[0001] The creation of asymmetry in a native antibody structure is a prerequisite for the generation of multispecific binding proteins having two (e.g., bispecific antibodies) or more binding specificities. For example, by separating one or more Fvs on different asymmetric binding arms or Fabs, a bispecific antibody can be made with the flexibility of binding two different antigens or epitopes simultaneously. Despite these advantages, however, a wide variety of multispecific antibody technologies suffer process and manufacturing problems due to mispairings of various asymmetric heavy and light chains. For example, many of these technologies suffer from the so-called“light-chain problem,” wherein random pairing of the two different light chains with heavy chains generates various combinations of chain pairings other than the desired combination. In some cases, the light-chain problem can be circumvented by the use of a common light chain, which enables binding to both antigens or epitopes. However, this might not be possible for many antibodies since this format requires de novo antibody generation in transgenic mice. Furthermore, rare antibodies like broadly neutralizing anti-HIV antibodies derived from human patients cannot be adapted to such a format. Accordingly, there remains a need for alternative and creative solutions to the mispairing problem.

SUMMARY OF THE INVENTION

[0002] The present disclosure is based upon the discovery of a novel heterodimerization domain termed a“stabilized knockout domain” which may be used to form a“pseudoFab”. As disclosed herein, a pseudoFab can be incorporated into a wide variety of binding proteins and binding

formats to confer multispecific binding properties. In certain aspects, the pseudoFab moiety can facilitate preferential production, synthesis or purification of a desired multispecific binding protein while minimizing or eliminating the undesired chain mispairings that are commonly generated with conventional multispecific binding protein formats. In one aspect, the present disclosure provides a binding protein comprising:

a first pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain;

wherein the stabilized knockout domain comprises (3) one or more inactivating mutations which abolish binding to a target antigen; and (4) one or more engineered interchain disulfide bonds.

[0003] In one aspect, the present disclosure provides a binding protein comprising:

a first pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain;

wherein the stabilized knockout domain comprises (3) one or more inactivating mutations which abolish its binding to a target antigen relative to wild type domains and (4) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

[0004] In some embodiments, the binding protein is a multispecific binding protein further comprising:

at least a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B.

[0005] In one aspect, the present disclosure provides a multispecific binding protein comprising

a) a first pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain;

b) a first Fab portion comprising (3) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B; (4) a first CH1 domain paired with a first CL domain; and

wherein the stabilized knockout domain comprises (5) one or more inactivating mutations which abolish its binding to a target antigen; and (6) one or more engineered interchain disulfide bonds; or alternatively wherein the stabilized knockout domain comprises (5) one or more inactivating mutations which abolish its binding to a target antigen relative to wild type domains and (6) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

[0006] In another aspect, the present disclosure provides a multispecific binding protein comprising

a) a first pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a stabilized knockout domain, with the proviso that the first pseudoFab portion does not comprise a CH1 domain paired with a CL domain;

wherein the stabilized knockout domain comprises (3) one or more inactivating mutations which abolish its binding to a target antigen; and (4) one or more engineered interchain disulfide bonds; or alternatively wherein the stabilized knockout domain comprises (3) one or more inactivating mutations which abolish its binding to a target antigen relative to wild type domains and (4) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains

of the reference Fab molecule are replaced with VHX and VLX domains;

b) a first Fab portion comprising (5) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B; (6) a first CH1 domain paired with a first CL domain; and

c) a linker portion which operably links the first Fab portion and the first pseudoFab portion.

[0007] In another aspect, the present disclosure provides a multispecific binding protein comprising

a) a first pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain;

b) a first Fab portion comprising (3) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B; (4) a first CH1 domain paired with a first CL domain; and

wherein the stabilized knockout domain comprises (5) one or more inactivating mutations which abolish its binding to a target antigen; and (6) one or more engineered interchain disulfide bonds; or alternatively wherein the stabilized knockout domain comprises (5) one or more inactivating mutations which abolish its binding to a target antigen relative to wild type domains and (6) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

c) a linker portion which operably links the first Fab portion and the first pseudoFab portion.

[0008] In some embodiments, the linker portion is on one or more heavy chains.

[0009] In some embodiments, the multispecific binding protein further comprises a third VL domain (VLc) paired with a third VH domain (VHc), to form a third functional antigen binding site that binds target antigen C.

[0010] In some embodiments, the multispecific binding protein comprises independently one or two pseudoFab portion(s) and one or two Fab portion(s).

[0011] In some embodiments, the linker portion is a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker of the formulation (Gly4Ser)n, wherein n is 1-10.

[0012] In some embodiments, the heterodimerization domain comprises a full-length IgG antibody. In some embodiments, the heterodimerization domain comprises the Fc domain of a full-length IgG antibody or a functional fragment thereof.

[0013] In some embodiments, the binding protein comprises comprising separate proteins chains and selected from one of the following group:

(a) VHa-CH1-L1-VHb-L2-VHX and VLa-CL and VLb-L3-VLX;

(b) VHa-L2-VHX-L1-VHb-CH1 and VLa-L3-VLX and VLb-CL;

(c) VHa-CH 1 -L1 -VHa-CH 1 and VHb-L2-VHX-L3-VHb-L4-VHX and two chains VLb-L5-VLX and two chains VLa-CL;

wherein the chains of (a) and (b) can be present once or twice, and wherein L1 , L2, L3, L4 and L5 are linkers, which may independently be the same or different .

[0014] The present disclosure provides a multispecific antibody comprising

a) a first pseudoFab portion comprising:

(1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first antigen binding site that binds target antigen A;

(2) a first stabilized knockout VL domain (VLX) paired with a first stabilized knockout VH domain (VHX) to form a first disulfide stabilized knockout (dsKO) domain;

(3) a first heterodimerization domain (HD1);

wherein the first dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen and (ii) one or more engineered interchain disulfide bonds; or alternatively comprises (i) one or more inactivating mutations which abolish its binding to a target antigen relative to wild type domains and (ii) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with

VHX and VLX domains.

b) a first Fab portion comprising

(1) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(2) a first CH1 domain paired with a first CL domain; and

(3) a second heterodimerization domain (HD2).

[0015] In some embodiments, the first heterodimerization domain (HD1) is operably linked to the C-terminus of the VHX domain of the pseudoFab portion.

[0016] In some embodiments, the second heterodimerization domain (HD2) is operably linked to the C-terminus of the first CH1 domain of the first Fab portion.

[0017] In some embodiments, the first and second heterodimerization domains comprise first and second Fc domains.

[0018] In some embodiments, the Fc domains comprise the general structure hinge-CH2 domain-CH3 domain.

[0019] In some embodiments, the Fc domains comprise one or more knob-in-hole (KIH) mutations.

[0020] In some embodiments, one of the Fc domains comprises a first CH3 domain comprising one or both of S354C and T366W mutations, and the other Fc domain comprises a second CH3 domain comprising one or both of Y349C, T366S, L368A, and Y407V mutations.

[0021] In some embodiments, the Fc domains comprise H435R and/or Y436F mutations.

[0022] In some embodiments, the first pseudoFab portion comprises of a first polypeptide chain having a structure represented by the formula:

(la) N-VHa-L1-VHX-C

and a second polypeptide chain having a structure represented by the formula:

(lla) N-VLa-L2-VLX-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

[0023] In some embodiments, the first pseudoFab portion comprises of a first polypeptide chain having a structure represented by the formula:

(lb) N-VHX-L1-VHa-C

and a second polypeptide chain having a structure represented by the formula:

(lib) N-VLX-L2-VLa-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

[0024] In some embodiments, the first pseudoFab portion comprises of a first polypeptide chain having a structure represented by the formula:

(lc) N-VLa-L1-VHX-C

and a second polypeptide chain having a structure represented by the formula:

(lie) N-VHa-L2-VLX-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

[0025] In some embodiments, the first pseudoFab portion comprises of a first polypeptide chain having a structure represented by the formula:

(Ld) N-VHX-L1-VLa-C

and a second polypeptide chain having a structure represented by the formula:

(lid) N-VLX-L2-VHa-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

[0026] In some embodiments, at least two of Target Antigen A, Target Antigen B and Target Antigen C are different target antigens.

[0027] In some embodiments, at least one of the target antigens is a ligand of a cell surface receptor and at least one of the target antigens is a cell surface receptor.

[0028] In some embodiments, the antigen binding sites are derived from different antibodies.

[0029] In some embodiments, Target Antigen A, Target Antigen B, and Target Antigen C are the same target antigen.

[0030] In some embodiments, the antigen binding sites bind different epitopes on the same target antigen.

[0031] In some embodiments, the antigen binding sites bind the same epitope on the same target antigen.

[0032] In some embodiments, the antigen binding sites are derived from the same antibody.

[0033] In some embodiments, the melting temperature (Tm) of the pseudoFab portion at least 4 degrees Celsius higher than the reference Fab molecule.

[0034] In some embodiments, the engineered interchain disulfide bond is VH44C-VL100C.

[0035] In some embodiments, the engineered interchain disulfide bond is VH105C-VL43C.

[0036] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the VHX domain of the pseudoFab portion.

[0037] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRH3 of the VHX domain.

[0038] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRH2 of the VHX domain.

[0039] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRH1 of the VHX domain.

[0040] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the VLX domain of the PseudoFab portion.

[0041] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRL3 of the VLX domain.

[0042] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRL2 of the VLX domain.

[0043] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRL1 of the VLX domain.

[0044] In some embodiments, the VHX domain of the pseudoFab portion comprises comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79.

[0045] In some embodiments, the VLX/VHX pair is selected from the group consisting of:

(i) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 77;

ii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 78; and iii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 79.

[0046] In some embodiments, the binding protein further comprises one or more additional binding domains operably linked to an N- or C-terminus of the binding protein.

[0047] In some embodiments, the one or more additional binding domains are operably linked to N-terminus of the first or second pseudoFab portion.

[0048] In some embodiments, the one or more additional binding domains are operably linked to N-terminus of the first or second Fab portion.

[0049] In another aspect, the present disclosure provides a multispecific binding protein comprising four polypeptide chains that form at least two antigen-binding sites, wherein

(a) a first polypeptide comprises a structure represented by the formula:

VLa-L1-VLX [I]

(b) a second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-FC1 [II]

(c) a third polypeptide comprises a structure represented by the formula:

VLb-CL [III]

(d) a fourth polypeptide comprises a structure represented by the formula:

VHb-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 and L2 are amino acid linkers, which may independently be the same or different;

wherein

(1) the first VL domain (VLa) is paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with a second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[0050] In another aspect, the present disclosure provides an antigen binding protein comprising six polypeptide chains that form four antigen-binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-L3-VHb-CH1-FC1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-L3-VHb-CH1-FC2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2 and L3 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[0051] In another aspect, the present disclosure provides an antigen binding protein comprising six polypeptide chains that form four antigen-binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHb-CH1-L3-VHa-L2-VHX-FC1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VH b-CH 1 - L3- VH a- L2-VHX- F C2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2 and L3 are amino acid linkers,

wherein

(1 ) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; (ii) one or more engineered interchain disulfide bonds.

[0052] In another aspect, the present disclosure provides an antigen binding protein comprising six polypeptide chains that form four antigen-binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHa- L2- VHX- L3- VHa- L4-VHX- FC 1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VHb-CH1-L5-VHb-CH1-FC2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4 and L5 are amino acid linkers,

wherein

(1 ) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[0053] In another aspect, the present disclosure provides an antigen-binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein:

(a) the polypeptide comprises a structure represented by the formula:

VLa-L1-VLX [I]

(b) the second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-FC1 [II]

(c) the third polypeptide comprises a structure represented by the formula:

VLb-L3-VLc-L4-CL [III]

(d) the fourth polypeptide comprises a structure represented by the formula:

VHc-L5-VHb-L6-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VLc is a third immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VHc is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

VLX is a first stabilized knockout light chain variable domain;

VHX is a first stabilized knockout heavy chain variable domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4, L5 and L6 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the third VL domain (VLc) is paired with the third VH domain (VHc) to form a first functional antigen binding site that binds target antigen C;

(4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV);

(5) the stabilised knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[0054] In another aspect, the present disclosure provides an antigen-binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein:

(a) the polypeptide comprises a structure represented by the formula:

VLa-L1-VLX [I]

(b) the second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-FC1 [II]

(c) the third polypeptide comprises a structure represented by the formula:

VLb-L3-VLc-L4-CL [III]

(d) the fourth polypeptide comprises a structure represented by the formula:

VHc-L5-VHb-L6-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VLc is a third immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VHc is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

VLX is a first stabilized knockout light chain variable domain;

VHX is a first stabilized knockout heavy chain variable domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4, L5 and L6 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the third VL domain (VLc) is paired with the third VH domain (VHc) to form a first functional antigen binding site that binds target antigen C;

(4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV);

(5) the stabilised knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen of a reference Fab molecule; and (ii) one or more engineered interchain disulfide bonds.

[0055] In other aspects, some embodiments are related to all the binding proteins described herein wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen relative to wild type domains and (ii) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains. In those embodiments, binding to a target antigen is measured by methods known in the art such as but not limited to surface plasmon resonance and thermal stability is measured by methods known in the art such as but not limited to Differential Scanning Calorimetry.

[0056] In some embodiments, the FC1 and FC2 domains comprise one or more knob-in-hole (KIH) mutations, wherein the mutations facilitate Fc domain heterodimerization the polypeptides.

[0057] In some embodiments, one of FC1 or FC2 comprises a first CH3 domain comprising one or both of S354C and T366W mutations, and the other of FC1 or FC2 comprises a second CH3 domain comprising one or both of Y349C, T366S, L368A, and Y407V mutations, wherein the mutations facilitate Fc domain heterodimerization.

[0058] In some embodiments, the FC1 or FC2 domains comprise H435R and/or Y436F mutations.

[0059] In some embodiments, at least two of Target Antigen A, Target Antigen B and Target Antigen C are different target antigens.

[0060] In some embodiments, at least one of the target antigens is a ligand of a cell surface receptor and at least one of the target antigens is a cell surface receptor.

[0061] In some embodiments, the antigen binding sites are derived from different antibodies.

[0062] In some embodiments, Target Antigen A, Target Antigen B, and Target Antigen C are the same target antigen.

[0063] In some embodiments, the antigen binding sites bind different epitopes on the same target antigen.

[0064] In some embodiments, the antigen binding sites bind the same epitope on the same target antigen.

[0065] In some embodiments, the antigen binding sites are derived from the same antibody.

[0066] In some embodiments, the melting temperature (Tm) of the pseudoFab portion at least 4 degrees Celsius higher than the reference Fab molecule.

[0067] In some embodiments, the engineered interchain disulfide bond is VH44C-VL100C.

[0068] In some embodiments, the engineered interchain disulfide bond is VH105C-VL43C.

[0069] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the VHX domain of the pseudoFab portion.

[0070] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRH3 of the VHX domain.

[0071] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRH2 of the VHX domain.

[0072] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRH1 of the VHX domain.

[0073] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the VLX domain of the pseudoFab portion.

[0074] T In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRL3 of the VLX domain.

[0075] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRL2 of the VLX domain.

[0076] In some embodiments, at least one of the one or more inactivating mutations which abolish binding to the target antigen are present in the CDRL1 of the VLX domain.

[0077] In some embodiments, the VHX domain of the pseudoFab portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79.

[0078] In some embodiments, the VLX/VHX pair is selected from the group consisting of:

(i) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 77;

ii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 78; and

iii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 79.

[0079] In another aspect, the present disclosure provides for the use of a stabilized knockout domain to reduce heavy chain- light chain mispairing in a multispecific binding protein wherein the stabilized knockout domain comprises VHX and VLX domains comprising (3) one or more inactivating mutations which abolish its binding to the target antigen; and (4) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

[0080] In some embodiments, the engineered interchain disulfide bond is VH44C-VL100C.

[0081] In some embodiments, the engineered interchain disulfide bond is VH105C-VL43C.

[0082] In some embodiments, the VHX domain of the pseudoFab portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79.

[0083] In some embodiments, the VLX/VHX pair is selected from the group consisting of:

(i) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 77;

ii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 78; and iii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 79.

[0084] In some embodiments, the pseudoFab lacks the CH1 and CL domains.

[0085] In some embodiments, an isolated nucleic acid molecule comprising a nucleotide sequence encoding one or more of the binding protein is provided. In some embodiments, a kit of isolated nucleic acid molecules comprising one or more nucleotide sequences encoding one or more of the binding proteins is provided.

[0086] In some embodiments, an expression vector comprising the nucleic acid molecule is provided. In some embodiments, a kit of expression vectors comprising the kit of nucleic acid molecules is provided.

[0087] In some embodiments, an isolated host cell comprising the nucleic acid molecule or the expression vector is provided. In some embodiments, an isolated host cell comprising the kit of nucleic acid molecules or the kit of expression vectors is provided.

[0088] In some embodiments, a method of producing the binding protein comprising culturing the host cell under conditions such that the binding protein is expressed; and purifying the binding protein from the host cell, is provided.

[0089] In some embodiments, a pharmaceutical composition comprising a pharmaceutically

acceptable carrier and a therapeutically effective amount of the multispecific binding protein, is provided. In some embodiments, a multispecific binding protein for use as a medicament is provided.

[0090] In some embodiments, a method of treating a disorder in which antigen activity is detrimental, the method comprising administering to a subject in need thereof an effective amount of a multispecific binding protein is provided.

[0091] The summary of the invention described above is non-limiting and other features and advantages of the disclosed composition and methods will be apparent from the following detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE SEQUENCES

[0092] SEQ ID NO: 1 : Variable light chain sequence of trastuzumab.

[0093] SEQ ID NO: 2: Variable heavy chain sequence of trastuzumab.

[0094] SEQ ID NO: 3: Variable heavy chain sequence of trastuzumab ko variant 1.

[0095] SEQ ID NO: 4: Variable heavy chain sequence of trastuzumab ko variant 2.

[0096] SEQ ID NO: 5: Variable heavy chain sequence of trastuzumab ko variant 3.

[0097] SEQ ID NO: 6: anti-IL13~VL-G4S-anti-Her2-(Trastuzumab-Q100C)-VL.

[0098] SEQ ID NO: 7: anti-IL13~VH-G4S-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[0099] SEQ ID NO: 8: anti-IL13-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL.

[00100] SEQ ID NO: 9: anti-IL13-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00101] SEQ ID NO: 10: anti-IL13-VL3-IGK.

[00102] SEQ ID NO: 1 1 : anti-IL13-VH2-IGHG1.

[00103] SEQ ID NO: 12: anti-TNFalpha-VL-G4S-anti-Her2-(Trastuzumab-Q100C)-VL.

[00104] SEQ ID NO: 13: anti-TNFalpha-VH-G4S-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgGl

[00105] SEQ ID NO: 14: anti-TNFalpha-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL.

[00106] SEQ ID NO: 15: anti-TNFalpha-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgd .

[00107] SEQ ID NO: 16: anti-TNFa-VL-hulGKC.

[00108] SEQ ID NO: 17: anti-TNFa-VH-hulgG1.

[00109] SEQ ID NO: 18: anti-IL6R-VL-G4S-anti-Her2-(Trastuzumab-Q100C)-VL

[00110] SEQ ID NO: 19: anti-IL6R-VH-G4S-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00111] SEQ ID NO: 20: anti-IL6R-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00112] SEQ ID NO: 21 : anti-IL6R-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00113] SEQ ID NO: 22: anti-IL6R-VL-hulGKC.

[00114] SEQ ID NO: 23: anti-IL6R-VH-hulgG1.

[00115] SEQ ID NO: 24: anti-CTLA4-VL-G4S-anti-Her2-(Trastuzumab-Q100C)-VL

[00116] SEQ ID NO: 25: anti-CTLA4-VH-G4S-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00117] SEQ ID NO: 26: anti-CTLA4-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00118] SEQ ID NO: 27: anti-CTLA4-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG

[00119] SEQ ID NO: 28: anti-CTLA4-VL-hulGKC.

[00120] SEQ ID NO: 29: anti-CTLA4-VH -hulgGI .

[00121] SEQ ID NO: 30: anti-PD1-VL-G4S-anti-Her2-(Trastuzumab-Q100C)-VL.

[00122] SEQ ID NO: 31 : anti-PD1-VH-G4S-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00123] SEQ ID NO: 32: anti-PD1-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00124] SEQ ID NO: 33: anti-PD1-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00125] SEQ ID NO: 34: anti-huPD-1-VL-hulGKC.

[00126] SEQ ID NO: 35: anti-huPD-1-VH-hulgG1.

[00127] SEQ ID NO: 36: anti-IL4-VL-G4S-anti-Her2-(Trastuzumab-Q100C)-VL

[00128] SEQ ID NO: 37: anti-IL4-VH-G4S-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00129] SEQ ID NO: 38: anti-IL4-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00130] SEQ ID NO: 39: anti-IL4-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C)-VH-Fc-hulgG1.

[00131] SEQ ID NO: 40: anti-IL4-VL1-IGKC.

[00132] SEQ ID NO: 41 : anti-IL4-VH1-lgG1.

[00133] SEQ ID NO: 42: anti-PD1-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00134] SEQ ID NO: 43: anti-PD1-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgG

[00135] SEQ ID NO: 44: anti-CTLA4-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00136] SEQ ID NO: 45: anti-CTLA4-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgG

[00137] SEQ ID NO: 46: anti-IL4-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00138] SEQ ID NO: 47: anti-IL4-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgG

[00139] SEQ ID NO: 48: anti-IL13-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00140] SEQ ID NO: 49: anti-IL13-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-DKTHT-His6.

[00141] SEQ ID NO: 50: anti-IL13-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00142] SEQ ID NO: 51 : anti-IL13-VH-(G4S)2-anti-Her2-(Trastuzumab-VH_Var1-G44C)-VH-Fc-hulgG

[00143] SEQ ID NO: 52: anti-IL4-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00144] SEQ ID NO: 53: anti-IL4-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgGI (knob).

[00145] SEQ ID NO: 54: anti-PD1-hulGKC.

[00146] SEQ ID NO: 55: anti-PD1-VH-hulgG1 (hole-RF).

[00147] SEQ ID NO: 56: anti-IL13-VL -(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL.

[00148] SEQ ID NO: 57: anti-IL13-VH -(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgGI (knob).

[00149] SEQ ID NO: 58: anti-PD1-VL-hulGKC.

[00150] SEQ ID NO: 59: anti-PD1-VH-hulgG1 (hole-RF).

[00151] SEQ ID NO: 60: anti-PD1-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00152] SEQ ID NO: 61 : anti-PD1-VH- (G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgGI (knob).

[00153] SEQ ID NO: 62: anti-IL13-VL hulGKC.

[00154] SEQ ID NO: 63: anti-IL13-VH -hulgGI (hole-RF).

[00155] SEQ ID NO: 64: anti-CTLA4-VL -(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL.

[00156] SEQ ID NO: 65: anti-CTLA4-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgGI (knob).

[00157] SEQ ID NO: 66: anti-PD1-VL-hulGKC.

[00158] SEQ ID NO: 67: anti-PD1-VH-hulgG1 (hole-RF).

[00159] SEQ ID NO: 68: anti-IL4-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00160] SEQ ID NO: 69: anti-IL4-VH-(G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgGI (knob).

[00161] SEQ ID NO: 70: anti-IL13-VL hulGKC.

[00162] SEQ ID NO: 71 : anti-IL13-VH -hulgGI (hole-RF).

[00163] SEQ ID NO: 72: anti-PD1-VL-(G4S)2-anti-Her2-(Trastuzumab-Q100C)-VL

[00164] SEQ ID NO: 73: anti-PD1-VH- (G4S)2-anti-Her2-(Trastuzumab-G44C-Var2)-VH-Fc-hulgGI (knob).

[00165] SEQ ID NO: 74: anti-PD1-VL-hulGKC.

[00166] SEQ ID NO: 75: anti-PD1-VH-hulgG1 (hole-RF).

[00167] SEQ ID NO: 76: Variable light chain sequence of ds ko trastuzumab.

[00168] SEQ ID NO: 77: Variable heavy chain sequence of ds ko trastuzumab variant 1.

[00169] SEQ ID NO: 78: Variable heavy chain sequence of ds ko trastuzumab variant 2.

[00170] SEQ ID NO: 79: Variable heavy chain sequence of ds ko trastuzumab variant 3.

[00171] SEQ ID NO: 80: anti-TCR a/b x anti-CD123 Wild Type

[00172] SEQ ID NO: 81 : anti-TCR a/b x anti-CD123 - dsTrasK02

[00173] SEQ ID NO: 82: anti-TCR a/b - dsTrasK02 x anti-CD123

[00174] SEQ ID NO: 83: anti-CD3c x anti-CD123 Wild Type

[00175] SEQ ID NO: 84: anti-CD3c x anti-CD123-dsTrasK02

[00176] SEQ ID NO: 85: anti-CD3£-dsTrasK02 x anti-CD123

[00177] SEQ ID NO: 86: anti-CD3c x anti-CD123 Wild Type

[00178] SEQ ID NO: 87: anti-CD3c x anti-CD123-dsTrasK02

[00179] SEQ ID NO: 88: anti-CD3£-dsTrasK02 x anti-CD123

[00180] SEQ ID NO: 89: anti-TCR a/b x anti-TNP Negative control - Wild Type

[00181] SEQ ID NO: 90: anti-TCR a/b x anti-TNP-dsTrasK02 Negative control

[00182] SEQ ID NO: 91 : anti-TCR a^-dsTrasK02 x anti-TNP Negative control

[00183] SEQ ID NO: 92: anti-TNP x anti-CD123 Negative control - Wild Type

[00184] SEQ ID NO: 93: anti-TNP x anti-CD123-dsTrasK02 Negative control

[00185] SEQ ID NO: 94: anti-TNP-dsTrasK02 x anti-CD123 Negative control

[00186] SEQ ID NO: 95: anti-CD3c x anti-TNP Negative control - Wild Type

[00187] SEQ ID NO: 96: anti-CD3c x anti-TNP-dsTrasK02 Negative control

[00188] SEQ ID NO: 97: anti-CD3£-dsTrasK02 x anti-TNP Negative control

[00189] SEQ ID NO: 98: anti-CD3c x anti-TNP Negative control - Wild Type

[00190] SEQ ID NO: 99: anti-CD3c x anti-TNP-dsTrasK02 Negative control

[00191] SEQ ID NO: 100: anti-CD3£-dsTrasK02 x anti-TNP Negative control

BRIEF DESCRIPTION OF THE DRAWINGS

[00192] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[00193] FIG. 1A - FIG. 1B schematically depict dimeric bispecific tandem molecules comprising pseudoFab fragments with replacement of a CH1/CL pair with a disulfide stabilized Knockout domain (dsKO). A Tandem-(Fv-Fab x Fv-pseudoFab) molecule is depicted in FIG. 1A, and a Tandem-(Fv-pseudoFab x Fv-Fab) molecule is depicted in FIG. 1 B.

[00194] FIG. 2 schematically depicts an exemplary dimeric bispecific IgG molecule ((Fv-pseudoFab) x (Fv-Fab)-Fc)). A disulfide stabilized knockout domain (dsKO) replaces the CH/CL domains of one Fab arm of the IgG molecule. Peptide linkers (e.g., G4S or (G4S)2) link a first antigen binding site (Fv) to the dsKO domain to form a pseudoFab portion which binds antigen Target A. An Fc heterodimerization domain with knob-into-hole (KIH) or RF mutations links the pseudoFab portion with a second Fab binding arm that binds antigen Target B.

[00195] FIG. 3A - FIG. 3C schematically depict the monomeric fraction of an antibody construct comprising Trastuzumab WT VH/VL replacement of CH1/CL (FIG. 3A) compared to an antibody construct comprising disulfide stabilized Trastuzumab knouck (“dsTrastKO”) VH/VL replacement of CH1/CL (FIG. 3B), as well as the thermostability of both constructs (FIG. 3C).

[00196] FIG. 4 schematically depicts the PDB structure 1 N8Z of trastuzumab binding to HER2. The location of the four inactivating mutations which abolish binding to HER2 (R50, R59, Y33, and Y103) are noted.

[00197] FIG. 5A - FIG. 5B graphically depict the results of binding experiments demonstrating that dsTrastuKO variants 1-3 no longer bind HER2.

[00198] FIG. 6A - FIG. 6B depict the pseudolgG and and pseudoFab constructs used as certain controls in experiments.

[00199] FIG. 7 A - FIG. 7D depict a representative bispecific format and purification results according to certain exemplary embodiments. FIG. 7A shows the arrangement of the individual domains within the IgG scaffold. Fv1 (anti-l L4) is fused to VL/VH of dsTrasK02 via (G4S)2 linker. Fv2 (anti-IL13) retains the wildtype configuration. FIG. 7B shows the reduced (one light and one heavy chain) and oxidized form of the antibody using 4-12% Bis/Tris MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the product is shown in FIG. 7C using analytical size exclusion chromatography. Molecule integrity was verified by intact mass analysis using Agilent 6540 ultra-high definition (UHD) Q-TOF equipped with a Jet Stream dual ESI interface and an Agilent 1290/1260 Infinity LC System (FIG. 7D).

[00200] FIG. 8 shows analytical hydrophobic-interaction chromatography (HIC) results indicating that dsTrasK02-bispecific constructs were correctly paired and did not contain unexpected species.

[00201] FIG. 9 schematically depicts the crystal structure of a Pseudo-Fab IL13-dsTrasK02 construct having the ultrastructure of Figure 6A. The structure was solved at 3.75A. The structure shows the superposition of IL13-VH/VL domains of TrasK02-IL13 pseudo-Fab (light grey) and Fab anti- IL13 (dark grey).

[00202] FIG. 10A - 10B schematically depict dimeric bispecific tandem molecules comprising pseudoFab fragments with replacement of a CH1/CL pair with a disulfide stabilized Knockout domain (dsKO). Tandem-(Fv-Fab x Fv-pseudoFab)-lgG molecule is depicted in FIG. 10A, and a Tandem-(Fv-pseudoFab x Fv-Fab)-lgG molecule is depicted in FIG. 10B.

[00203] FIG. 11A - 11B schematically depict dimeric bispecific tandem molecules comprising pseudoFab fragments with replacement of a CH1/CL pair with a disulfide stabilized Knockout domain (dsKO). (((Fv-pseudoFab)[HC]-(Fv-pseudoFab)) x ((Fv-Fab)[HC]-(Fv-Fab)))-Fc molecule is depicted in FIG. 11A with an Fc heterodimerization domain with knob-into-hole (KIH) and RF mutations, and in FIG. 11 B. with RF mutations only.

[00204] FIG. 12A - FIG. 12D depict a representative bispecific tandem IgG design in which a first pseudoFab comprising a Fv1 domain (having binding specificity for a first Target Antigen A, i.e., GITR) and a dsTrasKO domain is appended to each Fv2 domain (having binding specificity for a second Target antigen B, i.e., 0x40) of a conventional IgG antibody (Pogalizumab). FIG. 12A

shows the arrangement of the individual domains within the IgG scaffold. Fv1 (anti-GITR) is fused to VL/VH of dsTrasK02 via (G4S)2 linker. Fv2 (anti-Ox40) retains the wildtype configuration. Fv1-dsTrasK02 is fused to Fv2-Ck/CH1 via (G4S)2 linker. FIG. 12B shows the reduced (two light and one heavy chain) and oxidized form of the antibody using 4-12% Bis/Tris MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the product is shown in FIG. 12C using analytical size exclusion chromatography. Molecule integrity was verified by intact mass analysis using Agilent 6540 ultra-high definition (UHD) Q-TOF equipped with a Jet Stream dual ESI interface and an Agilent 1290/1260 Infinity LC System (FIG. 12D).

[00205] FIG. 13A - FIG. 13D depict a representative trispecific CODV IgG design in which a first pseudoFab comprising a Fv3 domain (having binding specificity for a first Target Antigen A, i.e., CD137) and a dsTrasKO domain is paired with a CODV arm (having a binding specificity (Fv1) for a second Target antigen B, i.e., 0x40 and a binding specificity (Fv2) for a third Target Antigen C, i.e., PD1). FIG. 13A shows the arrangement of the individual domains within the CODV IgG scaffold. Fv1 (anti-Ox40) and Fv2 (anti-PD1) on the CODV arm are fused to wildtype lambda and CH1 domains. Fv3 (anti-CD137) on the Fab arm is fused to VL/VH of dsTrasK02 via (G4S)2 linker. FIG. 13B shows the reduced (two light and two heavy chains) and oxidized form of the CODV antibody using 4-12% Bis/Tris MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the product is shown in FIG. 13C using analytical size exclusion chromatography. Molecule integrity was verified by intact mass analysis using Agilent 6540 ultra-high definition (UHD) Q-TOF equipped with a Jet Stream dual ESI interface and an Agilent 1290/1260 Infinity LC System (FIG. 13D).

[00206] FIG. 14 depicts a schematic of a two-step purification process used to ensure correct light chain pairing with dsTrasK02 knockout bispecific molecules.

[00207] FIG. 15A- FIG. 15B depict cytotoxicity assays of human panT cells against THP-1 target cells co-incubated with bispecific antibodies anti-CD3s x anti-CD123. FIG. 15A corresponds to bispecific antibodies with ID numbers 33, 34, and 35 with negative controls 45 and 46. FIG. 15B corresponds to bispecific antibodies with ID numbers 36, 37, and 38 with negative controls 47 and 48. T effector cells and CFSE-labeled THP-1 target cells were seeded in an effector to target ratio of 10:1 and co-incubated with serial dilutions of respective bispecific molecules (10 nM - 0 nM) for 20 hours at 37°C. Dead cells were stained with 7-AAD and measured by flow cytometry.

Cytotoxic activity was calculated based on percentage of dead THP-1 target cells (7-AAD/CFSE double positive). Data show dead target cells [%] against concentration of bispecific molecules [pM] as mean of two representative healthy donors.

DETAILED DESCRIPTION

I. DEFINITIONS

That the disclosure may be more readily understood, selected terms are defined below.

[00208] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino acids such as a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, c-N,N,N-trimethyllysine, c-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, u-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention. Naturally occurring residues may be divided into classes based on common side chain properties (see Table 1).

Table 1.

Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid residues. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

[00209] As used herein, the term“mutation” or“mutated” refers to an alteration of the amino acid sequence by deletion, insertion and/or substitution of one or more amino acids. A mutation is introduced with respect to a given sequence, e.g., the amino acid sequence of a VL1 and/or VH1 pair that specifically recognizes epitope 1. The term“non-mutated” refers to any amino acid sequence exhibiting functional properties, e.g., any sequence still showing binding properties. This is illustrated as follows: A VH1/VL1 is mutated in such that it does not specifically bind to an epitope. The non-mutated form of this VH1/VL1 still specifically binds to epitope 1. Every VH/VL domain of an antibody binding to any epitope is thus, suitable to be mutated to serve as a scaffold protein of the invention.

[00210] As used herein, the term“variant” refers to an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence it is derived from, for example SEQ ID NO: 1 or SEQ ID NO: 2. The determination of percent identity between two sequences is accomplished using the mathematical algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLAST programs,

the default parameters of the respective programs are used. Alternatively, a variant can also be defined as having up to 20, 15, 10, 5, 4, 3, 2, or 1 amino acid substitutions, in particular conservative amino acid substitutions. Conservative substitutions are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company). An overview of physical and chemical properties of amino acids is given in Table 1 above. In a particular embodiment, conservative substitutions are substitutions made with amino acids having at least one property according to Table 1 in common (i.e. , of column 1 and/or 2). The term“variant” also includes fragments. A fragment has an N-terminal and/or C-terminal deletion of up to 20, 15, 10, 5, 4, 3, 2, or 1 amino acid(s) in total. In addition or alternatively, the variant may be modified, for example by N-terminal and/or C-terminal amino acid additions of up to 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 amino acid(s) in total.

[00211] As used herein, the term“binding protein” or“binding polypeptide” refers to a polypeptide (e.g., an antibody or fragment thereof) that contains at least one binding site which is responsible for selectively binding to a target antigen of interest (e.g., a human antigen). Exemplary binding sites include, but are not limited to, an antibody variable domain, a ligand binding site of a receptor, or a receptor binding site of a ligand. In certain aspects, the binding polypeptides comprise multiple (e.g., two, three, four, or more) binding sites. In certain aspects, the binding protein is not a therapeutic enzyme.

[00212] As used herein, the term“Her2” or“HER2” refers to human epidermal growth factor receptor 2 which is a member of the epidermal growth factor receptor family.

[00213] As used herein, the term “binding protein” refers to a non-naturally occurring or recombinant or engineered molecule capable of specifically binding to at least one antigen. In a particular embodiment, a binding protein comprises at least one VH / VL pair that specifically binds to an antigen.

[00214] Production of a bispecific binding protein by co-expression of the two light and two heavy chains in a single host cell can be highly challenging because of the low yield of desired bispecific binding protein and the difficulty in removing closely related mispaired binding protein contaminants (Suresh et al., Proc. Natl. Acad. Sci. U.S.A.83, 7989-7993, 1986). This is because heavy chains form homodimers as well as the desired heterodimers, referred to herein as the

“heavy chain-pairing problem.” Additionally, light chains can mispair with non-cognate heavy chains, referred to herein as the“light chain pairing problem.” Consequently, co-expression of two antibodies can give rise to up to nine unwanted species in addition to the desired bispecific binding proteins.

[00215] As used herein a“heterodimerization domain” refers to a subunit of a bi- or a multi specific binding protein that facilitates, directs or forces the correct assembly of light chains and their cognate heavy chains to result in the desired protein while preventing mispairing of the respective light or heavy chains.

[00216] As used herein, the term “heterodimerizing Fc” or “functional fragment of a heterodimerizing Fc refers to a mutant form of the constant domain, e.g., the CH2-CH3 or CH2-CH3-CH4, that is mutated with regard to a naturally occurring Fc part in that it no longer forms homodimers but forms a heterodimer with a correspondingly mutated Fc part. Thus, the term refers to one part of the two chains that form a heterodimer. Several of such pairs are known in the art and comprise, e.g., knob-in-hole (KIH) variant or a EV-RWT variant.

[00217] Ridgeway and coworkers generated a CH3 interface favoring heterodimeric assembly by replacing small side chains on one CH3 interface with larger ones to create a knob and replacing large side chains on the other CH3 domain with smaller ones to generate a hole. Testing such variants demonstrated a preferential heterodimerization. This original knobs-into-holes mutations were further extended to identify further suitable combinations by phage display which were used to generate bispecific IgG antibodies testing additional substitutions allowing for disulfide bond formation. The knobs-in-hole variants are described further in U.S. Patent No. 5,732,168 and U.S. Patent No. 8,216,805, which are herein incorporated by reference. Accordingly, in an embodiment, the CH3 domain of one FC domain or heterodimerization domain contains the mutations Y349C, T366S, L368A, and Y407V, and the CH3 domain of another FC domain or heterodimerization domain contains the mutations S354C and T366W (amino acid position being indicated by reference to an lgG1 sequence).

[00218] As used herein, the term“homodimerization domain” refers to a domain mediating the homodimerization of two like domains, e.g., two heavy chains. Heavy chain pairing is mediated by the last domain of the constant region, i.e., CH3 in IgG molecules, which forms high-affinity homodimer complexes (KD ~ 10 pM). Further interactions reside in the hinge region responsible

for covalent linkage of two heavy chains, which form after heavy chain assembly. Interaction in a CH3 homodimer involves approximately 16 residues at the CH3-CH3 interface as shown for human y1 CH3 with patch formed by 6 residues (T366, L368, F405, Y407 and K409) at the center of the interface strongly contributing to stability. Homodimerization domains include, but are not limited to, Fc regions and effector modified variants thereof and fragments of either, CH2 domains or fragments thereof, CH3 domains or fragments thereof, CH4 domains or fragments or the like.

[00219] Naturally-occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length “light” chain (typically having a molecular weight of about 25 kDa) and one full-length“heavy” chain (typically having a molecular weight of about 50-70 kDa). The terms“heavy chain” and“light chain,” as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 1 10 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain IgG immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[00220] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, lgG1 , lgG2, lgG3, and lgG4. IgM has subclasses including, but not limited to, lgM1 and lgM2. IgA is similarly subdivided into subclasses including, but not limited to, lgA1 and lgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a“J” region of about 12 or more amino acids, with the heavy chain also including a“D” region of about 10 more amino acids. See, e.g., Fundamental Immunology (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.

[00221] As used herein, the term“CDR sets” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL I NTEREST (National Institutes of Health, Bethesda, MD (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Affol. Biol. 196: 901-17; Chothia et al., 1989, Nature 342: 877-83) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1 , L2, and L3 or H1 , H2, and H3 where the“L” and the “H” designate the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, 1995, FASEB J. 9: 13339; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C.“Protein sequence and structure analysis of antibody variable domains,” In Antibody Engineering, Vol. 2. Kontermann R., Dikel S., eds. Springer-Verlag, Berlin, p. 33-51 (2010). The amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other

heavy and light chain variable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.

[00222] In some embodiments, CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[00223] The term“Fc,” as used herein, refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is typically of human origin and can be any of the immunoglobulins, although lgG1 and lgG2 are used in exemplary embodiments. Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., lgG1 , lgG2, lgG3, lgA1 , and lgGA2). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term“native Fc,” as used herein, is generic to the monomeric, dimeric and multimeric forms.

[00224] An F(ab) fragment typically includes one light chain and the VH and CH1 domains of one heavy chain, wherein the VH-CH1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, an F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CH1 domain.

[00225] A F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CH1 and CH2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule. [00226] As used herein, the term“Tm” refers to the melting temperature of a binding protein, an antigen-binding protein, an antibody and is a parameter critical for the thermal stability of antigen binding proteins. The Tm commonly refers to the thermal stability of the Fv fragment, i.e., a variable region heavy and light chain (VH/VL). The Tm can be measured by differential scanning calorimetry (DSC).

[00227] One embodiment of the disclosure provides binding proteins having biological and immunological specificity to between one and three target antigens. Another embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the disclosure provides host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).

[00228] The term“antigen” or“target antigen” or“antigen target,” as used herein, refers to a molecule or a portion of a molecule (e.g., an epitope) that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[00229] As used herein, the term“epitope” or“target epitope” or“epitope target” refers to any determinant, e.g., a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. For example, but in no way limiting, a target epitope A may be a first epitope of an antigen and a target epitope B may be a second epitope of the antigen. Alternatively, the target epitope B may be a second epitope on a second antigen. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or by an antigen-binding fragment of an antibody or by a binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex

mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is < 108 M, when the equilibrium dissociation constant is < 1 9 M, or when the dissociation constant is < 10 10 M.

[00230] As used herein, the term“linker” refers to 0-100 contiguous amino acid residues. The linkers are, present or absent, and same or different. Linkers may all have the same amino acid sequence or may all have different amino acid sequences.

[00231] In some embodiments, the term“linker” refers to 1-15 contiguous amino acid residues. Typically, a linker provides flexibility and spatial separation between two amino acids or between two polypeptide domains. A linker may be inserted between VH, VL, CH and/or CL domains to provide sufficient flexibility and mobility for the domains of the light and heavy chains depending on the format of the molecule, e.g., to fold into cross over dual variable region immunoglobulins. A linker is typically inserted at the transition between variable domains between variable and knockout domain, or between variable and constant domains, respectively, at the amino sequence level. The transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be determined by techniques of modeling or secondary structure prediction. In certain exemplary embodiments, the linker may be inserted between Fab domains to create a tandem Fab antibody. In particular embodiments, the linker may be inserted between the N terminus of a VH domain of a first Fab and the C terminus of a CH1 domain of a second Fab.

[00232] The identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element(s) necessary to achieve in the linker. For example, glycine, serine and alanine are suitable for linkers having maximum flexibility. Certain combinations of glycine, proline, threonine and serine are useful if a more rigid and extended linker is desired. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as needed depending on the desired properties.

[00233] In some embodiments, a linker comprises: a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly- Gly-Gly; SEQ ID NO: x); a peptide with five glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x); and a peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: x).

[00234] In some embodiments, a linker comprises small amino acids, like Gly, Ala or Ser.

[00235] In some embodiments, a linker comprises Gly and Ser, or GS, GGS, GGGS or GGGGS. In some embodiments, a linker comprises (Gly-Gly-Gly-Gly-Ser)2 (i.e., (GGGGS)2). In some embodiments, a linker comprises (Gly-Gly-Gly-Gly-Ser)3 (i.e., (GGGGS)3).

[00236] In some embodiments, a linker comprises Gly-Gly- Gly-Gly-Ser (SEQ ID NO: x), the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly- Gly-Ser (SEQ ID NO: x), and the peptide Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO:x).

[00237] In some embodiments, a linker comprises a single Ser residue; a single Val residue; a dipeptide selected from Arg-Thr, Gin-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), Thr-Val-Ala-Ala-Pro (SEQ ID NO: x), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: x), Gln-Arg- lle-Glu-Gly (SEQ ID NO: x); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: x), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO:x), His-lle-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: x), and Asp-Lys-Thr-His-Thr (SEQ ID NO: x).

[00238] In some embodiments, two tandem Fabs are linked through a (Gly-Gly-Gly-Gly-Ser)2 linker. In some embodiments, the linker between a stabilized knocked out domain and a VH/VL pair is (Gly-Gly-Gly-Gly-Ser)2 linker.

[00239] In some embodiments having CODV-Fab portion wherein L1 and L2 are on the light chain and L3 and L4 are on the heavy chains, L1 is 3 to 12 amino acid residues in length, L2 is 3 to 14 amino acid residues in length, L3 is 1 to 8 amino acid residues in length, and L4 is 1 to 3 amino acid residues in length. In some embodiments, L1 is 5 to 10 amino acid residues in length, L2 is 5 to 8 amino acid residues in length, L3 is 1 to 5 amino acid residues in length, and L4 is 1 to 2 amino acid residues in length. In some embodiments, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length. In some embodiments, L1 is 10 amino acid residues in length, L2 is 10 amino acid residues in length, L3 is 0 amino acid residue in length, and L4 is 0 amino acid residues in length. In some embodiments, L1 , L2, L3, and L4 each have an independently selected length from 0 to 15 amino acids (e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids), wherein at least two of the linkers have a length of 1 to 15 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids). In some embodiments, L1 , L2, L3, and L4 are Asp-Lys-Thr-His-Thr (SEQ ID NO: x). In some embodiments, linker(s) comprise the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), L2 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: x), L3 comprises the sequence Ser, and L4 comprises the sequence Arg-Thr. In some embodiments, L3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x). In some embodiments, L1 comprises the sequence Ser, L2 comprises the sequence Arg-Thr, L3 comprises the sequence Gly-GIn-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x) and L4 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: x).

[00240] In some embodiments, L1 , L2, L3 and L4 each independently comprises a sequence selected from (Gly-Gly-Gly-Gly-Ser)n (wherein n is an integer between 0 and 5; SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Ser, Arg-Thr, Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x). In some embodiments, L1 comprises the sequence Gly-GIn-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), L2 comprises the sequence Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), L3 comprises the sequence Ser, and L4 comprises the sequence Arg-Thr. In some embodiments, L1 comprises the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), L2 comprises the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises

the sequence Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x), L2 comprises the sequence Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), L2 is 0 amino acids in length, L3 comprises the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), and L4 is 0 amino acids in length. In some embodiments, L1 and L2 are zero amino acids in length, and L3 and L4 each comprise a sequence independently selected from (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: x) (wherein n is an integer between 0 and 5; SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Ser, Arg-Thr, Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x). In some embodiments, L3 and L4 are zero amino acids in length, and L1 and L2 each comprise a sequence independently selected from (Gly-Gly-Gly-Gly-Ser)n (wherein n is an integer between 0 and 5; SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: x), Ser, Arg-Thr, Thr-Lys-Gly-Pro-Ser (SEQ ID NO: x), Gly-GIn-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: x), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: x).

[00241] In some embodiments, linker(s) comprise a sequence derived from a naturally occurring sequence at the junction between an antibody variable domain and an antibody constant domain (e.g., as described in WO2012/135345). For example, in some embodiments, the linker comprises a sequence found at the transition between an endogenous VH and CH1 domain, or between an endogenous VL and CL domain (e.g., kappa or lambda). In some embodiments, the linker comprises a sequence found at the transition between an endogenous human VH and CH1 domain, or between an endogenous human VL and CL domain (e.g., human kappa or lambda).

[00242] The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline are suitable for use in the binding proteins

described herein. For additional descriptions of linker sequences, see, e.g., WO2012135345, W02017/180913 incorporated by reference.

[00243] As used herein, the term“valency” refers to the number of binding sites of a binding protein, an epitope, an antigen-binding protein or an antibody. For example, the term“monovalent binding protein” refers to a binding protein that has one antigen binding site. The term“bivalent binding protein” refers to a binding protein that has two binding sites. The term“trivalent binding protein” refers to a binding protein that has three binding sites. The term“tetravalent binding protein” refers to a binding protein that has four binding sites. In particular embodiments the divalent binding protein can bind to one antigen target. In other embodiments, the divalent binding protein can bind to two different antigen targets. In particular embodiments the trivalent binding protein can bind to one antigen target, i.e., is monospecific. In other embodiments, the trivalent binding protein can bind to two different antigen targets, i.e., is bispecific. In other embodiments, the trivalent binding protein can bind to three different antigen targets, i.e., is trispecific. In particular embodiments the tetravalent binding protein can bind to one antigen target, i.e., is monospecific. In other embodiments, the tetravalent binding protein can bind to two different antigen targets, i.e., is bispecific. In other embodiments, the tetravalent binding protein can bind to three different antigen targets, i.e., is trispecific. In other embodiments, the tetravalent binding protein can bind to four different antigen targets, i.e., is tetraspecific.

[00244] As used herein, the term“specificity” refers to the number of binding specificities of a binding protein, an epitope, an antigen-binding protein or an antibody. For example, the term “monospecific binding protein” refers to a binding protein that specifically binds to one antigen target. The term“bispecific binding protein” refers to a binding protein that specifically binds to two different antigen targets. The term“trispecific binding protein”" refers to a binding protein that specifically binds to three different antigen targets. The term“tetraspecific binding protein” refers to a binding protein that specifically binds to four different antigen targets and so forth.

[00245] As used herein, the term“selective recognition site” refers to a modification in the binding protein allowing to be selectively recognized by an affinity reagent binding to the selective recognition site. Examples of a selective recognition site comprise the binding site for protein A in the Fc part of an immunoglobulin.

[00246] As used herein, the term“affinity reagent” refers to a reagent that contains a ligand that is immobilized on a matrix and specifically binds to surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Affinity reagents are tools in affinity chromatography, where purification is enabled by the specific interaction between the ligand and the product. “Protein L,” which is an example of an affinity reagent, refers to recombinant protein L that is immobilized on a matrix to form a ligand that has affinity for a subset of the variable domain of immunoglobulin kappa light chains. Such matrices can be resin. Another example of an affinity reagent is“KappaSelect,” which refers to a recombinant 13 kDa camelid-derived single chain antibody that is immobilized onto a matrix to form a ligand that has affinity for the constant domain of human immunoglobulin kappa light chains. Another example of an affinity reagent is Protein A. Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It has been shown via crystallographic refinement that the primary binding site for protein A is on the Fc region, between the CH2 and CH3 domains. In addition, protein A has been shown to bind human IgG molecules containing IgG F(ab')2 fragments from the human VH3 gene family. Protein A can bind with strong affinity to the Fc portion of immunoglobulin of certain species.

[00247] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term “KD,” as used herein, refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[00248] As used herein, the term“specifically binds” refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 10-6 M, 1 x 107 M, 1 x 10 M, 1 x 109 M, 1 x 10 10 M, 1 x 10 11 M, 1 x 10 12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. Binding affinity of an antigen to a binding protein or an antibody can be conducted by surface plasmon resonance (SPR) using a BIAcore instrument.

[00249] As used herein, the term“reference Fab molecule” refers to a molecule that is identical to the pseudoFab molecule except that, in a pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains. The“reference Fab molecule” refers to a molecule wherein variable domains are identical to the variable domains of the pseudoFab and has CH1 and CL domains. In the pseudoFab molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

[00250] As used herein, the term“nucleic acid” refers to polymeric or oligomeric macromolecules, or large biological molecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer strands coiled around each other to form a double helix. The DNA strand is also known as polynucleotides consisting of nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase as well as a monosaccharide sugar called deoxyribose or ribose and a phosphate group. Naturally occurring nucleobases comprise guanine (G), adenine (A), thymine (T), uracil (U) or cytosine (C). The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. If the sugar is deoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers.

[00251] As used herein, the term“polynucleotide” refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length. It is understood that the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term“polynucleotide” specifically includes single-stranded and double-stranded forms of DNA.

[00252] An“isolated polynucleotide” is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[00253] An“isolated polypeptide” is one that: (1 ) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g. , from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof. In exemplary embodiments, the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[00254] As used herein, the term“expression vector” also referred to as an expression construct, usually refers to a plasmid or virus designed for protein expression in cells. The term“vector” refers to a protein or a polynucleotide or a mixture thereof which is capable of being introduced or of introducing proteins and/or nucleic acids comprised therein into a cell. Examples of vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes. In particular, a vector is used to transport a gene product of interest, such as e.g. , foreign or heterologous DNA into a suitable host cell. Vectors may contain “replicon” polynucleotide sequences that facilitate the autonomous replication of the vector in a host cell. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell, which, for example, replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted DNA can be generated. In addition, the vector can also contain the necessary elements that permit transcription of the inserted DNA into an mRNA molecule or otherwise cause replication of the inserted DNA into multiple copies of RNA. Vectors may further encompass“expression control sequences” that regulate the expression of the gene of interest. Typically, expression control sequences are polypeptides or polynucleotides such as but not limited to promoters, enhancers, silencers, insulators, or repressors. In a vector comprising more than one polynucleotide encoding for one or more gene products of interest, the expression may be controlled together or separately by one or more expression control sequences. More specifically, each polynucleotide comprised on the vector may be control by a separate expression control sequence or all polynucleotides comprised on the vector may be controlled by a single expression control sequence. Polynucleotides comprised on a single vector controlled by a single expression control sequence may form an open reading frame. Some expression vectors additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and/or allow translation of the mRNA into a protein molecule. Many molecules of mRNA and polypeptide encoded by the inserted DNA can thus be rapidly synthesized.

[00255] As used herein, the term“host cell” refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term“host cell” as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To express a binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, in exemplary embodiments, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.

[00256] As used herein, the term “pharmaceutical composition” refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[00257] The term“pharmaceutically acceptable carrier” or“physiologically acceptable carrier,” as used herein, refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a binding protein.

[00258] The terms “effective amount” and “therapeutically effective amount,” when used in reference to a pharmaceutical composition comprising one or more binding proteins (e.g.,

antibodies or antigen-binding fragments thereof), refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein (e.g., an antibody or antigen-binding fragment thereof) sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific antibody-like binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein or multispecific binding protein is to be administered in vivo, factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.

[00259] As used herein, the term“method of production of a binding protein” refers to recombinant methods of protein expression using techniques well known in the art.

II. PSEUDOFAB MOIETIES

[00260] In certain embodiments, a binding molecule described herein comprises at least one pseudoFab moiety. As used herein, a“pseudoFab” moiety is analogous to a Fab moiety of a conventional antibody in that it comprises a functional antigen binding portion formed by the pairing of a variable light chain (VL) domain with a variable heavy chain (VH). However, whereas the VL and VH domains of a conventional Fab are directly fused with or linked to a constant light chain (CL) domain and a constant heavy chain 1 (CH1) domain, respectively, a pseudoFab moiety lacks CH1 and CL domains. Instead, the VL and VH domains of the pseudoFab are operatively linked to a second pair of stabilized knockout VL and VH domains (denoted herein as VLX and VHX) which form an inactive or non-functional binding portion (herein, a“stabilized knockout” portion or domain) that it is incapable of specifically binding to a target antigen (e.g., any target antigen). In certain embodiments, the pseudoFab moiety is incapable of binding the target antigen of the corresponding Fab moiety from which it is derived. The pseudoFab moiety lacks CH and CL domains.

[00261] While unable to selectively bind a target antigen, the VLX and VHX domains of a

pseudoFab nevertheless preferentially associate which each other to form a stable chain pairing. Therefore, by appending a pseudoFab to one or more additional binding domains of differing specificities, the inherent stability of the VLX/VHX chain pairing of a pseudoFab can drive heterodimerization of the chains of a desired multispecific binding molecule.

[00262] Accordingly, a pseudoFab of the present disclosure comprises or consists of a first polypeptide chain having a structure represented by the formula:

(I) VHa-L1-VHX; or

(II) VHb-L1-VHX

and second polypeptide chain having a structure represented by the formula:

(III) VLa-L2-VLX; or

(IV) VLb-L2-VLX

wherein

VHX associates with VLX to form a knockout domain,

VH associates with VL to form a first functional antigen binding domain, and

L1 and L2 are linkers, which may present or absent.

[00263] In certain embodiments, the first polypeptide chain of the pseudoFab has the structure VH-L1-VHX and the second polypeptide chain of the pseudoFab has the structure VL-L2-VLX.

[00264] In other embodiments, the first polypeptide of the pseudoFab has the structure VHX-L1-VH and the second polypeptide chain has the structure VLX-L2-VL.

[00265] In some embodiments, the binding protein comprises separate proteins chains selected from one of the following group:

(a) VHa-CH1-L1-VHb-L2-VHX and VLa-CL and VLb-L3-VLX;

(b) VHa-L2-VHX-L1-VHb-CH1 and VLa-L3-VLX and VLb-CL;

(c) VHa-CH1-L1-VHa-CH1 and VHb-L2-VHX-L3-VHb-L4-VHX and two chains VLb- L5-VLX and two chains VLa-CL;

wherein the chains of (a) and (b) can be present once or twice, and wherein L1 , L2, L3, L4 and L5 are linkers, which may independently be the same or different.

(a) Knockout Domains

[00266] The“knockout” domain of a pseudoFab can be generated by any means which results in abrogation or decrease of the binding affinity or specificity of a normally functional antigen binding site. In certain embodiments, the knockout domain of the pseudoFab has been rendered inactive or non-functional by one or mutations in one or both of the VLX and VHX domains of the knockout domain. In one embodiment, a knockout modification is an amino acid substitution. In other embodiments, a knockout modification is an amino acid insertion or deletion. In another embodiment, a knockout modification is a combination of one or more amino acid substitutions, amino acid insertions and amino acid deletions. In yet other embodiments, the knockout domain is rendered non-functional by covalent modification with, for example, a moiety which interferes with the ability of a variable domain to bind a target antigen.

[00267] In certain embodiments, the knockout modification abolishes binding by creating repulsion or disruption of stabilized antigen-binding protein complexes. In certain embodiments, the knockout modification may comprise replacing a residue which normally forms a contact with the target antigen with an amino acid that creates charge-charge repulsion with the target antigen. Additionally or alternatively, mutations which destabilize complexes of pi-pi interactions can be introduced.

[00268] In certain embodiments, the knockout modification is the substitution of a charged amino acid with an uncharged amino acid. In other embodiments, the knockout modification is the substitution of an uncharged amino acid with a charged amino acid. In other embodiments, the knockout modification is the substitution of a polar amino acid with a non-polar amino acid. In other embodiments, the knockout modification is the substitution of charged amino acids by polar and uncharged amino acids and polar uncharged amino acids with non-polar hydrophobic amino acids.

[00269] In certain embodiments, the knockout modification is introduced at an amino acid position which forms a binding interaction with an antigen. For example, the knockout modification may be located on the protein surface where an antigen-antibody interaction normally occurs. In certain exemplary embodiments, the modifications can be introduced in the complementary determining regions (CDR) of one or both VLX or VHX domains.

[00270] In certain embodiments, the knockout modification is a substitution of a residue in CDRH1 of the VHX domain. In another embodiment, the knockout modification is a substitution of a residue in CDRH2 of the VHX domain. In another embodiment, the knockout modification is a substitution of a residue in CDRH3 of the VHX domain.

[00271] In certain embodiments, the knockout modification is a substitution of a residue in CDRL1 of the VLX domain. In another embodiment, the knockout modification is a substitution of a residue in CDRL2 of the VLX domain. In another embodiment, the knockout modification is a substitution of a residue in CDRL3 of the VLX domain.

[00272] In certain exemplary embodiments, an arginine in the CDR of the VHX or VLX domains is mutated to glutamate. In other embodiments, one or more tyrosines in the CDR of the VHX or VLX domain is mutated to alanine.

[00273] In certain embodiments, the modification results in a knockout domain that is completely devoid the target antigen binding activity of the functional, non-knockout (i.e., wild-type) counterpart from which it is derived. Alternatively, the binding functionality of the knockout domain may be substantially reduced as compared to its functional, non-knockout counterpart, while nevertheless retaining some level of detectable binding.

[00274] Scaffolds for generating knockout domains can be obtained for example from the Protein Data Bank (PDB). The PDB is a crystallographic database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. Knockout domains can be generated by specifically mutating amino acids which are predicted to be involved in antigen binding by computer based modeling of antigen binding domain and its cognate target antigen. Subsequent binding studies can be conducted by using methods known in the art, e.g., surface plasmon resonance, and reveal with high accuracy whether the binding function is abolished and interaction between the binding domain and its target are disrupted.

(b) Stabilized Knockout Domains

[00275] In certain embodiments, the knockout domain is a stabilized knockout domain having increased thermal stability as compared to its wild-type counterpart. For example, in certain exemplary embodiments, the melting temperature (Tm) of the knockout domain is within at least

0.25 °C, at least 0.5°C, at least 0.75 °C, at least 1 °C, at least 2°C, at least 3°C, at least 4 °C, at least 5 °C, or at least 10 °C as compared to the wild-type counterpart from which it is derived. In other exemplary embodiments, the Tm of the knockout domain in increased with respect to its wild-type counterpart. For example, the Tm may be increase at least 0.25 °C, at least 0.5°C, at least 0.75 °C, at least 1 °C, at least 2°C, at least 3°C, at least 4 °C, at least 5 °C, or at least 10 °C as compared to the wild-type counterpart from which it is derived. Thermal stability may be measured by differential scanning fluorimetry (DSF) or other bioanalytic methods routinely used by those of skill in the art.

[00276] In certain embodiments, the pseudoFab comprises an engineered intrachain disulfide bond between the VHX or VLX domains of the knockout domain of the pseudoFab which confers enhanced stability. Typically, this modification is the substitution of at least one amino acid of the VHX domain with a cysteine (Cys) residue and at least one amino acid in the VLX domain with a Cys residue. The Cys residues may be introduced at positions of the VHX and VLX domains which allow formation of a disulfide bond after dimer formation. In certain embodiments, mutations may be made in the VH and VL interface to improve stability between the VH / VL interface. Specific sets of amino acid mutations in the VH domain and the VL domains may improve stability through the introduction of non-native cysteine residues that form disulfide bridges.

[00277] A first set of disulfide-stabilizing mutations may be made to amino acid residues in the VH and VL domains. In an exemplary embodiment, a disulfide bond is formed by a Cys at position 44 of the VHX domain and a Cys at position 100 of the VLX domain. This set of disulfide stabilizing mutations may be alternatively referred to as the“VH44C/VL100C” mutation set. The first set of disulfide stabilizing mutations are described in further detail in Reiter et al. Nature Biotechnology. Vol. 14. Pg. 1239-1245. 1996, incorporated herein by reference for all purposes.

[00278] A second set of disulfide stabilizing mutations may be made to amino acid residues in the VH and VL domain. In an exemplary embodiment, a disulfide bond is formed by a Cys at position 105 of the VHX domain and a Cys at position 43 of the VLX domain. The second set of disulfide stabilizing mutations may be alternatively referred to as the“VH105C/VL43C” mutation set. Other disulfide stabilizing mutations are described in further detail in U.S. Patent No. 9,527,927, which is incorporated herein by reference for all purposes.

[00279] In certain embodiments, the VLX domain of the pseudoFab comprises a variant of SEQ ID NO: 1 having at least one knockout modification. For example, the VLX domain may comprise an amino acid sequence that is at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 , but for the knockout modification.

[00280] In certain embodiments, the VHX domain of the pseudoFab comprises a variant of SEQ ID NO: 2 having at least one knockout modification. For example, the VHX domain may comprise an amino acid sequence that is at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, but for the knockout modification.

[00281] In one embodiment, the VLX domain of the pseudoFab comprises an amino acid sequence of SEQ ID NO: 76 and the VHX domain of the pseudoFab comprises an amino acid sequence selected from the group of SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79.

II. MULTISPECIFIC PSEUDOFAB-CONTAINING BINDING POLYPEPTIDES

[00282] In other aspects, multispecific binding proteins comprising a pseudoFab moiety described herein are provided. The highly modular nature of the pseudoFab moiety allows for a wide variety of multispecific structures to be formed.

[00283] In certain embodiments, the multispecific binding proteins of the disclosure comprise additional binding specificities appended to the N- terminus and/or C-terminus of one or both chains of a pseudoFab moiety to form a multivalent pseudoFab-containing binding protein.

[00284] In some embodiments, a multispecific binding protein comprises:

a) a first pseudoFab portion comprising:

(1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first antigen binding site that binds target antigen A;

(2) a first stabilized knockout VL domain (VLX) paired with a first stabilized knockout VH domain (VHX) to form a first disulfide stabilized knockout (dsKO) domain;

(3) a first heterodimerization domain (HD1);

wherein the first dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds ;

b) a first Fab portion comprising

(1) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(2) a first CH1 domain paired with a first CL domain; and

(3) a second heterodimerization domain (HD2).

[00285] In some embodiments, the first and second heterodimerization domains of the binding protein comprise first and second Fc domains. In some embodiments, the Fc domains comprise the general structure hinge-CH2 domain-CH3 domain.

(a)Fc-Heterodimerization domains

[00286] In certain embodiments the multispecific binding proteins of the disclosure may further comprises a Fc-heterodimerization C1 or C2 domain. In some embodiments, the Fc-heterodimerization domain is selected from the group consisting of a heterodimerizing Fc or fragments thereof, in particular a knob-in-hole (KIH) variant of a Fc-part and effector-modified variants thereof or a heterodimerizing Fc or fragments thereof, in particular an EV-RWT variant of a Fc and effector-modified variants thereof. In some embodiments the Fc comprises one or more amino acid mutations. One possibility is removing a selective recognition site for a first affinity reagent, e.g., by mutations selected from the group consisting of H435R, Y436F and introducing a selective recognition site for a second affinity reagent. Alternatively, solely a selective recognition site for a second affinity reagent can be introduced.

(b) Homodimerization domains

[00287] In certain embodiments the multispecific binding proteins of the disclosure may further comprise a homodimerization domain. In some embodiments the homodimerization domain is selected from the group consisting of an Fc region and effector-modified variants thereof; one or more CH2 domains, e.g., of IgG, IgE or IgM; one or more CH3 domains, e.g., of IgG, IgA or IgD; and one or more CH4 domains, e.g., of IgE or IgM.

III. MULTIMERIC PSEUDOFAB-CONTAINING BINDING PROTEINS

(a) Symmetrical tetravalent constructs

[00288] In another embodiment, the binding polypeptide comprises a pseudo Fab containing binding protein comprising additional polypeptide chains which associate with the polypeptide chains of a pseudoFab to form additional binding domains. These pseudo Fab containing binding polypeptides are further fused to an Fc heterodimerization domain to form a one half of conventional Y-shaped antibody.

[00289] In some embodiments, an antigen binding protein comprises six polypeptide chains that form four antigen-binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-L3-VHb-CH1-FC1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VHa- L2- VHX- L3- VH b-CH 1 - FC2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2 and L3 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[00290] These molecules may also be referred to as a“Tandem-(Fv-pseudo Fab x Fv-Fab)” (see FIG. 10B).

[00291] In some embodiments, an antigen binding protein comprises six polypeptide chains that form four antigen-binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHb-CH1-L3-VHa-L2-VHX-FC1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VH b-CH 1 - L3- VHa- L2-VHX- F C2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 andCH3 immunoglobulin heavy chain constant domains; and

L1 , L2 and L3 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[00292] These molecules may also be referred to as a“Tandem-(Fv-Fab x Fv-pseudoFab-lgG) (see FIG. 10A).

(b) Asymmetrical tetraspecific molecules

[00293] Dimerization of binding polypeptides wherein only one contains pseudoFab lead to asymmetrical constructs with additional specificities. These pseudoFab containing binding polypeptides are further fused to an Fc heterodimerization domain to form a one-half of conventional full-length Y-shaped IgG antibody.

[00294] In some embodiments, a multispecific binding protein comprises four polypeptide chains that form at least two antigen-binding sites, wherein

(a) a first polypeptide comprises a structure represented by the formula:

VLa-L1-VLX [I]

(b) a second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-FC1 [II]

(c) a third polypeptide comprises a structure represented by the formula:

VLb-CL [III]

(d) a fourth polypeptide comprises a structure represented by the formula:

VHb-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 and L2 are amino acid linkers, which may independently be the same or different, wherein

(1) the first VL domain (VLa) is paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with a second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[00295] These molecules may also be referred to as Dimeric Bispecific IgG molecules (Fv-pseudoFab)x(Fv-Fab)-Fc (see FIG. 2 and 7).

[00296] In another embodiment, the binding polypeptide comprises a pseudo Fab-containing binding protein comprising additional polypeptide chains which associate with the polypeptide chains of a pseudoFab to form additional binding domains. These pseudoFab-containing binding polypeptides are further fused to an Fc heterodimerization domain to form a one-half of a conventional Y-shaped antibody. Dimerization of such molecules leads to constructs with additional specificities.

[00297] In some embodiments, an antigen binding protein comprises six polypeptide chains that form four antigen-binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa- L1 -VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHa- L2-VHX- L3- VHa- L4- VHX- FC 1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VHb-CH1-L5-VHb-CH1-FC2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4 and L5 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; (ii) one or more engineered interchain disulfide bonds.

[00298] The C-terminal heterodimerization domain can be a conventional Fc-Knob-into-hole heterodimerization domain, a conventional Fc-RF heterodimerization domain or combinations thereof. These molecules may also be referred to as“Fv-pseudoFab([HC]-(Fv-pseudoFab)) x ((Fv-Fab)[HC]-(Fv-Fab)))-Fc” (see FIG. 11).

[00299] In some embodiments, an antigen binding protein comprises four polypeptide chains that form three antigen-binding sites, wherein:

(a) the first polypeptide comprises a structure represented by the formula:

VLa- L1 -VLX [I]

(b) the second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX- FC1 [II]

(c) the third polypeptide comprises a structure represented by the formula:

VLb-L3-VLc-L4-CL [III]

(d) the fourth polypeptide comprises a structure represented by the formula:

VHc-L5-VHb-L6-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VLc is a third immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VHc is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4, L5 and L6 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the third VL domain (VLc) is paired with the third VH domain (VHc) to form a third functional antigen binding site that binds target antigen C;

(4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV);

(5) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsK02) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

[00300] In some embodiments, an antigen binding protein comprises four polypeptide chains that form three antigen-binding sites, wherein:

(a) the first polypeptide comprises a structure represented by the formula:

VLa- L1 -VLX [I]

(b) the second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX- FC1 [II]

(c) the third polypeptide comprises a structure represented by the formula:

VLb-L3-VLc-L4-CL [III]

(d) the fourth polypeptide comprises a structure represented by the formula:

VHc-L5-VHb-L6-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VLc is a third immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VHc is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4, L5 and L6 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the third VL domain (VLc) is paired with the third VH domain (VHc) to form a third functional antigen binding site that binds target antigen C;

(4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV);

(5) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsK02) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen of a reference Fab molecule; and (ii) one or more engineered interchain disulfide bonds.

[00301] These molecules may also be referred to as“(CODV-Fab ) x (pseudoFab)-Fc” (see FIG. 13).

[00302] In particular embodiments of the first and second aspects of the present invention, the binding protein comprises a light chain and heavy chain pair selected from the group consisting of SEQ ID NO: 6 and 7; SEQ ID NO: 8 and 9; SEQ ID NO: 10 and 1 1 ; SEQ ID NO: 12 and 13; SEQ ID NO: 14 and 15; SEQ ID NO: 16 and 17; SEQ ID NO: 18 and 19; SEQ ID NO: 20 and 21 ;

SEQ ID NO: 22 and 23; SEQ ID NO: 24 and 25; SEQ ID NO: 26 and 27; SEQ ID NO: 28 and 29; SEQ ID NO: 30 and 31 ; SEQ ID NO: 32 and 33; SEQ ID NO: 34 and 35; SEQ ID NO: 36 and 37;

SEQ ID NO: 38 and 39; SEQ ID NO: 40 and 41 ; SEQ ID NO: 42 and 43; SEQ ID NO: 44 and 45;

SEQ ID NO: 46 and 47 and SEQ ID NO: 48 and 49; SEQ ID NO: 50 and 51 ; SEQ ID NO: 52 and

53 and SEQ ID NO: 54 and 55; SEQ ID NO: 56 and 57 and SEQ ID NO: 58 and 59; SEQ ID NO: 60 and 61 and SEQ ID NO: 62 and 63; SEQ ID NO: 64 and 65 and SEQ ID NO: 66 and 67; SEQ ID NO: 68 and 69 and SEQ ID NO: 70 and 71 ; SEQ ID NO: 72 and 73; and SEQ ID NO: 74 and 75.

[00303] In some embodiments, the first and second CL are independently selected from the group consisting of constant region light chain kappa (CLK) and constant region light chain lambda (Oίl).

[00304] In some embodiments, the HD1 and HD2 each comprise a Fc-region and effector-modified variants thereof; a heterodimerizing Fc-part, in particular a knob-in-hole (KIH) variant of a Fc-part and effector-modified variants thereof; one or more CH2 domains, e.g., of IgG, IgE or IgM, one or more CH3 domains, e.g., of IgG, IgA or IgD, or one or more CH4 domains, e.g., of IgE or IgM.

[00305] In some embodiments, one of the Fc domains comprises a first CH3 domain comprising one or both of S354C and T366W mutations, and the other Fc domain comprises a second CH3 domain comprising one or both of Y349C, T366S, L368A, and Y407V mutations.

[00306] In one embodiment the Fc region of HD1 or HD2 comprises one or more amino acid mutations which lead to removing a selective recognition site for a second affinity reagent, e.g., selected from the group consisting of H435R or Y436F, or lead to introducing a selective recognition site for a third affinity reagent.

CLAIMS

1. A binding protein comprising:

at least one pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain; and

wherein the stabilized knockout domain comprises (3) one or more inactivating mutations which abolish its binding to a target antigen; and (4) one or more engineered interchain disulfide bonds.

2. The binding protein of claim 1 , wherein the binding protein is a multispecific binding protein further comprising at least a second VL domain (VLb) paired with a second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B.

3. A multispecific binding protein comprising:

a) a first pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain; and

b) a first Fab portion comprising (3) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B; (4) a first CH1 domain paired with a first CL domain; and

wherein the stabilized knockout domain comprises (5) one or more inactivating mutations which abolish its binding to a target antigen; and (6) one or more engineered interchain disulfide bonds.

4. A multispecific binding protein comprising:

a) a first pseudoFab portion comprising (1) a first VL domain (VLa) paired with a first VH

domain (VHa) to form a first functional antigen binding site that binds target antigen A; (2) a first stabilized knockout VH domain (VHX) paired with a first stabilized knockout VL domain (VLX) to form a first stabilized knockout domain; and

b) a first Fab portion comprising (3) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B; (4) a first CH1 domain paired with a first CL domain; and

c) a linker portion which operably links the first Fab portion and the first pseudoFab portion,

wherein the stabilized knockout domain comprises (5) one or more inactivating mutations which abolish its binding to a target antigen; and (6) one or more engineered interchain disulfide bonds.

5. The binding protein of any one of the previous claims, wherein the linker portion is a peptide linker.

6. The binding protein of claim 5, wherein the peptide linker is a Gly-Ser linker of the formulation (Gly4Ser)n, wherein n is 1-10.

7. The binding protein of any one of the previous claims, wherein the linker portion is a heterodimerization domain.

8. The binding domain of any one of the previous claims, comprising independently one or two first pseudoFab portion(s) and one or two first Fab portion(s).

9. The binding protein of any one of the previous claims, comprising separate proteins chains selected from one of the following groups:

(a) VHa-CH1-L1-VHb-L2-VHX and VLa-CL and VLb-L3-VLX;

(b) VHa-L2-VHX-L1-VHb-CH1 and VLa-L3-VLX and VLb-CL;

(c) VHa-CH 1 -L1 -VHa-CH 1 and VHb-L2-VHX-L3-VHb-L4-VHX and two chains VLb-L5-VLX and two chains VLa-CL;

wherein the chains of (a) and (b) can be present once or twice, and wherein L1 , L2, L3, L4 and L5 are linkers, which may independently be the same or different .

10. The binding protein of any of the previous claims, wherein the first pseudoFab portion comprises a first polypeptide chain having a structure represented by the formula:

(la) N-VHa-L1-VHX-C

and a second polypeptide chain having a structure represented by the formula:

(lla) N-VLa-L2-VLX-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

11. The binding protein of any of the previous claims, wherein the first pseudoFab portion comprises of a first polypeptide chain having a structure represented by the formula:

(lb) N-VHX-L1-VHa-C

and a second polypeptide chain having a structure represented by the formula:

(lib) N-VLX-L2-VLa-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

12. The binding protein of any of the previous claims, wherein the first pseudoFab portion comprises of a first polypeptide chain having a structure represented by the formula:

(lc) N-VLa-L1-VHX-C

and a second polypeptide chain having a structure represented by the formula:

(lie) N-VHa-L2-VLX-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

13. The binding protein of any of the previous claims, wherein the first pseudoFab portion comprises of a first polypeptide chain having a structure represented by the formula:

(Ld) N-VHX-L1-VLa-C

and a second polypeptide chain having a structure represented by the formula:

(lid) N-VLX-L2-VHa-C

wherein L1 and L2 are linkers, which may independently be present or absent, and wherein N and C represent the N- and C-terminal ends, respectively.

14. The binding protein of any one of the previous claims, further comprising one or more additional binding domains operably linked to an N- or C-terminus of the binding protein.

15. The binding protein of any one of the previous claims, wherein the one or more additional binding domains are operably linked to N-terminus of the first or second pseudoFab portion.

16. A multispecific binding protein comprising:

a) a first pseudoFab portion comprising:

(1) a first VL domain (VLa) paired with a first VH domain (VHa) to form a first antigen binding site that binds target antigen A;

(2) a first stabilized knockout VL domain (VLX) paired with a first stabilized knockout VH domain (VHX) to form a first disulfide stabilized knockout (dsKO) domain;

(3) a first heterodimerization domain (HD1);

wherein the first dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds ;

b) a first Fab portion comprising

(1) a second VL domain (VLb) paired with second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(2) a first CH1 domain paired with a first CL domain; and

(3) a second heterodimerization domain (HD2).

17. The binding protein of claim 16, wherein the first and second heterodimerization domains comprise first and second Fc domains.

18. The binding protein of claim 16 or 17, wherein the Fc domains comprise the general structure hinge-CH2 domain-CH3 domain.

19. A multispecific binding protein comprising four polypeptide chains that form at least two antigen-binding sites, wherein

(a) a first polypeptide comprises a structure represented by the formula:

VLa-L1-VLX [I]

(b) a second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-FC1 [II]

(c) a third polypeptide comprises a structure represented by the formula:

VLb-CL [III]

(d) a fourth polypeptide comprises a structure represented by the formula:

VHb-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 and L2 are amino acid linkers, which may independently be the same or different, wherein

(1) the first VL domain (VLa) is paired with a first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with a second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

20. An antigen binding protein comprising six polypeptide chains that form four antigen binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-L3-VHb-CH1-FC1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VHa-L2-VHX-L3-VHb-CH1-FC2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2 and L3 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

21. An antigen binding protein comprising six polypeptide chains that form four antigen binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VH b-CH 1 - L3- VHa- L2-VHX- FC 1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VH b-CH 1 - L3- VHa- L2-VHX- F C2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2 and L3 are amino acid linkers,

wherein

(1 ) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

22. An antigen binding protein comprising six polypeptide chains that form four antigen binding sites, wherein

(a) the first and second polypeptides comprise a structure represented by the formula:

VLa-L1-VLX [I] and [II]

(b) the third and fourth polypeptides comprise a structure represented by the formula:

VLb-CL [III] and [IV]

(c) the fifth polypeptide comprises a structure represented by the formula:

VHa- L2-VHX- L3- VHa- L4- VHX- FC 1 [V]

(d) the sixth polypeptide comprises a structure represented by the formula:

VHb-CH1-L5-VHb-CH1-FC2 [VI]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin heavy chain constant domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4 and L5 are amino acid linkers,

wherein

(1 ) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsKO) domain;

wherein the dsKO domains comprise (i) one or more inactivating mutations which abolish its binding to a target antigen; (ii) one or more engineered interchain disulfide bonds.

23. An antigen-binding protein comprising four polypeptide chains that form three antigen binding sites, wherein:

(a) the first polypeptide comprises a structure represented by the formula:

VLa- L1 -VLX [I]

(b) the second polypeptide comprises a structure represented by the formula:

VHa-L2-VHX- FC1 [II]

(c) the third polypeptide comprises a structure represented by the formula:

VLb-L3-VLc-L4-CL [III]

(d) the fourth polypeptide comprises a structure represented by the formula:

VHc-L5-VHb-L6-CH1-FC2 [IV]

wherein:

VLa is a first immunoglobulin light chain variable domain;

VLb is a second immunoglobulin light chain variable domain;

VLc is a third immunoglobulin light chain variable domain;

VHa is a first immunoglobulin heavy chain variable domain;

VHb is a second immunoglobulin heavy chain variable domain;

VHc is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

VLX is a stabilized knockout light chain variable domain;

VHX is a stabilized knockout heavy chain variable domain;

FC1 and FC2 are Fc domains comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and

L1 , L2, L3, L4, L5 and L6 are amino acid linkers,

wherein

(1) the first VL domain (VLa) is paired with the first VH domain (VHa) to form a first functional antigen binding site that binds target antigen A;

(2) the second VL domain (VLb) is paired with the second VH domain (VHb) to form a second functional antigen binding site that binds target antigen B;

(3) the third VL domain (VLc) is paired with the third VH domain (VHc) to form a third functional antigen binding site that binds target antigen C;

(4) the polypeptide of formula III and the polypeptide of formula IV form a cross-over light chain-heavy chain pair (CODV);

(5) the stabilized knockout VL domain (VLX) is paired with the stabilized knockout VH domain (VHX) to form a disulfide stabilized knockout (dsK02) domain;

wherein the dsKO domain comprises (i) one or more inactivating mutations which abolish its binding to a target antigen; and (ii) one or more engineered interchain disulfide bonds.

24. The binding protein of any one of claims 17-23, wherein the Fc domains comprise one or more knob-in-hole (KIH) mutations.

25. The binding protein of any one of the previous claims, wherein the melting temperature (Tm) of the pseudoFab portion is at least 4 degrees Celsius higher than the reference Fab molecule.

26. The binding molecule of any one of the previous claims, wherein at least one of the one or more engineered interchain disulfide bonds is VH44C-VL100C.

27. The binding protein of any one of the previous claims, wherein target antigen A and target antigen B are different epitopes of the same antigen.

28. The binding protein of any one of the previous claims, wherein the VLX/VHX pair is selected from the group consisting of:

(i) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 77;

ii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 78; and

iii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 79.

29. Use of a stabilized knockout domain to reduce heavy chain- light chain mispairing in a multispecific binding protein wherein the stabilized knockout domain comprises VHK and VLX domains comprising (1) one or more inactivating mutations which abolish its binding to a target antigen relative to wild type domains and (2) one or more engineered interchain disulfide bonds which confer enhanced thermal stability (Tm) of the pseudoFab relative to a reference Fab molecule, wherein the reference Fab molecule is identical to the pseudoFab molecule except that, in a pseudoFab reference molecule, CH1 and CL domains of the reference Fab molecule are replaced with VHX and VLX domains.

30. Use according to claim 29, wherein the VLX/VHX pair is selected from the group consisting of:

(i) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 77;

ii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 78; and

iii) VLX comprising an amino acid sequence of SEQ ID NO: 76 and

VHX comprising an amino acid sequence of SEQ ID NO: 79.

31. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein of one of the previous claims.

32. An expression vector comprising the nucleic acid molecule of claim 31.

33. An isolated host cell comprising the nucleic acid molecule of claim 31 or the expression vector of claim 32.

34. A method of producing the binding protein of any one of claims 1-28 comprising culturing the host cell of claim 33 under conditions such that the binding protein is expressed; and purifying the binding protein from the host cell.

35. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the multispecific binding protein of any one of the claims 1-28.

36. A method of treating a disorder in which antigen activity is detrimental, the method comprising administering to a subject in need thereof an effective amount of a multispecific binding protein of any one of the previous claims.