Anti-ceacam6 t-cell engager bispecific antibodies and uses thereof

Abstract

Described herein are CEACAM6 targeting T cell engagers and uses thereof. The T cell engagers include CEACAM6 antigen binding domains and CD3 antigen binding domains. In some embodiments, the CEACAM6 antigen binding domains form low affinity and high avidity CEACAM6 targeting T cell engagers. In some embodiments, the CEACAM6 antigen binding domains and / or the CD3 antigen binding domains exhibit substantially improved binding affinity in an acidic pH environment compared to a neutral pH environment.

Description

Attorney Docket No. 55171.4010 / WOANTI-CEACAM6 T-CELL ENGAGER BTSPECIFTC ANTIBODIES AND USES THEREOFCROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63 / 790,576, filed on April 17, 2025, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 16, 2026, is named 55171_4010_WO_SL.xml and is 42,258,770 bytes in size.BACKGROUND

[0003] Bispecific therapeutics have significant promise as treatments for a multitude of hematologic and solid tumor cancers. Bispecific T cell engagers, for example, specifically bind to both an antigen on a cancer cell and a T cell receptor such as a CD3 on a T cell, which in turn, activates the T cell to eliminate the cancer cell.

[0004] A drawback of antibody therapies is that many tumor-associated antigens (TAAs) are not exclusively expressed on cancer cells. Non-specific antibody therapeutics can therefore bind to healthy cells expressing the TAAs and lead to off target toxi cities.

[0005] A unique characteristic of the tumor microenvironment relative to the environment of healthy tissue is its acidic pH, which is generally below pH 6.5. Therefore, if antibodies could selectively bind to antigens at acidic pH in the tumor microenvironment, this could circumvent the toxi cities associated with off target binding.

[0006] Carcinoembryonic antigen-related adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in solid tumor cancers including breast, non-small cell lung, pancreatic, and colorectal cancers. CEACAM6 acts as an oncogene in tumors and promotes cancer invasion, metastasis, and chemoresistance, and inhibits differentiation.14130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0007] There remains a need to develop effective and selective T cell engager therapies that amplify T cell activation to sustain the cancer cell killing capacity of the T cells.SUMMARY

[0008] Described herein are T cell-engaging molecules (also referred to as T cell engagers) that specifically bind to human CEACAM6 and human CD3.

[0009] In one aspect, provided herein is a CEACAM6 targeting T cell-engaging molecule comprising: (a) a first polypeptide chain comprising from N-terminus to C-terminus: (i) a first VHH antigen binding domain that binds human CEACAM6, (ii) a first flexible linker, (iii) a hinge domain, (iv) a first Fc domain; (b) a second polypeptide chain comprising from N-terminus to C-terminus: (i) a second VHH antigen binding domain that binds human CEACAM6, (ii) a second flexible linker, (iii) a variable heavy (VH) domain, (iv) a second hinge domain, (v) a second Fc domain; and (c) a third polypeptide chain comprising from N-terminus to C-terminus: a variable light (VL) domain and a constant light (CL) domain; wherein the VH domain and VL domain form an antigen binding domain that binds human CD3, and wherein the first and second Fc domains each comprises one or more amino acid substitutions for Fc heterodimerization. In some embodiments, the CEACAM6 targeting T cell-engaging molecule comprises any first polypeptide chain, second polypeptide chain, and third polypeptide chain described herein including the sequence listing such that the CEACAM6 targeting T cell-engaging molecule binds CEACAM6 and CD3.

[0010] In some embodiments of the CEACAM6 targeting T cell-engaging molecule, first VHH antigen binding domain or the second VHH antigen binding domain comprises: (i) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 43; (ii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 44, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 45, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 46; or (iii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 47, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 48, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 49.24130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0011] In some embodiments of the CEAC AM6 targeting T cell-engaging molecule, the first VHH antigen binding domain and the second VHH antigen binding domain each comprises: (i) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 43; (ii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 44, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 45, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 46; or (iii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 47, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 48, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 49.

[0012] In many embodiments, the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192. In some embodiments, the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

[0013] In many embodiments, the first VHH antigen binding domain and the second VHH antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, and 160-166. In some embodiments, the first VHH antigen binding domain and the second VHH antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

[0014] In some embodiments, the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13. In many embodiments, the first VHH antigen binding domain and the second VHH antigen binding domain each comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13. In some embodiments, the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOS: 10-13. In many embodiments, the first VHH antigen binding domain34130-6004-4904.10Attorney Docket No. 55171.4010 / WOand the second VHH antigen binding domain each comprises an amino acid sequence of any one of SEQ ID NOS: 10-13.

[0015] In some embodiments, the first flexible linker or the second flexible linker is selected from the group consisting of any one of SEQ ID NOS: 32-40. In some embodiments, the first flexible linker and the second flexible linker are each independently selected from the group consisting of any one of SEQ ID NOS: 32-40.

[0016] In some embodiments of the CEACAM6 targeting T cell-engaging molecule, the VH domain and the VL domain forming the antigen binding domain that binds human CD3 are selected from the group consisting of: (i) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; (ii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30; (iii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 61 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 62; (iv) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 67 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 68; (v) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 73 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 74; (vi) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 79 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 80; (vii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 85 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 86; (viii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 91 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 92; (ix) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 97 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 98; (x) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 103 and the VL domain comprising an 44130-6004-4904.10Attorney Docket No. 55171.4010 / WOamino acid sequence having at least 90% sequence identity to SEQ ID NO: 104; (xi) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 109 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 110; (xii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 115 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 116; (xiii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122; (xiv) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 127 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 128; (xv) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 133 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 134; (xvi) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 139 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 140; (xvii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 145 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 146; (xviii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 181 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 182; (xix) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 187 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 188; and (xx) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 193 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 194. In some embodiments of the CEACAM6 targeting T cell-engaging molecule, the VH domain and the VL domain forming the antigen binding domain that binds human CD3 are selected from the group consisting of: (i) the VH domain comprising an amino acid sequence of SEQ ID NO: 15 and the VL domain comprising an amino acid sequence of SEQ ID NO: 17; (ii) the VH domain comprising an amino acid sequence of SEQ ID NO: 28 and the VL domain comprising an amino acid sequence of SEQ ID NO: 30; (iii) the VH domain comprising an amino acid sequence of SEQ ID NO: 6154130-6004-4904.10Attorney Docket No. 55171.4010 / WOand the VL domain comprising an amino acid sequence of SEQ ID NO: 62; (iv) the VH domain comprising an amino acid sequence of SEQ ID NO: 67 and the VL domain comprising an amino acid sequence of SEQ ID NO: 68; (v) the VH domain comprising an amino acid sequence of SEQ ID NO: 73 and the VL domain comprising an amino acid sequence of SEQ ID NO: 74; (vi) the VH domain comprising an amino acid sequence of SEQ ID NO: 79 and the VL domain comprising an amino acid sequence of SEQ ID NO: 80; (vii) the VH domain comprising an amino acid sequence of SEQ ID NO: 85 and the VL domain comprising an amino acid sequence of SEQ ID NO: 86; (viii) the VH domain comprising an amino acid sequence of SEQ ID NO: 91 and the VL domain comprising an amino acid sequence of SEQ ID NO: 92; (ix) the VH domain comprising an amino acid sequence of SEQ ID NO: 97 and the VL domain comprising an amino acid sequence of SEQ ID NO: 98; (x) the VH domain comprising an amino acid sequence of SEQ ID NO: 103 and the VL domain comprising an amino acid sequence of SEQ ID NO: 104; (xi) the VH domain comprising an amino acid sequence of SEQ ID NO: 109 and the VL domain comprising an amino acid sequence of SEQ ID NO: 110; (xii) the VH domain comprising an amino acid sequence of SEQ ID NO: 115 and the VL domain comprising an amino acid sequence of SEQ ID NO: 116; (xiii) the VH domain comprising an amino acid sequence of SEQ ID NO: 121 and the VL domain comprising an amino acid sequence of SEQ ID NO: 122; (xiv) the VH domain comprising an amino acid sequence of SEQ ID NO: 127 and the VL domain comprising an amino acid sequence of SEQ ID NO: 128; (xv) the VH domain comprising an amino acid sequence of SEQ ID NO: 133 and the VL domain comprising an amino acid sequence of SEQ ID NO: 134; (xvi) the VH domain comprising an amino acid sequence of SEQ ID NO: 139 and the VL domain comprising an amino acid sequence of SEQ ID NO: 140; (xvii) the VH domain comprising an amino acid sequence of SEQ ID NO: 145 and the VL domain comprising an amino acid sequence of SEQ ID NO: 146; (xviii) the VH domain comprising an amino acid sequence of SEQ ID NO: 181 and the VL domain comprising an amino acid sequence of SEQ ID NO: 182; (xix) the VH domain comprising an amino acid sequence of SEQ ID NO: 187 and the VL domain comprising an amino acid sequence of SEQ ID NO: 188; and (xx) the VH domain comprising an amino acid sequence of SEQ ID NO: 193 and the VL domain comprising an amino acid sequence of SEQ ID NO: 194. In various embodiments of the CEACAM6 targeting T cell-engaging molecule, the VH domain comprises the amino acid sequence of SEQ ID NO: 15 and the VL domain comprises to the amino acid sequence of SEQ ID NO: 17.64130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0017] In some embodiments, the first hinge domain and the second hinge domain each is a human IgGl hinge domain or a variant thereof. In other embodiments, the first hinge domain and the second hinge domain each is a human IgG2 hinge domain, a human IgG3 hinge domain, a human IgG4 hinge domain, or a variant thereof. In some embodiments, the first Fc domain and the second Fc domain each is a CH2-CH3 domain of a human IgGl. In some embodiments, the first and second Fc domains each comprises a CH2-CH3 domain of a human IgG2, IgG3, or IgG4.

[0018] In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 14 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 23 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 14.

[0019] In some embodiments, the CL domain is a kappa light chain constant domain. In various embodiments, the CL domain is a lambda light chain constant domain.

[0020] In various embodiments, the CEACAM6 targeting T cell-engaging molecule described includes any one of the combinations of polypeptide chains: (i) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6; (ii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3; (iii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; (iv) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 63, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 64, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 65; (v) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence74130-6004-4904.10Attorney Docket No. 55171.4010 / WOidentity to SEQ ID NO: 69, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 70, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 71; (vi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 75, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 76, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 77; (vii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 81, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 82, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83; (viii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 87, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 88, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 89; (ix) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 94, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 95; (x) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 99, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 100, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 101; (xi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 105, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 106, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 107; (xii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 111, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 112, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 113; (xiii) the first polypeptide chain comprises an amino acid sequence having at least 90%84130-6004-4904.10Attorney Docket No. 55171.4010 / WOsequence identity to SEQ ID NO: 117, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 118, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 119; (xiv) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 124, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 125; (xv) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 129, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 130, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 131; (xvi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 135, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 136, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 137; (xvii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 141, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 142, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 143; (xviii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 147, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 148, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 149; (xix) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 183, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 184, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 185; (xx) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 189, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 190, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:94130-6004-4904.10Attorney Docket No. 55171.4010 / WO191; or (xxi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 195, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 196, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 197. In some embodiments, the CEACAM6 targeting T cell-engaging molecule described includes any one of the combinations of polypeptide chains: (i) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 4, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 5, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 6; (ii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 1, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 2, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 3; (iii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 7, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 8, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 9; (iv) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 63, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 64, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 65; (v) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 69, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 70, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 71; (vi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 75, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 76, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77; (vii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 81, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 82, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 83; (viii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 87, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89; (ix) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 93, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 94, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 95; (x) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 99, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:104130-6004-4904.10Attorney Docket No. 55171.4010 / WO100, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 101; (xi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 105, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 106, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 107; (xii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 111, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 112, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 113; (xiii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 117, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 118, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 119; (xiv) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 123, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 124, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 125; (xv) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 129, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 130, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 131; (xvi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 135, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 136, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 137; (xvii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 141, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 142, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 143; (xviii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 147, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 148, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 149; (xix) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 183, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 184, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 185; (xx) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 189, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 190, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 191; or (xxi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 195, the second polypeptide chain comprises114130-6004-4904.10Attorney Docket No. 55171.4010 / WOan amino acid sequence of SEQ ID NO: 196, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 197.

[0021] In some embodiments, provided herein is a nucleic acid composition comprising: (a) a first nucleic acid encoding any one of the first polypeptide chains disclosed herein; (b) a second nucleic acid encoding any one of the second polypeptide chains disclosed herein; and (c) a third nucleic acid encoding any one of the third polypeptide chains disclosed herein. In some embodiments, the expression vector composition comprises: a first expression vector comprising the first nucleic acid described herein; a second expression vector comprising the second nucleic acid described herein; and a third expression vector comprising the third nucleic acid described herein. In some embodiments, the host cell comprises any expression vector composition described herein. Also provided herein is a method of producing a CEACAM6 targeting T cell-engaging molecule comprising: (a) culturing any of the host cells described herein under conditions to express the CEACAM6 targeting T cell-engaging molecule, and (b) recovering the CEACAM6 targeting T cell-engaging molecule.

[0022] Also provided herein is a method of treating a cancer in a human subject in need thereof comprising administering to the human subject any of the CEACAM6 targeting T cell-engaging molecules described herein. In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colorectal cancer, and breast cancer.

[0023] In one aspect, provided herein is a VHH antibody or antibody fragment thereof comprising: (i) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 43; (ii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 44, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 45, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 46; or (iii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 47, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 48, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 49.

[0024] In some embodiments, the VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.124130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0025] In some embodiments, the VHH antibody or antibody fragment thereof comprises an amino acid sequence of any one of SEQ ID NOS: 10-13, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

[0026] In some embodiments, the VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13, and the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of SEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

[0027] In some embodiments, the VHH antibody or antibody fragment thereof comprises an amino acid sequence of SEQ ID NO: 13, and the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of SEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

[0028] In some embodiments, the VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-12, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192, and the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of SEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

[0029] In some embodiments, the VHH antibody or antibody fragment thereof comprises an amino acid sequence of any one of SEQ ID NOS: 10-12, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192, and the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of SEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

[0030] In some embodiments, the VHH antibody or antibody fragment has a higher binding affinity to human CEACAM6 compared to non-primate CEACAM6.134130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0031] In some embodiments, the VHH antibody or antibody fragment thereof, further comprises an Fc domain or a variant thereof.

[0032] In some embodiments, the VHH antibody or antibody fragment thereof comprising the Fc domain forms a VHH dimeric antibody or antibody fragment thereof by dimerization of the Fc domains.

[0033] Provided herein is a nucleic acid composition comprising a nucleic acid encoding any one of the VHH antibody or antigen binding fragment thereof described herein.

[0034] Provided herein is an expression vector comprising the nucleic acid composition described herein. Provided herein is a host cell comprising the expression vector described herein.

[0035] Provided herein is a method of producing a VHH antibody or antigen binding fragment comprising: (a) culturing the any of the host cells described herein under conditions to express the VHH antibody or antigen binding fragment, and (b) recovering the VHH antibody or antigen binding fragment.

[0036] Provided herein is a method of treating a cancer in a human subject in need thereof. In some embodiments, the method comprises administering to the human subject any one of the VHH antibody or antigen binding fragment described herein. In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colorectal cancer, and breast cancer.BRIEF DESCRIPTION OF DRAWINGS

[0037] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Fig.,” “FIG.,” “Figure,” “Figures,” “Figs.,” and “FIGs.” herein).

[0038] FIGS. 1A-1E depict monovalent and bivalent affinity of lead VHH by surface plasmon resonance (SPR) and flow cytometry and affinity of anti-CD3. Free VHHs and bivalent T-cell engagers (TCEs) were tested for binding to CEACAM6 substrates by SPR and flow cytometry. The lead VHH does not bind to CEACAM5, a related paralogue (FIG. 1A). The lead VHH binds to CEACAM6, KD 2.6 uM, when the Fc of the TCE is immobilized (FIG. IB). Avidity binding to144130-6004-4904.10Attorney Docket No. 55171.4010 / WOCEACAM6 by SPR when CEACAM6 is immobilized, KD 11.2 nM (FIG. 1C). Avidity binding to NCI-H2073 cells by flow cytometry, EC50 1.2 nM (Figure ID). Monovalent affinity of the CD3 Fab is 2.2e-07 at pH 7.4 and 3.17e-08 at pH 6.0 as measured by SPR (FIG. IE).

[0039] FIGS. 2A-2D depict machine-language (ML)-driven design improves size-exclusion chromatography (SEC) phenotype of the discovered VHH and hydrophobicity of the TCE. The unoptimized VHH exhibits strong non-specific interaction with the column matrix, exhibiting heterogeneous elution and broad peaks (FIG. 2A). The optimized VHH elutes as a single species with expected retention based on molecular weight (FIG. 2B). The unoptimized TCE elutes late as a broad, heterogeneous species due to hydrophobic interaction with the column matrix (FIG. 2C). The optimized TCE elutes earlier and mainly as a single species suggesting reduced overall hydrophobicity (FIG. 2D).

[0040] FIG. 3 depicts predicted MHC-II 9-mers. Y values represent the fraction of all MHC-II alleles that are predicted to bind to the 9-mer in the 5th or lower percentile of all possible peptides, where lower percentile indicates higher confidence. The predicted MHC-II epitopes for the affinity-optimized VHH are plotted as a sliding window of 9-mer derivatives of the primary sequence. The predicted MHC-11 epitopes of the de-immunized affinity-optimized VHH are plotted. A threshold of 20% predicted allele binding in the 1 st percentile was selected as significant based on prior literature. Major peaks represent the CDRs, some hashes represent 9-mers that are identical to human germline Ig sequences, other hashes represent 9-mer sequences found in clinical-stage VHHs, and yet other hashes represent 9-mers found in human proteins.

[0041] FIG. 4 depicts the humanness assessment of A-000-010-3936, showing the percent of 9-mers found in 50% or more of human patients in OAS (“OASis identity”) and the percent sequence identity with the nearest human heavy chain (“germline content”). The sequences depicted represent SEQ ID NOS: 167-172.

[0042] FIGS. 5A-5B show external biophysical analysis of A-000-010-3936. An analytical sizeexclusion chromatography (aSEC) profile after 2-step CH1-CHT purification (FIG. 5 A) and SDS-PAGE under reducing (R) and non-reducing (N-R) conditions (FIG. 5B) are shown.

[0043] FIGS. 6A-6D show internal biophysical characterization of A-000-010-3936. An aSEC profile (FIG. 6A) and an analytical hydrophobic interaction chromatography (aHIC or HIC) profile154130-6004-4904.10Attorney Docket No. 55171.4010 / WO(FIG. 6B) are shown. Barycentric mean (FIG. 6C) and SLS (FIG. 6D) traces across a temperature gradient

[0044] FIGS. 7A-7B depict stable cell line expression of A-000-010-3936, in particular, as shown in an aSEC profile (FIG. 7A) and intact mass spectrometry (MS) profile (FIG. 7B).

[0045] FIG. 8 depicts analytical SEC chromatograms of A-000-010-3936 following 21 day (21d) at 4°C, 25°C, and 40°C incubations. Indicated are monomeric purities of unstressed and 21d-incubated freeze-thawed antibodies.

[0046] FIGS. 9A-9C depict polyreactivity analysis of A-000-010-3936 by baculovirus particle (BVP) ELISA, antigen-based ELISA, and a cell-based flow cytometry assay. Signals were normalized to Adalimumab and benchmarked against a panel of commercial antibodies with known reactivities. Panels show normalized BVP binding (FIG. 9A), binding to antigens associated with polyreactivity (FIG. 9B), and non-specific binding to ExpiCHO cells (FIG. 9C). Top and bottom dotted lines denote thresholds of 5-fold and 2-fold signal increases over Adalimumab, respectively.

[0047] FIG. 10 depicts cytotoxicity of lead TCE candidate against cancer cell lines with various CEACAM6 densities. The negative control is represented as a diamond. The high affinity TCE control is represented as a circle. A-000-010-3936 is represented as a square.

[0048] FIGS. 11 A-l IB depict release of pro-inflammatory cytokines in vitro after co-culture with cell lines of varying CEACAM6 expression. Supernatants from TDCC assays were taken at 72h and analyzed for cytokine release by MSD V-plex T cell pro-inflammatory kit.

[0049] FIG. 12 depicts cytotoxicity of CEACAM6 TCEs against primary CEACAM6+ immune cells in healthy donor PBMCs. A high affinity CEACAM6xCD3 TCE (circle) was assessed versus the lead TCE A-000-006-3126 (square). CDllb+ and CD14+ populations with low levels of CEACAM6 were examined by flow cytometry. Upper and lower panels represent two unique PBMC donors.

[0050] FIG. 13 depicts cytokine release after co-culture of CEACAM6 TCEs with healthy donor PBMCs. A high-affinity CEACAM6xCD3 TCE (circle) was assessed versus the lead TCE A-000-006-3126 (square). Cytokines were measured by MSD. The dotted line indicates the upper limit of detection for IFNgamma (n=l, biological replicate).164130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0051] FIGS. 14A-14B depict T-cell activation in whole blood upon stimulation with CEACAM6 TCEs. A high-affinity CEACAM6xCD3 TCE (circle) was assessed versus the lead TCE candidate A-000-010-3936 (square). Data obtained from whole blood of donor 1 is shown in the top row of FIGS. 14A-14B and data from corresponding monocytes of donor 1 is shown in the top row of FIG. 14 A, and corresponding macrophages and neutrophils of donor 1 in the top row of FIG. 14B. Data from whole blood of donor 2 is shown in the bottom row of FIGS. 14A-14B and data from corresponding monocytes of donor 2 is shown in the bottom row of FIG. 14A and corresponding macrophages and neutrophils of donor 2 in the bottom row of FIG. 14B.

[0052] FIG. 15 shows release of pro-inflammatory cytokines in whole blood upon incubation with CEACAM6 TCEs. Supernatants from whole blood were analyzed for cytokine release by MSD. The lead TCE candidate A-000-010-3936 is represented as a square and the high-affinity CEACAM6xCD3 TCE (circle).

[0053] FIG. 16 depicts the effect of soluble CEACAM6 on TDCC activity. Soluble CEACAM6 was titrated into co-culture with GP2D (CEACAM6-med) cells and primary T-cells. A high-affinity CEACAM6xCD3 TCE and test TCE candidate A-000-010-4531 (SEQ ID NOS: 7-9) were assayed.

[0054] FIGS. 17A-17C show pharmacokinetic properties of lead TCE candidate A-000-010-3936 and in vivo cytokine release in NCG mice. Peripheral blood draws were taken from NCG mice implanted with human T cells and tumor cell line, NCI-H2073. Plasma samples were analyzed for inflammatory cytokines by MSD (FIG. 17A). Plasma samples were taken over the course of four weeks and analyzed for presence of A-000-010-3936 by meso scale discovery (MSD) assays (FIG.17B). NCG immunodeficient mice without tumors or T cells were dosed once weekly for four weeks and analyzed for the presence of drug by MSD (FIG. 17C).

[0055] FIGS. 18A-18B show in vivo activity of lead TCE candidate A-000-010-3936 in xenograft models of CEACAM6-expressing cancers. NCG mice with intraperitoneal engraftment of activated human T-cells were treated with the lead TCE as noted on a weekly basis. Groups minimally consisted of 5 animals. Treatment was initiated at tumor volumes exceeding 150 mm3. Mean and SEM is presented. FIG. 18A shows results from the SNU-C1 colon adenocarcinoma xenograft, NCI-H2170 NSCLC squamous cell carcinoma xenograft, and AsPCl pancreatic174130-6004-4904.10Attorney Docket No. 55171.4010 / WOadenocarcinoma xenograft. FIG. 18B shows results from the NCI-H2073 NSCLC adenocarcinoma xenograft.

[0056] FIG. 19 depicts ex vivo analysis of CEACAM6 expression on treated tumor cells. Five tumors in each group were excised, dissociated, and analyzed by flow cytometry for absolute receptor density by the Bangs Laboratories’ kit.

[0057] FIG. 20A depicts cell cytotoxicity of BxPC3 and MCF7 cell lines upon stimulation by CEACAM6 test TCE candidates and controls.

[0058] FIG. 20B depicts CD3 binding of various test CD3 antigen binding domains including SP34 (A-000-006-3116), 20G6 (A-000-006-3124), and OKT3 (A -000-005-2841).

[0059] FIG. 21 A depicts cell cytotoxicity of BxPC3 and MCF7 cell lines upon stimulation by CD3 antigen binding domain SP34 and CD3 variant antigen binding domains A-000-014-7708 (SEQ ID NOS: 152-153), A-000-014-7703 (SEQ ID NOS: 154-155), and A-000-014-7714 (SEQ ID NOS: 150-151).

[0060] FIG. 2 IB depicts CD3 binding of various test CD3 antigen binding domains including A-000-006-3116 (SP34, SEQ ID NOS: 73-74), A-000-006-3175 (SP34 variant, low affinity CD3 binder, SEQ ID NOS: 79-80), A-000-014-7703 (SP34 variant, medium affinity CD3 binding, SEQ ID NOS: 154-155), and A-000-006-3119 (SP34 variant, ultra-low affinity CD3 binder, SEQ ID NOS: 61-62).

[0061] FIG. 22A depicts cell cytotoxicity of BxPC3 and MCF7 cell lines upon stimulation by CD3 antigen binding domain 20G6 and CD3 variant antigen binding domains A-000-014-7715 (SEQ ID NOS: 158-159) and A-000-014-7709 (SEQ ID NOS: 156-157).

[0062] FIG. 22B depicts CD3 binding of various test CD3 antigen binding domains including A-000-006-3124 (20G6), BHB0962 (20G6 variant), and BHB0875 (20G6 variant).

[0063] FIG. 23 depicts % of cell viability of CEACAM6+ cells in human whole blood from donors 1-3 upon stimulation with CEACAM6 TCEs including lead TCE candidate A-000-010-3936 (SEQ ID NOS: 4-6) (circle), A-000-005-2841 (also referred to as BI00089; SEQ ID NOS: 195-197) (square), A-000-005-2486 (BHB0295; SEQ ID NOS: 189-191)(diamond), and A-000-007-0843 (SEQ ID NOS: 123-125) (triangle). TCEs were added to fresh human blood and cocultured for 72h and analyzed by flow cytometry.184130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0064] FIG. 24 depicts % of cell viability of CEACAM6+ cells in human whole blood from donors 1-3 upon stimulation with CEACAM6 TCEs including lead TCE candidate A-000-010-3936 (SEQ ID NOS: 4-6) (circle), A-000-005-2841 (SEQ ID NOS: 195-197) (square), A-000-005-2486 (BHB0295; SEQ ID NOS: 189-191) (diamond), and A-000-005-2485 (BHB0294; SEQ ID NOS: 183-185) (triangle). TCEs were added to fresh human blood and cocultured for 72h and analyzed by flow cytometry.

[0065] FIG. 25 depicts cell cytotoxicity (cell viability) of CEACAM3+ cells and T-cell activation in cells upon stimulation by CEACAM6 TCE test candidates A-000-006-3175 (SEQ ID NOS: 81-83), A-000-007-0843 (SEQ ID NOS: 123-125), and A-000-007-0842 (SEQ ID NOS: 129-131). The top panel shows cell viability (%) along y-axis and TCE concentration (M) along x-axis. The top panel shows median CD69 MFI along y-axis and TCE concentration (M) along x-axis.

[0066] FIG. 26 depicts cell cytotoxicity (cell viability) of CEACAM3+ cells and T-cell activation in cells upon stimulation by CEACAM6 TCE test candidates A-000-006-3175 (SEQ ID NOS: 81-83), A-000-007-0844 (SEQ ID NOS: 117-119), A-000-007-0846 (SEQ ID NOS: 105-107), and A-000-007-0845 (SEQ ID NOS: 111-113). The top panel shows cell viability (%) along y-axis and TCE concentration (M) along x-axis. The top panel shows median CD69 MFI along y-axis and TCE concentration (M) along x-axis.

[0067] FIG. 27 depicts cell cytotoxicity (cell viability) of CEACAM3+ cells and T-cell activation in the NCIH2073 cell line upon stimulation by CEACAM6 TCE test candidates A-000-006-3175 (SEQ ID NOS: 81-83), an anti-CD19x20G6 molecule (negative control), A-000-005-2485 (BHB0294; SEQ ID NOS: 183-185), and A-000-005-2841 (SEQ ID NOS: 195-197). The top panel shows cell viability (%) along y-axis and TCE concentration (M) along x-axis. The top panel shows median CD69 MFI along y-axis and TCE concentration (M) along x-axis.

[0068] FIGS. 28A-28B depict a CEACAM6 VHH antibody mutation coverage heatmap plot.

[0069] FIGS. 29A-29D depict a CD3 VHH antibody mutation coverage heatmap plot.

[0070] FIGS. 30A-30H depict a CEACAM6 VHH antibody mutation inference volcano plot. The parental sequence SEQ ID NO: 198 is shown in FIGS. 30C-30D and FIGS. 30G-30H.

[0071] FIGS. 31 A-31H depict a CD3 VHH antibody mutation inference volcano plot. The parental sequence SEQ ID NO: 199 is shown in FIGS. 31C-3 ID and FIGS. 31G-31H.194130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0072] FIG. 32 depicts a schematic diagram of the 2+1 asymmetric VHH2-Fab-Fc format.DETAILED DESCRIPTION

[0073] Disclosed herein are CEACAM6-specific T cell-engaging molecules that are useful to reduce or eliminate tumor cells by redirecting T cell cytotoxic effector function specifically to CEACAM6+ cancer cells and avoiding healthy cells. While CEACAM6 is expressed by myeloid hematopoietic precursor cells in the bone marrow and mature neutrophils in the periphery, these cell types express relatively low levels of CEACAM6 as compared to CEACAM6+ cancer cells. CEACAM6-specific T cell-engaging molecules provided herein have very low to low affinity and high avidity for CEACAM6 and are likely to spare healthy cells and specifically kill CEACAM6+ tumor cells.

[0074] Provided herein are T cell-engaging molecules containing anti-CEACAM6 antibodies or antibody fragments thereof and an anti-CD3s antibodies or antibody fragments thereof. In other words, the T cell-engaging molecules each include one or more CEACAM6 targeting antigen binding domains and one or more CD3 targeting antigen binding domains. Provided are high avidity and low affinity CEACAM6-directed T cell-engaging molecules that avoid binding to healthy cells and tissues expressing CEACAM6. The T cell-engaging molecules also include anti-CD3s antibodies and antibody fragments thereof that are pH selective such that they have high binding affinity for CD3 target antigens at an acidic pH relative to a neutral pH.

[0075] In some embodiments, the T cell-engaging molecules are bispecific antibodies. In some embodiments, the T cell-engaging molecules are bispecific, trivalent antibodies. The T cellengaging molecules bind the extracellular domain of CEACAM6 on cancer cells and the extracellular domain of CD3 on T cells. An exemplary format structure of a T cell-engaging molecule is depicted in FIG. 32. In some embodiments, the T cell-engaging molecules include monovalent anti-CD3 antigen binding domains and bivalent anti-CEACAM6 antigen binding domains.

[0076] Also provided herein are monospecific antibodies or antibody fragments thereof against human CEACAM6. In some embodiments, the anti-CEACAM6 antibodies or antibody fragments include single domain VHH antibodies or antibody fragments thereof.204130-6004-4904.10Attorney Docket No. 55171.4010 / WODefinitions

[0077] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0078] The term “carcinoembryonic antigen-related adhesion molecule 6” or “CEACAM6,” as used herein, refers to a glycosylphosphatidylinositol (GPI)-linked cell surface protein involved in cell-cell adhesion. CEACAM6 is also known as CD66c. The term “CEACAM6” as used herein includes human CEACAM6 (hCEACAM6), variants, isoforms, and species homologs of hCEACAM6, and analogs having at least one common epitope with hCEACAM6. A reference sequence for human CEACAM6 is available from UniProtKB / Swiss-Prot data base under accession number P40199.3. An exemplary amino acid sequence of the extracellular domain of a human CEACAM6 polypeptide is set forth as SEQ ID NO: 58. An exemplary amino acid sequence of the human CEACAM6 proprotein is set forth as SEQ ID NO: 59.

[0079] The term "cluster of differentiation 3” or “CD3,” as used herein, refers to a T cell coreceptor composed of four chains. In mammals, the CD3 protein complex contains a CD3y (gamma) chain, a CD35 (delta) chain, and two CD3s (epsilon) chains. These four chains associate with the T cell receptor (TCR) and the so-called q (zeta) chain to form the “T cell receptor complex” and to generate an activation signal in T lymphocytes. The CD3y (gamma), CD38 (delta), and CD3e (epsilon) chains are highly related cell-surface proteins of the immunoglobulin superfamily and each contain a single extracellular immunoglobulin domain. The intracellular tails of the CD3 molecules contain a single conserved motif known as an immunoreceptor tyrosinebased activation motif (ITAM), which is essential for the signaling capacity of the TCR. The CD3 epsilon (CD3s) molecule is a polypeptide, which in humans is encoded by the CD3E gene which resides on chromosome 11. Provided herein are antibodies, antibody fragments, and antigen binding polypeptides that bind to a CD3 polypeptide, such as to a human CD3s polypeptide, and more specifically, to the extracellular domain of a human CD3s polypeptide. An exemplary amino acid sequence of the extracellular domain of a human CD3s polypeptide is set forth as SEQ ID214130-6004-4904.10Attorney Docket No. 55171.4010 / WONO: 56. An exemplary amino acid sequence of the mature human CD3s polypeptide is set forth as SEQ ID NO: 57.

[0080] The term “T cell-engaging molecule” or “T cell engager” or “TCE” refers to a molecule that comprises at least one domain in which the structure is derived from or comprises the minimum structural features of an antibody, e.g., of a full-length immunoglobulin molecule, that allow for specific binding to an antigen on the surface of a T cell, such as CD3. Thus, a T cellengaging molecule according to the present disclosure comprises one or more antigen-binding domains, each of which will typically comprise the minimum structural requirements of an antibody that allow for specific target binding. The minimum structural requirement may, for example, be defined by the presence of the three heavy chain CDRs (i.e., HCDR1, HCDR2, and HCDR3) of a variable heavy domain (VHH) of a heavy-chain-only antibody. The minimum requirement may, for example, be defined by the presence of at least three light chain complementarity determining regions or CDRs (i.e., LCDR1, LCDR2 and LCDR3 of a VL region) and / or three heavy chain CDRs (i .e. HCDR1, HCDR2 and HCDR3 of a VH region), and preferably all six CDRs from both the light and heavy chain variable regions. In some aspect, provided herein are CEACAM6 targeting T cell-engaging molecules that specifically bind CEACAM6 (e.g., human CEACAM6) on the cell surface of a target cell (e.g., a tumor cell) and CD3 (e.g., human CD3) on the surface of a T cell.

[0081] An antibody includes a protein with an immunoglobulin fold that specifically binds to an antigen via its variable region or regions. An antibody may be a conventional type of antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. The light chain can be one of two types of immunoglobulin molecule, lambda (X) and kappa (K). The heavy chain can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. Each antibody chain contains distinct sequence domains (or regions). The light chain of a typical IgG antibody includes two regions, a variable region (VL) and a constant region (CL). The heavy chain of a typical IgG antibody includes four regions, namely a variable region (VH) and a constant region (CH), the latter being made up of three constant domains (CHI, CH2 and CH3). The variable regions of both light and heavy chains determine binding and specificity to the antigen. The constant regions of the light and heavy chains can confer important biological224130-6004-4904.10Attorney Docket No. 55171.4010 / WOproperties, such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

[0082] The term “antibody” as used herein, refer to and includes without limitation, polypeptides containing at least one antigen binding site (antigen binding domain), monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies including bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, hetero-conjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, nanobodies, VHH antibodies, intrabodies, affibodies, diabodies, common light chain antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, dual variable domains (DVD), and antigen binding fragments of any of the above. Antibodies may be camelid, murine, human, humanized, chimeric, or derived from other species. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunol. Biology, 5th Ed., Garland Publishing, New York).

[0083] The specificity of an antibody resides in the structural complementarity between the antigen binding site and the antigenic determinant. Antigen binding sites are made up of residues that are primarily from the so-called hypervariable or “complementarity determining regions” or “CDRs”. CDRs therefore refer to amino acid sequences which together define the binding affinity and specificity of the Fv region of an antibody. The light (L) and heavy (H) chains of an antibody each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, respectively. A conventional antibody’s antigen binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. See, e.g., Kindt et al., Kuby Immunology, 9th ed., W.H. Freeman & Co., 2018.

[0084] ‘ ‘Framework regions” or “FRs” refer to amino acid sequences interposed between CDRs, i.e., to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, FR1, FR2, FR3, and FR4 of the light chain and FR1, FR2,234130-6004-4904.10Attorney Docket No. 55171.4010 / WOFR3, and FR4 of the heavy chain, respectively. Accordingly, the CDR and FR sequences generally appear in the following order in VH: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4 or in VL: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, for instance 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to the framework region of a naturally occurring human antibody. In the context of the disclosure, CDR / FR definition in an immunoglobulin light or heavy chain is determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55- 77).

[0085] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) IMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme), Lefranc M. P. et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 2003 January; 27(l):55-77 (“IMGT” numbering scheme), and Honegger A. and Pluckthun A., “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J. Mol. Biol., 2001 Jun. 8; 309(3):657-70, (Aho numbering scheme).Table 1A.Kabat+Chothia IMGT Kabat Chothia Contact HCDR1 26-35 27-38 31-35 26-32 30-35 HCDR2 50-65 56-65 50-65 52-56 47-58 HCDR3 95-102 105-117 95-102 95-102 93-101 LCDR1 24-34 27-38 24-34 24-34 30-36 LCDR2 50-56 56-65 50-56 50-56 46-55 LCDR3 89-97 105-117 89-87 89-97 89-96244130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0086] Throughout the present specification, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g., Kabat et al., supra (1991)).

[0087] In the context of an IgG antibody, heavy chain domains including the constant heavy (CH) domains and the hinge domains are: “CHI domain” refers to positions 118-215 according to the EU index as in Kabat, “hinge domain” refers to positions 216-230 according to the EU numbering as in Kabat, “CH2 domain” refers to positions 231-340 according to the EU numbering as in Kabat, and “CH3 domain” refers to positions 341-447 according to the EU numbering as in Kabat. The exact numbering and placement of the heavy chain domains can be different among different numbering systems, such as, Kabat and Chothia, IMGT, Kabat, AbM, Chothia, and the like. As will be appreciated by those in the art, the exact numbering and placement of the heavy chain constant region domains (i.e., CHI, hinge, CH2 and CH3 domains) can be different among different numbering systems. A useful comparison of heavy constant region numbering according to EU and Kabat is as below, see Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, entirely incorporated by reference.Table IB.EU Numbering Kabat NumberingCHI 118-215 114-223Hinge 216-230 226-243CH2 231-340 244-360CH3 341-447 361-478

[0088] The term “CHI domain” refers to the first constant domain of an antibody heavy chain (e.g., amino acid positions 118-215 of human IgGl, according to the EU numbering). The term includes naturally occurring CHI domains and engineered variants of naturally occurring CHI domains (e.g., CHI domains comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring CHI domain). The N-terminal or254130-6004-4904.10Attorney Docket No. 55171.4010 / WOC-terminal of the CHI domain may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0089] The term “CH2 domain” refers to the second constant domain of an antibody heavy chain (e.g., amino acid positions 231-340 of human IgGl, according to the EU numbering). The term includes naturally occurring CH2 domains and engineered variants of naturally occurring CH2 domains (e.g., CH2 domains comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring CH2 domain). The N-terminal or C-terminal of the CH2 domain may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0090] The term “CH3 domain” refers to the third constant domain of an antibody heavy chain (e.g., amino acid positions 341-447 of human IgGl, according to the EU numbering). The term includes naturally occurring CH3 domains and engineered variants of naturally occurring CH3 domains (e.g., CH3 domains comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring CH3 domain). The N-terminal or C-terminal of the CH3 domain may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0091] The term “hinge region” or “hinge domain” refers to the portion of an antibody heavy chain comprising the cysteine residues (e.g., the cysteine residues at amino acid positions 226 and 229 of human IgGl, according to the EU numbering) that mediate disulfide bonding between two heavy chains in an intact antibody. An exemplary full-length IgGl hinge region comprises amino acid positions 216-230 of human IgGl, according to the EU index. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable regions and / or constant domains in a single polypeptide molecule. In some embodiments, the hinge region can be from the human IgGl subtype extending from amino acid 216 to amino acid 230 according to EU numbering, or from amino acid 226 to amino acid 243 according to Kabat numbering. The N-terminal or C-terminal of the hinge region may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0092] The term “antibody fragment” refers to a portion of a full-length antibody or a polypeptide that includes a portion of a full-length antibody, that retains antigen binding activity via its variable region or regions. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2,264130-6004-4904.10Attorney Docket No. 55171.4010 / WOscFv, sc(Fv)2, diabodies, linear antibodies, minibodies (Olafsen et al., Protein Eng. Design & Sei.17(4):315-323, 2004), fragments produced by a Fab expression library, single chain antibody molecules (e.g., scFv and scFab), and bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

[0093] The term “antigen binding domain” or “antigen binding region” is used to refer to one or more antibody variable domain(s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen(s). An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). In some aspects, an antigen binding domain comprises a heavy-chain-only antibody heavy chain variable domain (VHH domain). In some embodiments, an antigen binding domain of a VHH domain refers to a set of 3 Complementary Determining Regions (CDRs) that, when present as part of a polypeptide sequence, specifically binds a target antigen (e.g., CEACAM6). In some aspects, an antigen binding domain comprises an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL). In some aspects, an antigen binding domain refers to a set of six CDRs that, when present as part of a polypeptide sequence, specifically binds a target antigen (e.g., CD3). As is known in the art, these CDRs are generally present as a first set of variable heavy CDRs (HCDRs) and a second set of variable light CDRs (LCDRs), each comprising three CDRs: HCDR1, HCDR2, HCDR3 variable heavy CDRs and LCDR1, LCDR2 and LCDR3 variable light CDRs. The CDRs are present in the heavy chain variable domain (HCDR1-3) and light chain variable domain (LCDR1-3). In some embodiments, an antigen binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally-occurring antibodies.

[0094] The term “VHH” or “VHH domain” or “VHH region” refers to a single variable domain of a heavy chain only antibody (such as a camelid-derived heavy chain only antibody) that specifically binds to a target antigen. A VHH domain includes a set of 3 CDRs and 4 FRs, where the CDR and FR sequence generally appear in the following order: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. See US Patent No. 5,759,808 and Hamers-Casterman et al., Nature, 1993, 363(6428), 446-448.274130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0095] The term “Fab” denotes an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, in which about half of the N-terminal side of the heavy chain and the entire light chain are bound together through a disulfide bond. It is usually obtained among fragments by treating IgG with a protease, papain.

[0096] A term “single chain Fv” or “scFv” refers to a fusion polypeptide comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified otherwise, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0097] The term “monospecific antibody” refers to the ability of an antibody, such as an antibody, antibody fragment thereof, or antigen-binding polypeptide, to bind only one epitope.

[0098] The term “bispecific antibody” refers to the ability of an antibody, such as an antibody, antibody fragment thereof, or antigen binding polypeptide, to bind two different epitopes. In some embodiments, the term “bispecific antibody” refers to an antibody, such as an antibody, antibody fragment thereof, or antigen binding polypeptide, which comprises different antigen binding sites for different antigens. In some embodiments, a bispecific antibody is able to bind two different antigens simultaneously.

[0099] The term “multispecific antibody” refers to the ability of an antibody, such as an antibody, antibody fragment thereof, or antigen binding polypeptide, to bind more than one epitope. In certain embodiments, a multispecific antibody encompasses a bispecific antibody. For bispecific and multispecific antibodies provided herein, the epitopes can be on the same antigen, or each epitope can be on a different antigen. Therefore, in certain embodiments, a multispecific antibody provided herein, such as a bispecific antibody, binds to two different antigens. In certain embodiments, the multispecific antibody, such as a bispecific antibody, binds to different epitopes on one antigen. In some embodiments, a multispecific antibody includes an antibody which comprises two or more different antigen binding sites. In certain embodiments, a multispecific antibody provided herein binds to each epitope with a dissociation constant (Kd) of about < 1 M,284130-6004-4904.10Attorney Docket No. 55171.4010 / WOabout< 100 nM, about < 10 nM, about < 1 nM, about < 0.1 nM, about < 0.01 nM, or about < 0.001 nM (e.g., about 10'8M or less, from about 10'8M to about 10'13M, from about 10'8M to about 10’12M, from about 10’8M to about 1011M, from about 10’9M to about 1012M, from about IO10M to about 10'13M, or the like).

[0100] The term “specifically binds,” “specifically binding,” “binding specificity” or “binds” in the context of an antigen binding polypeptide or antigen-binding fragment thereof, refers that the antigen binding polypeptide or antigen-binding fragment thereof exhibits appreciable affinity for an antigen (e.g., CEACAM6 antigen or a CD3 antigen) and does not exhibit significant cross reactivity to a different target protein.

[0101] As used herein, the term “affinity” or “binding affinity” refers to the strength of the interaction between an antigen binding site or domain (paratrope) of an antibody or fragment thereof and the epitope on an antigen to which it binds. Factors influencing binding affinity include the complementarity in shape of the paratrope and epitope, electrostatic and hydrophobic interactions of the paratrope and epitope, and the binding conditions such as, but not limited to, temperature and pH. In some embodiments, affinity is measured by surface plasmon resonance (SPR) (e.g., B1ACORE®) or by biolayer interferometry (e.g., FORTEBIO®). As readily understood by those skilled in the art, a binding affinity may be reported as a dissociation constant (KD) in molarity (M). KD is a calculated ratio of the rate at which an antibody binds the target antigen and koff is the rate at which the antibody-antigen complex dissociates. Typically, high affinity antibodies have a low KD and are characterized by rapid target recognition (high kon) and strong stability of the resultant antibody-antigen complex (low koff). In some embodiments, antibodies or antibody fragments thereof described herein have KD values in the range of about I05M to about 1012M (i.e., low micromolar to picomolar range), about 107M to 1011M, about 10 M to about 1010M, about 109M. In certain embodiments, the antibody or antibody fragment thereof has a binding affinity of about 105M, 106M, 107M, 108M, 109M, 1010M, 1011M, or 1012M. In certain embodiments, the antibody or antibody fragment thereof has a binding affinity of about 107M to about 109M (nanomolar range).

[0102] The term “avidity” refers to the overall strength or stability of all the antibody-antigen interaction including the affinity, valency, and structural arrangement of the antibody and antigen. The term “valency” refers to the number of antigen binding sites on the antibody and the number294130-6004-4904.10Attorney Docket No. 55171.4010 / WOof amino acid residues contributing to the paratrope. Avidity refers to the cumulative strength or stability of multiple interactions between an antibody and an antigen that occur simultaneously, such as by way of multivalent binding.

[0103] The terms “identical” or “percent identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0104] For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two protein sequences with the default parameters is used. As an example, a sequence “at least 85% identical” to a reference sequence is a sequence having, over its entire length, 85% or more, for instance 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the entire length of the reference sequence. The percentage of “sequence identity” may thus be determined by comparing two such sequences over their entire length by global pairwise alignment using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), e.g. using the program Needle (EMBOSS) with the BLOSUM62 matrix and the following parameters: gap open=10, gap extend=0.5, end gap penal ty=false, end gap open=10, end gap extend=0.5 (which are standard settings).

[0105] The term “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different “replacement” amino acid residue. The replacement residue or residues may be “naturally occurring amino acid residues” (i.e., encoded by the genetic code) and selected from the group consisting of alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gin);304130-6004-4904.10Attorney Docket No. 55171.4010 / WOglutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (He); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Vai).

[0106] The term “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Conservative amino acid substitution groups can also be defined on the basis of amino acid size.CEACAM6 antigen binding domains

[0107] Provided are antigen binding domains, e.g., antibodies or antibody fragments thereof that specifically bind CEACAM6 protein, e.g., the extracellular domain (ECD) of human CEACAM6 protein. The antibodies can also be referred to as CEACAM6 targeting antibodies and anti-CEACAM6 antibodies. In some embodiments, the antibodies are monospecific and target a single epitope or a single antigen such as CEACAM6. In some embodiments, the antibodies are bispecific and target two epitopes or two antigens simultaneously (e.g., CEACAM6 and CD3). In some embodiments, the antibodies are multispecific and target two or more epitopes or target antigens simultaneously.

[0108] Provided herein are T cell-engaging molecules comprising CEACAM6 antigen binding domains and CD3 antigen binding domains. In some embodiments, the CEACAM6 antigen binding domains are CEACAM6 antigen binding domains of VHH antibodies (also referred to as nanobodies) or CEACAM6 targeting VHH domains. VHH antibodies are heavy chain-only antibodies that may be naturally isolated or derived from camelid species or engineered from human antibodies that are composed of one heavy chain variable domain (VHH domain) which lack CHI domains and are devoid of light chains. In other words, each heavy chain of a VHH314130-6004-4904.10Attorney Docket No. 55171.4010 / WOantibody has a variable heavy domain at the N-terminus and this domain is referred to as the VHH domain. The VHH domain includes 3 CDR regions of the heavy chain (HCDR1, HCDR2, and HCDR3) of the VHH immunoglobulin that confers antigen binding affinity and specificity. In some instances, a VHH antibody or antibody fragment thereof disclosed herein includes a camelid heavy chain antibody, a camelid VHH domain, a humanized VHH domain, a CDR-grafted VHH domain, an affinity matured VHH domain, a non-human VHH domain, or a human VHH domain. An isolated VHH domain can be combined with another antigen binding domain such as an scFv, a Fab, and the like to form a multispecific antibody (also referred to as a multispecific antigen binding polypeptide). An isolated VHH domain can be included in any type of antibody format including, but not limited to, bispecific antibodies, multispecific antibodies, bispecific antigen binding polypeptides, multispecific antigen binding polypeptides, antibodies comprising a Fab, antibodies comprising an scFv, antibodies comprising a single chain antibody fragment, and any other antibody formats described herein.

[0109] In some embodiments, an anti-CEACAM6 VHH domain includes an amino acid sequence set forth in any one ofSEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192. In some embodiments, an anti-CEACAM6 VHH domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to an amino acid sequence set forth in any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, and 160-166. In some embodiments, an anti-CEACAM6 VHH domain has from 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid modifications in an amino acid sequence set forth in any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192. In some embodiments, the amino acid modifications are in the CDRs and / or the FRs of the VHH domain. In some embodiments, the VHH domain includes one or more CDR modifications (e.g., one or more amino acid substitutions, deletions, or insertions in at least one CDR region) and / or one or more framework modifications (e.g., one or more amino acid substitutions, deletions, or insertions in at least one framework region) and / or one or more modifications to remove a T cell epitope to reduce the immunogenicity of the domain.

[0110] In some embodiments, provided herein is an antibody or antibody fragment thereof comprising an anti-CEACAM6 VHH domain. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof includes an amino acid sequence set forth in any one of 324130-6004-4904.10Attorney Docket No. 55171.4010 / WOSEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.[oni] In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 13 wherein Xi is N or Y, X2 is H or A, X3 is M or S. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 27. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 27. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 60. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 60. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 66. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 66. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 72. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment 334130-6004-4904.10Attorney Docket No. 55171.4010 / WOthereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 72. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 78. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 78. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 84. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 84. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 90. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 90. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 96. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 96. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 102. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 102. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 108. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 108. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 114. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 114. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises344130-6004-4904.10Attorney Docket No. 55171.4010 / WOthe amino acid sequence of SEQ ID NO: 120. In other embodiments, the anti-CEACAM6 VETH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 120. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 126. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 126. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 132. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 132. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 138. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 138. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 144. In other embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 144. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 160. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 161. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 161. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 162.354130-6004-4904.10Attorney Docket No. 55171.4010 / WOIn some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 163. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 163. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 164. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 164. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 165. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 165. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises the amino acid sequence of SEQ ID NO: 166. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 166.

[0112] In some embodiments, the anti-CEACAM6 antibody or antibody fragment thereof includes at least one anti-CEACAM6 VHH domain described herein. In some embodiments, the anti-CEACAM6 antibody includes more than one anti-CEACAM6 VHH domain described herein.

[0113] In some embodiments, provided herein is an antibody or antibody fragment thereof containing an anti-CEACAM6 VHH domain comprising a HCDR1, HCDR2, and HCDR3 as provided in the sequence listing. In some embodiments, the anti-CEACAM6 VHH domain comprises a HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 41-43, respectively. In some embodiments, the anti-CEACAM6 VHH domain comprises a HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 44-46, respectively. In some embodiments, the anti-CEACAM6 VHH domain comprises a HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 47-49, respectively. In some embodiments, the anti-CEACAM6 VHH domain includes one or more, e.g., 1, 2, 3, 4, 5, 6, or more amino acid substitutions in either HCDR1, HCDR2, HCDR3, or any combination thereof.

[0114] In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof includes a variant of the VHH domain described herein. In an embodiment, the variant VHH364130-6004-4904.10Attorney Docket No. 55171.4010 / WOdomain has from 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid modifications from a VHH domain that binds CEACAM6 described herein, including those in the sequence listing. In some embodiments, the variant VHH domain has from 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid modifications of one of the following VHH domains: VHH domain.1, VHH domain.2, VHH domain.3, VHH domain.4, and VHH domain. A, as provided in the sequence listing. In some embodiments, the one or more amino acid modifications are in the CDRs (CDR21, CDR2, and CDR3) and / or FRs (FR1, FR2, FR3, and FR4) of the VHH domain. In some embodiments, the one or more amino acid modifications are in one or more of the CDRs. In some embodiments, the one or more amino acid modifications are in one or more of the FRs. In some embodiments, the one or more amino acid modifications of the VHH domain alter binding affinity but not binding specificity to CEAC AM6. In one embodiment, the variant VHH domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the VHH domains described herein, including in the sequence listing. In some embodiments, the variant VHH domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to one of the following VHH domains: VHH domain.1, VHH domain.2, VHH domain.3, VHH domain.4, and VHH domain. A, as depicted as SEQ ID NOS: 10-13 and 27, respectively. In some embodiments, the CEACAM6 antigen binding domain including the VHH is capable of binding to CEACAM6, as measured by at least one of a Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assay. In some embodiments, the CEACAM6 antigen binding domain is capable of binding the extracellular domain of human CEACAM6 protein.

[0115] In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof has an altered affinity for CEACAM6 compared to a reference antibody or antibody fragment thereof, such as, but not limited to the antibody fragment of SEQ ID NO: 27. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof has a lower affinity for CEACAM6 compared to a reference antibody or antibody fragment thereof. In some embodiments, the anti-CEACAM6 VHH antibody or antibody fragment thereof has a significantly lower affinity for CEACAM6 compared to a reference antibody or antibody fragment thereof. In some embodiments, the reference antibody or antibody fragment thereof is anti-CEACAM6 VHH antibody or antibody fragment thereof exhibiting a high binding affinity for human CEAC AM6 as determined by standard binding assays known in the art. In other embodiments, the reference antibody or antibody fragment thereof is anti-CEACAM6 monoclonal antibody or antibody 374130-6004-4904.10Attorney Docket No. 55171.4010 / WOfragment thereof exhibiting a high binding affinity for human CEACAM6 as determined by standard binding assays known in the art. In other embodiments, the reference antibody or antibody fragment thereof is anti-CEACAM6 polyclonal antibody or antibody fragment thereof exhibiting a high binding affinity for human CEACAM6 as determined by standard binding assays known in the art.

[0116] Also provided herein are multispecific (such as bispecific) VHH-based antibodies containing one or more anti-CEACAM6 VHH domains described herein linked to one or more VHH domains that do not bind CEACAM6. In some embodiments, the multispecific VHH-based antibodies containing one or more anti-CEACAM6 VHH domain linked to one or more VHH domains that bind a different target antigen. In some embodiments, the multispecific VHH-based antibodies contain two or more anti-CEACAM6 VHH domains linked tandemly. In some embodiments, the multispecific VHH-based antibodies contain two or more VHH domains against a different target antigen that are linked tandemly.

[0117] The CEACAM6 antigen binding domains described herein can be included in T cellengaging molecules containing CD3 antigen binding domains. Such T cell-engaging molecules specifically bind CD3 proteins on the cell surface of T cells and CEACAM6 proteins on the cell surface of cancer cells (e.g., tumor cells). In some embodiments, the T cell-engaging molecule includes at least one CEACAM6 targeting VHH domain, e.g., 1, 2, 3, 4, 5, or more CEACAM6 targeting VHH domains. In some embodiments, each of the VHH domains have the same sequence. In some embodiments, one or more of the VHH domains have different sequences. In some embodiments, the VHH domains are arranged tandemly with an optional spacer between VHH domains. Non-limiting examples of a spacer include glycine-serine linkers known in the art including those described herein. In some embodiments, the VHH domain can be any described herein. In some embodiments, the VHH domain selectively binds to CEACAM6 at acidic pH (e.g., pH 5.8 to pH 6.5) in a tumor microenvironment. In some embodiments, the T cell-engaging molecule includes one or more CD3 antigen binding domains, e.g., 1, 2, 3, 4, 5, or more CD3 antigen binding domains. In some embodiments, the T cell-engaging molecule includes only one CD3 antigen binding domain. In some embodiments, the CD3 antigen binding domain selectively binds to CD3 at acidic pH (e.g., pH 5.8 to pH 6.5) in a tumor microenvironment. In some embodiments, the CD3 antigen binding domain includes any of the VH domains, VL domains, or any set of HCDRs and LCDRs described herein. In some embodiments, the CD3 antigen binding 384130-6004-4904.10Attorney Docket No. 55171.4010 / WOdomain is in a Fab format or a scFv format. Tn some embodiments, the T cell-engaging molecule is based on an antibody format containing an Fc region, such as a homodimeric Fc region or a heterodimeric Fc region. Exemplary T cell-engaging molecules containing CEACAM6 antigen binding domains and CD3 antigen binding domains can have any format including a 2+1 asymmetric VHH2-Fab-Fc format (see FIG. 32) and a 2+2 VHH2-Fab2-Fc format, both described in further detail below. In some embodiments, such T cell engager molecule formats include a naturally-occurring Fc region or a variant Fc region, which are described in detail below.

[0118] In various embodiments, the T cell-engaging molecule includes a CEACAM6 targeting VHH domain and a CD3 Fab. The CEACAM6 targeting VHH domain can be linked to the CD3 Fab region by way of a flexible linker. The CEACAM6 targeting VHH domain can be linked by way of a flexible linker to the N-terminus of the VH domain of the CD3 Fab. The CEACAM6 targeting VHH domain can be linked by way of a flexible linker to the C-terminus of the CHI domain of the CD3 Fab. In some embodiments, the T cell-engaging molecule comprises from N-terminus to C-terminus: a first monomer (a first polypeptide chain) comprising at least one CEACAM6 targeting VHH domain, an optional flexible linker, a VH domain of the CD3 Fab, and a CHI domain of the CD3 Fab, and a second monomer (a second polypeptide chain) comprising a VL domain of the CD3 Fab and a CL domain of the CD3 Fab. In some embodiments, the T cell engager molecule comprises from N-terminus to C-terminus: a first monomer comprising a VH domain of the CD3 Fab region, a CHI domain of the CD3 Fab region, an optional flexible linker, at least one VHH domain, and a second monomer comprising a VL domain of the CD3 Fab region and a CL domain of the CD3 Fab region. In some instances, the first monomer comprises an Fc region including a hinge region located at the C-terminus of the CHI domain of the CD3 Fab region. In some cases, the first monomer include a first Fc region, and an additional monomer comprises a second Fc region that forms a heterodimer with the first Fc region. In additional instances of the additional monomer, the N-terminus of the second Fc region is linked to the C-terminus of another (a second) CEACAM6 targeting VHH domain.

[0119] In some embodiments, the T cell-engaging molecule is based on an antibody format that does not include an Fc region. In some embodiments, the T cell-engaging molecule includes a CEACAM6 antigen binding domain such as a CEAC AM6 targeting VHH domain described herein and a CD3 antigen binding domain described herein. In some embodiments, the T cell-engaging molecule includes more than one CEAC AM6 targeting VHH domain and one CD3 antigen binding 394130-6004-4904.10Attorney Docket No. 55171.4010 / WOdomain such as a CD3 targeting scFv. The CEACAM6 targeting VHH domain can be linked to the CD3 scFv by way of an optional flexible linker. In some embodiments, the T cell-engaging molecule comprises from N-terminus to C-terminus: a CEACAM6 targeting VHH domain, an optional flexible linker, and a CD3 targeting scFv. In some embodiments, the T cell-engaging molecule comprises from N-terminus to C-terminus: a CD3 targeting scFv, an optional flexible linker, and a CEACAM6 targeting VHH domain.

[0120] In some embodiments, the T cell-engaging molecule includes two or more CEACAM6 targeting VHH domains and one or more CD3 antigen binding domains. In some embodiments, the T cell-engaging molecule includes two or more CEACAM6 targeting VHH domains and two or more CD3 targeting scFv. In some embodiments, two or more CEACAM6 targeting VHH domains is arranged in tandem and linked by an optional spacer. In some embodiments, the T cellengaging molecule comprises from N-terminus to C-terminus: a first CEACAM6 targeting VHH domain, an optional spacer, a second CEACAM6 targeting VHH domain, an optional flexible linker, and a CD3 targeting scFv. In some embodiments, the T cell-engaging molecule comprises from N-terminus to C-terminus: a CD3 targeting scFv, an optional flexible linker, a first CEACAM6 targeting VHH domain, an optional spacer, and a second CEACAM6 targeting VHH domain.CD3 antigen binding domains

[0121] Provided are antigen binding domains, e.g., antibodies or antibody fragments thereof that specifically bind CD3 protein, e.g., the mature human CD3s protein or the extracellular domain (ECD) of human CD3s protein. An exemplary amino acid sequence of the ECD of human CD3a protein is provided in SEQ ID NO: 56. An exemplary amino acid sequence of the mature human CD3s protein is provided in SEQ ID NO: 57.

[0122] Provided herein are T cell-engaging molecules comprising anti-CD3 antigen binding domains that bind CD3s with pH selectivity. Specifically, provided herein are T cell-engaging molecules comprising anti-CD3 antigen binding domains (particularly, anti-CD3s antigen binding domains) that bind CD3s (e.g., human CD3e) at an acidic pH, e.g., pH 5.8 to 6.5, and are devoid of CD3s binding at physiological or neutral pH, e g., pH 7.0 to 7.4. In some embodiments, the anti-CD3 antigen binding domains possess therapeutically effective levels of CD3s specific binding activity at pH 6.0 and significantly diminished or abolished CD3s specific binding activity at pH404130-6004-4904.10Attorney Docket No. 55171.4010 / WO7.4. In some embodiments, the anti-CD3 antigen binding domains possess therapeutically effective levels of CD3s specific binding activity at pH 6.0 and significantly lower CD3s specific binding activity at pH 7.4. Provided herein are T cell-engaging molecules comprising anti-CD3 antigen binding domains that selectively bind immune cells such as T cells in a tumor microenvironment. Provided herein are T cell-engaging molecules comprising anti-CD3 antigen binding domains with substantially higher binding affinity, e.g., more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold or higher binding affinity, in an acidic pH environment, e.g., pH 5.8 to 6.5, compared to a neutral pH environment, e.g., pH 7.0 to 7.4. Provided herein are T cell-engaging molecules comprising anti-CD3 antigen binding domains that do not bind or have negligible binding activity to immune cells, e.g., T cells in a neutral pH environment.

[0123] Provided herein are T cell-engaging molecules comprising anti-CD3 antigen binding domains that bind CD3e at a lower binding affinity compared to a wild-type or parental anti-CD3 antibody or antibody fragment thereof, such as, but not limited to, OKT3, SP34, 20G6, UCHT3, IT3, and SK7 antibodies and antibody fragments thereof. In some embodiments, the T cellengaging molecules comprising anti-CD3 antigen binding domains that bind CD3e at a moderate binding affinity compared to a wild-type or parental anti-CD3 antibody or antibody fragment thereof. In some instances, the binding affinity of wild-type OKT3 for CD3 represents high binding affinity to the antigen as determined by standard binding assays. In some instances, the binding affinity of wild-type SP34 for CD3 represents high binding affinity to the antigen as determined by standard binding assays. In particular instances, the binding affinity of wild-type 20G6 for CD3 represents high binding affinity to the antigen as determined by standard binding assays. In some particular instances, the binding affinity of wild-type UCHT3 for CD3 represents high binding affinity to the antigen as determined by standard binding assays. In other instances, the binding affinity of wild-type IT3 for CD3 represents high binding affinity to the antigen as determined by standard binding assays. In some other instances, the binding affinity of wild-type SK7 for CD3 represents high binding affinity to the antigen as determined by standard binding assays.

[0124] Provided herein are anti-CD3 antibodies or antibody fragments thereof comprising a set of HCDRs set forth in SEQ ID NOs: 50-52 and a set of LCDRs set forth in SEQ ID NOs: 53-55. In some embodiments, the anti-CD3 antibodies or antibody fragments thereof include a HCDR1 set forth in SEQ ID NO: 50, a HCDR2 set forth in SEQ ID NO: 51, a HCDR3 set forth in SEQ ID NO: 52, a LCDR1 set forth in SEQ ID NO: 53, a LCDR2 set forth in SEQ ID NO: 54, a LCDR3414130-6004-4904.10Attorney Docket No. 55171.4010 / WOset forth in SEQ ID NO: 55. Tn some embodiments, the anti-CD3 antibodies or antibody fragments thereof include a HCDR1 having an amino acid sequence having about 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as set forth in SEQ ID NO: 50, a HCDR2 having an amino acid sequence having about 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as set forth in SEQ ID NO: 51, a HCDR3 having an amino acid sequence having about 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as set forth in SEQ ID NO: 52, a LCDR1 having an amino acid sequence having about 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as set forth in SEQ ID NO: 53, a LCDR2 having an amino acid sequence having about 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as set forth in SEQ ID NO: 54, a LCDR3 having an amino acid sequence having about 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as set forth in SEQ ID NO: 55.

[0125] In some embodiments, the anti-CD3 antigen binding domains of the T cell-engaging molecules are in a fragment antigen-binding region (Fab) fragment antibody format. In some embodiments, the anti-CD3 antigen binding domain comprises heavy chain variable and constant regions and light chain variable and constant regions. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 15, a heavy chain constant CHI region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 16, a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 17, and a light chain constant CL region comprising at least 90% e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain constant CHI region comprising the amino acid sequence of SEQ ID NO: 16, a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain constant CL region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 28, a heavy chain constant CHI 424130-6004-4904.10Attorney Docket No. 55171.4010 / WOregion comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 29, a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 30, and a light chain constant CL region comprising at least 90% e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 28, a heavy chain constant CHI region comprising the amino acid sequence of SEQ ID NO: 29, a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 30, and a light chain constant CL region comprising the amino acid sequence of SEQ ID NO: 31.

[0126] In some embodiments, the anti-CD3 antigen binding domains of the T cell-engaging molecules are in a Fab antibody fragment (Fab) or a single-chain variable fragment (scFv) antibody format. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region and a light chain variable VL region.

[0127] In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 28, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-CD3434130-6004-4904.10Attorney Docket No. 55171.4010 / WOantigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 61, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 61 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 67, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 67 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 73, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 73 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 79, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 79 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the anti-CD3444130-6004-4904.10Attorney Docket No. 55171.4010 / WOantigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 85, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 85 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 91, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 91 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 97, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 97 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 103, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 103 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, the anti-CD3454130-6004-4904.10Attorney Docket No. 55171.4010 / WOantigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 109, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 109 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 115, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 115 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 116. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 121, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 121 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 127, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 128. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 127 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 128. In some464130-6004-4904.10Attorney Docket No. 55171.4010 / WOembodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 133, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 134. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 133 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 139, and a light chain variable VL region comprising at least 90%, e g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 139 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 145, and a light chain variable VL region comprising at least 90%, e g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 146. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 145 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 146. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 181, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 182. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 181 and a light chain474130-6004-4904.10Attorney Docket No. 55171.4010 / WOvariable VL region comprising the amino acid sequence of SEQ ID NO: 182. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 187, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 188. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 187 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 188. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 193, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 194. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 193 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 194.

[0128] In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises: a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 15, a heavy chain constant CHI region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 16, a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,484130-6004-4904.10Attorney Docket No. 55171.4010 / WOsequence identity to the amino acid sequence of SEQ ID NO: 17, and a kappa light chain constant (CL kappa) region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain constant CHI region comprising the amino acid sequence of SEQ ID NO: 16, a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 17, and a kappa light chain constant (CL kappa) region comprising the amino acid sequence of SEQ ID NO: 18.

[0129] In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises: a heavy chain variable VH region comprising at least 90%, e g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 28, a heavy chain constant CHI region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 29, a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 30, and a kappa light chain constant (CL kappa) region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-CD3 antigen binding domain of Fab format comprises: a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 28, a heavy chain constant CHI region comprising the amino acid sequence of SEQ ID NO: 29, a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 30, and a kappa light chain constant (CL kappa) region comprising the amino acid sequence of SEQ ID NO: 31.

[0130] In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising at least 90%, e.g.,494130-6004-4904.10Attorney Docket No. 55171.4010 / WO90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 27, and a light chain variable VL region comprising at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-CD3 antigen binding domain comprises a heavy chain variable VH region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable VL region comprising the amino acid sequence of SEQ ID NO: 29.

[0131] In some embodiments, an anti-CD3 scFv further comprises a scFv linker linking the VH region and the VL region. In some embodiments, an anti-CD3 scFv comprises from N-terminus to C-terminus: a VH region, an scFv linker, and a VL region. In some embodiments, an anti-CD3 scFv comprises from N-terminus to C-terminus: a VL region, an scFv linker, and a VH region.

[0132] In some embodiments, an scFv linker comprises a flexible linker, such as a flexible peptide linker. In some embodiments, an scFv linker comprises about more than 12 amino acid or about 15 to about 20 amino acids in length. In some embodiments, an scFv linker is a glycine-serine linker such as any of those described herein and in the sequence listing. In some embodiments, an scFv linker is a Whitlow linker as set forth in SEQ ID NO: 40 and disclosed in Whitlow et al., Protein Engineering, 1993, 6(8):989-995. Additional exemplary scFv linkers are disclosed in U.S. Patent No. 11,840,579, the disclosure is herein incorporated by reference.

[0133] In some aspects, T cell-engaging molecules provided herein comprise any one of the anti-CD3 antigen binding domains, e.g., anti-CD3 antibodies or antibody fragments thereof as set forth in PCT / US2023 / 067579, the disclosure is herein incorporated by reference in its entirety.

[0134] In some embodiments, the anti-CD3 antigen binding domains of the T cell-engaging molecules have at least one amino acid modification (e.g., substitution, deletion, or insertion) in the heavy chain, the light chain, or both, compared to the corresponding chain in a wild-type or parental anti-CD3 antibody or antibody fragment. In some embodiments, the anti-CD3 antigen binding domains of the T cell-engaging molecules have at least one amino acid modification in the heavy chain variable VH region, the light chain variable VL region, or both, compared to the corresponding region of a reference, wild-type or parental anti-CD3 antibody or antibody fragment. In some embodiments, the anti-CD3 antigen binding domains of the T cell-engaging molecules have at least one amino acid modification in the HCDR1, HCDR2, HDCR3, LCDR1, LCDR2, LDCR3, or any combination thereof, compared to the CDR of a reference, wild-type, or504130-6004-4904.10Attorney Docket No. 55171.4010 / WOparental anti-CD3 antibody or antibody fragment. Tn some embodiments, the anti-CD3 antigen binding domains of the T cell-engaging molecules have at least one amino acid modification in any of the heavy chain FRs, the light chain FRs, or any combination thereof, compared to the FRs of a reference, wild-type, or parental anti-CD3 antibody or antibody fragment. Non-limiting examples of a reference, wild-type, or parental anti-CD3 antibody or antibody fragment include 0KT3, UCHT3, SP34, HIT3, SK7, 20G6, and antibody fragments thereof.Antibody Fc regions

[0135] In some embodiments, any antigen binding polypeptide described herein, including antibodies, antibody fragments, and T cell-engaging molecules, which binds to the extracellular domain of the human CEACAM6 protein can contain an Fc region based on a naturally occurring human IgGl, IgG2, IgG3, or IgG4 molecule. In some embodiments, an Fc region based on a variant of a human IgGl, IgG2, IgG3, or IgG4 molecule. In some embodiments, the first and second Fc domains of the human Fc region are each a variant Fc domain containing one or more Fc mutations or modifications. Fc modifications can alter the effector functions attributed to the Fc region of an antibody including, but not limited to, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.

[0136] In some embodiments, the first and second Fc domains of the Fc region each include one or more amino acid modifications (e.g., substitutions, deletions, and insertions) that alter the glycosylation status of the antigen binding polypeptide. In some embodiments, the first and second Fc domains each include one or more amino acid modifications that alter the binding of the variant Fc region to one or more FcyR or to the FcRn, facilitate heterodimerization of the first and second Fc domains, prolong serum half-life, and / or improve effector functions. In some embodiments, the variant Fc region possesses one or more modified functional properties of the polypeptide such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity, compared to a corresponding polypeptide with a naturally occurring Fc region.

[0137] In some embodiments, a variant Fc region containing one or more mutations that preferentially enhances FcRn affinity to confer increased serum half-life of the antigen binding514130-6004-4904.10Attorney Docket No. 55171.4010 / WOpolypeptide. In some instances, the Fc modifications alter FcyR binding. In some instances, the Fc mutations reduce effector functions. In some instances, the Fc mutations confer increased affinity for FcyRIIIa. In some instances, the Fc modifications enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some instances, the Fc modifications result in altered Clq binding and / or complement dependent cytotoxicity (CDC). In some instances, the Fc mutations reduce or abolish CDC

[0138] In some embodiments, a variant Fc region contains RF mutations in the CH3 domains of the Fc region. In some embodiments, a variant Fc region does not contain RF mutations in the CH3 domains of the Fc region.

[0139] In some embodiments, any antigen binding polypeptide described herein which binds to the extracellular domain of human CEACAM6 protein includes one or more substitutions to promote heterodimerization of the Fc region including knob-in-hole modifications, electrostatic steering mutations, charge stabilization modifications, hydrophobic interaction modifications, and / or cysteine modifications.

[0140] In some embodiments of knob-in-hole modifications, one Fc domain comprises an amino acid substitution in the CH3 domain at one or more positions of 368, 394, 405, and 407, according to EU numbering, which produces a hole, and the other Fc domain comprises an amino acid substitution at position 366, according to EU numbering, which produces a knob. In some embodiments, one Fc domain comprises the amino acid substitution T366S, L368A, or Y407V, and the other Fc domain comprises the amino acid substitution T366W, according to EU numbering. In some embodiments, the amino acid substitutions to the CH3 domain include T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, and combinations thereof, according to EU numbering. In some embodiments, the amino acid substitutions to the CH3 domains includes T366Y or T366W for the knob mutation on a first CH3 domain and Y407T or Y407V for the hole mutation on a second CH3 domain, according to EU numbering. In some embodiments, the amino acid substitutions to the CH3 domains includes T366Y for the knob mutation on a first CH3 domain and Y407V for the hole mutation on a second CH3 domain, according to EU numbering. In some embodiments, the amino acid substitutions to the CH3 domains includes S354C / T366W for the knob mutation on a first CH3 domain and Y349C / T366S / L368A / Y407V for the hole mutation on a second CH3 domain, according to EU numbering.524130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0141] In some embodiments of electrostatic steering modifications, amino acid substitutions in the CH3 domains utilize electrostatic steering to promote and strengthen Fc domain pairing in the CH3 / CH3 domain interface. The charge complementarity between two CH3 domains is altered to favor heterodimerization (opposite charge pairing) over homodimerization (same charge pairing). Thus, the electrostatic repulsive forces prevent homodimerization. In some embodiments, amino acid substitutions to the Fc domain include K409D, K392D, K370D, or any combination thereof, and amino acid substitutions to the other Fc domain include D399K, E356K, E357K or any combination thereof, according to EU numbering. In some embodiments, amino acid substitutions to the first Fc domain include K409D / K392D, and amino acid substitutions to the other Fc domain include D399K / E356K, according to EU numbering. In some embodiments, amino acid substitutions to the first Fc domain include L351D / L368E, and amino acid substitutions to the other Fc domain include L351K / T366K, according to EU numbering.

[0142] In some embodiments of charge stabilization modifications, one Fc domain comprises the amino acid substitution N297K, and the other Fc domain comprises the amino acid substitution N297D, according to EU numbering. In an embodiment of a charge stabilization mutation, one Fc domain comprises the amino acid substitution T299K, and the other Fc domain comprises the amino acid substitution T299D, according to EU numbering. In an embodiment of a cysteine mutation, one Fc domain comprises the amino acid substitution Y349C, and the other Fc domain comprises the amino acid substitution S354C, according to EU numbering.

[0143] In some embodiments of hydrophobic interaction modifications, amino acid substitutions in the CH3 domains utilize hydrophobic interactions to promote and strengthen Fc domain pairing in the CH3 / CH3 domain interface. In some embodiments, amino acid substitutions to the first CH3 domain include K409W, K360E, Q347E, Y349S, S354C, or any combination thereof, and amino acid substitutions to the second CH3 domain include D399V, F405T, Q347R, E357W, Y349C or any combination thereof, according to EU numbering.

[0144] In some embodiments, amino acid substitutions to the first Fc domain and / or the second Fc domain include: L234A / L235A (LALA substitution), L234A / L235A / P329G (LALAPG substitution), L235A / G237A (LAGA substitution), M252Y / S254T / T256E (YTE substitution), K222R, M428L / N434S (LS substitution), M428L / N434A (LA substitution), H433K / N434F (KF substitution), and any combination thereof, according to EU numbering. Non-limiting amino acid534130-6004-4904.10Attorney Docket No. 55171.4010 / WOsubstitutions in the first Fc domain and / or the second Fc domain also include S239D / I332E, A330L, S239D / A330L / I332E, E430G, E430G / E435K, K326W / K333S, S267E, H268F, S324T, L235V / F243L / R292P / Y300L / P396L, S267E / L328F, L234F / L235E, L234F / L235E / P331S, and E233P / L234V / L235A, according to EU numbering. In some embodiments, pairs of amino acid substitutions between a first CH3 domain and a second CH3 domain (first CH3: second CH3) include K409W:D399V, K409W:F405T, K360E / K409W:Q347R / D399V / F405T, S364H / F405A:Y349T / T394F, K360E:Q347R, Y349S:E357W, and S354CY349C, according to EU numbering.

[0145] In some embodiments, the first and second hinge domains are each hinge domains based on a naturally-occurring hinge domain of an IgG antibody such as an IgGl, IgG2, IgG3, or IgG4 antibody. Structurally, the IgG CHI domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. For an IgG antibody, the hinge domain is defined to include positions 216 (E216 in IgGl) to 230 (P230 in IgGl), according to EU numbering. In some embodiments, the first and second hinge domains are each a variant hinge domain, which includes one or more fewer amino acids at either the N-terminus, the C-terminus, or both termini of the hinge domain. In some embodiments, the first and second hinge domains each include one or more amino acid modifications (e.g., substitutions, deletions, and insertions). In some embodiments, at least one cysteine residue of the hinge domain is substituted with a serine residue. The first and second hinge domains can include a modified hinge described in US 2024 / 0383990, the disclosure is herein incorporated by reference.Flexible linkers

[0146] Exemplary flexible linkers are used to attach relevant domains such as VHH domains, Fab domains, Fc domains, scFv domains, VH domains, VL domains, etc., of the antigen binding polypeptides (such as antibodies, antibody fragments thereof, and T cell-engaging molecules) described herein. In some embodiments, a flexible linker is a polypeptide linker. In some embodiments, the polypeptide linker may predominantly include one or more of the following amino acid residues: Gly, Ser, Ala, or Thr. A polypeptide linker should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another and they retain the desired activity. In some embodiments, the polypeptide linker has a length from about 1-50 amino acids, about 1-40 amino acids, about 1-30 amino acids, about 1-20 amino acids, about 1-10 amino acids, about 12-20 amino acids, about 12-50 amino acids, and 544130-6004-4904.10Attorney Docket No. 55171.4010 / WOthe like. Exemplary useful polypeptide linkers include glycine-serine containing peptides, glycinealanine containing peptides, alanine-serine containing peptides, and other flexible linkers known in the art. Non-limiting examples of flexible polypeptide linkers are shown in the sequence listing including SEQ ID NOS: 32-40. In some embodiments, a flexible linker is a non-protein containing polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers. In some embodiments, a flexible linker comprises the sequence (GS)n, where n is at least 1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, a flexible linker comprises one or more repeating amino acid sequence including any one of SEQ ID NOS: 36-39. In some embodiments, a flexible linker comprises one or more repeating amino acid sequence of SEQ ID NO: 35, such that there are 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) consecutive sequences of SEQ ID NO: 35. In some embodiments, a flexible linker comprises one or more repeating amino acid sequence of SEQ ID NO: 36, such that there are 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) consecutive sequences of SEQ ID NO: 36. In some embodiments, a flexible linker comprises one or more repeating amino acid sequence of SEQ ID NO: 37, such that there are 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) consecutive sequences of SEQ ID NO: 37. In some embodiments, a flexible linker comprises one or more repeating amino acid sequence of SEQ ID NO: 38, such that there are 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) consecutive sequences of SEQ ID NO: 38. In some embodiments, a flexible linker comprises one or more repeating amino acid sequence of SEQ ID NO: 39, such that there are 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) consecutive sequences of SEQ ID NO: 39. In some embodiments, a flexible linker comprises SEQ ID NO: 40.Exemplary CEACAM6 targeting T cell-engaging molecules

[0147] Provided herein are CEACAM6 targeting T cell-engaging molecules of various formats including: (a) a 2+1 asymmetric VHH2-Fab-Fc, (b) a 2+2 symmetric VHH2-Fab2-Fc, 1+1 Fab-VHH-Fc, and (d) scFv-VHH. In some embodiments, the T cell engager molecule includes one or more VHH domains that each bind CEACAM6 and a Fab domain that binds CD3. In some embodiments, the T cell-engaging molecule includes one or more VHH domains that each bind CEACAM6 and a scFv domain that binds CD3.

[0148] In some embodiments, the one or more VHH domains targeting CEACAM6 includes an amino acid sequence set forth in any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96,554130-6004-4904.10Attorney Docket No. 55171.4010 / WO103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192. In some embodiments, the VHH domain targeting CEACAM6 has at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to an amino acid sequence set forth in any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, and 160-166.

[0149] In some embodiments, the VHH domain targeting CEACAM6 has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications in an amino acid sequence set forth in any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192. In some embodiments, the amino acid modifications are in the CDRs, the FRs, or both the CDRs and FRs of the VHH domain. In some embodiments, the amino acid modifications are in a set of CDRs of the VHH domain comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 41-43, HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 44-46, or HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 47-49. In some embodiments, the amino acid modifications are in any one of the CDRs set forth in SEQ ID NOS: 41-43. In some embodiments, the amino acid modifications are in any one, or even one or more, of the CDRs set forth in SEQ ID NOS: 44-46. In some embodiments, the amino acid modifications are in any one, or even one or more, of the CDRs set forth in SEQ ID NOS: 47-49. In some embodiments, the amino acid modifications are in any one, or even one or more, of the CDRs of the VHH domains set forth in SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

[0150] In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth SEQ ID NO: 15 and a VL domain having at least 90% e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having an amino acid sequence set forth in SEQ ID NO: 15 and a VL domain having an amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, a CH domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, a VL domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 564130-6004-4904.10Attorney Docket No. 55171.4010 / WO99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 17, a CL domain having at least 90% e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having an amino acid sequence set forth in SEQ ID NO: 15, a CHI domain having an amino acid sequence set forth in SEQ ID NO: 16, a VL domain having an amino acid sequence set forth in SEQ ID NO: 17, a CL domain having an amino acid sequence set forth in SEQ ID NO: 18.

[0151] In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth SEQ ID NO: 28 and a VL domain having at least 90% e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having an amino acid sequence set forth in SEQ ID NO: 28 and a VL domain having an amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 28, a CHI domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 29, a VL domain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 30, a CL domain having at least 90% e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the scFv or Fab domain targeting CD3 includes a VH domain having an amino acid sequence set forth in SEQ ID NO: 28, a CHI domain having an amino acid sequence set forth in SEQ ID NO: 29, a VL domain having an amino acid sequence set forth in SEQ ID NO: 30, a CL domain having an amino acid sequence set forth in SEQ ID NO: 31.

[0152] In one aspect, provided herein are CEACAM6 targeting T cell-engaging molecules, in particular, an anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format (see FIG.32) comprising: (a) a first monomer (a first polypeptide chain), (b) a second monomer (a second polypeptide chain), and a light chain. The first monomer comprises, from N-terminal to C-terminal: a first VHH domain, an optional flexible linker, a first hinge domain, a first heavy chain 574130-6004-4904.10Attorney Docket No. 55171.4010 / WOconstant 2 (CH2) domain, and a first heavy chain constant 3 (CH3) domain. The second monomer comprises, from N-terminal to C-terminal: a second VHH domain, an optional flexible linker, a heavy chain variable (VH) domain, a constant heavy 1 (CHI) domain, an optional flexible linker, a hinge domain, a second CH2 domain, and a second CH3 domain. The light chain comprises from N-terminal to C-terminal: a light chain variable (VL) domain and a light chain constant (CL) domain. The VH domain of the first monomer and the VL domain of the light chain form a Fab molecule which is also the CD3 antigen binding domain. The Fab molecule is fused at the C-terminus of the second VHH domain. The first VHH domain binds a CEACAM6 protein such as a human CEACAM6 protein (SEQ ID NO: 59) or a portion thereof. In some embodiments, the first VHH domain binds a CEACAM6 protein such as the extracellular domain (e.g., extracellular N-terminal IgV-like domain) of human CEACAM6 protein (SEQ ID NO: 58) or a portion thereof. The second VHH domain binds a CEACAM6 protein. The first CH2 domain and first CH3 domain form a first Fc domain. The second CH2 domain and second CH3 domain form a second Fc domain. The first VHH is fused to the N-terminus of the hinge domain of the first Fc domain. The second VHH is fused to the N-terminus of the Fab heavy chain of the CD3 antigen binding domain. Any of the VHH domains described herein, any of the VH and VL domains described herein, any of the Fc domains and variant Fc domains described herein, and any of the flexible linkers described herein can be included in an anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format.

[0153] In some embodiments, the CEACAM6 targeting T cell-engaging molecule or anti-CEACAM6 x anti-CD3 antibody is a bispecific antibody. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody is a bispecific, trivalent antibody. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody includes two CEAC AM6 antigen binding domains and one CD3 antigen binding domain.

[0154] In some embodiments, the CEACAM6 targeting T cell-engaging molecule (the anti-CEACAM6 x anti-CD3 antibody) having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain (monomer 1) of SEQ ID NO: 1, a second polypeptide chain (monomer 2) of SEQ ID NO: 2, and a third polypeptide chain (monomer 3) of SEQ ID NO: 3. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 4, a second polypeptide chain of SEQ ID NO: 5, and a third polypeptide chain of SEQ ID NO: 6. In some embodiments, the anti-CEACAM6 x 584130-6004-4904.10Attorney Docket No. 55171.4010 / WOanti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 7, a second polypeptide chain of SEQ ID NO: 8, and a third polypeptide chain of SEQ ID NO: 9. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 63, a second polypeptide chain of SEQ ID NO: 64, and a third polypeptide chain of SEQ ID NO: 65. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 69, a second polypeptide chain of SEQ ID NO: 70, and a third polypeptide chain of SEQ ID NO: 71. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 75, a second polypeptide chain of SEQ ID NO: 76, and a third polypeptide chain of SEQ ID NO: 77. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 81, a second polypeptide chain of SEQ ID NO: 82, and a third polypeptide chain of SEQ ID NO: 83. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 87, a second polypeptide chain of SEQ ID NO: 88, and a third polypeptide chain of SEQ ID NO: 89. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 93, a second polypeptide chain of SEQ ID NO: 94, and a third polypeptide chain of SEQ ID NO: 95. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 99, a second polypeptide chain of SEQ ID NO: 100, and a third polypeptide chain of SEQ ID NO: 101. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 105, a second polypeptide chain of SEQ ID NO: 106, and a third polypeptide chain of SEQ ID NO: 107. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 111, a second polypeptide chain of SEQ ID NO: 112, and a third polypeptide chain of SEQ ID NO: 113. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 117, a second polypeptide chain of SEQ ID NO: 118, and a third polypeptide chain of SEQ ID NO: 119. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 123, a second polypeptide chain of SEQ ID NO: 124, and a third polypeptide chain of SEQ594130-6004-4904.10Attorney Docket No. 55171.4010 / WOID NO: 125. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 129, a second polypeptide chain of SEQ ID NO: 130, and a third polypeptide chain of SEQ ID NO: 131. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 135, a second polypeptide chain of SEQ ID NO: 136, and a third polypeptide chain of SEQ ID NO: 137. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 141, a second polypeptide chain of SEQ ID NO: 142, and a third polypeptide chain of SEQ ID NO: 143. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 147, a second polypeptide chain of SEQ ID NO: 148, and a third polypeptide chain of SEQ ID NO: 149. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 183, a second polypeptide chain of SEQ ID NO: 184, and a third polypeptide chain of SEQ ID NO: 185. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 189, a second polypeptide chain of SEQ ID NO: 190, and a third polypeptide chain of SEQ ID NO: 191. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain of SEQ ID NO: 195, a second polypeptide chain of SEQ ID NO: 196, and a third polypeptide chain of SEQ ID NO: 197.

[0155] In some embodiments, the CEACAM6 targeting T cell-engaging molecule (the anti-CEACAM6 x anti-CD3 antibody) having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 1, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 2, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 3. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 1-3 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,604130-6004-4904.10Attorney Docket No. 55171.4010 / WOsequence identity to SEQ ID NO: 4, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 5, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 6. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 7, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 8, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 9. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 63, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 64, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 65. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 69, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 70, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 71. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 75, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 76, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 77. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 81, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence614130-6004-4904.10Attorney Docket No. 55171.4010 / WOidentity to SEQ ID NO: 82, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 83. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 87, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 88, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 89. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 93, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 94, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 95. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 99, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 100, and a third polypeptide chain having at least 90%, e g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 101. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 105, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 106, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 107. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 111, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 112, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,624130-6004-4904.10Attorney Docket No. 55171.4010 / WOsequence identity to SEQ ID NO: 113. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 117, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 118, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 119. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 123, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 124, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 125. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 129, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 130, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 131. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 135, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 136, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 137. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 141, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 142, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 143. In some embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format634130-6004-4904.10Attorney Docket No. 55171.4010 / WOcomprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 147, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 148, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 149. In other embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 183, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 184, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 185. In other embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 189, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 190, and a third polypeptide chain having at least 90%, e g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 191. In certain embodiments, the anti-CEACAM6 x anti-CD3 antibody having a 2+1 VHH2-Fab-Fc format comprises a first polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 195, a second polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 196, and a third polypeptide chain having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO: 197.

[0156] In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 4-6 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 7-8 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 24-26 has substantially 644130-6004-4904.10Attorney Docket No. 55171.4010 / WOequivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In other embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 63-65 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 69-71 has substantially equivalent binding affinity to CEAC AM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 75-77 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 81-83 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 87-89 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 99-101 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 105-107 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In other embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 111-113 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 117-119 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 123-125 has substantially equivalent binding affinity to CEACAM6 and CD3 as654130-6004-4904.10Attorney Docket No. 55171.4010 / WOthe corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 129-131 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 135-137 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 141-143 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications. In some embodiments, CEACAM6 targeting TCE molecule containing amino acid modifications to one or more amino acid sequences of SEQ ID NOS: 147-149 has substantially equivalent binding affinity to CEACAM6 and CD3 as the corresponding CEACAM6 TCE without the modifications.

[0157] In some embodiments, the CEACAM6 targeting T cell-engaging molecule includes a low affinity and high avidity CEACAM6 antigen binding domain. In some embodiments, the CEACAM6 targeting T cell-engaging molecule includes a CEACAM6 antigen binding domain that is pH selective in which the antigen binding domain binds to CEACAM6 preferentially at an acidic pH as compared to a neutral pH. In some embodiments, the CD3 targeting T cell-engaging molecule includes a CD3 antigen binding domain that is pH selective.

[0158] In some embodiments, a high affinity CEACAM6 targeting T cell-engaging molecule having a 2+1 VHH2-Fab-Fc format and including high affinity anti-CEACAM6 VHH domains comprises a first polypeptide chain of SEQ ID NO: 24, a second polypeptide chain of SEQ ID NO: 25, and a third polypeptide chain of SEQ ID NO: 26.

[0159] In one aspect, provided herein are CEACAM6 targeting T cell-engaging molecules, in particular, an anti-CEACAM6 x anti-CD3 antibody has a 2+2 VHH2-Fab2-Fc format comprising: (a) a first monomer (first polypeptide chain), (b) a second monomer (second polypeptide chain), (c) a first light chain, and (d) a second light chain. The first monomer comprises, from N-terminal to C-terminal: a first VHH domain, an optional flexible linker, a first heavy chain variable (VH) domain, a first heavy chain constant 1 (CHI) domain, an optional flexible linker, a first hinge domain, a first heavy chain constant 2 (CH2) domain, and a first heavy chain constant 3 (CH3)664130-6004-4904.10Attorney Docket No. 55171.4010 / WOdomain. The second monomer comprises, from N-terminal to C-terminal: a second VHH domain, an optional flexible linker, a second VH domain, a second CHI domain, an optional flexible linker, a second hinge domain, a second CH2 domain, and a second CH3 domain. The first light chain comprises from N-terminal to C-terminal: a first light chain variable (VL) domain and a first light chain constant (CL) domain. The second light chain comprises from N-terminal to C-terminal: a second VL domain and a second CL domain. The VH domain of the first monomer and the VL domain of the first light chain form a Fab molecule which is also the CD3 antigen binding domain. The VH domain of the second monomer and the VL of the second light chain form another Fab molecule which is also another CD3 antigen binding domain. The first VHH domain binds CEACAM6. The second VHH domain binds CEACAM6. The first CH2 domain and first CH3 domain form a first Fc domain. The second CH2 domain and second CH3 domain form a second Fc domain. Any of the VHH domains described herein, any of the VH and VL domains described herein, any of the Fc domains and variant Fc domains described herein, and any of the flexible linkers described herein can be included in an anti-CEACAM6 x anti-CD3 antibody having a 2+2 VHH2-Fab2-Fc format.

[0160] In another aspect, provided herein is a CEACAM6 x CD3 antigen binding polypeptide has an scFv-VHH format comprising from N-terminus to C-terminus: a CD3 targeting scFv, a flexible linker, and an anti-CEACAM6 VHH domain. In other embodiments, a scFv-VHH format comprises from N-terminus to C-terminus: an anti-CEACAM6 VHH domain, a flexible linker, and a CD3 targeting scFv. In some embodiments, a scFv-VHH format comprises: one or more anti-CEACAM6 VHH domains, one or more flexible linkers, and one or more CD3 targeting scFvs. In some embodiments, an scFv-VHH format comprises from N-terminus to C-terminus: a CD3 targeting scFv, a flexible linker, and one or more anti-CEACAM6 VHH domains where the VHH domains are fused sequentially. In some embodiments, an scFv-VHH format comprises from N-terminus to C-terminus: a CD3 targeting scFv, a flexible linker, a first anti-CEACAM6 VHH domain, a flexible linker, and a second anti-CEACAM6 VHH domain where the flexible linker between the VHH domains allows each of the VHH domains to bind a CEACAM6 protein. In some embodiments, an scFv-VHH format comprises from N-terminus to C-terminus: a first anti-CEACAM6 VHH domain, a flexible linker, a second anti-CEACAM6 VHH domain, a flexible linker, a CD3 targeting scFv, where the flexible linker between the VHH domains allows each of the VHH domains to bind a CEACAM6 protein. The CD3 targeting scFv can be a pH selective 674130-6004-4904.10Attorney Docket No. 55171.4010 / WOCD3 scFv. Tn some embodiments, VH and VL pair of the CD3 targeting scFv can be any VH domain sequence and any VL domain sequence disclosed herein including in the tables. The anti-CEACAM6 VHH domain can be a pH selective VHH domain. In some embodiments, the VHH domain can be any VHH sequence disclosed herein including in the tables. Any of the VHH domains described herein, any of the VH and VL domains described herein, and any of the flexible linkers described herein can be included in a CEACAM6 x CD3 antigen binding polypeptide having an scFv-VHH format.

[0161] In another aspect, provided herein is a monomeric VHH-Fc antibody that includes a single domain variable region of the heavy chain (VHH) that binds CEACAM6 and is fused directly or indirectly to an Fc domain of an IgG molecule. Tn some embodiments, a monomeric VHH-Fc antibody includes from the N-terminus to the C-terminus: an anti-CEACAM6 VHH domain, an optional peptide linker, a hinge domain, and a CH2-CH3 domain of an IgG molecule such as a human IgGl molecule. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises any anti-CEAC AM6 VHH domain described herein including an amino acid sequence set forth in any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises the amino acid sequence of SEQ ID NO: 13 wherein Xi is N or Y, X2 is H or A, X3 is M or S. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In some embodiments, the anti-CEACAM6 VHH-Fc antibody includes a set of CDRs of the VHH domain comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 41-43, HCDR1, HCDR2, and HCDR3684130-6004-4904.10Attorney Docket No. 55171.4010 / WOas set forth in SEQ ID NOS: 44-46, or HCDR1 , HCDR2, and HCDR3 as set forth in SEQ ID NOS: 47-49.

[0162] In other embodiments, the anti-CEACAM6 VHH-Fc antibody comprises the amino acid sequence of SEQ ID NO: 27. In other embodiments, the anti-CEACAM6 VHH-Fc antibody comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 27. Any of the VHH domains described herein and any of the Fc domains described can be included in a monomeric VHH-Fc antibody.

[0163] In another aspect, provided herein is a VHH-Fc dimer antibody that includes two monomers (2 polypeptide chains), where each monomer contains a VHH that binds CEACAM6 that is fused directly or indirectly to an Fc domain of an IgG molecule. In some embodiments, each monomer includes from the N-terminus to the C-terminus: an anti-CEACAM6 VHH domain, an optional peptide linker, a hinge domain, and a CH2-CH3 domain of an IgG molecule such as a human IgGl molecule. In some embodiments, each monomer includes from the N-terminus to the C-terminus: a hinge domain, and a CH2-CH3 domain of an IgG molecule such as a human IgGl molecule, an optional peptide linker, and an anti-CEACAM6 VHH domain.

[0164] In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises any anti-CEACAM6 VHH domain described herein including an amino acid sequence set forth in any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 103, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192. In some embodiments, each monomer contains a VHH domain having the same amino acid sequence. In some embodiments, each monomer contains a VHH domain having different amino acid sequences. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing the amino acid sequence of SEQ ID NO: 13 wherein Xi is N or Y, X2 is H or A, X3 is M or S. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing an amino acid sequence having at least 90%, e.g., 90%, 91%,694130-6004-4904.10Attorney Docket No. 55171.4010 / WO92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing the amino acid sequence of SEQ ID NO: 27. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises a VHH domain containing an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 27. In some embodiments, the anti-CEACAM6 VHH-Fc antibody comprises an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In some embodiments, the anti-CEACAM6 VHH-Fc antibody includes a set of CDRs of the VHH domain comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 41-43, HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 44-46, or HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 47-49.

[0165] In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 13 wherein Xi is N or Y, X2 is H or A, X3 is M or S. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing an amino acid sequence having at least 90%, e g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the 704130-6004-4904.10Attorney Docket No. 55171.4010 / WOanti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a set of CDRs in the VHH domain comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 41-43, HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 44-46, or HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 47-49. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 27. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, each monomer includes a VHH domain containing an amino acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 27.

[0166] In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 13 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 13 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 13 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 12. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 13 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 10 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 10 and a second monomer includes a VHH domain containing the amino acid 714130-6004-4904.10Attorney Docket No. 55171.4010 / WOsequence of SEQ ID NO: 12. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 10 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 11 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 12. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 11 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 27. In other embodiments, the anti-CEACAM6 VHH-Fc dimer antibody comprises two monomers, where a first monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 12 and a second monomer includes a VHH domain containing the amino acid sequence of SEQ ID NO: 27. In some embodiments, the two Fc domains of the anti-CEACAM6 VHH-Fc dimer antibody heterodimerize through knob-in-hole pairing via protuberances (“knobs”) and cavities (“holes”) engineered into the interface of CH3 domains. In some embodiments, one Fc domain fused to a VHH contains a knob, and the other Fc domain fused to a VHH contains a hole. Detailed descriptions of knob-in-hole modifications of Fc regions can be found in, for example, U.S. Pat. No. 5,821,333; Ridgway et al., Protein Engineering 9:617-621 (1996); and Carter P., J. Immunol. Methods 248: 7-15 (2001). Any of the VHH domains described herein and any of the Fc domains described can be included in a VHH-Fc dimer antibody.Nucleic Acids

[0167] Provided herein are nucleic acid compositions encoding the anti-CEACAM6 x anti-CD3 antibodies provided herein. A nucleic acid composition may refer to one or multiple polynucleotides.

[0168] The nucleic acid compositions will depend on the format antibodies, antibody fragments, and T cell-engaging molecules. Thus, for example, when the format requires three amino acid sequences, such as for the 2+1 VHH2-Fab2-Fc format, three polynucleotides can be incorporated into one or more expression vectors for expression. In some embodiments, each polynucleotide is incorporated into a different expression vector.724130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0169] As is known in the art, the nucleic acids encoding the components of the antigen binding domains and antibodies disclosed herein can be incorporated into expression vectors as is known in the art, and depending on the host cells used to produce the antibodies of the invention. Generally, the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.). In some embodiments, polynucleotides encoding each monomer (each polypeptide chain) are each contained within a single expression vector, generally under different or the same promoter controls. In some embodiments, each of these polynucleotides are contained on different expression vectors.

[0170] The polynucleotides and / or expression vectors are then transformed into any number of different types of host cells as is well known in the art, including mammalian, bacterial, yeast, insect and / or fungal cells, with mammalian cells (e.g., CHO cells), finding use in many embodiments.Functionality of T cell-engaging molecules

[0171] The T cell-engaging molecules described herein are administered to patients with a CEACAM6-associated cancer, and efficacy is assessed, as recognized by those in the art and in a way as described herein such as in the Examples. Standard assays of efficacy can be performed, such as cancer load, size of tumor, evaluation of presence or extent of metastasis, etc., and also immuno-oncology therapies can be assessed on the basis of immune status evaluations. This can be performed in a number of ways, including both in vitro and in vivo assays. In some instances, biological activities of the T cell-engaging molecules may, for example, include the induction of proliferation of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine secretion by T cells, the induction of lysis of target cells such as tumor cells, and the induction of tumor regression and / or the improvement of survival.Administering Treatments

[0172] Provided herein are compositions of any of the antibodies, antibody fragments, and T cellengaging molecules that can be used in a number of immuno-oncology applications, by treating cancer, generally by enhancing immune responses (e.g., T cell activation and proliferation), particularly when used with anti-cancer therapies such as anti-tumor bispecific antibodies. In some734130-6004-4904.10Attorney Docket No. 55171.4010 / WOembodiments, the antibodies, antibody fragments, and T cell-engaging molecules provided herein enhance immune responses (e.g., T cell activation and proliferation) by providing co-stimulation of T cells in the microenvironment of tumors expressing CEACAM6. The compositions can be formulated for administration to a subject, e.g., a human subject, in accordance with known methods used for immuno-oncology based therapies.

[0173] In some embodiments, the CEACAM6 targeting antibodies, antibody fragments, and T cell-engaging molecules are useful for treating a cancer, including pancreatic cancer, pancreatic adenocarcinoma, adenosquamous carcinomas, squamous cell carcinomas, signet ring cell carcinomas, acinar cell carcinomas, undifferentiated carcinomas, undifferentiated carcinomas with giant cells, ampullary cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma, large cell neuroendocrine carcinoma (LCNEC), adenosquamous carcinoma, sarcomatoid carcinoma, small cell lung cancer (SCLC), lung carcinoid tumor, adenoid cystic carcinomas, adenoid cystic lymphoma, adenoid cystic sarcoma, colorectal cancer, colon cancer, rectal cancer, colorectal adenocarcinoma, colorectal polyp, primary colorectal lymphoma, gastrointestinal stromal tumor, colon and rectal leiomyosarcoma, carcinoid tumor, colon and rectal melanoma, colorectal squamous cell carcinoma, familial adenomatous polyposis, breast cancer, ductal carcinoma in situ (DCIS), invasive or infdtrating breast cancer, invasive ductal carcinoma, invasive lobular carcinoma, triple-negative breast cancer, angiosarcoma of the breast, inflammatory breast cancer, Paget disease of the breast, and phyllodes tumor.ExamplesExample 1. Discovery and characterization of CEACAM6 targeting T cell-engaging molecule A-000-010-3936

[0174] Described herein are potent, and differentiated anti-CEACAM6 T-cell engaging bispecific antibodies including A-000-010-3936. The example summarizes the computational, in vitro, and in vivo data of A-000-010-3936. A-000-010-3936 is a potent and differentiated anti-CEACAM6 T-cell engaging bispecific antibody. The antibody was shown to possess robust target engagement with in vitro and in vivo potency in culture-adapted cell-lines and models consistent with published clinical-stage TCEs. The antibody also showed minimal on-target engagement of healthy744130-6004-4904.10Attorney Docket No. 55171.4010 / WOexpressing cells as modeled in vitro. Moreover, the T-cell engaging bispecific antibody has excellent manufacturing properties from transient mammalian expression systems. A surrogate TCE A-000-017-2633 with similar biophysical properties and in vitro activity to the human test candidate was characterized in a non-human primate tolerability study with a clear demonstration of a reasonable therapeutic window between the dose leading to substantial anti-tumor response and that causing on-target toxicity.Methods and MaterialsTable 2. Molecular DefinitionsTest TCE molecule DescriptionidentifierA-000-010-3936 Bispecific, 2+1 T-cell engager with anti-CEACAM6 VHH and anti- CD3 Fab binding domainsA-000-005-2841 Bispecific, 2+1 T-cell engager with high affinity anti-CEACAM6 VHH (2A3 clone) (SEQ ID NO: 192) and anti-CD3 Fab binding domains (SEQ IDNOS: 193-194)A-000-019-8982 Isotype control (Pali + CD3 Fab) “2+1” T-cell engagerTable 3. Cell LinesCell Line Source Catalog Number CEACAM6 Receptor Density ABC (per cell) AsPCl (PDAC) ATCC CRL-1682 >315,000BxPC3 (PDAC) ATCC CRL-1687 >315,000 NCIH2073 (NSCLC) ATCC CRL-5918 >315,000 NCIH2170 (NSCLC) ATCC CRL-5928 >315,000SNUC1 (CRC) ATCC CRL-5972 117,800GP2d (CRC) Sigma 95090714 46,600MCF7 (BC) ATCC HTB-22 23,000A549 (NSCLC) ATCC CRL-2159 13,700CHO-hCEACAM6 BigHat n.a. 120,000754130-6004-4904.10Attorney Docket No. 55171.4010 / WOCHO-cyCEACAM6 BigHat n.a. 120,000Discovery of T cell engagers

[0175] The anti-CEACAM6 VHH in A-000-010-3936 (SEQ ID NOS: 4-6) was identified from in vitro phage display selections on a synthetic library based on the human IGHV3-23*01 germline VH gene. Briefly, three rounds of phage display selections were carried out against recombinant human CEACAM6 antigen (Aero CE6-H82E7). For round 1, 1013phage particles were diluted into PBS + 3% (w / v) milk powder at pH 6 and blocked for 30 minutes. Next, biotinylated CEACAM6 was added to the blocked phage to a final concentration of 1 pM and incubated for 1 hour. After 1 hour, 40 pL of streptavidin speed beads were added to the phage / CEACAM6 mixture and incubated for 30 minutes to pull down all biotinylated CEACAM6 and any phage bound. Beads were then separated using a magnet, and the beads were subsequently washed 4 times with PBS 6.0 + 0.1% Tween-20 (PBST) and 4 washes of PBS pH 6.0. After washing, 250 pL of PBS pH 7.4 was added and incubated for 30 minutes to elute pH conditional VHHs. The eluted phage were added to TGI cells, allowed to infect for 1 hour, then plated on carbenicillin containing agar plates and incubated overnight at 30°C. The next day, agar plates were scraped and glycerol stocks were made. To prepare phage for Round 2, 50 pL of the glycerol stock from Round 1 was added to 20 mL of 2xYT media containing 2% glucose and carbenicillin and grown until mid-log phase. Helper phage were added at an MOI of 10 to the bacteria and infected for 1 hour. The bacteria were then pelleted and resuspended in 2xYT media containing carbenicillin and kanamycin and grown overnight at 30°C. The next day, bacteria were pelleted and the phage containing supernatant was used for Round 2 of selection. 100 pL of 5x PBS pH 6 and 15% milk was added to 400 pL of phage and incubated for 30 minutes. During this time, biotinylated CEACAM6 was added to neutravidin coated plates at 200 nM and incubated for 30 minutes. Phage were then added to the CEACAM6 containing plate and incubated for 1 hour. Plates were washed 10 times with PBST, pH 6 and 10 times with PBS pH 6. 200 pL of PBS, pH 7.4 were added to each well and incubated for 30 minutes to elute phage. The eluted phage were then added to TGI cells and subsequently plated on 2xYT agar plates with carbenicillin and grown overnight at 30°C. This process was repeated for Round 3, but the concentration of CEACAM6 was lowered to 50 nM. After the final round of selection, single colonies were picked and VHH-encoding genes were sequenced by Sanger sequencing. Antibodies were subsequently expressed and characterized.764130-6004-4904.10Attorney Docket No. 55171.4010 / WOAntibody Optimization

[0176] Initially, sequential variants of the discovered VHH were generated and tested via large language models. After sufficient training data consisting of variant VHHs with measured true properties were accumulated, predictive models were trained on thermostability (Tm) and affinity (KD) data. These models were used to predict mutations that would further optimize affinity while preserving or enhancing thermostability (Tm) and improving drug-like properties such as polyreactivity and hydrophobicity. In each design cycle, the top ranked variants based on predicted improved properties and model prediction uncertainty were then selected for synthesis and characterized. This process was repeated to iteratively optimize the discovered VHH into an early test candidate.Structure-based analysis of sequence liabilities

[0177] Sequence-based post-translational modification liabilities were determined using the Liability Antibody Profiler tool (see, for example, Satlawa et al., PLoS Comput Bio, 2024, 20(3):el011881). Sequence motifs for the following liabilities were evaluated based on presence in the human germline, presence in clinical stage therapeutics, and solvent exposure: oxidation (Met and Trp), Asp isomerization (canonical and non-canonical), Asn deamidation (canonical and non-canonical), and N-linked glycosylation. The 9% threshold criteria for surface exposure was based on prior literature (Yang et al., 2017, mAbs, 9(4): 646-653). CDRs were annotated using the Kabat numbering scheme.Immunogenicity predictionsComparison to germlines and clinical stage antibodies.

[0178] The sequence was split into all possible 9-mers. The 9-mer was chosen as the core binding peptide to MHC-II and is 9 amino acids long. Each 9-mer was screened for 100% identity within the sequence of human germlines (IMGT.org) as well as marketed, under review and clinical stage antibodies passed phase 1 clinical trials (Jain et al., 2017, Proc Natl Acad Sci USA, 114(5): 944-949) with publicly available sequences. The dataset was curated to remove antibodies with the known presence of anti-drug antibodies (ADA) in patients (Mosch and Guchelaar, 2022, Front Immunol, 13: 885672), resulting in a total number of 184 antibodies. Sequence regions that were identical to either germline or this set of clinical-stage antibodies were considered “de-risked”, irrespective of their humanness or MHC-II binding predictions.774130-6004-4904.10Attorney Docket No. 55171.4010 / WOMHC-II binding predictions.

[0179] The sequence was split into 15-mer peptides, for each peptide a binding score to each of the 27 most common MHC-II alleles in the general population (covering an estimated 98% of major ethnicities worldwide, Greenbaum et al., 2011, Immunogenetics, 63(6): 325-35) were computed using the IEDB (IEDB.org) NetMHCIIpan 4.1 EL prediction method (Nielsen et al., 2010, Immunology, 130(3): 319-328). Peptides among the top 5, 2, and 1 - ranked binders were considered as binders, meaning that peptides are among the top 5, 2, and 1% of binders, respectively, when compared to a very large set of peptides. A total number of alleles with predicted ranks within those thresholds were plotted for each peptide.Humanness assessment.

[0180] Humanness was assessed by percent germline content as well as by the OASis humanness metric. The OASis humanness metric was described by Prihoda et al., 2022, Mabs, 14(1): 2020203 and was shown to be superior to all but one other published humanness metric in both its ability to predict ADA presentation rates in human patients, as well as its ability to discriminate between human, humanized, chimeric and mouse antibodies (Prihoda et al., 2022). The OASis score works by breaking the VH and VL sequences down into 9-mers, and comparing each of these to a large (>130M sequence) database of human BCR sequences (the Observed Antibody Space, OAS). It then calculates the fraction of 9-mers that were found in at least 50% of human subjects. Finally, this 9-mer prevalence is compared to a database of 544 therapeutic mAbs from the IMGT mAb DB (Manso et al., 2022, Nucleic Acids Research, 50(Dl): D1262-D1272; https: / / www.imgt.org / IMGTindex / IMGTmAb-DB.php), and the “OASis percentile” is calculated, giving the percent of these therapeutic antibodies which are less human than the analyzed sequence (i.e., an OASis percentile of 75% means the antibody is more human than 75% of therapeutic abs in the IMGT mAb DB).Generative humanization / de-immunization

[0181] Antibodies were analyzed for in silico immunogenicity risk as noted above. Identified putative immunogenicity “hot spots” or predicted MHC-II epitopes were iteratively removed or mitigated from the VHH sequence by the generation of mutations specifically targeting these positions that were simultaneously predicted to preserve affinity and stability properties (Gordon & Raghu, “Generative Humanization for Therapeutic Antibodies,” published at the GEM workshop, ICLR 2024).784130-6004-4904.10Attorney Docket No. 55171.4010 / WOAffinity measurements by biolayer interferometry (BLT) and surface plasmon resonance (SPR)Screening measurements of VHHs by BLI

[0182] For optimization of CFPS-expressed VHHs, BLI-based kinetics assays designed against human and cynomolgus CEACAM6 were used.

[0183] To characterize binding against human CEACAM6, Fc-tagged human CEACAM6 (AcroBiosystems Cat# CE6-H5254) was reconstituted according to the manufacturer’s specification, diluted to 50 nM in lOx KB buffer (lx PBS with 0.1% BSA and 0.02% Tween-20, BigHat Biosciences), and captured to 1.2 nm on Anti-hlgG Fc Capture (AHC) biosensors (Sartorius Cat# 18-5064). Antibody fragments were used as analytes and tested at 50-500 nM. 10 mM glycine pH 1.5 was used to regenerate the biosensors between runs. The binding kinetics were measured on the Octet RH96 platform (Sartorius) using lOx KB as the assay buffer and a data acquisition rate of 0.6 Hz. The experiment was performed at 30 °C and the biosensors were prewetted for 10 min with 1000 rpm shaking.Table 4. Data acquisition stepsStep Time (s) Speed (rpm)Regeneration 30 1000Baseline 120 1000Capture Up to 1.2 nm 400Association 300 1000Dissociation 600 1000

[0184] To characterize binding against cynomolgus CECAM6, His-tagged cynomolgus CEACAM6 (internal) was diluted to 50 nM in lOx KB buffer (lx PBS with 0.02% BSA and 0.02% Tween-20, BigHat Biosciences), and captured to 0.5 nm on Ni-NTA biosensors (Sartorius Cat# 18-5103). Antibody fragments were used as analytes and tested as noted above. 500 mM EDTA was used to regenerate the biosensors between runs, and a Tris solution containing 20 mM NiCh794130-6004-4904.10Attorney Docket No. 55171.4010 / WOwas used to recharge the sensors. The binding kinetics were measured on the Octet RH96 platform (Sartorius) using lOx KB02 as the assay buffer and a data acquisition rate of 0.6 Hz. The experiment was performed at 30 °C and the biosensors were pre- wetted for 10 min with 1000 rpm shaking.Table 5. Data acquisition stepsStep Time (s) Speed (rpm) Regeneration 30 1000 Baseline 60 1000 Capture Up to 0.5 nm 400 Association 300 1000 Dissociation 600 1000

[0185] Antibody-antigen binding kinetics data were analyzed in Octet® Analysis Studio vl3 software (Sartorius). Reference subtraction was first performed with the no analyte control sample. Data correction was then applied to the traces by aligning the data to the average of the baseline step and performing an inter-step correction to the dissociation step. Savitzky-Golay Filtering was applied to remove high-frequency noise from the traces. A 1 : 1 Langmuir binding model was used to obtain kon, koff, and KD values for each antigen antibody binding interaction.Full characterization affinity measurements of TCEs by SPR

[0186] To measure affinity against human CEACAM6, an anti-human Fc capture antibody (Cytiva, 29234600) was immobilized onto a Series S Sensor Chip CM5. Fc-containing TCEs were diluted to 2 pg / mL in lx HBS-EP+ (150mM sodium chloride, lOmM HEPES free acid, 3mM EDTA, 0.05% Tween-20, pH 7.4) and subsequently captured onto a Series S Sensor Chip CM5 (Cytiva, 29104988). Anti -human CEACAM6 was diluted to lOpM in lx HBS-EP+ and used as analyte at 4 concentrations in % dilutions. 3M MgCh (Cytiva, 29234600) was used to regenerate the sensor chip between runs. Binding kinetics were measured at 25°C using lx HBS-EP+ as the running buffer on a Biacore 8k+ platform (Cytiva) by multi-cycle kinetics with a data acquisition rate of 10 Hz. Data acquisition steps are detailed in the table below.804130-6004-4904.10Attorney Docket No. 55171.4010 / WOTable 6. Data acquisition stepsStep Time (s) Flow Rate (pL / min) Antibody capture 60 10 Association 180 30 Dissociation 300 30 Regeneration 30 10

[0187] To measure affinity against cynomolgus CEACAM6, an anti-human Fc capture antibody (Cytiva, 29234600) was immobilized onto a Series S Sensor Chip CM5. Fc-containing TCEs were diluted to 2 pg / mL in lx HBS-EP+ (150mM sodium chloride, lOmM HEPES free acid, 3mM EDTA, 0.05% Tween-20, pH 7.4) and subsequently captured onto a Series S Sensor Chip CM5 (Cytiva, 29104988). Anti-cynomolgus CEACAM6 was diluted to lOpM in lx HBS-EP+ and used as analyte at 4 concentrations in 'A dilutions. 3M MgCL (Cytiva, 29234600) was used to regenerate the sensor chip between runs. Binding kinetics were measured at 25°C using lx HBS-EP+ as the running buffer on a Biacore 8k+ platform (Cytiva) by multi-cycle kinetics with a data acquisition rate of 10 Hz. Data acquisition steps are detailed in the table below.Table 7. Data acquisition stepsStep Time (s) Flow Rate (pL / min) Antibody capture 60 10 Association 180 30 Dissociation 300 30 Regeneration 30 10

[0188] Antibody-antigen binding kinetics data were analyzed using the Biacore Insight Evaluation Software v5.0 (Cytiva). Double reference subtraction was performed with the no analyte control sample and a no ligand control flow cell surface. A 1:1 Langmuir binding model was used on smoothed and aligned data globally fitted by the Levenberg-Marquardt algorithm to obtain kon, koff, and KD values for each antigen antibody binding interaction.814130-6004-4904.10Attorney Docket No. 55171.4010 / WOAvidity measurements of TCEs against human CEACAM6 by SPR

[0189] To measure avidity measurements of our TCE against human CEACAM6, a biotinylated human CEACAM6 antigen was diluted to 0.3 pg / mL in lx HBS-EP+ (150mM sodium chloride, lOmM HEPES free acid, 3mM EDTA, 0.05% Tween-20, pH 7.4) and captured on a Series S Sensor Chip CAP (Cytiva, 29805820) following immobilization of the CAPture reagent (Cytiva, 29423383). TCEs were diluted to lOOnM in lx HBS-EP+ and used as analyte at 6 concentrations in % dilutions. A 3:1 mixture of Regeneration Stock 1 and Regeneration Stock 2 from the Series S Biotin CAPture Kit (Cytiva, 28920234) was used to regenerate the sensor chip between runs. Binding kinetics were measured at 25°C using lx HBS-EP+ as the running buffer on a Biacore 8k+ platform (Cytiva) by multi-cycle kinetics with a data acquisition rate of 10 Hz. Data acquisition steps are detailed in the table below.Table 8. Data acquisition stepsStep Time (s) Flow Rate (pL / min)CAPture Reagent capture 120 2Ligand capture 300 10Association 100 30Dissociation 600 30Regeneration 120 10

[0190] Antibody-antigen binding kinetics data were analyzed using the Biacore Insight Evaluation Software v5.0 (Cytiva). Double reference subtraction was performed with the no analyte control sample and a no ligand control flow cell surface. A 2:1 bivalent analyte model was used on smoothed and aligned data globally fitted to obtain kon, koir, and KD values for each antigen antibody binding interaction.Antibody expressionCell-free expression and purification824130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0191] During antibody optimization, VHH single-domain antibodies were expressed via cell-free protein synthesis and purified by affinity chromatography using standard platform methods. Transient mammalian expression and purification

[0192] For functional characterization, VHH fragments were cloned into a T-cell engager scaffold with three chains. The first polypeptide chain (also referred to as monomer 1) features the VHH fused to the human IgGl hinge, modified to replace endogenous cysteine residues that typically pair with CL with a non-reactive serine residue, followed by the IgGl CH2 (LALAPG) and CH3 (T366S) sequences. The second polypeptide chain (also referred to as monomer 2) features the VHH fused to a (Gly4Ser)x2 linker followed by the VH-CH1 assembly for the anti-CD3 Fab and native IgGl hinge, CH2 (LALAPG) and CH3 (T366W) sequences. The third polypeptide chain (also referred to as monomer 3) consists of the VL-CL fusion for the CD3 Fab.

[0193] The following steps were performed by our protein production contract research organization, BioIntron. Plasmids were transiently transfected and expressed at a IL scale in HEK293 cells using standard methods. Antibodies were purified from culture supernatants by CHI affinity chromatography followed by a CHT polishing step using standard binding, wash, and elution methods. Antibodies were then buffer exchanged into a standard formulation buffer (20mM Histidine-HCl, 150mM NaCl, pH 6.0). Post-purification, antibodies were assessed by analytical SEC and SDS-PAGE.Stable cell line expression and purification

[0194] Early development services were contracted to the contract development and manufacturing organization Lonza Group. Though more thoroughly detailed in the product report, the TCE was expressed with four different vector designs in stable pools using CHOK1SV GS-KO cells. Antibodies were purified from culture supernatants by CHI affinity chromatography followed by a semi-preparative polishing step using standard binding, wash, and elution methods. Post purification, antibodies were assessed by SEC, SDS-PAGE, and intact mass analysis.In vitro potencyTDCC Assay

[0195] TCEs were tested against various cell lines using a standard T-cell-dependent cellular cytotoxicity (TDCC) assay design. Briefly, CEACAM6+ target cells were seeded in white, opaque 96 well plates in complete RPMI (RPMI-1640 +10% FBS, 1% Glutamax, pH 7.4). Primary human834130-6004-4904.10Attorney Docket No. 55171.4010 / WOT-cells isolated from fresh leukopaks (AllCells) were added at a 10:1 ratio relative to the number of target cells in the well. The TCEs were serially diluted PBS and subsequently lOuL were incubated with cells for 72 hours, after which cells were lifted and analyzed by flow cytometry. Cells were stained with eFluor 450 viability dye, CD69-PE, CD3-APC-Cy7, and Tinurilimab (anti-CEACAM6)-APC reagents. Viable target cell counts were normalized to the untreated controls and plotted against drug concentration in PRISM (GraphPad). IC50s were derived from four parameter Hill function fits. A minimum of three biological replicates in technical duplicate were performed. T-cell activation (CD69+ cells) was similarly analyzed.Whole Blood TDCC

[0196] Whole blood was diluted 1 : 10 in RIO (RPMI-1640 +10% FBS, l% Glutamax, pH 7.4) and 90uL and were seeded in white, clear flat-bottom 96 well plates. The TCEs were serially diluted in PBS and subsequently lOuL were incubated with cells for 72 hours, after which cells were lifted and analyzed by flow cytometry. Cells were stained with CD69-PE, CD3-PerCP, CD1 lb-BV605, CD14-BV786, CDllc-APC, Tinurilimab (anti-CEACAM6)-FITC and 7AAD reagents. Viable cell counts were normalized to the untreated controls and plotted against drug concentration in PRISM (GraphPad). IC50s were derived from four parameter Hill function fits. A minimum of three biological replicates in technical duplicate were performed. T-cell activation (CD69+ cells) was similarly analyzed.PBMC TDCC

[0197] 10e-5 PBMCs in 90uL R10 (RPMI-1640 +10% FBS, 1% Glutamax, pH 7.4) were seeded in white, opaque 96 well plates. The TCEs were serially diluted in PBS and subsequently lOuL were incubated with cells for 72 hours, after which cells were lifted and analyzed by flow cytometry. Cells were stained with eFluor450 viability dye, CD69-PE, CD3-APC-Cy7, CDllb-BV605, CD14-BV786, CDllc-APC, Tinurilimab (anti-CEACAM6)-APC reagents. Viable cell counts were normalized to the untreated controls and plotted against drug concentration in PRISM (GraphPad). IC50s were derived from four parameter Hill function fits. A minimum of three biological replicates in technical duplicate were performed. T-cell activation (CD69+ cells) was similarly analyzed.Cytokine Analysis844130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0198] Supernatants from TDCC and whole blood assays were tested for cytokine release using the V-PLEX Proinflammatory Panel 1 Human Kit from MSD (K15049D).Pharmacokinetic Analysis

[0199] Serial peripheral blood draws were taken from CDX-bearing (NCI-H2170) mice with i.p. human T cells. Streptavidin plates were coated in 1 ug / mL biotinylated anti-Fc capture (Capture Select) for 1 hour, washed and diluted plasma samples were added and incubated for 2 hours. Anti-fab-sulfo-tag detection reagent was added at 1 ug / mL and incubated for 1 hour. Plates were subsequently washed and analyzed on MSD instrument.Biophysical characterizationsAnalytical size exclusion chromatography (SEC)

[0200] Analytical SEC was performed using an Agilent 1260 Infinity II Prime System. The TCE was normalized to Img / mL in formulation buffer and 10 pg was injected onto a TSKgel SuperSW3000 250A column (Tosoh Biosciences, 0018675). The sample was passed for 15 minutes at a flow rate of 0.35 pL / min with a mobile phase of lOOmM sodium phosphate, 250mM sodium chloride, 0.02% sodium azide, pH 6.8. A protein standard mix (Waters, 186006518) was used both for quality control and as a reference for ensuring our TCE eluted at the expected retention time according to its expected molecular weight.Analytical hydrophobic interaction chromatography (HIC)

[0201] Analytical HIC was performed using an Agilent 1260 Infinity II Prime System. The TCE was normalized to Img / mL in formulation buffer and then diluted 1 : 1 with mobile phase A (1 ,9M ammonium sulfate, lOOmM sodium phosphate, pH 6.8). 5pg was injected onto a TSKgel Butyl-NPR 250A column (Tosoh Biosciences, 0014947). The sample was passed for 12 minutes at a flow rate of 0.8 pL / min over a gradient from 60% mobile phase A to 100% mobile phase B (lOOmM sodium phosphate, pH 6.8). An internally developed protein standard mix (Adalimumab, Briakinumab, Rituximab, and Ixekizumab) containing commercially available monoclonal antibodies was used both for quality control and as a reference for understanding the relative hydrophobic character of our TCE.Thermostability

[0202] For melting temperature (Tm) and aggregation temperature (Tagg) determination, purified antibody was normalized to 1 mg / mL in formulation buffer (20mM Histidine-HCl, 150mM NaCl854130-6004-4904.10Attorney Docket No. 55171.4010 / WOpH 6.0) and subject to a temperature gradient from 25°C to 95°C with a thermal ramp of 0.3°C / min. The sample was loaded in duplicate onto an UNcle (Unchained Labs) and measurements of intrinsic fluorescence and static light scattering (SLS) at 266nm were used to obtain Tm and Tagg calls, respectively (averaged from the duplicate loadings).Accelerated stability

[0203] To predict shelflife and evaluate the effect of temperature stress on our TCE in accordance with 1CH accelerated stability stress conditions, the TCE was normalized to Img / mL in formulation buffer (20mM Histidine-HCl, 150mM NaCl pH 6.0) and aliquotted into 150pL aliquots sufficient to generate pull points at 4°C, 25°C, and 40°C following incubation periods of 7 days, 14 days, 21 days, 3 months, 6 months, 9 months, 12 months, 15 months, and 18 months. Following incubation, aliquots were flash frozen and stored at -80°C until analysis. Reference material was generated in parallel with aliquots available for each of the incubation periods, flash frozen, and stored at -80°C until analysis.Forced degradation

[0204] For post-translational modification (PTM) liability stress testing, the TCE was normalized to Img / mL in formulation buffer (20mM Histidine-HCl, 150mM NaCl pH 6.0) and 12x 500pg aliquots. One aliquot was buffer exchanged back into formulation buffer to account for exposure to the buffer exchange columns, flash frozen and stored at -80°C for the duration of the stress studies as an unstressed control. The other aliquots were stressed with the following methods to explore specified liabilities.Isomerization Stress

[0205] To probe sensitivity to isomerization, one 500pg aliquot was buffer exchanged into 50mM sodium acetate pH 5.0 and one into the formulation buffer. Both aliquots were stressed at 40°C for 14 days. Once incubation was complete, aliquots were buffer exchanged back into formulation buffer, flash frozen and stored at -80°C until analysis.Deamidation Stress

[0206] To probe sensitivity to deamidation, one 500pg aliquot was buffer exchanged into 20mM Tris pH 8.5 and one into the formulation buffer. Both aliquots were stressed at 40°C for 7 days. Once incubation was complete, aliquots were buffer exchanged back into formulation buffer, flash frozen and stored at -80°C until analysis.864130-6004-4904.10Attorney Docket No. 55171.4010 / WOOxidation Stress

[0207] To probe sensitivity to oxidation of methionine residues, one 500pg aliquot was buffer exchanged into 20mM Histidine-HCl 150mM NaCl pH 6.0 containing 0.5% H2O2. To probe sensitivity to oxidation of methionine residues, one 500pg aliquot was buffer exchanged into 20mM Histidine-HCl 150mM NaCl pH 6.0 containing ImM AAPH. As an oxidation control, one 500pg aliquot was buffer exchanged into formulation buffer. All samples were incubated for 24 hours at RT with ambient light. Once incubation was complete, aliquots were buffer exchanged back into formulation buffer, flash frozen and stored at -80°C until analysis.Antibody polyreactivity assessment

[0208] To assess the molecule (both unconjugated and conjugated) for the propensity for nonspecific binding, polyreactivity analyses were carried out using an ELISA against baculovirus particles (BVPs), an ELISA against a panel of antigens known to exhibit broad polyreactive characteristics, and a flow cytometry based assay evaluating binding against ExpiCHO cells. Analysis included benchmarking samples which were commercially obtained: Adalimumab (a well-behaved, low signal control), Tanezumab, Dinutuximab, and Palivizumab (mid-range polyreactivity controls) and Lenzilumab (a high-polyreactivity control).

[0209] For the BVP ELISA, BVPs (Curia, 25690) were diluted in 50mM sodium carbonate buffer, pH 9.5 and coated overnight at 4°C onto 96-well flat bottom plates. The next day unbound BVP was aspirated and wells blocked with blocking buffer (PBS with 0.5% BSA) for 1 hour. Plates were washed 3 times with PBS. Antibodies were diluted to 300 nM in blocking buffer and incubated on wells for Ih. Wells were washed 6 times with PBS and incubated with Goat-anti-human HRP (Jackson Immunoresearch Labs, 109-035-098) diluted 1:10,000 in blocking buffer for Ih. Wells were washed 6 times with PBS, incubated with TMB substrate for 60s, and 2M sulfuric acid stop solution added; plates were read on a Spectramax plate reader at 450 nm and A450 using signal was normalized to secondary-only wells. The BVP score was calculated as the normalized score of the test antibody divided by the normalized score of the well-behaved, low polyreactivity control, Adalimumab. An ideal polyreactivity cutoff was set as a score <2 while an acceptable cutoff was set as a score <5..

[0210] For the ELISA-based assay, a panel of standard antigens was used; these consisted of KLH, insulin, dsDNA, ssDNA, LPS, and cardiolipin. Antigens were diluted in PBS and coated overnight874130-6004-4904.10Attorney Docket No. 55171.4010 / WOat 4C onto 96 well flat bottom plates. The coating concentrations were 1 ug / mL for ssDNA and dsDNA, 5 ug / mL for insulin and KLH, 10 ug / mL for LPS, and 50 ug / mL for cardiolipin. The next day, plates were blocked with blocking buffer (PBS + 0.5% BSA) for Ih at room temperature, washed with PBS, and test samples diluted to 100 nM in blocking buffer were added. After Ih incubation at room temperature, plates were washed and incubated with Goat-anti-human HRP (Jackson ImmunoResearch, 109-035-098) diluted 1:10,000 in blocking buffer for Ih. Following Ih incubation at room temperature, the plate was washed, incubated with TMB substrate for 60s, and 2M sulfuric acid stop solution added; plates were read on a Spectramax plate reader at 450 nm and A450 using signal was normalized to secondary-only wells. Polyreactivity scores were determined by summing across the antigens and dividing by the summed scores of the well-behaved, low polyreactivity control, Adalimumab. An ideal polyreactivity cutoff was set as a score <2 while an acceptable cutoff was set as a score <5.

[0211] For the flow-based assay, expiCHO were cultured according to manufacturer recommendations. On the day of the assay, 1E5 cells were plated per well and washed with FACS buffer (PBS + 10% FBS). Cells in each well were incubated for 45min at RT with 700 nM of antibody diluted in FACS buffer. Following the incubation, cells were washed with FACS buffer and Alexa Fluor 647-conjugated Fc-targeting secondary (Jackson ImmunoResearch, 109-606-170) was added at a 1:100 dilution. Following a 30 min incubation at RT and washing, samples were analyzed on a Novocyte instrument where 10,000 events were collected and gated for live singlets. The polyreactivity scoring was determined by normalizing the sample median fluorescence intensity (MFI) to the low-polyreactivity control, An ideal polyreactivity cutoff was set as a score <2 while an acceptable cutoff was set as a score <5.Evaluation of in vivo activity

[0212] TCEs were administered via tail vein injection to tumor-bearing NCG mice (Charles River #572). These mice were injected with 3-5xl06cells / animals (determined per cell line) suspended in a 1 : 1 PBS / matrigel diluent into the subcutaneous tissue of the right flank region. These animals were monitored for tumor outgrowth with caliper measurements taken twice weekly (BIW). Once tumors reached ~150mm3, they were randomized into groups of 5-6 animals each. After randomization, all the animals were administered with weekly doses of A-000-019-8982 (isotype TCE), A-000-005-2841 (positive control), or A-000-010-3936 at various doses. Following this treatment, tumor volumes were measured with calipers twice weekly for 4-5 weeks, or until 884130-6004-4904.10Attorney Docket No. 55171.4010 / WOsacrifice due to humane criteria, by recording only the lengths and widths of the tumors according to standard procedures. Tumor volumes and body weights were recorded in conjunction. Animals were sacrificed at predefined endpoints including tumor volume exceeding 1500mm3, tumor ulceration, and / or significant change in BCS (body condition score).ResultsAntibody discovery, optimization, and affinity characterization

[0213] The discovered VHH fragment was iteratively optimized using the BigHat ML-enabled platform to a final monovalent affinity to human CEACAM6 of approximately 2.6 uM at (FIG.1). Using in silico models, the primary sequence was found to contain several intrinsic developability liabilities in HCDR1 and HCDR3 and a hydrophobic patch in HCDR3 which translated to substantial interaction with silica-based size-exclusion chromatography resin and, in the TCE form, late retention and heterogeneous elution on a butyl non-porous hydrophobic interaction resin. Through ML-design, the discovered VHH sequence was optimized to eliminate the majority of sequence liabilities and improve hydrophobicity without consequence to affinity, significantly improving the poor chromatography phenotype by both analytical SEC and analytical HIC (FIG. 2).

[0214] The optimized VHH was fused as described in the Methods above into a “2+1” TCE scaffold. The CD3 binding domain was derived from the publicly disclosed 20G6 sequence (US Patent No. 10,9069,78B2) and subsequently optimized to interact with recombinant and cell-presented CD3 in a pH dependent manner. The final CD3 binder has a monovalent affinity at pH 7.4 of approximately 200 nM and features 10-fold tighter binding to CD3 at pH 6.0, KD 20 nM (FIG. 1). The CD3 binder readily cross-reacts with cynomolgus CD3 with similar binding kinetics (not shown).

[0215] The final optimized VHH sequence is in the 69th humanness percentile according to an open source tool with experimentally-detected peptides corresponding to all 9mers in the VHH sequence found in a panel of healthy human donors, and 81% of the antibody sequence is identical to a human germline VH sequence (FIG. 3). The immunogenicity risk of A-000-010-3936 was examined by analyzing all potential 9-mers for predicted binding to the 20 most prevalent HLA class II alleles using the NetPanMHC II model and IEDB MHC-II prediction tools as noted. There were two 9-mers in the 5th or higher percentile for more than 25% of HLA alleles corresponding894130-6004-4904.10Attorney Docket No. 55171.4010 / WOto CDRH1 and CDRH3 9-mers in the 1st percentile for more than 5% of HLA alleles, suggesting limited immunogenicity risk (FIG. 4). Immunogenicity risk experimentally examined was using an in vitro PBMC-based assay with unremarkable outcomes. Results are shown below.Developability: de-risking liabilities and developmentExpression and biophysical attributes.

[0216] From a IL transient HEK293 expression, A-000-010-3936 yielded 242.7 mg / L postpurification. The protein was purified using a two-step CH1-CHT workflow and showed 98.25% monomeric purity by analytical SEC (FIG. 5 A) and >95% purity by reducing SDS-PAGE (FIG.5B) when characterized externally by BioIntron, the CRO responsible for protein production.

[0217] The antibody was further characterized internally and demonstrated a comparable 97.5% monomeric purity by analytical SEC (FIG. 6A) and acceptable retention and monomeric purity of 92.0% by analytical HIC (FIG. 6B). In an unoptimized storage buffer (20 mM Histidine-HCl, 150 mM NaCl, pH 6.0), A-000-010-3936 exhibited a Tml of 61.4 °C, Tm2 of 76.6 °C, and a Tagg of 73.9 °C, measured by intrinsic fluorescence and SLS at 266 nm, respectively (FIG. 6C).

[0218] Finally, stable cell lines were prepared from four different vector designs encoding A-000-010-3936 and 2 L production cultures were used to express the TCE, yielding between 2.1-2.3 g / L post CHI purification and 1.7-1.8 g / L post ProA purification. Protein prepared by CHI affinity purification was subject to an additional semi-preparative polishing step and then further characterized for purity by SEC and intact mass spectrometry. Two-step purified antibody was found to contain 94.7-97.6% monomeric content with 100% correct assembly with representative data shown for one vector design in FIG. 7.Accelerated stability & forced degradation

[0219] The TCE was incubated in an unoptimized storage buffer (20 mM Histidine-HCl, 150 mM NaCl, pH 6.0) at 4 °C, 25 °C, and 40 °C for up to 21 days, with samples taken on days 7, 14, and 21. After incubation, samples were flash-frozen and stored at -80 °C to ensure consistent freezethaw conditions across all materials and controls. Aggregation and degradation were assessed by analytical SEC relative to an unstressed -80 °C reference. A-000-010-3936 showed minimal loss in monomeric purity (<5%) under all conditions (Table 9), with 21 -day chromatograms shown alongside the unstressed control in FIG. 8. Table 9 shows monomeric purities measured at 7-day, 14-day, and 21 -day post incubation at 4°C, 25°C and 40°C.904130-6004-4904.10Attorney Docket No. 55171.4010 / WOTable 9.Stress Temperature 7d 14d 21d -80°C (unstressed control) - - 96.74°C 97.5 96.6 96.3 25°C 96.9 97.9 98.1 40°C 96.2 93.6 93.0

[0220] To evaluate susceptibility to common manufacturing-related PTMs, the TCE was exposed to stress conditions promoting oxidation (24 h, RT, ambient light with hydrogen peroxide or AAPH), deamidation (7 days at 40 °C, pH 8.5), and isomerization (14 days at 40 °C, pH 5.0). After each incubation, samples were flash-frozen and stored at -80 °C to ensure consistent freeze-thaw handling across stressed and control materials. Susceptibility to liabilities post stress were evaluated relative to respective controls (“stressed” controls) for each stress condition and to an unstressed control. Stability was assessed by analytical SEC and HIC, and functional impact was measured by SPR binding to both targets and by TDCC (Table 10). Although oxidation stress reduced stability and some SPR binding, no loss of function was observed by TDCC.

[0221] Table 10 depicts effects of forced degradation on monomeric purities assessed by analytical HIC and SEC and on Rmax against CEACAM6 and CD3 by SPR. Criteria for ideal (top), acceptable (middle), and unsuitable (bottom) responses are indicated based on assay variability and expected performance.Table 10.Ratio of Ratio of Ratio ofcytotoxic SEC HIC %Rmax %RmaxStress activity (CEACAM6) (CD3)Condition (IC50) (Unstressed - (Unstressed - (Stressed / (Stressed / (Stressed / Stressed) Stressed) Unstressed) Unstressed) Unstressed) <5 <5 >85 >85 <5 Performance5-10 5-10 50-85 50-85 5-10 Criteria>10 >10 <50 <50 >10 Deamidation -3.5 -0.3 104.4 64 1.20Isomerization 4.6 -0.9 107.1 82.4 0.98914130-6004-4904.10Attorney Docket No. 55171.4010 / WOTrp Oxidation 1.87 32.34 74 95.5 1.23Met Oxidation 4.88 0.14 76 82 1.67Polyreactivity

[0222] The polyreactivity of A-000-010-3936 was measured in ELIS As against baculovirus particles (BVPs) (FIG. 9A) and against a panel of antigens exhibiting properties common to prompt non-specific binding (FIG. 9B), and in a flow cytometry based assay against expiCHO cells (FIG. 9C). Binding to these polyreactivity reagents was compared to assessments of controls known to exhibit, low-, high- and sometimes midrange polyreactivity scores. In all assays, the candidate showed minimal difference versus the non-polyreactive control (adalimumab). A polyreactivity score of 2 (binding fold-change relative to adalimumab) was defined as the ideal threshold for polyreactivity based while a score of 5 is considered acceptable. Overall, the level of non-specific binding to all polyreactive reagents is similar to adalimumab, suggesting that the polyreactivity risk of A-000-010-3936 is low.In vitro potency

[0223] The capacity of A-000-010-3936 to elicit T-cell -dependent T-cellular cytotoxicity (TDCC) was demonstrated in vitro using co-cultures of primary human T-cells and culture-adapted cell lines expressing human CEACAM6 in a ratio of 10 T-cells per target cell. Potency to a variety of cell lines with decreasing CEACAM6 density as measured by the Bangs Lab MESF / QSC bead system. Representative antigen densities were determined based on panels of dissociated primary lung and colon tumor cells from human patients (not shown). Against the cells with the highest densities of CEACAM6, robust and reproducible activity with IC50s in the low pM range was noted. As the CEACAM6 density decreased, potency decreased with the most dramatic loss of potency occurring below 85,000 copies per cell (FIG. 10). Between the cells that express >300,000 copies per cell (ULOD of ABC assay) and 25,000 copies per cell, a 100-200-fold difference in IC50 was noted. A positive control, high affinity TCE with another VHH (clone 2A3) with monovalent KD of 2 nM displayed robust activity against all cell lines regardless of target density. An isotype control TCE displayed no activity against these cell lines.

[0224] Table 11 shows a summary of in vitro activity ± SEM of TCE to cell lines of various CEACAM6 densities.924130-6004-4904.10Attorney Docket No. 55171.4010 / WOTable 11.Cell Line Type TDCC IC50 CD69 EC50 Receptor MFI(% positive) DensityNCI-H2073 NSCLC 25 ± 12 pM 63 ± 25 pM >315,000 1,390,000 AsPCl PDAC 117 ± 61 pM 83 ± 33 pM >315,000 1,200,000 BxPC3 PDAC 202 ± 104 pM 820 ± 230 pM >315,000 1,230,000 SNU-C1 CRC 382 ± 152 pM 3.1 ± 1.6 nM 120,000 456,000 GP2D CRC 6.7 ± 2.6 nM 7.6± 2.2 nM 45,000 144,800 MCF7 BC 2.2 ± 1.36 nM 5.4 ± 2.1 nM 22,000 63,200A549 NSCLC 1.8 ± 0.7 nM 5.2 ± 1.9 nM 8,600 27,200

[0225] As an orthogonal measure, CD69 was also assessed by flow cytometry. EC50s in good agreement with the IC50s for the same cell line were observed (Table 11). Assay supernatants were analyzed for the presence of pro-inflammatory cytokines. Robust induction of fFNy, IL2 and IL6 after 72 hours against target cells that express high levels of CEACAM6 were observed. This was in agreement with the cytotoxicity and T cell activation results (FIG. 11, Table 12).

[0226] Table 12 shows cytokine release ± SEM after co-culture with cell lines of varying CEACAM6 expression.Table 12.ReceptorCell Line Density IFNy IL2 IL6 TNFa 3.27E-09 ± 4.65E-09± 3.26E-12 ± 1.91E-09± NCI-H2073 >315,000 3.12E-09 (n=2) 4.51E-09 (n=2) 4.50E-13 (n=2) 1.63E-09 (n=2)1.16E-08 ± 1.283e-008NCI-H2170 >315,000 1.45E-10 (n=2) (n=l) 1.24E-08 (n=l) 2.09E-09 (n=l) BxPC3 >315,000 1.53E-10 (n=l) N / A N / A N / A6.30E-09 ± 6.39E-09± 4.34E-09 ± 6.09E-09 ± AsPCl >315,000 5.75E-09 (n=2) 5.41E-09 (n=2) 3.25E-09 (n=2) 5.21E-09 (n=2)2.23E-08 ± 1.44E-08± SNU-C1 120,000 2.14E-08 (n=l) 2.08E-08 (n=2) N / A 1.27E-08 (n=2)1.91E-07 ± 8.54E-08 ± 1.69E-07± GP2D 45,000 1.59E-07 (n=2) 4.27E-08 (n=2) N / A 1.46E-07 (n=2)4.15E-08 ±MCF7 22,000 9.97E-09 (n=l) 2.52E-08 (n=1) 3.64E-08 (n=2) 8.38E-09 (n=l)2.80E-08 ± 3.99E-08 ± 4.38E-08 ± 4.97E-08 ±A549 8,600 2.53E-08 (n=2) 3.97E-08 (n=2) 3.58E-08 (n=2) 2.98E-08 (n=2)934130-6004-4904.10Attorney Docket No. 55171.4010 / WOTable 13.Cell Type MFI ABC valueJurkat cells (neg control) 2,840 1,606CD 14+ Monocyte 4,298 ± 607 2,430 ± 343CD11+ Ml Macrophage 9,779 ± 480 5,524 ± 270 CD 11+ M2 Macrophage 12,521 + 512 7,701 + 289iDC 14,528 + 745 8,203 ± 420mDC 16,970 + 1106 9,580 ± 624

[0227] Subsequently, antigen density of primary immune cell populations that express low levels of CEACAM6 from peripheral blood mononuclear cells (PBMC) were examined. Table 13 shows quantification of CEACAM6 density ± SEM on primary immune cells from healthy donors (n=3). Specifically, monocytes express about 2,000 copies per cell, macrophages express about 6,000 copies per cell, and dendritic cells express up to 12,000 copies per cell (Table 13). Activity of early lead A-000-006-3126 to these cells were tested. TCEs were added directly to PBMC isolates and incubated for 72 hours. Again, the high-affinity control TCE elicited potent T-cell redirection and loss of these cells with low pM IC50s similar to potency against cancer cell lines with substantially higher amounts of CEACAM6. However, A-000-006-3126 elicited no redirection against these populations across multiple donors (FIG. 12). Consistent with these results, no CD69 induction versus isotype control in the same assays up to 1 pM TCE were observed. Further supporting this, analysis of cytokine release showed no detectable levels of these cytokines whereas the high affinity control TCE elicited robust cytokine release (FIG. 13).

[0228] CEACAM6 is expressed on primary human neutrophils at higher levels than other myeloid cells found in PBMCs (Table 13). Potency of A-000-010-3936 against neutrophils in fresh whole blood was examined. TCEs were added to anti coagulated, fresh, peripheral whole blood that had been diluted 10-fold with culture medium to reduce viscosity and quantified cytotoxicity of neutrophils and monocytes as well as CD69 induction in the T-cell compartment. The high affinity TCE elicited potent neutrophil killing and T-cell activation at low pM EC50s within 72 hours, whereas A-000-010-3936 displayed substantially lower potency (FIG. 14). The EC50 across two944130-6004-4904.10Attorney Docket No. 55171.4010 / WOdonors were similar or slightly higher (less potent) than typical EC50s in TDCC against cancer cells with similar CEACAM6 densities. Proinflammatory cytokine analysis was performed on supernatants from peripheral whole blood at 72h post dose. The high affinity TCE elicited robust cytokine release compared to test molecule A-000-010-3936 (FIG. 15).

[0229] The effect of soluble CEACAM6 on potency in vitro was examined to understand whether the presence of soluble antigen in the blood may interfere with drug exposure and activity. A-000-010-3936 potency was unaffected up to 1500 ng / ml (50 nM) sCEACAM6 ECD whereas the high affinity TCE shows reduced potency (FIG. 16).Drug metabolism and pharmacokinetics

[0230] Serial peripheral blood draws for PK and cytokine detection were taken from CDX-bearing (NCI-H2170) mice with intraperitoneal (i.p.) human T cells (FIG. 17). The pharmacokinetic properties of A-000-010-3936 demonstrated dose-proportional clearance profile with 6-8 day terminal elimination half-life. Minimal peripheral cytokine release was detected versus isotype after the first dose at day 1. Repeat dose study for peak and trough concentrations showed minimal dose stacking and a clear cmax and cmin.In vivo efficacy

[0231] The activity of A-000-010-3936 was evaluated in NSCLC, CRC, and pancreatic CDX models in NCG mice. TCEs were delivered on a once weekly basis and led to significant antitumor responses in all models. NCI-H2073, NCI-H2170 and SNU-C1 showed near complete elimination at 0.2 and 2 mg / kg with reduced efficacy at the 0.02 mg / kg dose level (FIG. 18). The AsPCl pancreatic model displayed significant tumor growth inhibition at the 2.0 and 0.2 mg / kg dose levels with a drop in efficacy at 0.02 mg / kg as well (FIG. 18). Therefore, it was concluded that A-000-010-3936 was active in vivo in relevant models of CEACAM6+ cancers.

[0232] It was hypothesized that recrudescent tumors in mice treated with the avidity-engineered TCE would have lower CEACAM6 expression levels compared to tumors treated with the isotype TCE due to tumor outgrowth with lower expressing cells. The TCE was specifically engineered to have minimal activity against cells with less than 50,000 CEACAM6 receptors per cell in vitro. Upon excision, dissociation, and analysis, it was noted that over multiple models and experiments, the recrudescent tumors from mice treated with the avidity-engineered TCE have tumor escape at very low CEACAM6 levels compared to tumors from isotype control -treated animals (FIG. 19).954130-6004-4904.10Attorney Docket No. 55171.4010 / WODiscussion

[0233] In summary, the bispecific T-cell engager A-000-010-3936 for the treatment of diverse unmet human malignancies. The antibody is readily expressed and purified in mammalian systems and features robust stability in accelerated / forced degradation studies. The antibody is cross-reactive to the primate orthologue and potently eliminates CEACAM6+ cancer cells in vitro and in vivo. There is evidence that A-000-010-3936 may have low immunogenicity risk based on ex vivo primary cell coculture experiments. Unlike all prior CEACAM6-directed therapeutics A-000-010-3936 may have a remarkable safety profile and therapeutic window.

[0234] The TCE candidate A-000-010-3936 represents an attractive first-in-class candidate for advancement into IND-enabling studies and human studies.Example 2. Functional data testing TCEs with various CD3 and CEACAM6 antigen binding variants

[0235] Provided in the example is binding and functional data of various test CEACAM6 VHH antibodies paired with CD3 antigen binding domain variants including low affinity SP34 variants. Tables 14-18 describe the test candidates and control molecules.Table 14.TCE Ab ID Components Notesvhh-130_0013 + 20G6 variant: scfv-CD3- A-000-006-3126 026_0112 Early CEACAM6 TCE lead vhh-130_0485 + 20G6 variant: scfv-CD3- A-000-010-4531 026_0112 Late CEACAM6 TCE lead vhh-130_0414 + 20G6 variant: scfv-CD3- A-000-010-3936 026_0112 Late CEACAM6 TCE lead Clinical candidate, A-000- 010-3936 with RFA-000-021-7889 vhh-130_0414 + 20G6 variant: scfv-CD3- mutations in Fc domain (BHB299) 026_0112 removedParental CEACAM6 VHH A-000-005-2485 from discovery paired with (BHB294) vhh-130_0001 & WT SP34 SP34964130-6004-4904.10Attorney Docket No. 55171.4010 / WOParental CEACAM6 VHH A-000-005-2486 vhh-130_0001 & WT 20G6 (SEQ ID NOS: from discovery paired with (BHB295) 189-191) 20G6Early lead CEACAM6 A-000-006-3116 vvhh-CEACAM6-130_0013 & SP34 binder paired with wt SP34Early lead CEACAM6 vhh-CEACAM6-130_0013 & scfv-CD3- binder paired with SP34 A-000-006-3118 005_779 variantEarly lead CEACAM6 vhh-CEACAM6-130_0013 & scfv-CD3- binder paired with SP34 A-000-006-3119 005_1725 variantEarly lead CEACAM6 vhh-CEACAM6- 130 0013 & scfv-CD3- binder paired with SP34 A-000-006-3175 192_0157 variantvhh-CEACAM6-130_0013 & WT 20G6 - Early lead CEACAM6 A-000-006-3124 60 nM binder paired with wt 20G6Early lead CEACAM6 vhh-CEACAM6-130_0013 & scfv-CD3- binder paired with 20G6 A-000-006-3127 026J396 variantvhh-CEACAM6-130_0497 & scfv-CD3- late lead CEACAM6 binder A-000-007-0842 192_0157 paired with SP34 variant vhh-CEACAM6-130_0414 & scfv-CD3- late lead CEACAM6 binder A-000-007-0843 192_0157 paired with SP34 variant vhh-CEACAM6-130_0485 & scfv-CD3- CEACAM6 binder paired A-000-007-0844 192_0157 with SP34 variantvhh-CEACAM6-130_0467 & scfv-CD3- CEACAM6 binder paired A-000-007-0845 192 0157 with SP34 variant vhh-CEACAM6-130_458 & scfv-CD3- CEACAM6 binder paired A-000-007-0846 192 0157 with SP34 variantA-000-005-2841 High affinity CEACAM6 (SEQ IDNOS: positive control VHH from 195-197) vhh-CEACAM6-002 0001 + OKT3 WT literature paired with OKT3

[0236] The test CEACAM6 TCE candidates and TCE control molecules are described in Table 14 which highlights the CEACAM6 VHH antigen binding domains and the CD3 antigen binding domains.974130-6004-4904.10Attorney Docket No. 55171.4010 / WOTable 15.CEACAM6 CEACAM6 VHH variantAntibody ID name DescriptionOriginal (parental) CEACAM6 VHH from A-000-000-9348 vhh-130 or vhh-130_0001 discovery phase.Vhh-CEACAM6-130_0001 with a histidine mutation, used in A-000-010-3126 (early A-000-000-7443 vhh-130_0013 lead)Optimized for developability but showed A-000-016-9847 vhh-130_0497 reduced potencyCEACAM6 VHH in final lead BHB299 and A-000-006-4044 vhh-130_0414 A-000-010-3936CEACAM6 VHH in A-000-010-4531 (late A-000-016-9835 vhh-130_0485 lead)Optimized for developability but showed A-000-016-9817 vhh-130_0467 reduced potencyOptimized for developability but showed A-000-016-9808 vhh-130_0458 reduced potencyHigh affinity positive control CEACAM6 A-000-001-1545 CEAC AM6-002_0001 VHH

[0237] The test CEACAM6 VHH candidates for the test TCE molecules and control CEACAM6 VHH molecules are described in Table 15. The test CEACAM6 VHH candidate A-000-000-7443 was utilized to assess pH selectivity. The parental or original CEACAM6 VHH antigen binding domain used in the assays was referred to as vhh-130 or vhh-130_0001 (also known as A-000-000-9348). Test CEACAM6 VHH candidates A-000-016-9847, A-000-006-4044, A-000-016-9835, A-000-016-9817, and A-000-016-9808 possessed developability mutations. The CEACAM6 VHH control molecule (CEACAM6-002_0001) was a high affinity positive control CEACAM6 VHH obtained from literature.Table 16.Antibody ID CD3 scFv variant name DescriptionA-000-014-7715 scfv-CD3-026_0112 CD3 - 20G6 variantA-000-014-7709 scfv-CD3-026_1396 CD3 - 20G6 variantA-000-014-7703 scfv-CD3-005-1779 CD3 - SP34 variant984130-6004-4904.10Attorney Docket No. 55171.4010 / WOA-000-014-7708 scfv-CD3-192_0157 CD3 - SP34 variantA-000-014-7714 scfv-CD3-005_1725 CD3 - SP34 variant

[0238] The variant CD3 antigen binding domain candidates for the test TCE molecules are described in Table 16. Multiple test CD3 scFv candidates were utilized in test CEACAM6 TCE candidates as shown herein. Test CD3 scFv candidates A-000-014-7709 and A-000-014-7714 exhibited ultra-low binding affinity for human CD3 antigen. Test CD3 scFv candidates A-000-014-7715 and A-000-014-7708 exhibited low binding affinity for human CD3 antigen. Test CD3 scFv candidate A-000-014-7703 exhibited medium binding affinity for human CD3 antigen.

[0239] The example depicts data of early test CEACAM6 VHH antibodies paired with various CD3 antigen binding domain variants in dataset 1. FIGS. 20A, 21 A, and 22A depict cell cytotoxicity (% cell viability) of BxPC3 and MCF7 cell lines upon dose-dependent stimulation by CEACAM6 test TCE candidates and controls. FIGS. 23 and 24 depict cell cytotoxicity (% cell viability) of human whole blood from donor 1, donor 2, and donor 3 upon dose-dependent stimulation of CEACAM6 test TCE candidates and controls. Table 17 shows IC50 TDCC and IC50 whole blood data for the test candidates and controls as well as binding affinities for CD3 by the CD3 antigen binding domain.Table 17.IC50 IC50CD3 antigen binding KI) TDCC TDCC IC50 Whole Blood TCE Antibody ID domain name (nM) (BxPC3) (MCF7) (n=3 donors)OKT3 WT 2 2.25E-12 5.12E-09 Not tested 8.824E-10 ± 7.6E- A-000-006-3116 SP34 WT 2 1.28E-11 2.77E-10 1020G6 WT 9.44E-09 ± 2.70E- A-000-006-3124 (US 10,9069,78B2) 40 1.23E-11 3.91E-09 09SP34 variant: scfv- A-000-006-3118 CD3-005-1779 48 8.70E-12 2.52E-10 Not tested SP34 variant: scfv- 2.45E-09 ± 1.10E- A-000-006-3175 CD3-192_0157 200 1.41E-11 1.90E-09 09SP34 variant: scfv- A-000-006-3119 CD3-005-1725 1000 >1.0E-6 >1.0E-6 Not tested994130-6004-4904.10Attorney Docket No. 55171.4010 / WO20G6 variant: scfv- 5.364e-09 ± 2.648E- A-000-006-3126 CD3-026 0112 200 2.27E-11 1.18E-09 0920G6 variant: scfv- 5.537E-07 ± A-000-006-3127 CD3-026_1396 1000 2.48E-11 3.27E-09 3.329E-07

[0240] The examp e depicts data of early test CEACAM6 VHH antibodies paired with low affinity SP34 antigen binding domain variants in dataset 2.

[0241] FIG. 25 shows cell viability and T cell activation of cells upon stimulation by test TCE candidates A-000-006-3175 (SEQ ID NOS: 81-83), A-000-007-0842 (SEQ ID NOS: 129-131), and A-000-007-0843 (SEQ ID NOS: 123-125). Data from the experiment is shown in Table 18. Table 18.IC50 Yield SEC Antibody ID Protein name Cytotox IC50 CD69 (mg / L) % vhh-CEACAM6-130_0013 &A-000-006-3175 scfv-CD3-192_0157 1.15e-l 1 7.216e-ll 88.4 99 vhh-CEACAM6-130_0497 &A-000-007-0842 scfv-CD3-192_0157 6.183e-l 1 2.101e-l 1 64 94.5 vhh-CEACAM6-130_0414 &A-000-007-0843 scfv-CD3-192_0157 1.676e-l 1 9.437e-ll 76.1 97

[0242] FIG. 26 shows cell viability and T cell activation of cells upon stimulation by test TCE candidates A-000-006-3175, A-000-007-0844 (SEQ ID NO: 117-119), A-000-007-0846 (SEQ ID NOS: 105-107), and A-000-007-0845 (SEQ ID NOS: 111-113). Data from the experiment is shown in Table 19.Table 19.Yield SEC Antibody ID Protein name IC50 Cytotox IC50 CD69 (mg / L) % vhh-CE AC AM6- 130_0013A-000-006-3175 & scfv-CD3-192_0157 1.15e-l 1 7.216e-l 1 88.4 99 vhh-CEAC AM6- 130_0485A-000-007-0844 & scfv-CD3-192_0157 1.728e-l 1 1.836e-09 108.3 95 vhh-CEAC AM6- 130_0467A-000-007-0845 & scfv-CD3-192_0157 9.177e-10 1.509e-09 64.3 97 Vhh-CEAC AM6- 130_458A-000-007-0846 & scfv-CD3-192_0157 6.191e-l 1 2.076e-09 67 951004130-6004-4904.10Attorney Docket No. 55171.4010 / WO

[0243] FIG. 27 shows cell viability and T cell activation of NCI-H2073 cells upon stimulation by test TCE candidate A-000-006-3175 and control TCE molecules containing 20G3, SP34 and OKT3 antigen binding domains. Data from the experiment is shown in Table 20.Table 20.Antibody ID Protein IC50 Cytotox IC50 CD69 BHB0294 vhh-CEACAM6-130 & SP34 1.82e-l 1 4.009e-ll vhh-CEACAM6-130_0013 & scfv- A-000-006-3175 CD3-192 0157 1.15e-l 1 7.216e-l 1 A-000-005-2841(BIO-0089) 2+1 CEACAM6-002_0001 x OKT3 7.64e-12 1.041e-l 1Example 3. Developability characteristics of test TCE candidates

[0244] Test TCE candidates A-000-006-3126, A-000-010-3936, and A-000-010-4531 were subjected to full characterization studies. Sequence alignment between the early lead variant sequence (A-000-006-3126) and subsequent developability optimized variants (A-000-010-3936 and A-000-010-4531) are shown in Table 21. The amino acid sequences of the HCDR1, HCDR2, and HCDR3 of the TCE candidate A-000-006-3126 is provided in SEQ ID NOS: 41-43, respectively. The amino acid sequences of the HCDR1, HCDR2, and HCDR3 of the TCE candidate A-000-010-3936 is provided in SEQ ID NOS: 44-46, respectively. The amino acid sequences of the HCDR1, HCDR2, and HCDR3 of the TCE candidate A-000-010-4531 is provided in SEQ ID NOS: 47-49, respectively.Table 21.CEACAM6 Region CDR1 CDR2 CDR3A-000-006-3126 N H M FA-000-010-3936 Y A S EA-000-010-4531 Y H S EIMGT Position 36 68 109 113

[0245] The specific mutations were selected to remove sequence-based deamidation and oxidation motifs and had the positive effect of reducing the overall hydrophobicity profile. Relative to A-000-006-3126, A-000-010-3936 and A-000-010-4531 exhibited one fewer deamidation liability in 1014130-6004-4904.10Attorney Docket No. 55171.4010 / WOHCDR1, one fewer oxidation liability in HCDR3, and one fewer hydrophobic patch in HCDR3. The general developability profile, characterized in Tables 22A-22C, was superior for A-000-01 CI-3936 relative to either A-000-006-3126 and A-000-010-4531 with respect to hydrophobicity profile and non-specific interaction (polyreactivity) profile. All three candidates were subject to deeper characterization to further probe their relative stability (Table 23) and the effect of chemical stressors intended to cause post-translational modification on structure and function (Table 24). In all assays, A-000-010-3936 performed as well as or better than the other development candidates, leading to the candidate’s selection as a candidate for further developability.Antibody VHH CD3 In silico (VHH only) 293 Production optimized for? optimizedfor? Deamid. Isomer. Oxid. Yield SDS- SEC (mg / L) PAGE Purity (1- step) Ideal None None None >50 >95 >95 Acceptable Med Med Med 20-50 85-95 85-95 Risk High High High <20 <85 <85 A-000-006-3126 pH dependent pH Med None Med 529.2 >95 97 (130 0013 + binding dependent20G6 0112) bindingA-000-010-3936 Developability pH None None Med 242.7 >95 98 (130_0414 + dependentBHB0875) bindingA-000-010-4531 pH dependent pH None None Med 170.5 >95 96 (130_0442 + binding dependentBHB0875) bindingTable 22A.Table 22B.1024130-6004-4904.10Attorney Docket No. 55171.4010 / WOAntibody VHH CD3 Thermostability CE- Temp HIC optimized for? optimized SDS hold (MP%) for? Tm Tagg (AMP%)Ideal >55 >Tm >95 <5 >85 Acceptable 50-55 <Tm 85-95 5-10 70-85 Risk <50 <85 >10 <70 A-000-006-3126 pH dependent pH 60.0 72.1 >99% 0.2 89.3**, (130 0013 + binding dependent i 20G6 0112) bindingA-000-010-3936 Developability pH 61.1 73.4 >99% 3.2 96.5 (130 0414 + dependentBHB0875) bindingA-000-010-4531 pH dependent pH 60.0 72.0 >99% 4.5 78.2** (130_0442 + binding dependentBHB0875) bindingTable 22C.Antibody VHH CD3 optimized Polyreactivity (Fold-over-D2E7) optimized for? for? Flow Antigen BVP PanelIdeal <2Acceptable 2-5Risk >5A-000-006-3126 pH dependent pH dependent 1.24 1.04 0.59 (130 0013 + binding binding20G6 0112)A-000-010-3936 Developability pH dependent 1.13 1.56 1.37 (130 0414 + bindingBHB0875)A-000-010-4531 pH dependent pH dependent 13.34 4.4 1.38 (130_0442 + binding bindingBHB0875)Table 23.Aggregation RateFormulation Condition(ASLS266 Intensity / At)Buffer SaltCondi ti A-000-006- A-000-010- A-000-010- System Concentrati PHon 3126 3936 4531(20mM) on (mM)Histidine1 0 6.0 4.2 2.9 4.5-HC11034130-6004-4904.10Attorney Docket No. 55171.4010 / WO2 6.5 6.1 4.3 4.83 7.0 7.2 4.7 5.34 6.0 19.3 9.9 15.25 150 6.5 12.4 7.0 8.56 7.0 9.7 6.1 8.0

[0246] Table 23 shows the effect of isothermal hold on aggregate development under variable formulation conditions for all three development candidates.1044130-6004-4904.10Attorney Docket No. 55171.4010 / WORatio of Ratio of Ratio of SEC HIC X-max Aggregation %Rmax %Rmax Condition Rate (CEACAM6) (CD3) (Unstressed (Unstressed (Unstressed (Stressed / (Stressed / (Stressed / Candidate- Stressed) - Stressed) - Stressed) Unstressed) Unstressed) Unstressed) Ideal <100 >85 >85 Acceptable 5-10 5-10 5-10 100-125 50-85 50-85 Risk >10 >10 >10 >125 <50 <50 Deamidation 2.37 -0.8 -1 74.9 96.5 72.2 Isomerization -3.37 -0.5 0 16.8 50.9 82.7 A-000- 006-3126 Trp Oxidation 6.2 46.3 -2 83.2 73 110.6 Met Oxidation 3.9 -0.8 -1 76.5 73 93 Deamidation 1 -14.9 -1 27.8 103.5 69.7 Isomerization -4 5.8 -1 19.0 89.5 86.2 A-000- 010-3936 Trp Oxidation 0.8 42.1 -1 113 76 110.7 Met Oxidation 0.6 -2 -1 73.8 59 93.4 Deamidation -3.5 -0.3 2 26.5 104.4 64 Isomerization 4.6 -0.9 0 17.6 107.1 82.4 A-000- 010-4531 Trp Oxidation 1.87 32.34 -1 100.6 74 95.5 Met Oxidation 4.88 0.14 0 80.4 76 82Table 24.

[0247] Table 24 shows the impact of chemical stress on the structure and function of all three development candidates.Methods

[0248] To assess the relative aggregation propensity of antibodies under different formulation conditions, an isothermal hold was performed at 40°C for 72 hours using the UNchained Labs UNcle. Samples were formulated at 5 mg / mL and then diluted 1:5 in a set of buffer solutions for stability screening. Static light scattering (SLS) data was generated for the duration of the1054130-6004-4904.10Attorney Docket No. 55171.4010 / WOisothermal hold and aggregation rates were determined based on a linear regression of the linear aggregation mode for each antibody. Aggregation rates are reported as the slopes of the linear regression line of best fit.Example 4. Data about BHB299 (clinical candidate) and A-000-010-3936 (test candidate)

[0249] BHB299 is a clinical candidate molecule based on the test TCE candidate A-000-010-3936 but with RF mutations in the CH3 domain of the Fc domains removed. In other words, BHB299 includes H435 and Y436 in the CH3 domain of the Fc domains. The "RF mutation" generally refers to the mutation of the amino acids HY into RF in the CH3 domain of Fc domains, such as the mutation H435R and Y436F in CH3 domain as described by Jendeberg et al. (1997, J. Immunological Meth., 20: 25-34) and is described as advantageous for purification purposes as it abolishes binding to protein A.

[0250] Following production of BHB299 from early development studies, internal biophysical characterization was performed to compare the relative hydrophobicity by hydrophobic interaction chromatography (HIC) (Table 25), aggregation profile by size exclusion chromatography (Table 26), and thermostability profile (Table 27) of A-000-010-3936 and BHB299. While a difference in MP% was observed by HIC, the main hydrophobic species across both materials are consistent and considered to be aligned. The slight differences in thermostability between A-000-010-3936 and BHB299 appears to be likely attributable to the difference in formulation buffer and may not represent a meaningful difference in functional stability. Chromatograms were generated by hydrophobic interaction chromatography for A-000-010-3936 and BHB299 and the reported retention times (RT in minutes) and main peak percentages (MP%) are shown in Table 25. Chromatograms were generated by size exclusion chromatography for A-000-010-3936 and BHB299 and the reported retention times (RT in minutes) and main peak percentages (MP%) are shown in Table 26.Table 25.Variant MP% RT [min]A-000-010-393697.11 5.868BHB29992.57 6.0251064130-6004-4904.10Attorney Docket No. 55171.4010 / WOTable 26.Variant MP% RT [min]A-000-010-3936 97.86 8.030BHB29997.80 8.060

[0251] Thermostability profiles depicted thermal transitions and aggregation temperatures for A-000-010-3936 and BHB299. Barycentric mean traces were generated by nanoDSF showing onset melting temperatures and melting temperatures. Static light scattering traces were also generated with aggregation temperatures.Table 27.Variant Tmi (°C) Tonset (°C) Tagg (°C) A-000-010-3936 58.61 52.46 62.18BHB299 62.21 54.30 61.76

[0252] Table 27 shows the reported first melting temperature, onset melting temperature, and aggregation temperatures by nanoDSF and static light scattering of A-000-010-3936 to BHB299.Example 5. Kinetic data of anti-CEACAM6 TCE candidates representing amino acid variants and their impact on antigen binding

[0253] CEACAM6 and CD3 starter binders were iteratively optimized using the ML-enabled platform. During the optimization process thousands of sequence variants were expressed and characterized. Mutational coverage analysis identified specific amino acid residues that were most frequently mutated and their impact on antigen binding.

[0254] FIGS. 28A-28D and FIGS. 29A-29D illustrate the mutation coverage across the heavy chain variable region (VH) of CEACAM6 VHH antibody (CEACAM6-130; Vhh-130), and both the heavy chain variable region (VH) and light chain variable region (VL) of CD3 antibody (CD3-026; scFv-CD3-192). The heatmap displays the frequency of amino acid substitutions observed at each position along the VH sequence, organized by antibody structural region: Framework Region 1 (VH FR1), Complementarity Determining Region 1 (VH CDR1), Framework Region 2 (VH FR2), Complementarity Determining Region 2 (VH CDR2), Framework Region 3 (VH FR3),1074130-6004-4904.10Attorney Docket No. 55171.4010 / WOComplementarity Determining Region 3 (VH CDR3), and Framework Region 4 (VH FR4). The y-axis enumerates the twenty standard amino acids and the x-axis denotes the amino acid position within each respective structural region. Cell shading intensity corresponds to the number of unique constructs harboring a given substitution at a given position, with darker shading indicating higher representation.

[0255] FIGS. 30A-30H and FIGS. 31A-31H show mutation inference volcano plots for CEACAM6 VHH antibody CEACAM6-130 and CD3 antibody CD3-026, summarizing the statistical associations between individual amino acid substitutions and two functional readouts: Kinetics Assay Pass / Fail Likelihood (left panels) and Binding Affinity expressed as dissociation constant kd (right panels). Each panel is subdivided into substitutions that improve (upper panels) and substitutions that worsen (lower panels) the respective functional property. The x-axis denotes amino acid position along the full variable domain sequence (positions 1-280, encompassing both VH and VL regions), and the y-axis represents the negative loglO of the adjusted p-value ( loglO(adj. P-value)), providing a measure of statistical confidence for each observed mutational effect. The parental sequence of CEACAM6-130 (SEQ ID NO: 198) for the plot is shown in FIGS.3OC-3OD or FIGS. 30G-30H. The parental sequence of CD3-026 (SEQ ID NO: 199) for the plot is shown in FIGS. 30C-30D or FIGS. 30G-30H.1084130-6004-4904.10Attorney Docket No. 55171.4010 / WO Table 28.SEQ ID NO: Name1 A-000-006-3126 heavy chain.02 A-000-006-3126 heavy chain.13 A-000-006-3126 light chain. 24 A-000-010-3936 heavy chain.05 A-000-010-3936 heavy chain.16 A-000-010-3936 light chain. 27 A-000-010-4531 heavy chain.0 with LALAPG-knob8 A-000-010-4531 heavy chain.1 with LALAPG-hole9 A-000-010-4531 light chain.210 Anti-CEACAM6 VHH domain.111 Anti-CEACAM6 VHH domain.212 Anti-CEACAM6 VHH domain.313 Anti-CEACAM6 VHH domain.414 Fc domain - knob variant in chain 015 Anti-CD3 VH domain !16 Anti-CD3 CHI domain !17 Anti-CD3 VL domain !18 Anti-CD3 CL kappa domain.119 Human IgGl hinge domain20 Human CH2 IgGl domain - LALAPG variant21 Human CH3 IgGl domain - hole variant in chain 122 Human CH3 IgGl domain - knob variant in chain 023 Fc domain - hole variant in chain 124 A-000-005-2848 heavy chain.025 A-000-005-2848 heavy chain.126 A-000-005-2848 light chain.127 Anti-CEACAM6 VHH domain.A28 Anti-CD3 VH domain.A29 Anti-CD3 CHI domain.A30 Anti-CD3 VL domain.A31 Anti-CD3 CL domain.A32 Peptide linker.133 Peptide linker.234 Peptide linker.335 Peptide linker.41094130-6004-4904.10Attorney Docket No. 55171.4010 / WO 36 Peptide linker.537 Peptide linker.638 Peptide linker.739 Peptide linker.840 Whitlow linker41 3126 HCDR1 VHH CEACAM642 3126 HCDR2 VHH CEACAM643 3126 HCDR3 VHH CEACAM644 3936 HCDR1 VHH CEACAM645 3936 HCDR2 VHH CEACAM646 3936 HCDR3 VHH CEACAM647 4531 HCDR1 VHH CEACAM648 4531 HCDR2 VHH CEACAM649 4531 HCDR3 VHH CEACAM650 CD3 HCDR151 CD3 HCDR252 CD3 HCDR353 CD3 LCDR154 CD3 LCDR255 CD3 LCDR356 extracelluar domain of human CD3epsilon protein57 Human CD3 epsilon mature protein58 extracelluar domain of human CEAC AM6 protein59 Human CEACAM6 proprotein60 A-000-006-3119 VHH CEACAM661 A-000-006-3119 VH CD362 A-000-006-3119 VL CD363 A-000-006-3119 monomerl64 A-000-006-3119 monomer265 A-000-006-3119 monomer366 A-000-006-3118 VHH CEACAM667 A-000-006-3118 VH CD368 A-000-006-3118 VL CD369 A-000-006-3118 monomerl70 A-000-006-3118 monomer271 A-000-006-3118 monomer372 A-000-006-3116 VHH CEACAM61104130-6004-4904.10Attorney Docket No. 55171.4010 / WO 73 A-000-006-3116 VH CD374 A-000-006-3116 VL CD375 A-000-006-3116 monomerl76 A-000-006-3116 monomer277 A-000-006-3116 monomer378 A-000-006-3175 VHH CEACAM679 A-000-006-3175 VH CD380 A-000-006-3175 VL CD381 A-000-006-3175 monomerl82 A-000-006-3175 monomer283 A-000-006-3175 monomer384 A-000-006-3126 VHH CEACAM685 A-000-006-3126 VH CD386 A-000-006-3126 VL CD387 A-000-006-3126 monomerl88 A-000-006-3126 monomer289 A-000-006-3126 monomer390 A-000-006-3127 VHH CEACAM691 A-000-006-3127 VH CD392 A-000-006-3127 VL CD393 A-000-006-3127 monomerl94 A-000-006-3127 monomer295 A-000-006-3127 monomer396 A-000-006-3124 VHH CEACAM697 A-000-006-3124 VH CD398 A-000-006-3124 VL CD399 A-000-006-3124 monomerl100 A-000-006-3124 monomer2101 A-000-006-3124 monomer3102 A-000-007-0846 VHH CEACAM6103 A-000-007-0846 VH CD3104 A-000-007-0846 VL CD3105 A-000-007-0846 monomerl106 A-000-007-0846 monomer2107 A-000-007-0846 monomer3108 A-000-007-0845 VHH CEACAM6109 A-000-007-0845 VH CD31114130-6004-4904.10Attorney Docket No. 55171.4010 / WO 110 A-000-007-0845 VL CD3111 A-000-007-0845 monomerl112 A-000-007-0845 monomer2113 A-000-007-0845 monomer3114 A-000-007-0844 VHH CEACAM6115 A-000-007-0844 VH CD3116 A-000-007-0844 VL CD3117 A-000-007-0844 monomerl118 A-000-007-0844 monomer2119 A-000-007-0844 monomer3120 A-000-007-0843 VHH CEACAM6121 A-000-007-0843 VH CD3122 A-000-007-0843 VL CD3123 A-000-007-0843 monomerl124 A-000-007-0843 monomer2125 A-000-007-0843 monomer3126 A-000-007-0842 VHH CEACAM6127 A-000-007-0842 VH CD3128 A-000-007-0842 VL CD3129 A-000-007-0842 monomerl130 A-000-007-0842 monomer2131 A-000-007-0842 monomer3132 A-000-021-7889 VHH CEACAM6133 A-000-021-7889 VH CD3134 A-000-021 -7889 VL CD3135 A-000-021-7889 monomerl136 A-000-021-7889 monomer2137 A-000-021-7889 monomer3138 A-000-005-2485 VHH CEACAM6139 A-000-005-2485 VH CD3140 A-000-005-2485 VL CD3141 A-000-005-2485 monomerl142 A-000-005-2485 monomer2143 A-000-005-2485 monomer3144 A-000-005-2486 VHH CEACAM6145 A-000-005-2486 VH CD3146 A-000-005-2486 VL CD31124130-6004-4904.10Attorney Docket No. 55171.4010 / WO 147 A-000-005-2486 monomerl148 A-000-005-2486 monomer2149 A-000-005-2486 monomer3150 A -000-014-7714 VH CD3151 A-000-014-7714 VL CD3152 A-000-014-7708 VH CD3153 A-000-014-7708 VL CD3154 A-000-014-7703 VH CD3155 A-000-014-7703 VL CD3156 A-000-014-7709 VH CD3157 A-000-014-7709 VL CD3158 A-000-014-7715 VH CD3159 A-000-014-7715 VL CD3160 A-000-016-9808 VHH CEACAM6161 A-000-006-4044 VHH CEACAM6162 A-000-016-9847 VHH CEACAM6163 A-000-016-9817 VHH CEACAM6164 A-000-016-9835 VHH CEACAM6165 A-000-000-7443 VHH CEACAM6166 A-000-000-9348 vhh167 CEACAM6 vhh of FIG. 4168 IGH V3 -23 01 IGHJ 1 _01169 IGHV3 -23_02_IGHJ4_01170 IGHV3 -23 04 IGHJ5 01171 IGHV3 -66 01 IGHJ5 02172 IGHV3OR16-9 01 IGHJ2 01173 OKT3 VH174 OKT3 VL175 SP34 VH176 SP34 VL177 20G6 VH178 20G6 VL179 A-000-001-1545 CEACAM6-002 vhh180 A-000-005-2485 VHH CEACAM6181 A-000-005-2485 VH CD3182 A-000-005-2485 VL CD3183 A-000-005-2485 monomerl1134130-6004-4904.10Attorney Docket No. 55171.4010 / WO184 A-000-005-2485 monomer2185 A-000-005-2485 monomer3186 A-000-005-2486 VHH CEACAM6187 A -000-005-2486 VH CD3188 A-000-005-2486 VL CD3189 A-000-005-2486 monomerl190 A-000-005-2486 monomer2191 A-000-005-2486 monomer3192 A-000-005-2841 VHH CEACAM6193 A-000-005-2841 VH CD3194 A-000-005-2841 VL CD3195 A-000-005-2841 monomerl196 A-000-005-2841 monomer2197 A-000-005-2841 monomer3198 CEACAM6-130 of Example 4199 CD3-026 of Example 4

[0256] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0257] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.1144130-6004-4904.10

Claims

Attorney Docket No. 55171.4010 / WOCLAIMS1. A CEACAM6 targeting T cell-engaging molecule comprising:(a) a first polypeptide chain comprising from N-terminus to C-terminus: (i) a first VHH antigen binding domain that binds human CEACAM6, (ii) a first flexible linker, (iii) a hinge domain, (iv) a first Fc domain;(b) a second polypeptide chain comprising from N-terminus to C-terminus: (i) a second VHH antigen binding domain that binds human CEACAM6, (ii) a second flexible linker, (iii) a variable heavy (VH) domain, (iv) a second hinge domain, (v) a second Fc domain; and(c) a third polypeptide chain comprising from N-terminus to C-terminus: a variable light (VL) domain and a constant light (CL) domain;wherein the VH domain and VL domain form an antigen binding domain that binds human CD3, and wherein the first and second Fc domains each comprise one or more amino acid substitutions for Fc heterodimerization.

2. The CEACAM6 targeting T cell-engaging molecule of claim 1, wherein the first VHH antigen binding domain or the second VHH antigen binding domain comprises:(i) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 43;(ii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 44, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 45, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 46; or(iii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 47, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 48, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 49.

3. The CEAC AM6 targeting T cell-engaging molecule of claim 1 or 2, wherein the first VHH antigen binding domain and the second VHH antigen binding domain each comprises:1154130-6004-4904.10Attorney Docket No. 55171.4010 / WO(i) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 43;(ii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 44, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 45, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 46; or(iii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 47, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 48, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 49.

4. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

5. The CEACAM6 targeting T cell-engaging molecule of claim 1 or 2, wherein the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

6. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein the first VHH antigen binding domain and the second VHH antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

7. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein the first VHH antigen binding domain and the second VHH antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOS: 10-13, 27, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.1164130-6004-4904.10Attorney Docket No. 55171.4010 / WO8. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13.

9. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein the first VHH antigen binding domain and the second VHH antigen binding domain each comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13.

10. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein the first VHH antigen binding domain or the second VHH antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOS: 10-13.

11. The CEAC AM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein the first VHH antigen binding domain and the second VHH antigen binding domain each comprises an amino acid sequence of any one of SEQ ID NOS: 10-13.

12. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-11, wherein the first flexible linker or the second flexible linker is selected from the group consisting of any one of SEQ ID NOS: 32-40.

13. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-12, wherein the first flexible linker and the second flexible linker each is selected from the group consisting of any one of SEQ ID NOS: 32-40.

14. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-13, wherein the VH domain and the VL domain forming the antigen binding domain that binds human CD3 are selected from the group consisting of:(i) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17;1174130-6004-4904.10Attorney Docket No. 55171.4010 / WO(ii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30;(iii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 61 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 62;(iv) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 67 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 68;(v) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 73 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 74;(vi) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 79 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 80;(vii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 85 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 86;(viii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 91 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 92;(ix) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 97 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 98;(x) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 103 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 104;(xi) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 109 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 110;1184130-6004-4904.10Attorney Docket No. 55171.4010 / WO(xii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 115 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 116;(xiii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 121 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 122;(xiv) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 127 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 128;(xv) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 133 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 134;(xvi) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 139 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 140;(xvii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 145 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 146;(xviii) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 181 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 182;(xix) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 187 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 188; and(xx) the VH domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 193 and the VL domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 194.1194130-6004-4904.10Attorney Docket No. 55171.4010 / WO15. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-14, wherein the VH domain and the VL domain forming the antigen binding domain that binds human CD3 are selected from the group consisting of:(i) the VH domain comprising an amino acid sequence of SEQ ID NO: 15 and the VL domain comprising an amino acid sequence of SEQ ID NO: 17;(ii) the VH domain comprising an amino acid sequence of SEQ ID NO: 28 and the VL domain comprising an amino acid sequence of SEQ ID NO: 30;(iii) the VH domain comprising an amino acid sequence of SEQ ID NO: 61 and the VL domain comprising an amino acid sequence of SEQ ID NO: 62;(iv) the VH domain comprising an amino acid sequence of SEQ ID NO: 67 and the VL domain comprising an amino acid sequence of SEQ ID NO: 68;(v) the VH domain comprising an amino acid sequence of SEQ ID NO: 73 and the VL domain comprising an amino acid sequence of SEQ ID NO: 74;(vi) the VH domain comprising an amino acid sequence of SEQ ID NO: 79 and the VL domain comprising an amino acid sequence of SEQ ID NO: 80;(vii) the VH domain comprising an amino acid sequence of SEQ ID NO: 85 and the VL domain comprising an amino acid sequence of SEQ ID NO: 86;(viii) the VH domain comprising an amino acid sequence of SEQ ID NO: 91 and the VL domain comprising an amino acid sequence of SEQ ID NO: 92;(ix) the VH domain comprising an amino acid sequence of SEQ ID NO: 97 and the VL domain comprising an amino acid sequence of SEQ ID NO: 98;(x) the VH domain comprising an amino acid sequence of SEQ ID NO: 103 and the VL domain comprising an amino acid sequence of SEQ ID NO: 104;(xi) the VH domain comprising an amino acid sequence of SEQ ID NO: 109 and the VL domain comprising an amino acid sequence of SEQ ID NO: 110;(xii) the VH domain comprising an amino acid sequence of SEQ ID NO: 115 and the VL domain comprising an amino acid sequence of SEQ ID NO: 116;(xiii) the VH domain comprising an amino acid sequence of SEQ ID NO: 121 and the VL domain comprising an amino acid sequence of SEQ ID NO: 122;1204130-6004-4904.10Attorney Docket No. 55171.4010 / WO(xiv) the VH domain comprising an amino acid sequence of SEQ ID NO: 127 and the VL domain comprising an amino acid sequence of SEQ ID NO: 128;(xv) the VH domain comprising an amino acid sequence of SEQ ID NO: 133 and the VL domain comprising an amino acid sequence of SEQ ID NO: 134;(xvi) the VH domain comprising an amino acid sequence of SEQ ID NO: 139 and the VL domain comprising an amino acid sequence of SEQ ID NO: 140;(xvii) the VH domain comprising an amino acid sequence of SEQ ID NO: 145 and the VL domain comprising an amino acid sequence of SEQ ID NO: 146;(xviii) the VH domain comprising an amino acid sequence of SEQ ID NO: 181 and the VL domain comprising an amino acid sequence of SEQ ID NO: 182;(xix) the VH domain comprising an amino acid sequence of SEQ ID NO: 187 and the VL domain comprising an amino acid sequence of SEQ ID NO: 188; and(xx) the VH domain comprising an amino acid sequence of SEQ ID NO: 193 and the VL domain comprising an amino acid sequence of SEQ ID NO: 194.

16. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-15, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 15 and the VL domain comprises to the amino acid sequence of SEQ ID NO: 17.

17. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-16, wherein the first hinge domain and the second hinge domain each is a human IgGl hinge domain or a variant thereof.

18. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-15, wherein the first hinge domain and the second hinge domain each is a human IgG2 hinge domain, a human IgG3 hinge domain, a human IgG4 hinge domain, or a variant thereof.

19. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-18, wherein the first Fc domain and the second Fc domain each is a CH2-CH3 domain of a human IgGl.1214130-6004-4904.10Attorney Docket No. 55171.4010 / WO20. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-16 or 18, wherein the first and second Fc domains each comprises a CH2-CH3 domain of a human IgG2, IgG3, or IgG4.

21. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-19, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 14 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 23.

22. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-19, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 23 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 14.

23. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-22, wherein the CL domain is a kappa light chain constant domain.

24. The CEACAM6 targeting T cell-engaging molecule of any one of claims 1-22, wherein the CL domain is a lambda light chain constant domain.

25. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein :(i) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6;(ii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3;(iii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9;1224130-6004-4904.10Attorney Docket No. 55171.4010 / WO(iv) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 63, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 64, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 65;(v) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 69, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 70, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 71;(vi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 75, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 76, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 77;(vii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 81, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 82, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83;(viii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 87, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 88, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 89;(ix) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 94, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 95;(x) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 99, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 100, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 101;1234130-6004-4904.10Attorney Docket No. 55171.4010 / WO(xi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 105, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 106, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 107;(xii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 111, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 112, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 113;(xiii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 118, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 119;(xiv) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 123, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 124, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 125;(xv) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 129, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 130, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 131;(xvi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 135, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 136, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 137;1244130-6004-4904.10Attorney Docket No. 55171.4010 / WO(xvii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 141, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 142, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 143;(xviii) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 147, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 148, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 149;(xix) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 183, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 184, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 185;(xx) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 189, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 190, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 191; or(xxi) the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 195, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 196, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 197.

26. The CEACAM6 targeting T cell-engaging molecule of any one of the preceding claims, wherein:(i) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 4, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 5, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 6;1254130-6004-4904.10Attorney Docket No. 55171.4010 / WO(ii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 1, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 2, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 3;(iii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 7, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 8, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 9;(iv) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 63, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 64, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 65;(v) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 69, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 70, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 71;(vi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 75, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 76, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77;(vii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 81, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 82, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 83;(viii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 87, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89;(ix) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 93, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 94, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 95;(x) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 99, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 100, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 101;(xi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 105, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 106, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 107;1264130-6004-4904.10Attorney Docket No. 55171.4010 / WO(xii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 111, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 112, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 113;(xiii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 117, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 118, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 119;(xiv) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 123, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 124, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 125;(xv) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 129, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 130, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 131;(xvi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 135, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 136, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 137;(xvii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 141, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 142, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 143;(xviii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 147, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 148, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 149;(xix) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 183, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 184, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 185;(xx) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 189, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 190, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 191; or(xxi) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 195, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 196, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 197.1274130-6004-4904.10Attorney Docket No. 55171.4010 / WO27. A nucleic acid composition comprising:(a) a first nucleic acid encoding the first polypeptide chain of any one of the preceding claims;(b) a second nucleic acid encoding the second polypeptide chain of any one of the preceding claims; and(c) a third nucleic acid encoding the third polypeptide chain of any one of the preceding claims.

28. An expression vector composition comprising:(a) a first expression vector comprising the first nucleic acid of claim 27;(b) a second expression vector comprising the second nucleic acid of claim 27; and (c) a third expression vector comprising the third nucleic acid of claim 27.

29. A host cell comprising the expression vector composition of claim 28.

30. A method of producing a CEACAM6 targeting T cell-engaging molecule comprising:(a) culturing the host cell of claim 29 under conditions to express the CEACAM6 targeting T cell-engaging molecule, and(b) recovering the CEACAM6 targeting T cell-engaging molecule.

31. A method of treating a cancer in a human subject in need thereof comprising administering to the human subject the CEACAM6 targeting T cell-engaging molecule of any one of claims 1-26.

32. The method of claim 31, wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colorectal cancer, and breast cancer.

33. A VHH antibody or antibody fragment thereof comprising:(i) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 41, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 42, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 43;1284130-6004-4904.10Attorney Docket No. 55171.4010 / WO(ii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 44, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 45, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 46; or(iii) an HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 47, an HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 48, and an HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 49.

34. A VHH antibody or antibody fragment thereof comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

35. The VHH antibody or antibody fragment thereof of claim 34, comprising an amino acid sequence of any one of SEQ ID NOS: 10-13, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192.

36. The VHH antibody or antibody fragment thereof of any one of claims 34-35, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13, wherein the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of SEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

37. The VHH antibody or antibody fragment thereof of any one of claims 33-36, comprising an amino acid sequence of SEQ ID NO: 13, wherein the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of SEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

38. The VHH antibody or antibody fragment thereof of any one of claims 33-35, comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-12, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192, wherein the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of1294130-6004-4904.10Attorney Docket No. 55171.4010 / WOSEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

39. The VHH antibody or antibody fragment thereof of any one of claims 33-35 and 38, comprising an amino acid sequence of any one of SEQ ID NOS: 10-12, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 160-166, 179, 186, and 192, wherein the VHH antibody or antigen binding fragment thereof binds human CEACAM6 with a lower binding affinity compared to a reference VHH antibody or antibody fragment thereof of SEQ ID NO: 27 or compared to a high binding affinity anti-CEACAM6 VHH antibody or antibody fragment thereof.

40. The VHH antibody or antibody fragment thereof of any one of claims 33-39, wherein the VHH antibody or antibody fragment has a higher binding affinity to human CEACAM6 compared to non-primate CEACAM6.

41. The VHH antibody or antibody fragment thereof of any one of claims 33-40, further comprises an Fc domain or a variant thereof.

42. The VHH antibody or antibody fragment thereof of claim 41, wherein the VHH antibody or antibody fragment thereof comprising the Fc domain forms a VHH dimeric antibody or antibody fragment thereof by dimerization of the Fc domains.

43. A nucleic acid composition comprising a nucleic acid encoding the VHH antibody or antigen binding fragment thereof of any one of claims 33-42.

44. An expression vector comprising the nucleic acid composition of claim 43.

45. A host cell comprising the expression vector of claim 44.

46. A method of producing a VHH antibody or antigen binding fragment comprising:(a) culturing the host cell of claim 45 under conditions to express the VHH antibody or antigen binding fragment, and(b) recovering the VHH antibody or antigen binding fragment.

47. A method of treating a cancer in a human subject in need thereof comprising administering to the human subject the VHH antibody or antigen binding fragment of any one of claims 33-42.1304130-6004-4904.10Attorney Docket No. 55171.4010 / WO48. The method of claim 47, wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colorectal cancer, and breast cancer.1314130-6004-4904.10

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