CN121152803A

CN121152803A - CEACAM6 binding antibodies and antigen binding fragments thereof

Info

Publication number: CN121152803A
Application number: CN202480028990.1A
Authority: CN
Inventors: S·H·卡斯默-罗德里格斯; 陈韦豪
Original assignee: Hundred Jin Co
Current assignee: Hundred Jin Co
Priority date: 2023-05-25
Filing date: 2024-05-21
Publication date: 2025-12-16
Also published as: WO2024243217A1

Abstract

Provided herein are antibodies, including antigen-binding fragments thereof, that bind to all or a portion of CEACAM6, compositions containing such antibodies or antigen-binding fragments thereof, combinations of such antibodies or antigen-binding fragments thereof, and methods of use. In specific embodiments, the antibodies or antigen binding fragments thereof are used in methods of detecting the presence of CEACAM6 by imaging, including molecular, medical, and diagnostic imaging.

Description

CEACAM6 binding antibodies and antigen binding fragments thereof

Related patent application

The present patent application claims the benefit of U.S. provisional patent application No. 63/468,990, filed 5/25/2023, entitled CEACAM6 binding antibodies and antigen binding fragments thereof, identified Susannah Kassmer et al as inventors, and assigned by attorney docket No. 102738-1365417-002900 US. The entire contents of the foregoing patent application are incorporated herein by reference for all purposes, including all text, tables, and figures.

Technical Field

The present disclosure relates in some aspects to antibodies or antigen-binding fragments thereof that bind CEACAM6, and methods, systems, and kits for detecting CEACAM 6. In certain aspects, the disclosure relates to antibodies, or antigen-binding fragments thereof, for use in determining CEACAM6 levels in a sample containing or suspected of containing CEACAM 6. In some aspects, the disclosure relates to antibodies, or antigen-binding fragments thereof, for use in diagnosing or treating an individual having or suspected of having a disease or disorder associated with CEACAM 6.

Background

CEACAM6, also known as CD66c, is overexpressed in several cancer types (e.g., ovarian, colon, breast, and non-small cell lung cancer) and promotes cancer progression by inducing epithelial-to-mesenchymal transition and metastasis. CEACAM6 is an immune checkpoint inhibitor in hematological malignancies.

Disclosure of Invention

Provided herein are antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof that specifically bind CEACAM6, e.g., human CEACAM6, wherein the antibodies or antigen-binding fragments contain specific Complementarity Determining Regions (CDRs) including heavy chain CDRs (i.e., CDRH1, CDRH2, and/or CDRH 3) and light chain CDRs (i.e., CDRL1, CDRL2, and/or CDRL 3), e.g., any of the CDRs described herein. In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, such as any of the variable domains described herein.

Provided herein are antibodies or antigen binding fragments thereof that bind CEACAM6 or a portion thereof, comprising a) an immunoglobulin heavy chain variable domain comprising (I) a heavy chain complementarity determining region 1 (CDRH 1), the CDRH1 comprising the sequence GFX1X2SX3YGX X5 (SEQ ID NO: 18), wherein X1 is T or S, X2 is F or L, X3 is N or T, X4 is M or NO amino acid, and X5 is G or NO amino acid; (ii) a heavy chain complementarity determining region 2 (CDRH 2), the CDRH2 comprising the sequence IX1X2X3X4X5X6X7 (SEQ ID NO: 19), wherein X1 is a or W, X2 is N or W, X3 is S or D, X4 is G or D, X5 is G or D, X6 is T or K, and X7 is T or NO amino acid; and (iii) heavy chain complementarity determining region 3 (CDRH 3), the CDRH3 comprising the sequence X1X2X3X4X5GX6X7X8X9X10X11 (SEQ ID NO: 20), wherein X1 is T or A, X2 is T or R, X3 is L or I, X4 is K or L, X5 is F or L, X6 is A or F, X7 is G or D, X8 is G or Y, X9 is F or an amino acid-free, X10 is A or an amino acid-free, and X11 is Y or an amino acid-free, and b) an immunoglobulin light chain variable domain comprising (I) a light chain complementarity determining region 1 (CDRL 1), the CDRL1 comprising the sequence X1SX2X3X4X5X6X7X8X9X10X11 (SEQ ID NO: 21), wherein X1 is Q or K, X2 is L or I, X3 is L or S, X4 is G or Y, X5 is an amino acid-free, X5 is K or an amino acid-free, and X10 is an amino acid-free, and b) an immunoglobulin light chain variable domain comprising (I) light chain complementarity determining region 1 (CDRL 1, and X11 is Y or NO amino acid, (ii) a light chain complementarity determining region 2 (CDRL 2), said CDRL2 comprising the sequence X1X2S (SEQ ID NO: 22) wherein X1 is W or S and X2 is A or G, and (iii) a light chain complementarity determining region 3 (CDRL 3), said CDRL3 comprising the sequence QX1YX2X3X4PX5T (SEQ ID NO: 3) wherein X1 is Q or H, X2 is Y or N, X3 is I or E, X4 is F or Y, and X5 is N or L.

In some embodiments, the immunoglobulin heavy chain variable domain comprises a CDRH1 comprising an amino acid sequence shown in SEQ ID NO 6 or 12, or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 6 or 12, a CDRH2 comprising an amino acid sequence shown in SEQ ID NO 7 or 13, or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 6 or 12, and a CDRH3 comprising an amino acid sequence shown in SEQ ID NO 8 or 14, or an amino acid sequence showing at least 80%, 81%, 82%, 84%, 86%, 88%, 92%, 94%, 95%, 96%, 97%, 99% or more sequence identity to SEQ ID NO 8 or 14, or a CDRH 3.

In some embodiments, the immunoglobulin heavy chain variable domain comprises a CDRH1 comprising the amino acid sequence shown in SEQ ID NO. 6 or 12, a CDRH2 comprising the amino acid sequence shown in SEQ ID NO. 7 or 13, and a CDRH3 comprising the amino acid sequence shown in SEQ ID NO. 8 or 14.

In some embodiments, the immunoglobulin light chain variable domain comprises a CDRL1 comprising an amino acid sequence shown in SEQ ID NO 9 or 15, or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 9 or 15, a CDRL2 comprising an amino acid sequence shown in SEQ ID NO 10 or 16, or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 9 or 15, and a CDRL3 comprising an amino acid sequence shown in SEQ ID NO 11 or 17, or an amino acid sequence showing at least 80%, 81%, 82%, 84%, 86%, 88%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 11 or 17, or a CDRL2 comprising an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 95%, 96%, 98% or more sequence.

In some embodiments, the immunoglobulin light chain variable domain comprises a CDRL1 comprising the amino acid sequence shown in SEQ ID NO. 9 or 15, a CDRL2 comprising the amino acid sequence shown in SEQ ID NO. 10 or 16, and a CDRL3 comprising the amino acid sequence shown in SEQ ID NO. 11 or 17.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 6, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 6, CDRH2 comprises the sequence shown in SEQ ID NO. 7, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 7, and CDRH3 comprises the sequence shown in SEQ ID NO. 8, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 8.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 6, CDRH2 comprises the sequence shown in SEQ ID NO. 7, and CDRH3 comprises the sequence shown in SEQ ID NO. 8.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 12, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 12, CDRH2 comprises the sequence shown in SEQ ID NO. 13, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 13, and CDRH3 comprises the sequence shown in SEQ ID NO. 14, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 14.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 12, CDRH2 comprises the sequence shown in SEQ ID NO. 13, and CDRH3 comprises the sequence shown in SEQ ID NO. 14.

In some embodiments, CDRL1 comprises the sequence shown in SEQ ID NO. 9, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 9, CDRL2 comprises the sequence shown in SEQ ID NO. 10, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 10, and CDRL3 comprises the sequence shown in SEQ ID NO. 11, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 11.

In some embodiments, CDRL1 comprises the sequence shown in SEQ ID NO. 9, CDRL2 comprises the sequence shown in SEQ ID NO. 10, and CDRL3 comprises the sequence shown in SEQ ID NO. 11.

In some embodiments, CDRL1 comprises the sequence shown in SEQ ID NO. 15, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 15, CDRL2 comprises the sequence shown in SEQ ID NO. 16, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 16, and CDRL3 comprises the sequence shown in SEQ ID NO. 17, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 17.

In some embodiments, CDRL1 comprises the sequence shown in SEQ ID NO. 15, CDRL2 comprises the sequence shown in SEQ ID NO. 16, and CDRL3 comprises the sequence shown in SEQ ID NO. 17.

In some embodiments, CDRH1 comprises the amino acid sequence shown in SEQ ID NO. 6 or 12, CDRH2 comprises the amino acid sequence shown in SEQ ID NO. 7 or 13, CDRH3 comprises the amino acid sequence shown in SEQ ID NO. 8 or 14, CDRL1 comprises the amino acid sequence shown in SEQ ID NO. 9 or 15, CDRL2 comprises the amino acid sequence shown in SEQ ID NO. 10 or 16, and CDRL3 comprises the amino acid sequence shown in SEQ ID NO. 11 or 17.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID No. 6, or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 6; CDRH2 comprises the sequence shown in SEQ ID No. 7, or an amino acid sequence which shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 7; CDRH3 comprises the sequence shown in SEQ ID NO. 8, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 8, CDRL1 comprises the sequence shown in SEQ ID NO. 9, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 9, CDRL2 comprises the sequence shown in SEQ ID NO. 10, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86% or 86% of the sequence shown in SEQ ID NO. 10 96%, 97%, 98%, 99% or more sequence identity, and CDRL3 comprises the sequence shown in SEQ ID NO. 11 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 11.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 6, CDRH2 comprises the sequence shown in SEQ ID NO. 7, CDRH3 comprises the sequence shown in SEQ ID NO. 8, CDRL1 comprises the sequence shown in SEQ ID NO. 9, CDRL2 comprises the sequence shown in SEQ ID NO. 10, and CDRL3 comprises the sequence shown in SEQ ID NO. 11.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 12, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 12; CDRH2 comprises the sequence shown in SEQ ID No. 13, or an amino acid sequence which shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 13; CDRH3 comprises the sequence shown in SEQ ID NO. 14, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 14, CDRL1 comprises the sequence shown in SEQ ID NO. 15, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 15, CDRL2 comprises the sequence shown in SEQ ID NO. 16, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86% or more sequence identity to SEQ ID NO. 16 96%, 97%, 98%, 99% or more sequence identity, and CDRL3 comprises the sequence shown in SEQ ID NO: 17, or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 12, CDRH2 comprises the sequence shown in SEQ ID NO. 13, CDRH3 comprises the sequence shown in SEQ ID NO. 14, CDRL1 comprises the sequence shown in SEQ ID NO. 15, CDRL2 comprises the sequence shown in SEQ ID NO. 16, and CDRL3 comprises the sequence shown in SEQ ID NO. 17.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID NO. 2 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 2 or 4.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in any one of SEQ ID NOs 2 or 4.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID NO. 2, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 2.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in any one of SEQ ID NOs.2.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID NO. 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 4.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in any one of SEQ ID NOs.4.

In some embodiments, the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 3 or 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 3 or 5.

In some embodiments, the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 3 or 5.

In some embodiments, the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 3, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 3.

In some embodiments, the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 3.

In some embodiments, the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 5.

In some embodiments, the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 5.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID NO. 2 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 2 or 4, and the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 3 or 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 3 or 5.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID NO. 2 or 4, and the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 3 or 5.

In some embodiments, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO. 2, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 2, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO. 3, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 3.

In some embodiments, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO. 2, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO. 3.

In some embodiments, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO. 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 4, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO. 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO. 5.

In some embodiments, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO. 4, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO. 5.

In some embodiments, the antibody or antigen binding fragment comprises one immunoglobulin heavy chain variable domain and one immunoglobulin light chain variable domain.

In some embodiments, the antibody or antigen binding fragment comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.

In some embodiments, the antibody or antigen binding fragment thereof is isolated.

In some embodiments, the antibody or antigen binding fragment thereof is humanized.

In some embodiments, the antibody or antigen binding fragment thereof is conjugated.

In some embodiments, the antibody or antigen binding fragment further comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises a sample barcode sequence. In some embodiments, the oligonucleotide comprises binding sites for the primer and the anchor.

In some embodiments, the antibody or antigen binding fragment thereof is conjugated to a detectable marker or tag. In some embodiments, the detectable marker or tag is conjugated directly to the antigen or antigen binding fragment thereof. In some embodiments, the detectable marker or tag is conjugated to an oligonucleotide. In some embodiments, the detectable marker or tag comprises a detectable moiety. In some embodiments, the detectable moiety is a radioisotope, a fluorescent tag, or an enzyme-substrate tag.

In some embodiments, the antibody or antigen binding fragment thereof is non-diffusively immobilized on a solid support.

In some embodiments, the present disclosure provides an isolated antibody that specifically binds CEACAM6, wherein the isolated antibody competes with an antibody described herein for binding to CEACAM6.

In some embodiments of the antibodies described herein, the antibodies are monoclonal antibodies. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody comprises one or more human framework regions. In some embodiments, the antibody or antigen binding fragment is a single chain fragment. In some embodiments, the single-stranded fragment is a single-stranded variable fragment (scFv).

In some aspects, provided herein are combinations of antibodies or antigen-binding fragments thereof, wherein the combinations comprise two or more anti-CEACAM 6 antibodies or antigen-binding fragments described herein. In some embodiments, the two or more antibodies or antigen-binding fragments comprise one or more first antibodies or antigen-binding fragments thereof that bind a first epitope or region within CEACAM6 and one or more second antibodies or antigen-binding fragments thereof that bind a second epitope or region within CEACAM6. In some further embodiments, the one or more first antibodies or antigen-binding fragments thereof and the one or more second antibodies or antigen-binding fragments thereof bind to non-overlapping epitopes or regions of CEACAM6 (e.g., human CEACAM 6) and/or do not compete for binding to CEACAM6. Furthermore, in some embodiments, the antibody is conjugated to a detectable marker or tag. In some embodiments, at least one antibody or antigen-binding fragment of a combination of two or more anti-CEACAM 6 antibodies or antigen-binding fragments described herein, optionally one or more first antibodies or antigen-binding fragments thereof or one or more second antibodies or antigen-binding fragments thereof, is conjugated to a tag. In some embodiments, the at least one antibody or antigen-binding fragment, optionally one or more primary antibodies or antigen-binding fragments thereof, or one or more secondary antibodies or antigen-binding fragments thereof, is attached or immobilized to a solid support. In some embodiments, the one or more first or second antibodies or antigen binding fragments are attached or immobilized to a solid support, and the other of the one or more first or second antibodies or antigen binding fragments is conjugated to a tag. In some embodiments, the tag is a fluorescent dye, fluorescent protein, radioisotope, chromophore, metal ion, gold particle, silver particle, magnetic particle, polypeptide, enzyme, streptavidin, biotin, luminescent compound, or oligonucleotide. In some embodiments, the solid support is a bead, column, array, assay plate, microwell, rod, filter, or strip. In certain embodiments, the antibody is non-diffusively immobilized on a solid support. In further embodiments, the device is a rapid detection device or a rapid diagnostic device.

In another aspect, the disclosure features an isolated nucleic acid encoding an isolated antibody described herein. The present disclosure also provides expression vectors comprising the nucleic acids described herein. In addition, the present disclosure also provides isolated host cells comprising the expression vectors described herein.

In some embodiments, the antibodies or antigen binding fragments thereof provided herein can be used to detect CEACAM6 in a sample. In some embodiments, the antibody or antigen binding fragment thereof binds to CEACAM6 expressing cells in the sample. In some embodiments, the sample comprises immune cells. In some embodiments, the sample comprises a heterogeneous population of immune cells. In some embodiments, the immune cells are selected from the group consisting of B cells, plasmacytoid dendritic cells (pdcs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mdcs), resident lymphocytes, mast cells, eosinophils, basophils, natural killer cells, and Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, the sample comprises cells having a disease or disorder. In some embodiments, the disease or disorder is cancer, an autoimmune disorder, an inflammatory disorder, a neurological disorder, or an infection. In some embodiments, the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal cancer, ovarian cancer, breast cancer, gynecological cancer, liver cancer, glioblastoma, hodgkin's lymphoma, chronic lymphocytic leukemia, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, and melanoma. In some embodiments, the detecting comprises using a single antibody or antigen binding fragment thereof to bind a portion of CEACAM6. In some embodiments, the detection comprises the use of two antibodies or antigen binding fragments thereof, each capable of binding to a different portion of CEACAM6. In some embodiments, detection of CEACAM6 is performed on the cell surface. In some embodiments, detection of CEACAM6 is performed intracellularly. In some embodiments, detection of CEACAM6 indicates the presence or absence of a disease or disorder. In some embodiments, the detection is performed in vitro. In some embodiments, the detection is performed in vivo.

In some embodiments, the antibody or antigen binding fragment thereof binds to cells expressing CEACAM 6.

Provided herein is a diagnostic antibody or antigen-binding fragment thereof, comprising any one of the antibodies or antigen-binding fragments thereof described herein. Provided herein is a kit comprising an antibody or antigen-binding fragment thereof of any one of the embodiments described herein. In some embodiments, the kit is a diagnostic kit configured to detect CEACAM6 in a biological sample.

Provided herein is a composition comprising an antibody or antigen-binding fragment thereof of any one of the embodiments described herein and a pharmaceutically acceptable excipient. In some embodiments, the antibody or antigen binding fragment thereof is used as an adjuvant or in combination with an adjuvant.

Provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an agent of any of the embodiments described herein. Also provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of any of the reagents of any of the embodiments described herein. The sequence encoding the immunoglobulin heavy chain variable domain of the agent of any of the embodiments described herein and the sequence encoding the immunoglobulin light chain variable domain of the agent of any of the embodiments described herein can be on the same isolated nucleic acid or different isolated nucleic acids. Thus, also provided herein is an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of an antibody or antigen binding fragment thereof of any of the embodiments described herein.

Provided herein are recombinant expression vectors comprising an isolated nucleic acid of any one of the embodiments described herein. Provided herein are recombinant expression vectors comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of any one of the embodiments described herein, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of an antibody or antigen binding fragment thereof of any one of the embodiments described herein.

Provided herein are recombinant expression vectors comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence of any one of the embodiments described herein, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence of any one of the embodiments described herein. In some embodiments, the first and second expression cassettes comprise promoters.

Provided herein are host cells transfected with the recombinant expression vectors of any of the embodiments described herein.

Provided herein are reagent-drug conjugates comprising an antibody or antigen-binding fragment thereof of any one of the embodiments described herein. Provided herein are compositions comprising an antibody-drug conjugate and a pharmaceutically acceptable carrier.

Provided herein is a method of detecting CEACAM6 comprising a) contacting a sample with an antibody or antigen binding fragment thereof of any one of the embodiments described herein under conditions that allow binding of the antibody or antigen binding fragment thereof to a CEACAM6 receptor on the sample, wherein the binding results in the production of a receptor/antibody or antigen binding fragment complex thereof, b) detecting the presence of the receptor/antibody or antigen binding fragment complex thereof, and c) wherein the detecting comprises the presence or absence of a CEACAM6 receptor on the sample.

Provided herein is a method of treating or preventing a disease or disorder associated with CEACAM6 in a subject, comprising a) contacting a sample known or suspected to contain CEACAM6 with an antibody or antigen binding fragment thereof of any one of the embodiments described herein, b) detecting the presence of a complex comprising CEACAM6 and the antibody or antigen binding fragment thereof, wherein the presence of the complex is indicative of the presence of the disease or disorder, and c) administering to the subject the antibody or antigen binding fragment thereof of any one of the embodiments described herein.

Provided herein is a method of diagnosing a disease or disorder comprising a) isolating a sample from a subject, b) incubating the sample with an antibody or antigen-binding fragment thereof of any of the embodiments described herein for a time sufficient to produce CEACAM6: anti-CEACAM 6 complexes, c) detecting the presence or absence of CEACAM6: anti-CEACAM 6 complexes from the isolated tissue, and d) correlating the presence or abundance of CEACAM6 to a target location of the tissue sample. In some embodiments, an increase in CEACAM6 over a control level in a target location of a tissue sample is indicative of a disease or disorder in a subject.

In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the detection comprises an intracellular detection. In some embodiments, the detecting comprises detecting on the surface of a cell. In some embodiments, the detection comprises hybridization of a detectable moiety to the antibody or antigen binding fragment thereof. In some embodiments, the sample is contacted with a second antibody. In some embodiments, the second antibody is an antibody comprising a detectable moiety. In some embodiments, the detectable moiety comprises an oligonucleotide. In some embodiments, the detectable moiety comprises a fluorescent tag. In some embodiments, the detecting comprises sequencing. In some embodiments, the detectable moiety comprises immunofluorescence. In some embodiments, the sample is a formalin fixed paraffin embedded sample. In some embodiments, the sample comprises cells. In some embodiments, the sample comprises a tissue sample.

In some embodiments, the sample comprises immune cells. In some embodiments, the immune cells are selected from the group consisting of B cells, plasmacytoid dendritic cells (pdcs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mdcs), resident lymphocytes, mast cells, eosinophils, basophils, natural killer cells, and Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, the sample comprises tissue or cells associated with a disease or disorder. In some embodiments, the disease or disorder is cancer, an autoimmune disorder, an inflammatory disorder, or an infection. In some embodiments, the disease or disorder is selected from the group consisting of non-viral cancer, virus-associated cancer, cancer associated with HBV infection, cancer associated with epstein-barr virus (EBV) infection, cancer associated with polyomavirus infection, leprosy Erythema Nodosum (ENL), autoimmune disease, autoimmune inflammation, autoimmune thyroid disease, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemia, myelodysplastic syndrome, solid phase cancer, herpes virus infection, and/or transplanted tissue or organ rejection.

In some embodiments, the antibodies or antigen binding fragments thereof may be used in methods of correlating the presence or abundance of CEACAM6 to a target location of a tissue sample.

In some embodiments, the antibodies or antigen binding fragments thereof may be used in methods of detecting CEACAM6 in a tissue sample. In some embodiments, the method comprises generating a nucleic acid molecule comprising all or a portion of the sequence of the oligonucleotide or the complement thereof.

In some embodiments, the antibodies or antigen binding fragments thereof can be used to construct a protein library. In some embodiments, constructing the protein library includes sequencing. In some embodiments, constructing the protein library includes using flow cytometry.

The present disclosure provides an isolated antibody or antigen binding fragment that binds CEACAM6 or a portion thereof and comprises (i) an immunoglobulin heavy chain comprising a set of heavy chain Complementarity Determining Region (CDR) amino acid sequences CDRH1, CDRH2, and CDRH3, and (ii) an immunoglobulin light chain comprising a set of light chain CDR amino acid sequences CDRL, CDRL2, and CDRL3. The heavy and light chain CDRs of the set are each selected from the same set 1 or set 2:

In at least one embodiment, the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID NO. 4 or a sequence having at least 80% amino acid sequence identity to SEQ ID NO. 4, and wherein the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID NO. 5 or a sequence having at least 80% amino acid sequence identity to SEQ ID NO. 5.

In another embodiment, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO. 2 or a sequence having at least 80% amino acid sequence identity to SEQ ID NO. 2, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO. 3 or a sequence having at least 80% amino acid sequence identity to SEQ ID NO. 3.

The disclosure features a diagnostic antibody, or antigen-binding fragment thereof, comprising any one of the embodiments described herein.

In at least one embodiment, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof of any one of the embodiments described herein.

The present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of any one of the embodiments described herein and a pharmaceutically acceptable excipient.

Provided herein is an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an antibody or antigen binding fragment thereof of any one of the embodiments described herein.

Provided herein is a recombinant expression vector comprising an isolated nucleic acid of any one of the embodiments described herein.

The present disclosure further provides a host cell comprising a nucleic acid or expression vector of any one of the embodiments described herein.

In one embodiment, the isolated nucleic acid comprises a nucleotide sequence, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising SEQ ID NO. 4 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 4, or an immunoglobulin light chain comprising SEQ ID NO. 5 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 5.

In at least one embodiment, the isolated nucleic acid comprises a nucleotide sequence, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising SEQ ID NO.2 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO.2, or an immunoglobulin light chain comprising SEQ ID NO.3 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 3.

The disclosure features a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of any one of the embodiments described herein, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of an antibody or antigen binding fragment thereof of any one of the embodiments described herein.

Provided herein is a method of detecting CEACAM6, wherein the method comprises contacting a sample with an antibody or antigen binding fragment thereof of any one of the embodiments described herein under conditions that allow the antibody or antigen binding fragment thereof to bind to CEACAM6 receptors in the sample, wherein the binding results in the production of a receptor/antibody or antigen binding fragment complex thereof.

Drawings

The drawings illustrate certain embodiments of the technology and are not limiting. The figures are not drawn to scale for clarity and ease of illustration, and in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

FIG. 1 anti-CEACAM 6 antibodies AB1 and AB2 did not show any binding to CEACAM8 (CD 66 b) on RBL-1 cells transfected with CEACAM 8. The histograms of the negative control and anti-CEACAM 6 antibodies show a comparable single peak in the low fluorescence intensity range, indicating the absence of any bound anti-CEACAM 6 antibody. RBL-1 cells were transfected with CEACAM8 and stained with two different amounts of anti-CEACAM 6 antibody AB1, AB2 and commercially available anti-CEACAM 6 antibody REA414 and (iv) anti-CEACAM 1/5/6 antibody ASL-32, wherein the commercially available antibodies were used as negative controls. An Allophycocyanin (APC) -labeled anti-human IgG1 recombinant antibody (BioLegend catalog number 403506, clone QA16a 12) was used as isotype control. anti-CEACAM 6 antibodies were detected using APC-labeled anti-rat IgG secondary antibodies (APC-hCD 66c, X-axis).

FIG. 2 shows that no staining of lymphocytes by AB1 and AB2 was observed based on the very low number of fluorescent signals in the upper right quadrants Q2 and Q6, respectively. FIG. 2A-Co-staining of lymphocytes with anti-human CEACAM6 antibody AB1 detected using PE-labeled anti-rat IgG secondary antibody (PE-A, X-axis in left and right density panels) or anti-CD 19 antibody labeled with Brilliant Violet 605 (TM) (CD 19 BV605, Y-axis, left density panels) or anti-CD 3 antibody labeled with fluorescein isothiocyanate (CD 3 FITC, Y-axis, right density panels). FIG. 2B-lymphocyte co-staining with anti-human CEACAM6 antibody clone AB2 detected using PE-labeled anti-rat IgG secondary antibody (PE-A, X-axis in left and right density panels) or CD19 BV605 (left density panels) or CD3 FITC (right density panels).

FIG. 3 CEACAM6 on granulocytes and monocytes was detected by both anti-CEACAM 6 antibodies AB1 and AB2 based on high fluorescence signal density in the upper right Q2 quadrant. Peripheral blood leukocytes were stained with anti-human CEACAM6 antibody AB1 (fig. 3A) and anti-human CEACAM6 antibody clone AB2 (fig. 3B), which were detected using PE-labeled anti-rat IgG secondary antibody (PE-a, X-axis in left and right density plots). For detection of anti-CEACAM 6 antibodies binding to CEACAM6 on monocytes, cells were co-stained with APC-Cy 7-labeled anti-CD 14 antibodies (APC-Cy 7, Y-axis, left density plot). For detection of anti-CEACAM 6 antibodies that bind to CEACAM6 on granulocytes, the cells are gated against side scatter (SSC, Y-axis, right density plot).

FIG. 4A color dot plot from titration experiments shows that at 0.1 ug, both anti-CEACAM 6 antibodies AB1 and AB2 show higher fluorescence intensities than the two reference clones ASL-32 and REA414 (see black square box). Peripheral blood leukocytes were stained with 7 different amounts of anti-human CEACAM6 antibody AB1 (bottom row), AB2 (penultimate row), reference clone REA414 (second row) or reference clone ASL-32 (first row) ranging from 0.001 ug to 1 ug. anti-CEACAM 6 antibodies were detected using APC-labeled anti-rat IgG secondary antibodies (APC-hCD 66c, Y-axis).

FIG. 5 both anti-CEACAM 6 antibodies AB1 (FIG. 5B) and AB2 (FIG. 5A) blocked the binding of PE-labeled reference clone KOR-SA3544 (KOR-SA 3544-PE) to CEACAM6 on granulocytes as indicated by the left shift of the light gray peak. Peripheral blood leukocytes were stained directly with PE-labeled reference clone KOR-SA3544 (KOR-SA 3544-PE, comp-PE-A, X-axis, dark gray histogram), or first pre-incubated with anti-human CEACAM6 antibody AB1 (FIG. 5B) or AB2 (FIG. 5A), followed by staining with KOR-SA3544-PE (light gray histogram). The data shown are gated on granulocyte populations.

FIG. 6 both anti-CEACAM 6 antibodies AB1 and AB2 block CEACAM 1/5/6-specific reference antibody ASL-32 from binding to CEACAM6 as indicated by the left shift of the light gray peak in FIGS. 6C and 6D. AB1 and AB2 did not block CEACAM8 (CD 66B) -specific reference antibody 6/40c-PE from binding to CD66B, as indicated by the light gray and dark gray peaks stacked on top of each other in fig. 6A and 6B. Peripheral blood leukocytes were incubated with AB1 or AB2 for 15 minutes, followed by staining with PE-labeled reference antibody ASL-32 (ASL-32-PE) or PE-labeled reference antibody 6/40c (6/40 c-PE). The data shown are gated on granulocyte populations.

FIG. 7 shows that at a concentration of 1 antibody per million cells, the brightness of both anti-CEACAM 6 antibodies AB1 and AB2 is at least twice that of the reference antibody KOR-SA 3544. Peripheral blood leukocytes from two different donors were stained with PE-labeled AB1 (AB 1, light grey plot) or PE-labeled AB2 (AB 2, dark grey plot) or PE-labeled KOR-SA3544 (KOR-SA 3544, grey triangle data points). The data shown are gated on granulocyte populations.

FIG. 8 anti-CEACAM 6 antibody AB1 reacted with CEACAM6 on A549 cells as indicated by the right shift of the AB1-PE peak compared to the isotype control peak in FIG. 8A. AB1 was not cross-reactive with other CEACAM family members as indicated in fig. 8B-E by AB1-PE peaks and isotype control peaks stacked on top of each other. anti-CEACAM 6 antibody AB2 reacted with both CEACAM6 and CEACAM4 as indicated by the right shift of AB2-PE peak compared to isotype control peak in fig. 8A, 8B and 8D. Cell lines A549, U937, A431, THP-1 and LNCaP were stained with PE-labeled AB1 (AB 1-PE) or PE-labeled AB2 (AB 2-PE) or PE-labeled rat IgG2a, k isotype control (isotype control).

FIG. 9 anti-CEACAM 6 antibody AB1 was detected by Immunohistochemistry (IHC) for CEACAM6 expression in colon epithelial cells as indicated by fluorescent signal in the highlighted area (left fluorescent image, white squares). AB2 was unable to detect CEACAM6 expression as indicated by the absence of any fluorescent signal (right fluorescent image). Human paraffin-embedded colon tissue was subjected to heat-mediated antigen repair by sodium citrate and incubated with 5 μg/ml purified anti-human CEACAM6 antibodies AB1 (left side of fig. 9) and AB2 (right side of fig. 9), followed by staining with anti-rat IgG Alexa Fluor 555 secondary antibody. Nuclei were counterstained with DAPI dye. The image was captured by a 10x objective lens.

FIG. 10 detection of expression of CEACAM6 in human lung adenocarcinoma cell line A549 by Immunocytochemistry (ICC) by anti-CEACAM 6 antibody AB1, as indicated by fluorescence signals in the left and middle fluorescent images. A549 cells were grown on 96-well plates with coverslips bottom and fixed with fixing buffer (catalog number 420801) for 30 minutes. Cells were washed twice with PBS and stained with 5ug/ml Ultra-LEAFTM purified anti-human CEACAM6 antibody AB1 followed by Alexa Fluor 555-labeled anti-rat IgG secondary antibody (catalogue number 405420). Rat IgG2a, k isotype control antibody was used as negative control (isotype, right fluorescence image in fig. 10). Nuclei were counterstained with DAPI dye (catalog number 422801). Cells were imaged using a 40x objective.

FIG. 11 anti-CEACAM 6 antibody AB1 blocked lung adenocarcinoma cell invasion as indicated by a significant reduction in cell number in each field of view for all three tested AB1 concentrations. The effect of different concentrations of Ultra-LEAFTM purified anti-human CEACAM6 antibody AB1 on the ability to inhibit cell invasion by extracellular matrix was tested by matrigel invasion assay. 20A commercial available antibody 1H7-4B (reference) of ug/ml was used as CEACAM6 antibody reference. Human lung adenocarcinoma cell line a549 was serum starved for 24h and inoculated onto the top layer wells of Corning bio coat matrix invar. Rat IgG2a, k isotype antibodies were used as controls (isotypes). DMEM medium containing 20% serum was placed in the bottom wells as a chemoattractant. After 16h, cells that have migrated to the bottom of the membrane were counted.

FIG. 12 anti-CEACAM 6 antibody AB1 blocks lung adenocarcinoma cell migration. The effect of Ultra-LEAFTM purified anti-human CEACAM6 antibody AB1 on the ability to block cell migration was tested by a wound healing assay. Human lung adenocarcinoma cell line a549 grew to confluence and a line was drawn using a 10 ul pipette tip. Rat IgG2a, k isotype antibodies were used as controls (isotypes). After 16h, gap width was measured using Image J and the percentage of gap closure normalized to isotype control was calculated.

FIG. 13 crosslinking of CEACAM6 with AB1 induces signaling and increases actin polymerization by activating Akt phosphorylation, as indicated by a significant increase in fluorescence signal and intensity when comparing lower right and lower left fluorescence images and upper right and upper left fluorescence images. The effect of AB1 on cross-linking to induce CEACAM6 signaling was tested on human lung adenocarcinoma cell line a 549. Cells were grown on 96-well plates with coverslips bottom and treated with 5ug/ml Ultra-LEAFTM purified anti-human CEACAM6 antibody AB1 or rat IgG2a, k isotype control (isotype) for 15 minutes at 37 ℃, followed by 30 minutes at 37 ℃ with anti-rat IgG secondary antibody (5 ug/ml). Cells were fixed with the fixation buffer for 30 min. Cells were washed twice with 1x intracellular staining permeabilization wash buffer, then stained with anti-AKT Phospho (Ser 473) antibody (antibody a 21001C), followed by Alexa Fluor 555 anti-mouse IgG antibody, flash Phalloidin ^TM Red 594 dye (imaging filiform F-actin) and DAPI dye (imaging nuclei).

Detailed Description

CEACAM6 is part of the carcinoembryonic antigen (CEA) family and is a Glycosylphosphatidylinositol (GPI) anchored cell surface glycoprotein. CEACAM6 has an amino acid overlap of 84% with CEACAM1, 84-66% with CEACAM5, 79% with CEAMCAM, 68% with CEACAM7, 67% with CEACAM2 and 48% with CEACAM 4. CEACAM6 is a cell adhesion molecule that mediates homotypic binding to other CEA family members and heterotypic binding to integrin receptors. Biological effects of CEACAM6 include neural tissue development, inflammation, immune cell migration, and immune response. CEACAM6 is overexpressed in more than 50% of all human adenocarcinomas. CEACAM6 promotes abnormal cell differentiation, anti-apoptosis, cell growth and resistance to therapeutic agents, cell invasion and metastasis. As described herein, antibodies to CEACAM6 inhibit cell migration, invasion, and adhesion in vitro studies. Furthermore, BAY1834942 is a novel checkpoint inhibitor with the potential to treat patients with CEACAM6 expressing cancers (experiments were completed in 2021 with no published results). CEACAM6 is expressed on epithelial surfaces and is localized to myeloid cells in bone marrow and blood, and to granulocytes and monocytes. CEACAM6 is a myeloid marker with aberrant expression in pre-B cell ALL, strongly correlated with non-random genetic alterations (BCR/ABL rearrangement).

Provided herein are antibodies (including antigen-binding fragments thereof) that bind CEACAM6, nucleic acids encoding such antibodies and antigen-binding fragments, and cells (e.g., recombinant cells) for expressing and producing such antibodies and antigen-binding fragments that bind CEACAM6 under physiological and/or in vitro conditions. Methods of producing and using the antibodies and antigen-binding fragments are also provided, e.g., in methods for detecting CEACAM6 in a sample from an individual, including methods for laboratory/research purposes (e.g., flow cytometry, ELISA, and/or western blotting) and/or methods for treating and/or preventing various diseases or disorders using pharmaceutical or other compositions containing such antibodies or antigen-binding fragments thereof, and by delivering pharmaceutical or other compositions containing such antibodies or antigen-binding fragments thereof.

All references, including patent applications, patent publications, and scientific literature, and databases, cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference.

For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. definition of the definition

Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter belongs. 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 significant departure from the commonly understood meaning in the art. It is to be understood that the disclosure provided herein is not limited to a particular composition or biological system. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "antibody" as used herein includes antigen binding fragments thereof that retain binding specificity. For example, there are many well-characterized antigen binding fragments. Thus, for example, pepsin digests antibodies at the disulfide C-terminal end of the hinge region to produce F (ab)' 2, a dimer of Fab, which is itself a light chain linked to VH-CH1 by disulfide bonds. F (ab) '2 can be reduced under mild conditions to break disulfide bonds in the hinge region, thereby converting the (Fab ') 2 dimer into the Fab ' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (see Fundamental Immunology, w.e. Paul, ed., RAVEN PRESS, N.Y. (1993) for a more detailed description of other antigen binding fragments). Although various antigen binding fragments are defined in terms of digestion of intact antibodies, the skilled artisan will appreciate that the fragments may be synthesized from the new species by chemical means or by using recombinant DNA methods. Thus, the term antibody as used herein also includes antigen binding fragments produced by modification of an intact antibody or synthesized using recombinant DNA methods.

Antibodies as described herein may consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Putative immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes and a number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin types IgG, igM, igA, igD and IgE, respectively. In some embodiments, the antibody is IgG (e.g., igG1, igG2, igG3, igG 4), igM, igA, igD, or IgE.

Typical immunoglobulin (antibody) structural units are known to comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

In antibodies, a substitution variant has at least one amino acid residue removed and a different residue inserted at its position. For substitution mutagenesis, the sites of most interest include the hypervariable regions, but framework changes are also contemplated. Examples of conservative substitutions are described herein.

Significant modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the polypeptide backbone structure of the substituted region, e.g., as a β -sheet or helix conformation, (b) molecular charge or hydrophobicity at the target site, or (c) side chain volume. Naturally occurring residues are classified into the following groups based on the usual side chain properties:

(1) Nonpolar norleucine Met, ala, val, leu, ile;

(2) The polarity is not charged Cys, ser, thr, asn, gln;

(3) Acid (negative charge): asp, glu;

(4) Basic (positive charge) Lys, arg;

(5) Residues affecting chain orientation, gly, pro, and

(6) Aromatic Trp, tyr, phe, his.

Non-conservative substitutions are made by exchanging members of one of these classes for another class.

One type of substitution that may be made is to change one or more cysteines in the antibody that may be chemically reactive to another residue, such as, but not limited to, alanine or serine. For example, there may be substitutions of non-classical cysteines. Substitutions may be made in the CDRs or framework regions of the variable domains of the antibody or in the constant regions. In some embodiments, the cysteine is classical (e.g., involved in disulfide bond formation). Any cysteine residue that does not participate in maintaining the correct conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability, particularly where the antibody is an antigen binding fragment, such as an Fv fragment.

Antibodies include V _H-V_L dimers, which include single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv), in which variable heavy and variable light domains are linked together (either directly or through a peptide linker) to form a continuous polypeptide. A single chain Fv antibody is a covalently linked V _H-V_L that can be expressed from a nucleic acid comprising V _H -and V _L -coding sequences linked directly or through a peptide-coding linker (e.g., huston, et al, proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). Although V _H and V _L are linked to each other as a single polypeptide chain, the V _H and V _L domains associate non-covalently. Or the antibody may be another fragment. Other fragments may also be produced, for example using recombinant techniques, as soluble proteins or as fragments obtained from display methods. Antibodies may also include diabodies and minibodies. Antibodies of the present disclosure also include heavy chain dimers, e.g., antibodies from camels. In some embodiments, the antibody is a dimer. In other embodiments, the antibody may be in monomeric form with an active isotype. In some embodiments, the antibody is in a multivalent form, e.g., a trivalent or tetravalent form.

An "antibody fragment" or "antigen-binding fragment thereof" comprises a portion of an intact antibody, i.e., the antigen-binding and/or variable regions of an intact antibody. Antibody fragments or antigen binding fragments thereof, including but not limited to Fab fragments, fab ' fragments, F (ab ') ₂ fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd ' fragments, diabodies, linear antibodies (see U.S. Pat. No. 5,641,870, example 2; zapata et al, protein eng. 8 (10): 1057-1062 [1995 ]), single chain antibody molecules, including single chain Fv (scFv) or single chain Fab (scFab), antigen binding fragments of any of the above, and multispecific antibodies from antibody fragments.

"Fv" is composed of one heavy chain variable region domain and one light chain variable region domain connected by a non-covalent association. From the folding of these two domains, 6 Complementarity Determining Regions (CDRs) (3 each in the heavy and light chains) are obtained that promote antigen binding by amino acid residues and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half Fv comprising only 3 CDRs with specificity for an antigen) has the ability to recognize and bind antigen, although in some cases the affinity is lower than the entire binding site.

"DsFv" refers to Fv's having engineered intermolecular disulfide bonds that stabilize the VH-VL pair.

An "Fd fragment" is an antibody fragment that contains the variable domain (VH) and one constant region domain (CHI) of the antibody heavy chain.

"Fab fragments" are fragments of antibodies resulting from digestion of full-length immunoglobulin with papain, or synthetically produced, e.g., fragments of identical structure produced by recombinant means. The Fab fragment contains a light chain (containing VL and CL) and another chain containing the variable domain of the heavy chain (VH) and one constant region domain of the heavy Chain (CHI).

"F (ab') 2 fragments" are antibody fragments resulting from the digestion of immunoglobulins with pepsin at pH 4.0-4.5, or synthetically produced, e.g., fragments of the same structure produced by recombinant methods. The F (ab') 2 fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises additional amino acids, including cysteine residues that form disulfide bonds linking the two fragments.

"Fab 'fragments" are fragments containing half of the F (ab') 2 fragment (one heavy and one light chain).

An "Fd 'fragment" is an antibody fragment containing one heavy chain portion of a F (ab') 2 fragment.

An "Fv' fragment" is a fragment that contains only the VH and VL domains of an antibody molecule.

"ScFv fragment" refers to an antibody fragment containing a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker. The length of the linker is such that the two variable domains are bridged without significant interference. Exemplary linkers are (Gly-Ser) n residues, with some Glu or Lys residues interspersed throughout to increase solubility.

A "diabody" is a dimeric scFv, and diabodies typically have a shorter peptide linker than scFv and dimerize preferentially.

As used herein, the terms "variable region" and "variable domain" refer to the portion of an antibody light and heavy chain that includes the amino acid sequences of complementarity determining regions (CDRs, e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3) and Framework Regions (FR). The variable domains for the heavy and light chains are commonly referred to as V _H and V _L, respectively. Variable domains are included on Fab, F (ab') ₂, fv, and scFv antigen-binding fragments described herein and are involved in specific antigen recognition.

As used herein, "Complementarity Determining Regions (CDRs)" refer to three hypervariable regions that truncate in each chain the four framework regions established by the light and heavy chain variable regions. CDRs are mainly responsible for binding to epitopes. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, VH CDR3 is located in the variable domain of the antibody heavy chain in which it is present, while VL CDR1 is CDR1 from the variable domain of the antibody light chain in which it is present.

The framework region sequences of different light or heavy chains are relatively conserved in species. The antibody framework regions, which are the combined framework regions of the constituent light and heavy chains, are used to position and align CDRs in three-dimensional space.

The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., kabat, north methods (see, e.g., north et al, J Mol biol 406 (2): 228-256, 2011), chothia, international ImMunoGeneTics database (IMGT) and AbM (see, e.g., johnson et al, supra, definitions of the ; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J.Mol.Biol 1997, 273(4)). antigen binding sites are also described in ：Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268–9272 (1989); Martin, et al, Methods Enzymol., 203, 121–153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and infra Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141–172 1996).

As used herein, "chimeric antibody" refers to an immunoglobulin molecule in which (a) the constant region or a portion thereof is altered, substituted or exchanged such that the antigen-binding site (variable region) is altered, substituted or exchanged with a constant region of a different or altered class, effector function and/or species, or an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or (b) the variable region or a portion thereof is altered, substituted or exchanged by a variable region or a portion thereof having different or altered antigen specificity, or a corresponding sequence from another species or from another antibody class or subclass.

As used herein, a "humanized antibody" refers to an immunoglobulin molecule in which CDRs from a donor antibody are grafted onto a human framework sequence. Humanized antibodies may also comprise residues of donor origin in the framework sequences. Humanized antibodies may also comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies may also comprise residues that are not present in both the recipient antibody and the imported CDR or framework sequences. Humanization can be performed using methods known in the art (e.g., ,Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No.4,816,567), including techniques such as "super-humanization" antibodies (Tan et al, J. Immunol. 169:1119, 2002) and "surface remodeling" (e.g., STAELENS ET al, mol. Immunol. 43:1243, 2006; and Roguska et al, proc. Natl. Acad. Sci USA 91:969, 1994).

The term "recombinant" when used in reference to, for example, a cell or nucleic acid, protein or vector, means that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or alteration of the native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

The terms "antigen," "immunogen," "antibody target," "target analyte," and the like are used herein to refer to a molecule, compound, or complex that is recognized by an antibody (i.e., can be specifically bound by an antibody). The term may refer to any molecule specifically recognizable by an antibody, such as a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combination thereof (e.g., phosphorylated or glycosylated polypeptide, etc.). The skilled person will understand that the term does not indicate that the molecule is in all cases immunogenic, but only that it can be targeted by an antibody.

Antibodies bind to an "epitope" on an antigen. An epitope is a localized site on an antigen that is recognized and bound by an antibody. An epitope may comprise several amino acids or a part of several amino acids, e.g. 5 or 6 or more, e.g. 20 or more amino acids, or a part of these amino acids. In some cases, the epitope includes a non-protein component, such as from a carbohydrate, a nucleic acid, or a lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope may be composed of contiguous amino acids, or amino acids from different portions of the protein that are pulled into proximity by protein folding (e.g., discontinuous epitopes). The same is true for other types of target molecules that form three-dimensional structures. Epitopes typically comprise at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., epitope Mapping Protocols in Methods in Molecular Biology, vol.66, glenn e.Morris, ed (1996).

A "tag" or "detectable moiety" is a diagnostic agent or component that is detectable by spectroscopic, radiological, photochemical, biochemical, immunochemical, chemical or other physical means. Exemplary tags include radioactive tags (e.g., ¹¹¹In、^99mTc、¹³¹I、⁶⁷ Ga) and other FDA approved imaging agents. Additional tags include ³² P, fluorescent dyes, electron dense reagents, enzymes, biotin, digoxigenin or haptens and proteins or other entities that can be detected, for example, by incorporating a radioactive tag into the targeting agent. Any method known in the art for conjugating a nucleic acid or nanocarrier to a tag may be used, for example using the method described in Hermanson, bioconjugate Techniques 1996, ACADEMIC PRESS, inc.

A "labeled" or "tagged" antibody or reagent is one that binds to the tag covalently (via a linker or chemical bond) or non-covalently (via ionic, van der waals, electrostatic or hydrogen bonding) such that the presence of the antibody or reagent can be detected by detecting the presence of the tag bound to the antibody or reagent.

Techniques for conjugating detectable and therapeutic agents to antibodies are well known (see, e.g., ,Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery"in Controlled Drug Delivery (2^nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)).

The terms "specific for," "specifically binds," and the like refer to a molecule (e.g., an antibody or antigen binding fragment) that binds a target with at least 2-fold affinity, e.g., at least any one of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold affinity, of a non-target compound. For example, antibodies that specifically bind a target (e.g., TMPRSS 2) typically bind the target with at least 2-fold affinity to the non-target. Specificity can be determined Using standard methods, such as solid phase ELISA immunoassays (see, e.g., harlow & Lane, using Antibodies, A Laboratory Manual (1998)) for descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

The term "bind" with respect to an antibody target (e.g., antigen, analyte, immune complex) generally means that the antibody binds to most of the antibody target in a pure population (assuming the appropriate molar ratio). For example, an antibody that binds a given antibody target typically binds at least 2/3 of the antibody target in solution (e.g., at least any one of 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). The skilled artisan will recognize that some variability will occur depending on the method and/or threshold of assay binding.

A "control" sample or value refers to a sample that is used as a reference, typically a known reference, for comparison to a test sample. For example, a test sample may be obtained from test conditions, e.g., in the presence of a test compound, and compared to a sample from known conditions, e.g., in the absence of a test compound (negative control) or in the presence of a known compound (positive control). Controls may also represent averages or ranges collected from multiple tests or results. Those skilled in the art will recognize that the control may be designed to evaluate any number of parameters. For example, a control can be designed to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison benefits and/or side effects). The control may be designed for in vitro use. Those skilled in the art will understand what controls are valuable under given conditions and can analyze the data based on comparison to control values. The control is also valuable for determining data significance. For example, if the values for a given parameter vary greatly in the control, the differences in the test samples will not be considered significant.

In the case of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, within a specified region when compared and aligned for maximum correspondence in the comparison window or specified region), as measured using the BLAST 2.0 sequence comparison algorithm as well as default parameters described below or by manual alignment and visual inspection (see, e.g., NCBI website ncbi.n lm.nih.gov/BLAST/etc.). Such sequences are then considered "substantially identical". As described herein, a preferred algorithm may account for gaps, etc. Preferably, identity exists in a region of at least about 25 amino acids or nucleotides in length, or more preferably in a region of 50-100 or more amino acids or nucleotides in length.

For sequence comparison, typically one sequence is used as a reference sequence for comparison of test sequences. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" refers to a segment selected from any one of a number of consecutive positions from 20 to 600, typically from about 50 to about 200, more typically from about 100 to about 150, wherein after optimal alignment of two sequences, one sequence can be compared to a reference sequence for the same number of consecutive positions. Alignment methods for comparing sequences are well known in the art.

Algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, nuc. Acids Res.25:3389-3402 (1977) and Altschul et al, J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used according to the parameters described herein to determine percent sequence identity for the nucleic acids and proteins of the present disclosure. Software for performing BLAST analysis is publicly available through National Center for Biotechnology Information (//www.ncbi.nlm.nih.gov /). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits extend in both directions along each sequence, so long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of word hits in each direction stops when the cumulative alignment score drops by an amount X from its maximum value, the cumulative score drops to zero or below due to the accumulation of one or more negative scoring residue alignments, or the end of either sequence is reached. BLAST algorithm parameters W, T and X determine the alignment sensitivity and speed. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value I, M =5 of 10, n= -4, and a two strand comparison as default values. For amino acid sequences, the BLASTP program uses a word length of 3 and an expected value I of 10 as default values, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, proc. Natl. Acad. Sci. USA 89:10915 (1989)) uses an alignment of 50 (B), an expected value I, M = 5, an n= -4, and a two strand comparison as default values.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form, as well as their complements. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and/or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term includes amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon to which is bound a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Also, nucleotides may be referred to by their recognized single letter codes.

With respect to antibodies, the term "competing" as used herein means that a first antibody or antigen-binding portion thereof competes for binding with a second antibody or antigen-binding portion thereof, wherein binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody as compared to binding of the first antibody in the absence of the second antibody. This option may be, but is not necessarily, the case when binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody may inhibit the binding of the second antibody to its epitope, but the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, where each antibody detectably inhibits binding of another antibody to its cognate epitope or ligand, whether to the same extent, greater or lesser, the antibodies are said to "cross-compete" with each other for binding to their respective epitope. Both competing and cross-competing antibodies are included in the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof, etc.), the skilled artisan will appreciate from the teachings provided herein that such competing and/or cross-competing antibodies are included and are useful in the methods disclosed herein.

Many types of competitive binding assays are known, for example solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see Stahli et al, methods in Enzymology 9:242-253 (1983)), solid phase direct biotin-avidin EIA (see KIRKLAND ET al, J. Immunol. 137:3614-3619 (1986)), solid phase direct labeling assays, solid phase direct labeling sandwich assays (see Harlow and Lane, antibodies, A Laboratory Manual, cold Spring Harbor Press (1988)), solid phase direct labeling RIA using I-125 tags (see Morel et al, molecular. Immunol. 25 (1): 7-15 (1988)), solid phase direct biotin-avidin EIA (Cheung et al, virogy 176:546-552 (1990)), and direct labeling A (Moldenhauer et al, scutel. J immunol. 32-82:82 (1990)). Typically, such assays involve the use of purified antigens bound to a solid surface or cells carrying any of these, unlabeled test immunoglobulins and labeled reference immunoglobulins. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cell in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an epitope that binds to the reference antibody in sufficient proximity that steric hindrance occurs. Typically, when the competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 50 or 75%.

The term "CEACAM6" as used herein refers to a human CEACAM6 protein, subtype or variant thereof, including naturally occurring human CEACAM6 variants, such as splice variants or allelic variants. An exemplary amino acid sequence of human CEACAM6 is shown in SEQ ID NO. 1. In some embodiments, human CEACAM6 may refer to a variant, such as an allelic variant or splice variant, that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs 1. In some embodiments, it is understood that the provided antibodies or antigen binding fragments may exhibit cross-reactive binding to another mammalian CEACAM6 protein, such as murine CEACAM6 or primate CEACAM 6.

Human CEACAM6 Uniprot P40199 (SEQ ID NO: 1)

MGPPSAPPCR LHVPWKEVLL TASLLTFWNP PTTAKLTIES TPFNVAEGKE VLLLAHNLPQ NRIGYSWYKG ERVDGNSLIV GYVIGTQQAT PGPAYSGRET IYPNASLLIQ NVTQNDTGFY TLQVIKSDLV NEEATGQFHV YPELPKPSIS SNNSNPVEDK DAVAFTCEPE VQNTTTTYLWWV NGQSLPVSPR LQLSNGNMTL TLLSVKRNDA GSYECEIQNP ASANRSDPVT LNVLYGPDVP TISPSKANYR PGENLNLSCH AASNPPAQYS WFINGTFQQS TQELFIPNIT VNNSGSYMCQ AHNSATGLNR TTVTMITVSG SAPVLSAVAT VGITIGVLAR VALI

By "solid support" is meant a non-aqueous matrix to which antibodies according to the disclosure provided can adhere or attach. For example, solid supports include, but are not limited to, microtiter plates, membranes (e.g., nitrocellulose), beads, test strips, thin layer chromatography plates, or other solid media.

As used herein, an "individual" or "subject" is a mammal. "mammal" for therapeutic purposes includes humans, domestic and farm animals, as well as zoo, sports or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the individual or subject is a human.

Antibodies that bind CEACAM6

Provided herein are antibodies, including antigen binding fragments thereof, that specifically bind CEACAM 6. In some embodiments, provided antibodies include monoclonal antibodies and antigen-binding fragments thereof that bind CEACAM6 and provide superior targeting specificity, signal-to-noise ratio, and the like, as compared to other reported antibodies. Also provided herein are methods for producing anti-CEACAM 6 antibodies, as well as methods for detecting and using such antibodies.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAM) are a large family of proteins in humans. The CEACAM family consists of membrane-linked glycoproteins anchored to the cell surface by Glycosyl Phosphatidylinositol (GPI) anchors or transmembrane domains. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM 6) is a member of this family, which is normally expressed on the cell surface of epithelial and myeloid lines and is linked to the cell surface by GPI anchors (Johnson AND MAHADEVAN, CLINICAL CANCER Drugs 2, pp. 100-11 (2015)). CEACAM6 is associated with invasion and metastasis of several cancers, such as pancreatic, lung and colon cancer cells (Wu, et al, translational Oncology, 101057 (2021)). CEACAM6 regulates cancer progression through aberrant cell differentiation, anti-apoptosis, cell growth and resistance to therapeutic Drugs (Johnson AND MAHADEVAN, CLINICAL CANCER Drugs 2, pp. 100-11 (2015)).

The present disclosure provides CEACAM6 antibodies that are non-cross-reactive with other members of the CEACAM6 protein family and have the ability to block CEACAM6 function and inhibit cancer cell invasion.

In some embodiments, any of the antibodies provided herein, or antigen binding fragments thereof, bind to all or a portion of CEACAM 6. In some embodiments, any of the antibodies provided herein, or antigen binding fragments thereof, bind to all or a portion of the extracellular domain of CEACAM 6.

In some embodiments, any of the antibodies or antigen-binding fragments thereof is a CEACAM6 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is isolated (e.g., separate from components of its natural environment (e.g., animal, biological sample)). In some embodiments, the anti-antibody is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody is a derivative of a conjugated humanized antibody. In some embodiments, the antibody binds under laboratory conditions (e.g., in vitro, in a flow cytometry assay, in ELISA). In some embodiments, the antibody binds under physiological conditions (e.g., binds in a cell in a subject).

Generally, antibodies provided herein comprise at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain. In some embodiments, an antibody described herein comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. Typically, each immunoglobulin heavy chain variable domain of the antibody comprises first, second and third heavy chain complementarity determining regions (CDRs; CDRH1, CDRH2 and CDRH 3), and each immunoglobulin light chain variable domain of the antibody comprises first, second and third light chain CDRs (CDRL 1, CDRL2 and CDRL 3).

In some embodiments, the antibody is an antigen binding fragment, such as Fab, F (ab') 2, fv, or scFv. Antigen binding fragments can be produced using any means known in the art, including chemical digestion (e.g., papain or pepsin) and recombinant methods. Methods for isolating and preparing recombinant nucleic acids are known to those skilled in the art (see Sambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)). for antibodies that can be expressed in a variety of host cells, including E.coli, other bacterial hosts, yeast, and a variety of higher eukaryotic cells, such as COS, CHO, and HeLa cell lines, and myeloma cell lines.

The present disclosure provides an isolated antibody or antigen binding fragment thereof that specifically binds CEACAM6 or a portion thereof, comprising a) an immunoglobulin heavy chain variable domain comprising (I) a heavy chain complementarity determining region 1 (CDRH 1) comprising the sequence GFX1X2SX3YGX4X5 (SEQ ID NO: 18), wherein X1 is T or S, X2 is F or L, X3 is N or T, X4 is M or NO amino acid, and X5 is G or NO amino acid; (ii) heavy chain complementarity determining region 2 (CDRH 2) comprising the sequence IX1X2X3X4X5X6X7 (SEQ ID NO: 19), wherein X1 is A or W, X2 is N or W, X3 is S or D, X4 is G or D, X5 is G or D, X6 is T or K, and X7 is a T or amino acid-free, and (iii) heavy chain complementarity determining region 3 (CDRH 3) comprising the sequence X1X2X3X4X5GX6X7X8X9X10X11 (SEQ ID NO: 20), wherein X1 is T or A, X2 is T or R, X3 is L or I, X4 is K or L, X5 is F or L, X6 is A or F, X7 is G or D, X8 is G or Y, X9 is F or amino acid-free, X10 is A or amino acid-free, and X11 is Y or amino acid-free, and (b) immunoglobulin domain light chain complementarity determining region 3 (SEQ ID NO: 20), wherein X1 is T or R, X3 is L or I, X4 is K or K, X5 is F or L, X6 is A or F, X7 is G or D, X8 is G or Y or amino acid-free, X9 is X11, and X11 is Y or amino acid-free, and b) light chain complementarity determining region 1 (SEQ ID NO:20, 1 or amino acid-containing amino acid-free, 1X 1 or 5 is X6 is N or L or amino acid-free, and X11 is Y or NO amino acid, (ii) a light chain complementarity determining region 2 (CDRL 2) comprising the sequence X1X2S (SEQ ID NO: 22), wherein X1 is W or S and X2 is A or G, and (iii) a light chain complementarity determining region 3 (CDRL 3) comprising the sequence QX1YX2X3X4PX5T (SEQ ID NO: 3), wherein X1 is Q or H, X2 is Y or N, X3 is I or E, X4 is F or Y, and X5 is N or L.

In some embodiments, an antibody of the present disclosure may comprise the sequences of heavy chain complementarity determining region 1 (CDRH 1), CDRH2, CDRH3, light chain complementarity determining region 1 (CDRL 1), CDRL2, CDRL 3. Exemplary CDR amino acid sequences and related SEQ ID NOs and exemplary heavy chain variable domain (VH) and light chain variable domain (VL) amino acid sequences and related SEQ ID NOs are shown in table 1.

TABLE 1 amino acid sequences of CEACAM6, heavy and light chain variable domains and CDRs of anti-CEACAM 6 antibodies

In some embodiments, the antibody or antigen binding fragment thereof comprises (i) an immunoglobulin heavy chain variable domain comprising a set of heavy chain Complementarity Determining Region (CDR) amino acid sequences CDRH1, CDRH2, and CDRH3, and (ii) an immunoglobulin light chain variable domain comprising a set of light chain CDR amino acid sequences CDRL, CDRL2, and CDRL3, wherein the set of heavy and light chain CDRs are each selected from the same set 1 or set 2 shown in table 2.

TABLE 2 CDR sets of anti-CEACAM 6 antibodies

In some embodiments, the immunoglobulin heavy chain variable domain comprises CDRH1 comprising an amino acid sequence shown in SEQ ID NO 6 or 12 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 6 or 12, CDRH2 comprising an amino acid sequence shown in SEQ ID NO 7 or 13 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 7 or 13, and CDRH3 comprising an amino acid sequence shown in SEQ ID NO 8 or 14 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 92%, 98%, 99% or more sequence identity to SEQ ID NO 8 or 14.

In some embodiments, the immunoglobulin light chain variable domain comprises CDRL1 comprising an amino acid sequence shown in SEQ ID NO 9 or 15 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 9 or 15, CDRL2 comprising an amino acid sequence shown in SEQ ID NO 10 or 16 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 10 or 16, and CDRL3 comprising an amino acid sequence shown in SEQ ID NO 11 or 17 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 92%, 98%, 99%, or more sequence identity to SEQ ID NO 11 or 17.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID No. 6 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 6, CDRH2 comprises the sequence shown in SEQ ID No. 7 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 6, and CDRH3 comprises the sequence shown in SEQ ID No. 8 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 8.

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO. 12 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 12, CDRH2 comprises the sequence shown in SEQ ID NO. 13 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 13, and CDRH3 comprises the sequence shown in SEQ ID NO. 14 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 14.

In some embodiments, CDRL1 comprises the sequence shown in SEQ ID NO. 9 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 9, CDRL2 comprises the sequence shown in SEQ ID NO. 10 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 10, and CDRL3 comprises the sequence shown in SEQ ID NO. 11 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 11.

In some embodiments, CDRL1 comprises the sequence shown in SEQ ID NO. 15 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 15, CDRL2 comprises the sequence shown in SEQ ID NO. 16 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 15, and CDRL3 comprises the sequence shown in SEQ ID NO. 17 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 17.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in any of SEQ ID NOs 2 or 4.

AB2

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID No. 6 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 6, CDRH2 comprises the sequence shown in SEQ ID No. 7 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 7; CDRH3 comprises the sequence shown in SEQ ID NO. 8 or an amino acid sequence which shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 8, CDRL1 comprises the sequence shown in SEQ ID NO. 9 or an amino acid sequence which shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 9, CDRL2 comprises the sequence shown in SEQ ID NO. 10 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 88% or more sequence identity to SEQ ID NO. 10 98%, 99% or more, and CDRL3 comprises the sequence shown in SEQ ID No. 11 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 11.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in any of SEQ ID NOs.2.

AB1

In some embodiments, CDRH1 comprises the sequence shown in SEQ ID No. 12 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 12; CDRH2 comprises the sequence shown in SEQ ID No. 13 or an amino acid sequence which shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 13; CDRH3 comprises the sequence shown in SEQ ID NO. 14 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 14, CDRL1 comprises the sequence shown in SEQ ID NO. 15 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 15, CDRL2 comprises the sequence shown in SEQ ID NO. 16 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 88% or more sequence identity to SEQ ID NO. 16 98%, 99% or more, and CDRL3 comprises the sequence shown in SEQ ID No. 17 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 17.

In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in any of SEQ ID NOs.4.

Fc polypeptides

Antibodies provided herein may comprise a fragment crystallizable region (Fc region), also referred to herein as an Fc polypeptide. An Fc polypeptide is a portion of each of the two heavy chains of an antibody and can interact with certain cell surface receptors and certain components of the complement system. Fc polypeptides typically include a CH2 domain and a CH3 domain, which are immunoglobulin constant region domain polypeptides. In some embodiments, the Fc polypeptide in an antibody described herein can be a wild-type Fc polypeptide, such as a human IgG1 Fc polypeptide.

In other embodiments, antibodies described herein can comprise variants of a wild-type Fc polypeptide that have at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity and at least one amino acid substitution relative to the sequence of the wild-type Fc polypeptide (e.g., SEQ ID NO: 87).

In some embodiments, the Fc polypeptide comprises one or more modifications (e.g., one or more amino acid substitutions, insertions, or deletions relative to a comparable wild-type Fc region). Antibodies comprising modified Fc polypeptides generally have altered phenotypes relative to antibodies comprising wild-type Fc polypeptides. For example, antibodies comprising modified Fc polypeptides may have altered serum half-life, altered stability, altered susceptibility to cellular enzymes, and/or altered effector function (e.g., as determined in NK-dependent or macrophage-dependent assays).

In some embodiments, the Fc polypeptides in the antibodies described herein may include amino acid substitutions that modulate effector function. In certain embodiments, the Fc polypeptides in the antibodies described herein may include amino acid substitutions that reduce or eliminate effector function.

In some embodiments, the Fc polypeptide comprises one or more modifications that alter the ratio of affinity of the modified Fc polypeptide to activate fcγr (e.g., fcγriia or fcγriiia) relative to inhibiting fcγr (e.g., fcγriib), relative to a wild-type Fc polypeptide:

Where the modified Fc polypeptide has an affinity ratio of greater than 1, the antibodies herein may be particularly useful in providing therapeutic or prophylactic treatment of a disease, disorder, or infection, or amelioration of symptoms thereof, wherein increased potency of effector cell function (e.g., ADCC) mediated by fcγr is desirable, e.g., cancer or an infectious disease. Where the modified Fc region has an affinity ratio of less than 1, the antibodies herein may be particularly useful in providing therapeutic or prophylactic treatment of a disease or disorder, or amelioration of symptoms thereof, wherein reduced efficacy of effector cell function mediated by fcγr is desired, e.g., autoimmune or inflammatory disorders. Table 3 lists examples of single, two, three, four and five amino acid substitutions in Fc polypeptides that provide an affinity ratio of greater than 1 or less than 1 (see, e.g., PCT publication No. WO 04/063351; WO 06/088494; WO 07/024249; WO 06/113665; WO 07/021841; WO 07/106707; WO 2008/140603). amino acid positions numbered according to the EU numbering scheme).

TABLE 3 Table 3

Antibodies that competitively bind to anti-CEACAM 6 antibodies

Also provided herein are antibodies that competitively bind or are capable of competitively binding (e.g., competitor antibodies) with one or more CEACAM6 antibodies described herein. In certain instances, an antibody (e.g., a competitor antibody) may be considered to compete for binding to CEACAM6 when the competitor binds to the same general region of CEACAM6 as the antibodies described herein. In certain instances, an antibody (e.g., a competitor antibody) may be considered to compete for binding to CEACAM6 when the competitor binds to an identical CEACAM6 region (e.g., an identical peptide (linear epitope) or an identical surface amino acid (conformational epitope)) as the antibody described herein. In certain instances, an antibody (e.g., a competitor antibody) may be considered capable of competing for CEACAM6 binding when, under suitable assay conditions, the competitor binds to the same CEACAM6 general region (e.g., extracellular region or leucine-rich binding domain) as the antibody described herein. In certain instances, an antibody (e.g., a competitor reagent) may be considered capable of competing for CEACAM6 binding when, under suitable assay conditions, the competitor binds to an identical CEACAM6 region (e.g., an identical peptide (linear epitope) or an identical surface amino acid (conformational epitope)) as the antibody described herein.

In certain instances, an antibody (e.g., a competitor antibody) may be considered to compete for binding to CEACAM6 when the competitor blocks binding of one or more antibodies described herein to CEACAM6 (e.g., under suitable assay conditions). Whether a competitor blocks the binding of one or more antibodies described herein to CEACAM6 may be determined using a suitable competition assay or blocking assay, e.g., a blocking assay described herein. In a competition or blocking assay, a competitor antibody may block 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%) of the binding of one or more antibodies described herein to CEACAM6, and conversely, in a competition or blocking assay, one or more antibodies described herein may block about 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%) of the binding of a competitor antibody to CEACAM6.

In certain instances, an antibody (e.g., a competitor antibody) may be considered to compete for binding to CEACAM6 when the competitor binds to CEACAM6 with a similar affinity as the one or more antibodies described herein (e.g., under suitable assay conditions). In some embodiments, an antibody (i.e., a competitor antibody) is considered to compete for binding to CEACAM6 when the competitor binds to CEACAM6 with an affinity of at least about 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the affinity of one or more antibodies described herein.

Also provided herein are antibodies that bind or are capable of binding to the same epitope as one or more antibodies described herein. In particular, provided herein are antibodies that compete with one or more antibodies described herein for binding to the same epitope (e.g., the same peptide (linear epitope) or the same surface amino acid (conformational epitope)) on CEACAM 6. Such antibodies that bind the same epitope may be referred to as epitope competitors.

Polyclonal and monoclonal antibodies

Polyclonal antibodies can be raised in animals (vertebrates or invertebrates, including mammals, birds, and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to use a bifunctional or derivatizing agent, such as maleimide benzoylsulfosuccinimidyl ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, SOCl2 or r1n=c=nr, where R and R1 are different alkyl groups, to conjugate the relevant antigen with a protein or other carrier that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor. Non-protein carriers (e.g., colloidal gold) may also be used for antibody production.

Animals can be immunized against an antigen, immunogenic conjugate or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbit or mouse, respectively) with three volumes of freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals were boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injections at multiple sites. After 7-14 days, animals were bled and serum antibody titers were determined. Animals were boosted until the titers reached the plateau. Typically, animals are boosted with conjugates of the same antigen but conjugated to different proteins and/or by different crosslinking agents. Conjugates can also be prepared as protein fusions in recombinant cell cultures. In addition, aggregating agents such as aluminum adjuvants are suitable for enhancing immune responses.

Monoclonal antibodies can be prepared using hybridomas, for example, the hybridoma method first described by Kohler et al, nature, 256:495 (1975), or can be prepared by other methods, such as recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal, such as hamster or macaque, is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Or lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (see, e.g., goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pp.59-103 (ACADEMIC PRESS, 1986)).

The hybridoma cells thus prepared are inoculated and grown in a suitable medium that may contain one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are highly potent fusions, support stable high level production of antibodies by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as SP-2 or X63-Ag8-653 cells available from AMERICAN TYPE Culture Collection, rockville, md. USA. Human myeloma and mouse-human heterologous myeloma cell lines have also been described for use in the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Wherein the medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation, by in vitro binding assays, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), or by flow cytometry analysis of cells expressing the membrane antigen. The binding affinity of monoclonal antibodies can be detected, for example, by Scatchard analysis of Munson et al, anal biochem, 107:220 (1980).

After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and/or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see, e.g., goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pp.59-103 (ACADEMIC PRESS, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, hybridoma cells can be grown in vivo in animals as ascites tumors. Monoclonal antibodies secreted by subcloning are suitably separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Alternatively, cDNA may be prepared from mRNA, which is then subjected to DNA sequencing. Hybridoma cells are used as a preferred source of such genomic DNA or RNA for cDNA preparation. Once isolated, the DNA can be placed in an expression vector well known in the art, and the expression vector is then transfected into a host cell, such as an e.coli cell, simian COS cell, chinese Hamster Ovary (CHO) cell, or myeloma cell, that would not otherwise produce immunoglobulin protein, to obtain synthesis of monoclonal antibodies in the recombinant host cell.

V. humanization and amino acid variants

General methods for humanization of antibodies are described, for example, in U.S. patent nos. 5861155、6479284、6407213、6639055、6500931、5530101、5585089、5693761、5693762、6180370、5714350、6350861、5777085、5834597、5882644、5932448、6013256、6129914、6210671、6329511、5225539、6548640 and 5624821. In certain embodiments, it may be desirable to produce amino acid sequence variants of these humanized antibodies, particularly where the variants improve the binding affinity or other biological properties (e.g., half-life) of the antibody.

In some embodiments, the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. This can be accomplished, for example, by retaining non-human CDR regions and replacing the remaining portions of the antibody with their human counterparts. See, e.g., ,Morrison et al., PNAS USA, 81 :6851-6855 (1984) ; Morrison and Oi, Adv. Immunol., 44 :65-92 (1988) ; Verhoeyen et al., Science, 239 :1534-1536 (1988) ; Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994). for techniques for humanizing antibodies are well known in the art and are described, e.g., in U.S. Pat. nos. 4,816,567, 5,530,101, 5,859,205, 5,585,089, 5,693,761, 5,693,762, 5,777,085, 6,180,370, 6,210,671, and 6,329,511, WO 87/02671, EP patent application 0173494, jones et al (1986) Nature 321:522, and Verhoyen et al (1988) Science 239:1534. Humanized antibodies are further described, for example, in WINTER AND MILSTEIN (1991) Nature 349:293. For example, a polynucleotide comprising a first sequence encoding a humanized immunoglobulin framework region and a second sequence set encoding a desired immunoglobulin complementarity determining region may be synthetically produced or produced by combining appropriate cDNA and genomic DNA segments. The human constant region DNA sequences can be isolated from a variety of human cells according to well known procedures. CDRs for use in producing immunoglobulins of the present disclosure may similarly be obtained from monoclonal antibodies capable of specifically binding CEACAM 6.

Amino acid sequence variants of antibodies can be prepared by introducing appropriate nucleotide changes into the antibody DNA or by peptide synthesis. For the examples herein, such variants include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions is performed to achieve the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also alter the post-translational processes of the humanized or variant antibodies, e.g., alter the number or position of glycosylation sites.

One method for identifying certain residues or regions of an antibody as preferred mutagenesis positions is referred to as "alanine scanning mutagenesis" as described by, for example, cunningham and Wells, science, 244:1081-1085 (1989). Here, a residue or set of residues of interest (e.g., charged residues such as Arg, asp, his, lys and Glu) are identified and replaced by neutral or negatively charged amino acids (most preferably Ala or polyAla) to affect the interaction of the amino acids with the antigen. These amino acid positions, which demonstrate functional sensitivity to substitution, are then refined by introducing further or other variants at or for the substitution site. Thus, although the site for introducing the amino acid sequence change is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of mutations at a given site, alanine scanning or random mutagenesis is performed at the target codon or region and the expressed antibody variants are screened for the desired activity. Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include N-terminal methionyl residues or antibodies fused to epitope tags. Other insertional variants include fusions of enzymes or polypeptides to the N-or C-terminus of an antibody that increase the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted at its position. For substitution mutagenesis, the most interesting sites include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred, but more significant changes may be introduced and the product may be screened. Examples of substitutions are listed below:

(1) Ala (A):Val;Leu;Ile;Val

(2) Arg €:Lys;Gln;Asn;Lys

(3) Asn (N):Gln;His;Asp;Lys;Gln;Arg

(4) Asp (D):Glu;Asn

(5) Cys €:Ser;Ala

(6) Gln (Q):Asn;Glu

(7) Glu €:Asp;Gln

(8) Gly (G):Ala

(9) His (H):Asn;Gln;Lys;Arg

(10) Ile (I) Leu, val, met, ala, leu, phe, norleucine

(11) Leu (L) is norleucine, ile, val, ile, met, ala, phe

(12) Lys (K):Arg;Gln;Asn

(13) Met (M):Leu;Phe;Ile

(14) Phe (F):Leu;Val;Ile;Ala;Tyr

(15) Pro (P):Ala

(16) Ser (S):Thr

(17) Thr (T):Ser

(18) Trp (W):Tyr;Phe

(19) Tyr (Y):Trp;Phe;Thr;Ser

(20) Val (V) is Ile, leu, met, phe, ala, norleucine

Significant modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the substitution region, e.g., as a folded or helical conformation, (b) molecular charge or hydrophobicity at the target site, or (c) side chain volume. Naturally occurring residues are divided into the following groups based on common side chain properties:

(1) Hydrophobic norleucine Met, ala, val, leu, ile;

(2) Neutral hydrophilic Cys, ser, thr;

(3) Acid, asp, glu;

(4) Basicity Asn, gln, his, lys, arg;

(5) Residues affecting chain orientation, gly, pro, and

(6) Aromatic Trp, tyr, phe

Non-conservative substitutions require exchanging a member of one of the above classes for another class.

Any cysteine residues that do not participate in maintaining the correct conformation of the antibody may also be substituted to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly where the antibody is an antigen binding fragment such as an Fv fragment).

One type of substitution variant comprises substitution of one or more hypervariable region residues of the parent antibody. In general, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were derived. A convenient way to generate such substitution variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus produced can be displayed in a monovalent manner from filamentous phage particles as a fusion with the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity), as disclosed herein.

To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and the antigen. Such contact residues and nearby residues are substitution candidates according to the techniques set forth herein. Once such variants are produced, the set of variants is subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development.

Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. By altered is meant that one or more carbohydrate moieties present in the antibody are deleted, and/or one or more glycosylation sites not present in the antibody are added. Glycosylation of antibodies is typically N-linked and/or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are the most common recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The addition of a glycosylation site to an antibody can be accomplished by altering the amino acid sequence such that it contains one or more of the tripeptide sequences described above (for an N-linked glycosylation site). The alteration may also be made by adding one or more serine or threonine residues to the original antibody sequence, or by substitution with one or more serine or threonine residues (for O-linked glycosylation sites).

VI. other modifications

Other modifications of the antibody are contemplated. For example, the technology herein also relates to immunoconjugates comprising an antibody described herein conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragment thereof) or a radioisotope (e.g., a radio conjugate) or a cytotoxic drug. Such conjugates are sometimes referred to as "antibody-drug conjugates" or "ADCs". Conjugates can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridinedithiol) propionate (SPDP), iminothiolane (IT), difunctional derivatives of imidoesters (e.g., adipimide HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis- (p-azidobenzoyl) hexamethylenediamine), bis-diazonium derivatives (e.g., bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (e.g., toluene 2, 6-diisocyanate), and bis-active fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene).

In some of any of the embodiments, any of the antibodies disclosed herein or antigen-binding fragments thereof can be formulated as an immunoliposome. Liposomes containing antibodies are prepared by methods known in the art, for example, as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. patent nos. 4,485,045 and 4,544,545. Liposomes with extended circulation times are disclosed in U.S. Pat. No. 5,013,556. For example, liposomes can be produced by reverse phase evaporation methods using lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to obtain liposomes having the desired diameter. The Fab' fragments of the antibodies provided herein can be conjugated to liposomes by disulfide exchange reactions, as described in Martin et al, j. Biol. Chem. 257:286-288 (1982). Optionally containing another active ingredient within the liposome.

Enzymes or other polypeptides may be covalently bound to antibodies by techniques well known in the art, for example, using the heterobifunctional crosslinking reagents discussed above. In some embodiments, fusion proteins comprising at least the antigen binding region of an antibody provided herein linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., neuberger et al, nature 312:604-608 (1984)).

In certain embodiments, it may be desirable to use antigen binding fragments, rather than whole antibodies, to, for example, increase penetration of target tissues and cells. In such cases, it may be desirable to modify the antigen binding fragment to increase its serum half-life. This may be achieved, for example, by incorporating a rescue receptor binding epitope into the antigen binding fragment (e.g. by mutation of an appropriate region in the antigen binding fragment or by incorporating the epitope into a peptide tag which is then fused at either end or in the middle to the antigen binding fragment, e.g. by DNA or peptide synthesis; see, for example, WO96/32478, disclosed in 10, 17, 1996).

In some embodiments, any of the antibodies or antigen fragments thereof disclosed herein are conjugated or hybridized to an oligonucleotide. In some embodiments, the oligonucleotides include a sample barcode sequence, a primer binding site, and an anchor. In some embodiments, the oligonucleotide may be conjugated or hybridized to any of the detectable markers or tags disclosed herein. In some embodiments, the oligonucleotide is a polymer sequence. In some embodiments, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to single-stranded polymers of nucleotides ranging from about 2 to about 500 nucleotides in length. In some embodiments, any of the oligonucleotides described herein can be synthetic, enzymatically prepared (e.g., by polymerization), or using a "split-pool" approach. In some embodiments, any of the oligonucleotides described herein can include ribonucleotide monomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some embodiments, any of the oligonucleotides described herein can include a combination of deoxyribonucleotide monomers and ribonucleotide monomers (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers) in the oligonucleotide. In some embodiments, the oligonucleotide may be 4-10、10-20、21-30、31-40、41-50、51-60、61-70、71-80、80-100、100-150、150-200、200-250、250-300、300-350、350-400 or 400-500 nucleotides in length. In some embodiments, any of the oligonucleotides described herein can include one or more functional moieties attached (e.g., covalently or non-covalently) to another structure. In some embodiments, any of the oligonucleotides described herein can include one or more detectable labels (e.g., a radioisotope or a fluorophore). In some embodiments, the anchor is a defined polymer, such as a polynucleotide or oligonucleotide sequence, that is designed to hybridize to a complementary oligonucleotide sequence. In some embodiments, the anchors are designed for the purpose of generating double stranded construct oligonucleotide sequences. In some embodiments, the anchor is located 3' to the oligonucleotide sequence of the construct. In other embodiments, the anchor is located 5' to the oligonucleotide sequence of the construct. Each anchor is specific for its intended complementary sequence.

In some embodiments, the sample barcode sequence is a polymer, e.g., a polynucleotide, that is specific for a single ligand as a functional element. In some embodiments, the sample barcode sequence can be used to identify a particular cell or substrate, such as Drop-seq microbeads. In some embodiments, the sample barcode sequence may be formed from DNA, RNA, modified bases, or combinations of these bases of a specified sequence, as well as any other polymer defined above. In some embodiments, the sample barcode sequence is about 2-4 monomer components, e.g., nucleotide base lengths. In other embodiments, the barcode is at least about 1-100 monomer components, e.g., nucleotides in length. Thus, in various embodiments, the barcode is formed from a sequence of at least 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、80、91、92、93、94、95、96、97、98、99 or up to 100 monomer components, e.g., nucleic acids. In some embodiments, the sample barcode sequence is a particular barcode that may be unique relative to other barcodes.

In some of any of the embodiments, the sample barcode sequence can have a variety of different forms. For example, sample barcode sequences may include polynucleotide barcodes, random nucleic acids and/or amino acid sequences, and synthetic nucleic acids and/or amino acid sequences. The sample barcode sequence may be linked to the analyte or another moiety or structure in a reversible or irreversible manner. Sample barcode sequences may be added to fragments of, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples prior to or during sample sequencing. Sample barcode sequences may allow for the identification and/or quantification of individual sequencing reads (e.g., a barcode may be or may include a unique molecular identifier or "UMI").

The sample barcode sequence may spatially resolve molecular components present in the biological sample, for example, at single cell resolution (e.g., the barcode may be or may include a "spatial barcode"). In some embodiments, the bar code includes both UMI and spatial bar codes. In some embodiments, the bar code comprises two or more sub-bar codes that together function as a single bar code. For example, a polynucleotide barcode may include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more non-barcode sequences.

In some embodiments, the primer binding site is a functional component of an oligonucleotide, which is itself an oligonucleotide or polynucleotide sequence, providing an annealing site for oligonucleotide amplification. The primer binding site may be formed of DNA, RNA, PNA, modified bases, or polymers or polyamides of combinations of these bases, or the like. In some embodiments, the primer binding site is about 10 such monomer components, e.g., nucleotide base lengths. In other embodiments, the primer binding site is at least about 5-100 monomer components, e.g., nucleotides in length. Thus, in various embodiment components, the primer binding site is formed from at least 5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、80、91、92、93、94、95、96、97、98、99 or up to 100 monomer components, e.g., the sequence of a nucleic acid. In certain embodiments, the primer binding site may be a universal sequence suitable as an annealing site for various amplification techniques. Amplification techniques include, but are not limited to, DNA-polymerase based amplification systems such as Polymerase Chain Reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP, MALBAC), strand Displacement Amplification (SDA), multiple Displacement Amplification (MDA), recombinase Polymerase Amplification (RPA) and polymerization by any number of DNA polymerases (e.g., T4 DNA polymerase, sulfulobus DNA polymerase, klenow DNA polymerase, bst polymerase, phi29 polymerase), and RNA-polymerase based amplification systems (e.g., T7-, T3-, and SP 6-RNA-polymerase amplification), nucleic Acid Sequence Based Amplification (NASBA), self-sustained sequence replication (3 SR), rolling Circle Amplification (RCA), ligase Chain Reaction (LCR), helicase dependent amplification (I), branched amplification methods, and RNA-seq. The method for conjugating or hybridizing oligonucleotides may be performed in the manner shown in WO/2018/144813, WO/2016/018960, WO/2018/089438, WO/2014/182528, WO/2018/026873, WO/2021/188838.

In some embodiments, modifications may optionally be introduced into the antibody (e.g., within the polypeptide chain or at the N or C terminus), e.g., to extend in vivo half-life, e.g., pegylation, or incorporation of long chain polyethylene glycol Polymers (PEG). The introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the polypeptide, e.g., to prevent rapid filtration into urine. In some embodiments, the lysine residues in the sequence are conjugated to PEG directly or through a linker. Such a linker may be, for example, a Glu residue or an acyl residue containing a thiol functionality for attachment to a suitably modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue, e.g. a substitution of Arg or Lys residue, at the C-terminus or at a solvent exposed residue. The Cys residue is then site-specifically linked to a PEG chain containing, for example, maleimide functionality. Methods for introducing PEG or long chain polymers of PEG are known in the art (e.g., as described in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, M. J., et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)),, the contents of which are incorporated herein by reference).

Covalent modification of antibodies is also included within the scope of the technology. For example, the modification may be performed by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent modification of antibodies are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N or C terminal residues. Exemplary covalent modifications of polypeptides are described in U.S. Pat. No. 5,534,615, which is expressly incorporated herein by reference. Preferred types of covalent modification of antibodies include attaching the antibody to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyalkylene oxide, in a manner such as shown in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337.

VII nucleic acids, vectors, host cells and recombinant methods

The disclosure also provides isolated nucleic acids encoding antibodies, vectors and host cells comprising the nucleic acids, and recombinant techniques for producing antibodies. The nucleic acids herein may include one or more subsequences, each referred to as a polynucleotide.

Provided herein are nucleic acids (e.g., isolated nucleic acids) comprising a nucleotide sequence encoding an antibody or fragment thereof. In some embodiments, provided herein are nucleic acids encoding immunoglobulin heavy chain variable domains of antibodies. In some embodiments, provided herein are nucleic acids encoding immunoglobulin light chain variable domains of antibodies. In some embodiments, provided herein are nucleic acids encoding immunoglobulin heavy chain variable domains and immunoglobulin light chain variable domains of antibodies. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID No. 1-23. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin heavy chain comprising SEQ ID NO. 4 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 4 or an immunoglobulin light chain comprising SEQ ID NO. 5 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 5. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin heavy chain comprising SEQ ID NO. 4 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 4 and an immunoglobulin light chain comprising SEQ ID NO. 5 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 5. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin heavy chain comprising SEQ ID NO. 2 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 2 or an immunoglobulin light chain comprising SEQ ID NO. 3 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 3. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin heavy chain comprising SEQ ID NO. 2 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 2 and an immunoglobulin light chain comprising SEQ ID NO. 3 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 3.

Provided herein are the is a nucleotide sequences that encoding the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of the antibody or antigen binding fragment thereof of any of the antibodies or antigen binding fragments thereof.

For recombinant production of antibodies, the nucleic acid encoding the antibody may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. In some cases, antibodies can be produced by homologous recombination. DNA encoding an antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components typically include, but are not limited to, one or more of a signal sequence and an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

Suitable host cells for cloning or expressing the DNA in the vectors herein may be prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as E.coli, enterobacter, erwinia, klebsiella, proteus, salmonella, such as Salmonella typhimurium, serratia, such as Serratia marcescens, and Shigella, and Bacillus, such as Bacillus subtilis and Bacillus licheniformis, pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces. A preferred E.coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E.coli B, E.coli X1776 (ATCC 31,537) and E.coli W3110 (ATCC 27,325) may also be suitable. These examples are illustrative and not limiting.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies. Saccharomyces cerevisiae or Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms. Many other genera, species and strains are commonly available and may be used herein, such as Schizosaccharomyces pombe, kluyveromyces hosts, such as Kluyveromyces lactis, kluyveromyces fragilis (ATCC 12,424), kluyveromyces bulgaricus (ATCC 16,045), kluyveromyces weighanii (ATCC 24,178), kluyveromyces Wo Erdi (ATCC 56,500), kluyveromyces drosophila (ATCC 36,906), kluyveromyces thermotolerans and Kluyveromyces marxianus, yarrowia (EP 402,226), pichia pastoris (EP 183,070), candida, trichoderma reesei (EP 244,234), neurospora crassa, such as Schwanella wegenensis, and filamentous fungi, such as Neurospora, penicillium, curvulus and Aspergillus hosts, such as Aspergillus nidulans and Aspergillus niger.

Suitable host cells for expressing antibodies may also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants have been identified as well as corresponding tolerant insect host cells from hosts such as spodoptera frugiperda (trichostrongyloides), aedes aegypti (mosquitoes), aedes albopictus (mosquitoes), drosophila melanogaster (drosophila) and bombyx mori (silkworm moth). Various strains for transfection are publicly available, for example, L-1 variants of Spodoptera frugiperda NPV and Bm-5 strain of Bombyx mori NPV, and such viruses are useful as viruses herein, in particular for transfection of Spodoptera frugiperda cells, in accordance with the present technology. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

Suitable host cells for expressing the antibodies may also include vertebrate cells (e.g., mammalian cells). Vertebrate cells can be proliferated in culture (tissue culture). Examples of useful mammalian host cell lines include the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney (293 cells or 293 cells subcloned for growth in suspension culture, graham et al, J.Gen. Virol. 36:59 (1977)), baby hamster kidney (BHK, ATCC CCL 10), chinese hamster ovary cells/-DHFR (CHO, urlaub et al, proc. Natl. Acad. Sci. USA 77:4216 (1980))), mouse Sertoli cells (TM 4, mather, biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV 1 ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, 8065), breast cancer cells (Table 6:3, mr. SCL 5, mr. 4, mr. 6, and Mr. 6:1980)), liver cancer cells (Mather 1 ATCC CCL 70).

The host cells can be transformed with the above-described expression or cloning vectors for antibody production and, where appropriate, cultured in conventional nutrient media modified as a result of induction of promoters, selection of transformants or amplification of the gene encoding the desired sequence. Host cells for producing antibodies provided herein can be cultured in a variety of media. Commercially available media such as Ham's F (Sigma), minimal essential media ((MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's modified Eagle media ((DMEM), sigma) are suitable for culturing host cells in addition, ham et al, meth Enz 58:44 (1979), barnes et al, anal biochem.102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 are suitable for culturing host cells, any of which may be supplemented as desired with hormones and/or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCINTM), as a micro-molar concentration of the final element (e.g., glucose) is also known to those of ordinary skill in the art, and the appropriate conditions for the expression of the host cells, and the like, and the appropriate conditions for the medium to be selected to be used for the appropriate conditions for the expression of such as those of the host cells.

When recombinant techniques are used, antibodies may be produced intracellularly, in the pericellular space, or directly secreted into the medium. If the antibody is produced intracellularly, particulate debris, whether host cells or lysed fragments, is first removed, for example by centrifugation or ultrafiltration. Carter et al, bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell pellet was thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When antibodies are secreted into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors, such as PMSF, may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein a as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify human heavy chain-based antibodies (LINDMARK ET al, J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isoforms and for human gamma 3 (Guss et al, EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most typically agarose, but other matrices are also useful. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl) benzene, allow for faster flow rates and shorter processing times than achieved by agarose. Bakerbond abx.tm. Resins (j.t. Baker, philips burg, n.j.) can be used for purification when the antibodies comprise a CH3 domain. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sephadetm chromatography, anion or cation exchange resin chromatography (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, may also be used depending on the antibody to be recovered.

After any preliminary purification steps, the mixture comprising the antibody of interest and the contaminant may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of, for example, about 2.5-4.5, and may be performed at low salt concentrations (e.g., about 0-0.2 m salt).

Pharmaceutical formulations, routes of administration and administration

The present disclosure provides antibodies and related compositions that are useful, for example, for eliminating expressed pathogens from the body, and for, for example, identifying and quantifying the amount of CEACAM6 expressed pathogens in a biological sample.

In some embodiments, any antibody or antigen-binding fragment thereof may be formulated in a pharmaceutical composition that may be used for a variety of purposes, including the treatment of a disease or disorder. Pharmaceutical compositions comprising one or more antibodies may be administered to a patient in need thereof using a pharmaceutical device, and according to one embodiment of the technology, kits comprising such devices are provided. Such devices and kits can be designed for conventional administration, including self-administration, of the pharmaceutical compositions herein.

Therapeutic formulations of antibodies may be prepared for storage by mixing an agent or antibody of the desired purity with an optional physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16 ^th edition, osol, a. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt forming counterions such as sodium, metal complexes such as Zn-protein complexes, and/or nonionic complexes such as polyethylene glycol 35 or PEG TWEENTM, PLURONICSTM.

In some embodiments, the disease or disorder is associated with CEACAM6 expression. In some embodiments, the disease or disorder is associated with aberrant CEACAM6 expression. In some embodiments, the disease or disorder is associated with Natural Killer (NK), αβ T cells, γδ T cells, cd8+ T cells, monocytes or dendritic cells. In some embodiments, the disease or disorder is associated with Natural Killer (NK) cells. In some embodiments, the disease or disorder is associated with αβ T cells. In some embodiments, the disease or disorder is associated with γδ T cells. In some embodiments, the disease or disorder is associated with cd8+ T cells. In some embodiments, the disease or disorder is associated with monocytes. In some embodiments, the disease or disorder is associated with dendritic cells.

In some embodiments, the disease or disorder is cancer, an infectious disease, or an autoimmune disorder.

In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is, for example, metastatic melanoma, solid tumor, bladder cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, colon-derived liver metastasis, papillary thyroid carcinoma, acute myeloid leukemia, or asymptomatic myeloma.

In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the infectious disease is, for example, human Immunodeficiency Virus (HIV), chronic hepatitis C, cytomegalovirus, or hantavirus.

In some embodiments, the disease or disorder is an autoimmune disorder. In some embodiments, the autoimmune disorder is, for example, crohn's disease, multiple sclerosis, systemic sclerosis, myasthenia gravis of the eye muscle, psoriasis, or rheumatoid arthritis.

In some embodiments, any of the antibodies described herein, or antigen-binding fragments thereof, can be used to reduce androgen production in prostate cancer cells.

In some embodiments, any of the antibodies described herein, or antigen binding fragments thereof, can be used to inhibit or reduce cleavage of a coronavirus spike glycoprotein. In some embodiments, any of the antibodies described herein, or antigen binding fragments thereof, can be used to inhibit or reduce viral uptake in a host cell.

The formulations herein may also contain more than one active compound for the particular indication being treated as desired, preferably those compounds having complementary activity that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

Formulations for in vivo administration are typically sterile. This can be achieved, for example, by filtration through sterile filtration membranes.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the agent/antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamic acid, nondegradable ethylene-vinyl acetate, degradable lactic-glycolic acid copolymers such as Lupron Depot (injectable microspheres composed of lactic-glycolic acid copolymer and leuprorelin acetate), and poly-D- (-) -3-hydroxybutyric acid. Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for more than 100 days, certain hydrogel release proteins last for a shorter period of time. When encapsulated agents/antibodies are maintained in vivo for prolonged periods of time, they may denature or aggregate as a result of exposure to moisture at 37 ℃, resulting in loss of biological activity and possible immunogenic changes. A rational strategy may suggest stabilization according to the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation through thiol-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, using suitable additives, and developing specific polymer matrix compositions.

For therapeutic use, the antibodies provided herein are administered to a mammal (e.g., a human) in pharmaceutically acceptable dosage forms, such as those discussed above, including those that can be administered intravenously as a bolus or continuously infused over a period of time, or to a human by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. For preventing or treating a disease, the appropriate dosage of the agent or antibody will depend on the type of disease to be treated as defined above, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapies, the clinical history of the patient and the response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody may be the initial candidate dose to be administered to the patient, whether by one or more separate administrations or by continuous infusion, for example. Typical daily or weekly dosage ranges may be from about 1 μg/kg to about 20 mg/kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired inhibition of disease symptoms occurs. However, other dosage regimens may also be useful. The progress of the therapy is readily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods for determining TMPRSS2 levels in body fluids or tissues using the antibodies can be used to optimize patient exposure to therapeutic antibodies.

In some embodiments, a composition comprising an antibody herein may be administered as monotherapy, and in some embodiments, a composition comprising the antibody may be administered as part of a combination therapy. In some cases, the effectiveness of an antibody in preventing or treating a disease may be improved by administering the antibody continuously or in combination with another drug that is effective for these purposes (e.g., a chemotherapeutic drug for treating cancer or microbial infection). In other cases, the antibodies can be used to enhance or sensitize cells to chemotherapeutic treatments, allowing efficacy at lower doses and having lower toxicity. In addition to administering a composition comprising an antibody that reduces the number of expressing cells, certain combination therapies further comprise delivering a second therapeutic regimen selected from the group consisting of chemotherapeutic agents, radiation therapy, surgery, and combinations of any of the foregoing. Such other agents may be present in the composition to be administered, or may be administered separately. In addition, the antibodies may be suitably administered sequentially or in combination with other agents or forms (e.g., chemotherapeutic drugs or radiation for the treatment of cancer, infection, etc., or immunosuppressive drugs).

IX. research and diagnosis

Also provided herein are diagnostic reagents comprising the antibodies described herein. For example, the antibodies provided herein may be used to detect and/or purify CEACAM6 from a bodily fluid or tissue. Also provided herein are methods for detecting CEACAM6. For example, a method can comprise contacting a sample (e.g., a biological sample known or suspected of containing) with an antibody provided herein, and detecting CEACAM6: antibody complex if the sample contains CEACAM6. Reagents comprising the antibodies described herein and detection methods for research purposes are also provided herein.

Any of the antibodies or antigen binding fragments disclosed herein can be used in a diagnostic assay to detect its presence in a particular cell, tissue or body fluid. Such diagnostic methods may be used to diagnose, for example, hyperproliferative diseases or disorders. Thus, clinical diagnostic uses and research uses are included herein. In some embodiments, the antibody comprises a detectable marker or tag. In some embodiments, the antibody is conjugated to a detectable marker or tag. For example, for research and diagnostic applications, antibodies may be labeled with a detectable moiety. Many tags are available, which generally fall into the following categories:

(a) Radioisotopes such as 35S, 14C, 125I, 3H and 131I. The antibodies may be labeled with a radioisotope using techniques such as those described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), and radioactivity may be measured using scintillation counting.

(B) Fluorescent labels, such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, texas red and light violet (TM) are available. Fluorescent tags may be conjugated to antibodies using techniques such as those disclosed in Current Protocols in Immunology (supra). Fluorescence can be quantified using a flow cytometer, imaging microscope, or fluorometer.

Various enzyme-substrate tags are available. Enzymes typically catalyze chemical alterations of chromogenic substrates, which can be measured using a variety of techniques. For example, enzymes can catalyze a color change of a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying fluorescence change are described above. Chemiluminescent substrates become electronically excited by chemical reactions, and can then emit measurable light (e.g., using a chemiluminescent meter) or transfer energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g., firefly luciferases and bacterial luciferases), luciferins, 2, 3-dihydrophthalazinediones, malate dehydrogenases, ureases, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidases, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRP) with hydrogen peroxide as substrate, wherein hydrogen peroxide oxidizes dye precursors (e.g., o-phenylenediamine (OPD) or 3,3', 5' -tetramethyl benzidine hydrochloride (TMB));

(ii) Alkaline Phosphatase (AP) with p-nitrophenyl phosphate as chromogenic substrate, and

(Iii) beta-D-galactosidase (beta-D-Gal) has a chromogenic substrate (e.g., p-nitrophenyl-beta-D-galactosidase) or a fluorogenic substrate 4-methylumbelliferyl-beta-D-galactosidase.

In some cases, the tag is indirectly conjugated to the agent or antibody. The skilled artisan will appreciate various techniques for accomplishing this. For example, the antibody may be conjugated to biotin, and any three broad categories of tags mentioned above may be conjugated to avidin, or vice versa. Biotin selectively binds to avidin and thus the tag can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the tag to the antibody, the antibody is conjugated to a small hapten (e.g., digoxin), and one of the different types of tags mentioned above is conjugated to an anti-hapten antibody (e.g., an anti-digoxin antibody). Thus, indirect conjugation of the tag to the antibody may be achieved.

In some embodiments, the antibody or antigen binding fragment thereof need not be labeled, and its presence can be detected, for example, using a labeled antibody that binds to the antibody.

In some embodiments, the antibodies herein are immobilized on a solid support or substrate. In some embodiments, the antibodies herein are non-diffusively immobilized on a solid support (e.g., the antibodies do not detach from the solid support). The solid support or substrate may be any physically separable solid to which the antibodies may be directly or indirectly attached, including, but not limited to, surfaces provided by microarrays and wells, as well as particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. Solid supports may also include, for example, chips, posts, optical fibers, wipes, filters (e.g., planar filters), one or more capillaries, glass and modified glass or functionalized glass (e.g., controlled Pore Glass (CPG)), quartz, mica, diazonium salt films (paper or nylon), paraformaldehyde, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrenes, copolymers of styrene with other materials, polybutenes, polyurethanes, TEFLON (TM), polyethylenes, polypropylenes, polyamides, polyesters, polyvinylidene difluoride (PVDF), etc.), polysaccharides, nylons or nitrocellulose, resins, silica gel or silica gel-based materials (including silicon, silica gel and modified silicon), sephadex, sepharose, carbon, metals (e.g., steel, gold, silver, aluminum, silicon and copper), inorganic glass, conductive polymers (including polymers such as polypyrrole and polybenzazole), microstructured or nanostructured surfaces such as nucleic acid spreading arrays, nanotubes, nanowires or nanoparticle modified surfaces, or porous surfaces or gels such as methacrylates, acrylamides, sugar polymers, celluloses, silicates or other fibrous or chain polymers. In some embodiments, the solid support or substrate may be coated with any number of materials, including polymers, such as dextran, acrylamide, gelatin, or agarose, using inert coatings or chemically derivatized coatings. The beads and/or particles may be free or attached to each other (e.g., sintered). In some embodiments, the solid support or substrate may be an aggregate of particles. In some embodiments, the particles may comprise silica gel, and the silica gel may comprise silica. In some embodiments, the silica gel may be porous, and in certain embodiments, the silica gel may be non-porous. In some embodiments, the particles further comprise an agent that imparts paramagnetic properties to the particles. In certain embodiments, the agent comprises a metal, and in certain embodiments, the agent is a metal oxide (e.g., iron or iron oxide, wherein the iron oxide contains a mixture of fe2+ and fe3+). Antibodies may be attached to a solid support by covalent bonds or by non-covalent interactions, and may be attached directly or indirectly to a solid support (e.g., by an intermediate agent, such as a spacer molecule or biotin).

The antibodies and antigen-binding fragments thereof provided herein can be used in any known assay, such as flow cytometry, immunohistochemistry, immunofluorescence, mass flow cytometry (e.g., cytof instruments), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, monoclonal Antibodies: A Manual of Techniques, pp. -158 (CRC Press, inc. 1987). Flow cytometry and mass flow cytometry assays typically involve the use of a single primary antibody to specifically identify the presence of a target molecule expressed on the surface of a dispersion suspension of individual cells. The dispersed cells are typically obtained from a biological fluid sample, such as blood, but may also be obtained from single cell suspensions prepared from solid tissue samples (e.g., spleen or tumor biopsy). The primary antibody may be conjugated directly to a detectable moiety, such as a fluorophore, e.g., phycoerythrin, for flow cytometry, or to a heavy metal chelate for mass spectrometry. Alternatively, the first antibody may be unlabeled or labeled with an undetectable tag (e.g., biotin) and then detected by a detectably labeled second antibody that specifically recognizes the first antibody itself or a tag on the first antibody. The labeled cells are then analyzed in an instrument capable of single cell detection (e.g., flow cytometer, mass cytometer, fluorescence microscope, or bright field optical microscope) to identify those single cells in the dispersed population or tissue sample that express the target recognized by the primary antibody. For a detailed description of the technical basis and practical application of the principles of flow cytometry, see, for example, shapiro, PRACTICAL FLOW CYTOMETRY, 4 ^th Edition, wiley, 2003.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion or epitope of the protein being detected. In a sandwich assay, the analyte of the test sample is bound by a first antibody immobilized on a solid support, followed by a second antibody binding to the analyte, thereby forming an insoluble three-part complex. See, for example, U.S. Pat. No. 4,376,110. The secondary antibody itself may be labeled with a detectable moiety (direct sandwich assay), or may be measured using a detectable partially labeled anti-immunoglobulin antibody (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. In a cell ELISA, a target cell population may be attached to a solid support using antibodies that first bind to the support and recognize different cell surface proteins. These primary antibodies capture cells to a support. CEACAM6 on the cell surface can then be detected by adding any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof described herein to the captured cells and detecting the amount of anti-CEACAM 6 antibodies or antigen binding fragments thereof attached to the cells. In some cases, immobilized and permeabilized cells can be used, and in such cases, both surface CEACAM6 and intracellular CEACAM6 can be detected.

In some embodiments, any of the antibodies provided herein, or antigen binding fragments thereof, are formulated for immunohistochemical analysis. In some embodiments, the immunohistochemical analysis includes the use of a sample. In some embodiments, immunohistochemical analysis includes the use of blood and/or tissue samples. In some embodiments, the sample may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative (e.g., formaldehyde). In some embodiments, the sample is a Formalin Fixed Paraffin Embedded (FFPE) sample. In some embodiments, FFPE samples are saturated with formalin (i.e., formaldehyde) and then embedded in paraffin blocks. In some embodiments, the FFPE sample is stable at room temperature. In some embodiments, all structures in the FFPE sample are preserved. In some embodiments, intracellular and surface proteins in FFPE samples are preserved. In some embodiments, mRNA in the FFPE sample is preserved. In some embodiments, mRNA, intracellular and surface proteins in the FFPE sample are preserved. In some embodiments, the surface proteins in the FFPE sample are denatured.

In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein is capable of detecting CEACAM6 in a formalin fixed paraffin embedded sample. In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein is capable of detecting CEACAM6 on the cell surface of a formalin fixed paraffin embedded sample. In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein is capable of detecting intracellular CEACAM6 in a formalin fixed paraffin embedded sample. In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein are capable of detecting CEACAM6 in cells and CEACAM6 on the surface of formalin fixed paraffin embedded samples.

In some embodiments, the sample is a fresh sample that has been frozen. In some embodiments, the sample is a fresh sample that has been cryogenically frozen. In some embodiments, the sample is flash frozen. In some embodiments, the sample is flash frozen and stored at 80 ℃. In some embodiments, all structures in the flash frozen sample are preserved. In some embodiments, intracellular and surface proteins in the flash frozen sample are preserved. In some embodiments, mRNA in the flash frozen sample is preserved. In some embodiments, mRNA, intracellular proteins, and surface proteins in the flash-frozen sample are preserved. In some embodiments, the surface proteins in the flash frozen sample are denatured.

In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein is capable of detecting CEACAM6 in a frozen sample. In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein is capable of detecting CEACAM6 on the surface of a frozen sample. In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein is capable of detecting intracellular CEACAM6 in a frozen sample. In some embodiments, any of the anti-CEACAM 6 antibodies or antigen binding fragments thereof provided herein are capable of detecting CEACAM6 in a cell and CEACAM6 on the surface of a frozen sample.

Antibodies herein may also be used in vivo diagnostic assays. Typically, the antibodies are labeled with a radionuclide (e.g., 111In, 99Tc, 14C, 131I, 125I, 3H, 32P, or 35S) such that the bound target molecule can be localized using immunoscintigraphy.

Detection of CEACAM6

Provided herein are antibodies and methods for detecting CEACAM6. In some embodiments, antibodies and methods for detecting CEACAM6 in a biological sample are provided. In some embodiments, the biological sample is a solid tissue, fluid, or cell. In some embodiments, CEACAM6 is detected on the cell surface. In some embodiments, CEACAM6 is detected intracellularly. In some embodiments, the detection of CEACAM6 is in vitro. In some embodiments, the detection of CEACAM6 is in vivo.

The solid tissue may include solid tissue from one or more of adipose tissue, bladder, bone, brain, breast, cervical, endothelial, gall bladder, kidney, liver, lung, lymph, ovary, prostate, salivary gland, stomach, testis, thyroid, urinary tract, uterus, vagina, and vulva. In some embodiments, the fluid comprises one or more of amniotic fluid, bile, blood, breast milk, breast fluid, cerebrospinal fluid, lavage fluid, lymph, mucus, plasma, saliva, semen, serum, spinal fluid, sputum, tears, cord blood, urine, and vaginal fluid.

In some embodiments, the sample comprises immune cells. In some embodiments, the sample comprises a heterogeneous population of immune cells. In some embodiments, the immune cells are selected from the group consisting of B cells, plasmacytoid dendritic cells (pdcs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mdcs), resident lymphocytes, mast cells, eosinophils, basophils, natural killer cells, and Peripheral Blood Mononuclear Cells (PBMCs).

In some of any of the embodiments, any of the antibodies provided herein or antigen binding fragments thereof can be used to characterize a single cell by measuring the level of gene expression and cellular protein. Known single cell sequencing platforms suitable for integration with the antibodies or antigen binding fragments thereof described herein include Drop-Seq methods including, but not limited to, microfluidic, plate-based or microwell, modified versions of the Seq-WellTM method and basic protocols, and InDropTM method. In another embodiment, a single cell sequencing platform suitable for integration with an antibody or antigen binding fragment thereof described herein is the l0x genomics single cell 3' protocol or single cell V (D) J protocol, which runs on a Chromium controller or a dedicated Chromium single cell controller. Other suitable sequencing methods include WAFERGEN ICELL. TM. Methods, microwell-seq methods, fluidigm CITM methods, and equivalent single cell products. Still other known sequencing protocols that may be used with the antibodies or antigen binding fragments thereof described herein include BD ResolveTM single cell analysis platform and ddSeq (from Illumina Bio-Rad SureCellTM WTA 3' library preparation kit for ddSEQTM systems, 2017, pub No. 1070-2016-014-B, illumina inc., bio-Rad Laboratories, inc.). In still other embodiments, the antibodies or antigen binding fragments thereof described herein can be used in combinatorial indexing-based methods (sci-RNA-seqTM methods or SPLiT-seqTM methods) and spatial transcriptomics, or comparable spatially resolved sequencing methods. The methods and compositions described herein can also be used as additional information layers for standard index sorting (FACS) and mRNA sequencing-based methods.

In some of any of the embodiments, any of the antibodies described herein or antigen binding fragments thereof can be used to detect the presence, absence, or amount of the various nucleic acids, proteins, targets, oligonucleotides, amplification products, and barcodes described herein.

In some embodiments, the biological sample is from a healthy subject. In some embodiments, the sample is from a subject having a disease or condition. In some embodiments, detection of CEACAM6 indicates the presence or absence of a disease or disorder. In some embodiments, the disease or disorder is cancer, an autoimmune disorder, an inflammatory disorder, a neurological disorder, or an infection. In some embodiments, the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecological, liver, glioblastoma, hodgkin's lymphoma, chronic lymphocytic leukemia, esophageal, gastric, pancreatic, colon, renal, head-neck, lung, and melanoma.

In some embodiments, the disease or disorder is associated with CEACAM6 expression. In some embodiments, the disease or disorder is associated with aberrant CEACAM6 expression. In some embodiments, the disease or disorder is associated with Natural Killer (NK), αβ T cells, γδ T cells, cd8+ T cells, monocytes or dendritic cells. In some embodiments, the disease or disorder is associated with Natural Killer (NK) cells. In some embodiments, the disease or disorder is associated with αβ T cells. In some embodiments, the disease or disorder is associated with γδ T cells. In some embodiments, the disease or disorder is associated with cd8+ T cells. In some embodiments, the disease or disorder is associated with monocytes. In some embodiments, the disease or disorder is associated with dendritic cells. In some of any of the embodiments, the disease or disorder is selected from the group consisting of non-viral cancer, virus-associated cancer, cancer associated with HBV infection, cancer associated with Epstein Barr Virus (EBV) infection, cancer associated with polyomavirus infection, leprosy nodular Erythema (ENL), autoimmune disease, autoimmune inflammation, autoimmune thyroid disease, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemia, myelodysplastic syndrome, solid phase cancer, herpes virus infection, and/or transplanted tissue or organ rejection.

In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is metastatic melanoma, solid tumor, bladder cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, colon-derived liver metastasis, papillary thyroid carcinoma, acute myeloid leukemia, or asymptomatic myeloma.

In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the infectious disease is Human Immunodeficiency Virus (HIV), chronic hepatitis C, cytomegalovirus, or hantavirus.

In some embodiments, the disease or disorder is an autoimmune disorder. In some embodiments, the autoimmune disorder is crohn's disease, multiple sclerosis, systemic sclerosis, myasthenia gravis of the eye muscle, psoriasis, or rheumatoid arthritis. In some embodiments, the autoimmune disorder is crohn's disease, multiple sclerosis, systemic sclerosis, myasthenia gravis of the eye muscle, psoriasis, or rheumatoid arthritis.

In some embodiments, any of the antibodies or antigen binding fragments thereof may be used to generate a nucleic acid molecule comprising all or a portion of the oligonucleotide sequence or the complement thereof. In some of any of the embodiments, the antibody or antigen binding fragment thereof may be used in a method of correlating the presence or abundance of CEACAM6 to a target location of a tissue sample.

In some embodiments, any of the antibodies or antigen binding fragments thereof may be used to construct a protein library. In some of any of the embodiments, the construction of the protein library comprises sequencing. In some of any of the embodiments, the construction of the protein library comprises using flow cytometry.

In some of any of the embodiments, provided herein is a method of detecting CEACAM6 comprising

A) Contacting a sample with any of said antibodies or antigen-binding fragments thereof under conditions that allow binding of said antibodies or antigen-binding fragments thereof to CEACAM6 receptors on said sample, wherein said binding results in the production of receptor/antibody or antigen-binding fragment complexes thereof;

b) Detecting the presence of a receptor/antibody or antigen binding fragment complex thereof;

c) Wherein the detecting comprises the presence or absence of CEACAM6 receptors on the sample.

In some of any of the embodiments, provided herein is a method of treating or preventing a disease or disorder associated with CEACAM6 in a subject, comprising a) contacting a sample known or suspected to contain CEACAM6 with any of the antibodies or antigen binding fragments thereof, b) detecting the presence of a complex comprising CEACAM6 and the antibodies or antigen binding fragments thereof, wherein the presence of the complex is indicative of the presence of the disease or disorder, and c) administering any of the antibodies or antigen binding fragments thereof to the subject.

In some of any of the embodiments, provided herein is a method of diagnosing a disease or disorder comprising a) isolating a sample from a subject, b) incubating the sample with any of the antibodies or antigen-binding fragments thereof for a time sufficient to generate CEACM: anti-CEACAM 6 complexes, c) detecting the presence or absence of CEACAM6: anti-CEACAM 6 complexes from the isolated tissue, and d) correlating the presence or abundance of CEACAM6 to a target location of the tissue sample.

In some of any of the embodiments, an increase in CEACAM6 relative to a control level in a target location of the tissue sample is indicative of a disease or disorder in the subject.

In some of any of the embodiments, hybridization of the detectable moiety to the antibody or antigen binding fragment thereof is detected. In some of any of the embodiments, the sample is contacted with a second antibody. In some of any of the embodiments, the second antibody is an antibody comprising a detectable moiety. In some of any of the embodiments, the detectable moiety comprises an oligonucleotide. In some of any of the embodiments, the detectable moiety comprises a fluorescent tag. In some of any of the embodiments, measuring comprises sequencing. In some of any of the embodiments, the detectable moiety comprises immunofluorescence. In some of any of the embodiments, the sample is a formalin fixed paraffin embedded sample. In some of any of the embodiments, the sample comprises cells. In some of any of the embodiments, the sample comprises a tissue sample.

XI kit comprising anti-antibodies

The antibodies herein may be provided in a kit, for example, a predetermined amount of reagents in combination with packaging for instructions for use (e.g., instructions for performing a diagnostic assay; instructions for performing a laboratory assay). In some embodiments, the kit is a diagnostic kit configured to detect CEACAM6 in a sample (e.g., a biological sample). In the case of antibodies labeled with fluorophores, the kit may include negative control unrelated antibodies of the same isotype to exclude non-specific binding of the antibodies. In the case of antibodies labeled with an enzyme, the kit may include the substrate and cofactor required by the enzyme (e.g., a substrate precursor providing a detectable chromophore or fluorophore). Additional additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), and the like. The relative amounts of the various reagents can be widely varied to provide concentrations in the solution of the reagents that significantly optimize the sensitivity of the assay. In some cases, the reagents may be provided as a dry powder (e.g., lyophilized powder), including excipients that, when dissolved, will provide a reagent solution having a suitable concentration.

Examples

Example 1 production of hybridomas expressing anti-CEACAM 6 antibodies

This example describes the generation and characterization of hybridomas secreting monoclonal antibodies reactive with CEACAM 6.

Briefly, animals were immunized with CEACAM6 immunogen (recombinant protein of amino acid numbers 35-320 of SEQ ID NO: 1) and myeloma cells were fused with spleen to form hybridomas using standard protocols, and lymph node cells were drained and harvested. Successful fusions were selected into HAT medium and cloned into microtiter plates to approximately 1 cell/well, after which culture supernatants were tested by flow cytometry for CEACAM6 expressing cell transfectants. Wells were selected by evaluating staining characteristics and then subcultured into larger containers and subcloned. Hybridoma subclones were further characterized by flow cytometry using CEACAM 6-transfected cells. Candidate clones expressing exemplary anti-CEACAM 6 antibodies are selected and screened using various methods, including by flow cytometry for human blood cells (e.g., lymphocytes, monocytes, etc.) separated into different subsets and for one or more cell lines generated from disease and/or infected human cells. The percentage of positive cells in each blood cell subset was quantified compared to isotype control.

Example 2 sequencing of variable regions of exemplary anti-CEACAM 6 antibodies

This example describes the sequencing of the exemplary anti-CEACAM 6 antibodies generated in example 1 above.

Cells from the anti-CEACAM 6 hybridoma cell line described in example 1 above were grown in standard mammalian tissue culture medium. Total RNA was isolated from hybridoma cells from various clones expressing anti-CEACAM 6 monoclonal antibodies using a RNEASY MINI kit (Qiagen) based procedure. Sequencing hybridoma cells were used for heavy and light chain VDJ rearrangements. Briefly, RNA is used to generate the first strand of cDNA of the light and heavy chain variable domains. Both light and heavy chain variable domain cdnas were amplified by template switching techniques using gene-specific C-primers to enrich for variable gene sequences. Amplified libraries were sequenced using Illumina next generation sequencing on a 500 cycle kit using single ended reads from Read 1.

The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the respective variable domains (CDRs and framework regions) comprising the heavy and light chains of two different antibodies (clones) are shown in table 1 and the CDR sets of the anti-CEACAM 6 antibodies are shown in table 2.

EXAMPLE 3 detection of CEACAM6 expressing cells Using exemplary anti-CEACAM antibodies

This example describes the ability of generated exemplary anti-CEACAM 6 antibodies to detect CEACAM6 expressing cells by flow cytometry and Immunohistochemistry (IHC).

In the first experiment, cross-reactivity of exemplary anti-CEACAM 6 antibodies with another CEACAM family was evaluated on cells from an immortalized rat colorectal lymphoblastic cell line (RBL-1, ATCC CRL-1378) transfected with human CEACAM8 (also known as CD66 b).

RBL-1 cells were grown to about 80% confluency in EMEM medium supplemented with 10% FBS in T75 flasks. Cells were suspended in cell staining buffer and 1 ug or 0.1 ug anti-CEACAM 6 antibodies AB1 and AB2 were added and allowed to incubate for 15 minutes. The cells were then washed twice with FACS wash buffer and stained with anti-rat IgG-APC secondary antibody for 15 minutes. Cells were washed with FACS buffer and analyzed on a BD LSRII flow cytometer. Two commercially available anti-CEACAM 6 antibodies were used as negative controls (REA 414 and ASL-32). As shown in fig. 1, the exemplary test anti-CEACAM 6 antibodies AB1 and AB2 did not show any binding to CEACAM8 and thus may be considered to lack cross-reactivity with CEACAM 8.

Next, exemplary antibodies were assayed for their ability to bind CEACAM6 on leukocytes (lymphocytes, monocytes, and granulocytes) isolated from healthy volunteer donors. In whole blood, leukocytes were incubated with anti-CEACAM 6 antibodies AB1 and AB2, respectively, followed by lysis of erythrocytes. The lysed blood was washed twice with FACS wash buffer and anti-CEACAM 6 antibodies were detected with PE-labeled anti-rat IgG secondary antibodies (PE-a, in fig. 2 and 3) or with APC-labeled anti-rat IgG secondary antibodies (fig. 4) for 15 minutes. Cells were washed with FACS buffer and analyzed on a BD LSRII flow cytometer. As shown in fig. 2, exemplary test anti-CEACAM 6 antibodies AB1 (fig. 2A) and AB2 (fig. 2B) did not stain lymphocytes, depending on the very low or no signal density in quadrants Q2 and Q6. As shown in fig. 3, antibodies AB1 (fig. 3A) and AB2 (fig. 3B) both detected CEACAM6 expression on monocytes and granulocytes based on the high signal density in quadrant Q2. As shown in fig. 4, both antibodies AB1 and AB2 showed significantly brighter signal intensity on granulocytes at 0.1 ug compared to the two reference antibodies REA414 and ASL-32.

Example 4 evaluation of antibody blocking ability of exemplary anti-CEACAM 6 antibodies

This example describes the ability of an exemplary anti-CEACAM 6 antibody to block binding of a commercially available anti-CEACAM 6 antibody.

Leukocytes (lymphocytes, monocytes and granulocytes) were isolated from healthy volunteer donors. The leukocytes were incubated with anti-CEACAM 6 antibody for 15 min, followed by the addition of PE-labeled reference antibody KOR-SA3544. After erythrocyte lysis, the cells were washed twice with FACS buffer and analyzed on a BD LSRII flow cytometer. As shown in FIG. 5, both antibodies AB1 (FIG. 5B) and AB2 (FIG. 5A) were able to block binding of reference antibody KOR-SA3544. In a similar experiment, leukocytes were incubated with anti-CEACAM 6 antibodies AB1 and AB2 for 15 minutes, followed by the addition of PE-labeled reference antibody ASL-32, which binds CD66a/c/e or 6/40c. As shown in FIG. 6, both antibodies blocked the binding of reference antibody ASL-32 to CEACAM6 (FIGS. 6C-D), but did not block the binding of reference antibody 6/40C to CEACAM8 (FIGS. 6A-B). FIG. 7 shows the mean fluorescence intensities (Y-axis) of PE-labeled anti-CEACAM 6 antibodies AB1 and AB2 at different concentrations (X-axis: concentration of antibody per million cells) compared to the mean fluorescence intensity of PE-labeled reference antibody KOR-SA3544 (measured at a single concentration). At a concentration of 1 antibody per million cells, both anti-CEACAM 6 antibodies AB1 and AB2 showed a higher average fluorescence intensity than KOR-SA3544.

To confirm the specificity of the two anti-CEACAM 6 antibodies for CEACAM6 and to evaluate cross-reactivity with other CEACAM family members, anti-CEACAM 6 antibodies AB1 and AB2 were tested on human lung cancer cell line a549 (ATCC a549 CCL-185) expressing CEACAM6 and human monocyte line U-937 (ATCC U-937 CRL-1593.2) expressing CEACAM3, epidermoid carcinoma cell line a431 (ATCC CRL-1555) expressing CEACAM3, human monocyte leukemia cell line THP-1 expressing CEACAM4, and human prostate cancer cell line LNCaP (ATCC CRL-1740) expressing CEACAM 5. As shown in fig. 8, the anti-CEACAM 6 antibody AB1 reacted specifically with CEACAM6 according to the right shift of the peak compared to the isotype control in fig. 8A, and did not cross-react with any other CEACAM family members according to the absence of any right shift of the peak compared to the isotype control in fig. 8B-D. According to the right shift of the peak compared to isotype control in fig. 8A, B and D, antibody AB2 reacted specifically with both CEACAM6 and CEACAM 4.

In another experiment, the ability of exemplary anti-CEACAM 6 antibodies AB1 and AB2 to stain CEACAM6 protein in Formalin Fixed Paraffin Embedded (FFPE) samples was evaluated by Immunohistochemistry (IHC). 5 μm sections of FFPE samples of human colon were dewaxed with xylene and reconstituted in gradient ethanol. Heat-mediated antigen retrieval was performed using sodium citrate pH 6.0 at 90 ℃ for 30 minutes. Samples were permeabilized with 0.1% Triton X-100 in PBS for 30min and blocked with 5% FBS in PBS for 1 h. Samples were stained overnight with 5 μg/ml of purified exemplary anti-human CEACAM6 antibodies AB1 or AB2 at 4 ℃. The tissue sections were then washed with PBS and stained with 2.5ug/ml Alexa-555 conjugated anti-rat IgG dye in a dark room for 1 hour at room temperature, followed by two washes in PBS. Samples were imaged using Antifade gold seals with DAPI and using metagraph software and analyzed using Image J. As shown in fig. 9, according to the fluorescence signal detected in the left fluorescence image of fig. 9, the anti-CEACAM 6 antibody AB1 was able to stain CEACAM6 protein in the human colon paraffin section, whereas the anti-CEACAM 6 antibody AB2 was unable to stain CEACAM6 in the human colon paraffin section (right fluorescence image).

In another experiment, the ability of an exemplary anti-CEACAM 6 antibody AB1 to stain CEACAM6 in fixed cells was evaluated by Immunohistochemistry (ICC). Human lung adenocarcinoma cell line a549 was grown on 96-well plates with coverslips bottom and fixed with fixing buffer for 30 minutes. Cells were washed twice with PBS and stained with antibody AB1, followed by Alexa Fluor 555 anti-rat IgG antibody. Nuclei were counterstained with DAPI dye. Cells were imaged using a 40x objective. As shown in fig. 10, anti-CEACAM 6 antibody AB1 detected CEACAM6 protein expression in a549 cells based on the fluorescent signal in the fluorescent images on the left and middle of fig. 10, compared to no fluorescent signal in the isotype control (fig. 10, right).

Example 5 evaluation of functional Activity of exemplary anti-CEACAM 6 antibodies

This example describes functional evaluation of an exemplary anti-CEACAM 6 antibody AB1 as measured by inhibition of CEACAM6 activity.

This example describes the functional evaluation of an exemplary anti-CEACAM 6 antibody AB1 as measured by its effect on cancer cell invasion compared to the commercially available antibody (reference 1H 7-4B). The effect of anti-CEACAM 6 on the ability to block cell invasion through the extracellular matrix was tested by matrigel invasion assay. Human lung cancer cell line a549 (ATCC a549 CCL-185) was serum starved for 24h, after which 5x10 ⁵ cells were resuspended in serum-free DMEM medium containing 20, 10 and 5ug/ml AB1 or isotype control antibody (isotype) and plated on top wells of Corning BioCoat Matrigel invasion chamber. DMEM medium containing 20% serum was placed in the bottom wells as a chemoattractant. After 16h, cells were removed from the top layer wells with cotton swabs and cells that had migrated to the bottom of the membrane were counted. Each experiment was performed in duplicate and 3 images were counted using 910xo objective lens for each condition using the cell counter function of Image J software. As shown in fig. 11, AB1 blocked a549 cells from invading matrigel and migrating to the bottom of the membrane at 20, 10, and 5ug/ml, according to significantly reduced cell count per field for all three concentrations. In contrast, the commercially available antibody 1H7-4B did not block invasion at 20ug/ml (reference 20 ug/ml) based on the equally high number of cells per field counted, compared to the isotype control (isotype 20 ug/ml).

The ability of anti-CEACAM 6 antibody AB1 to inhibit cancer cell migration was tested by a wound healing assay. Human lung adenocarcinoma cell line a549 was grown to confluence and streaked out using a 10 ul pipette tip. anti-CEACAM 6 antibody AB1 was added at 20, 10 or 5 ug/ml. Rat IgG2a, k isotype control antibodies were used as controls. After 16h, gap width was measured using Image J and the percentage of gap closure normalized to isotype control was calculated. As shown in FIG. 12, the anti-CEACAM 6 antibody AB1 inhibited lung cancer cell migration by more than 50% at 20 and 10 ug/ml.

In another experiment, the anti-CEACAM 6 antibody AB1 was tested for its ability to induce intracellular signaling through CEACAM6 activation. The effect of antibody AB1 on inducing intracellular signaling by cross-linking was tested on human lung adenocarcinoma cell line a 549. Cells were grown on 96-well plates with coverslips bottom and treated with 5 ug/ml of anti-human CEACAM6 antibody AB1 or rat IgG2a, k isotype control antibody at 37 ℃ for 15 minutes followed by anti-rat IgG secondary antibody (5 ug/ml) at 37 ℃ for 30 minutes. Cells were fixed with the fixation buffer for 30 min. Cells were washed twice with 1x intracellular staining permeabilization wash buffer, then stained with anti-AKT Phospho (Ser 473) antibody from mice, followed by Alexa Fluor 555 anti-mouse IgG dye, flash Phalloidin ^TM Red 594 dye and DAPI dye. Flash Phalloidin ^TM Red 594 dye is a probe for imaging and stabilizing filiform F-actin in fixed and permeabilized cells, thereby providing a background for cell structure and volume determination. As shown in fig. 13, cross-linking CEACAM6 with anti-CEACAM 6 antibody AB1 induced intracellular signaling, which resulted in increased Akt kinase phosphorylation and increased actin polymerization. The conclusion is based on the increase in fluorescence signal and intensity around the nucleus when comparing the AB1 fluorescence image (top left and bottom left of fig. 13) with the corresponding isotype control fluorescence image (top right and bottom right of fig. 13).

The examples herein are provided to illustrate embodiments of the disclosure, but not to limit the scope thereof. Other variations of the present disclosure will be apparent to those of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, internet resources, patents, patent applications, and search numbers cited herein are incorporated by reference in their entirety for all purposes.

Claims

1. An isolated antibody or antigen binding fragment thereof that binds CEACAM6 or a portion thereof, wherein the antibody comprises (i) an immunoglobulin heavy chain comprising a set of heavy chain Complementarity Determining Region (CDR) amino acid sequences CDRH1, CDRH2, and CDRH3, and (ii) an immunoglobulin light chain comprising a set of light chain CDR amino acid sequences CDRL, CDRL2, and CDRL3, wherein the set of heavy and light chain CDRs are each selected from the same set 1 or set 2 as follows:

。

2. The antibody or antigen binding fragment thereof of claim 1, wherein the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID No. 4 or a sequence having at least 80% amino acid sequence identity to SEQ ID No. 4, and wherein the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID No. 5 or a sequence having at least 80% amino acid sequence identity to SEQ ID No. 5.

3. The antibody or antigen binding fragment thereof of claim 1, wherein the immunoglobulin heavy chain comprises an amino acid sequence shown in SEQ ID No. 2 or a sequence having at least 80% amino acid sequence identity to SEQ ID No. 2, and wherein the immunoglobulin light chain comprises an amino acid sequence shown in SEQ ID No. 3 or a sequence having at least 80% amino acid sequence identity to SEQ ID No. 3.

4. A diagnostic antibody or antigen-binding fragment thereof comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.

5. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-4.

6. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4 and a pharmaceutically acceptable excipient.

7. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of the antibody or antigen binding fragment thereof of any one of claims 1-4.

8. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of the antibody or antigen binding fragment thereof of any one of claims 1-4.

9. A recombinant expression vector comprising the isolated nucleic acid of claim 7 and/or the isolated nucleic acid of claim 8.

10. A host cell comprising the nucleic acid of claim 7 and/or the nucleic acid of claim 8, or the expression vector of claim 8.

11. An isolated nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising SEQ ID NO. 4 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 4, or an immunoglobulin light chain comprising SEQ ID NO. 5 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 5.

12. A recombinant expression vector comprising the isolated nucleic acid of claim 11.

13. A host cell comprising the nucleic acid of claim 11 or the expression vector of claim 12.

14. An isolated nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising SEQ ID NO.2 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO.2, or an immunoglobulin light chain comprising SEQ ID NO. 3 or a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO. 3.

15. A recombinant expression vector comprising the isolated nucleic acid of claim 14.

16. A host cell comprising the nucleic acid of claim 14 or the expression vector of claim 15.

17. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain according to any one of claims 1-4, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of the antibody or antigen binding fragment thereof according to any one of claims 1-4.

18. A host cell comprising the recombinant expression vector of claim 17.

19. A method of detecting CEACAM6, the method comprising contacting a sample with the antibody or antigen binding fragment thereof of any one of claims 1-4 under conditions that allow the antibody or antigen binding fragment thereof to bind to CEACAM6 receptors in the sample, wherein the binding results in the production of a receptor/antibody or antigen binding fragment complex thereof.