (Stavenhagen et al. (2007) Cancer Res. 67:8882; U.S. Patent No. 8,652,466; Nordstrom et al. (2011) Breast Cancer Res. 13:R123).

[0249] Various IgG isotypes also exhibit differential CDC activity (IgG3 > IgG1 >> IgG2 ≒ IgG4). Dangl et al. (1988) EMBO J. 7:1989. For applications where enhanced CDC is desired, mutations that increase C1q binding can be introduced. The ability to recruit complement (CDC) can be enhanced by mutations at K326 and / or E333 in IgG2, such as K326W (which reduces ADCC activity) and E333S, which increase binding to C1q, the first component of the complement cascade. Idusogie et al. (2001) J. Immunol. 166:2571. Introduction of S267E / H268F / S324T (alone or in any combination) into human IgG1 enhances C1q binding. Moore et al. (2010) mAbs 2:181. The Fc region of the IgG1 / IgG3 hybrid isotype antibody "113F" (Figure 1 therein) of Natsume et al. (2008) Cancer Res. 68:3863 also contributes to enhanced CDC. See also Michaelsen et al. (2009) Scand. J. Immunol. 70:553 and Redpath et al. (1998) Immunology 93:595.

[0250] Additional mutations that can increase or decrease effector function are disclosed in Dall'Acqua et al. (2006) J. Immunol. 177: 1129. See also Carter (2006) Nat. Rev. Immunol. 6:343; Presta (2008) Curr. Op. Immunol. 20:460.

[0251] Fc variants that enhance affinity to the inhibitory receptor FcyRIIb may also be used, for example, to induce apoptosis or enhance adjuvant activity. Li & Ravetch (2011) Science 333:1030; Li & Ravetch (2012) Proc. Nat'l Acad. Sci (USA) 109:10966; U.S. Patent Publication 2014 / 0010812. Such variants are useful for enhancing the affinity of FcyRIIb, including, for example, B cells and monocytes. + The present invention provides antibodies with cell-associated immunomodulatory activity. In some embodiments, the Fc variants selectively enhance affinity for FcyRllb relative to one or more activating receptors. Modifications for improved binding to FcyRllb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332 according to the EU index. Examples of substitutions for enhancing FcyRllb affinity include, but are not limited to, 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Other Fc variants for enhanced binding to FcyRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F. In particular, the S267E, G236D, S239D, L328F, and I332E variants, including the S267E+L328F double variant of human IgG1, are particularly valuable for specifically enhancing affinity for the inhibitory FcyRIIb receptor. Chu et al. (2008) Mol. Immunol. 45:3926; U.S. Patent Publication No. 2006 / 024298; WO2012 / 087928. Enhanced specificity for FcyRIIb (FcyRIIa) R131(distinguished from) can be obtained by adding the P238D substitution. Mimoto et al. (2013) Protein. Eng. Des. & Selection 26:589; WO2012 / 115241.

[0252] Glycosylation Glycosylation of antibodies can be modified to increase or decrease effector function. For example, mutation of the conservative asparagine residue at position 297 (e.g., N297A) can produce an aglycosylated antibody that lacks all effector function, thereby eliminating complement and FcγRI binding. Bolt et al. (1993) Eur. J. Immunol. 23:403. See also Tao & Morrison (1989) J. Immunol. 143:2595 (using N297Q to remove glycosylation at position 297 in IgG1).

[0253] Aglycosylated antibodies generally lack effector function, but mutations can be introduced to retain that function. Aglycosylated antibodies, such as those with N297A / C / D / or H mutations or derived from systems that do not glycosylate proteins (e.g., E. coli), can be further mutated to retain FcγR binding, for example, S298G and / or T299A / G / or H (WO2009 / 079242) or E382V and M428I (Jung et al. (2010) Proc. Nat'l Acad. Sci (USA) 107:604).

[0254] Furthermore, antibodies with enhanced ADCC can be produced by modifying glycosylation. For example, removal of fucose from the heavy chain Asn297-linked oligosaccharides has been shown to enhance ADCC, based on improved binding to FcγRIIIa. Shields et al. (2002) JBC 277:26733; Niwa et al. (2005) J. Immunol. Methods 306: 151; Cardarelli et al. (2009) Clin. Cancer Res. 15:3376 (MDX-1401); Cardarelli et al. (2010) Cancer Immunol. Immunotherap. 59:257 (MDX-1342). Such low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87:614) or other cells that produce afucosylated antibodies. See, for example, Zhang et al. (2011) mAbs 3:289 and Li et al. (2006) Nat. Biotechnol. 24:210 (both describing antibody production in glycoengineered Pichia pastoris); Mossner et al. (2010) Blood 115:4393; Shields et al. (2002) J. Biol. Chem. 277:26733; Shinkawa et al. (2003) J. Biol. Chem. 278:3466; EP1176195B1. ADCC can also be enhanced as described in PCT Publication WO 03 / 035835, which discloses the use of a variant CHO cell line, Lec13, with reduced capacity for fucose to Asn(297)-linked carbohydrates and the resulting hypofucosylation of antibodies expressed in the host cells (Shields, RL et al. (2002) J. Biol. Chem. 277(See also WO2009 / 135181.) Alternatively, fucose analogs can be added to the culture medium during antibody production to inhibit fucose incorporation into the carbohydrate of the antibody.

[0255] Increasing bisecting GlcNac structures in antibody-linked oligosaccharides also enhances ADCC. Umana et al., PCT Publication WO 99 / 54342, describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (Umana et al. (1999) Nat. Biotech. 17 :176-180).

[0256] Additional glycosylation variants lacking galactose, sialic acid, fucose, and xylose residues (so-called GNGN glycoforms) that exhibit enhanced ADCC and ADCP but reduced CDC, and others lacking sialic acid, fucose, and xylose (so-called G1 / G2 glycoforms) that exhibit enhanced ADCC, ADCP, and CDC have been developed (U.S. Patent Application Publication No. 2013 / 0149300). Antibodies with these glycosylation patterns are optionally produced in genetically modified Nicotiana benthamiana plants in which the endogenous xylosyl- and fucosyltransferase genes have been knocked out.

[0257] Glycoengineering can also be used to modify the anti-inflammatory properties of IgG constructs by altering the α2,6 sialylated content of the carbohydrate chain attached to Asn297 in the Fc region, where increasing the proportion of the α2,6 sialylated form results in enhanced anti-inflammatory effects. See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513. Conversely, decreasing the proportion of antibodies with α2,6 sialylated carbohydrates can be useful when anti-inflammatory properties are not desired. For example, methods for modifying the α2,6 sialylated content of antibodies by selective purification or enzymatic modification of the α2,6 sialylated form are described in U.S. Patent Application Publication No. 2008 / 0206246. In other embodiments, the amino acid sequence of the Fc region is modified to mimic the effects of α2,6 sialylation, for example, by inclusion of an F241A modification. WO2013 / 095966.

[0258] The antibodies described herein may contain one or more glycosylation sites in the light or heavy chain variable region. Such glycosylation sites may increase the immunogenicity of the antibody or alter the antibody's pK by altering antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J. Immunol 172:5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing the NXS / T sequence.

[0259] Biological half-life In some embodiments, antibodies are modified to extend their biological half-life. Various approaches are possible. For example, this can be achieved by increasing the binding affinity of the Fc region to FcRn. In some embodiments, the antibody is modified in the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents 5,869,046 and 6,121,022 to Presta et al. Other examples of Fc variants with increased binding to FcRn and / or improved pharmacokinetics include substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants with increased binding to FcFcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, and 434A (Shields et al., Journal of Biological Chemistry, 2001). 276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Del'Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). See U.S. Patent 8,367,805.

[0260] Modifications of certain conserved residues in IgG Fc (I253 / H310 / Q311 / H433 / N434), such as the N434A variant (Yeung et al. (2009) J. Immunol. 182:7663), have been proposed as a way to enhance FcRn affinity and thus extend the half-life of antibodies in circulation. WO98 / 023289. A combined Fc variant containing M428L and N434S has been shown to increase FcRn binding by up to five-fold and extend serum half-life. Zalevsky et al. (2010) Nat. Biotechnol. 28:157. A combined Fc variant containing T307A, E380A, and N434A modifications also extends the half-life of IgG1 antibodies. Petkova et al. (2006) Int. Immunol. 18:1759. Additionally, combination Fc variants including M252Y / M428L, M428L / N434H, M428L / N434F, M428L / N434Y, M428L / N434A, M428L / N434M, and M428L / N434S variants have also been shown to extend half-life. WO2009 / 086320.

[0261] Furthermore, a combined Fc variant containing M252Y, S254T, and T256E also extends half-life by approximately fourfold. (Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514). A related IgG1 modification (M252Y / S254T / T256E / H433K / N434F) that increases FcRn affinity but decreases pH dependence has been used to create an IgG1 construct ("MST-HN Abdeg") for use as a competitor to block binding of other antibodies to FcRn, resulting in increased clearance of other antibodies, either endogenous IgG (e.g., in autoimmune situations) or other exogenous (therapeutic) mAbs. (Vaccaro et al. (2005) Nat. Biotechnol. 23:1283; WO2006 / 130834).

[0262] Other modifications that increase FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; 6,277,375; 6,821,505; WO97 / 34631; WO2002 / 060919.

[0263] In certain embodiments, hybrid IgG isotypes may be used to increase FcRn binding and possibly extend half-life. For example, IgG1 / IgG3 hybrid variants may be constructed by replacing IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions where the two isotypes differ. In this manner, hybrid variant IgG antibodies may be constructed that contain one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments described herein, IgG1 / IgG2 hybrid variants may be constructed by replacing IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgG1 at positions where the two isotypes differ. In this way, hybrid variant IgG antibodies can be constructed that contain one or more substitutions, for example, one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to the insertion of glycine at position 236), and 327A. See U.S. Patent 8,629,113. Hybrid IgG1 / IgG2 / IgG4 sequences have been produced that purportedly have extended serum half-lives and improved expression. U.S. Patent 7,867,491 (SEQ ID NO: 18 therein).

[0264] The serum half-life of the antibodies of the present invention can also be extended by pegylation. Antibodies can be pegylated, for example, to extend the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or its fragment is generally reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass all forms of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP 0154316 to Nishimura et al. and EP 0401384 to Ishikawa et al.

[0265] Alternatively, in some situations, it may be desirable to shorten rather than extend the half-life of the antibodies of the invention. Modifications such as I253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A / R, I253A, or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819) in the Fc of human IgG1 may inhibit FcRn binding for use in situations where rapid clearance is preferred, such as medical imaging. reductionThis can shorten the half-life (increased clearance). See also Kenanova et al. (2005) Cancer Res. 65:622. Another means of increasing clearance includes modifying the antigen-binding domain of the present invention as an antibody fragment, such as a Fab fragment, that lacks the ability to bind to FcRn. Such modifications can reduce the circulating half-life of the antibody from several weeks to a few hours at most. If necessary, selective pegylation of the antibody fragment can be used to fine-tune (extend) the half-life of the antibody fragment. Chapman et al. (1999) Nat. Biotechnol. 17:780. Antibody fragments can be fused to human serum albumin, for example, in a fusion protein construct, to extend half-life. Yeh et al. (1992) Proc. Nat'l Acad. Sci. 89:1904. Alternatively, a bispecific antibody can be constructed with a first antigen-binding domain of the present invention and a second antigen-binding domain that binds to human serum albumin (HSA). See International Patent Application WO2009 / 127691 and the patents cited therein. Alternatively, specialized polypeptide sequences, such as "XTEN" polypeptide sequences, can be added to antibody fragments to extend their half-life. Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; International Patent Application WO2010 / 091122.

[0266] stability Potential protease cleavage sites in the hinge of the IgG1 construct can be removed by D221G and K222S modifications to increase antibody stability. WO2014 / 043344.

[0267] In certain embodiments, the antibodies described herein do not contain asparagine isomerism sites. Deamidation of asparagine can occur at NG or DG sequences and result in the creation of isoaspartic acid residues, which can cause a kink in the polypeptide chain and reduce stability (the isoaspartic acid effect).

[0268] Each antibody has a unique isoelectric point (pI), which generally falls within the pH range of 6 to 9.5. The pI of IgG1 antibodies generally falls within the pH range of 7 to 9.5, and the pI of IgG4 antibodies generally falls within the pH range of 6 to 8. There is speculation that antibodies with pIs outside the normal range may be partially denatured and unstable under in vivo conditions. Therefore, it is preferable for antibodies to have a pI value within the normal range. This can be achieved by selecting antibodies with a pI within the normal range or by modifying charged surface residues.

[0269] Each antibody has a characteristic melting temperature; the higher the melting temperature, the greater the overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). M1 (initial denaturation temperature) is preferably greater than 60° C., preferably greater than 65° C., and even more preferably greater than 70° C. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

[0270] In a preferred embodiment, antibodies are selected that do not rapidly degrade. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0271] When an IgG4 constant domain is used, it is generally preferred to include the substitution 228P, which mimics the hinge sequence in IgG1, thereby stabilizing the IgG4 molecule by, for example, reducing Fab arm exchange between the therapeutic antibody and the treated patient's endogenous IgG4. Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy et al. (2000) J. Immunol. 164:1925. Similarly, in IgG2 hinge-containing antibodies, the C219S and / or C220S mutations stabilize IgG2 hinge-containing antibodies.

[0272] agglomeration In another preferred embodiment, antibodies are selected that have minimal aggregation effects, which may induce unwanted immune responses and / or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, and even more preferably 5% or less. Aggregation can be measured by several techniques, including size exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

[0273] V. Non-Antibody Proteins and Antibody Derivatives The invention described herein can also be applied to molecules other than full-length antibodies, so long as they contain a hinge. For example, IgG fusion proteins with enhanced biological activity or lacking effector function can be produced. Thus, provided herein are fusion proteins comprising an IgG2 hinge and, optionally, an active portion of an IgG constant region, e.g., an Fc region, including the CH2 and CH3 domains or a portion thereof, linked, e.g., covalently linked, to an IgG (e.g., IgG1) or portion thereof with reduced effector function, e.g., a mutation at P238, e.g., P238K. The Fc can be any Fc of a modified heavy chain constant region described herein, such as the Fc portion of a modified heavy chain constant region shown in Tables 5 and 6 or the Sequence Listing.

[0274] The antibodies described herein can also be used to form bispecific molecules or molecules for CAR-T therapy. Antibodies or antigen-binding portions thereof can be derivatized or conjugated with other functional molecules, such as other peptides or proteins (e.g., other antibodies or ligands for receptors), to produce bispecific molecules that bind to at least two different binding sites or target molecules. The antibodies described herein can be derivatized or conjugated with more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create bispecific molecules, the antibodies described herein can be functionally conjugated (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as other antibodies, antibody fragments, peptides, or binding mimetics, to produce bispecific molecules.

[0275] VI. Composition Also provided are compositions, e.g., pharmaceutical compositions, comprising one or a combination of the antibodies or antigen-binding portions thereof described herein, formulated with a pharmaceutically acceptable carrier. Such compositions can include one or a combination (e.g., two or more different) of the antibodies, immunoconjugates, or bispecific molecules described herein. For example, a pharmaceutical composition described herein can include a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes of a target antigen or have complementary activities.

[0276] In some embodiments, the composition comprises an antibody described herein at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 1-300 mg / ml, or 100-300 mg / ml.

[0277] The pharmaceutical compositions described herein can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include an antibody described herein in combination with at least one other anti-cancer and / or T cell stimulating (e.g., activating) agent. Examples of therapeutic agents that can be used in combination therapy are described in further detail below in the section on uses of the antibodies described herein.

[0278] In some embodiments, the therapeutic compositions disclosed herein may contain other compounds, drugs, and / or agents for use in the treatment of cancer. Such compounds, drugs, and / or agents include, for example, chemotherapy drugs, small molecule drugs, or antibodies that stimulate immune responses to certain cancers. In some examples, the therapeutic compositions may include, for example, one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L) antibody, or an anti-LAG-3 antibody.

[0279] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0280] The pharmaceutical compounds described herein include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like, and non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, and non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0281] The pharmaceutical compositions described herein may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0282] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0283] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above-mentioned sterilization procedures and the addition of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to add isotonic agents such as sugars and sodium chloride to the compositions. Furthermore, prolonged absorption of the injectable formulation can be achieved by adding absorption delaying agents such as aluminum monostearate and gelatin.

[0284] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersion media and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Unless conventional media or agents are incompatible with the active compounds, they are intended to be used in the pharmaceutical compositions described herein.Additional active compounds may also be included in the compositions.

[0285] Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to add isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride to the compositions. Prolonged absorption of injectable compositions can be achieved by adding to the composition agents that delay absorption, for example, monostearate salts and gelatin.

[0286] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed ingredients, as needed, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which produces a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0287] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, when combined with a pharmaceutically acceptable carrier, this amount, out of 100 percent, is about 0.01 percent to about 99 percent of the active ingredient, preferably about 0.1 percent to about 70 percent, and most preferably about 1 percent to about 30 percent of the active ingredient.

[0288] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary administration to the subject, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the necessary pharmaceutical carrier. The specifications of the dosage unit forms described herein are dictated by and directly depend on (a) the unique properties of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating active compounds to treat susceptible individuals.

[0289] For administration of antibodies, dosages range from about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg, of the host body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg, or within the range of 1 to 10 mg / kg body weight. Exemplary treatment regimens involve weekly, biweekly, triweekly, 4 weekly, monthly, trimonthly, or 6 monthly administration. Preferred dosing regimens for the antibodies described herein include intravenous administration of 1 mg / kg or 3 mg / kg body weight, with the antibody being given using one of the following dosing schedules: (i) six doses every four weeks, followed by every three months; (ii) every three weeks; or (iii) one dose of 3 mg / kg body weight, followed by 1 mg / kg body weight every three weeks.

[0290] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, with the dosage of each antibody administered falling within the indicated range. Antibodies are typically administered multiple times. The interval between doses can be, for example, weekly, monthly, every three months, or yearly. The interval can also be indicated by measuring the blood level of antibody against the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods about 25-300 μg / ml.

[0291] The antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients receive treatment until death. In therapeutic applications, relatively high dosages at relatively short intervals may be required until the progression of the disease is reduced or stopped, preferably until the patient shows partial or complete improvement in disease symptoms. The patient can then be administered a preventive regimen.

[0292] Actual dosage levels of the active ingredients of the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition described herein or its ester, salt, or amide employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0293] A "therapeutically effective dose" of an antibody described herein preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom intervals, or prevention of functional impairment or disability due to disease affliction. In the context of cancer, a therapeutically effective dose preferably prevents further progression of physical symptoms associated with the cancer. Symptoms of cancer are well known in the art and include, for example, abnormal lentigo features, changes in appearance of lentigines including asymmetry, border, color and / or diameter, newly pigmented skin areas, abnormal lentigines, dark areas under the fingernails, breast masses, nipple changes, breast cysts, breast pain, death, weight loss, weakness, extreme fatigue, difficulty eating, loss of appetite, chronic cough, worsening shortness of breath, hemoptysis, blood in the urine, blood in the stool, nausea, vomiting, liver metastasis, lung metastasis, bone metastasis, abdominal distension, flatulence, ascites, vaginal bleeding, constipation, abdominal bloating, colon perforation, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle spasms, colon metastasis, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, kidney metastasis and pancreatic metastasis, difficulty swallowing, etc.

[0294] A therapeutically effective dose can prevent or delay the onset of cancer, as may be desired at the early stage of the disease or when the symptoms are present. Clinical tests used to diagnose cancer include chemistry, hematology, serology, and radiology. Therefore, any clinical or biochemical assay that monitors any of the above can be used to determine whether a particular treatment is a therapeutically effective dose for cancer treatment. Those skilled in the art can determine such amounts based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or administration route selected.

[0295] The compositions described herein can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or method of administration will vary depending on the desired results. Preferred routes of administration for the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0296] Alternatively, the antibodies described herein may be administered by topical, epithelial, or mucosal routes of administration, such as non-parenteral routes, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0297] Active compound can be formulated with carriers that prevent compound from rapid release, such as controlled release preparations, including implants, transdermal patches and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used.Many methods for producing such preparations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0298] Therapeutic compositions can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic compositions described herein can be administered using a needleless hypodermic injector, such as the devices disclosed in U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use with the antibodies described herein include U.S. Patent 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent 4,447,233, which discloses an infusion pump that delivers drugs at a precise infusion rate; U.S. Patent 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; U.S. Patent 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0299] In some embodiments, the antibodies described herein are formulated for proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes contain one or more moieties that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, e.g., VV Ranade (1989) J. Clin. Pharmacol. 29:685). Examples of targeting moieties include folate or biotin (e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; See also JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0300] VII. Uses and Methods The antibodies, antibody compositions, and methods described herein have numerous in vitro and in vivo utilities, including, for example, the treatment of various disorders, e.g., cancer. For example, the antibodies described herein can be applied to cultured cells in vitro or ex vivo, or to human subjects, e.g., in vivo. Accordingly, provided herein are methods of treating a subject, comprising administering to the subject an antibody comprising a modified heavy chain constant region, such that treatment is achieved. Also provided herein are methods of modifying a subject's immune response, comprising administering to the subject an antibody, such that the immune response in the subject is modified. Preferably, the response is enhanced, stimulated, or upregulated. However, in other embodiments, the immune response is inhibited.

[0301] A preferred subject is a human patient in whom an enhanced immune response is desired. The method is particularly suitable for treating human patients with disorders that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). In certain embodiments, the method is particularly suitable for in vivo cancer treatment. In certain embodiments, the subject is a tumor-bearing subject, and an immune response against the tumor is stimulated. The tumor can be a solid tumor or a liquid tumor, for example, a hematopoietic tumor. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-L1 positive. In certain embodiments, the tumor is PD-L1 negative. The subject can also be a virus-bearing subject, and an immune response against the virus is stimulated.

[0302] Further provided are methods of inhibiting tumor cell growth in a subject, comprising administering to the subject an antibody described herein, such that tumor growth is inhibited in the subject. Also provided are methods of treating a viral infection in a subject, comprising administering to the subject an antibody described herein, such that the viral infection is treated in the subject.

[0303] Also included herein are T regand administering to the subject a therapeutically effective amount of an antibody described herein comprising an Fc that stimulates cell depletion, thereby removing T cells from the tumor microenvironment of a subject having a tumor, e.g., a cancerous tumor. reg The Fc can be an Fc with an effector function or enhanced effector function, such as, for example, binding or enhanced binding to one or more activating Fc receptors.

[0304] In some embodiments, the antibody comprising the modified heavy chain constant region binds to and inhibits the activity of a stimulatory molecule, i.e., is an antagonist of the stimulatory molecule, or the antibody binds to and stimulates the activity of an inhibitory molecule, i.e., is an agonist of the inhibitory molecule. Such antibodies can be used to treat diseases in which the immune system or immune response should be downregulated, such as autoimmune diseases or to prevent transplant rejection.

[0305] cancer Provided herein are methods of treating a subject with cancer, comprising administering to the subject an antibody described herein, such that the subject is treated, e.g., cancerous tumor growth is inhibited or reduced and / or tumor regression occurs. For example, GITR activation by an anti-GITR antibody can stimulate an immune response against cancer cells in the patient. The antibody can be used alone to inhibit cancerous tumor growth. Alternatively, the antibody can be used in combination with other agents, e.g., other immunogenic agents described below, standard cancer treatments, or other antibodies.

[0306] Cancers whose growth can be inhibited using the antibodies described herein are generally cancers that respond to immunotherapy.Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or cancer), gastric cancer, reproductive cancer, ... Cell tumors, pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastatic melanoma, e.g., cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma epithelial cell carcinomas, T-cell lymphomas, environmentally induced cancers including those induced by asbestos, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and hematological tumors derived from either of the two major blood cell lineages, i.e., myeloid cell lineages (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lineages (producing B, T, NK, and plasma cells), including all types of leukemia, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myeloid leukemias. , such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome) and Waldenstrom's macroglobulinemia lymphoplasmacytic lymphoma (LPL) with hematologic malignancies; myelomas, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called low-grade myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic tumors, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; central and peripheral nerve tumors, including seminoma, teratocarcinoma, astrocytoma, Schwann's lymphoma, tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including T-cell and B-cell neoplasms, including, but not limited to, melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma, lymphoid hematopoietic tumors, such as T-cell disorders, e.g., T-prolymphocytic leukemia (T-PLL), including small cell and cerebrospinal cell types; large granular lymphocyte leukemia (LGL), preferably of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer;The methods described herein may also be used to treat metastatic cancers, refractory cancers (e.g., cancers refractory to prior immunotherapy, e.g., CTLA-4 or PD-1 blocking antibodies), and recurrent cancers.

[0307] Combination therapy In addition to the above treatments, the antibodies described herein can also be used in combination with other treatments. For example, to treat cancer, the antibodies described herein can be administered to a subject who has already received other cancer treatments, such as chemotherapy, radiation, surgery, or gene therapy.

[0308] Methods of treatment include co-administration of the antibodies described herein (e.g., antagonistic antibodies, agonistic antibodies, and ADCs with modified heavy chain constant regions) with other molecules, e.g., antibodies (e.g., antagonistic antibodies, agonistic antibodies, and ADCs). Antibodies described herein that stimulate the immune system can be administered with other molecules that stimulate the immune system, e.g., molecules that are costimulatory molecules for inhibitory molecules or agonists of inhibitors.

[0309] The antibodies described herein can be combined with standard cancer treatments, either alone or in combination with one or more additional immunostimulatory antibodies (e.g., CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade). For example, the antibodies described herein can be effectively combined with chemotherapy regimens, either alone or in combination with one or more additional antibodies. In these cases, it may be possible to reduce the dose of other chemotherapeutic agents administered with the combination of the present disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is the further combination of the antibodies described herein, alone or in combination with additional antibodies, with dacarbazine or IL-2 for the treatment of melanoma.

[0310] The antibodies described herein may be combined with anti-neoplastic antibodies such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexar® (tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab), Lymphocide® (epratuzumab), Avastin® (bevacizumab), and Tarceva® (erlotinib).The antibodies described herein were administered in combination with the following chemotherapeutic agents: camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-FU, or camptothecin plus apo2l / TRAIL (6X combo); proteasome inhibitors (e.g., bortezomib or MG132); Bcl-2 inhibitors (e.g., BH3I-2' (bcl-xl inhibitors), indoleamine dioxygenase-1 (IDO1) inhibitors (e.g., INCB24360), AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax) or MCL-1 (myeloid leukemia cell differentiation protein-1) antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g., smac7, smac4, small molecule smac mimetics, synthetic smac peptides (Fulda et al., Nat Med 2002;8:808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic factors targeting VEGF and VEGFR (e.g., Avastin), synthetic triterpenoids (Hyer et al., Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE inhibitory protein) modulators (e.g., natural and synthetic ligands of PPARγ (peroxisome proliferator-activated receptor gamma) 5809354 or 5569100), kinase inhibitors (e.g., sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalidomide, GSK3β inhibitors, IAP inhibitors and / or genotoxic drugs.

[0311] The antibodies and combination antibody therapies described herein may further be used in combination with one or more antiproliferative cytotoxic agents. Classes of compounds that may be used as antiproliferative cytotoxic agents include, but are not limited to: Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan TM ) phosphamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide. Antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin and gemcitabine.

[0312] Suitable antiproliferative agents for combination with the antibodies described herein include the taxanes, paclitaxel (paclitaxel is taxol TM(commercially available as), docetaxel, discodermolide (DDM), dictyostatin (DCT), peloruside A, epothilone, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone Bl,

[17] -dehydrodesoxyepothilone B,

[18] dehydrodesoxyepothilone B, C12,13-cyclopropyl-epothilone Epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10, discodermolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (discodermolide), TZT-1027 (sobridotin), ILX- 651 (tacidotin hydrochloride), halichondrin B, eribulin mesylate (E-7389), hemiasterin (HTI-286), E-7974, cryptophycin, LY-355703, maytansinoid immunoconjugate (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, In addition to eleutherobin, 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didecyl-(+)-discodermolide, and cryptotylon 1, other microtubule-stabilizing agents known in the art include, but are not limited to, erytherobin, 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didecyl-(+)-discodermolide, and cryptotylon 1, as well as other microtubule-stabilizing agents known in the art.

[0313] The combination treatment may be administered simultaneously or sequentially. In some instances, the combination is a fixed dose combination.

[0314] When it is desirable to render normally proliferating cells quiescent, in combination with or prior to the use of an antibody described herein, hormones and steroids (including synthetic analogs), such as 17a-ethynylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, zoladex TM When using the methods or compositions described herein, other agents used in clinical settings in modulating tumor growth or metastasis, such as antiemetic agents, may also be administered to the patient, if desired.

[0315] Methods for safely and effectively administering chemotherapeutic agents are known to those skilled in the art. Furthermore, the administration is described in standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., the 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), the disclosure of which is incorporated herein by reference.

[0316] Chemotherapeutic agents and / or radiation therapy can be administered according to treatment protocols well known in the art.It is clear to those skilled in the art that the administration of chemotherapeutic agents and / or radiation therapy can vary depending on the disease to be treated and the known effects of chemotherapeutic agents and / or radiation therapy on that disease.In addition, according to the knowledge of a skilled clinician, treatment protocols (e.g., dosage and administration time) can be changed in view of the effect of the administered therapeutic agent observed in the patient and the response of the administered therapeutic agent observed on the disease.

[0317] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents, and published patent applications cited throughout this specification are incorporated herein by reference. In particular, the disclosures of PCT applications WO09 / 045957, WO09 / 073533, WO09 / 073546, WO09 / 054863, PCT / US2013 / 072918, PCT / US15 / 61632, and U.S. Patent Publication No. 2011 / 0150892 are expressly incorporated herein. [Example]

[0318] Example 1: Enhanced internalization of anti-CD73 antibodies with IgG2 hinges relative to the same antibodies with non-IgG2 hinges It was observed that the hybridoma-derived anti-CD73 antibody 11F11 having an IgG2 constant region was more potent in a cellular CD73 inhibition assay than the 11F11 antibody as an IgG1 or IgG1.1 (effectorless IgG1), and more potent than other anti-CD73 antibodies having an IgG1 constant region. Based at least on this observation, it was hypothesized that the increased inhibitory activity of anti-CD73 antibodies having an IgG2 hinge relative to those having a non-IgG2 hinge, such as an IgG1 hinge, is due to increased internalization of the antibody. To test this hypothesis, anti-CD73 antibodies having an IgG1 or IgG2 constant region, or portions thereof, were tested in an internalization assay. The antibodies used are listed in Table 7, which provides the identity of each of the domains of the constant region (all human) of each antibody, including the specific mutation, if present. [Table 7] 1 Full-length heavy chain sequence number 2 Full-length light chain sequence number

[0319] Antibodies were produced by heavy and light chain expression in HEK293-6E cells, and culture medium was collected 5 days after transfection. Binding of the constructs to FcγR was measured. hCD64 and hCD32a-H131 binding data for IgG1.1 and IgG2 molecules were consistent with predictions for the various Fc. IgG1.1f was the most inactive Fc. IgG2 and IgG2-C219S exhibited FcR binding typical of IgG2. As expected, data for IgG2-C219S-G1.1f showed significantly weaker binding than wild-type IgG1 or IgG2, but increased binding compared to IgG1.1f. The affinity of the antibody for human CD73 was measured to determine whether changes in the constant region had an effect. Affinity was measured by surface plasmon resonance (SPR) as follows. CD73 binding kinetics and affinity were examined by surface plasmon resonance (SPR) using a Biacore T100 instrument (GE Healthcare) at 25°C. This experiment examined the binding of the N-terminal domain of hCD73 (consisting of residues 26-336 of human CD73; designated N-hCD73) to an antibody captured on an immobilized Protein A surface. For these experiments, Protein A (Pierce) was immobilized onto flow cells 1-4 of a CM5 sensor chip (GE Healthcare) using standard ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking at a density of 3000-4000 RU in a running buffer of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20. Kinetic experiments were performed by first capturing antibody (5-10 μg / ml) on the Protein A surface using a 30-second contact time at 10 μl / min, and then measuring binding of 600 nM, 200 nM, 66.7 nM, 22.2 nM, 7.4 nM, and 2.5 nM N-hCD73-his using a 180-second association time and a 360-second dissociation time at a flow rate of 30 μl / min. The running buffer for kinetic experiments was 10 mM sodium phosphate, 130 mM sodium chloride, 0.05% Tween 20, pH 7.1. After each cycle, the surface was regenerated using two 30-second pulses of 10 mM glycine pH 1.5 at a flow rate of 30 μl / min. Sensogram data were double-referenced and then fitted to a 1:1 Langmuir model using Biacore T100 evaluation software v2.0.4 to determine the association rate constant (ka), dissociation rate steady state (kd), and equilibrium dissociation constant (K D ) was decided.

[0320] The results are shown in Table 8. The table compiles data from different experiments. For antibodies with two sets of numbers, each set corresponds to data obtained in a separate experiment. [Table 8] The results show that the presence of a different constant region in the antibody, eg, CD73.10, did not alter the affinity of the antibody for human CD73.

[0321] Internalization of anti-CD73 antibodies was measured in two different assays. A. High-content internalization assay (2-hour routine assay) Anti-CD73 antibody-dependent CD73 internalization in Calu6 cells was tested using an anti-CD73 antibody by assessing cellular expression 2 hours after antibody incubation. Cells (2,000 cells / well) in 20 μl of complete medium (Gibco RPMI Media 1640 supplemented with 10% heat-inactivated fetal bovine serum) were seeded into 384 BD Falcon plates and grown overnight at 37°C, 5% CO2, and 95% humidity. Anti-CD73 antibody was serially diluted in PBS buffer containing 0.2% BSA and added to the cell plate at 5 μl / well. Cells were incubated with the antibody for 2 hours at 37°C, 5% CO2, and 95% humidity, followed by a single wash with PBS buffer. Formaldehyde (final 4% in PBS) was then added to the cell plate at 20 μl / well, and the plate was incubated at room temperature for 10 minutes. Afterwards, all liquid was aspirated, and the cells were washed once with 30 μl of PBS. Detection antibody (2.5 μg / well anti-CD73 Ab CD73.10.IgG2C219S) was added to the fixed cell plate at 15 μg / well. The cells were incubated overnight at 4°C. The next day, the plate was washed twice with PBS buffer, followed by the addition of secondary antibodies containing Alexa-488 goat anti-human and DAPI for 1 hour at room temperature. After washing three times with PBS buffer, the plate was imaged on an Arrayscan Vti (Cellomics, Pittsburgh, PA). IC 50 and Ymax were measured. Ymax was determined by comparison with a 100 nM dose of 11F11 as the internal maximum. All calculations were determined as a percentage of internalization compared to this control, which was set at 100%. The results are provided in Table 9. [Table 9] ND = Not Detected NA = Not applicable

[0322] The results showed that anti-CD73 antibodies with IgG2 hinges had low EC 50 and high Ymax. An internalization kinetics study was performed to evaluate the internalization kinetics. Several cell lines were tested: H2228, HCC15, Calu6, and NCI-H2030. Cells (2,000 cells / well) in 20 μl of complete medium (Gibco RPMI Media 1640 supplemented with 10% heat-inactivated fetal bovine serum) were seeded into 384 BD Falcon plates and grown overnight at 37°C, 5% CO2, and 95% humidity. CD73 antibody was diluted to 10 μg / ml in PBS buffer containing 0.2% BSA, and 5 μl / well was added to the cell plate. Cells were incubated with the antibody for a 0-2 hour time course at 37°C, followed by a single wash with PBS buffer. Cells were then fixed with formaldehyde (final 4% in PBS) for 10 minutes at room temperature and then washed once with 30 μl PBS. The detection antibody (2.5 μg / well anti-CD73 Ab CD73.10.IgG2C219S) was diluted in 0.2% BSA-containing PBS buffer and added to the fixed cell plate at 15 μl / well. The plate was incubated overnight at 4°C. The following day, after washing three times with PBS buffer, secondary antibody Alexa488 goat anti-human and DAPI were added. The cells were stained for 60 minutes at room temperature, washed three times, and then images were acquired using an Arrayscan Vti (Cellomics, Pittsburgh, PA). The results are presented in Figures 1A-J and Tables 10 and 11. The values ​​in Table 10 are derived from the data shown in Figures 1A-J. [Table 10]

[0323] [Table 11] The results show that 11F11 (an IgG2 antibody) was internalized within minutes, reaching a plateau at 30 minutes, while 6E11 (an IgG1 antibody) was internalized more slowly, reaching a plateau at approximately 1 hour (Figures 1A-J). Similarly, 11F11 with an IgG1 constant region was internalized more slowly than 11F11 with an IgG2 constant region. This trend was observed in several cell lines (Tables 10 and 11 and Figures 1A-BJ).

[0324] B. Internalization measured by flow cytometry Anti-CD73 antibody-mediated internalization of CD73 was also examined by flow cytometry. The indicated cells were incubated with 10 μg / mL of the indicated antibody for 30 minutes on ice, washed several times, and transferred to 37°C for the indicated time. Cells were harvested at the same times after the indicated incubation periods. Cells were again stained with the primary antibody (the same antibody used for the initial incubation), followed by an anti-human secondary antibody. Cells were then assayed for CD73 expression by flow cytometry. The results shown in Figure 1E and Table 11 are consistent with those obtained in the internalization assay described above, demonstrating that all antibodies with an IgG2 hinge and CH1 induce rapid and complete internalization. CD73 levels remained low 22 hours after washout, indicating persistent internalization. Similar results, shown in Figure 1F and Table 11, were obtained with the NCI-H292 cell line in which the antibody was maintained in culture (without washout) for the entire incubation period. Similarly, these data demonstrate rapid and significant internalization and sustained downregulation of endogenous CD73.

[0325] Internalization assays were also performed in human SNU-C1 (colon cancer cell line) and NCI-H1437 (non-small cell lung cancer cell line) cells. The results, shown in Figure 1I and J, also demonstrated rapid internalization, reaching maximum levels within 5 h, with maximum internalization levels of CD73.4.IgG2-C219S-IgG1.1f reaching approximately 50% in SNU-C1 and 60% in NCI-H1437 cells. Figure 1G and H show similar kinetics of internalization of CD73.4.IgG2-C219S-IgG1.1f in Calu6 and NCI-H292 cells. For the graph showing the % of internalized CD73, this value was obtained as follows:

number

[0326] [Table 12] Therefore, anti-CD73 antibodies with IgG2 hinges are internalized faster and to a greater extent than anti-CD73 antibodies with IgG1 hinges.

[0327] Example 2: Enhanced agonist activity of GITR antibodies with an IgG2 hinge compared to the same antibody with an IgG1 hinge This example demonstrates that an anti-GITR antibody containing an IgG2 hinge has an increased ability to induce IL-2 and IFN-γ secretion from T cells compared to the same antibody with an IgG1 hinge. In the CHO-OKT3 and 3A9 assays described above, we observed that hybridoma-derived antibodies with IgG2 constant regions stimulated cytokine secretion more potently than the same antibodies in which the heavy chain constant region was converted to IgG1 or effector-less IgG1 (IgG1.1). Therefore, the effect of the IgG2 constant region or hinge was further tested on anti-GITR antibodies in these assays. The heavy chain variable region of the anti-human GITR antibody (SEQ ID NO: 75) was combined with the heavy chain constant region shown in Table 13. The light chain of the anti-GITR antibody comprised SEQ ID NO: 77. Table 13 shows the affiliation of each domain of the constant region. [Table 13] * SEQ ID NO of the full-length heavy chain constant region

[0328] First, the binding affinities of these GITR antibodies were compared with those of a GITR antibody with an IgG1 hinge. The binding affinities of the anti-GITR antibodies to soluble GITR were determined by Biacore as follows: Anti-GITR antibodies were captured on a human kappa-coated chip (approximately 5 kRU; Southernbiotech cat#2060-01), and recombinant human GITR (rHGITR / Fc: R&D systems, CAT#689-GR) was flowed across the chip at concentrations of 500 nM, 250 nM, 125 nM, 62 nM, and 31 nM. The capture concentration of mAb / volume was 2–40 μg / mL (5 μL at 10 μL / min). The antigen association time was 5 min at 15 μL / min, and the antigen dissociation time was 6 min. Regeneration was performed with 50 mM HCl / 50 mM NaOH (12 μL each at 100 μL / min). The results shown in Figure 2 indicate that GITR antibodies with all three IgG2 hinges have similar affinities for activated T cells as GITR antibodies with IgG1 or IgG1.1 constant regions.

[0329] Next, we tested the ability of GITR antibodies bearing an IgG1 constant region or an IgG2 hinge / IgG1 Fc domain to induce IL-2 and IFN-γ secretion from human donor T cells stimulated with anti-CD3 scFv (OKT3)-expressing CHO cells. CHO cells express low levels of OKT3, promoting suboptimal stimulation and allowing observation of agonism by anti-GITR antibodies. CD4+ T cells from donors were stimulated with OKT3-expressing CHO cells and anti-GITR antibodies, and IL-2 and IFN-γ secretion was measured. Experiments were performed as follows: For CD4+ T cell experiments, CD4+ T cells were obtained from human PBMCs using RosetteSep Human CD4+ T Cell Enrichment Cocktail (StemCell Technology #15062) according to the manufacturer's protocol. Anti-CD3 scFv (OKT3)-expressing CHO cells (CHO-OKT3) were washed twice with RPMI medium and irradiated at a dose of 50 KRad. The cells were harvested and cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 55 nM β-mercaptoethanol, 1 mM sodium pyruvate, and 100 U / mL penicillin / streptomycin at 2.5 × 10 5 Resuspended at 2.5 x 10 / mL per well. 4 CHO-OKT3 cells and 1 × 10 5 T cells were seeded in 96-well TC-grade flat-bottom plates (Costar). Cells were incubated with an eight-point, four-fold titration of GITR antibody, starting at 40 μg / mL. Irrelevant hIgG1 was added at 40 μg / mL as an isotype control. A cell-only sample was included to represent baseline activity without any treatment. Supernatants from each sample were obtained on day 3 (for assays with CD4+ T cells only) for IL-2 measurement (BD optEIA Human IL-2 ELISA kit; BD Bioscience #555190) and day 3 for IFN-γ measurement (BD optEIA human IFN-γ ELISA Kit; BD Bioscience #555142). As shown in Figure 3A and B, an antibody with an IgG2 hinge / IgG1 Fc domain (anti-GITR.g2.g1) induced IL-2 and IFN-γ secretion from T cells to a greater extent than an antibody with an IgG1 constant region (anti-GITR.g1). Similar results were obtained with effector-less versions of these constant domains (Figure 3C).

[0330] To further confirm the enhancement of T cell activation with anti-GITR antibodies containing an IgG2 hinge, IL-2 secretion was examined in a different experimental format. In this experiment, the ability of GITR antibodies to induce IL-2 secretion from 3A9-hGITR cells (a murine T cell hybridoma 3A9 cell line ectopically expressing human GITR) was examined as follows: 3A9 murine T cell hybridoma cell line ectopically expressing human GITR (3A9-hGITR) was cultured on anti-CD3 monoclonal antibody-coated plates in the presence of increasing amounts of the indicated antibodies. 5 x 10 4 3A9-hGITR cells were cultured on plates coated with 1 μg / ml anti-CD3 antibody (Clone 145-2C11; BD Biosciences) and treated with the indicated concentrations of antibody for 7 hours. As shown in Figure 4, all antibodies with an IgG2 hinge (anti-GITR.g2, anti-GITR.g2.g1f, and anti-GITR.g2.g1.f) induced IL-2 secretion from 3A9-hGITR cells to a greater extent than their IgG1 constant region-containing counterparts (anti-GITR.g1f and anti-GITR.g1.1f). Collectively, these results suggest that GITR antibodies with an anti-IgG2 hinge and a g1 or g1.1 constant region are more potent than the same antibodies with an IgG1 hinge.

[0331] Example 3: Effect of different hinge / Fc combinations on the size of antibody / antigen complexes As shown in the above examples, anti-CD73 antibodies with IgG2 hinges are better inhibitors of CD73 cellular activity and are better internalized than the same antibodies with IgG1 hinges, and anti-GITR antibodies with IgG2 hinges are more potent agonists than the same antibodies with IgG1 hinges. Based on this observation and the fact that IgG2 hinges are more rigid than IgG1 hinges, it was hypothesized that larger complexes would form between antigens and antibodies with IgG2 hinges compared to antibodies with IgG1 hinges. The following experiments were performed to analyze this hypothesis. The structure and oligomeric state of CD73 / antibody complexes in solution were examined by SEC-MALS and DLS. For these studies, antibodies containing IgG1 or IgG2 constant regions were mixed at various molar ratios with recombinant proteins containing the full-length extracellular domain of human CD73 containing a C-terminal polyhistidine tag (amino acid residues 26–546 of human CD73, designated hCD73-his) or a fragment corresponding to the N-terminal domain of human CD73 (amino acid residues 26–336, designated N-hCD73-his). The oligomeric state of CD73 / antibody complexes was examined by size-exclusion chromatography coupled with an in-line multi-angle light scattering detector (SEC-MALS). Isocratic separation was performed on a Shodex PROTEIN KW-803 column coupled to a Prominence Shimadzu UFLC, with a flow rate of 0.5 mL / min in a buffer containing 200 mM KHPO, 150 mM NaCl, pH 6.8, containing 0.02% sodium azide (0.1 μm filtered). Samples were injected onto the column using a SIL-20AC Prominence Shimadzu autosampler, and data were analyzed on a Prominence SPD-20AD diode array UV / vis spectrophotometer followed by a Wyatt miniDAWN TMData were acquired from three sequentially connected online detectors: a TREOS Multi-Angle Light Scattering Detector, followed by a Wyatt Optilab T-rEX Refractive Index Detector. Data were collected and analyzed using Astra (Wyatt) and Labsolutions (Shimadzu) software.

[0332] Dynamic light scattering (DLS) experiments were performed in 384-well plates on a Wyatt DynaPro plate reader at 25°C. Experimental parameters were 20 acquisitions of 5 seconds per measurement, measurements were recorded in quadruplicate, and the mean and standard deviation were reported. The intensity autocorrection function was fitted using the "regularization" algorithm in Dynamics software (Wyatt Technologies). A summary of the SEC-MALS and DLS data is provided in Figures 6 and 7. Analysis of the antibodies alone shows, for each antibody, retention times (approximately 16-17 min), masses (140-150 kDa), and hydrodynamic radii (5.0-5.4 nm) typical of monomeric monoclonal antibodies. The data for the hCD73-his protein are consistent with the protein adopting the predicted dimeric structure in solution. In particular, the mass determined from the SEC-MALS data (120 kDa) is consistent with that predicted for the CD73-his dimer (117 kDa) and inconsistent with that predicted for the hCD73-his monomer (58.5 kDa). The N-hCD73 data are consistent with the recombinant N-domain protein being monomeric in solution (SEC-MALS measured mass = 38 kDa compared to predicted monomer mass = 35.0 kDa), as expected since the region of the full-length CD73 extracellular domain responsible for protein dimerization is contained within the C-terminal domain without contribution from N-domain residues. An equimolar mixture of an antibody and N-hCD73-his elutes as a single species by SEC with a shorter retention time than either the antibody or N-hCD73-his alone and with a large hydrodynamic radius (Rh) by DLS, consistent with the formation of a complex. MALS data indicate a mass of approximately 210 kDa for these complexes. This is consistent with one N-hCD73-his molecule binding to each of the two Fab domains of an antibody, forming a 1:2 antibody:N-hCD73-his complex.

[0333] SEC-MALS data for mixtures of anti-CD73 antibodies and hCD73-his dimers showed that the mixture eluted faster than hCD73-his or the antibody alone, suggesting the formation of a complex. Comparison with data from mAbs containing the same variable region but different constant domains showed that the elution time of the complex between hCD73-his and mAbs containing an IgG2 constant domain (IgG2-C219S, IgG2-C219S-IgG1.1f) was faster than that of the complex between hCD73-his and mAbs containing an IgG1.1f constant domain. Furthermore, the MALS-determined mass of the complex between hCD73-his and mAbs containing an IgG2 constant domain was larger than that of the complex between hCD73-his and mAbs containing an IgG1 constant domain. DLS data further showed that the hydrodynamic radius of the complex between hCD73-his and mAbs containing an IgG2 constant domain was larger than that of the complex between hCD73-his and mAbs containing an IgG1 constant domain. For example, SEC-MALS and DLS data for CD73.4 with three different constant domains (IgG2-C219S, IgG2-C219S-IgG1.1f, or IgG1.1f) are shown in Figure 5. Here, it can be seen that the complex of hCD73-his and CD73.4 containing the IgG2 constant domain has a shorter retention time (Figure 5A), a larger hydrodynamic radius (Figure 5B), and a larger MALS-determined mass (Figure 5C) compared to the complex of hCD73-his and CD73.4-IgG1.1f. Based on the MALS masses, a schematic model of the structure and stoichiometry of the hCD73-his and antibody complexes is shown in Figure 5D, where complexes containing CD73.4-IgG1.1f formed primarily small 2:2 (peak 1 = ∼550 kDa) or 4:4 mAb / CD73 dimeric complexes (peak 2 = ∼1300 kDa), whereas CD73.4-IgG2-C219S or CD73.4-IgG2-C219S-IgG1.1f formed much larger complexes with hCD73-his (>3000 kDa), the exact structure and stoichiometry of which could not be confidently modeled. Collectively, the SEC-MALS and DLS data indicate that larger complexes are formed between hCD73-his and mAbs containing an IgG2 hinge region (IgG2-C219S or IgG2-C219S-IgG1.1f) compared with those containing an IgG1 hinge region (IgG1.1f).

[0334] Example 4: CH1 of IgG2 isotype further improves antibody-mediated CD73 internalization Additional internalization assays were performed in Calu6 and H292 cells to further explore the role of isotype in internalization. Internalization assays were performed as described in Examples 1A and 1B (flow cytometry protocol without antibody washout steps), and antibodies of various hybrid isotypes, as shown in Table 14, were maintained in culture at 10 μg / mL throughout the incubation period. For flow cytometry experiments, the method of Example 1B was adapted for 96-well plates (rather than 48-well plates) and high-throughput analysis of 50,000 cells / well.

[0335] [Table 14]

[0336] FcγR binding was shown to be as expected for each construct, ie, FcγR binding is driven by the lower hinge / CH2 region. The results are shown in Figures 8A, B, and C and in Tables 15 and 16. The data shown in Table 15 was generated using the same protocol as described in Example 1B (without washing out the antibody). The data shown in Table 16 was generated using the same protocol as described in Example 1A. [Table 15]

[0337] [Table 16]

[0338] Figures 8A-C and Tables 15 and 16 show that antibodies with hinge and CH1 domains of the IgG2 isotype are most efficient at driving CD73 internalization, while antibodies with IgG1 hinge and CH1 domains correspond to the lower curve in the figure, i.e., have a lower degree of internalization. Furthermore, antibodies with only a hinge from IgG2 show increased internalization compared to human IgG1 hinges. Therefore, antibodies with hinge and CH1 domains of the IgG2 isotype have superior internalization properties compared to antibodies with the IgG1 isotype. Therefore, the anti-CD73 antibody mAb-CD73.4-IgG2CS-IgG1.1f (containing an IgG2 hinge and IgG2 CH1 domain with a C219S substitution) induces rapid internalization depending on the cell line tested. 1 / 2 The T ranges from a few minutes to less than an hour. Most cell lines tested have T values ​​of less than 10 minutes. 1 / 2 Near-complete internalization was induced in some cell lines, and most tested showed at least a 50% reduction in surface CD73 expression, which generally reached maximal levels by 5 hours, although in some cases at much shorter times.

[0339] Example 5: IgG2 CH1 enhances GITR Ab-induced IL-2 secretion by CD4+ T cells This example demonstrates that a CH1 domain of the IgG2 isotype enhances anti-GITR antibody-induced T cell activity compared to an antibody having a CH1 domain of the IgG1 isotype. The same modified heavy chain constant region used in Example 4 was combined with the variable region of the anti-GITR antibody (from Example 2). Donor CD4+ T cells were incubated with OKT3-scFv-expressing CHO cells and various anti-GITR antibodies, and secreted IL-2 levels were measured, as described in Example 2. The results shown in Figure 9 indicate that whole anti-GITR antibodies with an IgG2 isotype hinge plus an IgG2 isotype CH1 domain are more effective at inducing IL-2 secretion from CD4+ T cells than those with an IgG1 hinge and CH1. Therefore, this example shows that the presence of an IgG2 hinge and an IgG2 CH1 domain in an agonist anti-GITR antibody further enhances the agonist activity of the antibody compared to the same antibody without the hinge and / or CH1 domain of the IgG2 isotype. Antibodies with both the hinge and CH1 domain of the IgG2 isotype have a stronger agonist effect than antibodies with an IgG2 isotype hinge but a different CH1. Furthermore, this example shows that antibodies with a CH1 domain from IgG2 have stronger agonist activity than antibodies with a CH1 domain from IgG1 isotype. Antibodies with a hinge from IgG2 and a CH1 domain from IgG1 have stronger agonist activity than antibodies with a CH1 and hinge of the IgG1 isotype.

[0340] Example 6: Involvement of certain amino acid residues in IgG2 CH1 and hinge in improving antibody-mediated CD73 internalization An anti-CD73 antibody (CD73.4) having the heavy chain constant region shown in Table 17 was prepared and tested in the antibody-mediated CD73 internalization assay as described above. [Table 17]

[0341] The results shown in Figure 10 provide the following information on the context of CD73 internalization: The CH2 domain appears to have no effect, as shown below. a) The slight difference in internalization capacity was observed in the form "AY" (IgG2 hinge ERKCCVECPPCPAP PVAG (SEQ ID NO: 8)) and an antibody of the form "KH" (ERKCCVECPPCPAP ELLGG (SEQ ID NO: 22) observed in (sets 5, 6 and 7); ○ b) CH2 exchange is equivalent to wild-type G1 or G2 (sets 5 and 6); and c) Residue 237 does not affect internalization: neither the addition of a “G” residue to the IgG2 hinge nor the deletion of the C-terminal “G” to the IgG1 hinge affected internalization (set 9).

[0342] This suggests that the CH2 domain does not affect internalization (ie, the CH2 domain can be from IgG1 or IgG2). • Exchange of the CH1 regions shown in set 3 in IgG1 (KRGEGSSNLF; KRGEGS; SNLF; ITNDRTPR and SNLFPR) into IgG2 provides little benefit, i.e., internalization remains similar to IgG1 (see set 3); • The impact of exchanging the CH1 regions shown in Set 4 (RKEGSGNSFL; RKEGSG; NSFL; TIDNTRRP and NSFLRP) in IgG2 with those of IgG1 is variable: exchange of NSFL has no effect, but the other two regions (RKEGSG and RP) are involved (see Set 4). Based on the results of Sets 3 and 4, there is an interaction between the CH1 region and the hinge, and the RKEGSG and RP regions appear to be more important than the NSFL region; ● The hinge region affects internalization, i.e., IgG2 hinges provide better internalization than IgG1 hinges (see Sets 7 and 8). Furthermore, IgG1 with a deletion (G1-delta-hinge) improves internalization over IgG1. IgG2 with a deletion (G2-delta-hinge) provides an internalization level similar to that of the IgG2 hinge. This indicates that the hinge region affects internalization, and this effect is enhanced by IgG2 CH1 (G2-G1-G2-G2-AY is equivalent to G1-G2-G1-G1-AY); IgG2.4(C220S) shows similar or reduced internalization compared to IgG2.3(C219S). IgG2.3 / 4(C219S / C220S) shows much reduced internalization compared to IgG2.3 or IgG2.4 alone (see Set 10). This suggests that the internalization of antibodies with an IgG2 hinge and C219S is nearly the same as that of an IgG2 hinge with C220S, and both are much better than an IgG2 hinge with both C219S and C220S; • IgG2.5 (C131S mutation) has reduced internalization compared to constructs with C131 (see sets 1, 6 and 7).

[0343] Therefore, these results indicate that the CH1 domain and hinge are both involved in antibody-mediated CD73 internalization, and that antibodies possessing IgG2 sequences from these domains internalize more efficiently than antibodies possessing these regions from IgG1.

[0344] Example 7: Antibodies with IgG2 hinge and / or CH1 domains form high molecular weight complexes CD73.4 antibodies with heavy chain constant regions shown in Table 14 were tested for high molecular weight complex formation by SEC-MALS and DLS experiments as described in Example 3. Two of the 16 antibodies in this study were previously tested: CD73.4-IgG1.1f, CD73.4-IgG2-C219S (also referred to as CD73.4-IgG2.3), and CD73.4-IgG2-C219S-IgG1.1f (also referred to as CD73.4-IgG2.3G1.1f-KH). SEC-MALS and DLS data for the antibodies alone showed retention times, masses, and hydrodynamic radii for each antibody that were typical of monomeric monoclonal antibodies. Equimolar complexes of each antibody (5.5 μM) with hCD73-his (5.5 μM) showed delayed retention times for the total complex compared to the antibody or hCD73-his alone, indicating complex formation. An overlay of the SEC chromatogram data from each of the 16 complexes is shown in Figure 11A. The chromatogram data can be resolved into four distinct peaks, which are shown in Figure 11B. Peak 1 contains the largest species, with a MALS-determined mass suggesting a complex of greater mass equivalent than the 4:4 hCD73-his:mAb complex. Peak 2 contains a species with a MALS-determined mass suggesting a complex of approximately 2:2 hCD73-his:mAb complex. Peak 3 contains a minor species with a low signal and MALS-determined mass suggesting a complex of approximately 1:1 hCD73-his:mAb complex. Peak 4 corresponds to the elution of mAb alone, with a MALS-determined mass consistent with free antibody. To quantify the relative amounts of each species, four peaks for each signal were integrated: Peak 1 (<12.9 min), Peak 2 (12.9–15.1 min), Peak 3 (15.1–16.7 min), and Peak 4 (16.7–19.3 min). The integration also included an additional integration range, designated Peak 5 (>19.3 min), which may account for some lower molecular weight species, but this was found to be negligible (<3.5% of the total complex). The percentages of each species from this integral are summarized in Table 18. Total complex c contained a similar small percentage of peak 3 (approximately 6-9%), while the amounts of the other peaks were variable. Most notably, the total complex of antibody containing hCD73-his and the CH1 domain from hIgG1 had a significantly greater percentage of small complexes (peak 2), while the one with the CH1 domain from hIgG2 had a greater percentage of large complexes (peak 1) (Table 18 and Figure 11C).This suggests the importance of not only the hinge region but also the CH1 domain in higher-order complex formation.

[0345] [Table 18]

[0346] Example 8: Fc receptor binding to antibodies with engineered constant domains This example shows that antibodies with modified heavy chain constant regions containing the CH1 and hinge of IgG2 bind to FcγR when they contain the CH2 and CH3 domains of IgG1. In addition to antigen binding via the variable domain, antibodies can bind to Fc gamma receptors (FcgR) through interactions with the constant domain. These interactions mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Effector function activity is high for the IgG1 isotype, but is very low or absent for IgG2 and IgG4 due to the low affinity of these isotypes for FcgR. Furthermore, the effector function of IgG1 can be modified by mutating amino acid residues within the constant region to alter FcgR affinity and selectivity. Antibody binding to Fc gamma receptors (FcγR or FcgR) was tested using biosensor technologies, including Biacore surface plasmon resonance (SPR) and Fortebio Biolayer Interferometry (BLI). SP testing was performed at 25°C on a Biacore T100 instrument (GE Healthcare). Fab fragments from a mouse anti-6xHis antibody were immobilized to a CM5 sensor chip using EDC / NHS to a density of approximately 3000 RU. Various his-tagged FcgRs (7 μg / ml) were captured via their C-terminal his tags using a 30-second contact time at 10 μl / min. Binding of 1.0 μM antibody was assessed in a running buffer of 10 mM NaPO, 130 mM NaCl, 0.05% p20 (PBS-T), pH 7.1. The FcgRs used in these experiments included CD64 (FcgRI), CD32a-H131 (FcgRIIa-H131), CD32a-R131 (FcgRIIa-R131), CD32b (FcgRIIb), CD16a-V158 (FcgRIIIa-V158), CD16b-NA1 (FcgRIIIb-NA1), and CD16B-NA2 (FcgRIIIb-NA2). BLI experiments were performed on a Fortebio Octet RED instrument (Pall, Fortebio) at 25°C in 10 mM NaPO, 130 mM NaCl, 0.05% p20 (PBS-T), pH 7.1. Antibodies were captured from undiluted expression supernatants on protein A-coated sensors followed by binding of 1 μM hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158 or 0.1 μM hCD64 analytes.

[0347] First, we investigated the binding of modified IgG1 Fc domains containing substitutions 267E (SE) and S267E / L328F (SELF) and antibodies containing various combinations of mutations P238D, P271G, H268D, A330R, G237D, and E233D, designated V4, V7, V8, V9, and V12, to various targets. Binding of these antibodies was tested by Biacore SPR and compared with IgG1f, IgG2.3 (IgG2-C219S), and IgG4.1 (IgG4-S228P) antibodies, as well as an IgG1.1f antibody engineered for reduced binding to all FcgRs. The results, shown in Figure 12, show the predicted FcgR binding properties of IgG1f, IgG2.3, and IgG4.1 and mutant IgG1 antibodies, including increased CD32a-H131, CD32a-R131, and CD32b binding for SE and SELF, and increased selectivity for CD32b over CD32a-H131 and CD32a-R131 for the V4, V7, V8, V9, and V12 mutants, Figure 12. The next set of constructs was used to engineer effector function into the otherwise effector-function-negative IgG2 isotype. For this study, the above mutations were introduced into the IgG2.3 constant region or an IgG2.3 / IgG1f hybrid designated IgG2.3G1-AY (Table 19). The antibodies were expressed at small scale in the supernatant and tested for binding to FcgR using Fortebio Octet BioLayer Interferometry biosensor technology. Because the antibodies are present at low concentrations in the supernatant, experiments were performed using a protein A-coated sensor to capture the antibodies from the supernatant, followed by binding of the FcgR analyte in solution. Purified and supernatant control IgG1f antibodies, including wild-type IgG1, SE, P238D, V4, and V12 antibodies, were also included for comparison; each of these control antibodies exhibited the predicted FcgR binding properties (Figure 13). The IgG2.3 antibodies also exhibited the predicted binding profile, with appreciable binding only to CD32a-H131. However, all mutations introduced into IgG2.3 to introduce the S267E, L328F, P238D, P271G, H268D, A330R, G237D, or E233D mutations failed to reproduce the FcgR affinity of the corresponding engineered IgG1 mAb (Figure 13). In contrast, the IgG2.3G1-AY construct, while retaining the IgG2.3 CH1 and hinge regions, was able to fully preserve the FcgR-binding properties of wild-type IgG1. Furthermore, all IgG2.3G1-AY mutants, including S267E, L328F, P238D, P271G, H268D, A330R, G237D, and E233D, exhibited FcgR-binding properties comparable to those of the IgG1-version mAb with the same mutations (Figure 13). This demonstrates the successful engineering of antibodies with the CH1 and hinge regions of IgG2 combined with the effector functions of wild-type or mutant IgG1.

[0348] [Table 19]

[0349] This engineering strategy was further explored by producing other antibodies shaped like IgG2.3G1-AY, IgG2.3G1-AY-S267E (IgG2.3G1-AY-V27), as well as IgG2-B form variants (IgG2.5G1-AY and IgG2.5G1-AY-V27) and hybrid antibodies containing various combinations of other IgG1 and IgG2 constant domains, and testing the binding of these antibodies to anti-his Fab-captured his-tagged FcgR using Biacore SPR technology. Consistent with the Octet supernatant data, SPR data showed that the IgG2.3G1-AY and IgG2.3G1-AY-V27 antibodies had FcgR-binding properties comparable to IgG1f and IgG1f-S267E, respectively, despite containing the CH1 and hinge regions of the A form IgG2 antibody (IgG2.3) (Figures 14A and B and Table 20). Similar data were obtained using the IgG2.5G1-AY and IgG2.5G1-AY-V27 antibodies, demonstrating successful engineering of a B-form IgG2 antibody (containing the C131S mutation, designated IgG2.5) with IgG1f or modified IgG1f-like effector function. Data from several other antibodies with different variable regions, including the IgG2.3G1-AY, IgG2.3G1-AY-V27, IgG2.5G1-AY, or IgG2.5G1-AY-V27 constant regions, indicate that this engineering strategy is applicable to other antibodies, regardless of the variable domain (Figures 14A and B and Table 20). Other constructs that displayed IgG1f-like FcgR binding properties were IgG1-G2.3G1-AY and IgG1 delta THT, whereas several modified constant region constructs, including IgG2.3G1-KH, IgG2.5G1-KH, IgG2.3+THT, IgG2.5+THT, and IgG2.3+GGG constructs, failed to retain IgG1f-like FcgR binding properties (Figures 14A and B and Table 20).

[0350] [Table 20] [Table 21] [Table 22]

[0351] Together, these data indicate that sequences immediately C-terminal to the conserved CPPCPAP motif in the hinge region contribute to FcgR-mediated effector function, while the CH1 and upper portion of the hinge of an antibody can be replaced with IgG2 or modified IgG2 sequences, potentially combining the effector functions of IgG1 and modified IgG1 with the superior internalization or signaling properties of antibodies containing the IgG2 CH1 and / or hinge region.

[0352] Example 9: GITR agonist Ab internalization is enhanced with antibodies possessing IgG2 hinge and CH1 domains To induce GITR expression, cells were incubated with 20 ng / ml anti-CD3 and 1000 ng / ml CD28 for 72 hours at 37°C. Alternatively, large batches of activated CD4+ T cells were prepared using a three-step culture protocol: CD4+ T cells were stimulated with plate-bound CD3 (1.5 μg / ml) supplemented with 1 μg / ml soluble CD28 for 72 hours at 37°C, expanded in the presence of 20 μg / ml IL2 for 14 days, and finally subjected to an additional round of activation with the addition of 10 μg / ml PHA, 2 μg / ml IL2, and 1 μg / ml CD28 for 72 hours at 37°C. Stimulated T cells were seeded into 384-well PDL imaging plates for 2 hours to allow cell attachment, cooled to 4°C for 15 minutes, and then Alexa 488-labeled GITR antibody was added separately for 1 hour. Finally, the plate was imaged by HCS and the results were quantified as total intensity per cell. Three different GITR antibodies were monitored using the T cell activation method described above: the G1 isotype, an inactive (IgG1.1) isotype that cannot bind to Fc receptors, and the GITR.6 antibody as a chimera with an IgG2 hinge instead of the IgG1 hinge.

[0353] GITR antibody-induced internalization was assessed using an Alexa quenching assay format in CD3-stimulated CD4+ T cells. Freshly obtained CD4+ T cells were incubated as described above to induce GITR expression. After stimulation, cells were resuspended in fresh medium and plated for the internalization assay as follows: Cells were incubated with antibody as described above, washed with warm medium, and incubated at 37°C for the indicated times before fixation and quenching. Internalized antibody was measured as the increase in fluorescence above the small, unquenchable signal observed at time 0, and total fluorescence was normalized to the "unquenched" control that was initially bound to the cells. As shown in Figure 15, GITR ligation resulted in rapid internalization, peaking between 30 and 60 minutes for each antibody tested, while the control antibody was found to maintain localization to the plasma membrane. The results demonstrate that the IgG2 hinge region induces GITR ligation-induced internalization.

[0354] To further probe the detailed mechanisms of internalization and associated kinetics, we analyzed antibody endocytosis and delivery to early endosomal compartments. In this experiment, cells were subjected to pulse-chase analysis with unlabeled antibodies. Cells were permeabilized by fixation, stained with the early endosomal marker EEA1 (cell signaling technology), washed, and then detected with an Alexa Fluor-488-conjugated anti-rabbit secondary antibody (EEA1) and an Alexa Fluor-647-conjugated anti-human antibody (GITR). Plates were imaged with an Opera confocal system using a 60x water-immersion objective. Results showed a clear separation of membrane-bound anti-GITR antibody staining and intracellular EEA1 signal. By warming the cultures, we detected clustering of certain antibodies, which appeared to colocalize with endosomal proteins. Quantification of endosomal colocalization was performed using HCS Studio software, and the results were plotted as the ratio of colocalized pixel intensity to total staining (Figure 16). Colocalization of GITR antibody and early endosomes was most pronounced at 30 minutes. At this time point, GITR.6.G2.G1f showed a higher fraction of colocalization than GITR.6.G1f antibody. The colocalization results are consistent with the observations made using the alexa quenching method described above, supporting the model and suggesting that the G2 hinge may be more favorable than the G1 for inducing GITR internalization.

[0355] Example 10: GITR agonist Ab signaling in T cell receptor-activated CD4+ and CD8+ T cells is enhanced with antibodies bearing IgG2 hinge and CH1 domains To further examine the mechanism of anti-GITR agonist antibodies, several signaling pathways involved in T cell activation, such as the NFkB and p38 signaling pathways, were monitored. CD4+ and CD8+ T cells from a healthy donor (M6576) were activated with plate-coated 0.4 μg / ml anti-CD3 and 0.4 μg / ml anti-CD28. Three days later, cells were harvested and seeded into 384-well imaging plates for signaling activation. Cells were allowed to settle on the plate for 2 hours, then treated with GITR antibody for 15 minutes. Signaling events were stopped by adding 10% final formaldehyde to the assay plate. Cells were then permeabilized and stained with a phosphoro-p65 NFKB antibody for signaling detection. As shown in Figure 17, the GITR.6.G2 and GITR.6.G2.G1f antibodies had a higher signaling response compared to GITR.6.G1f in both CD4+ and CD8+ T cells. While there is no direct evidence linking internalization to signaling pathway activation, it is interesting to note that the G2 isotype appears to improve both antibody functional activity against GITR.6 compared to IgG1. To quantify the signaling activity of each antibody, the EC 50 Both Emax and Emax were calculated as both parameters are important for capturing the full range of signaling events. The response level of GITR.6.G2.G1f was chosen to be the 100% control, to which all other antibodies were normalized. Potency (EC) was measured for both CD4+ and CD8+ T cell populations activated by anti-CD3 and anti-CD28 antibodies, as shown in Table 21. 50 There was a range of activity of GITR antibodies in terms of both potency (ECmax%) and efficacy (Emax%). GITR.6.G2, GITR.6.G2.G1f, and GITR.6.G1f had similar potencies (ECmax%) in the approximately 10 nM range. 50 ), however, efficacy (Emax) varied considerably between the different isotypes, suggesting that the G1 antibody does not signal as efficiently as the G2 or chimeric isotypes.

[0356] [Table 23]

[0357] To further confirm whether the signaling differences between GITR.6.G2 and GITR.6.G2.G1f compared to GITR.6.G1f were limited to NFkB signaling alone or applied to other signaling events as well, p38 MAPK signaling readouts were examined. As shown in Figure 18, the GITR.6.G2 and GITR.6.G2.G1f antibodies had enhanced signaling responses compared to the GITR.6.G1f antibody in a CD4+ cell p38 MAPK activation assay. Therefore, the superior signaling activity of the GITR.6.G2 isotype compared to the G1 isotype was not limited to NFkB signaling. In addition to enhanced agonist activity and internalization, it has also been shown that modified heavy chain constant regions can confer enhanced ADCC (e.g., against agonists of stimulatory receptors), as well as provide novel activities to antibodies. For example, changing the constant heavy chain domain of an antibody that binds to an inhibitory cell surface molecule and blocks the inhibitory activity of the cell surface molecule (antagonist) to a modified heavy chain constant region described herein resulted in an antibody that lost its ability to be antagonistic, but rather was endowed with agonist activity (inhibitory activity).

[0358] Example 11: Identification of disulfide bonds in IgG2.3 and IgG2.5 constructs The disulfide bond structure of antibodies containing constant domains IgG2.3 (A form), IgG2.3G1 (A form) and IgG2.5 (B form) was confirmed as correct by comparison of non-reduced versus reduced Lys-C digestion. Antibody samples were digested with Lys-C, which selectively cleaves peptide bonds on the carboxyl-terminal side of lysine (K, Lys) residues. Peptides from the digest were separated using a Waters ACQUITY BEH C18 column, 1.7 μm, 2.1 × 150 mm, reverse-phase HPLC column, and detected with an ultraviolet (UV) detector at 214 nm and a Thermo LTQ mass spectrometer.

[0359] Lys-C enzyme digestion and disulfide bond reduction: To a vial containing 100 μg of antibody sample, 120 μL of denaturing buffer was added to obtain a 3.7 M GuHCl, 0.2 M Tris pH 7.0 solution. The mixture was incubated at 55°C for 30 minutes. Protein alkylation was performed by adding 1 μL of 50 mM iodoacetamide to the above solution, followed by a 30-minute incubation at room temperature in the dark. The alkylated sample was diluted with 80 μL of dH2O, and Waco Lys-C was added at a 1:10 enzyme-to-substrate ratio. The antibody was digested overnight at room temperature in the dark. After digestion, a 100 μL aliquot was removed from the Lys-C digested sample, and 10 μL of 0.5 M DTT was added. The sample was incubated at room temperature for 1 hour to reduce disulfide bonds.

[0360] The results obtained are as follows: Disulfide structure of IgG2.3 and IgG2.3G1 antibodies (A form): In the heavy chain Fab region, Cys22 (H) is bonded to Cys98 (H), and Cys151 (H) is bonded to Cys207 (H). In the heavy chain Fc region, Cys265 (H) is bonded to Cys325 (H), and Cys371 (H) is bonded to Cys429 (H). In the light chain Fab region, Cys23 (L) is bonded to Cys88 (L), and Cys134 (L) is bonded to Cys194 (L). The C-terminus of the light chain, Cys214 (L), is bonded to the heavy chain at Cys138 (H). The hinge region of the heavy chain contains three cysteine ​​residues, Cys227(H), Cys230(H), and Cys233(H), which provide three interchain disulfide bonds. The most likely bonds are Cys227(H) to Cys227(H), Cys230(H) to Cys230(H), and Cys233(H) to Cys233(H), which is the correct theoretical disulfide configuration for the IgG2 A form.

[0361] Disulfide structure of IgG2.5 antibody (B form): In the heavy chain Fab region, Cys22 (H) is bonded to Cys98 (H), and Cys151 (H) is bonded to Cys207 (H). In the heavy chain Fc region, Cys264 (H) is bonded to Cys324 (H), and Cys370 (H) is bonded to Cys428 (H). In the light chain Fab region, Cys23 (L) is bonded to Cys88 (L), and Cys134 (L) is bonded to Cys194 (L). The heavy chain hinge region contains four cysteine ​​residues: Cys226 (H), Cys227 (H), Cys230 (H), and Cys233 (H). The C-terminal Cys214(L) of the light chain binds to a cysteine ​​residue in the heavy chain in the hinge region, and the remaining three cysteine ​​residues provide three interchain disulfide bonds. The most likely bonds are Cys214(L) and Cys226(H), followed by Cys227(H) and Cys227(H), Cys230(H) and Cys230(H), and Cys233(H) and Cys233(H), which is the correct theoretical disulfide configuration for the IgG2 B form. Furthermore, the disulfide bonds in the hinge region were confirmed using electron transfer dissociation (ETD)-induced tandem mass spectrometry using an ion trap mass spectrometer.

[0362] Example 12: Involvement of certain amino acid residues in IgG2 CH1 and hinge in improving GITR agonism in T cells An anti-GITR antibody (GITR.6) having the heavy chain constant region shown in Table 17 was prepared and tested in an IL-2 production assay as described in Example 2, except that supernatants were obtained at 40 hours instead of 48 hours. The results shown in Figures 20A to 20D were almost identical to the CD73 internalization results obtained with an anti-CD73 antibody having the same heavy chain constant region as used in this example (see Figure 10).

[0363] Example 13: Elimination of effector function with the P238K mutation The variable region of the antibody was fused to an IgG1 Fc, P238K (SEQ ID NO: 198), which differs from wild-type IgG1 Fc by a single amino acid residue. With this single mutation, the antibody exhibited loss of effector function and essentially no detectable binding signal to the low-affinity FcγRs hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, or hCD16b-NA2 (see data in Example 14). Furthermore, the antibody with IgG1 P238K exhibited reduced binding affinity to the high-affinity FcγR CD64 (see data in Example 14). The antibody's binding to CD64 showed a faster cleavage rate (dissociation constant) than that of an antibody with a wild-type IgG1 constant domain. The lack of effector function of IgG1 Fc with the P238K mutation (SEQ ID NO: 198) was also confirmed in the antibody variant. Therefore, for example, a human IgG1 Fc containing the heavy chain constant region amino acid sequence of SEQ ID NO: 198 and having a single mutation (P238K) can be used in any antibody for which effector function is undesirable.

[0364] Example 14: Elimination of effector function by P238K and additional mutations Additional antibodies were generated with Fc mutations to further reduce effector function, preferably both ADCC and CDC. As shown in Table 22, mutants were created to further reduce FcR binding. In particular, as shown above, P238K eliminates detectable FcR binding other than to CD64, and therefore the goal was to combine P238K with additional mutations to reduce CD64 binding. Mutations were tested in the context of IgG1, IgG2.3 and IgG2.5 isotypes, and IgG2.3G1 isotype formats. The Fc used in these antibodies comprises one of the amino acid sequences having SEQ ID NOs: 234-245 and 247-262. The locations of the mutations are shown in FIG. Binding of human FcγRs to antibodies was tested by surface plasmon resonance using a Biacore 8K system (GE Healthcare). For these studies, Protein A was immobilized to a density of approximately 3000 RU on flow cells 1-4 of a CM5 sensor chip using standard ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking in a running buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant p20. Purified antibodies (10 μg / mL) or expression supernatants (diluted to approximately 10 μg / mL) were captured onto Protein A surfaces to a density of approximately 1000–1200 RU, and binding of FcγR analytes was tested in a running buffer consisting of 10 mM NaPO, 130 mM NaCl, 0.05% P20, and buffer (PBS-T) pH 7.1 at 25°C with a flow rate of 20 μL / min and a 120-second association and dissociation time. Data were analyzed using Biacore 8K evaluation software by measuring the measured binding response (%Rmax) as a percentage of the theoretical maximum binding response for each antibody, based on the level of captured antibody assumed to be 100% fractional activity, and taking into account only the unglycosylated protein mass, as follows: For comparison of FcγR binding of various molecules, SPR binding data were analyzed by calculating the maximum binding response (%Rmax) as a percentage of the theoretical maximum binding response, as generally shown in Equation 1:

number

number

[0365] "%Rmax analysis" is particularly useful for evaluating the binding of "low affinity" FcgRs, e.g., hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, hCD16a-F158, hCD16b-NA1, and hCD16b-NA2, which have relatively fast association and dissociation kinetics and affinities near or below test analyte concentrations (1 micromolar (μM)), such that surface saturation is generally not achieved under these conditions. In contrast, the "high affinity" FcgR hCD64 binds with higher affinity and slower dissociation kinetics than other FcgRs, particularly to IgG1 and IgG4; therefore, these isotypes generally saturate the hCD64 surface at micromolar analyte concentrations, making affinity discrimination using %Rmax more difficult. For these interactions, differences between antibodies can be easily observed by comparing dissociation kinetics in sensorgram data.

[0366] The results are shown in Table 22 and example sensorgram data are provided in Figures 21A-L. [Table 24]

[0367] As shown in Table 22 and Figure 22, the combination mutants exhibited very weak FcR binding. Enrichment of the L235 mutation to the P238K isotype reduced CD64 binding to levels similar to IgG1.3f. L235E was superior to the L235A mutation in reducing CD64 binding. Addition of the P238K mutation to IgG2 (IgG2.3-P238K) resulted in a completely inactive isotype with no detectable binding to any of the FcR proteins. The mutations also showed similar trends in the IgG1 and IgG2.xG1 formats. The K322A mutation, which reduced c1q binding (CDC activity) and was added to some constructs, had minimal effect on FcR binding, and therefore, the effect of K322A was not observed to any significant extent.

[0368] Example 14: Elimination of effector functions with IgG1.3 Fc This example is described in Examples 2 and 3 of the co-filed and commonly owned PCT application entitled "MODIFIED IgG1 Fc DOMAINS AND ANTI-CD40 DOMAIN ANTIBODY FUSIONS THEREWITH." This example demonstrates that antibodies or polypeptides having IgG1.3 Fc essentially lack binding to CD16, CD32a, CD32b, and CD64. This has also been observed when IgG1.3 Fc is bound to the variable domain of an anti-TIM3 antibody (see WO 2018 / 013818). IgG1.3 was derived from "IgG1.1" Fc ("IgG1.1" is an IgG1 with L234A, L235E, G237A, A330S, and P331S substitutions) by removing A330S and P331S, thereby retaining three of the five mutations, i.e., L234A, L235E, and G237A. It was a surprising discovery that the deletion of A330S and P331S in IgG1.1 Fc did not significantly affect the inactivity of this Fc. The following are examples of FcγR binding measurements of IgG1.1 and IgG1.3 (and other Fc for comparison) containing antibodies and fusion proteins, comparing the inactivity of IgG1.1 and IgG1.3 in antibodies and in non-antibody proteins. The materials and methods used in this example include the following:

[0369] FcgR binding SPR: FcgR binding was measured in vitro using purified FcγR and Biacore TM Surface plasmon resonance (SPR) can be used to measure this. Two methods were used here. One method tests the binding of purified antibodies or dAb-Fc proteins to His-tagged FcgR proteins (FcgR-His ("FcgR" is used interchangeably with "FcγR") captured on immobilized Fab fragments of anti-His antibodies). These experiments were performed using Biacore™. TM T100 or Biacore TMTests were performed on a T200 instrument (GE Healthcare) at 25°C. Fab fragments from a mouse anti-6xHis antibody (produced in-house) were immobilized to a CM5 sensor chip using standard ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking to approximately 3000 resonance units (RU) in a running buffer of 10 millimolar (mM) HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant p20 (HBS-EP+). All remaining tests were performed at pH 7.1 using a running buffer of 10 mM NaPO4, 130 mM NaCl, 0.05% p20 (PBS-T). Various FcgR proteins containing a C-terminal 6x polyhistidine tag (generated in-house) were captured to this surface using a contact time of 30 seconds (s) at 10 μl / min (typically using an FcgR-His protein concentration of approximately 7 μg / ml). Various concentrations of purified antibody or dAb-Fc protein are tested for binding using, for example, an association time of 120 seconds at 30 μl / min and a dissociation time of 120 seconds at 30 μl / min. The FcgR proteins tested in these studies include the "high affinity" FcgR hCD64 (hFcgRI), and the "low affinity" FcgR hCD32a-H131 (FcgRIIa-H131), hCD32a-R131 (FcgRIIa-R131), hCD32b (FcgRIIb), hCD16a-V158 (FcgRIIIa-V158), hCD16a-F158 (FcgRIIIa-F158), hCD16b-NA1 (FcgRIIIb-NA1), and hCD16b-NA2 (FcgRIIIb-NA2).

[0370] To quantitatively analyze binding responses and compare FcgR binding of various molecules, SPR binding data can generally be analyzed by calculating the maximum binding response as a percentage of the theoretical maximum binding response (%Rmax) as shown in Equation 1:

number

number

[0371] "%Rmax analysis" is particularly useful for evaluating the binding of "low affinity" FcgRs, e.g., hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, hCD16a-F158, hCD16b-NA1, and hCD16b-NA2, which have relatively fast association and dissociation rates and affinities near or below test analyte concentrations (1 micromolar (μM)), such that surface saturation is generally not achieved under these conditions. In contrast, the "high affinity" FcgR hCD64 binds with higher affinity and slower dissociation rates than other FcgRs, particularly to IgG1 and IgG4; therefore, these isotypes generally saturate the hCD64 surface at micromolar analyte concentrations, making affinity discrimination using %Rmax more difficult. For these interactions, differences between antibodies can be easily observed by comparing dissociation rates in sensorgram data.

[0372] A second SPR assay to test the interaction of antibody or dAb-Fc proteins with FcgR proteins is the Protein A capture method. These experiments were also performed using Biacore TM T100 or Biacore TMThese experiments were performed on a T200 instrument (GE Healthcare) at 25°C. For these experiments, Protein A was immobilized onto flow cells 1-4 of a CM5 sensor chip using standard ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking at a density of approximately 3000 RU in a running buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant p20. Antibody or dAb-Fc proteins (typically 3-10 μg / ml) were captured on the Protein A surface, and FcγR analyte binding was tested, for example, using a 120-second association time and a 180-second dissociation time at a flow rate of 30 μL / min in a running buffer consisting of 10 mM NaPO4, 130 mM NaCl, 0.05% surfactant p20, and buffer (PBS-T), pH 7.1, at 25°C. Protein A capture assays can also be used to analyze unpurified supernatants containing antibody or dAb-Fc molecules. For this analysis, antibody or dAb-Fc proteins can be captured from undiluted supernatants or supernatants diluted with running buffer. To quantitatively analyze the binding response and compare FcgR binding of various molecules, SPR binding data are analyzed by calculating %Rmax using Equation 1 above, where the analyte is purified FcgR protein and the ligand is the captured antibody or dAb-Fc protein. In addition to %Rmax analysis, quantitative analysis of binding kinetics and affinity can be performed by testing titrations of FcgR analytes for binding to Protein A-captured antibodies or dAb-Fc proteins. For example, 3:1 serial dilutions of FcgR can be titrated down from 10 μM to 0.15 nM (hCD64) or 1.5 nM (all other FcgRs). These kinetic data can be analyzed by Biacore™. TM Kinetic and affinity values ​​can be obtained using the T200 evaluation software to fit a 1:1 Langmuir model or a steady-state binding model.

[0373] dAb-Fc: The dAb-Fc tested in this example are shown in Table 23. In these sequences, the single variable domain 3h56-269 residues are amino acids 1 to 118 (underlined). The linker AST is double underlined. [Table 25] The sequences of IgG1.1f and IgG1.3f shown in Table 23, each beginning with "EPK" (ie, the sequences of SEQ ID NOs: 77 and 78), are identical to the sequences of SEQ ID NOs: 83 and 248, respectively, which also begin with EPK.

[0374] Control mAb: A control monoclonal antibody (1F4) was also generated using similar Fc domain mutations. The sequences of the individual chains are shown in Table 24, including the sequence of a portion of the 1F4 heavy chain (SEQ ID NO: 268), including the variable and CH1 regions. This sequence is underlined in the heavy chain sequence (SEQ ID NOs: 269-275). The heavy and light chain sequence pairs for each 1F4 mAb variant are shown in Table 25. [Table 26] [Table 27]

[0375] [Table 28]

[0376] Results: dAb-Fc molecules were generated with mutations in the Fc domain to reduce FcgR binding. Specifically, the anti-CD40 domain antibody 3h56-269 was produced using the following Fc domain variants: IgG1.1f, IgG1.3f, and IgG1-D265A. In 3h-56-269-IgG1.1f (SEQ ID NO: 77), 3h-56-269-IgG1.3f (SEQ ID NO: 78), and 3h-56-269-IgG1-D265A (SEQ ID NO: 79), amino acids 1-116 are the 3h-56-269 dAb, amino acids 117-119 are a linker, and amino acids 120-351 are the Fc domain.

[0377] Each of these dAb-Fc fusion proteins, as well as 3h56-269-IgG4.1 and 3h56-269-CT, were analyzed by Biacore TM High affinity binding to purified human CD40 monomer (hCD40 monomer, produced in-house) was confirmed as measured by SPR. As shown in Table 26, K D Values ​​range from 7.3 nM to 11.5 nM for the various Fc variants. Each of the dAb-Fc molecules also binds to human CD40 with high avidity, as measured by SPR using hCD40-Fc on the surface of a sensor chip and the dAb-Fc molecule as a soluble analyte in solution, where data from 250 nM and 25 nM dAb-Fc analyte injections were fitted to a 1:1 Langmuir model to determine the apparent K as influenced by avidity. D Value (K Dapparent ) was estimated as <1 nM for all dAb-Fc. See Table 26. [Table 29] * 3h-56-269-CT was expressed and purified from UCOE-CHO cells.

[0378] The FcgR binding properties of dAb-Fc molecules and various control monoclonal 1F4 antibodies were characterized by SPR. Initial assays involved binding of 1 μM or 10 μM dAb-Fc or a human-IgG1f antibody control (1F4-IgG1f) to an anti-His Fab-captured FcgR-His surface. These data are shown in Table 27. [Table 30]

[0379] In other assays, FcgR analytes (1 μM or 10 μM) were tested for binding to Protein A captured dAb-Fc surfaces (data shown in Table 28) and antibody surfaces (data shown in Table 29). [Table 31]

[0380] [Table 32]

[0381] Based on the binding response, or lack thereof, in these experiments, a subset of high affinity dAb-Fc / FcgR or Ab / FcgR interactions and strongest binding responses were selected for kinetic / affinity characterization using analyte titration (FcgR analytes bound to Protein A capture antibody or dAb-Fc). These data are shown in Table 30. [Table 33]

[0382] Collectively, these FcgR binding SPR data indicate that IgG1f and IgG4.1 isotype molecules have significantly higher FcgR affinity across the entire FcgR compared to the modified Fc variants IgG1-D265A, IgG1.1f, IgG1.3f, or CT molecules. Among the modified Fc variants, hCD64 binding affinity was higher for 3h56-269-CT (K D =4.6 nM) was the strongest, and 3h56-269-IgG1-D265A (K D = 62 nM), and 3h56-269-IgG1.1f and 3h56-269-IgG1.3f were the weakest, and their affinities were too weak to be quantified under the test conditions (K D >5 μM, which is half of the highest analyte concentration tested). All other FcgR interactions (hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, hCD16b-NA2) for IgG1-D265A, IgG1.1f, IgG1.3f, and CT variants also showed reliable K D It was too weak to obtain a value (K D >5 μM). However, differences in relative binding responses could be observed in the %Rmax data. For example, the IgG1-D265A variant has a stronger binding response to hCD32a-H131 than the IgG1.1f, IgG1.3f, or CT variants (Table 28). In contrast, the IgG1.1f and IgG1.3f variants have a stronger binding response to hCD32a-R131 than the IgG1-D265A and CT variants (Table 28).

[0383] IgG1.3-containing fusion proteins or antibodies were evaluated by DSC, icIEF and mass spectrometry. Materials and methods are described below. Differential Scanning Calorimetry: DSC experiments were performed on a MicroCal VP-Capillary DSC instrument (Malvern Instruments, Malvern, UK) in 10 mM NaPO, 130 mM NaCl pH 7.1. 1 mg / ml dAb-Fc or antibody samples were tested using a scan range of 10-110 °C and a scan rate of 90 °C / hr. Data were analyzed using MicroCal-Origin 7.0 software.

[0384] Imaged capillary isoelectric focusing: icIEF experiments performed with ProteinSimple iCE3 TM The assay was performed on a ProteinSimple System (ProteinSimple, San Jose, CA). For these studies, dAb-Fc or antibody samples, typically at a concentration of 2 mg / ml, were mixed with a carrier ampholyte mixture consisting of 2 M urea, 0.35% methylcellulose, 1% Pharmalyte 5-8, 3% Pharmalyte 8-10.5, and pI markers 5.85 and 10.10 to a final protein concentration of 0.20 mg / mL and analyzed using a 1-minute prefocusing time at 1.5 kV and a 10-minute focusing time at 3 kV.

[0385] Mass Spectrometry: For mass spectrometric (mass spec) analysis, samples were reduced using 100 mM DTT and N-deglycosylated using peptide:N-glycosidase (FPNGaseF). The liquid chromatography-mass spectrometry (LC / MS) system used was a Waters Synapt® G2 (Waters Corporation, Milford, MA) with a Waters Acquity® UPLC (ultra-performance liquid chromatography). The UPLC column was a Waters Acquity® BEH (ethylene-bridged hybrid particles) C4 (2.1 × 150 mm, 300 Å, 1.7 μm particles). The gradient was 10% to 38% (mobile phase B) at a 200 μL / min flow rate for 10 min. Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetonitrile. The column temperature was 60 °C. Data analysis was performed using Waters MassLynx. TM It was performed manually with the aid of software; spectral deconvolution was performed with the MaxEnt1 algorithm.

[0386] Accelerated Stability Studies: Accelerated stability studies were performed by first exhaustively dialyzing the dAb-Fc molecules against the target formulation buffer at 4°C. Samples were collected, concentrated using Amicon® Ultra Centrifugal Filter Units (Merck KgaA, Germany), and adjusted to various target concentrations in dialysis buffer. These samples were incubated at various temperatures, typically 4°C, 25°C, 32°C, and / or 40°C, for several weeks, and aliquots were removed and analyzed by analytical size exclusion chromatography. Analytical size exclusion chromatography was performed using a Shodex TM Analysis was performed on an Agilent 1260 HPLC using a K403-4F column (Showa Denko America, Inc., New York, NY) with a mobile phase of 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3, at a flow rate of 0.3 ml / min.

[0387] Results - Differential Scanning Calorimetry: DSC can be used to measure the thermal stability of proteins. The best fit Tm values ​​are summarized in Table 31. [Table 34]

[0388] Based on the characteristic thermal denaturation profiles of IgG Fc domains, the Fc CH3 domain transition of 3h56-269-IgG4.1 was assigned as a transition with a midpoint (Tm) value of 69.6 °C, and the Fc CH3 domains of various IgG1 molecules were assigned as transitions with Tm around 82-83 °C. The denaturation of the dAb domain and CH2 domain for dAb-Fc was assigned as a transition below 65 °C, which is the onset of thermal denaturation (T onset ), both the shape of the unfolding transitions and the best-fit Tm values ​​differed between the various constructs. For example, the thermal transitions of the dAb and CH2 domains of 3h56-269-IgG4.1 were seen as single overlapping or cooperative transitions, with a Tm of 62.8°C. The unfolding profiles of the dAb and CH2 domains of 3h56-269-IgG1-D265A, 3h56-269-IgG1.1f, and 3h56-269-IgG1.3f all conformed to a more asymmetric transition, best represented by two transitions with Tm values ​​of approximately 56-63°C. 3h56-269-CT had the lowest T onset It began to denature at approximately 40°C, with a broad thermal transition and the lowest compatible Tm values, Tm1 = 55.4°C and Tm2 = 60.4°C.

[0389] Results - Imaging Capillary Isoelectric Focusing (icIEF): Imaging capillary isoelectric focusing (icIEF) can be used to characterize sample homogeneity or heterogeneity. The ability to produce a homogeneous product is another important viability criterion. As a result, during the discovery and optimization of novel protein therapeutics, various analytical methods are utilized to characterize and quantify sample heterogeneity and to select the most homogeneous molecules. The charge profiles of the dAb-Fc molecules were characterized by icIEF. The data are shown in Figure 23. The icIEF profiles of 3h56-269-IgG4.1 (Figure 23A), 3h56-269-IgG1.1f (Figure 23E), and 3h56-269-IgG1.3f (Figure 23F) were all relatively simple, consisting of a clear main peak with an area of ​​69–86%, respectively, and two to four less abundant charge variants. This icIEF profile resembles the typical profile obtained with antibodies. The main peak of 3h56-269-IgG1-D265A (Figure 23D) is somewhat less abundant (49%), with correspondingly high levels of acidic variants and at least six detectable species. In contrast, the profile of 3h56-269-CT (Figure 23B) is highly heterogeneous, consisting of at least 16 different species and lacking a clear main peak. The icIEF profile of 3h56-269-CT expressed in a different cell line (UCOE-CHO) was equally heterogeneous (FIG. 23C), but the distribution of charge variants differed considerably from the HEK293-expressed material.

[0390] Results - Mass Spectrometry: Typical glycosylation of the Fc domain of IgG or Fc-containing proteins is a mixture of G0F, G1F, and some G2F species. Other glycoforms, such as sialylated or nonfucosylated forms, are generally found at much lower abundance or at undetectable levels. Mass spectrometry experiments were performed to characterize the glycosylation profile of the dAb-Fc protein and compare it to a control antibody with similar Fc mutations. The data are shown in Table 32. [Table 35] Mass spectrometry data for the control ant...

Claims

1. 1. A composition for treating a subject, comprising an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a modified heavy chain constant domain comprising a human IgG heavy chain constant domain; where: (a) the amino acid at position 238 is lysine (K); (b) the modified heavy chain constant domain has a reduced effector function relative to the same IgG heavy chain constant domain in which the amino acid at position 238 is a proline (P); (c) the IgG heavy chain constant domain is a human IgG1 heavy chain constant domain and comprises the amino acid sequence of SEQ ID NO: 198; and where: (i) binding to CD32a or CD16a by the antibody or antigen-binding fragment thereof is reduced compared to an antibody having the same IgG heavy chain constant domain in which amino acid 238 is P; and (ii) the binding of the antibody or antigen-binding fragment thereof to CD64 has a faster dissociation rate compared to an antibody having a wild-type IgG1 constant region; composition.

2. The composition of claim 1, wherein the binding of the antibody or its antigen-binding fragment to CD32a is lower than that of an antibody having the same IgG heavy chain constant domain in which the amino acid at position 238 is P.

3. The composition of claim 1 or 2, wherein the subject has cancer.

4. 3. The composition of claim 1 or 2, wherein the subject has an autoimmune disease and the antibody or antigen-binding fragment thereof inhibits an immune response in the subject.

5. The composition of any of claims 1 to 4, wherein the modified heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

198.

6. (a) the antibody or antigen-binding fragment thereof is an antigen-binding fragment attached to a modified heavy chain constant region; or (b) the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain linked to a modified heavy chain constant domain and a light chain variable domain linked to a light chain constant domain; The composition according to any one of claims 1 to 5.

7. The composition of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is a full-length antibody, wherein the heavy and light chains are each full-length.

8. The composition of any one of claims 1 to 7, wherein the effector function of the antibody or antigen-binding fragment thereof is approximately the same as that of an IgG2 antibody.

9. The composition of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof has a lower binding affinity to a low-affinity FcγR compared to an antibody having wild-type IgG1.

10. The composition of claim 9, wherein the antibody or antigen-binding fragment thereof has no detectable binding to a low affinity FcγR.

11. The composition of claim 9 or 10, wherein the low affinity FcγRs are hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, and hCD16b-NA2.

12. 12. The composition of claim 11, wherein the antibody or antigen-binding fragment thereof has no detectable binding to the low affinity FcγRs hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, or hCD16b-NA2 at an antibody concentration of 10 μM.

Images