BISPECIFIC ANTIBODIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No.

62/644,281, filed March 16, 2018, which is hereby incorporated by reference herein in its entirety.

FIELD

[0002] The present disclosure generally relates to multispecific binding agents, such as bispecific antibodies, methods of making the multispecific binding agents, and compositions comprising the multispecific binding agents.

BACKGROUND

[0003] Antibodies and/or antibody-based agents are now commonly seen as a therapeutic option for a wide variety of diseases and disorders. Currently there are at least 70 antibodies approved in the United States and/or European Union, with large numbers of new molecules in preclinical studies and clinical trials. However, there is a continual search for new, better, and safer therapeutic agents within the research and clinical communities.

[0004] A naturally occurring antibody is monospecific and binds to one epitope or antigen. Bispecific antibodies combine specificities of two antibodies and have the capability to bind different antigens or epitopes. Many technical hurdles have hampered development of bispecific antibodies over the years. However, rapidly advancing technologies have enabled the progression of a wide variety of formats and strategies for the engineering and the production of bispecific antibodies (for a review, see for example, Brinkmann and Kontermann, Mabs , 2017,

9: 182-212). Although the bispecific antibody field is moving forward at a fast pace, there are few bispecific antibodies that have been approved as therapeutics. There is still a need for better methods to efficiently produce functional and stable bispecific antibodies.

BRIEF SUMMARY

[0005] The present disclosure generally relates to multispecific binding agents, such as bispecific antibodies, and methods for making the agents. In one aspect, the multispecific binding agents, such as bispecific antibodies, comprise a modified heavy chain and a modified light chain leading to formation of an alternative disulfide bond and resulting in more efficient association of a specific heavy chain/light chain pair. In certain embodiments, the multispecific binding agents ( e.g ., bispecific antibodies) further comprise modifications in the CH3 region and/or the Fc region of each heavy chain to promote heterodimer formation and/or alter functional capabilities of the antibody.

[0006] In some embodiments, a multispecific binding agent is a bispecific antibody. In some embodiments, a bispecific antibody comprises: (a) a first IgGl heavy chain polypeptide associated with a first light chain polypeptide, wherein the first heavy chain/light chain pair comprises an alternative interchain disulfide bond; and wherein (i) a CH1 and hinge region of the first heavy chain polypeptide comprises a cysteine at position 126 and a non-cysteine amino acid at position 220 according to EU numbering, and the constant region of the first light chain polypeptide comprises a cysteine at position 124 (kappa chain) or position 125 (lambda chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering; or (ii) a CH1 and hinge region of the first heavy chain polypeptide comprises a cysteine at position 173 and a non-cysteine amino acid at position 220 according to EEG numbering; and the constant region of the first light chain polypeptide comprises a cysteine at position 162 (kappa chain) and a non cysteine amino acid at position 214 according to Kabat/EEG numbering; and (b) a second IgGl heavy chain polypeptide associated with a second light chain polypeptide. In some

embodiments, a bispecific antibody comprises: (a) a first IgGl heavy chain polypeptide associated with a first light chain polypeptide, wherein the first heavy chain/light chain pair comprises an alternative interchain disulfide bond; and wherein the CH1 and hinge region of the first heavy chain polypeptide comprises a cysteine at position 126 and a non-cysteine amino acid at position 220 according to EU numbering, and the constant region of the first light chain polypeptide comprises a cysteine at position 124 (kappa chain) or position 125 (lambda chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering; and (b) a second IgGl heavy chain polypeptide associated with a second light chain polypeptide. In some embodiments, a bispecific antibody comprises: (a) a first IgGl heavy chain polypeptide associated with a first light chain polypeptide, wherein the first heavy chain/light chain pair comprises an alternative interchain disulfide bond, and wherein the CH1 and hinge region of the first heavy chain polypeptide comprises a cysteine at position 173 and a non-cysteine amino acid at position 220 according to EU numbering; and the constant region of the first light chain polypeptide comprises a cysteine at position 162 (kappa chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering; and (b) a second IgGl heavy chain polypeptide associated with a second light chain polypeptide. In some embodiments, the constant region of the first light chain polypeptide comprises a cysteine at position 124 (kappa chain). In other embodiments, the constant region of the first light chain polypeptide comprises a cysteine at position 125 (lambda chain). In some embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an arginine (R), a lysine (K), a glutamic acid (E), or an aspartic acid (D) at position 220. In some embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an arginine (R) at position 220. In some embodiments, the constant region of the first light chain polypeptide comprises a glutamic acid (E), an aspartic acid (D), an arginine (R), or a lysine (K) at position 214. In some embodiments, the constant region of the first light chain polypeptide comprises a glutamic acid (E) at position 214. In some

embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an arginine (R) at position 220 and the constant region of the first light chain polypeptide comprises a glutamic acid (E) at position 214. In some embodiments, the first light chain and the second light chain are both kappa chains. In some embodiments, the first light chain and the second light chain are both lambda chains. In some embodiments, the first light chain is a kappa chain and the second light chain is a lambda chain. In some embodiments, the first light chain is a lambda chain and the second light chain is a kappa chain. In some embodiments, the two heavy chain polypeptides each comprise a heavy chain variable region, a CH1 domain, a hinge region, and an Fc region. In some embodiments, the two heavy chain polypeptides each comprise a heavy chain variable region and an IgGl constant region. In some embodiments, the two heavy chain polypeptides each comprise a heavy chain variable region and an IgG2 constant region. In some embodiments, the two light chain polypeptides each comprise a light chain variable region and a light chain constant region. In some embodiments, the IgGl constant region is from an IgGl allotype. In some embodiments, the CH1 and hinge region of an IgGl is from allotype Glml, nGlm2, Glm3, Glml7,l, Glml7,l,2, Glm3,l, or Glml7.

[0007] In another aspect, the disclosure provides a multispecific binding agent ( e.g ., a bispecific antibody) comprising: (a) a first IgGl heavy chain polypeptide comprising a CH1 domain and a hinge region, and a first light chain polypeptide comprising a light chain constant region, wherein the first IgGl heavy chain polypeptide is associated with the first light chain polypeptide thereby forming a first heavy chain/light chain pair, wherein the first heavy chain/light chain pair comprises an alternative interchain disulfide bond than that found between a heavy chain polypeptide and light chain polypeptide in a wild type IgGl antibody; and wherein (i) the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:5 and comprises a cysteine at position 126 and a non-cysteine amino acid at position 220 according to EU numbering, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8 and comprises a cysteine at position 124 (kappa chain) or position 125 (lambda chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering; or (ii) the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:6 and comprises a cysteine at position 173 and a non-cysteine amino acid at position 220 according to EU numbering; and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:9 and comprises a cysteine at position 162 (kappa chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering; and (b) a second IgGl heavy chain polypeptide associated with a second light chain polypeptide.

[0008] In another aspect, the disclosure provides a multispecific binding agent ( e.g ., a bispecific antibody) comprising (a) a first IgGl heavy chain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-103 (or 9-103) of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 9 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine; and (b) (i) a first light chain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12, wherein the amino acid corresponding to position 17 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is a cysteine and the amino acid corresponding to position 107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is not a cysteine; or (ii) a first light chain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acids 1-106 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: l3, SEQ ID NO:l4, or SEQ ID NO: l5, wherein the amino acid corresponding to position 18 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is a cysteine and the amino acid corresponding to position 105 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is not a cysteine. In some embodiments, a multispecific binding agent ( e.g ., a bispecific antibody) comprises (a) a first IgGl heavy chain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-103 (or 9-103) of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 56 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine; and (b) a first light chain polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to amino acids 1-107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12, wherein the amino acid corresponding to position 55 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:l 1, or SEQ ID NO: 12 is a cysteine and the amino acid corresponding to position 107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is not a cysteine.

[0009] In another aspect, the disclosure provides a multispecific binding agent (e.g., a bispecific antibody) comprising a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region and the first light chain variable region when paired form a first antigen-binding site that binds to an target antigen, wherein the first heavy chain variable region is either (i) directly linked or (ii) linked via a linker to a first polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to amino acids 1-103 of SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 9 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine; and wherein the light chain variable region is either (i) directly linked or (ii) linked via a linker to a second polypeptide comprising (i) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to amino acids 1-107 of any one of SEQ ID NO:2, SEQ ID NO: l 1, or SEQ ID NO: 12, wherein the amino acid corresponding to position 17 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is a cysteine and the amino acid corresponding to position 107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is not a cysteine, or (ii) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to amino acids 1-106 of any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, wherein the amino acid corresponding to position 18 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is a cysteine and the amino acid corresponding to position 105 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is not a cysteine. In some embodiments, a multispecific binding agent ( e.g ., a bispecific antibody) comprises a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region and the first light chain variable region when paired form a first antigen-binding site that binds to an target antigen, wherein the first heavy chain variable region is either (i) directly linked or (ii) linked via a linker to a first polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to amino acids 1-103 of SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 56 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine; and wherein the light chain variable region is either (i) directly linked or (ii) linked via a linker to a second polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to amino acids 1-107 of any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12, wherein the amino acid corresponding to position 55 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is a cysteine and the amino acid corresponding to position 107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is not a cysteine.

[0010] In some embodiments, the constant region of the first heavy chain polypeptide and the constant region of the second heavy chain polypeptide are each modified at one or more amino acid positions wherein the modifications promote heterodimer formation. In some embodiments, an Fc region of the first heavy chain polypeptide and an Fc region of the second heavy chain polypeptide are each modified at one or more amino acid positions relative to a wild type human IgGl Fc region. In some embodiments, the modifications promote heterodimer formation. In some embodiments, an Fc region of the first heavy chain polypeptide and an Fc region of the second heavy chain polypeptide are modified, relative to a wild type human IgGl Fc region, using a knobs-into-holes technique using methods known to those skilled in the art. In some embodiments, an Fc region of the first heavy chain polypeptide comprises a tryptophan (W) at position 366 and an Fc region of the second heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407. In some embodiments, an Fc region of the first heavy chain polypeptide comprises a tyrosine (Y) at position 366 and an Fc region of the second heavy chain polypeptide comprises a threonine (T) at position 407. In some embodiments, an Fc region of the first heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407, and an Fc region of the second heavy chain polypeptide comprises a tryptophan (W) at position 366. In some embodiments, an Fc region of the first heavy chain polypeptide comprises a threonine at position 407 and an Fc region of the second heavy chain polypeptide comprises a tyrosine (Y) at position 366. In some embodiments, an Fc region of the first heavy chain polypeptide and an Fc region of the second heavy chain polypeptide are modified, relative to a wild type human IgGl Fc region, based upon electrostatic effects using methods known to those skilled in the art.

[0011] In some embodiments, the two heavy chain polypeptides each comprise a heavy chain variable region, a CH1 region, a CH2 region, and a CH3 region. In some embodiments, the two light chain polypeptides each comprise a light chain variable region and a light chain constant region. In some embodiments, the CH3 region of the first heavy chain polypeptide and the CH3 region of the second heavy chain polypeptide are each modified at one or more amino acid positions. In some embodiments, the modifications promote heterodimer formation. In some embodiments, the CH3 region of the first heavy chain polypeptide and the CH3 region of the second heavy chain polypeptide are modified using a knobs-into-holes technique using methods known to those skilled in the art. In some embodiments, the CH3 region of the first heavy chain polypeptide comprises a tryptophan (W) at position 366 and the CH3 region of the second heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407. In some embodiments, the CH3 region of the first heavy chain polypeptide comprises a tyrosine (Y) at position 366 and the CH3 region of the second heavy chain polypeptide comprises a threonine (T) at position 407. In some embodiments, the CH3 region of the first heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407, and the CH3 region of the second heavy chain polypeptide comprises a tryptophan (W) at position 366. In some embodiments, the CH3 region of the first heavy chain polypeptide comprises a threonine (T) at position 407 and the CH3 region of the second heavy chain polypeptide comprises a tyrosine (Y) at position 366. In some embodiments, the CH3 region of the first heavy chain polypeptide and the CH3 region of the second heavy chain polypeptide are modified based upon electrostatic effects using methods known to those of skill in the art.

[0012] In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the CH2, CH3, or Fc region of the first heavy chain polypeptide and the CH2, CH3, or Fc region of the second heavy chain polypeptide comprise additional amino acid modifications. In some embodiments, the additional amino acid modifications affect the functional capabilities of the bispecific antibody. In some embodiments, the additional amino acid modifications result in decreased antibody-dependent cell-mediated cytotoxicity (ADCC). In other embodiments, the additional amino acid modifications result in decreased complement-dependent cytotoxicity (CDC). In other embodiments, the additional amino acid modifications result in decreased ADCC and CDC. In other embodiments, the additional amino acid modifications result in increased ADCC. In other embodiments, the additional amino acid modifications result in increased CDC. In other embodiments, the additional amino acid modifications result in increased ADCC and CDC.

[0013] In some embodiments, the bispecific antibody is a monoclonal antibody. In some embodiments, the bispecific antibody is a chimeric antibody. In some embodiments, the bispecific antibody is a humanized antibody. In some embodiments, the bispecific antibody is a human antibody.

[0014] In some embodiments, the bispecific antibody is isolated. In some embodiments, the bispecific antibody is substantially pure.

[0015] In another aspect, the disclosure provides compositions comprising a multispecific binding agent described herein. In some embodiments, the disclosure provides compositions comprising a bispecific antibody described herein. [0016] In another aspect, the disclosure provides pharmaceutical compositions comprising a multispecific binding agent described herein and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides pharmaceutical compositions comprising a bispecific antibody described herein and a pharmaceutically acceptable carrier.

[0017] In another aspect, the disclosure provides an isolated polynucleotide or polynucleotides encoding a multispecific binding agent described herein. In some embodiments, the disclosure provides an isolated polynucleotide or polynucleotides encoding a bispecific antibody described herein.

[0018] In another aspect, the disclosure provides a vector or vectors comprising a

polynucleotide or polynucleotides encoding a multispecific binding agent described herein. In some embodiments, the disclosure provides a vector or vectors comprising a polynucleotide or polynucleotides encoding a bispecific antibody, described herein. In some embodiments, the vector is an expression vector.

[0019] In another aspect, the disclosure provides a host cell or cells comprising a

polynucleotide or polynucleotides encoding a multispecific binding agent described herein. In some embodiments, the disclosure provides a host cell or cells comprising a polynucleotide or polynucleotides encoding a bispecific antibody described herein.

[0020] In another aspect, the disclosure provides a host cell or cells comprising a vector or vectors comprising a polynucleotide or polynucleotides encoding a multispecific binding agent described herein. In some embodiments, the disclosure provides a host cell or cells comprising a vector or vectors comprising a polynucleotide or polynucleotides encoding a bispecific antibody described herein.

[0021] In another aspect, the disclosure provides a method of producing a multispecific binding agent, the method comprising culturing a host cell under conditions wherein a polynucleotide or polynucleotides (or expression vector or expression vectors) encoding a multispecific binding agent described herein is expressed. In some embodiments, the disclosure provides a method of producing a bispecific antibody, the method comprising culturing a host cell under conditions wherein a polynucleotide or polynucleotides (or expression vector or expression vectors) encoding a bispecific antibody described herein is expressed. In some embodiments, a host cell produces a bispecific antibody described herein. [0022] In another aspect, methods of making a bispecific antibody described herein are provided. In some embodiments, a method comprises making a bispecific antibody, wherein the bispecific antibody comprises a first IgGl heavy chain/light chain pair of polypeptides and a second IgGl heavy chain /light chain pair of polypeptides, wherein the first heavy chain polypeptide and the first light chain polypeptide are each modified to promote an alternative interchain disulfide bond, and wherein the method comprises: (a) substituting a cysteine at position 126 and substituting a non-cysteine amino acid at position 220 of the CH1 region of the first heavy chain polypeptide according to EU numbering, and substituting a cysteine at position 124 (kappa chain) or position 125 (lambda chain) and substituting a non-cysteine amino acid at position 214 (kappa or lambda chain) of the constant region of the first light chain according to Kabat/EU numbering; or (b) substituting a cysteine at position 173 and substituting a non cysteine amino acid at position 220 of the CH1 region of the first heavy chain polypeptide according to EEG numbering, and substituting a cysteine at position 162 (kappa chain) and substituting a non-cysteine amino acid at position 214 of the constant region of the first light chain according to Kabat/EEG numbering. In some embodiments, a method comprises substituting a cysteine at position 126 and substituting a non-cysteine amino acid at position 220 of the CH1 region of the first heavy chain polypeptide according to EU numbering, and substituting a cysteine at position 124 (kappa chain) or position 125 (lambda chain) and substituting a non-cysteine amino acid at position 214 (kappa or lambda chain) of the constant region of the first light chain according to Kabat/EU numbering. In some embodiments, a method comprises substituting a cysteine at position 173 and substituting a non-cysteine amino acid at position 220 of the CH1 region of the first heavy chain polypeptide according to EU numbering, and substituting a cysteine at position 162 (kappa chain) and substituting a non cysteine amino acid at position 214 of the constant region of the first light chain according to Kabat/EU numbering.

[0023] In some embodiments of a method of making a bispecific antibody described herein, the non-cysteine amino acid substituted at position 220 of the CH1 region of the first heavy chain polypeptide is an arginine (R), a lysine (K), a glutamic acid (E), or an aspartic acid (D). In some embodiments, the non-cysteine amino acid substituted at position 220 of the CH1 region of the first heavy chain polypeptide is an arginine (R). In some embodiments of a method of preparing a bispecific antibody described herein, the non-cysteine amino acid substituted at position 214 of the first light chain polypeptide is a glutamic acid (E), an aspartic acid (D), an arginine (R), or a lysine (K). In some embodiments, the non-cysteine amino acid substituted at position 214 of the first light chain polypeptide is a glutamic acid (E). In some embodiments, the non-cysteine amino acid substituted at position 220 of the CH1 region of the first heavy chain polypeptide is an arginine (R) and the non-cysteine amino acid substituted at position 214 of the first light chain polypeptide is a glutamic acid (E). In some embodiments of a method of preparing a bispecific antibody described herein, the first light chain and the second light chain of the bispecific antibody are both kappa chains. In some embodiments of a method of preparing a bispecific antibody described herein, the first light chain and the second light chain of the bispecific antibody are both lambda chains. In some embodiments of a method of preparing a bispecific antibody described herein, the first light chain is a kappa chain and the second light chain of the bispecific antibody is a lambda chain. In some embodiments of a method of preparing a bispecific antibody described herein, the first light chain is a lambda chain and the second light chain of the bispecific antibody is a kappa chain.

[0024] In some embodiments of a method of preparing a bispecific antibody described herein, the two heavy chain polypeptides of the bispecific antibody each comprise a heavy chain variable region, a CH1 domain and an Fc region. In some embodiments of a method of preparing a bispecific antibody described herein, the two light chain polypeptides of the bispecific antibody each comprise a light chain variable region and a light chain constant region. In some embodiments of a method of making a bispecific antibody described herein, an Fc region of the first heavy chain polypeptide and an Fc region of the second heavy chain polypeptide of the bispecific antibody are each modified at one or more amino acid positions relative to a wild type human IgGl Fc region. In some embodiments, the modifications promote heterodimer formation. In some embodiments of a method of making a bispecific antibody described herein, an Fc region of the first heavy chain polypeptide and an Fc region of the second heavy chain polypeptide of the bispecific antibody are modified, relative to a wild type human IgGl Fc region, using a knobs-into-holes technique. In some embodiments of a method of making a bispecific antibody described herein, an Fc region of the first heavy chain polypeptide comprises a tryptophan (W) at position 366 and an Fc region of the second heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407. In some embodiments of a method of making a bispecific antibody described herein, an Fc region of the first heavy chain polypeptide comprises a tyrosine (Y) at position 366 and an Fc region of the second heavy chain polypeptide comprises a threonine (T) at position 407. In some embodiments of a method of making a bispecific antibody described herein, an Fc region of the first heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407, and an Fc region of the second heavy chain polypeptide comprises a tryptophan (W) at position 366. In some embodiments of a method of making a bispecific antibody described herein, an Fc region of the first heavy chain polypeptide comprises a threonine (T) at position 407 and an Fc region of the second heavy chain polypeptide comprises a tyrosine (Y) at position 366. In some embodiments of a method of making a bispecific antibody described herein, an Fc region of the first heavy chain polypeptide and an Fc region of the second heavy chain polypeptide of the bispecific antibody are modified, relative to a wild type human IgGl Fc region, based upon electrostatic effects.

[0025] In some embodiments of a method of making a bispecific antibody described herein, the two heavy chain polypeptides each comprise a heavy chain variable region, a CH1 region, a CH2 region, and a CH3 region. In some embodiments of a method of making a bispecific antibody described herein, the two light chain polypeptides each comprise a light chain variable region and a light chain constant region. In some embodiments of a method of making a bispecific antibody described herein, the CH3 region of the first heavy chain polypeptide and the CH3 region of the second heavy chain polypeptide are each modified at one or more amino acid positions. In some embodiments, the modifications promote heterodimer formation. In some embodiments of a method of making a bispecific antibody described herein, the CH3 region of the first heavy chain polypeptide and the CH3 region of the second heavy chain polypeptide are modified using a knobs-into-holes technique. In some embodiments of a method of making a bispecific antibody described herein, the CH3 region of the first heavy chain polypeptide comprises a tryptophan (W) at position 366 and the CH3 region of the second heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407. In some embodiments of a method of making a bispecific antibody described herein, the CH3 region of the first heavy chain polypeptide comprises a tyrosine (Y) at position 366 and the CH3 region of the second heavy chain polypeptide comprises a threonine (T) at position 407. In some embodiments of a method of making a bispecific antibody described herein, the CH3 region of the first heavy chain polypeptide comprises a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407, and the CH3 region of the second heavy chain polypeptide comprises a tryptophan (W) at position 366. In some embodiments of a method of making a bispecific antibody described herein, the CH3 region of the first heavy chain polypeptide comprises a threonine (T) at position 407 and the CH3 region of the second heavy chain polypeptide comprises a tyrosine (Y) at position 366. In some embodiments of a method of making a bispecific antibody described herein, the CH3 region of the first heavy chain polypeptide and the CH3 region of the second heavy chain polypeptide are modified based upon electrostatic effects.

[0026] In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the CH2, CH3, or Fc region of the first heavy chain polypeptide and the CH2, CH3, or Fc region of the second heavy chain polypeptide comprise additional amino acid modifications.

[0027] In some embodiments of the methods described herein, the bispecific antibody is produced in a host cell. In some embodiments, the host cell is a mammalian cell.

[0028] Where aspects or embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in the claimed disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0029] Figure 1. Representative diagrams of a wild-type Fab (WT Fab), a Fab comprising an alternative disulfide bond (Alt Cys Fab), and a Fab comprising an alternative disulfide bond and a charged pair (Alt Cys/Charged pair Fab).

[0030] Figure 2A. Representative IgGl heavy chain CH1 and hinge region sequence and a kappa light chain sequence each aligned with a parallel sequence comprising mutations. Figure 2B. Representative IgGl heavy chain CH1 and hinge region sequence and a lambda light chain sequence each aligned with a parallel sequence comprising mutations. Figure 2C. Representative IgGl heavy chain CH1 and hinge region sequence and a kappa light chain sequence each aligned with a parallel sequence comprising mutations. [0031] Figure 3. Formation of intact antibody with alternative disulfide bonds between heavy chains and light chains.

[0032] Figure 4. Formation of intact bispecific antibody comprising a heavy chain/light chain pair with an alternative disulfide bond.

[0033] Figure 5A. SDS-PAGE analysis of bispecific antibodies from Transfections 1 and 2 after purification steps. Figure 5B. SDS-PAGE analysis of bispecific antibodies from

Transfections 3 and 4 after purification steps.

[0034] Figure 6. Non-reducing mass spectrometry of final purified bispecific antibodies.

DETAILED DESCRIPTION

[0035] The present disclosure relates to a platform technology for multispecific binding agents having alternative disulfide bond formation between polypeptide chains ( e.g ., heavy and light chains). This platform permits efficient generation of multispecific binding agents with correct assembly of polypeptide chain pairs (e.g., heavy chain/light chain pairs in a bispecific antibody). Thus, the present disclosure provides multispecific binding agents, such as bispecific antibodies, that comprise one or more substituted amino acid residues that can result in alternative disulfide bond formation between polypeptide chains. Related polynucleotides encoding the multispecific binding agents (e.g, bispecific antibodies), vectors comprising the related polynucleotides encoding the multispecific binding agents (e.g, bispecific antibodies), host cells for producing the multispecific binding agents, compositions (e.g., pharmaceutical compositions) comprising the multispecific binding agents, and methods of making the multispecific binding agents are also provided.

I. Definitions

[0036] ETnless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control. [0037] The term“antibody” as used herein refers to an immunoglobulin molecule, or a fragment thereof, that recognizes and specifically binds a target through at least one antigen binding site.“Antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to, polyclonal antibodies, recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, diabodies, tribodies, tetrabodies, single chain Fv (scFv) antibodies, and antibody fragments as long as they exhibit the desired antigen-binding activity.

[0038] The term“intact antibody” or“full-length antibody” refers to an antibody having a structure substantially similar to a native antibody structure. This includes an antibody comprising two light chains each comprising a variable region and a light chain constant region (CL) and two heavy chains each comprising a variable region and at least heavy chain constant regions CH1, CH2, and CH3 and a hinge region ( e.g ., an upper hinge, a core hinge, and/or a lower hinge). As used herein the term“CH1 and hinge region” of an IgGl antibody refers to amino acids 118 to 230 (according to EU numbering) of an IgGl antibody or an allotype or isoallotype of IgGl.

[0039] As used herein the term“alternative interchain disulfide bond” in the context of a heavy and light chain pair refers to a disulfide bond other than the disulfide bond that forms between C220 of IgGl and C214 of the light chain (kappa or lambda) in wild type IgGl/kappa or IgGl/lambda heavy and light chain pairs.

[0040] The term“antibody fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and generally an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, disulfide- linked Fv (sdFv), Fd, linear antibodies, single chain antibody molecules (e.g., scFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies, and multispecific antibodies formed from antibody fragments.

[0041] The term“variable region” as used herein refers to the region of an antibody light chain or the region of an antibody heavy chain that is involved in binding the antibody to antigen. The variable region of an antibody heavy chain and an antibody light chain have similar structures, and generally comprise four framework regions and three complementarity determining regions (CDRs) (also known as hypervariable regions). [0042] The term“monoclonal antibody” as used herein refers to a substantially homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. The term“monoclonal antibody” encompasses intact and full-length monoclonal antibodies as well as antibody fragments ( e.g ., Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody fragment, and any other modified immunoglobulin molecule comprising an antigen-binding site. Furthermore,“monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0043] The term“chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

[0044] The term“humanized antibody” as used herein refers to a chimeric antibody that includes human immunoglobulins in which the native CDR amino acid residues are replaced by amino acid residues from corresponding CDRs of an antibody from a nonhuman species such as mouse, rat, rabbit, or nonhuman primate, wherein the nonhuman antibody has the desired specificity, affinity, and/or activity. In some instances, one or more (e.g., 1, 2, 3, 4, 5) framework region residues of a human immunoglobulin are replaced by corresponding amino acid residues from a nonhuman antibody. Furthermore, humanized antibodies can comprise residues (e.g, 1, 2, 3, 4, 5) that are not found in the original human antibody or in the original nonhuman antibody. These modifications may be made to further refine and/or optimize antibody characteristics. A humanized antibody may comprise variable regions containing all or substantially all of the CDRs that correspond to those of a nonhuman immunoglobulin and all or substantially all of the framework regions that correspond to those of a human immunoglobulin. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0045] The term“human antibody” as used herein refers to an antibody that possesses an amino acid sequence that corresponds to an antibody produced by a human and/or an antibody that has been made using any of the techniques that are known to those of skill in the art for making human antibodies. These techniques include, but not limited to, phage display libraries, yeast display libraries, transgenic animals, and B-cell hybridoma technology. [0046] The terms“epitope” and“antigenic determinant” are used interchangeably herein and refer to that portion of an antigen or target capable of being recognized and specifically bound by a particular antibody. When the antigen or target is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. In some cases, X-ray crystallography is used to predict potential epitopes on a target protein. In some cases, X-ray crystallography is used to characterize an epitope on a target protein by analyzing the amino acid interactions of an antigen/antibody complex.

[0047] The terms“selectively binds” or“specifically binds” as used herein mean that an agent ( e.g ., an antibody) interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to a particular antigen, epitope, protein, or target molecule than with alternative substances. A binding agent that specifically binds an antigen can be identified, for example, by immunoassays, ELISAs, Biacore assays, FACS, or other techniques known to those of skill in the art.

[0048] The terms“polypeptide” and“peptide” and“protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a“polypeptide” can occur as a single chain or as two or more associated chains.

[0049] The terms“polynucleotide” and“nucleic acid” and“nucleic acid molecule” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0050] The terms“identical” or percent“identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the disclosure are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 nucleotides or amino acid residues, at least about 60-80 nucleotides or amino acid residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 nucleotides or amino acid residues, such as at least about 80-100 nucleotides or amino acid residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, for example, (i) the coding region of a nucleotide sequence or (ii) an amino acid sequence.

[0051] The phrase“conservative amino acid substitution” as used herein refers to a

substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including 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), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is considered to be a conservative substitution. Generally, conservative substitutions in the sequences of polypeptides and/or antibodies do not abrogate the binding of the polypeptide or antibody to the target binding site. Methods of identifying nucleotide and amino acid conservative substitutions that do not eliminate binding are well-known in the art.

[0052] The term“vector” as used herein means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid vectors, cosmid vectors, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0053] The term“isolated” as used herein refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. For examples, an“isolated” antibody is substantially free of material from the cellular source from which it is derived. In some embodiments, isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions are those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition may be isolated from a natural source or from a source such as an engineered cell line.

[0054] The term“substantially pure” as used herein refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0055] The term“subject” refers to any animal ( e.g ., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rabbits, rodents, and the like, which is to be the recipient of a particular treatment or therapy. Typically, the terms“subject” and“patient” are used interchangeably herein in reference to a human subject.

[0056] The term“pharmaceutically acceptable” as used herein refers to a substance approved or approvable by a regulatory agency or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, including humans.

[0057] The terms“pharmaceutically acceptable excipient, carrier, or adjuvant” or“acceptable pharmaceutical carrier” as used herein refer to an excipient, carrier, or adjuvant that can be administered to a subject, together with at least one therapeutic agent ( e.g ., an antibody), and which does not affect the pharmacological activity of the therapeutic agent. In general, those of skill in the art and regulatory agencies consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation or composition.

[0058] The term“pharmaceutical formulation” or“pharmaceutical composition” as used herein refers to a preparation which is in such form as to permit the biological activity of the active ingredient (e.g., an antibody) to be effective. A pharmaceutical formulation/composition generally comprises additional components, such as a pharmaceutically acceptable excipient, carrier, adjuvant, and/or buffer.

[0059] The term“effective amount” or“therapeutically effective amount” as used herein refers to the amount of an agent (e.g, an antibody) which is sufficient to reduce and/or ameliorate the severity and/or duration of a disease, disorder or condition and/or a symptom in a subject. The term also encompasses an amount of an agent necessary for the (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development, or onset of a given disease, disorder, or condition, and/or (iii) the improvement or enhancement of the prophylactic or therapeutic effect(s) of another agent or therapy (e.g, an agent other than the binding agents provided herein).

[0060] As used herein, reference to“about” or“approximately” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, a description referring to“about X” includes description of“X”.

[0061] As used in the present disclosure and claims, the singular forms“a”,“an” and“the” include plural forms unless the context clearly dictates otherwise.

[0062] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of“consisting of’ and/or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase“consisting essentially of’ otherwise analogous

embodiments described in terms of“consisting of’ are also provided. [0063] The term“and/or” as used in a phrase such as“A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

II. Multispecific binding agents

[0064] Multispecific binding agents are generated to bind more than one target, antigen, or epitope. A multispecific binding agent can comprise two or more antigen-binding sites. In some embodiments, the binding agent is an antibody. In other embodiments, the antibody is an antigen-binding fragment thereof. In some embodiments, a multispecific binding agent comprises at least one antibody or an antigen-binding fragment thereof. In some embodiments, the multispecific binding agent comprises two or more antibodies, with each antibody having specificity for a different epitope.

[0065] In some embodiments, the multispecific binding agent is a bispecific antibody.

Bispecific antibodies can specifically recognize and bind at least two different targets, antigens, or epitopes. The different epitopes can be within the same molecule ( e.g ., two epitopes on one protein) or on different molecules (e.g., one epitope on a first protein and one epitope on a second protein). In some embodiments, a bispecific antibody has enhanced potency as compared to an individual antibody or a combination of more than one antibody. In some embodiments, a bispecific antibody has reduced toxicity as compared to an individual antibody or a combination of more than one antibody. It is known to those of skill in the art that any therapeutic agent may have unique pharmacokinetics (PK) (e.g, circulating half-life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active binding agents wherein the two individual binding agents have different PK profiles. In some embodiments, a bispecific antibody has the ability to concentrate the actions of two agents in a common area in a subject.

In some embodiments, the common area is a tissue in the subject. In some embodiments, a bispecific antibody concentrates the actions of two agents to a common target. In some embodiments, the common target is a specific cell type. In some embodiments, a bispecific antibody targets the actions of two agents to more than one biological pathway or function. In some embodiments, a bispecific antibody targets two different cells and brings them closer together.

[0066] In some embodiments, a bispecific antibody has decreased toxicity and/or side effects. In some embodiments, a bispecific antibody has decreased toxicity and/or side effects as compared to a mixture of the two individual antibodies or the antibodies as single agents. In some embodiments, a bispecific antibody has an increased therapeutic index. In some embodiments, a bispecific antibody has an increased therapeutic index as compared to a mixture of the two individual antibodies or the antibodies as single agents.

[0067] A variety of techniques for making bispecific antibodies have been developed.

However, there are still problems producing sufficient quantities of properly assembled functional antibodies. For example, there are problems with obtaining the correct association of each heavy chain/light chain pair and with efficient production of an intact, functional bispecific antibody. To solve the problem of the heavy chain/light chain mispairing, several strategies have been proposed including, for example, the use of two antibodies of different specificities that share a common light chain. One drawback of this approach is the difficulty in identifying different antibodies with good binding affinities that have a common light chain.

[0068] This disclosure is based, in part, on the identification of sites in the IgGl CH1 region and the light chain constant region that may be modified ( e.g ., substituted) to produce a disulfide bond between the IgGl CH1 region and the light chain constant region (i.e., an alternative interchain disulfide bond). When combined with additional modifications to the IgGl CH1 and hinge region and light chain constant region to destroy a native interchain disulfide bond (e.g., cysteines that interact with each other to form a disulfide bond in a wild type IgGl antibody), the resulting multispecific binding agents achieve efficient association of correct heavy chain and light chain pairs. Thus, described herein are multispecific binding agents (e.g, bispecific antibodies) comprising a first heavy chain polypeptide and a first light chain polypeptide, each of which comprises one or more amino acid mutations in their respective constant regions and/or in the heavy chain hinge region, relative to a wild type IgGl antibody, wherein the amino acid mutations allow for: (i) formation of an alternative interchain disulfide bond (i.e., an interchain disulfide bond not found in wild type IgGl antibodies) and (ii) destruction of a native interchain disulfide bond (i.e., an interchain disulfide bond found in a wild type IgGl antibody). The multispecific binding agents (e.g, bispecific antibodies) further comprise a second heavy chain polypeptide and a second light chain polypeptide. The second heavy chain polypeptide and the second light chain polypeptide form a native interchain disulfide bond. As a result, the first light chain cannot form an interchain disulfide bond with the second heavy chain and the first heavy chain cannot form an interchain disulfide bond with the second light chain. This method allows for efficient association of each heavy chain/light chain pair. See, e.g. , Table 1 for exemplary modifications to the heavy and light chains that produce alternative interchain disulfide bonds and exemplary modifications to the heavy and light chains that destroy a native interchain disulfide bond.

Table 1

sequence

² - According to Kabat/EU numbering for human kappa and lambda light chain constant region sequence

[0069] IgG antibodies exist as various allotypes and isoallotypes and each of these are encompassed by the present disclosure. In certain embodiments, a multispecific binding agent (e.g., a bispecific antibody) described herein comprises an IgGl heavy chain constant region or CH1 and hinge region having an allotype selected from: Glml, nGlm2, Glm3, Glml7,l, Glml7,l,2, Glm3,l, or Glml7. These allotypes or isoallotypes are characterized by the following amino acid residues at the indicated positions within the IgGl heavy chain constant region (according to EU numbering): Glml : D356, L358; nGlml : E356, M358; Glm3: R214, E356, M358, A431; Glml7,l : K214, D356, L358, A431; Glml7,l,2: K214, D356, L358, G431; Glm3,l : R214, D356, L358, A431; and Glml7: K214, E356, M358, A431. In certain embodiments, a bispecific antibody described herein comprises an IgGl heavy chain constant region or CH1 and hinge region selected from the allotype nGlml, Glm3, Glml7,l,

Glml7,l,2, Glm3,l, or Glml7. In certain embodiments, a bispecific antibody described herein comprises an IgGl heavy chain constant region or CH1 and hinge region of the allotype Glml7.

[0070] The kappa light chains of IgG antibodies exist as various allotypes and each of these are encompassed by the present disclosure. In certain embodiments, a multispecific binding agent ( e.g ., a bispecific antibody) described herein comprises a kappa light chain having an allotype selected from: Kml, Kml,2, or Km3. Each of these allotypes is characterized by the following amino acid residues at the indicated positions within the kappa light chain (according to

Kabat/EU numbering): Kml : V153, L191; Kml, 2: A153, L191; and Km3: A153, V191.

[0071] The lambda light chains of IgG antibodies exist as various allotypes and each of these are encompassed by the present disclosure. In certain embodiments, a multispecific binding agent (e.g., a bispecific antibody) described herein comprises a lambda light chain having an allotype selected from: Lambdal, Lambda2, Lambda3, Lambda4, Lambda5, Lambda6, or Lambda7. In some embodiments, a bispecific antibody comprises a lambda light chain selected from Lambdal, Lambda2, Lambda3, or Lambda 7. Each of these allotypes is characterized by the following amino acid residues at the indicated positions within the lambda light chain (according to Kabat/EU numbering): Lambda 1 : N113, T115, 1137, G153, V156, A158, K164, R190, Q195, T210; K164, Lambda 2: A113, Sl 15, 1137, S153, V156, A158, T164, R190, Q195, T210; Lambda 3: A113, Sl 15, 1137, S153, A156, A158, T164, K190, Q195, T210; Lambda 7:

Al 13, Sl 15, V137, G153, V156, V158, K164, R190, R195, A210.

[0072] In certain embodiments, a multispecific binding agent (e.g, a bispecific antibody) described herein comprises: (a) a first IgGl heavy chain polypeptide comprising a CH1 domain and a hinge region, and a first light chain polypeptide comprising a light chain constant region, wherein the first IgGl heavy chain polypeptide is associated with the first light chain polypeptide thereby forming a first heavy chain/light chain pair, wherein the first heavy chain/light chain pair comprises an alternative interchain disulfide bond than that found between a heavy chain polypeptide and a light chain polypeptide found in a wild type IgGl antibody; and (b) a second IgGl heavy chain polypeptide associated with a second light chain polypeptide.

[0073] In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises a cysteine at position 126 and a non-cysteine amino acid at position 220 according to EU numbering, and the light chain constant region of the first light chain polypeptide comprises a cysteine at position 124 (with respect to a kappa light chain) or position 125 (with respect to a lambda light chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering.

[0074] In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises a cysteine at position 173 and a non-cysteine amino acid at position 220 according to EEG numbering, and the light chain constant region of the first light chain polypeptide comprises a cysteine at position 162 (with respect to a kappa light chain) and a non-cysteine amino acid at position 214 according to Kabat/EEG numbering.

[0075] In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5 and comprises a cysteine at position 126 and a non-cysteine amino acid at position 220 according to EU numbering, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8 and comprises a cysteine at position 124 (kappa chain) or position 125 (lambda chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering. In some embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:5 and comprises a cysteine at position 126 and a non-cysteine amino acid at position 220 according to EU numbering, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 and comprises a cysteine at position 124 and a non-cysteine amino acid at position 214 according to Kabat/EU numbering. In some embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5 and comprises a cysteine at position 126 and a non-cysteine amino acid at position 220 according to EU numbering, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8 and comprises a cysteine at position 125 and a non-cysteine amino acid at position 214 according to Kabat/EU numbering.

[0076] In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6 and comprises a cysteine at position 173 and a non-cysteine amino acid at position 220 according to EU numbering; and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9 and comprises a cysteine at position 162 (kappa chain) and a non-cysteine amino acid at position 214 according to Kabat/EU numbering.

[0077] In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-103 (or 9-103) of SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 9 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to: (i) amino acids 1-107 of any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12, wherein the amino acid corresponding to position 17 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is a cysteine and the amino acid corresponding to position 107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is not a cysteine, or (ii) amino acids 1-106 of any one of SEQ ID NO:3, SEQ ID NO:l3, SEQ ID NO: l4, or SEQ ID NO: l5, wherein the amino acid corresponding to position 18 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is a cysteine and the amino acid corresponding to position 105 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: l4, or SEQ ID NO:l5 is not a cysteine. In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-103 (or 9-103) of SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 9 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-107 of any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12, wherein the amino acid corresponding to position 17 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is a cysteine and the amino acid corresponding to position 107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is not a cysteine. In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-103 (or 9-103) of SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 9 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-106 of any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, wherein the amino acid corresponding to position 18 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is a cysteine and the amino acid corresponding to position 105 of the sequence set forth in any one of SEQ ID NO:3, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is not a cysteine. In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-103 (or 9-103) of SEQ ID NO:4 or SEQ ID NO: 10, wherein the amino acid corresponding to position 56 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is a cysteine and the amino acid corresponding to position 103 of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 10 is not a cysteine, and the light chain constant region of the first light chain polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 1-107 of any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12, wherein the amino acid corresponding to position 55 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is a cysteine and the amino acid corresponding to position 107 of the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12 is not a cysteine.

[0078] In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an arginine (R), a lysine (K), a glutamic acid (E), or an aspartic acid (D) at position 220 (according to EU numbering). In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an arginine (R) at position 220 (according to EU numbering). In certain embodiments, the constant region of the first light chain polypeptide comprises a glutamic acid (E), an aspartic acid (D), an arginine (R), or a lysine (K) at position 214 (according to Kabat/EU numbering). In certain embodiments, the constant region of the first light chain polypeptide comprises a glutamic acid (E) at position 214 (according to Kabat/EU numbering).

In certain embodiments, the CH1 and hinge region of the first heavy chain polypeptide comprises an arginine (R) at position 220 (according to EU numbering) and the constant region of the first light chain polypeptide comprises a glutamic acid (E) at position 214 (according to Kabat/EU numbering).

[0079] The skilled artisan will appreciate that the second IgGl heavy chain polypeptide and the second light chain polypeptide do not comprise the alternative interchain disulfide bond of the first IgGl heavy chain polypeptide and the first light chain polypeptide. For example, in certain embodiments, the first IgGl heavy chain polypeptide and first light chain polypeptide may comprise the substitutions for producing an alternative interchain disulfide bond pair as shown in Table 1 {i.e., Phel26Cys in the IgGl CH1 and hinge region (according to EU numbering) and Glnl24Cys (according to Kabat/EU numbering for a kappa constant region) or Glul25Cys (according to Kabat/EU numbering for a lambda constant region)) and the substitutions for destroying the native interchain disulfide bond as shown in Table 1 (i.e., Cys220(Non-Cys) in the IgGl CH1 and hinge region (according to EU numbering) and

Cys2l4(Non-Cys) (according to Kabat/EU numbering for kappa or lamba constant region)). In such embodiments, the second IgGl heavy chain polypeptide and second light chain polypeptide will not have the substitutions for producing an alternative interchain disulfide bond pair.

Rather, the second IgGl heavy chain polypeptide and the second light chain polypeptide may retain the native interchain disulfide bond pair of Table 1 (i.e., Cys220 in the IgGl CH1 and hinge region (according to EU numbering) and Cys2l4 (according to Kabat/EU numbering for a kappa or lambda light chain constant region)).

[0080] In certain embodiments, the multispecific binding agent ( e.g ., a bispecific antibody) comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) modifications (e.g, substitutions, deletions, additions) described herein (e.g, one or more modifications to the constant region or the Fc region of the multispecific binding agent (e.g, bispecific antibody)).

In some instances, the multispecific binding agent (e.g, bispecific antibody) includes an antibody Fc region. In some embodiments, the Fc region is from human IgGl antibody, human IgG2 antibody, human IgG3 antibody, or human IgG4 antibody. In certain embodiments, the Fc region is aglycosylated (“agly”) (e.g, N297Q, or T299A). In certain embodiments, the Fc region is a human IgGl agly Fc region. In certain embodiments, the Fc region contains a human IgG4P agly CH2 domain and a human IgGl CH3 domain. In certain instances, the Fc region has reduced effector function relative to its wild type counterpart.

[0081] In some embodiments, the Fc region may include one or mutations relative to the wild type Fc region. In certain embodiments, the one or more mutations may be made to lower effector function, increase stability, improve pharmacokinetics, and/or increase half-life. In certain embodiments, the very C-terminal residue of each IgGl Fc region is deleted (i.e., the final lysine is removed) because there can be splicing and/or enzymatic cleavage at this C- terminal lysine which can lead to product heterogeneity. In some embodiments, the very C- terminal residue of the IgGl Fc region is a lysine residue. In certain embodiments, the very C- terminal residue of the IgGl Fc region is changed from a lysine to a cysteine residue (which can allow for C-terminal site conjugation). In some embodiments, the Fc region comprises a S442C mutation (according to EEG numbering).

[0082] In some embodiments, a multispecific binding agent (e.g, bispecific antibody) comprises additional modifications or mutations within the heavy chain constant regions or Fc region, wherein the modifications affect the interface between the two heavy chains and enhance heterodimer formation. In some embodiments, a bispecific antibody comprises“knobs” and “holes” modifications (Ridgway et al, Protein Eng., 1996, 9:617-621). In some cases, the terms “knobs” and“holes” are used interchangeably herein with the terms“protuberances” or “protrusions” and“cavities”, respectively. Nonlimiting examples of knob-into-hole

modifications are provided in Tables 2 and 3 herein. In some embodiments, a bispecific antibody comprises hinge region modifications, wherein disulfide linkages are formed only between heterodimer molecules. In some embodiments, a bispecific antibody comprises modifications that result in altered electrostatic interactions between the two heavy chains. In some embodiments, a bispecific antibody comprises modifications that result in altered hydrophobic/hydrophilic interactions between the two heavy chains.

[0083] In some embodiments, a bispecific antibody is engineered using the“knob-into-hole” technology as known to those of skill in the art. This strategy has been used to increase the yield of heterodimer formation over unwanted end products such as homodimers. CH3 modifications using knob-into-hole methods include, but are not limited to, those listed in Table 2.

Table 2

¹ - According to EU numbering for human immunoglobulin heavy chain constant region sequence

[0084] In some embodiments, a bispecific antibody is engineered using other technologies known to those of skill in the art including, but not limited to, lysine repositioning, ZW1, ZW2, ZW3, KiH+S-S stabilization, electrostatic steering, ionic electrostatic, ionic electrostatic DD- KK, mixed HA-TF, SEEDbody, and LUZ-Y (see, e.g. , Table 3 for exemplary modifications to Fc chains). These strategies have been used to increase the yield of heterodimer formation over unwanted end products such as homodimers.

Table 3

¹ - According to EU numbering for human immunoglobulin heavy chain constant region sequence

[0085] It will be understood by one of skill in the art that any of the CH3 or Fc modifications to enhance heterodimerization described herein can be on either chain of the bispecific antibody, so long as one chain has one set of modifications and the other chain has the compensatory modifications.

[0086] In some embodiments, bispecific antibodies are intact antibodies. In some

embodiments, bispecific antibodies comprise antibody fragments comprising antigen-binding sites. In some embodiments, antibodies are multispecific antibodies with more than two valencies or specificities.

[0087] In certain embodiments, a multispecific binding agent ( e.g ., a bispecific antibody) comprises at least a portion of one or more“parental” antibodies (e.g., the CDRs or variable domains). In some embodiments, a parental antibody is a recombinant antibody. In some embodiments, a parental antibody is a monoclonal antibody. In some embodiments, a parental antibody is a chimeric antibody. In some embodiments, a parental antibody is a humanized antibody. In some embodiments, a parental antibody is a human antibody. In some

embodiments, a parental antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In certain embodiments, a parental antibody is an IgGl antibody. In certain embodiments, a parental antibody is an IgG2 antibody. In some embodiments, a parental antibody is an IgG4 antibody.

[0088] In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) is isolated. In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) is substantially pure.

[0089] In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) is derived from at least one monoclonal antibody. In some embodiments, a monoclonal antibody is prepared using hybridoma methods known to one of skill in the art. For example, using the hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above to elicit the production of antibodies that specifically bind the immunizing antigen. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a mouse protein or a fragment thereof.

[0090] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g, flow cytometry, FACS, ELISA, and radioimmunoassay). Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution techniques. The hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal. The monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

[0091] In certain embodiments, monoclonal antibodies can be made using recombinant DNA techniques as known to one skilled in the art. For example, in certain examples, polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques.

The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli , simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

[0092] In certain other embodiments, recombinant monoclonal antibodies, or fragments thereof, can be isolated from phage display libraries expressing variable domains or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

[0093] In some embodiments, a monoclonal antibody is modified, for example, by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light chain and heavy chain of, for example, a mouse monoclonal antibody can be substituted for constant regions of, for example, a human antibody to generate a chimeric antibody, or for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region(s) is used to optimize specificity and/or affinity of a monoclonal antibody.

[0094] In some embodiments, a multispecific binding agent ( e.g ., a bispecific antibody) is derived from a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody. In some embodiments, humanized antibodies are generated by substituting all six CDRs of a parent non-human antibody ( e.g ., rodent) for the corresponding CDR sequences of a human antibody.

[0095] The choice of which human heavy chain variable region and light chain variable region to be used in generating humanized antibodies can be made based on a variety of factors and by a variety of methods. In some embodiments, the“best-fit” method is used where the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences. The human sequence that is most similar to that of the non-human sequence is selected as the human variable region backbone for the humanized antibody. In some embodiments, a method is used wherein a particular variable region backbone derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains is selected. In some embodiments, the framework is derived from the consensus sequences of the most abundant human subclasses. In some embodiments, human germline genes are used as the source of the variable region framework sequences.

[0096] Other methods for humanization include, but are not limited to, a method called “superhumanization” which is described as the direct transfer of CDRs to a human germline framework, a method called Human String Content (HSC) which is based on a metric of antibody“humanness”, methods based on generation of large libraries of humanized variants (including phage, ribosomal, and yeast display libraries), and methods based on framework region shuffling.

[0097] In certain embodiments, a multispecific binding agent (e.g, a bispecific antibody) is derived from a human antibody. Human antibodies can be directly prepared using various techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In any case, cells that produce an antibody directed against a target antigen can be generated and isolated. In some embodiments, a human antibody is selected from a phage library, where that phage library expresses human antibodies. Alternatively, phage display technology may be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, may be employed to generate higher affinity human antibodies.

[0098] In some embodiments, human antibodies are produced in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

[0099] In some embodiments, the multispecific binding agents described herein are derived from antibodies ( e.g ., full-length antibodies or fragments thereof) that comprise modifications in at least one or more of the constant regions. In some embodiments, the antibodies comprise modifications to one or more of the three heavy chain constant regions (CH1, CH2 or CH3), to the hinge region, and/or to the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibodies comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibodies comprises more than one human constant region. In some embodiments, modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the entire CH2 domain has been removed from an antibody (ACH2 constructs). In some embodiments, an omitted constant region is replaced by a short amino acid spacer (e.g., 10 amino acid residues) that provides some of the molecular flexibility typically imparted by the absent constant region.

In some embodiments, a modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In other embodiments, a modified antibody comprises a peptide spacer inserted between the hinge region and modified CH2 and/or CH3 domains.

[00100] It is known in the art that the constant region(s) of an antibody mediates several effector functions. For example, binding of the Cl component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR). There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).

Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin

production.

[00101] In certain embodiments, the modified antibodies provide for altered effector functions that, in turn, affect the biological profile of the multispecific binding agent that comprises the modified antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region reduces Fc receptor binding of the circulating modified antibody. In other embodiments, the constant region modifications increase the serum half-life of the antibody. In other embodiments, the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region modifications decrease or remove ADCC and/or CDC activities of the antibody. For example, in some embodiments, specific amino acid substitutions in a human IgGl Fc region with corresponding IgG2 or IgG4 residues reduce effector functions (e.g, ADCC and CDC) in the modified antibody. Thus, in certain embodiments, a multispecific binding agent (e.g, a bispecific antibody) derived from a modified antibody does not have one or more effector functions. In some embodiments, the multispecific binding agent has no ADCC activity and/or no CDC activity. In certain embodiments, the multispecific binding agent does not bind an Fc receptor and/or complement factors. In certain embodiments, the multispecific binding agent has no effector function(s) (e.g, an“effectorless” antibody). In some embodiments, the constant region is modified to eliminate one or more disulfide linkages or oligosaccharide moieties. In certain embodiments, the constant region is modified to add one or more amino acids to provide, for example, one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.

[00102] In certain embodiments, one or more heavy chain modifications are selected from the following amino acid substitutions (according to EU numbering) or combinations thereof:

L234F; L235E; G236A; S239D; F243L; D265E; D265A; S267E; H268F; R292P; N297Q;

N297A; S298A; S324T; I332E; S239D; A330L; L234F; L235E; P331S; F243L; Y300L; V305I; P396L; S298A; E333A; K334A; E345R; L235V; F243L; R292P; Y300L; P396L; M428L;

E430G; N434S; G236A, S267E, H268F, S324T, and I332E; G236A, S239D, and I332E; S239D, A330L, I332E; L234F, L235E, and P331S; F243L, R292P, Y300L, V305I, and P396L; G236A, H268F, S324T, and I332E; S239D, H268F, S324T, and I332E; S298A, E333A, and K334A; L235V, F243L, R292P, Y300L, and P396L; S239D, I332E; S239D, S298A, and I332E; G236A, S239D, I332E, M428L, and N434S; G236A, S239D, A330L, I332E, M428L, and N434S;

S239D, I332E, G236A and A330L; M428L and N4343S; M428L, N434S; G236A, S239D, A330L, and I332E; and G236A and I332E. In some embodiments, the one or more

modifications are selected from the group consisting of: N297A, D265A, L234F, L235E,

N297Q, and P331 S. In certain embodiments, the one or more modifications is N297A or D265A. In certain embodiments, the one or more modifications are L234F and L235E. In certain embodiments, the one or more modifications are L234F, L234E, and D265A. In certain embodiments, the one or more modifications are L234F, L234E, and N297Q. In certain embodiments, the one or more modifications are L234F, L235E, and P331 S. In certain embodiments, the one or more modifications are D265A and N297Q. In certain embodiments, the one or more modifications are L234F, L235E, D265A, N297Q, and P331S.

[00103] Mutations that reduce Fc-receptor binding include, but are not limited to, N297A, N297Q, D265A, L234F/L235E, L234F/L235E/N297Q, L234F/L235E/P331S, D265A/N297Q, and L234F/L235E/ D265A/N297Q/P331 S (according to EEG numbering). In certain

embodiments, the bispecific antibodies disclosed herein comprise L234F and L235E mutations. In certain embodiments, the bispecific antibodies disclosed herein comprise L234F, L235E, and D265A mutations. In certain embodiments, the bispecific antibodies disclosed herein comprise L234F, L235E, and D265A mutations. In certain embodiments, the bispecific antibodies disclosed herein comprise an N297A or N297Q mutation. In certain embodiments, the bispecific antibodies disclosed herein comprise an N297A or N297Q mutation as well as L234F, L235E, and D265A mutations. In certain embodiments, one, two, three, four, or more amino acid substitutions are introduced into a Fc region to alter the effector function of the bispecific antibody. In some embodiments, these substitutions are located at positions selected from the group consisting of amino acid residues 234, 235, 236, 237, 265, 297, 318, 320, and 322

(according to EEG numbering). These positions can be replaced with a different amino acid residue such that the antibody has an altered (e.g, reduced) affinity for an effector ligand (e.g, an Fc receptor or the Cl component of complement), but retains the antigen-binding ability of the parent antibody. In certain embodiments, the bispecific antibodies disclosed herein comprise E233P, L234V, L235A, and G236A mutations (according to EEG numbering). In some embodiments, the bispecific antibodies comprise A327G, A330S, and P331S mutations (according to EU numbering). In some embodiments, the bispecific antibodies comprise K322A mutations (according to EU numbering). In some embodiments, the bispecific antibodies comprise E318 A, K320A, and K322A (according to EU numbering) mutations. In certain embodiments, the bispecific antibodies comprise a L235E (according to EU numbering) mutation.

[00104] Mutations that increase the half-life of an antibody ( e.g ., a bispecific antibody) are known in the art. In one embodiment, the constant region of a bispecific antibody described herein comprises a methionine to tyrosine substitution at position 252 (according to EU numbering), a serine to threonine substitution at position 254 (according to EU numbering), and a threonine to glutamic acid substitution at position 256 (according to EU numbering). This type of mutant is referred to as a“YTE mutant”. In certain embodiments, a bispecific antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 (according to EU numbering). In other embodiments, a bispecific antibody described herein comprises a M428L and N434S substitution (according to EU numbering). In other embodiments, a bispecific antibody described herein comprises T250Q and M428L (according to EU numbering) mutations. In other embodiments, a bispecific antibody described herein comprises H433K and N434F (according to EU numbering) mutations.

[00105] In particular embodiments, the bispecific antibodies comprise two or more, three or more, four or more, five or more, six or more, six or fewer, five or fewer, four or fewer, three or fewer, two or fewer, or one modified Fc amino acid residue(s). In certain embodiments, the bispecific antibodies comprise the L234F, L235E, D264A mutations, which are collectively referred to as“FEA.” In certain embodiments, the bispecific antibodies comprise the L234F, L235E, D264A, and F405L mutations, which are collectively referred to as“FEAL.” In certain embodiments, the bispecific antibodies comprise the L234F, L235E, D264A, and a mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the bispecific antibodies comprise the L234F, L235E, D264A, and K409R mutations, which are collectively referred to as“FEAR.” In certain embodiments, the bispecific antibodies comprise the M428L and N434S mutations, which are collectively referred to as“LS”. In certain embodiments, the bispecific antibodies comprise the L234F, L235E, D264A, F405L, M428L, and N434S mutations, which are collectively referred to as“FEALLS.” In certain embodiments, the bispecific antibodies comprise the L234F, L235E, D264A, M428L, and N434S mutations along with one further mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the bispecific antibodies comprise the L234F, L235E, D264A, K409R, M428L, and N434S mutations which are collectively referred to as“FEARLS.” In certain embodiments, the bispecific antibodies comprise the S239D, I332E, G236A, A330L mutations, which are collectively referred to as“DEAL”. In certain embodiments, the bispecific antibodies comprise the S239D, I332E, G236A, A330L, M428L and N434S mutations, which are collectively referred to as“DEALLS”. The FEA mutations decrease or abrogate effector function while the DEAL mutations increase or enhance effector function by enhancing the binding of the Fc to activating FcyRs. The LS mutations increase the pharmacokinetic half-life of the bispecific antibody.

[00106] Modifications to the constant region of antibodies ( e.g ., parental antibody) and/or multispecific binding agents (e.g., a bispecific antibody) described herein may be made using well known biochemical or molecular engineering techniques. In some embodiments, variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by direct synthesis of the desired polypeptide or agent. In this respect it may be possible to disrupt the activity or effector function provided by a specific sequence or region while substantially maintaining the structure, binding activity, and other desired characteristics of the modified binding agent.

[00107] The present disclosure further embraces additional variants and equivalents which are substantially homologous to the multispecific binding agents described herein. In some embodiments, it is desirable to improve the binding affinity and/or other biological properties of the agent, including but not limited to, specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of a polypeptide, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

[00108] Variations may be a substitution, deletion, or insertion of one or more nucleotides encoding a multispecific binding agent that results in a change in the amino acid sequence as compared with the sequence of the parental binding agent. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. In some embodiments, insertions or deletions are in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. Variations in the amino acid sequence that are biologically useful and/or relevant may be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parental protein.

[00109] In some embodiments, variants may include the addition of amino acid residues at the amino- and/or carboxyl-terminal end of one or more polypeptides that make up the multispecific binding agent. The length of additional amino acids residues may range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, the variant comprising an additional polypeptide/protein, i.e ., a fusion protein. In certain embodiments, a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g, an enzyme).

[00110] In some embodiments, a cysteine residue not involved in maintaining the proper conformation of a binding agent (e.g, bispecific antibody) is substituted or deleted to modulate the agent’s characteristics, for example, to improve oxidative stability and/or prevent aberrant disulfide crosslinking. Conversely, in some embodiments, one or more cysteine residues are added to create disulfide bond(s) to improve stability.

[00111] In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) of the present disclosure is“deimmunized”. The deimmunization of agents such as antibodies generally consists of introducing specific mutations to remove T-cell epitopes without significantly reducing the binding affinity or other desired activities of the agent.

[00112] The variant multispecific binding agents or polypeptides described herein may be generated using methods known in the art, including but not limited to, site-directed

mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis. [00113] In some embodiments, a multispecific binding agent described herein is chemically modified. In some embodiments, a multispecific binding agent is a bispecific antibody that has been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications may be carried out by known techniques,

[00114] Generally speaking, antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces. When describing the strength of an antigen-antibody complex, affinity and/or avidity are usually mentioned. The binding of an antibody to its antigen is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD). KD is the ratio of an antibody dissociation rate (k ₀ ff or kd) (how quickly it dissociates from its antigen) to the antibody association rate (k ₀ n or k _a ) (how quickly it binds to its antigen). In some embodiments, KD values are determined by measuring the k ₀ n and k _0ff rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the KD value. KD values may be used to evaluate and rank order the strength of individual antibody/antigen interactions. The lower the KD of an antibody, the higher the affinity of the antibody for its target. Avidity gives a measure of the overall strength of an antibody-antigen complex. It is dependent on three major parameters: (i) affinity of the antibody for the epitope, (ii) valency of both the antibody and antigen, and (iii) structural arrangement of the parts that interact.

[00115] In certain embodiments, a multispecific binding agent ( e.g ., a bispecific antibody) binds one or more targets, antigens, or epitopes with a dissociation constant (KD) of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) binds a target, antigen, or epitope with a KD of about 20 nM or less. In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) binds a target, antigen, or epitope with a KD of about 10 nM or less. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) binds a target, antigen, or epitope with a KD of about 1 nM or less. In some embodiments, a

multispecific binding agent (e.g, a bispecific antibody) binds a target, antigen, or epitope with a KD of about 0.5 nM or less. In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) binds a target, antigen, or epitope with a KD of about 0.1 nM or less. In some embodiments, a multispecific binding agent ( e.g ., a bispecific antibody) binds a target, antigen, or epitope with a KD of about 50 pM or less. In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) binds a target, antigen, or epitope with a KD of about 25 pM or less. In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) binds a target, antigen, or epitope with a KD of about 10 pM or less. In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) binds a target, antigen, or epitope with a KD of about 1 pM or less. In some embodiments, the dissociation constant of a multispecific binding agent (e.g, a bispecific antibody) to a target is the dissociation constant determined using a fusion protein comprising at least a portion of the target protein immobilized on a Biacore chip. In some embodiments, the dissociation constant of a multispecific binding agent (e.g, a bispecific antibody) to a target is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and a soluble target protein.

[00116] In certain embodiments, a multispecific binding agent (e.g, a bispecific antibody) binds a target, antigen, or epitope with a half maximal effective concentration (EC50) of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In certain embodiments, a binding agent (e.g, a bispecific antibody) binds a target, antigen, or epitope with an EC50 of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

[00117] The polypeptides that make up the multispecific binding agents described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site- specific mutagenesis to provide functional variants thereof. In some embodiments, a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest.

For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[00118] Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host. Thus, provided herein are vectors comprising polynucleotide sequences encoding a multispecific binding agent ( e.g ., bispecific antibody) described herein, or portions thereof.

[00119] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding multispecific binding agents described herein. For example,

recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of a binding agent or fragment thereof, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of

transformants can additionally be incorporated. DNA regions are“operatively linked” when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In other embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide may include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[00120] The choice of an expression control sequence and an expression vector generally depends upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli , including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as Ml 3 and other filamentous single-stranded DNA phages.

[00121] The multispecific binding agents ( e.g ., a bispecific antibody) of the present disclosure can be expressed from one or more vectors. For example, in some embodiments, a heavy chain polypeptide is expressed by one vector and a light chain polypeptide is expressed by a second vector. In some embodiments, a heavy chain polypeptide and a light chain polypeptide are expressed by one vector. In some embodiments, a first heavy chain polypeptide is expressed by one vector, a second heavy chain polypeptide is expressed by a second vector, a first light chain polypeptide is expressed by a third vector, and a second light chain is expressed by a fourth vector. In some embodiments, a first heavy chain polypeptide and a first light chain polypeptide are expressed by a first vector and a second heavy chain polypeptide and a second light chain polypeptide are expressed by a second vector.

[00122] Suitable host cells for expression of a multispecific binding agent (e.g., a bispecific antibody) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, for example A. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well known in the art.

[00123] Various mammalian culture systems may be used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney- derived), L-929 (murine fibroblast-derived), Cl 27 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney- derived) cell lines and variants thereof. Mammalian expression vectors can comprise non- transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non- translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

[00124] Expression of recombinant proteins in insect cell culture systems ( e.g ., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

[00125] Thus, the present disclosure provides cells comprising the multispecific binding agents described herein. In some embodiments, the cells produce the multispecific binding agents described herein. In some embodiments, the cell or cells comprise one or more polynucleotides encoding a multispecific binding agent (e.g., bispecific antibody) described herein. In some embodiments, the cell or cells comprise one or more vectors encoding a multispecific binding agent (e.g, bispecific antibody) described herein. In certain embodiments, the cells produce a bispecific antibody. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is an eukaryotic cell. In some embodiments, the cell is a mammalian cell.

[00126] Proteins produced by a host cell can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Affinity chromatography used for purifying immunoglobulins can include Protein A, Protein G, and Protein L chromatography. Isolated proteins can be physically characterized using such techniques as proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC), and x-ray crystallography. The purity of isolated proteins can be determined using techniques known to those of skill in the art, including but not limited to, SDS-PAGE, SEC, capillary gel

electrophoresis, IEF, and capillary isoelectric focusing (cIEF). In some embodiments, purified proteins are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[00127] In some embodiments, supernatants from expression systems which secrete

recombinant protein into culture media are first concentrated using a commercially available protein concentration filter, for example, an Ami con or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin is employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. In some embodiments, a cation exchange step is employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite media is employed, including but not limited to, ceramic hydroxyapatite (CHT). In certain embodiments, one or more reverse-phase HPLC steps employing hydrophobic RP- HPLC media, e.g. , silica gel having pendant methyl or other aliphatic groups, are employed to further purify a recombinant protein. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a homogeneous recombinant protein.

[00128] In some embodiments, supernatants comprising a bispecific antibody described herein are purified using (i) an affinity column (e.g., Protein A), (ii) a cation exchange column, and (iii) a hydroxyapatite column (e.g, CHT).

[00129] Multispecific binding agents (e.g, bispecific antibodies) of the present disclosure may be characterized for their physical/chemical properties and/or biological activities by various assays known in the art. In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) is tested for its ability to bind a first target and/or a second target which has been engineered to be expressed on the surface of a cell. Binding assays may include, but are not limited to, Biacore and FACS.

[00130] In some embodiments, assays are provided for identifying binding agents that modulate a targeted biological activity.

[00131] The present disclosure also provides conjugates comprising any one of the multispecific binding agents ( e.g ., bispecific antibodies) described herein. In some embodiments, a bispecific antibody is attached to an additional molecule. In some embodiments, a bispecific antibody is conjugated to a cytotoxic agent or moiety. In some embodiments, a bispecific antibody is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate). In some

embodiments, the cytotoxic moiety is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs), or other intercalating agents. In some

embodiments, the cytotoxic moiety is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DMI and DM4), and tubulysins. In some embodiments, the cytotoxic moiety is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes. In some

embodiments, a bispecific antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065. The derivatives of any one of these toxins can be used in a conjugate as long as the derivative retains the cytotoxic activity.

[00132] Conjugates comprising an antibody may be made using any suitable methods as known in the art. In some embodiments, conjugates are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene).

[00133] In some embodiments, a multispecific binding agent (e.g., a bispecific antibody) described herein is conjugated to detectable substances or molecules that allow the antibodies to be used for diagnosis and/or detection. The detectable substances may include but not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine(s); fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine,

tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials, such as luciferase;

radioactive materials, such as ²¹² Bi, ¹⁴ C, ⁵⁷ Co, ⁵¹ Cr, ⁶⁷ Cu, ¹⁸ F, ⁶⁸ Ga, ⁶⁷ Ga, ¹⁵³ Gd, ¹⁵⁹ Gd, ⁶⁸ Ge, ³ H, ¹⁶⁶ HO, ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I, ¹¹⁵ In, ¹¹³ In, ¹¹² In, ^m In, ¹⁴⁰ La, ¹⁷⁷ Lu, ⁵⁴ Mn, "Mo, ³² P, ¹⁰³ Pd, ¹⁴⁹ Pm,

¹⁴² Pr, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁰⁵ Rh, ⁹⁷ Ru, ³⁵ S, ⁴⁷ Sc, ⁷⁵ Se, ¹⁵³ Sm, ¹¹³ Sn, ¹¹⁷ Sn, ⁸⁵ Sr, ^99m Tc, ²⁰¹ Ti, ¹³³ Xe, ⁹⁰ Y, ⁶⁹ Yb, ¹⁷⁵ Yb, ⁶⁵ Zn; positron emitting metals; and magnetic metal ions.

[00134] In some embodiments, a multispecific binding agent (e.g, a bispecific antibody) is conjugated to a macromolecular substance such as a polymer including but not limited to, polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG),

polyglutamic acid (PGA), (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, hyaluronic acid, a hapten, an enzyme (e.g, glucose oxidase), a metal chelate, biotin, avidin, or a drug.

[00135] In some embodiments, a multispecific binding agent described herein is attached to a solid support, that are particularly useful for immunoassays or purification of a target antigen(s). Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

III. Polynucleotides

[00136] In certain embodiments, the disclosure encompasses polynucleotides comprising polynucleotides that encode a multispecific binding agent ( e.g ., bispecific antibody) described herein. The term“polynucleotides that encode a polypeptide” encompasses a polynucleotide that includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

[00137] In certain embodiments, a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell ( e.g ., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide). The polypeptide can have the leader sequence cleaved by the host cell to form a“mature” form of the

polypeptide.

[00138] In certain embodiments, a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a marker or tag sequence. For example, in some embodiments, a marker sequence is a hexa-histidine tag supplied by a vector that allows efficient purification of the polypeptide fused to the marker in the case of a bacterial host. In some embodiments, a marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG™ tag. In some embodiments, a marker is used in conjunction with other affinity tags.

[00139] The present disclosure further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of a polypeptide. In certain embodiments, the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a multispecific binding agent (e.g., a bispecific antibody) described herein.

[00140] As used herein, the phrase“a polynucleotide having a nucleotide sequence at least, for example, 95%“identical” to a reference nucleotide sequence” is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[00141] The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli). In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

[00142] In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

[00143] In certain embodiments, a polynucleotide is isolated. In certain embodiments, a polynucleotide is substantially pure.

[00144] Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, an expression vector comprises a polynucleotide molecule. In some embodiments, a host cell comprises an expression vector comprising the polynucleotide molecule. In some embodiments, a host cell comprises one or more expression vectors comprising polynucleotide molecules. In some embodiments, a host cell comprises a polynucleotide molecule. In some embodiments, a host cell comprises one or more polynucleotide molecules.

EXAMPLES

Example 1

Identification of potential sites for amino acid substitutions

[00145] The co-crystal structure of a Fab (anti-B Fab) in complex with its target (Antigen B) was analyzed to identify residues that form the interface between the heavy chain CH1 and hinge region (CH1 -hinge) and the light chain constant region (CL) using the MOE software package (Chemical Computing Group). The MOE software was queried for all CHl-hinge:CL residue pairs that were within 5 angstroms of each other to identify CHl-hinge:CL residue pairs that could potentially form a disulfide bond if the residues of each pair were changed to cysteine.

The interaction and the energy contribution to structural stability from the interaction for each potential CHl-hinge:CL pair was calculated. A representative set of the inter-residue distances and energy differences for the CHl-hinge:CL pairs is shown in Table 4.

Table 4

1 - According to EU numbering for human immunoglobulin IgGl heavy chain constant region sequence

2 - According to Kabat/EU numbering for human light chain kappa constant region

[00146] For placement of an alternative disulfide bond between the CH1 region of the heavy chain and the constant region of the light chain, the phenylalanine (Phe/F) at position 126 of the heavy chain constant region (EU numbering) and the glutamine (Gln/Q) at position 124 of a kappa light chain constant region (Kabat/EU numbering) were chosen for a prototype antibody. Alternatively, the valine (Val/V) at position 173 of the heavy chain constant region and the serine (Ser/S) at position 162 of a kappa light chain constant region were used.

[00147] The interchain disulfide bond between a human IgGl heavy chain CH1 region and a human light chain constant region in a native antibody comprises the cysteine at position 220 in the heavy chain constant region and the cysteine at position 214 of the light chain constant region. In parallel with identifying a new/altemative cysteine pair, in silico mutagenesis of the cysteine residues at these two positions was undertaken. One strategy for mutating these residues was to identify complementary charged residue pairs that best compensated for the loss of stability that could result from removal of the native interchain disulfide bond. The charged residue pairs evaluated were lysine (Lys/K) or arginine (Arg/R) on one chain to interact with aspartic acid (Asp/D) or glutamic acid (Glu/E) on the other chain. After modeling the anti-B Fab with different charged pairs, the energy destabilization values were calculated and are shown in Table 5.

Table 5

[00148] The charged pair mutation set of heavy chain C220R with light chain C214E was chosen for inclusion in the constructs for further analysis. A representative diagram of a native Fab (WT Fab), a Fab with an alternative cysteine disulfide bond (Alt Cys Fab), and a Fab with an alternative cysteine disulfide bond with a charged pair (Alt Cys/Charged pair) are shown in FIG. 1. FIG. 2A depicts the sequence of a representative human IgGl heavy chain CH1 region (SEQ ID NO:4) and parallel sequence with the selected mutation F126C/C220R (SEQ ID NO:5) and the sequence of a representative kappa light chain (SEQ ID NO:2) and parallel sequence with the selected mutation Q124C/C214E (SEQ ID NO:7). FIG. 2B depicts the sequence of a

representative human IgGl heavy chain CH1 region (SEQ ID NO:4) and parallel sequence with the selected mutation F126C/C220R (SEQ ID NO:5) and the sequence of a representative lambda light chain (SEQ ID NO:3) and parallel sequence with the selected mutation

E125C/C214E (SEQ ID NO:8). FIG. 2C depicts the sequence of a representative human IgGl heavy chain CH1 region (SEQ ID NO:4) and parallel sequence with the selected mutation V173C/C220R (SEQ ID NO:6) and the sequence of a representative kappa light chain (SEQ ID NO:2) and parallel sequence with the selected mutation S162C/C214E (SEQ ID NO:9).

Example 2

Formation of antibody with alternative disulfide bonds

[00149] The alternative cysteine/charged pair heavy and light chain mutations, HC

F126C/C220R and LC Q124C/C214E, were introduced into a full-length heavy chain and light chain polypeptide, respectively. Similarly the HC V173C/C220R and LC S162C/C214E mutations were introduced into full-length heavy chain and light chain polypeptides. These polypeptides were used to analyze the ability of the heavy and light chains comprising the alternative cysteines and charged pairs to selectively associate. [00150] In order to determine whether the alternative cysteine heavy and light chain pairs were able to preferentially associate, competition experiments were performed. Four transfections were set up using Expi293F™ cells (ThermoFisher Scientific) and plasmids encoding: (1) a heavy chain containing the F126C/C220R mutations (HC F126C/C220R), the corresponding light chain containing the Q124C/C214E mutations, (LC Q124C/C214E), and a competitor light chain, a non-mutated light chain from the parent antibody (WT LC), (2) a heavy chain containing the V173C/C220R mutations (HC V173C/C220R), the corresponding light chain containing the S162C/C214E mutation (LC S162C/C214E), and a competitor light chain, a non-mutated light chain from the parent antibody (WT LC), (3) a non-mutated heavy chain (WT HC), the corresponding non-mutated light chain (WT LC), and a competitor light chain containing the Q124C/C214E mutation (LC Q124C/C214E), or (4) a non-mutated heavy chain (WT HC), the corresponding non-mutated light chain (WT LC), and a competitor light chain containing the S162C/C214E mutation (LC S162C/C214E). The transfections were done following the manufacturer’s protocols and the cells were incubated at 37°C. After six days, culture supernatants were removed, clarified by centrifugation and filtration, and subjected to Protein A chromatography (MabSelect™, GE Healthcare Life Sciences).

[00151] Purified proteins captured on the Protein A column were analyzed by non-reducing SDS-PAGE using Stain-free gels (BioRad) and quantified following fluorography on a Gel Doc system using Image Lab software (BioRad).

[00152] A heavy chain comprising an alternative cysteine is incompatible with a native non- mutated light chain for disulfide bond formation. Thus, if HC F126C/C220R or HC

V173C/C220R associates with the competitor LC, the resulting heavy chain and light chain complex does not form a disulfide bond, the complex dissociates, and the proteins are resolved separately by SDS-PAGE. In contrast, when HC F126C/C220R associates with LC

Q124C/C214E or HC V173C/C220R associates with LC S162C/C214E, the chains are covalently linked by a disulfide bond. These complexes are SDS and heat-stable and are resolved as one band by non-reducing SDS-PAGE. As shown in FIG. 3, a band at -150 kDa indicates assembly of an IgG molecule with two heavy chains each disulfide-linked to a light chain, a band at -125 kDa (termed 3/4 IgG) indicates assembly of an IgG molecule with two heavy chains and only one disulfide-linked light chain, and a band at -100 kDa indicates a heavy chain dimer (with no light chains, HC dimer). Light chains that are dissociated from the heavy chain complexes are seen as a band at ~25 kDa.

[00153] The percentage of heavy chain/light chain pairs is calculated by first compensating for lost integrated band intensity due to dissociated light chain(s) from 3/4 IgG (loss of one light chain) and heavy chain dimer (loss of two light chains). Using the adjusted values, %

Compatible Pairing = 100% x (IgG + (0.5 x 3/4 IgG)) / (IgG +3/4 IgG + HC dimer).

[00154] Theoretically, if the probability of an alternative cysteine light chain and a non- mutated light chain associating with a heavy chain was equal, then 50% Compatible Pairing would be expected. As shown in FIG. 3 and Table 6, alternative cysteine light chains demonstrated selective association with the corresponding alternative cysteine heavy chain when challenged by co-expression of the incompatible non-mutated light chain (lanes 1 and 2 represent Transfection 1 and 2, respectively).

Table 6

[00155] The pair of HC F126C/C220R and LC Q124C/C214E demonstrated superior selectivity as compared to HC V173C/C220R and LC S162C/C214E (89% Compatible pairing vs 79% Compatible pairing).

Example 3

Formation of prototype bispecific antibody comprising a heavy chain/light chain pair with an alternative disulfide bond [00156] It was investigated whether the heavy chain/light chain pair comprising an alternative disulfide bond described herein could be used in a bispecific antibody format. There were four polypeptides chains: (1) a heavy chain with a native cysteine at position 220 (WT HC), (2) a light chain with a native cysteine at position 214 (WT LC), (3) a heavy chain with an alternative cysteine at position 126 and an arginine at position 220 (HC F126C/C220R), and (4) a light chain with an alternative cysteine at position 124 and an glutamic acid at position 214 (LC Q124C/C214E for kappa chain or LC E125C/C214E for lambda chain). For this experiment, the variable regions forming the antigen-binding site for each arm were identical. Thus the only differences between the two heavy chain/light chain interfaces were at the sites of the alternative cy steines and the charged pair residues. To facilitate the formation of heavy chain

heterodimerization, the knob-into-hole technique was used to modify the two heavy chains. In one set of constructs, the HC F126C/C22GR polypeptide comprised an additional mutation in the CH3 region to form the knob and the WT HC polypeptide comprised an additional mutation in the CH3 to form the hole. In a second set of constructs, the HC F126C/C220R polypeptide comprised an additional mutation in the CH3 region to form the hole and the WT HC

polypeptide comprised an additional mutation in the CH3 to form the knob. The light chain constructs were the WT LC polypeptide and the LC Q124C/C214E (kappa) or LC E125C/C214E

(lambda) polypeptides.

[00157] Four plasmids were co-transfected into Expi293F™ cells. Each plasmid encoded a single polypeptide chain: a WT HC, a WT LC, a modified HC, or a modified LC as outlined in Table 7.

Table 7

[00158] The transfections were done following the manufacturer’s protocols and the cells were incubated at 37°C. After six days, culture supernatants were removed, clarified by centrifugation and filtration, and subjected to Protein A chromatography (Mab Select™, GE Healthcare Life Sciences).

[00159] An equal amount of purified protein from each transfection was denatured in buffer containing 1% SDS and heated to 95°C for 5 minutes. The samples were analyzed by non reducing SDS-PAGE using Stain-free gels (BioRad). Protein bands were visualized and quantified by fluorography on a Gel Doe system and Image Lab software (BioRad).

[00160] A fully assembled IgG is a heterodimer comprising disulfide-linked heavy and light chain pairs on both arms. A 3/4 IgG is a heterodimer comprising two heavy chains with only one heavy chain disulfide-linked to a light chain. A 1/2 IgG is a monomer comprising one heavy chain disulfide-linked to a light chain. Representative diagrams are shown in FIG. 4. The formation of a disulfide bond between two heavy chains is treated as independent of the formation of a disulfide bond between a heavy chain and a light chain, and thus for the purpose of calculating efficiency of heavy chain/light chain pairing, 1/2 IgG molecules are considered to be properly paired whereas disulfide-linked heavy chains without light chains are not. The percentage of heavy chain/light chain pairings is calculated by the equation: IgG + 1/2 IgG + (0.6 X 3/4 IgG).

[00161] The proteins as resolved by SDS-PAGE are shown in FIG. 4, wherein Lanes 1, 2, and 3 are proteins from Transfections 1 , 2, and 3, respectively. The percentage of each protein band as quantified is shown in Table 8. In addition, the calculated % HC/LC pairings are included in Table 8.

Table 8

[00162] These results demonstrated that the selected alternative cysteine and charged pair residues were effective at guiding assembly of distinct heavy chain/light chain pairing within a bispecific antibody format with results ranging from 87.3 to 91.0% HC/LC. In addition, these results showed that the alternative cysteine disulfide bond formation was effective using both kappa and lambda light chains.

Example 4

Purification of hispecific antibodies

[00163] The alternative cysteine/charged pair mutations were used in polypeptides to form a bispecific antibody wherein each arm of the bispecific antibody bound a unique antigen.

Bispecific antibodies were expressed in Expi293F cells after transient co-transfection of four plasmids as described in Example 3. The four chain antibody format was set up so that assembly of a native heavy chain with a native light chain would result in an antigen-binding site for a first antigen and assembly of a heavy chain comprising an alternative cysteine/charged pair with a light chain comprising an alternative cysteine/charged pair would result in an antigen-binding site for a second antigen. In each transfection as outlined in Table 9, one of the heavy chains comprised a CH3 knob mutation and the other heavy chain comprised a CH3 hole mutation to facilitate heavy chain heterodimerization. Table 9

[00164] The transfections were done following the manufacturer’s protocols and the cells were incubated at 37°C. After six days, antibodies were purified from culture supernatants using a series of chromatography steps, a Protein A affinity column, a cation exchange column, and a ceramic hydroxyapatite column. For Protein A affinity chromatography, the supernatant was applied to Mab Select SuRe™ cartridges (GE Healthcare Life Sciences), washed with HEPES buffered saline, and eluted with 50 mM sodium acetate, pH 3.5. The resulting elution pool (Pool M) was adjusted to pH 5.2 using 1 M Tris pH 8.0 and diluted to approximately 5 mg/ml protein with 20 mM sodium acetate pH 5.2. The pool was applied to a HiTrap® SP HP cation exchange column (GE Healthcare), washed with 20 mM sodium acetate, pH 5.2 and eluted with a 150 ml linear gradient of 50 mM to 600 mM sodium chloride in 20 mM sodium acetate, pH 5.2.

Antibody-enriched fractions were pooled (Pool C) and diluted with 4 volumes of 10 mM sodium phosphate pH 6.5. This pool was applied to a CHT™ Ceramic Hydroxyapatite Type II column (BioRad), washed with 10 mM sodium phosphate, pH 6.5, and eluted with a 300 ml linear gradient of 0 to 1 M sodium chloride in 10 mM sodium phosphate, pH 6.5. Fractions enriched for intact antibodies were pooled (Pool H). [00165] Following each chromatographic step, individual elution fractions were analyzed by SDS-PAGE and mass spectrometry to identify those that were enriched for fully assembled antibodies. As described above, these fractions were pooled at each step. An equal amount of purified protein from each sample was denatured in buffer containing 1% SDS and heated to 95°C for 5 minutes. The samples were analyzed by non-reducing SDS-PAGE using Stain-free gels (BioRad). Protein bands were visualized and quantified by fluorography on a Gel Doc system and Image Lab software (BioRad).

[00166] fhe proteins as resolved by SDS-PAGE are shown in FIGS. 5A and 5B. The percentage of each protein band as quantified is shown in Tables 10-13, as well as the calculated % HC/LC pairings.

Table 10

Table 11

Table 12

Table 13

[00167] In addition, the identity of the final purified antibody from each transfection was confirmed by non-reducing mass spectrometry (FIG. 6). Example 5

Binding capability of bispecific antibodies

[00168] Surface plasmon resonance (SPR) was used to evaluate the antigen-binding capabilities of a bispecific antibody generated with the alternative cysteines/charged pair mutations described herein. One of the antibodies described in Example 4 was used to determine the antigen-binding kinetic parameters. The bispecific antibody (Transfection 2) comprised (i) a heavy chain with a native cysteine with a hole modification in the CH3 region and a light chain with a native cysteine which forms a binding site for Antigen B; and (ii) a heavy chain with an alternative cysteine/charged pair with a knob modification in the CH3 region with a light chain with an alternative cysteine/charged pair which form a binding site for Antigen C. As controls for the binding assays,“one-armed” antibodies were constructed. These antibodies consist of a heavy chain with a CH3 knob mutation, its compatible light chain, and an“arm-less” IgG Fc region (no antigen-binding site) with a CH3 hole mutation. One-armed antibodies were prepared for both the alternative cysteine/charged pair and native cysteine versions of each antibody for Antigen B and Antigen C. An additional control for the binding assays was the parental antibody that specifically binds Antigen B or Antigen C.

[00169] The binding capabilities of the bispecific antibody and controls were measured using a Biacore SPR system (GE Healthcare LifeSciences). The kinetic analyses were performed using low antibody density on the chip surface to reduce potential avidity effects that may occur when analyzing a bivalent molecule. Briefly, anti-Fc antibody (Sigma-Aldrich) was immobilized on all four flow cells of a CM5 chip using amine coupling reagents (GE Healthcare LifeSciences). The bispecific antibody and control antibodies were captured on flow cells 2, 3, and 4 using flow cell 1 as a reference. Antigen B or Antigen C was injected at a flow rate of 50 pL/min at 37°C. Kinetic data were collected over time and fit using the simultaneous global fit equation to yield affinity constants (KD values) for each antibody.

[00170] The results are shown in Table 14 and Table 15. Table 14

Table 15

[00171] As shown in Table 14, antigen-binding kinetic parameters for the bispecific antibody and the one-armed antibodies were very similar to the parental anti-C antibody. Similarly, as shown in Table 15, the antigen-binding kinetic parameters for the bispecific antibody and the one armed antibodies were very similar to the parental anti-B antibody with the exception of the one- armed anti-B antibody with an alternative cysteine/charged pair. The parental anti-B antibody forms a very stable antibody/antigen complex, and determining an accurate dissociation rate requires a longer washout phase than was performed in this experiment.

[00172] These results indicate that the alternative cysteine/charged pair modifications exemplified herein do not diminish the antigen-binding activity of either arm of a bispecific antibody.

[00173] Although the foregoing present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present disclosure. The embodiments of the present disclosure described herein are intended to be merely exemplary, and those skilled in the art will recognize numerous equivalents to the specific procedures described herein. All such equivalents are considered to be within the scope of the present disclosure and are covered by the embodiments.

[00174] All publications, patents, patent applications, internet sites, and accession

numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession

number/database sequence were specifically and individually indicated to be so incorporated by reference.

[00175] Following are the sequences disclosed in the application:

Human IgGl constant region (SEQ ID NO: 1)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GG

PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YN

STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DE

LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human Kappa (Km3) light chain constant region (SEQ ID NO:2)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Human Lambda (Lambda 2) light chain constant region (SEQ ID NO:3)

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSK

QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Human IgGl CH1 and hinge region (SEQ ID NO:4)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP

Human IgGl CH1 and hinge region - HC F126C/C220R (SEQ ID NO:5)

ASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSRDKTHTCPPCP

Human IgGl CH1 and hinge region - HC V173C/C220R (SEQ ID NO:6)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPACLQSS

GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSRDKTHTCPPCP

Human Kappa light chain constant region - LC Q124C/C214E (SEQ ID NO:7)

RTVAAPSVFIFPPSDECLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEE

Human Lambda light chain constant region - LC E125C/C214E (SEQ ID NO:8)

GQPKAAPSVTLFPPSSECLQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT PSK

QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEES

Human Kappa light chain constant region - LC S162C/C214E (SEQ ID NO:9)

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQECVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEE

Human IgGlm3 CH1 and hinge region (SEQ ID NO: 10)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP

Human Kappa (Kml) light chain constant region (SEQ ID NO: 11)

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNVLQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC

Human Kappa (Km 1,2) light chain constant region (SEQ ID NO: 12)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC

Human Lambda (Lambda 1) light chain constant region (SEQ ID NO: 13)

GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK

QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Human Lambda (Lambda 3) light chain constant region (SEQ ID N014)

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPAKAGVETTTPSK

QSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

Human Lambda (Lambda 7) light chain constant region (SEQ ID NO: 15)

GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSK

QSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS