Multispecific antigen-binding proteins and uses thereof

Abstract

The present invention relates to multispecific antigen-binding proteins (MSAPs) (e.g., trispecific antigen-binding proteins (TSAPs)) that specifically bind to CD3, CD8, and a target antigen (e.g., a tumor-associated antigen or a tumor-specific antigen). The present invention also provides antibodies or antigen-binding fragments thereof that target CD8 or CD3, and MSAPs comprising the same. Also provided are compositions, kits, methods of use (e.g., for treating cancer), and methods of making the same. In some embodiments according to any of the above MSAPs, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from the group consisting of Fab, Fab', Fab'-SH, F(ab')2, Fv, sdAb, scFv, and any combination thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from International Patent Application No. PCT / CN2023 / 082015 (filed March 16, 2023) and International Patent Application No. PCT / CN2023 / 127757 (filed October 30, 2023), the contents of each of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (221992000742SEQLIST.xml, size: 190,159 bytes, created date: March 13, 2024) are incorporated herein by reference in their entirety.

[0003] The present invention relates to multispecific antigen-binding proteins (MSAPs, e.g., trispecific antigen-binding proteins (TSAPs)) that specifically bind to CD3, CD8, and a target antigen (e.g., a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA)). Further provided herein are antibodies or antigen-binding fragments thereof that target CD3 or CD8, pharmaceutical compositions comprising MSAP (e.g., TSAP), methods for treating cancer using MSAP (e.g., TSAP), methods for producing the same, and kits comprising MSAP (e.g., TSAP). [Background technology]

[0004] Many abnormal cells and tissues, as well as many diseases, display unique antigens that can be exploited for immune cell-mediated clearance. Exemplary diseases include, but are not limited to, cancer (e.g., solid or liquid cancers), autoimmune disorders, bacterial diseases, viral diseases, or fungal diseases, each of which may display one or more different target antigens or one or more different epitopes of the same target antigen. For example, some antigens are overexpressed, mutated, or selectively mutated in tumor tissue. Therefore, antibodies targeting specific antigens on the surface of cancer cells can be used as cancer therapeutics.

[0005] CD3, which contains three distinct polypeptide chains (ε, δ, and γ chains), is an antigen expressed by T cells. The three CD3 polypeptide chains associate with the T cell receptor (TCR) and ζ chain to form the TCR complex, which functions to activate signaling cascades in T cells. Currently, many therapeutic strategies target TCR signaling for the treatment of diseases using anti-human CD3 monoclonal antibodies. The CD3-specific antibody OKT3 was the first monoclonal antibody approved for therapeutic use in humans and is used clinically as an immunomodulatory agent for the treatment of allograft rejection.

[0006] Although bispecific antibodies targeting CD3 and a target antigen (e.g., a tumor antigen) have shown the potential to effectively kill cancer cells, serious adverse effects, including systemic immune activation, immunogenicity (anti-drug antibody effect), and the generally poor manufacturability of these molecules, have significantly limited the widespread application of this type of drug.

[0007] Shortcomings in current forms of bispecific antibodies remain a major challenge to their widespread application in the treatment of cancer patients with good efficacy and safety. Summary of the Invention

[0008] The present invention provides multispecific antigen-binding proteins ("MSAPs," e.g., trispecific antigen-binding proteins, "TSAPs") that specifically bind to CD3 and CD8 and a target antigen (e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA)), pharmaceutical compositions comprising MSAPs, and methods of using MSAPs (e.g., methods of treating cancer). The present invention further provides antibodies and antigen-binding fragments thereof that target CD8 or CD3.

[0009] In one aspect of the present invention, there is provided an MSAP comprising: i) an anti-CD3 portion that specifically binds to CD3; ii) an anti-CD8 portion that specifically binds to CD8; and iii) an anti-target epitope portion that specifically binds to a target epitope (e.g., a TSA or TAA, e.g., CD19, EpCAM, or WT1), wherein a first of the three portions is a Fab fragment, the Fab fragment comprising a second polypeptide comprising a heavy chain variable region (VH) and a heavy chain constant region (CH1), and a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL); a second of the three portions is a first antigen-binding fragment (e.g., scFv); and a third of the three portions is a second antigen-binding fragment (e.g., scFv). (a) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment; (b) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment; (c) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment; (d) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment; or (e) the first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment.

[0010] In some embodiments according to any of the above MSAPs, (a) the anti-CD3 moiety is a Fab fragment ("anti-CD3 Fab fragment"), the anti-CD8 moiety is a first antigen-binding fragment ("first antigen-binding fragment of anti-CD8"), and the anti-target epitope moiety is a second antigen-binding fragment ("second antigen-binding fragment of anti-target epitope"); (b) the anti-CD3 moiety is a Fab fragment, the anti-target epitope moiety is a first antigen-binding fragment ("first antigen-binding fragment of anti-target epitope"), and the anti-CD8 moiety is a second antigen-binding fragment ("second antigen-binding fragment of anti-CD8"); (c) the anti-CD8 moiety is a Fab fragment ("anti-CD8 (d) the anti-CD8 portion is a Fab fragment, the anti-target epitope portion is a first antigen-binding fragment, and the anti-CD3 portion is a second antigen-binding fragment ("anti-CD3 second antigen-binding fragment"); (e) the anti-target epitope (e.g., a TSA or TAA, e.g., CD19, EpCAM, or WT1) portion is a Fab fragment ("anti-target epitope Fab fragment"), the anti-CD3 portion is a first antigen-binding fragment, and the anti-CD8 portion is a second antigen-binding fragment; or (f) the anti-target epitope portion is a Fab fragment, the anti-CD8 portion is a first antigen-binding fragment, and the anti-CD3 portion is a second antigen-binding fragment.

[0011] In some embodiments according to any of the above MSAPs, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from the group consisting of Fab, Fab', Fab'-SH, F(ab')2, Fv, sdAb, scFv, and any combination thereof. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are both scFv.

[0012] In some embodiments according to any of the above MSAPs, the anti-CD8 moiety specifically binds to CD8α. In some embodiments, the anti-CD8 moiety comprises: i) a heavy chain hypervariable region 1 (HVR-H1) comprising the amino acid sequence of SEQ ID NO: 13, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, an light chain hypervariable region 1 (HVR-L1) comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; ii) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; iii) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. HVR-L3 comprising the amino acid sequence of SEQ ID NO: 1; iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; v) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12; or vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23.In some embodiments, the anti-CD8 portion comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; iv) a VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 25; v) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 33; or vi) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD8 portion is an anti-CD8 scFv, and the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 21, 44-49, and 51-55.

[0013] In some embodiments according to any of the above MSAPs, the anti-CD8 portion comprises i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-CD8 portion comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD8 moiety is an anti-CD8 scFv, and the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55.

[0014] In some embodiments according to any of the above MSAPs, the anti-CD3 portion comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 160, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 161, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 162; or (c) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-CD3 portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 43; (b) a VH comprising the amino acid sequence of SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 167 and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD3 moiety is an anti-CD3 scFv, and the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184.

[0015] In some embodiments according to any of the above MSAPs, the anti-target epitope portion specifically recognizes a target epitope of a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In some embodiments, the TAA is CD19 or EpCAM. In some embodiments, the TSA is WT1.

[0016] In some embodiments according to any of the above MSAPs, the anti-target epitope portion specifically recognizes CD19 (anti-CD19 portion). In some embodiments, the anti-CD19 portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD19 portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 portion is an anti-CD19 scFv, and the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the anti-CD19 portion is an anti-CD19 Fab, and the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169.

[0017] In some embodiments according to any of the above MSAPs, the anti-target epitope portion specifically recognizes EpCAM (anti-EpCAM portion). In some embodiments, the anti-EpCAM portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-EpCAM portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-EpCAM portion is an anti-EpCAM scFv, and the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78.

[0018] In some embodiments according to any of the above MSAPs, the anti-target epitope portion specifically recognizes WT1 (anti-WT1 portion). In some embodiments, the anti-WT1 portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 79, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 80, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 81, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the anti-WT1 portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 85, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the anti-WT1 portion is an anti-WT1 scFv, and the anti-WT1 scFv comprises the amino acid sequence of SEQ ID NO: 87 or 88.

[0019] In some embodiments according to any of the above MSAPs, CH1 comprises the amino acid sequence of SEQ ID NO:93 and CL comprises the amino acid sequence of SEQ ID NO:94 or 165.

[0020] In some embodiments according to any of the above MSAPs, the first antigen-binding fragment is fused to the Fab fragment via a first linker, and / or the second antigen-binding fragment is fused to the Fab fragment via a second linker. In some embodiments, the first linker and / or the second linker is about 2 to about 30 amino acid residues in length. In some embodiments, the first linker and / or the second linker comprises an amino acid residue selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the first linker and / or the second linker comprises an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0021] In some embodiments according to any of the above MSAPs, the MSAP comprises i) an anti-CD3 Fab fragment, an anti-CD8 first scFv, and an anti-WT1 second scFv; or ii) an anti-CD3 Fab fragment, an anti-WT1 first scFv, and an anti-CD8 second scFv. In some embodiments, the anti-CD3 Fab fragment comprises (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 89 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the MSAP comprises a first fusion polypeptide comprising a first anti-WT1 scFv fused to the N-terminus of the VH of an anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising a second anti-CD8 scFv fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 95, and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0022] In some embodiments according to any of the above MSAPs, the MSAP comprises i) an anti-EpCAM Fab fragment, an anti-CD3 first scFv, and an anti-CD8 second scFv; or ii) an anti-EpCAM Fab fragment, an anti-CD8 first scFv, and an anti-CD3 second scFv. In some embodiments, the anti-EpCAM Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 76 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the multispecific antigen-binding protein comprises a first fusion polypeptide comprising an anti-CD3 first scFv fused to the N-terminus of the VH of the anti-EpCAM Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD8 second scFv fused to the N-terminus of the VL of the anti-EpCAM Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 97 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 98.

[0023] In some embodiments according to any of the above MSAPs, the MSAP comprises i) an anti-CD8 Fab fragment, an anti-EpCAM first scFv, and an anti-CD3 second scFv; or ii) an anti-CD8 Fab fragment, an anti-CD3 first scFv, and an anti-EpCAM second scFv. In some embodiments, the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 147 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 149 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 150; iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; iv) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 151 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; v) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 153 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 154; or vi) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments, the multispecific antigen binding protein comprises a first fusion polypeptide comprising a first scFv of anti-EpCAM fused to the N-terminus of the VH of an anti-CD8 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD3 fused to the N-terminus of the VL of the anti-CD8 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO:99 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:100.

[0024] In some embodiments according to any of the above MSAPs, the MSAP comprises i) an anti-CD3 Fab fragment, an anti-EpCAM first scFv, and an anti-CD8 second scFv; or ii) an anti-CD3 Fab fragment, an anti-CD8 first scFv, and an anti-EpCAM second scFv. In some embodiments, the anti-CD3 Fab fragment comprises (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 89 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the multispecific antigen binding protein comprises a first fusion polypeptide comprising a first scFv of anti-EpCAM fused to the N-terminus of the VH of an anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 101 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 102.

[0025] In some embodiments according to any of the above MSAPs, the MSAP comprises i) an anti-CD3 Fab fragment, an anti-CD19 first scFv, and an anti-CD8 second scFv; or ii) an anti-CD3 Fab fragment, an anti-CD8 first scFv, and an anti-CD19 second scFv. In some embodiments, the anti-CD3 Fab fragment comprises (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 89 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the multispecific antigen binding protein comprises a first fusion polypeptide comprising a first scFv of anti-CD19 fused to the N-terminus of the VH of an anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein i) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 104; ii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 106; or iii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 161 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 106.

[0026] In some embodiments according to any of the above MSAPs, the MSAP comprises i) an anti-CD19 Fab fragment, an anti-CD8 first scFv, and an anti-CD3 second scFv; or ii) an anti-CD19 Fab fragment, an anti-CD3 first scFv, and an anti-CD8 second scFv. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the anti-CD3 first scFv fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising the anti-CD8 second scFv fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 170 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 171. In some embodiments, the MSAP comprises a first fusion polypeptide comprising an anti-CD8 first scFv fused to the C-terminus of the CH1 of an anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD3 second scFv fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a second linker, wherein i) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises SEQ ID NO: 173; ii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 174 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 175; iii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 176; or iv) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, the MSAP comprises a first fusion polypeptide comprising a first scFv of anti-CD3 fused to the N-terminus of the VH of an anti-CD19 Fab fragment via a first linker and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the C-terminus of the CL of the anti-CD19 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 177 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 178.In some embodiments, the MSAP comprises: i) a fusion polypeptide comprising an anti-CD3 first scFv fused to the N-terminus of the VH of an anti-CD19 Fab fragment via a first linker and an anti-CD8 second scFv fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via a second linker; and ii) an anti-CD19 Fab fragment second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 180 and the anti-CD19 Fab fragment second polypeptide comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the MSAP comprises a fusion polypeptide comprising: i) a first polypeptide of an anti-CD19 Fab fragment; and ii) a first scFv of anti-CD3 fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a first linker, and a second scFv of anti-CD8 fused to the C-terminus of the CL of the anti-CD19 Fab fragment via a second linker, wherein the first polypeptide of the anti-CD19 Fab fragment comprises the amino acid sequence of SEQ ID NO: 137, and the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 181.

[0027] Also provided are isolated nucleic acids encoding any of the MSAPs described herein, vectors (e.g., viral vectors, e.g., lentiviral vectors or AAV) containing such nucleic acids, and host cells containing such nucleic acids or vectors.

[0028] Also provided is a method for producing any of the above MSAP, comprising: i) culturing a host cell containing the isolated nucleic acid or any of the above vectors, or any of the above host cells, under conditions suitable for expression of MSAP; and ii) obtaining the expressed MSAP from the host cell.

[0029] Also provided are pharmaceutical compositions comprising i) any of the MSAPs described herein, or an isolated nucleic acid or vector encoding same (e.g., a viral vector, e.g., a lentiviral vector or AAV), and optionally ii) a pharmaceutically acceptable carrier.

[0030] Also provided are methods for treating a disease associated with a target epitope in an individual, comprising administering to the individual an effective amount of any of the above-described MSAP or any of the above-described pharmaceutical compositions (e.g., viral vector pharmaceutical compositions). In some embodiments, the disease associated with the target epitope is a cancer that expresses the target epitope (e.g., a CD19+ cancer, an EpCAM+ cancer, or a WT1+ cancer). In some embodiments, the cancer is a solid cancer or a liquid cancer. In some embodiments, the cancer is Burkitt's lymphoma, Wilms' tumor, or colorectal cancer. In some embodiments, the MSAP or pharmaceutical composition is administered subcutaneously or intravenously. In some embodiments, the individual is human.

[0031] In another aspect, the present invention provides an anti-CD8 antibody or antigen-binding fragment thereof that specifically binds to CD8, the anti-CD8 antibody or antigen-binding fragment thereof comprising: HVR-H1 comprising the amino acid sequence of any of SEQ ID NOs: 1, 7, 13, and 20, or a variant thereof comprising up to about five amino acid variations; HVR-H2 comprising the amino acid sequence of any of SEQ ID NOs: 2, 8, 14, 16, and 18, or a variant thereof comprising up to about five amino acid variations; HVR-H3 comprising the amino acid sequence of any of SEQ ID NOs: 3, 9, 15, 17, 19, and 22, or a variant thereof comprising up to about five amino acid variations; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4 or 10, or a variant thereof comprising up to about five amino acid variations; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5 or 11, or a variant thereof comprising up to about five amino acid variations; and HVR-L3 comprising the amino acid sequence of any of SEQ ID NOs: 6, 12, and 23, or a variant thereof comprising up to about five amino acid variations.In some embodiments, the anti-CD8 antibody or antigen-binding fragment thereof is selected from the group consisting of: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; v) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12; or vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23.

[0032] In some embodiments according to any of the above-described anti-CD8 antibodies or antigen-binding fragments thereof, the anti-CD8 antibody or antigen-binding fragment thereof comprises: i) a VH comprising the amino acid sequence of SEQ ID NO:24 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:24, and a VL comprising the amino acid sequence of SEQ ID NO:25 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:25; ii) a VH comprising the amino acid sequence of SEQ ID NO:26 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:26, and a VL comprising the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:27; iii) a VH comprising the amino acid sequence of SEQ ID NO:28 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:28, and a VL comprising the amino acid sequence of SEQ ID NO:29 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:29. iv) a VH comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 30, and a VL comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 25; v) a VH comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 32, and a VL comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 33; or vi) a VH comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 34, and a VL comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 35.In some embodiments, the anti-CD8 antibody or antigen-binding fragment thereof comprises: i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; iv) a VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 25; v) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 33; or vi) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35.

[0033] In some embodiments according to any of the above anti-CD8 antibodies or antigen-binding fragments thereof, the anti-CD8 antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv. In some embodiments, the anti-CD8 antigen-binding fragment is an anti-CD8 scFv. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any of SEQ ID NOs: 21, 44-49, and 51-55. In some embodiments, the anti-CD8 antigen-binding fragment is an anti-CD8 Fab fragment. In some embodiments, the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 147 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 149 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 150; iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; iv) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 151 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; v) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 153 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 154; or vi) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156.

[0034] Also provided are MSAPs (eg, bispecific antigen-binding proteins (BSAPs), TSAPs) comprising any of the above anti-CD8 antibodies or antigen-binding fragments thereof.

[0035] Also provided are isolated nucleic acids encoding any of the above-described anti-CD8 antibodies or antigen-binding fragments thereof, or MSAP, vectors (e.g., viral vectors, e.g., lentiviral vectors or AAV) containing such nucleic acids, and host cells containing such nucleic acids or vectors.

[0036] Also provided is a method for producing an anti-CD8 antibody, or antigen-binding fragment thereof, or MSAP thereof, comprising: i) culturing a host cell containing any of the isolated nucleic acids or vectors encoding the anti-CD8 antibody, antigen-binding fragment thereof, or MSAP, or a host cell encoding the anti-CD8 antibody, antigen-binding fragment thereof, or MSAP, under conditions suitable for expression of the anti-CD8 antibody, or antigen-binding fragment thereof, or MSAP; and ii) obtaining the expressed anti-CD8 antibody, antigen-binding fragment thereof, or MSAP from the host cell.

[0037] Also provided are pharmaceutical compositions comprising i) any of the above-described anti-CD8 antibodies or antigen-binding fragments thereof, or any of the above-described MSAPs, or an isolated nucleic acid or vector (e.g., a viral vector, e.g., a lentiviral vector or AAV) encoding same, and optionally ii) a pharmaceutically acceptable carrier. Also provided are methods for treating a disease in an individual, comprising administering to the individual an effective amount of any of the above-described MSAPs or any of the above-described pharmaceutical compositions (e.g., a viral vector pharmaceutical composition). In some embodiments, the disease (e.g., cancer) expresses a target epitope recognized by MSAP (e.g., CD19, EpCAM, or WT1).

[0038] In another aspect, the present invention provides an anti-CD3 antibody or antigen-binding fragment thereof that specifically binds to CD3, the anti-CD3 antibody or antigen-binding fragment thereof comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof comprising up to about 5 amino acid variations; HVR-H2 comprising the amino acid sequence of any of SEQ ID NOs: 37, 157, and 166 or a variant thereof comprising up to about 5 amino acid variations; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38 or 158 or a variant thereof comprising up to about 5 amino acid variations; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof comprising up to about 5 amino acid variations; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof comprising up to about 5 amino acid variations; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof comprising up to about 5 amino acid variations.

[0039] In some embodiments according to any of the above-described anti-CD3 antibodies or antigen-binding fragments thereof, the anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 157, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 158. -H2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; or (c) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 42, and a VL comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 43; (b) a VH comprising the amino acid sequence of SEQ ID NO: 159 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 159, and a VL comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 43; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 167 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 43.In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 43; (b) a VH comprising the amino acid sequence of SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 167 and a VL comprising the amino acid sequence of SEQ ID NO: 43.

[0040] In some embodiments according to any of the above anti-CD3 antibodies or antigen-binding fragments thereof, the anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv. In some embodiments, the anti-CD3 antigen-binding fragment is an anti-CD3 scFv. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-CD3 antigen-binding fragment is an anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 89 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; or (c) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179.

[0041] Also provided are MSAPs (eg, BSAPs, TSAPs) comprising any of the above anti-CD3 antibodies or antigen-binding fragments thereof.

[0042] Also provided are isolated nucleic acids encoding any of the above-described anti-CD3 antibodies or antigen-binding fragments thereof, or MSAP, vectors (e.g., viral vectors, e.g., lentiviral vectors or AAV) containing such nucleic acids, and host cells containing such nucleic acids or vectors.

[0043] Also provided is a method for producing an anti-CD3 antibody, or an antigen-binding fragment thereof, or an MSAP thereof, comprising: i) culturing a host cell containing any of the isolated nucleic acids or vectors encoding the anti-CD3 antibody, antigen-binding fragment thereof, or MSAP, or a host cell encoding the anti-CD3 antibody, antigen-binding fragment thereof, or MSAP, under conditions suitable for expression of the anti-CD3 antibody, or antigen-binding fragment thereof, or MSAP; and ii) obtaining the expressed anti-CD3 antibody, antigen-binding fragment thereof, or MSAP from the host cell.

[0044] Also provided are pharmaceutical compositions comprising i) any of the above-described anti-CD3 antibodies or antigen-binding fragments thereof or MSAP, or an isolated nucleic acid or vector (e.g., a viral vector, e.g., a lentiviral vector or AAV) encoding same, and optionally ii) a pharmaceutically acceptable carrier. Also provided are methods for treating a disease in an individual, comprising administering to the individual an effective amount of any of the above-described MSAP or any of the above-described pharmaceutical compositions (e.g., a viral vector pharmaceutical composition). In some embodiments, the disease (e.g., cancer) expresses a target epitope recognized by MSAP (e.g., CD19, EpCAM, or WT1).

[0045] These and other aspects and advantages of the present invention will become apparent from the following detailed description and the appended claims. It is understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention.

[0046] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Brief explanation of the drawings]

[0047] [Figure 1]A shows the structure of a first exemplary MSAP (e.g., TSAP construct 002), B shows the structure of a second exemplary MSAP (e.g., TSAP construct 003), and C shows the structure of a third exemplary MSAP (e.g., TSAP construct 004).

[0048] [Figure 2A] 1 shows the EC50 of exemplary EpCAM×CD8 BSAP constructs 007-012 (anti-EpCAM scFv fused to the N-terminus of anti-CD8 Fab) binding to human CD8α in an ELISA binding assay. [Figure 2B] EC2 of exemplary EpCAMxCD8 BSAP constructs 007-012 (anti-EpCAM scFv fused to the N-terminus of anti-CD8 Fab) binding to cynomolgus monkey CD8α in an ELISA binding assay.

[0049] [Figure 3] A representative panel of flow cytometry data for binding of the exemplary WT1xCD8xCD3 TSAP construct 001 (structure in Figure 1A) to CD8+ and CD4+ T cells is shown. FSC: forward scatter; SSC: side scatter.

[0050] [Figure 4] Binding curves of an exemplary WT1xCD8xCD3 TSAP construct 001 (see structure in Figure 1A) to CD8+ and CD4+ T cells are shown. Conc: concentration.

[0051] [Figure 5-1] A representative panel of flow cytometry data for binding of exemplary CD19xCD8xCD3 TSAP constructs 005 and 006 (structures in Figure 1A) to CD8+ and CD4+ T cells is shown compared to the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of anti-CD19 Fab). FSC: forward scatter; SSC: side scatter. [Figure 5-2] Same as above.

[0052] [Figure 6] Binding curves for BSAP control construct 014 and exemplary CD19xCD8xCD3 TSAP constructs 005 and 006 (structures in Figure 1A) to CD8+ and CD4+ T cells are shown. Conc: concentration.

[0053] [Figure 7] Figure 1 shows specific cytotoxicity of target SW620 cancer cells by T cells incubated with either the exemplary WT1 x CD8 x CD3 TSAP construct 001 (structure in Figure 1A) or the corresponding BSAP control construct 013 (anti-WT1 scFv fused to the N-terminus of anti-CD3 Fab). Conc: concentration.

[0054] [Figure 8] Figure 1 shows specific cytotoxicity of target Raji cancer cells by T cells incubated with either the exemplary CD19xCD8xCD3 TSAP construct 006 (structure in Figure 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of anti-CD19 Fab). Conc: concentration.

[0055] [Figure 9A] The levels of CD4+ (FIG. 9A) and CD8+ (FIG. 9B) T cell activation levels of T cells co-cultured with Raji cells are shown by the extent of CD69+ staining via flow cytometry of CD4+ T cells incubated with either the exemplary CD19xCD8xCD3 TSAP construct 006 (structure in FIG. 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of anti-CD19 Fab). Conc.: concentration. [Figure 9B]The levels of CD4+ (FIG. 9A) and CD8+ (FIG. 9B) T cell activation levels of T cells co-cultured with Raji cells are shown by the extent of CD69+ staining via flow cytometry of CD4+ T cells incubated with either the exemplary CD19xCD8xCD3 TSAP construct 006 (structure in FIG. 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of anti-CD19 Fab). Conc.: concentration.

[0056] [Figure 9C] The T cell activation levels of CD4+ versus CD8+ T cells treated with either BSAP control construct 014 or TSAP construct 006 are compared. The CD4+ (FIG. 9C) and CD8+ (FIG. 9D) T cell activation levels of human PBMCs are shown by the extent of CD69+ staining via flow cytometry of CD4+ or CD8+ T cells incubated with either the exemplary CD19×CD8×CD3 TSAP construct 006 (structure in FIG. 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab). [Figure 9D] The T cell activation levels of CD4+ versus CD8+ T cells treated with either BSAP control construct 014 or TSAP construct 006 are compared. The CD4+ (FIG. 9C) and CD8+ (FIG. 9D) T cell activation levels of human PBMCs are shown by the extent of CD69+ staining via flow cytometry of CD4+ or CD8+ T cells incubated with either the exemplary CD19×CD8×CD3 TSAP construct 006 (structure in FIG. 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab). [Figure 9E]Figure 9 compares the levels of T cell activation in CD4+ versus CD8+ T cells treated with either BSAP control construct 014 or TSAP construct 006. Figure 9 shows T cell activation of CD4+ versus CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells are incubated with either BSAP control construct 014 (Figure 9E) or TSAP construct 006 (Figure 9F). [Figure 9F] Figure 9 compares the levels of T cell activation in CD4+ versus CD8+ T cells treated with either BSAP control construct 014 or TSAP construct 006. Figure 9 shows T cell activation of CD4+ versus CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells are incubated with either BSAP control construct 014 (Figure 9E) or TSAP construct 006 (Figure 9F).

[0057] [Figure 10] Flow cytometry shows the extent of TNFα production in CD4+ T cells (A) and CD8+ T cells (B) incubated with either the exemplary CD19xCD8xCD3 TSAP construct 006 (structure in Figure 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of anti-CD19 Fab). SSC: side scatter.

[0058] [Figure 11] Flow cytometry shows the extent of IFNγ production in CD4+ T cells (A) and CD8+ T cells (B) incubated with either the exemplary CD19xCD8xCD3 TSAP construct 006 (structure in Figure 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of anti-CD19 Fab). SSC: side scatter.

[0059] [Figure 12]The extent of IL-2 production in CD4+ T cells (A) and CD8+ T cells (B) incubated with either the exemplary CD19xCD8xCD3 TSAP construct 006 (structure in Figure 1A) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of anti-CD19 Fab) is shown by flow cytometry. SSC: side scatter.

[0060] [Figure 13A] EC50 curves for TNFα production in CD4+ T cells ( FIG. 13A ) and CD8+ T cells ( FIG. 13B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of TNFα production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 13C ). [Figure 13B] EC50 curves for TNFα production in CD4+ T cells ( FIG. 13A ) and CD8+ T cells ( FIG. 13B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of TNFα production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 13C ). [Figure 13C]EC50 curves for TNFα production in CD4+ T cells ( FIG. 13A ) and CD8+ T cells ( FIG. 13B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of TNFα production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 13C ).

[0061] [Figure 14A] EC50 curves for IFNγ production in CD4+ T cells ( FIG. 14A ) and CD8+ T cells ( FIG. 14B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of IFNγ production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 14C ). [Figure 14B] EC50 curves for IFNγ production in CD4+ T cells ( FIG. 14A ) and CD8+ T cells ( FIG. 14B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of IFNγ production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 14C ). [Figure 14C]EC50 curves for IFNγ production in CD4+ T cells ( FIG. 14A ) and CD8+ T cells ( FIG. 14B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of IFNγ production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 14C ).

[0062] [Figure 15A] EC50 curves for IL-2 production in CD4+ T cells ( FIG. 15A ) and CD8+ T cells ( FIG. 15B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of IL-2 production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 15C ). [Figure 15B] EC50 curves for IL-2 production in CD4+ T cells ( FIG. 15A ) and CD8+ T cells ( FIG. 15B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of IL-2 production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 15C ). [Figure 15C]EC50 curves for IL-2 production in CD4+ T cells ( FIG. 15A ) and CD8+ T cells ( FIG. 15B ) incubated with either exemplary CD19×CD8×CD3 Fab TSAP constructs 005 and 006 (structures in FIG. 1A ) or the corresponding BSAP control construct 014 (anti-CD3 scFv fused to the N-terminus of an anti-CD19 Fab), or a comparison of IL-2 production in CD4+ versus CD8+ T cells treated with either exemplary CD19×CD8×CD3 Fab TSAP construct 006 or the corresponding CD19 Fab×CD3 BSAP control construct 014 ( FIG. 15C ).

[0063] [Figure 16A] 16A-16C show EC50 curves for secretion of the pro-inflammatory cytokines IL-6 (FIG. 16A), IL-2 (FIG. 16B), and IFNγ (FIG. 16C) in human PBMCs incubated with either the exemplary CD19×CD8×CD3 Fab TSAP construct 015 (structure in FIG. 1A) or the corresponding CD19 Fab×CD3 BSAP control construct 014. [Figure 16B] 16A-16C show EC50 curves for secretion of the pro-inflammatory cytokines IL-6 (FIG. 16A), IL-2 (FIG. 16B), and IFNγ (FIG. 16C) in human PBMCs incubated with either the exemplary CD19×CD8×CD3 Fab TSAP construct 015 (structure in FIG. 1A) or the corresponding CD19 Fab×CD3 BSAP control construct 014. [Figure 16C] 16A-16C show EC50 curves for secretion of the pro-inflammatory cytokines IL-6 (FIG. 16A), IL-2 (FIG. 16B), and IFNγ (FIG. 16C) in human PBMCs incubated with either the exemplary CD19×CD8×CD3 Fab TSAP construct 015 (structure in FIG. 1A) or the corresponding CD19 Fab×CD3 BSAP control construct 014.

[0064] [Figure 17]FIG. 1 shows the levels of B cells circulating in the peripheral blood of cynomolgus monkeys after treatment with either a single dose of a 2-hour intravenous infusion (A) or multiple doses of subcutaneous injection (SC) administered at 0, 48, and 96 hours (B) of an exemplary CD19×CD8×CD3 Fab TSAP construct 015 (structure in FIG. 1A ), or the corresponding CD19 Fab×CD3 BSAP control construct 014.

[0065] [Figure 18A] 18A and 18C) or CD8+ T cell (FIGS. 18B and 18D) redistribution levels and recovery from peripheral blood of cynomolgus monkeys after treatment with either a single IV dose (FIGS. 18A-18B) or multiple SC doses administered at 0, 48, and 96 hours (FIGS. 18A-18D) of the exemplary CD19xCD8xCD3 Fab TSAP construct 015 (structure in FIG. 1A), or the corresponding CD19 FabxCD3 BSAP control construct 014. [Figure 18B] 18A and 18C) or CD8+ T cell (FIGS. 18B and 18D) redistribution levels and recovery from peripheral blood of cynomolgus monkeys after treatment with either a single IV dose (FIGS. 18A-18B) or multiple SC doses administered at 0, 48, and 96 hours (FIGS. 18A-18D) of the exemplary CD19xCD8xCD3 Fab TSAP construct 015 (structure in FIG. 1A), or the corresponding CD19 FabxCD3 BSAP control construct 014. [Figure 18C] 18A and 18C) or CD8+ T cell (FIGS. 18B and 18D) redistribution levels and recovery from peripheral blood of cynomolgus monkeys after treatment with either a single IV dose (FIGS. 18A-18B) or multiple SC doses administered at 0, 48, and 96 hours (FIGS. 18A-18D) of the exemplary CD19xCD8xCD3 Fab TSAP construct 015 (structure in FIG. 1A), or the corresponding CD19 FabxCD3 BSAP control construct 014. [Figure 18D] 18A and 18C) or CD8+ T cell (FIGS. 18B and 18D) redistribution levels and recovery from peripheral blood of cynomolgus monkeys after treatment with either a single IV dose (FIGS. 18A-18B) or multiple SC doses administered at 0, 48, and 96 hours (FIGS. 18A-18D) of the exemplary CD19xCD8xCD3 Fab TSAP construct 015 (structure in FIG. 1A), or the corresponding CD19 FabxCD3 BSAP control construct 014.

[0066] [Figure 19A] 1 shows the levels of secreted pro-inflammatory cytokines circulating in the peripheral blood of cynomolgus monkeys after treatment with vehicle (PBS), the exemplary CD19xCD8xCD3 Fab TSAP construct 015 (structure in FIG. 1A ), or the corresponding CD19 FabxCD3 BSAP control construct 014. IL-6 production levels are compared in cynomolgus monkeys treated with CD19 FabxCD3 BSAP 014 at 30 μg / kg IV (n=3) or 150 μg / kg SC (n=3), or with CD19xCD8xCD3 Fab TSAP 015 at 40 μg / kg IV (n=3) or 400 μg / kg SC (n=6). [Figure 19B] 1 shows the levels of secreted pro-inflammatory cytokines circulating in the peripheral blood of cynomolgus monkeys after treatment with vehicle (PBS), the exemplary CD19xCD8xCD3 Fab TSAP construct 015 (structure in FIG. 1A ), or the corresponding CD19 FabxCD3 BSAP control construct 014. IL-6 production levels are compared in cynomolgus monkeys treated with CD19 FabxCD3 BSAP 014 at 30 μg / kg IV (n=3) or 150 μg / kg SC (n=3), or with CD19xCD8xCD3 Fab TSAP 015 at 40 μg / kg IV (n=3) or 400 μg / kg SC (n=6). [Figure 19C]19C , 19D , and 19E show the levels of secreted pro-inflammatory cytokines circulating in the peripheral blood of cynomolgus monkeys after treatment with vehicle (PBS), the exemplary CD19×CD8×CD3 Fab TSAP construct 015 (structure in FIG. 1A ), or the corresponding CD19 Fab×CD3 BSAP control construct 014. The levels of IL-2 ( FIG. 19C ), IFNγ ( FIG. 19D ), and TNFα ( FIG. 19E ) production are shown in cynomolgus monkeys treated with CD19×CD8×CD3 Fab TSAP 015 at 40 μg / kg IV (n=3) or 400 μg / kg SC (n=6). [Figure 19D] 19C , 19D , and 19E show the levels of secreted pro-inflammatory cytokines circulating in the peripheral blood of cynomolgus monkeys after treatment with vehicle (PBS), the exemplary CD19×CD8×CD3 Fab TSAP construct 015 (structure in FIG. 1A ), or the corresponding CD19 Fab×CD3 BSAP control construct 014. The levels of IL-2 ( FIG. 19C ), IFNγ ( FIG. 19D ), and TNFα ( FIG. 19E ) production are shown in cynomolgus monkeys treated with CD19×CD8×CD3 Fab TSAP 015 at 40 μg / kg IV (n=3) or 400 μg / kg SC (n=6). [Figure 19E] 19C , 19D , and 19E show the levels of secreted pro-inflammatory cytokines circulating in the peripheral blood of cynomolgus monkeys after treatment with vehicle (PBS), the exemplary CD19×CD8×CD3 Fab TSAP construct 015 (structure in FIG. 1A ), or the corresponding CD19 Fab×CD3 BSAP control construct 014. The levels of IL-2 ( FIG. 19C ), IFNγ ( FIG. 19D ), and TNFα ( FIG. 19E ) production are shown in cynomolgus monkeys treated with CD19×CD8×CD3 Fab TSAP 015 at 40 μg / kg IV (n=3) or 400 μg / kg SC (n=6).

[0067] [Figure 20]FIG. 1 shows serum concentrations of CD19×CD8×CD3 Fab TSAP 015 over time in cynomolgus monkeys when administered SC at 400 μg / kg TSAP 015 (N=5) or IV at 40 μg / kg TSAP 015 (N=3).

[0068] [Figure 21] Figures 1A-1D show additional trispecific T cell engager formats. A shows the structure of a fourth exemplary MSAP (e.g., TSAP construct 017). B shows the structure of a fifth exemplary MSAP (e.g., TSAP constructs 018, 019, 020, and 022). C shows the structure of a sixth exemplary MSAP (e.g., TSAP construct 021). D shows the structure of a seventh exemplary MSAP (e.g., TSAP construct 023). E shows the structure of an eighth exemplary MSAP (e.g., TSAP construct 024).

[0069] [Figure 22A] Figure 22 shows binding of exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E) to CD4+ and CD8+ T cells: TSAP construct 017 (Figure 22A), TSAP construct 018 (Figure 22B), TSAP construct 019 (Figure 22C), TSAP construct 020 (Figure 22D), TSAP construct 021 (Figure 22E), TSAP construct 023 (Figure 22F), and TSAP construct 024 (Figure 22G). Conc: concentration. [Figure 22B]Figure 22 shows binding of exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E) to CD4+ and CD8+ T cells: TSAP construct 017 (Figure 22A), TSAP construct 018 (Figure 22B), TSAP construct 019 (Figure 22C), TSAP construct 020 (Figure 22D), TSAP construct 021 (Figure 22E), TSAP construct 023 (Figure 22F), and TSAP construct 024 (Figure 22G). Conc: concentration. [Figure 22C] Figure 22 shows binding of exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E) to CD4+ and CD8+ T cells: TSAP construct 017 (Figure 22A), TSAP construct 018 (Figure 22B), TSAP construct 019 (Figure 22C), TSAP construct 020 (Figure 22D), TSAP construct 021 (Figure 22E), TSAP construct 023 (Figure 22F), and TSAP construct 024 (Figure 22G). Conc: concentration. [Figure 22D] Figure 22 shows binding of exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E) to CD4+ and CD8+ T cells: TSAP construct 017 (Figure 22A), TSAP construct 018 (Figure 22B), TSAP construct 019 (Figure 22C), TSAP construct 020 (Figure 22D), TSAP construct 021 (Figure 22E), TSAP construct 023 (Figure 22F), and TSAP construct 024 (Figure 22G). Conc: concentration. [Figure 22E]Figure 22 shows binding of exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E) to CD4+ and CD8+ T cells: TSAP construct 017 (Figure 22A), TSAP construct 018 (Figure 22B), TSAP construct 019 (Figure 22C), TSAP construct 020 (Figure 22D), TSAP construct 021 (Figure 22E), TSAP construct 023 (Figure 22F), and TSAP construct 024 (Figure 22G). Conc: concentration. [Figure 22F] Figure 22 shows binding of exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E) to CD4+ and CD8+ T cells: TSAP construct 017 (Figure 22A), TSAP construct 018 (Figure 22B), TSAP construct 019 (Figure 22C), TSAP construct 020 (Figure 22D), TSAP construct 021 (Figure 22E), TSAP construct 023 (Figure 22F), and TSAP construct 024 (Figure 22G). Conc: concentration. [Figure 22G] Figure 22 shows binding of exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E) to CD4+ and CD8+ T cells: TSAP construct 017 (Figure 22A), TSAP construct 018 (Figure 22B), TSAP construct 019 (Figure 22C), TSAP construct 020 (Figure 22D), TSAP construct 021 (Figure 22E), TSAP construct 023 (Figure 22F), and TSAP construct 024 (Figure 22G). Conc: concentration.

[0070] [Figure 23A]Figure 23 shows specific cytotoxicity against target CD20+ B cells and CD8+ T cells by T cells incubated with exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21B): TSAP construct 017 (Figure 23A), TSAP construct 018 (Figure 23B), TSAP construct 019 (Figure 23C), and TSAP construct 020 (Figure 23D). [Figure 23B] Figure 23 shows specific cytotoxicity against target CD20+ B cells and CD8+ T cells by T cells incubated with exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21B): TSAP construct 017 (Figure 23A), TSAP construct 018 (Figure 23B), TSAP construct 019 (Figure 23C), and TSAP construct 020 (Figure 23D). [Figure 23C] Figure 23 shows specific cytotoxicity against target CD20+ B cells and CD8+ T cells by T cells incubated with exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21B): TSAP construct 017 (Figure 23A), TSAP construct 018 (Figure 23B), TSAP construct 019 (Figure 23C), and TSAP construct 020 (Figure 23D). [Figure 23D] Figure 23 shows specific cytotoxicity against target CD20+ B cells and CD8+ T cells by T cells incubated with exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21B): TSAP construct 017 (Figure 23A), TSAP construct 018 (Figure 23B), TSAP construct 019 (Figure 23C), and TSAP construct 020 (Figure 23D).

[0071] [Figure 24A]Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24B] Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24C]Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24D] Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24E]Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24F] Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24G]Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24H] Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration. [Figure 24I]Figure 2 shows the levels of T cell activation of CD4+ and CD8+ T cells, as indicated by the levels of the T cell activation marker CD69, when T cells were incubated with either BSAP control construct 014 (Figure 24A) or exemplary CD19xCD8xCD3 TSAP constructs (format shown in Figures 21A-21E): TSAP construct 017 (Figure 24B), TSAP construct 018 (Figure 24C), TSAP construct 019 (Figure 24D), TSAP construct 020 (Figure 24E), TSAP construct 021 (Figure 24F), TSAP construct 022 (Figure 24G), TSAP construct 023 (Figure 24H), and TSAP construct 024 (Figure 24I). Conc: concentration.

[0072] [Figure 25A] 1 shows the levels of CD4+ and CD8+ T cell proliferation as assessed by levels of Ki-67, a nuclear protein associated with the cell cycle, after incubation with TSAP construct 020. A representative panel of flow cytometry data from CD8+ T cells, CD4+ T cells, and human PBMCs stained for Ki-67 as a proliferation marker is shown. [Figure 25B] Figure 1 shows the levels of CD4+ and CD8+ T cell proliferation, assessed by levels of Ki-67, a nuclear protein associated with the cell cycle, after incubation with TSAP construct 020. The percentages of CD8+ and CD4+ T cells that are Ki-67+ are shown. Dose-response curves were fitted using Graphpad Prism software, and the corresponding EC50 values ​​were calculated. SSC: side scatter; FSC: forward scatter.

[0073] [Figure 26] Figure 1 shows the tumor growth inhibitory effect of CD19xCD8xCD3 TSAP construct 020 on Raji xenograft tumors implanted subcutaneously in immune-reconstituted NOG mice inoculated with human PBMCs. Vehicle injection (PBST) served as a negative control. SC: subcutaneous; N: number of mice.

[0074] [Figure 27] The mean number of human CD4+ T cells (A) and human CD8+ T cells (B) per milligram of tissue in the spleens of immune-reconstituted NOG mice inoculated with human PBMCs and subcutaneously implanted with Raji xenograft tumors 28 days after Raji implantation is shown. TSAP construct 020 was administered at the indicated dose. Vehicle injection (PBST) served as a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0075] After detailed investigation, the inventors of the present application discovered that the target x CD8 x CD3 MSAP (e.g., TSAP) format described herein has several unexpected advantages over other multispecific proteins. First, the target x CD8 x CD3 MSAP described herein has enhanced cytotoxic activity against target (e.g., cancer) cells, such as CD19-positive Raji Burkitt's lymphoma cancer cells and WT1-positive SW620 colon cancer cells. Second, the half-life of the target x CD8 x CD3 MSAP described herein can be extended by using Fab fragments, thereby allowing for less frequent administration and shorter infusion times, providing additional convenience to patients. Third, the target × CD8 × CD3 MSAP described herein significantly increased CD8+ T cell activation and target antigen-directed binding, did not induce cytotoxicity against CD8+ T cells, and minimized side effects induced by CD4+ T cell activation (e.g., cytokine storm) compared with, for example, target × CD3 BSAP or TSAP (without an anti-CD8 moiety) that do not discriminate between CD4+ and CD8+ T cell binding. Fourth, the target × CD8 × CD3 MSAP described herein is cross-reactive with both human and cynomolgus monkey CD8, which may facilitate extrapolation of toxicity and efficacy results obtained from cynomolgus monkeys to human clinical trials. Fifth, the target × CD8 × CD3 MSAP described herein demonstrated in vivo clearance of target cells (e.g., CD19+ B cells) in cynomolgus monkeys, as well as rapid CD8+ T cell redistribution and recovery (indicating in vivo CD8+ T cell activation). Sixth, the Targeted x CD8 x CD3 MSAP described herein exhibited robust pharmacokinetic properties upon both subcutaneous (SC) and intravenous (IV) administration to cynomolgus monkeys.Seventh, the targeted × CD8 × CD3 MSAPs described herein exhibited comparable in vivo efficacy when administered SC or IV, and, more surprisingly, demonstrated improved safety when administered SC compared to BSAP controls, based on the in vivo production and systemic circulation of pro-inflammatory cytokines (e.g., IL-6, TNFα) that can induce cytokine release syndrome or cytokine storm. These surprising effects were demonstrated with MSAPs of various structures, sequences, and target antigens tested. Taken together, these findings demonstrate the unique pharmacokinetic and pharmacodynamic properties of the targeted × CD8 × CD3 MSAPs described herein and their improved therapeutic efficacy compared to bispecific antibodies (i.e., BSAPs).

[0076] Thus, in one aspect, the present invention provides an MSAP (e.g., TSAP) comprising: i) an anti-CD3 portion that specifically binds to CD3; ii) an anti-CD8 portion that specifically binds to CD8; and iii) an anti-target epitope portion that specifically binds to a target epitope (e.g., a TSA or TAA, e.g., CD19, EpCAM, or WT1), wherein a first of the three portions is a Fab fragment, the Fab fragment comprising a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; a second of the three portions is a first antigen-binding fragment (e.g., scFv); and a third of the three portions is a second antigen-binding fragment (e.g., scFv); one antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment; 2) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment; 3) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment; 4) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment; or 5) the first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment.In some embodiments, a) the anti-CD3 portion is a Fab fragment ("anti-CD3 Fab fragment"), the anti-CD8 portion is a first antigen-binding fragment ("anti-CD8 first antigen-binding fragment"), and the anti-target epitope portion is a second antigen-binding fragment ("anti-target epitope second antigen-binding fragment"); b) the anti-CD3 portion is a Fab fragment, the anti-target epitope portion is a first antigen-binding fragment ("anti-target epitope first antigen-binding fragment"), and the anti-CD8 portion is a second antigen-binding fragment ("anti-CD8 second antigen-binding fragment"); c) the anti-CD8 portion is a Fab fragment ("anti-CD8 Fab fragment"), the anti-CD3 portion is a first antigen-binding fragment ("anti-CD3 first antigen-binding fragment"), and the anti-target epitope portion is a second antigen-binding fragment; d) the anti-CD8 portion is a Fab fragment, the anti-target epitope portion is a first antigen-binding fragment, and the anti-CD3 portion is a second antigen-binding fragment ("anti-CD3 second antigen-binding fragment"); e) the anti-target epitope portion is a Fab fragment ("anti-target epitope Fab fragment"), the anti-CD3 portion is a first antigen-binding fragment, and the anti-CD8 portion is a second antigen-binding fragment; or f) the anti-target epitope portion is a Fab fragment, the anti-CD8 portion is a first antigen-binding fragment, and the anti-CD3 portion is a second antigen-binding fragment. In some embodiments, the anti-target epitope portion specifically recognizes a target epitope of a TAA or TSA. In some embodiments, the TAA is CD19 or EpCAM. In some embodiments, the TSA is WT1. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are both scFvs. Exemplary MSAPs are shown in Figures 1A-1C and 21A-21E.

[0077] In another aspect, the present invention provides a novel anti-CD8 antibody or antigen-binding fragment thereof, a novel anti-CD3 antibody or antigen-binding fragment thereof, and an MSAP (e.g., BSAP, TSAP) comprising the same.

[0078] Also provided are pharmaceutical compositions and kits comprising any of the target x CD8 x CD3 MSAPs (e.g., TSAPs), anti-CD8 antibodies or antigen-binding fragments thereof (or MSAPs thereof), or anti-CD3 antibodies or antigen-binding fragments thereof (or MSAPs thereof) described herein. Additionally, provided are methods of using MSAPs or pharmaceutical compositions thereof, for example, to treat cancer (e.g., CD19-positive cancers, EpCAM-positive cancers, and WT1-positive cancers).

[0079] I. Definition The practice of the present invention will employ, unless expressly indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the skill of those in the art, many of which are described below by way of example, and such techniques are fully explained in the literature. For example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley&Sons, New York, NY (2009), Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley&Sons, 1995, Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), DNA Cloning: A Practical Approach, vol. I&II (D. Glover, ed.), Oligonucleotide Synthesis (N. Gait, ed., 1984), Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985), Transcription and Translation (B. Hames & S. Higgins, eds., 1984), Animal Cell See Culture (R. Freshney, ed., 1986), Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0080] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during clinical pathology. Desirable effects of treatment include slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated (including, but not limited to, reducing (or destroying) the proliferation of cancerous cells, reducing symptoms caused by the disease, improving the quality of life of a patient with the disease, reducing the dose of other medications required to treat the disease, and / or extending the individual's survival).

[0081] As used herein, "effective amount" refers to an amount of a drug or agent effective in treating a disease or disorder in a subject. In the case of cancer, an effective amount of a drug may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if a desired result can be or is achieved in conjunction with one or more other agents.

[0082] As used herein, "individual" or "subject" refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0083] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (so long as they exhibit the desired biological activity or function). As used herein, the terms "immunoglobulin (Ig)" and "antibody" are used interchangeably.

[0084] The terms "native antibody," "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies in a substantially intact form, rather than antibody fragments, as defined below. This term specifically refers to antibodies having heavy chains, including an Fc region. Naturally occurring antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by multiple constant domains. Each light chain has a variable domain (V L ) and a constant domain at its other end, with the light-chain constant domain aligned with the first constant domain of the heavy chain and the light-chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0085] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain is the C H 1. C H 2 and C H It contains three domains (collectively CH) as well as the CHL (or CL) domain of the light chain.

[0086] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain is sometimes referred to as "VH." The variable domain of the light chain is sometimes referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0087] The term "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are responsible for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs, also called CDRs) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain four FR regions, which primarily adopt a beta-sheet configuration with three HVRs connected to the beta-sheet structure, forming loops that in some cases form part of the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0088] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0089] The term IgG "isotype" or "subclass" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0090] Depending on the amino acid sequence in the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known and are generally described, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody may be part of a larger fusion molecule formed by covalent or noncovalent association of the antibody with one or more other proteins or peptides.

[0091] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding region thereof. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments (e.g., single-chain variable fragments, scFv), diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0092] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments (each with a single antigen-binding site), and a residual "Fc" fragment (this name reflects its ability to crystallize readily). Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0093] An "Fv" is the minimum antibody fragment containing a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains can associate in a "dimeric" structure similar to that of a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0094] Fab fragments have two polypeptide chains, containing heavy and light chain variable domains (VH, VL), as well as a light chain constant domain (CL) and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein to represent Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0095] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies. Springer Berlin Heidelberg, 1994, pp. 269-315.

[0096] The "Fc" fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain cell types.

[0097] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous antibody population, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of distinct antibodies. In some embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, where the target-binding polypeptide sequence was obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a pool of multiple clones, e.g., hybridoma clones, phage clones, or recombinant DNA clones. The selected target-binding sequence can be further modified, e.g., to improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and it is understood that antibodies comprising modified target-binding sequences are also monoclonal antibodies of the present invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0098] The modifier "monoclonal" indicates the character of the antibody as a population obtained from substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), phage display technology (e.g., Clackson et al., Nature 352:624-628 (1991); Marks et al. al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);andLeet al.,J.Immunol.Methods 284(1-2):119-132 (2004)), as well as techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO1998 / 24893, WO1996 / 34096, WO1996 / 33735, WO1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993), Jakobovits et al.,Nature 362:255-258(1993),Bruggemann et al.,Year in Immunol.7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al.,Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-813 (1994), Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0099] As used herein, the term "monoclonal antibody" expressly includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include the PRIMATTZED® antibody, the antigen-binding region of which is derived from an antibody produced, for example, by immunizing macaque monkeys with an antigen of interest.

[0100] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and / or capacity. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. Usually, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0101] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies, such as those disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991). Methods available for preparing human monoclonal antibodies are also described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomice, that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been inactivated (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006), for human antibodies generated by human B cell hybridoma technology.

[0102] The terms "hypervariable region," "HVR," or "HV," as used herein, refer to regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Antibodies generally contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural antibodies, H3 and L3 exhibit the highest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring antibody fine specificity. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable even in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). HVRs are also referred to as "CDRs" or "complementarity-determining regions."

[0103] The structure and location of immunoglobulin variable regions may be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and the latest edition thereof currently available on the Internet (immuno.bme.nwu.edu).

[0104] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0105] The term "covalently linked," as used herein, refers to a direct linkage through one or more chemical bonds or an indirect linkage through one or more linkers. Any suitable chemical bond can be used to create the direct linkage, including, but not limited to, a covalent bond, such as a peptide bond and a disulfide bond, or a non-covalent bond, such as a hydrogen bond, a hydrophobic bond, an ionic bond, or a van der Waals bond.

[0106] As used herein, a "covalent bond" refers to a stable bond between two atoms that share one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, a "peptide bond" refers to a covalent bond formed between a carboxyl group of an amino acid and an amine group of an adjacent amino acid. A "disulfide bond" refers to a covalent bond formed between two sulfur atoms (e.g., the combination of a heavy chain fragment CH1 and a light chain fragment CL via one or more disulfide bonds). One or more disulfide bonds can be formed between two fragments by linking thiol groups within the two fragments. In some embodiments, one or more disulfide bonds can be formed between one or more cysteines of the heavy chain fragment and the light chain fragment, respectively. A disulfide bond can be formed by oxidation of two thiol groups. In some embodiments, the covalent linkage is a direct covalent bond. In some embodiments, the covalent linkage is a direct peptide bond or a disulfide bond.

[0107] As used herein, the terms "binds to," "specifically binds to," or "specific for" refer to a measurable and reproducible interaction (e.g., binding between a target and an antibody) that determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may also be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or with a longer duration than to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of that antibody to its target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the dissociation constant (K d ) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0108] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences to be compared.

[0109] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into an antibody of interest, and the product can be screened for a desired activity, such as retention or improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. [Table 1]

[0110] Amino acids may be grouped by common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) acidic: Asp, Glu, (4) basic: His, Lys, Arg, (5) residues that affect chain orientation: Gly, Pro, (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0111] The term "multispecific" when used in conjunction with an antibody or antigen-binding protein (e.g., multispecific antigen-binding protein, MSAP) refers to an antibody or antigen-binding protein that has polyepitopic specificity (i.e., capable of specifically binding to two, three, or more different epitopes on a single biological molecule, or capable of specifically binding to epitopes on two, three, or more different biological molecules). Unless otherwise indicated, the antigens to which the multispecific antibody or MSAP binds can be listed in any order in the name of the multispecific antibody or MSAP. That is, these terms can be used interchangeably to refer to a multispecific antibody (e.g., MSAP) that specifically binds to CD3, CD8, and a target epitope (e.g., a particular TSA or TAA (e.g., CD19, EpCAM, or WT1)). In some embodiments, the MSAP is a TSAP.

[0112] The term "bispecific" when used in conjunction with an antibody or antigen-binding protein (e.g., bispecific antigen-binding protein, BSAP) refers to an antibody or antigen-binding protein capable of specifically binding to two different epitopes on one biological molecule or to epitopes on two different biological molecules. Unless otherwise indicated, the antigens to which the bispecific antibody or BSAP binds can be listed in any order in the name of the bispecific antibody or BSAP. In some embodiments, the BSAP is bivalent. In some embodiments, the BSAP is multivalent (e.g., trivalent).

[0113] The term "trispecific" when used in conjunction with an antibody or antigen-binding protein (e.g., trispecific antigen-binding protein, TSAP) refers to an antibody or antigen-binding protein that can specifically bind to three different epitopes on one biological molecule or that can specifically bind to epitopes on three different biological molecules. Unless otherwise indicated, the antigens to which the trispecific antibody or TSAP binds can be listed in any order in the name of the trispecific antibody or TSAP. That is, these terms can be used interchangeably to refer to a trispecific antibody (e.g., a TSAP) that specifically binds to CD3, CD8, and a target epitope (e.g., a particular TSA or TAA (e.g., CD19, EpCAM, or WT1)); for example, any of the terms "TAA(TSA) x CD3 x CD8," "TAA(TSA) x CD8 x CD3," "CD3 x CD8 x TAA(TSA)," "CD8 x CD3 x TAA(TSA)," "CD8 x TAA(TSA) x CD3," and "CD3 x TAA(TSA) x CD8" can be used. In some embodiments, the TSAP is bivalent. In some embodiments, the TSAP is multivalent (e.g., trivalent).

[0114] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide that donates its amine group to form a peptide bond with the carboxyl group of the adjacent amino acid residue. The "N-terminus" of a polypeptide, as used herein, refers to the first amino acid of the polypeptide that donates its carboxyl group to form a peptide bond with the amine group of the adjacent amino acid residue.

[0115] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked to them. Such vectors are referred to herein as "expression vectors."

[0116] The term "cell" includes the primary subject cell and its progeny.

[0117] The term "cytokine storm" (also known as "cytokine cascade" or "hypercytokinemia") refers to a potentially lethal immune response that typically consists of a positive feedback loop between cytokines and immune cells, resulting in highly elevated levels of various cytokines (e.g., INF-γ, IL-10, IL-6, CCL2, etc.).

[0118] Those skilled in the art will understand that uracil and thymine can both be represented by a "t" instead of a "u" for uracil and a "t" for thymine. In the context of ribonucleic acids, it will be understood that "t" is used to represent uracil unless otherwise indicated.

[0119] It is to be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0120] Reference herein to "about" a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, a reference to "about X" includes the reference to "X."

[0121] As used herein, reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancer types other than X.

[0122] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0123] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the content clearly dictates otherwise.

[0124] II. Multispecific antigen-binding proteins (MSAPs) In one aspect, the present invention provides a multispecific antigen-binding protein (MSAP) comprising: i) an anti-CD3 portion that specifically binds to CD3; ii) an anti-CD8 portion that specifically binds to CD8; and iii) an anti-target epitope that specifically binds to a target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein a first of the three portions is a Fab fragment, the Fab fragment comprising a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; a second of the three portions is a first antigen-binding fragment (e.g., an scFv); and a third of the three portions is a second antigen-binding fragment (e.g., an scFv). In some embodiments, the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment, and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment, and the second antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment, and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment, and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment, and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment, and the second antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment, and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment, and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment. In some embodiments, the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment, and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment.In some embodiments, a first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment, and a second antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment. In some embodiments, a first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment, and a second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment. In some embodiments, a first antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment, and a second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the MSAP comprises two or more first antigen-binding fragments (e.g., scFvs) and / or two or more second antigen-binding fragments (e.g., scFvs). In some embodiments, two or more first antigen-binding fragments (e.g., scFvs) are fused to each other in tandem and then fused to a Fab fragment (e.g., the N' of the VH, the N' of the VL, the C' of the CH1, and / or the C' of the CL). In some embodiments, two or more second antigen-binding fragments (e.g., scFvs) are fused in tandem to each other and then to a Fab fragment (e.g., N' of VH, N' of VL, C' of CH1, and / or C' of CL). In some embodiments, two or more first antigen-binding fragments (e.g., scFvs) are fused to the N-terminus of a Fab fragment (e.g., one at the N' of VH and / or the other at the N' of VL). In some embodiments, two or more second antigen-binding fragments (e.g., scFvs) are fused to the C-terminus of a Fab fragment (e.g., one at the C' of CH1 and / or the other at the C' of CL). In some embodiments, two or more second antigen-binding fragments (e.g., scFvs) are fused to the N-terminus of a Fab fragment (e.g., one at the N' of VH and / or the other at the N' of VL). In some embodiments, a first antigen-binding fragment (eg, an scFv) is fused to the C-terminus of a Fab fragment (eg, one at the C' of CH1 and / or the other at the C' of CL).

[0125] In some embodiments, a MSAP is provided that includes: i) an anti-CD3 portion that specifically binds to CD3; ii) an anti-CD8 portion that specifically binds to CD8; and iii) an anti-target epitope that specifically binds to a target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein a first of the three portions is a Fab fragment, the Fab fragment comprising a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; a second of the three portions is a first antigen-binding fragment (e.g., an scFv); and a third of the three portions is a second antigen-binding fragment (e.g., an scFv), wherein: 1) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment; 2) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment; 3) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment; 4) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment; or 5) the first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment.In some embodiments, a) the anti-CD3 portion is a Fab fragment ("anti-CD3 Fab fragment"), the anti-CD8 portion is a first antigen-binding fragment ("anti-CD8 first antigen-binding fragment"), and the anti-target epitope portion is a second antigen-binding fragment ("anti-target epitope second antigen-binding fragment"); b) the anti-CD3 portion is a Fab fragment, the anti-target epitope portion is a first antigen-binding fragment ("anti-target epitope first antigen-binding fragment"), and the anti-CD8 portion is a second antigen-binding fragment ("anti-CD8 second antigen-binding fragment"); c) the anti-CD8 portion is a Fab fragment ("anti-CD8 d) the anti-CD8 portion is a Fab fragment, the anti-target epitope portion is a first antigen-binding fragment, and the anti-CD3 portion is a second antigen-binding fragment ("anti-CD3 second antigen-binding fragment"); e) the anti-target epitope portion is a Fab fragment ("anti-target epitope Fab fragment"), the anti-CD3 portion is a first antigen-binding fragment, and the anti-CD8 portion is a second antigen-binding fragment; or f) the anti-target epitope portion is a Fab fragment, the anti-CD8 portion is a first antigen-binding fragment, and the anti-CD3 portion is a second antigen-binding fragment. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from the group consisting of Fab, Fab', Fab'-SH, F(ab'), Fv, sdAb, scFv, and any combination thereof. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are both scFvs. In some embodiments, CH1 comprises the amino acid sequence of SEQ ID NO: 93, and CL comprises the amino acid sequence of SEQ ID NO: 94 or 165. In some embodiments, the first antigen-binding fragment is fused to a Fab fragment via an optional first linker, and / or the second antigen-binding fragment is fused to a Fab fragment via an optional second linker. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.In some embodiments, the anti-CD8 portion comprises: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. VR-L3; iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; v) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 10, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12; or vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23.In some embodiments, the anti-CD8 moiety comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; iv) a VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 25; v) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 33; or vi) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD8 moiety is an anti-CD8 scFv, and the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 21, 44-49, and 51-55 (e.g., SEQ ID NOs: 46, 49, 52, or 55). In some embodiments, the anti-CD8 moiety is an anti-CD8 Fab fragment, and the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 147 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 149 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 150; iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; iv) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 151 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; v) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 153 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 154; or vi) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156.In some embodiments, the anti-CD3 portion comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, or (c) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-CD3 portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 43; (b) a VH comprising the amino acid sequence of SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 167 and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD3 portion is an anti-CD3 scFv, and the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-CD3 portion is an anti-CD3 Fab fragment, and the anti-CD3 Fab fragment comprises (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 89 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the anti-target epitope portion specifically recognizes CD19 (anti-CD19 portion).In some embodiments, the anti-CD19 portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD19 portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 portion is an anti-CD19 scFv, wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the anti-CD19 portion is an anti-CD19 Fab portion, wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the anti-target epitope portion specifically recognizes EpCAM (anti-EpCAM portion). In some embodiments, the anti-EpCAM portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-EpCAM portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-EpCAM portion is an anti-EpCAM scFv, and the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78. In some embodiments, the anti-EpCAM portion is an anti-EpCAM Fab fragment, and the anti-EpCAM Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 76 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the anti-target epitope portion specifically recognizes WT1 (anti-WT1 portion). In some embodiments, the anti-WT1 portion is HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, SEQ ID NO:80. HVR-H2 comprising the amino acid sequence of SEQ ID NO: 81, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 81, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the anti-WT1 moiety comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 85, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the anti-WT1 moiety is an anti-WT1 scFv, wherein the anti-WT1 scFv comprises the amino acid sequence of SEQ ID NO: 87 or 88. In some embodiments, the anti-WT1 moiety is an anti-WT1 Fab moiety, wherein the anti-WT1 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 145 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 146. In some embodiments, the MSAP is trivalent and trispecific, hereinafter referred to as a "TSAP," or, if the anti-target epitope portion recognizes an epitope of a target TAA or TSA, hereinafter referred to as a "TAA (TSA) x CD8 x CD3 TSAP." Exemplary TAA (TSA) x CD8 x CD3 MSAPs (e.g., TSAPs) are shown in Figures 1A-1C and 21A-21E and in the Examples.

[0126] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD8; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0127] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD8 is fused to the N-terminus of the VL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, the first antigen-binding fragment of the anti-target epitope and the second antigen-binding fragment of the anti-CD8 are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0128] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0129] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD3; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD3 is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, the first antigen-binding fragment of the anti-target epitope and the second antigen-binding fragment of the anti-CD3 are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0130] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD3 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD8 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0131] In some embodiments, an MSAP (e.g., a TSAP) is provided that comprises: i) an anti-target epitope Fab fragment; ii) a first antigen-binding fragment of anti-CD8 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD3 (e.g., an scFv), wherein the anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD3 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0132] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD3 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD8 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the C-terminus of the CH1 of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0133] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD8 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD3 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD8 is fused to the C-terminus of the CH1 of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD3 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0134] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD8; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD8 is fused to the C-terminus of the CH1 of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of the anti-target epitope are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0135] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CH1 of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD8 is fused to the N-terminus of the VL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, the first antigen-binding fragment of the anti-target epitope and the second antigen-binding fragment of the anti-CD8 are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0136] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the C-terminus of the CH1 of the anti-CD8 Fab fragment via an optional first linker, and the second antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of the anti-target epitope are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0137] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD3; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CH1 of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD3 is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, the second antigen-binding fragment of anti-CD3 and the first antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0138] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD3 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD8 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD8 is fused to the C-terminus of the CL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0139] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD8 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD3 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD3 is fused to the C-terminus of the CL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD3 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0140] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-CD8; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker, and the second antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of the anti-target epitope are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0141] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD8 is fused to the C-terminus of the CL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, the second antigen-binding fragment of anti-CD8 and the first antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0142] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker, and the second antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of the anti-target epitope are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0143] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD3; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD3 is fused to the C-terminus of the CL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, the second antigen-binding fragment of anti-CD3 and the first antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0144] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD3 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD8 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD8 is fused to the C-terminus of the CH1 of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0145] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD8 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD3 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD3 is fused to the C-terminus of the CH1 of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD3 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0146] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD8; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CH1 of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0147] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD8 is fused to the C-terminus of the CH1 of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, the second antigen-binding fragment of anti-CD8 and the first antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0148] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CH1 of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of the anti-target epitope are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0149] In some embodiments, an MSAP (e.g., a TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., an scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., an scFv) of anti-CD3; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD3 is fused to the C-terminus of the CH1 of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, the second antigen-binding fragment of anti-CD3 and the first antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0150] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD3 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD8 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VL of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD8 is fused to the C-terminus of the CL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0151] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) Fab fragment; ii) a first antigen-binding fragment of anti-CD8 (e.g., an scFv); and iii) a second antigen-binding fragment of anti-CD3 (e.g., an scFv), wherein the anti-target epitope Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VL of the anti-target epitope Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD3 is fused to the C-terminus of the CL of the anti-target epitope Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD3 are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0152] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-CD8; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VL of the anti-CD3 Fab fragment via an optional first linker, and the second antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of the anti-target epitope are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0153] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD3 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VL of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD8 is fused to the C-terminus of the CL of the anti-CD3 Fab fragment via an optional second linker. In some embodiments, the second antigen-binding fragment of anti-CD8 and the first antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0154] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and iii) a second antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, wherein the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional first linker, and the second antigen-binding fragment of the anti-target epitope is fused to the C-terminus of the CL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, both the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of the anti-target epitope are scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0155] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: i) an anti-CD8 Fab fragment; ii) a first antigen-binding fragment (e.g., scFv) of an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM); and iii) a second antigen-binding fragment (e.g., scFv) of anti-CD3; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of the anti-target epitope is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of the anti-CD3 is fused to the C-terminus of the CL of the anti-CD8 Fab fragment via an optional second linker. In some embodiments, the second antigen-binding fragment of anti-CD3 and the first antigen-binding fragment of the anti-target epitope are both scFvs. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0156] In some embodiments, an MSAP (or TSAP) comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-optional L1-anti-target epitope VL-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VH-optional L3-anti-CD8 VL-optional L4-anti-CD3 VL-CL-C'. In some embodiments, an MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-optional L1-anti-target epitope VL-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VL-optional L3-anti-CD8 VH-optional L4-anti-CD3 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VL-optional L1-anti-target epitope VH-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VH-optional L3-anti-CD8 VL-optional L4-anti-CD3 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VL-optional L1-anti-target epitope VH-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VL-optional L3-anti-CD8 VH-optional L4-anti-CD3 VL-CL-C'. In some embodiments, the MSAP (or TSAP) comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 VH-optional L1-anti-CD8 VL-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) VH-optional L3-anti-target epitope VL-optional L4-anti-CD3 VL-CL-C'.In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 VH-optional L1-anti-CD8 VL-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VL-optional L3-anti-target epitope VH-optional L4-anti-CD3 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 VL-optional L1-anti-CD8 VH-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-optional L3-anti-target epitope VL-optional L4-anti-CD3 VL-CL-C'. In some embodiments, an MSAP (e.g., a TSAP) comprises a first fusion polypeptide having the structure: N'-anti-CD8 VL-optional L1-anti-CD8 VH-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide having the structure: N'-anti-target epitope (e.g., TAA or TSA) VL-optional L3-anti-target epitope VH-optional L4-anti-CD3 VL-CL-C'. In some embodiments, an MSAP (or TSAP) comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VH-optional L1-anti-CD3 VL-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-optional L3-anti-target epitope VL-optional L4-anti-CD8 VL-CL-C'. In some embodiments, an MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VH-optional L1-anti-CD3 VL-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VL-optional L3-anti-target epitope VH-optional L4-anti-CD8 VL-CL-C'.In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VL-optional L1-anti-CD3 VH-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-optional L3-anti-target epitope VL-optional L4-anti-CD8 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VL-optional L1-anti-CD3 VH-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VL-optional L3-anti-target epitope VH-optional L4-anti-CD8 VL-CL-C'. In some embodiments, an MSAP (or TSAP) comprises a first fusion polypeptide having the structure: N'-anti-CD3 VH-optional L1-anti-CD3 VL-optional L2-anti-target epitope (e.g., a TAA or TSA) VH-CH1-C', and a second fusion polypeptide having the structure: N'-anti-CD8 VH-optional L3-anti-CD8 VL-optional L4-anti-target epitope VL-CL-C'. In some embodiments, an MSAP comprises a first fusion polypeptide having the structure: N'-anti-CD3 VH-optional L1-anti-CD3 VL-optional L2-anti-target epitope (e.g., a TAA or TSA) VH-CH1-C', and a second fusion polypeptide having the structure: N'-anti-CD8 VL-optional L3-anti-CD8 VH-optional L4-anti-target epitope VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VL-optional L1-anti-CD3 VH-optional L2-anti-target epitope (e.g., TAA or TSA) VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VH-optional L3-anti-CD8 VL-optional L4-anti-target epitope VL-CL-C'.In some embodiments, an MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VL-optional L1-anti-CD3 VH-optional L2-anti-target epitope (e.g., a TAA or TSA) VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VL-optional L3-anti-CD8 VH-optional L4-anti-target VL-CL-C'. In some embodiments, an MSAP (or TSAP) comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-optional L1-anti-target epitope VL-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VH-optional L3-anti-CD3 VL-optional L4-anti-CD8 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-optional L1-anti-target epitope VL-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VL-optional L3-anti-CD3 VH-optional L4-anti-CD8 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VL-optional L1-anti-target epitope VH-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VH-optional L3-anti-CD3 VL-optional L4-anti-CD8 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) VL-optional L1-anti-target epitope VH-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VL-optional L3-anti-CD3 VH-optional L4-anti-CD8 VL-CL-C'.In some embodiments, an MSAP (or TSAP) comprises a first fusion polypeptide having the structure: N'-anti-CD8 VH-optional L1-anti-CD8 VL-optional L2-anti-target epitope (e.g., a TAA or TSA) VH-CH1-C', and a second fusion polypeptide having the structure: N'-anti-CD3 VH-optional L3-anti-CD3 VL-optional L4-anti-target epitope VL-CL-C'. In some embodiments, an MSAP comprises a first fusion polypeptide having the structure: N'-anti-CD8 VH-optional L1-anti-CD8 VL-optional L2-anti-target epitope (e.g., a TAA or TSA) VH-CH1-C', and a second fusion polypeptide having the structure: N'-anti-CD3 VL-optional L3-anti-CD3 VH-optional L4-anti-target epitope VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 VL-optional L1-anti-CD8 VH-optional L2-anti-target epitope (e.g., TAA or TSA) VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VH-optional L3-anti-CD3 VL-optional L4-anti-target epitope VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 VL-optional L1-anti-CD8 VH-optional L2-anti-target epitope (e.g., TAA or TSA) VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VL-optional L3-anti-CD3 VH-optional L4-anti-target epitope VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising: N'-anti-CD8 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-target epitope (e.g., TAA or TSA) VH-CH1-C'; and a second fusion polypeptide comprising: N'-anti-target epitope VL-CL-optional L3-anti-CD3 scFv(VH-optional L4-VL or VL-optional L4-VH)-C'.In some embodiments, the MSAP comprises a first fusion polypeptide comprising: N'-anti-CD3 scFv (VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-target epitope (e.g., TAA or TSA) VH-CH1-C'; and a second fusion polypeptide comprising: N'-anti-target epitope VL-CL-optional L3-anti-CD8 scFv (VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VL-CL-optional L3-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv (VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD3 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 VL-CL-optional L3-anti-CD8 scFv (VH-optional L4-VL or VL-optional L). In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VL-CL-optional L3-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD8 VH-CH1-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 VL-CL-optional L3-anti-CD3 scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) VH-CH1-optional L1-anti-CD3 scFv (VH-optional L2-VL or VL-optional L2-VH)-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 scFv (VH-optional L3-VL or VL-optional L3-VH)-optional L4-anti-target epitope VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) VH-CH1-optional L1-anti-CD8 scFv (VH-optional L2-VL or VL-optional L2-VH)-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 scFv (VH-optional L3-VL or VL-optional L3-VH)-optional L4-anti-target epitope VL-CL-C'.In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VH-CH1-optional L1-anti-CD8 scFv(VH-optional L2-VL or VL-optional L2-VH)-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L3-VL or VL-optional L3-VH)-optional L4-anti-CD3 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD3 VH-CH1-optional L1-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L2-VL or VL-optional L2-VH)-C', and a second fusion polypeptide comprising the structure: N'-anti-CD8 scFv(VH-optional L3-VL or VL-optional L3-VH)-optional L4-anti-CD3 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 VH-CH1-optional L1-anti-CD3 scFv(VH-optional L2-VL or VL-optional L2-VH)-C', and a second fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L3-VL or VL-optional L3-VH)-optional L4-anti-CD8 VL-CL-C'. In some embodiments, the MSAP comprises a first fusion polypeptide comprising the structure: N'-anti-CD8 VH-CH1-optional L1-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L2-VL or VL-optional L2-VH)-C', and a second fusion polypeptide comprising the structure: N'-anti-CD3 scFv(VH-optional L3-VL or VL-optional L3-VH)-optional L4-anti-CD8 VL-CL-C'.In some embodiments, the MSAP comprises a fusion polypeptide comprising the structure: N'-anti-CD8 scFv (VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-target epitope (e.g., TAA or TSA) VH-CH1-optional L3-anti-CD3 scFv (VH-optional L4-VL or VL-optional L4-VH)-C', and a polypeptide comprising the structure: N'-anti-target epitope VL-CL-C'. In some embodiments, the MSAP comprises a fusion polypeptide comprising the structure: N'-anti-CD3 scFv (VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-target epitope (e.g., TAA or TSA) VH-CH1-optional L3-anti-CD8 scFv (VH-optional L4-VL or VL-optional L4-VH)-C', and a polypeptide comprising the structure: N'-anti-target epitope VL-CL-C'. In some embodiments, the MSAP comprises a fusion polypeptide comprising the structure: N'-anti-CD8 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD3 VH-CH1-optional L3-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L4-VL or VL-optional L4-VH)-C', and a polypeptide comprising the structure: N'-anti-CD3 VL-CL-C'. In some embodiments, the MSAP comprises a fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD3 VH-CH1-optional L3-anti-CD8 scFv(VH-optional L4-VL or VL-optional L4-VH)-C', and a polypeptide comprising the structure: N'-anti-CD3 VL-CL-C'.In some embodiments, the MSAP comprises a fusion polypeptide comprising the structure: N'-anti-CD3 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD8 VH-CH1-optional L3-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L4-VL or VL-optional L4-VH)-C', and a polypeptide comprising the structure: N'-anti-CD8 VL-CL-C'. In some embodiments, the MSAP comprises a fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD8 VH-CH1-optional L3-anti-CD3 scFv(VH-optional L4-VL or VL-optional L4-VH)-C', and a polypeptide comprising the structure: N'-anti-CD8 VL-CL-C'. In some embodiments, the MSAP comprises a polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-CH1-C' and a fusion polypeptide comprising the structure: N'-anti-CD8 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-target epitope VL-CL-optional L3-anti-CD3 scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a polypeptide comprising the structure: N'-anti-target epitope (e.g., a TAA or TSA) VH-CH1-C' and a fusion polypeptide comprising the structure: N'-anti-CD3 scFv (VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-target epitope VL-CL-optional L3-anti-CD8 scFv (VH-optional L4-VL or VL-optional L4-VH)-C'.In some embodiments, the MSAP comprises a polypeptide comprising the structure: N'-anti-CD3 VH-CH1-C' and a fusion polypeptide comprising the structure: N'-anti-CD8 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD3 VL-CL-optional L3-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a polypeptide comprising the structure: N'-anti-CD3 VH-CH1-C' and a fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD3 VL-CL-optional L3-anti-CD8 scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a polypeptide comprising the structure: N'-anti-CD8 VH-CH1-C' and a fusion polypeptide comprising the structure: N'-anti-CD3 scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD8 VL-CL-optional L3-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. In some embodiments, the MSAP comprises a polypeptide having the structure: N'-anti-CD8 VH-CH1-C' and a fusion polypeptide comprising the structure: N'-anti-target epitope (e.g., TAA or TSA) scFv(VH-optional L1-VL or VL-optional L1-VH)-optional L2-anti-CD8 VL-CL-optional L3-anti-CD3 scFv(VH-optional L4-VL or VL-optional L4-VH)-C'. L1, L2, L3, and L4 are any possible linkers (e.g., peptide linkers) described herein, such as GG and any of SEQ ID NOs: 108-110. L1-L4 may be the same or different. L1-L4 may independently be present or absent.

[0157] In some embodiments, there is provided an MSAP (e.g., TSAP) comprising: (i) an anti-CD3 portion that specifically binds to CD3; (ii) an anti-CD8 portion that specifically binds to CD8; and (iii) an anti-target epitope that specifically binds to a target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM), wherein the first of the three portions is a Fab fragment, the Fab fragment comprising a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; The second of the three portions is a first antigen-binding fragment (e.g., scFv), and the third of the three portions is a second antigen-binding fragment (e.g., scFv), wherein (a) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment via an optional first linker, and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional second linker; (b) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional first linker, and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional second linker. (c) a first antigen-binding fragment is fused to the N-terminus of the VH of a Fab fragment via an optional first linker and a second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment via an optional second linker; (d) a first antigen-binding fragment is fused to the N-terminus of the VH of a Fab fragment via an optional first linker and a second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional second linker; or (e) a first antigen-binding fragment is fused to the N-terminus of the VH of a Fab fragment via an optional first linker and a second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional second linker. the original antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional first linker, the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment via an optional second linker, and the anti-CD8 portion is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6;or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are both scFvs. In some embodiments, the anti-CD8 portion comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD8 portion is an anti-CD8 scFv, and the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD8 moiety is an anti-CD8 Fab fragment, and the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; or ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0158] In some embodiments, there is provided an MSAP (e.g., TSAP) comprising: i) an anti-CD3 portion that specifically binds to CD3; ii) an anti-CD8 portion that specifically binds to CD8; and iii) an anti-target epitope (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) portion that specifically binds to a target epitope, wherein the first of the three portions is a Fab fragment, the Fab fragment comprising a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the second of the three portions is a first antigen-binding fragment (e.g., scFv), and the third of the three portions is a second antigen-binding fragment (e.g., scFv), wherein (a) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment via an optional first linker, and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional second linker; (b) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional first linker, and the second antigen-binding fragment is fused to the C-terminus of the VL of the Fab fragment via an optional second linker. (c) a first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment via an optional first linker and a second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment via an optional second linker; (d) a first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment via an optional first linker and a second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional second linker; or (e) a first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional first linker, and a second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment via an optional second linker, and the anti-CD3 portion is selected from the group consisting of: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;(2) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; or (3) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are both scFvs. In some embodiments, the anti-CD3 portion comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD3 moiety is an anti-CD3 scFv, and the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-CD3 moiety is an anti-CD3 Fab fragment, and the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 89, 160, and 168, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110.

[0159] In some embodiments, there is provided an MSAP (e.g., TSAP) comprising: (1) an anti-CD3 portion that specifically binds to CD3; (2) an anti-CD8 portion that specifically binds to CD8; and (3) an anti-target epitope portion (e.g., a TAA or TSA, e.g., CD19, WT1, or EpCAM) that specifically binds to a target epitope, wherein the first of the three portions is a Fab fragment, the Fab fragment comprising a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; and the second of the three portions is a first antigen-binding fragment. and a third of the three portions is a second antigen-binding fragment (e.g., scFv), in which (a) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment via an optional first linker, and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional second linker; (b) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional first linker, and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional second linker; (c) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional first linker, and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional second linker. (d) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment via an optional first linker and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment via an optional second linker; (e) the first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment via an optional first linker and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment via an optional second linker; and the anti-CD8 portion is fused to the C-terminus of the CL of the Fab fragment via a linker of (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22,the anti-CD3 portion comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment are both scFvs. In some embodiments, the anti-CD8 portion comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD3 portion comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD8 portion is an anti-CD8 scFv, wherein the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 portion is an anti-CD3 scFv, wherein the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-CD8 portion is an anti-CD8 Fab fragment, wherein the anti-CD8 Fab fragment isThe anti-CD3 fragment comprises: i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; or ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments, the anti-CD3 portion is an anti-CD3 Fab portion, and the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 89, 160, and 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, the anti-target epitope portion specifically recognizes CD19 (anti-CD19 portion). In some embodiments, the anti-CD19 portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD19 portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 portion is an anti-CD19 scFv, wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the anti-CD19 portion is an anti-CD19 Fab portion, wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the anti-target epitope portion specifically recognizes EpCAM (anti-EpCAM portion). In some embodiments, the anti-EpCAM portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71,and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-EpCAM portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-EpCAM portion is an anti-EpCAM scFv, wherein the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78. In some embodiments, the anti-EpCAM portion is an anti-EpCAM Fab fragment, wherein the anti-EpCAM Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 76 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the anti-target epitope portion specifically recognizes WT1 (anti-WT1 portion). In some embodiments, the anti-WT1 portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 79, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 80, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 81, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the anti-WT1 portion comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 85, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the anti-WT1 portion is an anti-WT1 scFv, and the anti-WT1 scFv comprises the amino acid sequence of SEQ ID NO: 87 or 88. In some embodiments, the anti-WT1 portion is an anti-WT1 Fab portion, and the anti-WT1 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 145 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 146.

[0160] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a) an anti-CD3 Fab fragment, b) a first antigen-binding fragment (e.g., scFv) of anti-CD8, and c) a second antigen-binding fragment (e.g., scFv) of anti-CD19, wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, and the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD19 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional second linker. a first antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment, comprising: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; The Fab fragments are (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the second antigen-binding fragment of anti-CD19 comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the first antigen-binding fragment of anti-CD8 comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding fragment of anti-CD19 comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD3 Fab fragment comprises (i) a first polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 89, 160, and 179, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD19 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0161] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD3 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD19; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD19 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-CD3 Fab fragment via an optional second linker. a second antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment, comprising: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; The Fab fragments are (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the first antigen-binding fragment of anti-CD19 comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the second antigen-binding fragment of anti-CD8 comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the first antigen-binding fragment of anti-CD19 comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD3 Fab fragment comprises (i) a first polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 89, 160, and 168, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first antigen-binding fragment of anti-CD19 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided comprising a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 or 161 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 104 or 106. In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a first fusion polypeptide and a second fusion polypeptide, wherein i) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 104; ii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 106; or iii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 161 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 106;

[0162] In some embodiments, an MSAP (e.g., a TSAP) is provided, the MSAP comprising a first fusion polypeptide comprising an anti-CD19 first scFv fused to the N-terminus of an anti-CD3 Fab fragment via a first linker, and a second polypeptide comprising an anti-CD8 second scFv fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein i) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 103) and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 104 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 104); ii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 103). or iii) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 161 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 161) and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 106 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 106).

[0163] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD8 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD19; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD19 is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker; The Fab fragment comprises i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and is an anti-CD3 first antibody. antigen-binding fragments of the present invention include (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 second antigen-binding fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 Fab fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding fragment of anti-CD19 comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; or ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD19 are both scFvs. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0164] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD8 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD19; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD3; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD19 is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; the second antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker; The Fab fragment comprises i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and is a second Fab fragment of anti-CD3. antigen-binding fragments of the present invention include (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 first antigen-binding fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 Fab fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the second antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the first antigen-binding fragment of anti-CD19 comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; or ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments, the first antigen-binding fragment of anti-CD19 and the second antigen-binding fragment of anti-CD3 are both scFvs. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0165] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD19 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD19 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-CD19 Fab fragment via an optional second linker. The second antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and HVR-L3, wherein the first antigen-binding fragment of anti-CD3 comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 second antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD19 Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0166] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD19 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD8; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD3; wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-CD19 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD3 is fused to the N-terminus of the anti-CD19 Fab fragment via an optional second linker. The first antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and the second antigen-binding fragment of anti-CD3 comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 first antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the second antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD19 Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD3 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0167] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD19 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via an optional second linker; and the second antigen-binding fragment of anti-CD8 is fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via an optional second linker. The second antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and HVR-L3, wherein the first antigen-binding fragment of anti-CD3 comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 second antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD19 Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., a TSAP) is provided that comprises a first fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 170 and a second fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 171.

[0168] In some embodiments, an MSAP (e.g., a TSAP) is provided, comprising a first fusion polypeptide comprising an anti-CD3 first scFv fused to the C-terminus of the CH1 of an anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD8 second scFv fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 170 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 170), and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 171 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 171).

[0169] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a) an anti-CD19 Fab fragment, b) a first antigen-binding fragment (e.g., scFv) of anti-CD8, and c) a second antigen-binding fragment (e.g., scFv) of anti-CD3, wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, the first antigen-binding fragment of anti-CD8 is fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD3 is fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via an optional second linker. The first antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and the second antigen-binding fragment of anti-CD3 comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 first antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the second antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD19 Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the second antigen-binding fragment of anti-CD3 and the first antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a first fusion polypeptide and a second fusion polypeptide, wherein: (a) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 173; b) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 174 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 175; c) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 176; or d) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 182.

[0170] In some embodiments, an MSAP (e.g., a TSAP) is provided, comprising a first fusion polypeptide comprising an anti-CD8 first scFv fused to the C-terminus of the CH1 of an anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD3 second scFv fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 or 174 (or an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 172 or 174), and the second fusion polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 173, 175, 176, and 182 (or an amino acid sequence having at least about 85% sequence identity to any of SEQ ID NOs: 173, 175, 176, and 182).In some embodiments, an MSAP (e.g., a TSAP) is provided, the MSAP comprising a first fusion polypeptide comprising an anti-CD8 first scFv fused to the C-terminus of the CH1 of an anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD3 second scFv fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a second linker, wherein (a) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 172) and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 173 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 173); b) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 174 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 174) and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 175 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 175). c) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 172) and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 176 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 176); or d) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 172) and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 182 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 182).

[0171] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD19 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD19 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-CD19 Fab fragment via an optional second linker. The second antigen-binding fragment of anti-CD8 fused to the C-terminus of the CL of the Fab fragment is selected from the group consisting of: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and HVR-L3, wherein the first antigen-binding fragment of anti-CD3 comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 second antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD19 Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a first fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 177 and a second fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 178.

[0172] In some embodiments, an MSAP (e.g., a TSAP) is provided, comprising a first fusion polypeptide comprising an anti-CD3 first scFv fused to the N-terminus of the VH of an anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD8 second scFv fused to the C-terminus of the CL of the anti-CD19 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 177 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 177), and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 178 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 178).

[0173] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD19 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD19 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-CD19 Fab fragment via an optional second linker. The second antigen-binding fragment of anti-CD8 fused to the C-terminus of CH1 of the Fab fragment is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and HVR-L3, wherein the first antigen-binding fragment of anti-CD3 comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 second antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD19 Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 180 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 169.

[0174] In some embodiments, an MSAP (e.g., a TSAP) is provided, which comprises: i) a fusion polypeptide comprising an anti-CD3 first scFv fused to the N-terminus of the VH of an anti-CD19 Fab fragment via a first linker and an anti-CD8 second scFv fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via a second linker; and ii) an anti-CD19 Fab fragment second polypeptide, wherein the fusion polypeptide comprises SEQ ID NO: 180 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 180), and the anti-CD19 Fab fragment second polypeptide comprises the amino acid sequence of SEQ ID NO: 169 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 169).

[0175] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD19 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD19 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VL of the anti-CD19 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-CD19 Fab fragment via an optional second linker. The second antigen-binding fragment of anti-CD8 fused to the C-terminus of the CL of the Fab fragment is selected from the group consisting of: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and HVR-L3, wherein the first antigen-binding fragment of anti-CD3 comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-CD19 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD8 second antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-CD19 Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 63, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 181.

[0176] In some embodiments, an MSAP (e.g., a TSAP) is provided, the MSAP comprising i) a first polypeptide of an anti-CD19 Fab fragment, and ii) a fusion polypeptide comprising an anti-CD3 first scFv fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a first linker and an anti-CD8 second scFv fused to the C-terminus of the CL of the anti-CD19 Fab fragment via a second linker, wherein the first polypeptide of the anti-CD19 Fab fragment comprises SEQ ID NO: 137 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 137), and the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 181 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 181).

[0177] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD3 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD8; and c) a second antigen-binding fragment (e.g., scFv) of anti-EpCAM; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-EpCAM is fused to the N-terminus of the anti-CD3 Fab fragment via an optional second linker. a first antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment, comprising: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; The Fab fragments are (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the second antigen-binding fragment of anti-EpCAM comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the first antigen-binding fragment of anti-CD8 comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding fragment of anti-EpCAM comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 73, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 89, 160, and 168, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-EpCAM are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0178] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD3 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-EpCAM; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-EpCAM is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-CD3 Fab fragment via an optional second linker. a second antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment, comprising: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; The Fab fragments are (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the first antigen-binding fragment of anti-EpCAM comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second antigen-binding fragment of anti-CD8 comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the first antigen-binding fragment of anti-EpCAM comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 73, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 89, 160, and 168, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first antigen-binding fragment of anti-EpCAM and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided comprising a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 101 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 102.

[0179] In some embodiments, an MSAP (e.g., a TSAP) is provided, comprising a first fusion polypeptide comprising a first scFv of anti-EpCAM fused to the N-terminus of the VH of an anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 101 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 101), and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 102 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 102).

[0180] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD8 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-EpCAM; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-EpCAM is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker; The Fab fragment comprises i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and is an anti-CD3 first antibody. antigen-binding fragments of the present invention include (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the second antigen-binding fragment of anti-EpCAM comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD8 Fab fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding fragment of anti-EpCAM comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 73, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; or ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-EpCAM are both scFvs. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0181] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD8 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-EpCAM; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD3; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-EpCAM is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; the second antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VL of the anti-CD8 Fab fragment via an optional second linker; The Fab fragment comprises i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and is a second Fab fragment of anti-CD3. antigen-binding fragments of the present invention include (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the first antigen-binding fragment of anti-EpCAM comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD8 Fab fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the second antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the first antigen-binding fragment of anti-EpCAM comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 73, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-CD8 Fab fragment comprises i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92;or ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments, the first antigen-binding fragment of anti-EpCAM and the second antigen-binding fragment of anti-CD3 are both scFvs. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided comprising a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 99 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 100.

[0182] In some embodiments, an MSAP (e.g., a TSAP) is provided, comprising a first fusion polypeptide comprising a first scFv of anti-EpCAM fused to the N-terminus of the VH of an anti-CD8 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD3 fused to the N-terminus of the VL of the anti-CD8 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO:99 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO:99), and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:100 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO:100).

[0183] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a) an anti-EpCAM Fab fragment, b) a first antigen-binding fragment of anti-CD3 (e.g., scFv), and c) a second antigen-binding fragment of anti-CD8 (e.g., scFv), wherein the anti-EpCAM Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, and the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-EpCAM Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-EpCAM Fab fragment via an optional second linker. The second antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and HVR-L3, wherein the first antigen-binding fragment of anti-CD3 comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-EpCAM Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD8 second antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-EpCAM Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 73, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-EpCAM Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 76 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the first antigen-binding fragment of anti-CD3 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110. In some embodiments, an MSAP (e.g., TSAP) is provided comprising a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 97 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 98.

[0184] In some embodiments, an MSAP (e.g., a TSAP) is provided, the MSAP comprising a first fusion polypeptide comprising an anti-CD3 first scFv fused to the N-terminus of the VH of an anti-EpCAM Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD8 second scFv fused to the N-terminus of the VL of the anti-EpCAM Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO:97 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO:97), and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:98 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO:98).

[0185] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises a) an anti-EpCAM Fab fragment, b) a first antigen-binding fragment (e.g., scFv) of anti-CD8, and c) a second antigen-binding fragment (e.g., scFv) of anti-CD3, wherein the anti-EpCAM Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL, and the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-EpCAM Fab fragment via an optional first linker, and the second antigen-binding fragment of anti-CD3 is fused to the N-terminus of the anti-EpCAM Fab fragment via an optional second linker. The first antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment is selected from the group consisting of i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23. and the second antigen-binding fragment of anti-CD3 comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the anti-EpCAM Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CD8 first antigen-binding fragment comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the second antigen-binding fragment of anti-CD3 comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-EpCAM Fab fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 73, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-EpCAM Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 76 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-CD3 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 50, 56, 163, 164, 183, and 184. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0186] In some embodiments, there is provided an MSAP (e.g., TSAP) comprising: a) an anti-CD3 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD8; and c) a second antigen-binding fragment (e.g., scFv) of anti-WT1; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD8 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-WT1 is fused to the N-terminus of the anti-CD3 Fab fragment via an optional second linker. a first antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment, comprising: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; The Fab fragments are (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the second antigen-binding fragment of anti-WT1 comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 79, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 80, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 81, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the first antigen-binding fragment of anti-CD8 comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding fragment of anti-WT1 comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 85, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 89, 160, and 168, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first antigen-binding fragment of anti-CD8 and the second antigen-binding fragment of anti-WT1 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-WT1 scFv comprises the amino acid sequence of SEQ ID NO: 87 or 88. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108-110;

[0187] In some embodiments, an MSAP (e.g., TSAP) is provided that comprises: a) an anti-CD3 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-WT1; and c) a second antigen-binding fragment (e.g., scFv) of anti-CD8; wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-WT1 is fused to the N-terminus of the VH of the anti-CD3 Fab fragment via an optional first linker; and the second antigen-binding fragment of anti-CD8 is fused to the N-terminus of the anti-CD3 Fab fragment via an optional second linker. a second antigen-binding fragment of anti-CD8 fused to the N-terminus of the VL of the Fab fragment, comprising: i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23; The Fab fragments are (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;or (iii) comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41, and the first antigen-binding fragment of anti-WT1 comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 79, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 80, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 81, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the second antigen-binding fragment of anti-CD8 comprises i) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;or ii) a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising the amino acid sequence of any of SEQ ID NOs: 42, 159, and 167, and a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the first antigen-binding fragment of anti-WT1 comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 85, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 89, 160, and 168, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179. In some embodiments, the first antigen-binding fragment of anti-WT1 and the second antigen-binding fragment of anti-CD8 are both scFvs. In some embodiments, the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 46, 49, 52, and 55. In some embodiments, the anti-WT1 scFv comprises the amino acid sequence of SEQ ID NO: 87 or 88. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence independently selected from the group consisting of GG and SEQ ID NOs: 108 to 110. In some embodiments, an MSAP (e.g., TSAP) is provided comprising a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 95 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 96.

[0188] In some embodiments, an MSAP (e.g., a TSAP) is provided, comprising a first fusion polypeptide comprising an anti-WT1 first scFv fused to the N-terminus of the VH of an anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD8 second scFv fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 95 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 95), and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 96 (or an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID NO: 96).

[0189] In some embodiments, there is provided an MSAP (e.g., TSAP) comprising: a) an anti-CD8 Fab fragment; b) a first antigen-binding fragment (e.g., scFv) of anti-CD3; and c) a second antigen-binding fragment (e.g., scFv) of anti-WT1; wherein the anti-CD8 Fab fragment comprises a first polypeptide comprising a VH and a CH1 and a second polypeptide comprising a VL and a CL; the first antigen-binding fragment of anti-CD3 is fused to the N-terminus of the VH of the anti-CD8 Fab fragment via an optional first linker; and the se...

Claims

1. i) an anti-CD3 moiety that specifically binds to CD3; ii) an anti-CD8 moiety that specifically binds to CD8, and iii) a multispecific antigen-binding protein comprising an anti-target epitope portion that specifically binds to a target epitope, a first of the three portions is a Fab fragment, the Fab fragment comprising a first polypeptide comprising a heavy chain variable region (VH) and a heavy chain constant region (CH1), and a second polypeptide comprising a light chain variable region (VL) and a light chain constant region (CL); a second of the three portions is a first antigen-binding fragment; a third of the three portions is a second antigen-binding fragment; (a) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment; (b) the first antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment and the second antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment; (c) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment; (d) the first antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CH1 of the Fab fragment; or (e) the first antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment and the second antigen-binding fragment is fused to the C-terminus of the CL of the Fab fragment; The multispecific antigen-binding protein.

2. (a) the anti-CD3 portion is the Fab fragment ("anti-CD3 Fab fragment"), the anti-CD8 portion is the first antigen-binding fragment ("anti-CD8 first antigen-binding fragment"), and the anti-target epitope portion is the second antigen-binding fragment ("anti-target epitope second antigen-binding fragment"); (b) the anti-CD3 portion is the Fab fragment, the anti-target epitope portion is the first antigen-binding fragment (the "anti-target epitope first antigen-binding fragment"), and the anti-CD8 portion is the second antigen-binding fragment (the "anti-CD8 second antigen-binding fragment"); (c) the anti-CD8 portion is the Fab fragment ("anti-CD8 Fab fragment"), the anti-CD3 portion is the first antigen-binding fragment ("anti-CD3 first antigen-binding fragment"), and the anti-target epitope portion is the second antigen-binding fragment; (d) the anti-CD8 portion is the Fab fragment, the anti-target epitope portion is the first antigen-binding fragment, and the anti-CD3 portion is the second antigen-binding fragment ("anti-CD3 second antigen-binding fragment"); (e) the anti-target epitope portion is the Fab fragment (the "anti-target epitope Fab fragment"), the anti-CD3 portion is the first antigen-binding fragment, and the anti-CD8 portion is the second antigen-binding fragment; or (f) the anti-target epitope portion is the Fab fragment, the anti-CD8 portion is the first antigen-binding fragment, and the anti-CD3 portion is the second antigen-binding fragment; 2. The multispecific antigen-binding protein of claim 1.

3. The first antigen-binding fragment and the second antigen-binding fragment are independently Fab, Fab', Fab'-SH, F(ab') ２ 3. The multispecific antigen-binding protein of claim 1 or 2, selected from the group consisting of: Fv, sdAb, scFv, and any combination thereof.

4. The multispecific antigen-binding protein of any one of claims 1 to 3, wherein the first antigen-binding fragment and the second antigen-binding fragment are both scFvs.

5. the anti-CD8 moiety specifically binds to CD8α, i) heavy chain hypervariable region 1 (HVR-H1) comprising the amino acid sequence of SEQ ID NO: 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, light chain hypervariable region 1 (HVR-L1) comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; v) HVR-H1 comprising the amino acid sequence of SEQ ID NO:7, HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10, HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:12; or vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23 5. The multispecific antigen-binding protein of claim 1, comprising:

6. the anti-CD8 moiety is i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; iv) VH comprising the amino acid sequence of SEQ ID NO: 30 and VL comprising the amino acid sequence of SEQ ID NO: 25; v) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 33; or vi) A multispecific antigen-binding protein according to claim 5, comprising a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO:

35.

7. 7. The multispecific antigen-binding protein of any one of claims 1 to 6, wherein said anti-CD8 portion is an anti-CD8 scFv, and said anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 21, 44-49, and 51-55.

8. the anti-CD3 moiety is (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; or (c) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:

41.

8. The multispecific antigen-binding protein of any one of claims 1 to 7, comprising:

9. the anti-CD3 moiety is (a) a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (b) a VH comprising the amino acid sequence of SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 167 and a VL comprising the amino acid sequence of SEQ ID NO: 43; 9. The multispecific antigen-binding protein of claim 8.

10. 10. The multispecific antigen-binding protein of claim 8 or 9, wherein the anti-CD3 portion is an anti-CD3 scFv, and the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184.

11. 15. The multispecific antigen-binding protein of any one of claims 1 to 14, wherein said target epitope portion specifically recognizes a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA), and optionally (i) said TAA is CD19 or EpCAM, or (ii) said TSA is WT1.

12. The multispecific antigen-binding protein of claim 11, wherein the anti-target epitope portion specifically recognizes CD19 (anti-CD19 portion), and the anti-CD19 portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:

62.

13. 13. The multispecific antigen-binding protein of claim 12, wherein the anti-CD19 portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO:

64.

14. 14. The multispecific antigen-binding protein of claim 12 or 13, wherein the anti-CD19 portion is an anti-CD19 scFv, and the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 65 or 66.

15. 15. The multispecific antigen-binding protein of any one of claims 12 to 14, wherein said anti-CD19 portion is an anti-CD19 Fab and said anti-CD19 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 139 or 169.

16. 12. The multispecific antigen-binding protein of claim 11, wherein the anti-target epitope portion specifically recognizes EpCAM (anti-EpCAM portion), and the anti-EpCAM portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:

72.

17. 17. The multispecific antigen-binding protein of claim 16, wherein the anti-EpCAM portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO:

74.

18. 18. The multispecific antigen-binding protein of claim 16 or 17, wherein the anti-EpCAM portion is an anti-EpCAM scFv, and the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 75 or 78.

19. The multispecific antigen-binding protein of claim 11, wherein the anti-target epitope portion specifically recognizes WT1 (anti-WT1 portion), and the anti-WT1 portion comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, HVR-H3 comprising the amino acid sequence of SEQ ID NO:81, HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:

84.

20. 20. The multispecific antigen-binding protein of claim 19, wherein the anti-WT1 portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO:

86.

21. 22. The multispecific antigen-binding protein of claim 20 or 21, wherein the anti-WT1 portion is an anti-WT1 scFv, and the anti-WT1 scFv comprises the amino acid sequence of SEQ ID NO: 87 or 88.

22. the multispecific antigen-binding protein comprising: i) an anti-CD3 Fab fragment, an anti-CD8 first scFv, and an anti-WT1 second scFv; ii) an anti-CD3 Fab fragment, an anti-WT1 first scFv, and an anti-CD8 second scFv; iii) an anti-CD3 Fab fragment, an anti-EpCAM first scFv, and an anti-CD8 second scFv; iv) an anti-CD3 Fab fragment, an anti-CD8 first scFv, and an anti-EpCAM second scFv; v) an anti-CD3 Fab fragment, an anti-CD19 first scFv, and an anti-CD8 second scFv; or vi) an anti-CD3 Fab fragment, an anti-CD8 first scFv, and an anti-CD19 second scFv 22. The multispecific antigen-binding protein of any one of claims 1 to 9 and 11 to 21, comprising:

23. the anti-CD3 Fab fragment (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 89 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; 23. The multispecific antigen-binding protein of claim 22, comprising:

24. 24. The multispecific antigen-binding protein of claim 22 or 23, wherein the multispecific antigen-binding protein comprises a first polypeptide comprising an anti-WT1 first scFv fused to the N-terminus of the VH of the anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising an anti-CD8 second scFv fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO:95 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

96.

25. 24. The multispecific antigen-binding protein of claim 22 or 23, wherein the multispecific antigen-binding protein comprises a first polypeptide comprising a first scFv of anti-EpCAM fused to the N-terminus of the VH of the anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 101 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

102.

26. the multispecific antigen-binding protein comprises a first fusion polypeptide comprising a first scFv of anti-CD19 fused to the N-terminus of the VH of an anti-CD3 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the N-terminus of the VL of the anti-CD3 Fab fragment via a second linker; i) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 104; i) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 103 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 106; or i) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 161 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 106; 24. A multispecific antigen-binding protein according to claim 22 or 23.

27. the multispecific antigen-binding protein comprising: i) an anti-CD19 Fab fragment, an anti-CD8 first scFv, and an anti-CD3 second scFv; or ii) an anti-CD19 Fab fragment, an anti-CD3 first scFv, and an anti-CD8 second scFv 16. The multispecific antigen-binding protein of any one of claims 1 to 9, 11 to 13, and 15, comprising:

28. 28. The multispecific antigen-binding protein of claim 27, wherein said multispecific antigen-binding protein comprises a first polypeptide comprising a first scFv of anti-CD3 fused to the C-terminus of the CH1 of the anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a second linker, wherein said first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 170 and said second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

171.

29. the multispecific antigen-binding protein comprises a first fusion polypeptide comprising a first scFv of anti-CD8 fused to the C-terminus of the CH1 of an anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD3 fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a second linker; a) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 173; b) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 174 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 175; c) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 176; or d) the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 182; 28. The multispecific antigen-binding protein of claim 27.

30. 28. The multispecific antigen-binding protein of claim 27, wherein said multispecific antigen-binding protein comprises a first polypeptide comprising a first scFv of anti-CD3 fused to the N-terminus of the VH of the anti-CD19 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the C-terminus of the CL of the anti-CD19 Fab fragment via a second linker, wherein said first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 177 and said second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

178.

31. 28. The multispecific antigen-binding protein of claim 27, wherein said multispecific antigen-binding protein comprises i) a fusion polypeptide comprising an anti-CD3 first scFv fused to the N-terminus of the VH of said anti-CD19 Fab fragment via a first linker and an anti-CD8 second scFv fused to the C-terminus of the CH1 of said anti-CD19 Fab fragment via a second linker; and ii) a second polypeptide of said anti-CD19 Fab fragment, wherein said fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 180 and said second polypeptide of said anti-CD19 Fab fragment comprises the amino acid sequence of SEQ ID NO:

169.

32. 28. The multispecific antigen-binding protein of claim 27, wherein said multispecific antigen-binding protein comprises i) a first polypeptide of the anti-CD19 Fab fragment; and ii) a fusion polypeptide comprising a first scFv of anti-CD3 fused to the N-terminus of the VL of the anti-CD19 Fab fragment via a first linker, and a second scFv of anti-CD8 fused to the C-terminus of the CL of the anti-CD19 Fab fragment via a second linker, wherein said first polypeptide of the anti-CD19 Fab fragment comprises the amino acid sequence of SEQ ID NO: 137, and said fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

181.

33. the multispecific antigen-binding protein comprising: i) an anti-EpCAM Fab fragment, an anti-CD3 first scFv, and an anti-CD8 second scFv; or ii) anti-EpCAM Fab fragment, anti-CD8 first scFv, and anti-CD3 second scFv 18. The multispecific antigen-binding protein of any one of claims 1 to 11, 16 and 17, comprising:

34. 34. The multispecific antigen-binding protein of claim 33, wherein the anti-EpCAM Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 76 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

77.

35. 35. The multispecific antigen-binding protein of claim 33 or 34, wherein the multispecific antigen-binding protein comprises a first polypeptide comprising a first scFv of anti-CD3 fused to the N-terminus of the VH of the anti-EpCAM Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD8 fused to the C-terminus of the VL of the anti-EpCAM Fab fragment via a second linker, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO:97 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

98.

36. the multispecific antigen-binding protein comprising: i) an anti-CD8 Fab fragment, an anti-EpCAM first scFv, and an anti-CD3 second scFv; or ii) an anti-CD8 Fab fragment, an anti-CD3 first scFv, and an anti-EpCAM second scFv 19. The multispecific antigen-binding protein of any one of claims 1 to 6, 8 to 11, and 16 to 18, comprising:

37. the anti-CD8 Fab fragment i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 147 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 149 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 150; iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; iv) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 151 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; v) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 153 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 154; or vi) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

156.

37. The multispecific antigen-binding protein of claim 36, comprising:

38. 38. The multispecific antigen-binding protein of claim 36 or 37, wherein said multispecific antigen-binding protein comprises a first polypeptide comprising a first scFv of anti-EpCAM fused to the N-terminus of the VH of the anti-CD8 Fab fragment via a first linker, and a second fusion polypeptide comprising a second scFv of anti-CD3 fused to the N-terminus of the VL of the anti-CD8 Fab fragment via a second linker, wherein said first fusion polypeptide comprises the amino acid sequence of SEQ ID NO:99 and said second fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

100.

39. 39. A pharmaceutical composition comprising the multispecific antigen-binding protein of any one of claims 1 to 38, and optionally a pharmaceutically acceptable carrier.

40. 40. A method of treating a disease associated with a target epitope in an individual, the method comprising administering to said individual an effective amount of a multispecific antigen-binding protein of any one of claims 1 to 38 or a pharmaceutical composition of claim 39.

41. 41. The method of claim 40, wherein the disease associated with the target epitope is a cancer that expresses the target epitope.

42. 41. The method of claim 39 or 40, wherein the cancer is Burkitt's lymphoma, Wilms' tumor, or colon cancer.

43. 43. The method of any one of claims 40 to 42, wherein said multispecific antigen-binding protein or said pharmaceutical composition is administered subcutaneously or intravenously.

44. an HVR-H1 comprising the amino acid sequence of any of SEQ ID NOs: 1, 7, 13, and 20, or a variant thereof comprising up to about 5 amino acid variations; an HVR-H2 comprising the amino acid sequence of any of SEQ ID NOs: 2, 8, 14, 16, and 18, or a variant thereof comprising up to about 5 amino acid variations; an HVR-H3 comprising the amino acid sequence of any of SEQ ID NOs: 3, 9, 15, 17, 19, and 22, or a variant thereof comprising up to about 5 amino acid variations; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4 or 10, or a variant thereof comprising up to about 5 amino acid variations; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5 or 11, or a variant thereof comprising up to about 5 amino acid variations; and an HVR-L3 comprising the amino acid sequence of any of SEQ ID NOs: 6, 12, and 23, or a variant thereof comprising up to about 5 amino acid variations.

45. the anti-CD8 antibody or antigen-binding fragment thereof i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; iii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; v) HVR-H1 comprising the amino acid sequence of SEQ ID NO:7, HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10, HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:12; or vi) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23 45. The anti-CD8 antibody or antigen-binding fragment thereof of claim 44, comprising:

46. the anti-CD8 antibody or antigen-binding fragment thereof i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; iv) VH comprising the amino acid sequence of SEQ ID NO: 30 and VL comprising the amino acid sequence of SEQ ID NO: 25; v) a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 33; or vi) VH comprising the amino acid sequence of SEQ ID NO: 34 and VL comprising the amino acid sequence of SEQ ID NO: 35 46. ​​The anti-CD8 antibody or antigen-binding fragment thereof of claim 44 or 45, comprising:

47. The anti-CD8 antibody or antigen-binding fragment thereof may be a full-length antibody, Fab, Fab', Fab'-SH, F(ab') ２ 47. The anti-CD8 antibody or antigen-binding fragment thereof according to any one of claims 44 to 46, which is selected from the group consisting of Fv, scFv, and any combination thereof.

48. The anti-CD8 antibody or antigen-binding fragment thereof according to claim 47, wherein the anti-CD8 antibody or antigen-binding fragment thereof is an anti-CD8 scFv, and the anti-CD8 scFv comprises the amino acid sequence of any one of SEQ ID NOs: 21, 44 to 49, and 51 to 55.

49. the anti-CD8 antigen-binding fragment is an anti-CD8 Fab fragment, i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 147 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 149 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 150; iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 91 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 92; iv) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 151 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 148; v) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 153 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 154; or vi) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

156.

48. The anti-CD8 antibody or antigen-binding fragment thereof of claim 47, comprising:

50. an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof comprising up to about 5 amino acid variations; an HVR-H2 comprising the amino acid sequence of any of SEQ ID NOs: 37, 157, and 166 or a variant thereof comprising up to about 5 amino acid variations; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38 or 158 or a variant thereof comprising up to about 5 amino acid variations; an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof comprising up to about 5 amino acid variations; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof comprising up to about 5 amino acid variations; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof comprising up to about 5 amino acid variations.

51. the CD3 antibody or antigen-binding fragment thereof (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; (b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 157, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 158, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41; or (c) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 166, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:

41.

51. The anti-CD3 antibody or antigen-binding fragment thereof of claim 50, comprising:

52. the CD3 antibody or antigen-binding fragment thereof (a) a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 43; (b) a VH comprising the amino acid sequence of SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 43; or (c) VH comprising the amino acid sequence of SEQ ID NO: 167 and VL comprising the amino acid sequence of SEQ ID NO: 43 52. The anti-CD3 antibody or antigen-binding fragment thereof of claim 50 or 51, comprising:

53. The anti-CD3 antibody or antigen-binding fragment thereof may be a full-length antibody, Fab, Fab', Fab'-SH, F(ab') ２ 53. The anti-CD3 antibody or antigen-binding fragment thereof according to any one of claims 50 to 52, selected from the group consisting of Fv, scFv, and any combination thereof.

54. The anti-CD3 antibody or antigen-binding fragment thereof of claim 53, wherein the anti-CD3 antibody or antigen-binding fragment thereof is an anti-CD3 scFv, and the anti-CD3 scFv comprises the amino acid sequence of any of SEQ ID NOs: 50, 56, 163, 164, 183, and 184.

55. the anti-CD3 antigen-binding fragment is an anti-CD3 Fab fragment, (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 89 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179; or (c) an anti-CD3 antibody or antigen-binding fragment thereof according to claim 53, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 90 or 179.

56. 56. A multispecific antigen-binding protein comprising an anti-CD8 antibody or antigen-binding fragment thereof according to any one of claims 44 to 49, and / or an anti-CD3 antibody or antigen-binding fragment thereof according to any one of claims 50 to 55.

57. 56. An isolated nucleic acid encoding i) an anti-CD8 antibody or antigen-binding fragment thereof of any one of claims 44 to 49, ii) an anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 50 to 55, or iii) a multispecific antigen-binding protein of any one of claims 1 to 38 and 56.

58. 58. A vector comprising the isolated nucleic acid of claim 57.

59. 59. A host cell comprising the isolated nucleic acid of claim 57 or the vector of claim 58.

60. 1. A method of making an anti-CD8 antibody or antigen-binding fragment thereof, an anti-CD3 antibody or antigen-binding fragment thereof, or a multispecific antigen-binding protein, comprising: i) culturing a host cell comprising the isolated nucleic acid of claim 57 or the vector of claim 58, or the host cell of claim 59, under conditions suitable for expression of said anti-CD8 antibody or antigen-binding fragment thereof, said anti-CD3 antibody or antigen-binding fragment thereof, or a multispecific antigen-binding protein thereof; ii) obtaining the expressed anti-CD8 antibody or antigen-binding fragment thereof, the anti-CD3 antibody or antigen-binding fragment thereof, or the multispecific antigen-binding protein from the host cell; and The method comprising:

61. (a) i) the anti-CD8 antibody or antigen-binding fragment thereof of any one of claims 44 to 49, and optionally ii) a pharmaceutically acceptable carrier; and / or (b)i) 57. A pharmaceutical composition comprising the anti-CD3 antibody or antigen-binding fragment thereof of any one of claims 50 to 55, or the multispecific antigen-binding protein of claim 56, and optionally ii) a pharmaceutically acceptable carrier.

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