Bispecific CD3 and CD19 Antigen Binding Constructs

1 . An isolated bispecific antigen binding construct comprising a first antigen-binding polypeptide construct which monovalently and specifically binds a CD19 antigen and is a Fab;

a second antigen-binding polypeptide construct which monovalently and specifically binds a CD3 antigen and is an scFv; and

a heterodimeric Fc comprising first and second Fc polypeptides each comprising a modified CH3 domain, wherein each modified CH3 domain comprises asymmetric amino acid modifications that promote the formation of a heterodimeric Fc and the dimerized CH3 domains having a melting temperature (Tm) of about 68° C. or higher, wherein the first Fc polypeptide is linked to the first antigen-binding polypeptide construct, with or without a first linker, and the second monomeric Fc polypeptide is linked to the second antigen-binding polypeptide construct with or without a second linker.

2 . (canceled)

3 . The isolated bispecific antigen binding construct of claim 1 , comprising at least three, at least six, or at least 12 CDRs of variant 6754, 6751, 1853, 10151, 6475, 6749, 10152, 10153, 6476 5850, 5851, 5852, or 6325.

4 . The isolated bispecific antigen binding construct of claim 1 , wherein at least one polypeptide comprises an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one polypeptide of Variant 6754, 6751, 1853, 10151, 6475, 6749, 10152, 10153, 6476, 5850, 5851, 5852, or 6325.

5 . The isolated bispecific antigen binding construct of claim 1 , wherein

a. the first antigen-binding polypeptide construct comprises the antigen-binding polypeptide construct specific for CD19 derived from an antibody selected from the group consisting of 4G7; B4; B43; BU12; CLB-CD19; Leu-12; SJ25-C1; J4.119, B43, SJ25C1, FMC63 (IgG2a) HD237 (IgG2b), Mor-208, MEDI-551, and MDX-1342;

b. and the second antigen-binding polypeptide construct comprises the binding polypeptide construct specific for CD3 derived from an antibody selected from OKT3; Teplizumab™ (MGA031, Eli Lilly); Micromet, Blinatumomab™; UCHT1; NI0401; visilizumab; X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2 and F101.01;

c. and/or the antigen binding construct competes with an antibody described in a or b

d. and/or a humanized version thereof.

6 .- 7 . (canceled)

8 . The isolated bispecific antigen binding construct of claim 1 , wherein at least one Fc polypeptide comprises an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one Fc polypeptide of a heterodimeric Fc of Table A or variant 6754, 6751, 1853, 10151, 6475, 6749, 10152, 10153, 6476, 5850, 5851, 5852, or 6325.

9 . The isolated bispecific antigen binding construct of claim 1 , wherein the heterodimeric Fc

is a human Fc; and/or

is a human IgG1 Fc or IgG4 Fc; and/or

comprises one or more modifications in at least one of the CH3 domains; and/or

comprises one or more modifications in at least one of the CH3 domains that promote formation of a heterodimer with stability comparable to a wild-type homodimeric Fc; and/or

comprises one or more modifications in at least one of the CH3 domains as described in Table A;

further comprises at least one CH2 domain; and/or

further comprises at least one CH2 domain comprising one or more modifications; and/or

further comprises at least one CH2 domain comprising one or more modifications in at least one of the CH2 domains as described in Table B; and/or

comprises one or more modifications to promote selective binding of Fc-gamma receptors and/or complement.

10 . The isolated bispecific antigen binding construct of claim 1 , wherein the dimerized CH3 domains have a melting temperature (Tm) of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, 83, 84, or 85° C. or higher.

11 . The isolated bispecific antigen binding construct of claim 1 , wherein each heterodimeric Fc polypeptide is fused to each antigen-binding polypeptide construct by a linker, optionally wherein the linker is a polypeptide linker, or optionally wherein the linker comprises an IgG1 hinge region.

12 .- 13 . (canceled)

14 . The isolated bispecific antigen binding construct of claim 1 , displaying reduced Fc gamma receptor binding and no associated immune-cell mediated effector activity.

15 . The isolated bispecific antigen binding construct of claim 1 , wherein the bispecific antigen binding construct

is capable of synapse formation and bridging between CD19+ Raji B-cells and Jurkat T-cells as assayed by FACS and/or microscopy; and/or

mediates T-cell directed killing of CD20+ B cells in human whole blood; and/or

displays improved biophysical properties compared to v875; and/or

displays improved yield compared to v875, e.g., expressed at >10 mg/L after SEC (size exclusion chromatography); and/or

displays 10-fold better yield of the desired homogeneous species under comparable expression conditions, and/or

displays heterodimer purity, e.g., >95%.

16 . The isolated bispecific antigen binding construct of claim 1 , wherein the antigen-binding construct is conjugated to a drug.

17 . A pharmaceutical composition comprising the isolated bispecific antigen binding construct of claim 1 and a pharmaceutical carrier.

18 .- 20 . (canceled)

21 . A method of treating a cancer in a subject, the method comprising administering an effective amount of the isolated antigen-binding construct of claim 1 to the subject.

22 .- 23 . (canceled)

24 . A method of treating a condition in a subject, the method comprising administering an effective amount of the isolated antigen-binding construct of claim 1 to the subject, wherein the condition is an inflammatory condition, a proliferative disease, a minimal residual cancer, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, parasitic reaction, a graft-versus-host disease or host-versus-graft disease or a cell malignancies, a disease associated with B cells, a disease not responsive to treatment with at least one of an anti-CD19 antibody and an anti-CD20 antibody.

25 . (canceled)

26 . A method of producing the bispecific antigen binding construct of claim 1 comprising culturing a host cell under conditions suitable for expressing the bispecific antigen binding construct wherein the host cell comprises a polynucleotide encoding the isolated bispecific antigen binding construct of claim 1 , and purifying the bispecific antigen binding construct.

27 . A method of detecting or measuring CD3 and/or CD19 in a sample comprising contacting the sample with the bispecific antigen binding construct of claim 1 and detecting or measuring the bound complex.

28 . A method of inhibiting, reducing or blocking CD3 and/or CD19 signaling in a cell comprising administering an effective amount of the bispecific antigen binding construct of claim 1 to the cell, and optional administering small molecule or a second antibody

29 . An isolated polynucleotide or set of isolated polynucleotides comprising at least one nucleic acid sequence that encodes at least one polypeptide of the isolated bispecific antigen binding construct of claim 1 .

30 .- 31 . (canceled)

32 . A vector or set of vectors comprising one or more of the polynucleotides or sets of polynucleotides according to claim 29 .

33 . (canceled)

34 . An isolated cell comprising a polynucleotide or set of polynucleotides according to claim 29 .

35 .- 36 . (canceled)